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WO2022140399A1 - Altering plant calcium transport to improve plant anoxia tolerance - Google Patents

Altering plant calcium transport to improve plant anoxia tolerance Download PDF

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Publication number
WO2022140399A1
WO2022140399A1 PCT/US2021/064628 US2021064628W WO2022140399A1 WO 2022140399 A1 WO2022140399 A1 WO 2022140399A1 US 2021064628 W US2021064628 W US 2021064628W WO 2022140399 A1 WO2022140399 A1 WO 2022140399A1
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Prior art keywords
plants
plant
genetically modified
progeny
cax1
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French (fr)
Inventor
Kendal HIRSCHI
Jian Yang
Christine DOBBINS
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Baylor College of Medicine
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Baylor College of Medicine
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/12—Processes for modifying agronomic input traits, e.g. crop yield
    • A01H1/122—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance

Definitions

  • the field of the disclosure includes plant biology, yeast biology, agriculture, agronomy, molecular biology, and/or cell biology, for example.
  • ERFs Ethylene Response Factors
  • ROS Reactive Oxygen Species
  • Ca signaling differs among plants with marked differences between Arabidopsis thaliana and rice in the spatiotemporal parameters of the observed Ca signatures (10). Ca signaling differences appear to precede gene expression differences and metabolic fluxes, although precisely how Ca is triggering downstream events remains unclear (1). Conceptualizing signaling events involved in anoxia perception and tolerance is important to improve plant performance both during and after anoxia conditions.
  • Calcium is at the nexus between many facets of plant biology (11, 12). Calcium transporters on various membranes help to orchestrate responses to stresses such as changes in temperature and anoxia (11, 13-15). Calcium pumps and exchangers are found in multiple membrane systems, including the endoplasmic reticulum (ER), vacuole, and plasma membrane (PM). Calcium pumps (such as the Ca2+-ATPases) are considered low-capacity, high-affinity efflux systems, but within this broad group of transporters, H+/Ca2+ exchangers (CAXs) are distinct because they have high capacity and low affinity. While both Ca pumps and CAXs remove Ca from the cytoplasm, their individual contributions to the control of the magnitude or duration of different stimulus-specific Ca signals remains enigmatic.
  • CAX1 and CAX3 are expressed in aerial tissue and is the dominant mechanism for Ca accumulation in the leaf (17); CAX3 is primarily expressed in the roots (18). Expression of CAX3 is increased in aerial tissue during stresses which may help remove increased Ca levels from the cytosol.
  • CAX1 and CAX3 may function as heterodimers (18); furthermore, the two transporters are autoinhibited and co-expressed in guard cells where they may function singly or together in intracellular signaling (19). Absence of CAX1 induces ectopic expression of other CAXs, which is often sufficient to maintain mesophyll cellular uptake of Ca from the apoplast (20).
  • CAX transporters There is also interplay between CAX transporters and other transporters; deletions in CAX1 reduce vacuolar H+/Ca2+ antiport activity by 50%, decrease vacuolar V- type proton ATPase activity by 40% and increase vacuolar Ca-ATPase activity by 36% (21). Given the functional connectivity among the various transporters, the prevailing wisdom suggests that CAXs are important for viability (22).
  • the present disclosure concerns methods and compositions that address the management of plant tolerance to anoxia and production of plants with an increased tolerance to anoxia.
  • Anoxic conditions can be detrimental to plant development and yield. Recovery post-anoxia is also an important determinant of tolerance, yet mechanisms regulating this remain largely unknown. Anoxic responses have been extensively studied (4); however, less is known about the processes governing recovery from this stress and the signals that mediate these responses. Traditional approaches to study these events include differential gene expression studies and comparative genomic analysis (23).
  • an Arabidopsis thaliana mutant defective in endomembrane calcium transport that dramatically improves recovery from anoxic conditions has been identified and characterized.
  • the present disclosure is directed to compositions and methods for regulating such calcium transporters during anoxic conditions as a means of improving plant development and survival during periods of oxygen deprivation.
  • such compositions and methods are useful as a means of improving crop performance during periods of oxygen deprivation.
  • Embodiments of the disclosure include methods and compositions related to tolerance to anoxia, including tolerance that is acquired because of one or more genetic modifications by the hand of man.
  • the genetic modification may be applied to any kind of plant.
  • expression of one or more CAX genes is genetically modified by the hand of man to impart anoxia tolerance to the modified plant as compared to a plant of the same kind that lacks the recombinantly-introduced genetic modification.
  • expression of one or more CAX genes is genetically modified by the hand of man to impart anoxia tolerance to the modified plant as compared to a plant of the same kind that lacks the genetic modification.
  • expression of one or more CAX genes is genetically modified by the hand of man to impart anoxia tolerance to the modified plant as compared to a plant of the same kind that lacks the synthetic genetic modification.
  • the one or more CAX genes that is modified is CAX1.
  • the one or more CAX genes that is modified is CAX1 and one or more other genes, including one or more other CAX genes, such as CAX2, CAX3, and/or CAX4.
  • expression of a gene capable of synthesis of one or more activators of one or more CAX genes is genetically modified by the hand of man to impart anoxia tolerance to the modified plant as compared to a plant of the same kind that lacks the recombinantly-introduced genetic modification.
  • the gene capable of synthesis of one or more activators of one or more CAX genes that is modified is a gene capable of synthesis of one or more activators of CAX1.
  • the gene capable of synthesis of one or more activators of one or more CAX genes that is modified is a gene capable of synthesis of one or more activators of CAX1 and one or more activators of one or more other genes, including one or more other CAX genes, such as CAX2, CAX3, and/or CAX4.
  • the at least one activator of CAX1 is selected from the group consisting of SOS2 protein kinase, CXIP1, and CXIP4.
  • the disclosure encompasses anoxia tolerance for monocots and/or dicots and may encompass bryophytes (mosses), pteridophytes (ferns), gymnosperms (conifers), and angiosperms (flowering, seed-bearing plants), at least.
  • one or more crop plants are modified as disclosed herein so that they may be tolerant to anoxia as compared to crop plants without the recombinantly-introduced genetic modification or they may have enhanced tolerance compared to the level of tolerance they naturally had without the genetic modification.
  • a composition of the disclosure includes a genetically modified plant, part, and/or progeny thereof, excluding Arabidopsis thaliana, including a plurality of plant cells including: synthetically suppressed and/or eliminated expression of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4, in said plurality of plant cells.
  • the plurality of plant cells include: one or more agents capable of driving gene silencing of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4, in said plurality of plant cells; and, optionally, a regulatory sequence capable of inducing said agent(s) to drive said gene silencing of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4, in said plurality of plant cells.
  • the agent(s) include a nucleic acid sequence and/or a protein.
  • the nucleic acid sequence includes exogenous DNA, intragenic DNA, and/or exogenous RNA.
  • the protein includes a nuclease and/or a protease.
  • the agent(s) and, optionally, the regulatory sequence are (a) naked; and/or (b) comprised in (i) a complex; (ii) a carrier system; (iii) a particle gun system; (iv) a viral vector; (v) an Agrobacterium vector; and/or (vi) a CRISPR vector.
  • the agent(s) are capable of driving RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4.
  • the agent(s) are capable of expressing double stranded RNA (dsRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), small RNA (sRNA), microMRNA (mRNA), pre-miRNA, and/or pri-miRNA, capable of RNA interference (RNAi) of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4.
  • dsRNA double stranded RNA
  • hpRNA hairpin RNA
  • siRNA small interfering RNA
  • sRNA small RNA
  • mRNA microMRNA
  • pre-miRNA pre-miRNA
  • pri-miRNA capable of RNA interference (RNAi) of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4.
  • the agent(s) include double stranded RNA (dsRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), small RNA (sRNA), microMRNA (mRNA), pre-miRNA, and/or pri-miRNA, capable of RNA interference (RNAi) of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4.
  • dsRNA double stranded RNA
  • hpRNA hairpin RNA
  • siRNA small interfering RNA
  • sRNA small RNA
  • mRNA microMRNA
  • pre-miRNA pre-miRNA
  • pri-miRNA capable of RNA interference (RNAi) of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4.
  • regulator sequence is capable of inducing said agent(s) to drive RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4.
  • RNAi RNA interference
  • TGS transcriptional gene silencing
  • VIGS virus induced gene silencing
  • the regulatory sequence is capable of inducing said agent(s) to express double stranded RNA (dsRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), small RNA (sRNA), microMRNA (mRNA), pre-miRNA, and/or pri-miRNA, capable of RNA interference (RNAi) of CAX1 and, optionally, RNAi of at least one of CAX2, CAX3, or CAX4.
  • dsRNA double stranded RNA
  • hpRNA hairpin RNA
  • siRNA small interfering RNA
  • sRNA small RNA
  • sRNA small RNA
  • mRNA microMRNA
  • pre-miRNA pre-miRNA
  • pri-miRNA capable of RNA interference (RNAi) of CAX1 and, optionally, RNAi of at least one of CAX2, CAX3, or CAX4.
  • the regulatory sequence is tissue specific and/or cell-type specific.
  • the regulatory sequence is low oxygen-induced,
  • the plurality of plant cells includes suppressed or eliminated expression of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4 before, during, and/or after anoxic conditions.
  • the agent(s) are capable of driving RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4 before, during, and/or after anoxic conditions.
  • the plurality of plant cells include transient and/or stable suppressed or eliminated expression of CAX1 and, optionally, RNAi of at least one of CAX2, CAX3, or CAX4.
  • the agent(s) are capable of driving transient and/or stable RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of CAX1 and, optionally, RNAi, TGS, and/or VIGS of at least one of CAX2, CAX3, or CAX4.
  • RNAi transient and/or stable RNA interference
  • TGS transcriptional gene silencing
  • VIGS virus induced gene silencing
  • the plurality of plant cells have upregulated expression during anoxia of at least one gene selected from the group consisting of: AT 1G01355; AT1G02610; AT1G02620; AT1G03090; AT1G03610; AT1G06570; AT1G08630;
  • AT1G08930 AT1G10070; AT1G10140; AT1G10550; AT1G11260; AT1G12780;
  • AT1G32910 AT1G33050; AT1G33055; AT1G34140; AT1G34760; AT1G35140;
  • AT1G60750 AT1G62480; AT1G62510; AT1G63090; AT1G65970; AT1G66860;
  • AT3G1331O AT3G13450; AT3G15440; AT3G15450; AT3G15620; AT3G16770;
  • AT5G26200 AT5G27893; AT5G28610; AT5G28630; AT5G28770; AT5G34830;
  • the plurality of plant cells have upregulated expression during recovery from anoxia of at least one gene selected from the group consisting of:
  • AT1G19620 AT1G21340; AT1G21940; AT1G22110; AT1G26800; AT1G27730;
  • AT1G28760 AT1G30070; AT1G30135; AT1G31370; AT1G32910; AT1G32928;
  • AT1G71000 AT1G71520; AT1G72060; AT1G72416; AT1G72660; AT1G72760;
  • AT2G46830 AT2G47180; AT2G47520; AT3G02550; AT3G06435; AT3G07090;
  • AT3G14200 AT3G15440; AT3G15450; AT3G15500; AT3G16050; AT3G17400;
  • AT3G24500 AT3G25250; AT3G27220; AT3G28210; AT3G28740; AT3G29370;
  • AT4G02690 AT4G09150; AT4G10250; AT4G10265; AT4G10270; AT4G 11660;
  • AT4G12400 AT4G12410; AT4G13395; AT4G15280; AT4G 15420; AT4G15760;
  • AT4G27450 AT4G27652; AT4G27654; AT4G27657; AT4G27670; AT4G28811;
  • AT4G38030 AT5G01740; AT5G02170; AT5G02810; AT5G03210; AT5G03720;
  • AT5G22680 AT5G22920; AT5G25450; AT5G27660; AT5G35320; AT5G37340;
  • AT5G45640 AT5G47220; AT5G47590; AT5G47830; AT5G48570; AT5G49920;
  • AT5G53680 AT5G54165; AT5G57260; AT5G57550; AT5G57560; AT5G57660;
  • ATMG00400 ATMG00410; ATMG00480; ATMG00510; ATMG00513; ATMG00516;
  • the genetically modified plant, part, and/or progeny thereof is selected from the group consisting of whole plants, seedlings, leaves, stems, flowers, roots, fruits, seeds, spores, grafts comprising genetically modified scions and/or rootstocks, buddings comprising genetically modified buds and/or rootstocks, cuttings, bulbs, tubers, rhizomes, regenerable cells, tissue cultures of regenerable cells, regenerable protoplasts, tissue cultures of regenerable protoplasts, and other plant germplasms.
  • the genetically modified plant, part, and/or progeny thereof is selected from the group consisting of the Viridiplantae, the Glaucophyta, and the Rhodophyta.
  • the Viridiplantae are selected from the group consisting of green algae, hornworts, liverworts, mosses, ferns, lycophytes, gymnosperms, and angiosperms.
  • the genetically modified plant, part, and/or progeny thereof is monocotyledonous or dicotyledonous.
  • the genetically modified plant, part, and/or progeny thereof is selected from the group consisting of herbs, shrubs, trees, and vines. In specific embodiments, the genetically modified plant, part, and/or progeny thereof, excludes research plants, parts, and/or progeny thereof.
  • the genetically modified plant, part, and/or progeny thereof is a crop plant capable of producing a crop product.
  • the crop plant is selected from the group consisting of food crop plants, forage crop plants, fodder crop plants, medicinal crop plants, industrial crop plants, energy crop plants, and ornamental crop plants.
  • the crop product of said ornamental crop plant is selected from the group consisting of foliage-, flower-, and/or fruit-producing herbs, shrubs, trees, vines, and regenerable parts thereof.
  • the crop product is selected from the group consisting of cereal grains, legumes, vegetables, nuts, seeds, roots, tubers, rhizomes, flowers, fruits, timber, plant leaves, plant oils, plant fats, plant fibers, plant juices, plant extracts, and combinations thereof.
  • the crop plant is selected from the group consisting of rice plants, maize plants, barley plants, oat plants, rye plants, wheat plants, com plants, sorghum plants, soybean plants, pea plants, lentil plants, curcurbit plants, coffee plants, cocoa plants, rapeseed plants, sunflower plants, sugar cane plants, potato plants, palm plants, grape plants, apple plants, banana plants, plantain plants, cassava plants, sugar beet plants, tomato plants, sweet potato plants, yam plants, tobacco plants, cotton plants, rubber plants, tea plants, lettuce plants, pepper plants, onion plants, grape plants, pecan trees, timber trees, cannabis plants, poppy plants, and combinations thereof.
  • the genetically modified plant, part, and/or progeny thereof is not cultivated for harvest of crops.
  • the genetically modified plant, part, and/or progeny thereof, not cultivated for harvest crops is selected from the group consisting of Arabidopsis halleri, Arabidopsis lyrata, Eutrema salsugineum, Cardamine hirsute, the Viridiplantae, the Glaucophytes, and the Rhodophytes.
  • a composition of the disclosure includes an inbred, hybrid, or varietal plant, part, or progeny thereof, of the genetically modified plant, part, or progeny thereof.
  • a composition of the disclosure includes a genetically modified plant, part, and/or progeny thereof, propagated and/or regenerated from the seeds, spores, grafts comprising genetically modified scions and/or rootstocks, buddings comprising genetically modified buds and/or rootstocks, cuttings, bulbs, tubers, rhizomes, regenerable cells, tissue cultures of regenerable cells, regenerable protoplasts, tissue cultures of regenerable protoplasts, and/or other plant germplasms of the genetically modified plant, part, and/or progeny thereof.
  • a composition of the disclosure includes a genetically modified seed produced by the genetically modified plant, part, and/or progeny thereof.
  • a composition of the disclosure includes a genetically modified plant, part, and/or progeny thereof, excluding Arabidopsis thaliana, including a plurality of plant cells including: synthetically suppressed and/or eliminated synthesis of an activator of CAX1 and, optionally, an activator of at least one of CAX2, CAX3, or CAX4, in said plurality of plant cells.
  • the plurality of plant cells include: one or more agents capable of driving gene silencing of a gene capable of synthesis of said activator of CAX1 and, optionally, a gene capable of synthesis of said activator of at at least one of CAX2, CAX3, or CAX4, in said plurality of plant cells; and, optionally, a regulatory sequence capable of inducing said agent(s) to drive said gene silencing of said gene capable of synthesis of said activator of CAX1 and, optionally, said gene capable of synthesis of said activator of at at least one of CAX2, CAX3, or CAX4, in said plurality of plant cells.
  • the agent(s) include a nucleic acid sequence and/or a protein.
  • activator of CAX1 is capable of binding to the N-terminus of CAX1, and wherein said activator of at least one of CAX2, CAX3, or CAX4 is capable of binding to the N-terminus of at least one of CAX2, CAX3, or CAX4.
  • activator of CAX1 is selected from the group consisting of SOS2 protein kinase, CXIP1, and CXIP4.
  • the nucleic acid sequence includes exogenous DNA, intragenic DNA, and/or exogenous RNA.
  • the protein includes a nuclease and/or a protease.
  • the agent(s) and, optionally, said regulatory sequence are (a) naked; and/or (b) comprised in (i) a complex; (ii) a carrier system; (iii) a particle gun system; (iv) a viral vector; (v) an Agrobacterium vector; and/or (vi) a CRISPR vector.
  • the agent(s) are capable of driving RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of said gene capable of synthesis of said activator of CAX1 and, optionally, said gene capable of synthesis of said activator of at at least one of CAX2, CAX3, or CAX4.
  • the agent(s) are capable of expressing double stranded RNA (dsRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), small RNA (sRNA), microMRNA (mRNA), pre-miRNA, and/or pri-miRNA, capable of RNA interference (RNAi) of said gene capable of synthesis of said activator of CAX1 and, optionally, said gene capable of synthesis of said activator of at at least one of CAX2, CAX3, or CAX4.
  • dsRNA double stranded RNA
  • hpRNA hairpin RNA
  • siRNA small interfering RNA
  • sRNA small RNA
  • mRNA microMRNA
  • pre-miRNA pre-miRNA
  • pri-miRNA capable of RNA interference (RNAi) of said gene capable of synthesis of said activator of CAX1 and, optionally, said gene capable of synthesis of said activator of at at least one of CAX2, CAX3, or CAX4.
  • the agent(s) include double stranded RNA (dsRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), small RNA (sRNA), microMRNA (mRNA), pre-miRNA, and/or pri-miRNA capable of RNA interference (RNAi) of said gene capable of synthesis of said activator of CAX1 and, optionally, said gene capable of synthesis of said activator of at at least one of CAX2, CAX3, or CAX4.
  • dsRNA double stranded RNA
  • hpRNA hairpin RNA
  • siRNA small interfering RNA
  • sRNA small RNA
  • mRNA microMRNA
  • pre-miRNA pre-miRNA
  • pri-miRNA capable of RNA interference (RNAi) of said gene capable of synthesis of said activator of CAX1 and, optionally, said gene capable of synthesis of said activator of at at least one of CAX2, CAX3, or CAX4.
  • the regulatory agent is capable of inducing said agent(s) to drive RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of said gene capable of synthesis of said activator of CAX1 and, optionally, said gene capable of synthesis of said activator of at at least one of CAX2, CAX3, or CAX4.
  • RNAi RNA interference
  • TGS transcriptional gene silencing
  • VIGS virus induced gene silencing
  • the regulatory sequence is capable of inducing said agent(s) to express double stranded RNA (dsRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), small RNA (sRNA), microMRNA (mRNA), pre-miRNA, and/or pri-miRNA, capable of RNA interference (RNAi) of said gene capable of synthesis of said activator of CAX1 and, optionally, said gene capable of synthesis of said activator of at at least one of CAX2, CAX3, or CAX4.
  • the said regulatory sequence is tissue specific and/or cell-type specific.
  • the said regulatory sequence is low oxygen-induced, chemical-induced, temperature-induced, and/or light-induced.
  • the plurality of plant cells have suppressed and/or eliminated synthesis of said activator of CAX1 and, optionally, said activator of at least one of CAX2, CAX3, or CAX4, before, during, and/or after anoxic conditions.
  • the agent(s) are capable of driving RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of said gene capable of synthesis of said activator of CAX1 and, optionally, said gene capable of synthesis of said activator of at least one of CAX2, CAX3, or CAX4, before, during, and/or after anoxic conditions.
  • the plurality of plant cells have transient and/or stable suppressed or eliminated synthesis of said activator of CAX1 and, optionally, said activator of at least one of CAX2, CAX3, or CAX4.
  • the agent(s) are capable of driving transient and/or stable RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of said gene capable of synthesis of said activator of CAX1 and, optionally, said activator of at least one of CAX2, CAX3, or CAX4.
  • the plurality of plant cells have upregulated expression during anoxia of at least one gene selected from the group consisting of: AT 1G01355; AT1G02610; AT1G02620; AT1G03090; AT1G03610; AT1G06570; AT1G08630;
  • AT1G08930 AT1G10070; AT1G10140; AT1G10550; AT1G11260; AT1G12780;
  • AT1G24880 AT1G25560; AT1G27045; AT1G28330; AT1G30135; AT1G30720;
  • AT1G32910 AT1G33050; AT1G33055; AT1G34140; AT1G34760; AT1G35140;
  • AT1G60750 AT1G62480; AT1G62510; AT1G63090; AT1G65970; AT1G66860;
  • AT3G1331O AT3G13450; AT3G15440; AT3G15450; AT3G15620; AT3G16770;
  • AT5G26200 AT5G27893; AT5G28610; AT5G28630; AT5G28770; AT5G34830;
  • the plurality of plant cells have upregulated expression during recovery from anoxia of at least one gene selected from the group consisting of:
  • AT1G19620 AT1G21340; AT1G21940; AT1G22110; AT1G26800; AT1G27730;
  • AT1G28760 AT1G30070; AT1G30135; AT1G31370; AT1G32910; AT1G32928;
  • AT1G71000 AT1G71520; AT1G72060; AT1G72416; AT1G72660; AT1G72760;
  • AT2G46830 AT2G47180; AT2G47520; AT3G02550; AT3G06435; AT3G07090;
  • AT3G14200 AT3G15440; AT3G15450; AT3G15500; AT3G16050; AT3G17400;
  • AT3G24500 AT3G25250; AT3G27220; AT3G28210; AT3G28740; AT3G29370;
  • AT4G02690 AT4G09150; AT4G10250; AT4G10265; AT4G10270; AT4G11660;
  • AT4G12400 AT4G12410; AT4G13395; AT4G15280; AT4G15420; AT4G15760;
  • AT4G27450 AT4G27652; AT4G27654; AT4G27657; AT4G27670; AT4G28811;
  • AT4G38030 AT5G01740; AT5G02170; AT5G02810; AT5G03210; AT5G03720;
  • AT5G22680 AT5G22920; AT5G25450; AT5G27660; AT5G35320; AT5G37340;
  • AT5G45640 AT5G47220; AT5G47590; AT5G47830; AT5G48570; AT5G49920;
  • AT5G53680 AT5G54165; AT5G57260; AT5G57550; AT5G57560; AT5G57660;
  • ATMG00400 ATMG00410; ATMG00480; ATMG00510; ATMG00513; ATMG00516;
  • the genetically modified plant, part, and/or progeny thereof is selected from the group consisting of whole plants, seedlings, leaves, stems, flowers, roots, fruits, seeds, spores, grafts comprising genetically modified scions and/or rootstocks, buddings comprising genetically modified buds and/or rootstocks, cuttings, bulbs, tubers, rhizomes, regenerable cells, tissue cultures of regenerable cells, regenerable protoplasts, tissue cultures of regenerable protoplasts, and other plant germplasms.
  • the genetically modified plant, part, and/or progeny thereof is selected from the group consisting of the Viridiplantae, the Glaucophyta, and the Rhodophyta.
  • the Viridiplantae are selected from the group consisting of green algae, hornworts, liverworts, mosses, ferns, lycophytes, gymnosperms, and angiosperms.
  • the genetically modified plant, part, and/or progeny thereof is monocotyledonous or dicotyledonous.
  • the genetically modified plant, part, and/or progeny thereof is selected from the group consisting of herbs, shrubs, trees, and vines.
  • the genetically modified plant, part, and/or progeny thereof excludes research plants, parts, and/or progeny thereof.
  • the genetically modified plant, part, and/or progeny thereof is a crop plant capable of producing a crop product.
  • the crop plant is selected from the group consisting of food crop plants, forage crop plants, fodder crop plants, medicinal crop plants, industrial crop plants, energy crop plants, and ornamental crop plants.
  • the crop product of said ornamental crop plant is selected from the group consisting of foliage-, flower-, and/or fruit-producing herbs, shrubs, trees, vines, and regenerable parts thereof.
  • the crop product is selected from the group consisting of cereal grains, legumes, vegetables, nuts, seeds, roots, tubers, rhizomes, flowers, fruits, timber, plant leaves, plant oils, plant fats, plant fibers, plant juices, plant extracts, and combinations thereof.
  • the crop plant is selected from the group consisting of rice plants, maize plants, barley plants, oat plants, rye plants, wheat plants, com plants, sorghum plants, soybean plants, pea plants, lentil plants, curcurbit plants, coffee plants, cocoa plants, rapeseed plants, sunflower plants, sugar cane plants, potato plants, palm plants, grape plants, apple plants, banana plants, plantain plants, cassava plants, sugar beet plants, tomato plants, sweet potato plants, yam plants, tobacco plants, cotton plants, rubber plants, tea plants, lettuce plants, pepper plants, onion plants, grape plants, pecan trees, timber trees, cannabis plants, poppy plants, and combinations thereof.
  • the genetically modified plant, part, and/or progeny thereof is not cultivated for harvest of crops.
  • the genetically modified plant, part, and/or progeny thereof, not cultivated for harvest crops is selected from the group consisting of Arabidopsis halleri, Arabidopsis lyrata, Eutrema salsugineum, Cardamine hirsute, the Viridiplantae, the Glaucophytes, and the Rhodophytes.
  • a composition of the disclosure includes an inbred, hybrid, or varietal plant, part, or progeny thereof, of the genetically modified plant, part, or progeny thereof.
  • a composition of the disclosure includes a genetically modified plant, part, and/or progeny thereof, propagated and/or regenerated from the seeds, spores, grafts comprising genetically modified scions and/or rootstocks, buddings comprising genetically modified buds and/or rootstocks, cuttings, bulbs, tubers, rhizomes, regenerable cells, tissue cultures of regenerable cells, regenerable protoplasts, tissue cultures of regenerable protoplasts, and/or other plant germplasms of the genetically modified plant, part, and/or progeny thereof.
  • a composition of the disclosure includes a genetically modified seed produced by the genetically modified plant, part, and/or progeny thereof.
  • a method of the disclosure includes a method of producing a crop from the genetically modified crop plant, part, and/or progeny thereof, including the steps of: cultivating said genetically modified crop plant, part, and/or progeny thereof, to produce said crop; and harvesting said crop from said genetically modified crop plant, part, and/or progeny thereof.
  • a method of the disclosure includes a method of producing a genetically modified plant, part, and/or progeny thereof, excluding Arabidopsis thaliana, comprising a plurality of plant cells, wherein the method includes the step of: synthetically driving suppression and/or elimination of expression of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4, in said plurality of plant cells, wherein said synthetically driving step improves the anoxia tolerance of said genetically modified plant, part and/or progeny thereof.
  • the synthetically driving step further includes the steps of: introducing into said plurality of plant cells one or more agents, said agent(s) driving gene silencing of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4, in said plurality of plant cells; and, optionally, introducing into said plurality of plant cells a regulatory sequence, said regulatory sequence inducing said agent(s) to drive said gene silencing of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4, in said plurality of plant cells.
  • the one or more agents is a nucleic acid sequence and/or a protein.
  • the nucleic acid sequence comprises exogenous DNA, intragenic DNA, and/or exogenous RNA.
  • the protein comprises a nuclease and/or a protease.
  • the introducing steps are accomplished by: direct transfer of exogenous DNA, intragenic DNA, exogenous RNA, or combinations, complexes, carrier systems, and/or particle gun systems thereof; and/or viral vector-mediated, Agrobacterium vector-mediated, and/or CRISPR vector-mediated gene transformation of exogenous and/or intragenic DNA.
  • the direct transfer step is accomplished by: passive uptake; electroporation; polyethylene glycol treatment; electrophoresis; cell fusion with liposomes or spheroplasts; injection, silicon carbide whiskers, particle gun bombardment, spraying, soaking, pipetting, brushing, cationic nanoparticle carriers, clay nanosheet carriers, surfactant complexes, and/or peptide-based carriers.
  • the driving step further includes the step of driving RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) in expression of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4.
  • the step of driving RNA interference (RNAi) further includes the step of expressing double stranded RNA (dsRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), small RNA (sRNA), microMRNA (mRNA), pre-miRNA, and/or pri-miRNA capable of RNA interference (RNAi) of expression of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4.
  • the agent(s) comprise double stranded RNA (dsRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), small RNA (sRNA), microMRNA (mRNA), pre-miRNA, and/or pri-miRNA capable of RNA interference (RNAi) of expression of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4.
  • dsRNA double stranded RNA
  • hpRNA hairpin RNA
  • siRNA small interfering RNA
  • sRNA small RNA
  • sRNA small RNA
  • mRNA microMRNA
  • pre-miRNA pre-miRNA
  • pri-miRNA capable of RNA interference (RNAi) of expression of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4.
  • the inducing step further includes the step of inducing said agent(s) to drive RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) in expression of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4.
  • RNAi RNA interference
  • TGS transcriptional gene silencing
  • VIGS virus induced gene silencing
  • the step of inducing said agent(s) to drive RNA interference (RNAi) further includes the step of inducing said agent(s) to express double stranded RNA (dsRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), small RNA (sRNA), microMRNA (mRNA), pre-miRNA, and/or pri-miRNA capable of RNA interference (RNAi) of expression of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4.
  • the regulatory sequence is tissue specific and/or cell-type specific. In specific embodiments, the regulatory sequence is low oxygen-induced, chemical-induced, temperature-induced, and/or light-induced.
  • the said step of suppressing and/or eliminating expression of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4, occurs before, during, and/or after anoxic conditions.
  • the step of driving RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of expression of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4, occurs before, during, and/or after anoxic conditions.
  • the step of suppressing and/or eliminating expression of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4, is transient and/or stable.
  • the step of driving RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of expression of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4, is transient and/or stable.
  • the plurality of plant cells upregulate expression during anoxia of at least one gene selected from the group consisting of: AT 1G01355;
  • AT1G08930 AT1G10070; AT1G10140; AT1G10550; AT1G11260; AT1G12780;
  • AT1G24880 AT1G25560; AT1G27045; AT1G28330; AT1G30135; AT1G30720;
  • AT1G32910 AT1G33050; AT1G33055; AT1G34140; AT1G34760; AT1G35140;
  • AT1G60750 AT1G62480; AT1G62510; AT1G63090; AT1G65970; AT1G66860;
  • AT3G1331O AT3G13450; AT3G15440; AT3G15450; AT3G15620; AT3G16770; AT3G17225; AT3G18530; AT3G19680; AT3G20395; AT3G21352; AT3G22640;
  • AT5G26200 AT5G27893; AT5G28610; AT5G28630; AT5G28770; AT5G34830;
  • the plurality of plant cells upregulate expression during recovery from anoxia of at least one gene selected from the group consisting of: AT1G01720; AT1G03070; AT1G03090; AT1G03610; AT1G05575; AT1G06030; AT1G07330; AT1G07350; AT1G07400; AT1G07500; AT1G08630; AT1G09950;
  • AT1G19620 AT1G21340; AT1G21940; AT1G22110; AT1G26800; AT1G27730;
  • AT1G28760 AT1G30070; AT1G30135; AT1G31370; AT1G32910; AT1G32928;
  • AT1G71000 AT1G71520; AT1G72060; AT1G72416; AT1G72660; AT1G72760;
  • AT2G46830 AT2G47180; AT2G47520; AT3G02550; AT3G06435; AT3G07090;
  • AT3G14200 AT3G15440; AT3G15450; AT3G15500; AT3G16050; AT3G17400;
  • AT3G24500 AT3G25250; AT3G27220; AT3G28210; AT3G28740; AT3G29370;
  • AT4G02690 AT4G09150; AT4G10250; AT4G10265; AT4G10270; AT4G 11660;
  • AT4G12400 AT4G12410; AT4G13395; AT4G15280; AT4G 15420; AT4G15760;
  • AT4G27450 AT4G27652; AT4G27654; AT4G27657; AT4G27670; AT4G28811;
  • AT4G38030 AT5G01740; AT5G02170; AT5G02810; AT5G03210; AT5G03720;
  • AT5G22680 AT5G22920; AT5G25450; AT5G27660; AT5G35320; AT5G37340;
  • AT5G45640 AT5G47220; AT5G47590; AT5G47830; AT5G48570; AT5G49920;
  • AT5G53680 AT5G54165; AT5G57260; AT5G57550; AT5G57560; AT5G57660;
  • ATMG00400 ATMG00410; ATMG00480; ATMG00510; ATMG00513; ATMG00516;
  • a method of the disclosure includes a method of producing a genetically modified plant, part, and/or progeny thereof, excluding Arabidopsis thaliana, including a plurality of plant cells, wherein the method includes the step of: synthetically driving suppression and/or elimination of expression of a gene capable of synthesis of an activator of CAX1 and, optionally, a gene capable of synthesis of an activator of at least one of CAX2, CAX3, or CAX4, in said plurality of plant cells, wherein said synthetically driving step improves the anoxia tolerance of said genetically modified plant, part and/or progeny thereof.
  • the synthetically driving step further includes the steps of: introducing into said plurality of plant cells one or more agents, said agent(s) driving gene silencing of said gene capable of synthesis of an activator of CAX1 and, optionally, said gene capable of synthesis of an activator of at least one of CAX2, CAX3, or CAX4, in said plurality of plant cells; and, optionally, introducing into said plurality of plant cells a regulatory sequence, said regulatory sequence inducing said agent(s) to drive gene silencing of said gene capable of synthesis of an activator of CAX1 and, optionally, said gene capable of synthesis of an activator of at least one of CAX2, CAX3, or CAX4, in said plurality of plant cells.
  • the activator of CAX1 is capable of binding to the N- terminus of CAX1, and wherein said activator of at least one of CAX2, CAX3, or CAX4 is capable of binding to the N-terminus of at least one of CAX2, CAX3, or CAX4.
  • the activator of CAX1 is selected from the group consisting of SOS2 protein kinase, CXIP1, and CXIP4.
  • the introducing steps are accomplished by: direct transfer of exogenous DNA, intragenic DNA, exogenous RNA, or combinations, complexes, carrier systems, and/or particle gun systems thereof; and/or viral vector-mediated, Agrobacterium vector-mediated, and/or CRISPR vector-mediated gene transformation of exogenous and/or intragenic DNA.
  • the direct transfer step is accomplished by: passive uptake; electroporation; polyethylene glycol treatment; electrophoresis; cell fusion with liposomes or spheroplasts; injection, silicon carbide whiskers, particle gun bombardment, spraying, soaking, pipetting, brushing, cationic nanoparticle carriers, clay nanosheet carriers, surfactant complexes, and/or peptide-based carriers.
  • the driving step further includes the step of driving RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of a gene capable of synthesis of an activator of CAX1 and, optionally, a gene capable of synthesis of an activator of at least one of CAX2, CAX3, or CAX4.
  • RNAi RNA interference
  • TGS transcriptional gene silencing
  • VIGS virus induced gene silencing
  • the step of driving RNA interference further includes the step of expressing double stranded RNA (dsRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), small RNA (sRNA), microMRNA (mRNA), pre-miRNA, and/or pri-miRNA capable of RNA interference (RNAi) of a gene capable of synthesis of at least one activator of CAX1 and, optionally, a gene capable of synthesis of an activator of at least one of CAX2, CAX3, or CAX.
  • dsRNA double stranded RNA
  • hpRNA hairpin RNA
  • siRNA small interfering RNA
  • sRNA small RNA
  • mRNA microMRNA
  • pre-miRNA pre-miRNA
  • pri-miRNA capable of RNA interference (RNAi) of a gene capable of synthesis of at least one activator of CAX1 and, optionally, a gene capable of synthesis of an activator of at least one of CAX2, CA
  • the agent(s) includes double stranded RNA (dsRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), small RNA (sRNA), microMRNA (mRNA), pre-miRNA, and/or pri-miRNA capable of RNA interference (RNAi) of a gene capable of synthesis of an activator of CAX1 and, optionally, a gene capable of synthesis of an activator of at least one of CAX2, CAX3, or CAX4.
  • dsRNA double stranded RNA
  • hpRNA hairpin RNA
  • siRNA small interfering RNA
  • sRNA small RNA
  • mRNA microMRNA
  • pre-miRNA pre-miRNA
  • pri-miRNA capable of RNA interference (RNAi) of a gene capable of synthesis of an activator of CAX1 and, optionally, a gene capable of synthesis of an activator of at least one of CAX2, CAX3, or CAX4.
  • the inducing step further includes the step of inducing said agent(s) to drive RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of a gene capable of synthesis of an activator of CAX1 and, optionally, a gene capable of synthesis of an activator of at least one of CAX2, CAX3, or CAX4.
  • RNAi RNA interference
  • TGS transcriptional gene silencing
  • VIGS virus induced gene silencing
  • the step of inducing said agent(s) to drive RNA interference (RNAi) further includes the step of inducing said agent to express double stranded RNA (dsRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), small RNA (sRNA), microMRNA (mRNA), pre-miRNA, and/or pri-miRNA capable of RNA interference (RNAi) of a gene capable of synthesis of an activator of CAX1 and, optionally, a gene capable of synthesis of an activator of at least one of CAX2, CAX3, or CAX4, wherein said inducing step is accomplished using said regulatory sequence.
  • the regulatory sequence is tissue specific and/or cell-type specific.
  • the regulatory sequence is low oxygen-induced, chemical-induced, temperature-induced, and/or light- induced.
  • the step of suppressing and/or eliminating expression of a gene capable of synthesis of an activator of CAX1 and, optionally, a gene capable of synthesis of an activator of at least one of CAX2, CAX3, or CAX4, occurs before, during, and/or after anoxic conditions.
  • the step of driving RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of a gene capable of synthesis of an activator of CAX1 and, optionally, a gene capable of synthesis of an activator of at least one of CAX2, CAX3, or CAX4, occurs before, during, and/or after anoxic conditions.
  • the step of suppressing and/or eliminating synthesis of at least one activator of CAX1 and, optionally, at least one activator of at least one of CAX2, CAX3, or CAX4, is transient and/or stable.
  • the step of driving RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of a gene capable of synthesis of an activator of CAX1 and, optionally, a gene capable of synthesis of an activator of at least one of CAX2, CAX3, or CAX4, is transient and/or stable.
  • the plurality of plant cells upregulate expression during anoxia of at least one gene selected from the group consisting of: AT 1G01355; AT1G02610; AT1G02620; AT1G03090; AT1G03610; AT1G06570; AT1G08630;
  • AT1G08930 AT1G10070; AT1G10140; AT1G10550; AT1G11260; AT1G12780;
  • AT1G24880 AT1G25560; AT1G27045; AT1G28330; AT1G30135; AT1G30720;
  • AT1G32910 AT1G33050; AT1G33055; AT1G34140; AT1G34760; AT1G35140;
  • AT1G60750 AT1G62480; AT1G62510; AT1G63090; AT1G65970; AT1G66860;
  • AT3G1331O AT3G13450; AT3G15440; AT3G15450; AT3G15620; AT3G16770;
  • AT5G26200 AT5G27893; AT5G28610; AT5G28630; AT5G28770; AT5G34830;
  • the plurality of plant cells upregulate expression during recovery from anoxia of at least one gene selected from the group consisting of: AT1G01720; AT1G03070; AT1G03090; AT1G03610; AT1G05575; AT1G06030;
  • AT1G19620 AT1G21340; AT1G21940; AT1G22110; AT1G26800; AT1G27730;
  • AT1G28760 AT1G30070; AT1G30135; AT1G31370; AT1G32910; AT1G32928;
  • AT1G71000 AT1G71520; AT1G72060; AT1G72416; AT1G72660; AT1G72760;
  • AT2G46830 AT2G47180; AT2G47520; AT3G02550; AT3G06435; AT3G07090;
  • AT3G14200 AT3G15440; AT3G15450; AT3G15500; AT3G16050; AT3G17400;
  • AT3G24500 AT3G25250; AT3G27220; AT3G28210; AT3G28740; AT3G29370;
  • AT4G02690 AT4G09150; AT4G10250; AT4G10265; AT4G10270; AT4G 11660;
  • AT4G12400 AT4G12410; AT4G13395; AT4G15280; AT4G 15420; AT4G15760;
  • AT4G27450 AT4G27652; AT4G27654; AT4G27657; AT4G27670; AT4G28811;
  • AT4G38030 AT5G01740; AT5G02170; AT5G02810; AT5G03210; AT5G03720;
  • AT5G22680 AT5G22920; AT5G25450; AT5G27660; AT5G35320; AT5G37340;
  • AT5G45640 AT5G47220; AT5G47590; AT5G47830; AT5G48570; AT5G49920;
  • AT5G53680 AT5G54165; AT5G57260; AT5G57550; AT5G57560; AT5G57660; AT5G58070; AT5G59720; AT5G59820; AT5G62020; AT5G62520; AT5G63130;
  • ATMG00400 ATMG00410; ATMG00480; ATMG00510; ATMG00513; ATMG00516;
  • the progeny is produced by propagating said genetically modified plant or said part thereof, using asexual or sexual reproduction.
  • composition of the disclosure includes a genetically modified seed produced by the genetically modified plant, part, and/or progeny thereof, prepared according to any one of the methods of the disclosure.
  • a composition of the disclosure includes a genetically modified plant, part, and/or progeny thereof, propagated and/or regenerated from the seeds, spores, grafts comprising genetically modified scions and/or rootstocks, buddings comprising genetically modified buds and/or rootstocks, cuttings, bulbs, tubers, rhizomes, regenerable cells, tissue cultures of regenerable cells, regenerable protoplasts, tissue cultures of regenerable protoplasts, and/or other plant germplasms of the genetically modified plant, part, and/or progeny thereof, produced according to any one of the methods of the disclosure.
  • a composition of the disclosure includes a crop produced by the genetically modified plant, part, and/or progeny thereof, prepared according to any one of the methods of the disclosure.
  • FIGS. 1A-1E illustrate particular embodiments of the disclosure showing the effect of CAX mutation on anoxia tolerance of Arabidopsis thaliana plants.
  • FIG. 1A shows improved anoxia tolerance (reduced wilting and necrosis) of the caxl mutant line versus the Col-0 (wild-type, aka “WT”) line or the cax3 mutant line compared to normoxia.
  • Anoxia tolerance of caxl mutant line was enhanced when CAX3 was also mutated, e.g., the caxl/3 (aka “double mutant knockout”, aka “caxl/3DKO”) line.
  • Photographs are representative of more than 15 biological replicate plates for each condition.
  • FIG. 1A shows improved anoxia tolerance (reduced wilting and necrosis) of the caxl mutant line versus the Col-0 (wild-type, aka “WT”) line or the cax3 mutant line compared to normoxia.
  • Anoxia tolerance of caxl mutant line was enhanced when CAX
  • FIG. 1C shows improved post-anoxia reoxygenation tolerance (reduced cell death, measured by Trypan blue staining) of the caxl and caxl/3 mutant lines versus the Col-0 line or the cax3 mutant line. The images are representative of three independent experiments.
  • FIG. 1C shows improved postanoxia reoxygenation tolerance (reduced chlorophyll loss; reduced induction of lipid peroxidation, measured as malondialdehyde (MDA) level in leaves) of the caxl and caxl/3 mutant lines versus the Col-0 line or the cax3 mutant line
  • FIG. 1 shows reduced plant stress (reduced chlorophyll loss, measured by fluorescence; reduced H2O2 levels, measured by H2DCFDA dye fluorescence) of the caxl and caxl/3 mutant lines versus the Col-0 line or the cax3 mutant line in post-anoxia reoxygenation conditions compared to normoxia.
  • the images are representative of three independent experiments.
  • IE shows reduced plant stress (reduced hydrogen peroxide (H2O2) levels, via staining with 3,3 '-diaminobenzidine (DAB)) of the caxl and caxl/3 mutant lines versus the Col-0 line or the cax3 mutant line in 1 hr and 8 hr post-anoxic reoxyganation conditions compared to normoxia.
  • the images are representative of three independent experiments.
  • FIGS. 2A-2F illustrate particular embodiments of the disclosure showing RNAseq transcriptomic data for Arabidopsis thaliana plants.
  • the caxl mutant line and the caxl/3 mutant line were compared with the cax3 mutant line and the Col-0 line. Plants were sampled prior to, during, and after exposure to anoxic conditions.
  • FIG. 2A is a multi-dimension scaling (MDS) representation of Col-0, caxl, cax3, and caxl/3 transcriptomes before during and after anoxic treatment. Colors highlight the factor/time point and the shape defines the genotype.
  • MDS multi-dimension scaling
  • FIG. 2B shows Genome Ontology (GO) groups identified in caxl and caxl/3 preanoxia that coincide with genes differentially expressed in Col-0 and cax3 during anoxia and recovery (1-hour post 7-hour anoxia).
  • FIG. 2C shows GO enrichment in caxl and caxl/3 (caxl/3>caxl) after 4 hours of anoxia.
  • FIG. 2D shows GO enrichment in caxl and caxl/3 (caxl/3>caxl) during recovery.
  • FIG. 2E shows a scheme depicting timing of leaf tissue harvesting for RNAseq analysis.
  • FIG. 2F shows Metascape network visualization of the enriched genome ontology groups that were significantly different in both caxl and caxl/3 compared to cax3 and Col-0 under anoxia and recovery conditions.
  • the nodes are color coded to delineate the similarities and differences among caxl and caxl/3 during anoxia and in the recovery phase.
  • the size of the connecting dots represent the -loglO of the P value for that GO cluster.
  • FIGS. 3A-3C illustrate particular embodiments of the disclosure showing differentially abundant proteins (DAP) of WT, caxl mutant, and caxl/3 mutant genotypes of Arabidopsis thaliana plants based on normalized spectral abundance factor (NSAF).
  • FIG. 3A shows a summary table of differentially abundant proteins (DAP) showing number of proteins which increased or decreased in abundance in the different genotypes.
  • DAP differentially abundant proteins
  • GO enrichment analysis was performed on the DAPs in caxl and caxl/3 post anoxia (recovery).
  • Enrichment was performed using DAVID employing the hypergeometric test to determine significance. Rich factor is the ratio of identified protein numbers annotated in the given GO term pathway to the total number of input annotated proteins.
  • FIG. 3C shows a schematic diagram of the glycolytic pathway showing those proteins (P) and transcripts (T) regulated post anoxia. Red/Up arrow - increased; Blue/Down arrow - decreased. Similar responses for data from both transcriptomic analysis and protein abundance analysis are overlaid onto KEGG pathways.
  • FIGS 4A-4C illustrate particular embodiments of the disclosure showing the effect of Ca, ROS, temperature, and phytohormones on the anoxic tolerance.
  • FIG. 4A shows the effect of exogenous application of chemicals or heat treatment on anoxia tolerance.
  • EGTA is ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid, a chelating agent that binds calcium and thus lowers the levels found in the plant;
  • ABA is abscisic acid, a plant hormone that is important in plant stress responses.
  • FIG. 4B shows leaf death 4 days post anoxia for lines treated with water, EGTA, ABA, and heat. A leaf with > 50% necrosis was counted as an anoxia damaged leaf.
  • ABA abscisic acid
  • BA benzoic acid, a compound that elicits plant growth and development responses
  • CA trans-cinnamic acid
  • COUMA p-Coumaric acid which is a phenylpropaid in plants that can act as an antioxidant
  • IAA is indole-3-acetic acid, which has many effects including inducing cell elongation and cell division
  • SA is salicylic acid.
  • FIGS. 5A-5E illustrate particular embodiments of the disclosure showing Ca imaging studies of Col-0 and caxl mutant lines of Arabidopsis thaliana plants which express the Ca biosensor GCamP3.
  • Col-0 and caxl plants expressing a stable genetically encoded Ca indicator GCamP3 were grown on * strength MS medium with 0.5% sucrose for 12 days before they were treated with anoxia stress for 4h.
  • FIG. 5A shows fluorescence of GCamP3 recorded by streaming video immediately after the plants were re-exposed to oxygen. Images are representative of at least 6 independent experiments.
  • White bar 2 mm. Plants (12 days old) were surface sprayed with either water control (FIG. 5B), lOmM EGTA (FIG.
  • FIG. 6 illustrates a model comparing anoxia responses in CAX1 and caxl deficient lines (e.g., suppressed and/or eliminated expression).
  • CAX1 plants appear to respond to anoxia with increased ROS production, and minor metabolic and hormone signaling changes.
  • Ca levels in the cytosol remain tightly regulated.
  • caxl there appears to be dampened ROS production, heightened hormone signaling and changes in metabolism.
  • Calcium signaling is altered in the caxl lines, and we speculate that pH levels fluctuate less than in CAX1.
  • FIGS. 7A-7D illustrates a particular embodiment of the disclosure showing the effect of CAX mutation on anoxia tolerance of Arabidopsis thaliana plants.
  • FIG. 7A is a diagrammatic representation of the GasPak system used for generating anoxic conditions.
  • FIG. 7C shows improved anoxia tolerance (reduced wilting and necrosis) compared to nomoxia for the caxl mutant line versus either the Col-0 (wild-type, aka “WT”) line or the cax3 mutant line (the experiment was repeated at least three times with 10-12 plants in each replicate).
  • the caxl mutant line was more tolerant to anoxic conditions and this phenotype was clearly visible three days after being removed from the GasPak. This tolerance was lost when transgenic CAX1 expressed sCAXl (FIG. 7B, the experiment was repeated at least three times with 10-12 plants in each replicate).
  • the cax2, cax3 or cax4 had similar anoxia phenotypes as Col-0 (wild-type) plants (FIG. 7C). Meanwhile, the tolerance of caxl mutant line was enhanced if CAX3 was also mutated (FIG. 7C). Additionally, every double mutant analyzed was tolerant if one of the mutations was in CAX1 (FIG. 7C).
  • V-type ATPases like vha-a2 and vha-a3 and mutants regulating cation dependent proton transport activity such as nhxl-1 and nhx2-3 were susceptible to anoxia (FIG. 7D, two-week old plants grown on half-strength MS medium were used for the study. The experiment was repeated at least two times with 10-12 plants in each replicate).
  • FIGS. 8A-8C illustrate particular embodiments of the disclosure showing RNAseq transcriptomic data for Arabidopsis thaliana plants sampled after anoxia (FIG. 8A, A1-A7); recovery (FIG. 8B, B1-B12) and pre-conditions (FIG. 8C, C1-C7).
  • the caxl mutant line and the cax 1/3 mutant line were compared with the cax3 mutant line and the Col-0 line.
  • FIGS. 8A-8C illustrate genome ontology (GO) analysis which correlates the expressed transcripts in the samples with biologic function.
  • Gene categories have been identified by Metascape and are numbered, e.g., groups A1-A7, groups B1-B13, and groups C1-C7.
  • Heatmaps were created in R using pheatmap. Genes present in multiple categories are represented only once in the heat map. Red and blue represent up and downregulated gene expression. Color density indicating levels of fold change.
  • FIGS. 9A-9D illustrate particular embodiments of the disclosure showing enriched GO categories in Col-0 and cax3 for Arabidopsis thaliana plants sampled after anoxia and recovery.
  • FIG. 9A shows GO categories among the genes commonly up regulated in Col- 0 and cax3 following anoxia.
  • FIG. 9B shows GO categories among the genes commonly down regulated in Col-0 and cax3 following anoxia.
  • FIG. 9C shows GO categories among the genes commonly up regulated in Col-0 and cax3 during recovery.
  • FIG. 9D shows GO categories among the genes commonly down regulated in Col-0 and cax3 during recovery. Gene categories have been identified by Metascape.
  • FIG. 10 illustrates particular embodiments of the disclosure showing RNAseq data using real-time PCR (qRT-PCR) revealing a global change in the transcript abundances of Ca transporters, Ca sensor/effectors in both caxl and caxl/3 mutant lines.
  • qRT-PCR real-time PCR
  • FIGS. 11A-11B illustrate particular embodiments of the disclosure showing a hierarchically clustered heatmap showing the expression of approximately 30 different Ca sensors (FIG. 11A) and Ca transporters (FIG. 11B). Red and blue represent up and downregulated gene expression. Color density indicating levels of fold change.
  • FIGS. 11A- 11B show that the caxl and caxl/3 specific DEGs here were enriched for GO categories associated with various stress and signaling pathways including calcium, heat, hydrogen peroxide, carbohydrates, ethylene, chitin, and wounding.
  • FIGS. 12A-12D illustrate particular embodiments of the disclosure showing enriched temperature-responsive genes in caxl and caxl/3 during pretreatment conditions (FIGS. 12A-12B) and during recovery (FIGS. 12C-12D).
  • FIG. 12A shows Metascape output of genes which are highly down regulated in caxl and caxl/3 during the pre-treatment conditions.
  • FIG. 12B shows expression analysis of the cold responsive genes highly down regulated in caxl and caxl/3 during the pre-treatment conditions.
  • FIG. 12C shows Metascape output of genes which are highly up regulated in caxl and caxl/3 during recovery.
  • FIG. 12D shows expression analysis of the heat responsive genes highly up regulated in caxl and caxl/3 during recovery. Red and blue represent up and downregulated gene expression. Color density indicating levels of fold change
  • FIG. 13 illustrates particular embodiments of the disclosure showing expression pattern of genes involved in ABA and SA pathways. Heatmap was created in R using pheatmap. Red and blue represent up and downregulated gene expression. Color density indicating levels of fold change.
  • FIG. 14 illustrates particular embodiments of the disclosure showing transcript analysis of CAX1 and CAX3 RNAs in caxl and cax3.
  • Semi-quantitative RT-PCR was conducted in 2.5 week old Col-0, caxl, cax3 and caxl/3 to assess transcripts of CAX1 and CAX3.
  • RNA fragments of CAX1 and CAX3 before, across and after the T-DNA insertion were investigated in the various genetic backgrounds.
  • UBQ10 was used as an endogenous control.
  • T-DNA-span primers anneal to regions that span across the T-DNA insertion site.
  • Primers anneal to fragments before and after the T-DNA insertion were used to test the effect of T-DNA insertion on the transcription of these regions. This data shows that while CAX1 and CAX3 RNAs are present in the mutants, they are partial transcripts containing only the 5’ region before the T-DNA.
  • FIGS. 15A-15C illustrate particular embodiments of the disclosure showing pie charts of subcellular distributions of DAPs in Col-0 (FIG. 15A), caxl (FIG. 15B), and caxl/3 (FIG. 15C).
  • Subcellular locations of proteins were suggested by SUBA consensus, “Mix” indicates the proteins with multiple predicted locations. Numbers in the figure represent the regulated protein counts for the corresponding subcellular compartments.
  • FIG. 16 illustrates particular embodiments of the disclosure showing a schematic overview of cellular H2O2 removal and oxidative stress relief in caxl and caxl/3 as evidenced by proteomic studies.
  • SOD Superoxide dismutase
  • CAT Catalase
  • APX L- ascorbate peroxidase 1
  • GPX phospholipid hydroperoxide glutathione peroxidase
  • GLYR1 Glyoxylate succinic semialdehyde reductase
  • GSTUJ Glutathione S-transferase
  • PDX is Pyridoxal 5'-phosphate synthase
  • TRXM1 is Thioredoxin Ml
  • CDSP Thioredoxin-like protein
  • PRX2E Peroxiredoxin-2E
  • PRX2F Peroxiredoxin-2F
  • ACO3 is Aconitate hydratase 3.
  • FIG. 17 illustrates particular embodiments of the disclosure showing cytosolic calcium signals under dark conditions without anoxia in Col-0 and caxl.
  • Col-0 and caxl expressing a stable genetically encoded Ca indicators GCamP3 were grown on half-strength MS with 0.5% sucrose for 12 day before they were wrapped in foil and placed in anaerobic bags without the GasPak for 4h. This was done as a negative control for anoxia. Fluorescence of GCamP3 was recorded by streaming video immediately after the plants were exposed to light. Images are representative of at least 4 independent experiments. White bar: 2 mm. [0077] FIGS.
  • FIG. 18A-18I illustrates a particular embodiment of the disclosure showing the effect of CAX mutations on anoxia tolerance of Arabidopsis thaliana plants when assessed using submergence in water (e.g., fresh water) as an anoxic condition generating stimulus.
  • FIG. 18A is a diagrammatic representation of the submergence tolerance assay used for generating anoxic conditions.
  • FIG. 18B shows Col-0 line (wild-type, aka “WT”) plant wilting and/or necrosis seven days after plants have been removed from 38 hours of submergence in water.
  • FIG. 18C, 18D, and 18E represent results from various cax-1 mutant lines (caxl-1 (18C), caxl -2 (18D), and cax-13 (18E)) that exhibited reduced wilting and/or necrosis following submergence when compared to control lines.
  • FIG. 18F represent results from cax-1/3 double mutant lines that showed reduced wilting and/or necrosis following submergence when compared to control lines.
  • FIG. 18G show that complement based rescue of CAX1 expression in cax-1 mutant lines (“cax-1 complemented line”) restores wilting and/or necrosis phenotypes following submergence when compared to control cax-1 mutant lines that have not been rescued.
  • a” or “an” may mean one or more.
  • the words “a” or “an” when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one.
  • another may mean at least a second or more.
  • the terms “having”, “including”, “containing” and “comprising” are interchangeable and one of skill in the art is cognizant that these terms are open ended terms.
  • aspects of the disclosure may “consist essentially of’ or “consist of’ one or more sequences of the disclosure, for example.
  • Some embodiments of the invention may consist of or consist essentially of one or more elements, method steps, and/or methods of the disclosure.
  • x, y, and/or z can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” It is specifically contemplated that x, y, or z may be specifically excluded from an embodiment.
  • the present disclosure relates to methods of using the genetically modified plants described herein.
  • the genetically modified plants may exhibit improved properties over a control plant.
  • the genetically modified plants may have improved tolerance to anoxia as compared to a control plant.
  • anoxia or “anoxic conditions” refer to conditions in which there is total oxygen depletion to plant cells, plants or parts of such genetically modified plants.
  • hypooxia or “hypoxic conditions” refer to conditions in which there is an oxygen deficiency but not total oxygen depletion.
  • Anoxia resistance may be measured using assays known in the art such as, for example, subjecting the plants to low oxygen-stress over a period of a certain number of days (e.g., via methods as described herein, e.g., via hypoxia generating apparatus, via submergence, via tight packing of soil, etc.,).
  • a “control plant” is a plant that has not been modified as described herein.
  • exemplary control plants may include those from a natural plant species for a particular plant being modified.
  • a control plant is a plant that has not been genetically modified by the hand of man to have improved anoxia tolerance, including a plant that has not been genetically modified with respect to one or more CAX genes, including at least CAX1 or a combination of CAX1 and one or more of CAX2, CAX3, and CAX4.
  • a control plant and/or condition is relative to a test plant and/or condition, one of skill in the art can determine if a certain genotype, assay condition, etc. is an appropriate control relative to the question(s) asked or comparisons being made.
  • a control plant is one that has been genetically modified by the hand of man for the purposes of determining necessity and/or sufficiency of a particular biological phenomenon.
  • a control plant is a plant that has not been genetically modified by the hand of man to have improved anoxia tolerance, including a plant that has not been genetically modified with respect to one or more genes capable of synthesis of one or more activators of one or more CAX genes, including at least one or more activators of CAX1 or a combination of one or more activators of CAX1 and one or more activators of one or more of CAX2, CAX3, and CAX4.
  • methods described herein include planting any of the genetically modified plants described herein in a suitable area.
  • the area may possess low oxygen levels, for example but not limited to, conditions such as areas of high elevation, extremely tight soil, submersion by water and/or ice, etc.
  • the area may generally have normal oxygen levels but is at risk of acute anoxia.
  • the area may have normal oxygen levels but is at risk of submersion.
  • the area may be an area that is subject to flooding, such as near any body of water and including a flood plain (e.g., a river basin, near a reservoir and/or other standing body of fresh water, near an estuary and/or swamp, near a tidal flood zone, etc.).
  • a flood plain e.g., a river basin, near a reservoir and/or other standing body of fresh water, near an estuary and/or swamp, near a tidal flood zone, etc.
  • genetically modified plants described herein have increased tolerance to anoxic stress and may be useful for planting in areas where low oxygen levels are possible, predictable, and/or expected.
  • the genetically modified plants may be suitably stored and/or their seeds may be suitably stored.
  • the genetically modified plants and/or their progeny including seeds may be used for commercial purposes and/or research purposes.
  • the genetically modified plants and/or their progeny including seeds are planted for the purpose of being used as a crop or to produce crops and harvesting thereof.
  • plants are provided for genetic modification.
  • the plants may include any one of the plant cells, plant parts, or entire plants encompassed herein.
  • the plants may include plants in which every cell of the plant is a plant cell modified as described herein.
  • the plants may include plants in which only certain tissues within the plant include the plant cells described herein.
  • the plants may only have plant cells including modified expression of CAX genes in certain tissues of the plant.
  • the plants may only have plant cells including modified synthesis of activators of CAX genes in certain tissues of the plant.
  • a “plant” includes any multicellular organism of the Kingdom Plantae.
  • Clades of Kingdom Plantae include the Viridiplantae, the Glaucophytes, and the Rhodophytes.
  • the Viridiplantae includes green algae and embryophytes (hornworts, liverworts, mosses, ferns, lycophytes, gymnosperms, and angiosperms).
  • the Glaucophytes include fresh water algae.
  • the Rhodophytes include red algae.
  • a “plant, part, or progeny thereof’ includes any portion of the plant including, without limitation, a whole plant or a portion of a plant such as a part of a root, leaf, stem, seed, pod, flower, tissue, plant germplasm, asexual propagate, or any progeny thereof.
  • a corn plant refers to the whole com plant or portions thereof including, without limitation, the leaves, flowers, fruits, stems, roots, or otherwise.
  • a “plant germplasm” is any living tissue from which new plants can be grown, such as a seed or another plant part - a leaf, a piece of stem, pollen, spores, or even just a few cells that can be turned into a whole plant.
  • Plant germplasms contains the genetic information for the plant.
  • Plant germplasms may include whole plants, seedlings, leaves, stems, flowers, roots, fruits, seeds, grafts, buddings, cuttings, bulbs, tubers, rhizomes, regenerable cells, tissue cultures of regenerable cells, regenerable protoplasts, and tissue cultures of regenerable protoplasts.
  • Suitable plants may include, without limitation, a rice plant, a maize plant, a barley plant, an oat plant, a rye plant, a wheat plant, a corn plant, a sorghum plant, a soybean plant, a pea plant, a lentil plant, a curcurbit plant, coffee plant, a cocoa plant, a rapeseed plant, sunflower plant, a sugar cane plant, a potato plant, a palm plant, a grape plant, an apple plant, a banana plant, a plantain plant, a cassava plant, a sugar beet plant, a tomato plant, a sweet potato plant, a yam plant, a tobacco plant, a cotton plant, a natural rubber plant, a tea plant, a lettuce plant, a pepper plant, an onion plant, a grape plant, a pecan tree, a timber tree, a cannabis plant, or a poppy plant.
  • Plants may be sexually or asexually propagated to produce progeny. Plants may be cultivated from seeds, seedlings, grafts, cuttings, spores, rhizomes, tubers, or whole plants regenerated from cultured plant tissues or cells. Plants may be cultivated in natural or artificial culture medium. Cultivation may be performed in hydroponic systems or in soil media. Cultivation may occur in a container or in an open area. Indoor cultivation may occur in greenhouses while cultivation on open land may occur on farmland, orchards, vineyards, gardens, and/or forests, for example.
  • Genetic modification as encompassed herein includes any modification made to the genetic material of an organism. As described herein, genetic modifications are generally synthetic genetic modifications. A synthetic genetic modification is a genetic modification performed by the hand of man and does not encompass naturally produced mutations.
  • Genetic modification may be accomplished using genetic engineering techniques or by plant breeding (selection, hybridization, and induced mutation). Genetic modification as encompassed herein includes altered levels of expression of one or more genes normally present in an organism’s genome. Genetic modification may include removal of one or more genes normally present in an organism’s genome. Genetic modification may involve one or more mutations of one or more genes normally present in an organism’s genome. Genetic modification may involve insertion of genes into an organism’ s genome which are not normally present. Genetic modification may involve insertion into the cells of an organism of agents capable of gene silencing of one or more genes normally present in an organism’s genome. Genetic modification may involve insertion into and/or deletion out of the cells of an organism of nucleic acid sequences (DNA and/or RNA), nucleases (DNAses and/or RNAses), and/or proteases.
  • DNA and/or RNA nucleic acid sequences
  • DNAses and/or RNAses nucleases and/or RNAses
  • GMO genetically modified organism
  • a whole GMO plant may be regenerated from genetically modified regenerable plant parts (e.g. tissue cultures of plant cells or seeds).
  • transgenic plants Plants with exogenous DNA incorporated within are generally referred to as transgenic plants.
  • intragenic DNA may be used to achieve the same specific result.
  • Intragenic DNA may be produced by identifying functional equivalents of vector components from within the genome of a specific target plant species and using such DNA sequences to assemble vectors for transformation of that species.
  • plants with intragenic DNA incorporated within are not considered transgenic plants.
  • the terms “genetically modified plants” and “GMO plants” encompass both transgenic and non-transgenic plants produced according to the methods disclosed herein. Techniques that may be used to produce GMO plants with heritable genetic modifications include but are not limited to, physical (direct) gene transfer, Agrobacterium-mediated gene transfer, and/or CRISPR systems.
  • RNA molecules may also be used to achieve the same specific result as transgenic or intragenic gene modification.
  • viral vectors and exogenous application of RNA molecules generally achieve non-heritable modifications in gene expression which are present only during the lifetime of the plant, as the underlying genetic code remains unaffected.
  • physical (direct) and/or Agrobacterium-mediated gene transformation techniques may be used to achieve nonintegrative genetic modification.
  • GMO plants refer to plants possessing transgenic or intragenic gene modification as well as plants with modified gene expression based on insertion of nonintegrative nucleic acid sequences such as viral vectors and/or exogenous RNA into plant cells.
  • RNAi RNA interference
  • dsRNA double- stranded RNA
  • hpRNA hairpin RNA
  • dsRNA molecules are cut into shorter double stranded RNA fragments or single-stranded hairpin RNA fragments of 21-27 nucleotides by an RNase Ill-related enzyme (Dicer). These fragments, called small interfering RNAs (siRNAs), get incorporated into the RNA-induced silencing complex (RISC). After additional processing, the siRNAs are transformed into single-stranded RNAs that act as guide sequences to eventually cleave target messenger RNAs.
  • RISC RNA-induced silencing complex
  • RNAi is typically performed by producing transgenic plants that overexpress an exogenous DNA fragment that is transcribed to produce a dsRNA.
  • intragenic DNA fragments may also be used to avoid the controversy surrounding transgenic plants.
  • dsRNA may be directly introduced into plant cells rather than being indirectly expressed from DNA.
  • this dsRNA is then processed into siRNAs that mediate the cleavage and silencing of target genes, typically by targeting cleavage of the target gene by an RNA Induced Silencing Complex (RISC) or by translational repression.
  • RISC RNA Induced Silencing Complex
  • dsRNA are utilized to silence genes of interest as described herein (e.g., CAX1, CAX2, CAX3, CAX4, regulators of CAX gene activity, etc.).
  • miRNA regulation is a specialized branch of the RNA silencing pathway that evolved towards gene regulation, diverging from conventional RNAi.
  • miRNAs are a specific class of small RNAs that are encoded in gene-like elements organized in a characteristic partial inverted repeat. When transcribed, microRNA genes give rise to partially basepaired stem-looped precursor RNAs (pri-miRNAs and pre-miRNAs) from which the microRNAs are subsequently processed. miRNAs are typically about 21 nucleotides in length. The released miRNAs are incorporated into RISC-like complexes containing a particular subset of Argonaute proteins that exert sequence- specific gene repression. In some embodiments, miRNAs are utilized to silence genes of interest as described herein e.g., CAX1, CAX2, CAX3, CAX4, regulators of CAX gene activity, etc.).
  • genetic engineering techniques that may be used to achieve the specific result of gene silencing in plants. Certain techniques employ foreign (exogenous) DNA transfer (physical, viral, bacterial, or CRISPR systems) while other techniques avoid exogenous DNA (intragenic DNA transfer, exogenous RNA application). Genetic engineering techniques may result in stable modification involving integration of DNA into the host genome (physical, bacterial, CRISPR systems) and/or transient modification which is nonintegrative (physical, bacterial, viral, exogenous RNA application). In some embodiments, genetic engineering techniques such as, but not limited to, those described herein are utilized to silence genes of interest as described herein (e.g., CAX1, CAX2, CAX3, CAX4, regulators of CAX gene activity, etc.).
  • the modification of a CAX gene or a gene capable of synthesis of an activator of a CAX gene is carried out using physical (direct) gene transfer.
  • Direct gene transfer involves the uptake of naked DNA by plant cells and its subsequent integration into the genome.
  • the target cells can include: isolated protoplasts or cells; cultured tissues, organs or plants; intact pollen, seeds, and plants.
  • Direct DNA transfer methods are entirely physical processes with no biological interactions to introduce the DNA into plant cells and therefore no “host range” limitations associated with Agrobacterium-mediated transformation.
  • Methods to effect direct DNA transfer can involve a wide range of approaches, including: passive uptake; the use of electroporation; treatments with polyethylene glycol; electrophoresis; cell fusion with liposomes or spheroplasts; microinjection, silicon carbide whiskers, and particle bombardment.
  • particle bombardment is almost exclusively used because there are no limitations to the target tissue.
  • particle bombardment is the overall length of the DNA. Longer DNA molecules are likely to shear either upon particle acceleration or impact.
  • Vectors for direct DNA uptake only need to be standard bacterial plasmids to allow propagation of the vector. It is usual for such vectors to be small, high-copy plasmids capable of propagation in Escherichia coli. This allows convenient construction of plasmids using well-established molecular biology protocols and ensures high yields of vector upon plasmid isolation and purification for subsequent use in transformation. Various authors claim a preference to use DNA of a specific form (circular or linear, double- or single-stranded). However, comparisons of all four combinations of DNA conformation in parallel experiments resulted in similar transformation frequencies and integration patterns.
  • the modification of a CAX gene or a gene capable of synthesis of an activator of a CAX gene is carried out using a viral-mediated gene transformation.
  • a viral-mediated gene transformation The use of plant viruses as vehicles to introduce and express nonviral genes in plants is well known.
  • suitable modifications can be made to the virus itself.
  • the virus DNA can first be cloned into a bacterial plasmid for ease of constructing the desired viral vector with the foreign DNA. The virus can then be excised from the plasmid. A bacterial origin of replication can be attached to the viral DNA, which is then replicated by the bacteria. Transcription and translation of this DNA will produce the coat protein which will encapsulate the viral DNA.
  • RNA virus the virus is generally cloned as a cDNA and inserted into a plasmid.
  • the DNA of the plasmid is then used to make all of the constructions.
  • the RNA virus is then produced by transcribing the viral sequence of the plasmid and translation of the viral genes to produce the coat protein(s) which encapsulate the viral RNA.
  • Viral vectors have been engineered for delivery of genetic material and expression of recombinant proteins in plants.
  • Viral expression systems are considered transient expression systems as the viral vectors are not integrated into the genome of the host, however, depending on which virus is used, virus multiplication and gene expression can persist for long periods (up to several weeks or months).
  • Plant virus vectors provide advantages in quick and high level of foreign gene expression in plants due to the infection nature of plant viruses.
  • the full length of the plant viral genome can be used as a vector, but often a viral component is deleted, for example the coat protein, and transgenic ORFs are subcloned in that place.
  • the ICK motif protein expression ORF can be subcloned into such a site to create a viral vector.
  • These viral vectors can be introduced into plants mechanically since they are infectious themselves, for example through plant wound, spray-on etc.
  • viruses can also be transformed into plants by agroinfection by cloning the virus vector into the T-DNA of the crown gall bacterium, Agrobacterium tumefaciens, or the hairy root bacterium, Agrobacterium rhizogenes.
  • the expression of the ICK motif protein in this vector is controlled by the replication of the RNA virus, and the virus translation to mRNA for replication is controlled by a strong viral promoter, for example, 35S promoter from Cauliflower mosaic virus.
  • Viral vectors with ICK motif protein expression ORF are usually cloned into T-DNA region in a binary vector that can replicate itself in both E. coli strains and Agrobacterium strains.
  • the transient transformation of a plant can be done by infiltration of the plant leaves with the Agrobacterium cells which contain the viral vector for ICK motif protein expression.
  • the transient transformed plant it is common for the foreign protein expression to be ceased in a short period of time due to the post-transcriptional gene silencing (PTGS).
  • PTGS post-transcriptional gene silencing
  • Sometimes a PTGS suppressing protein gene is necessary to be cotransformed into the plant transiently with the same type of viral vector that drives the expression of with the ICK motif protein expression ORF. This improves and extends the expression of the ICK motif protein in the plant.
  • the most commonly used PTGS suppressing protein is P19 protein discovered from tomato bushy stunt virus (TBSV).
  • the modification of a CAX gene or a gene capable of synthesis of an activator of a CAX gene is carried out using Agrobacterium-mediated gene transfer.
  • Agrobacterium strains induce crown galls or hairy roots on plants by the natural transfer of a discrete segment of DNA (T-DNA) to plant cells.
  • T-DNA region contains genes that induce tumor or hairy root formation and opine biosynthesis in plant cells.
  • the T-DNA resides on the Ti or Ri plasmids along with several virulence loci with key vir genes responsible for the transfer process.
  • co-integrate vectors With co-integrate vectors the foreign DNA is integrated into the resident Ti plasmid.
  • the tumor-inducing genes of the T-DNA are first removed leaving the right border and left border sequences.
  • the foreign DNA is then inserted into a vector that cannot replicate in Agrobacterium cells, but can recombine with the Ti plasmids through a single or double recombination event at a homologous site previously introduced between the right border and left border sequences. This results in a co-integration event between the two plasmids.
  • a later refinement resulted in the split-end vector system in which only the left border is retained on the Ti plasmid and the right border is restored by the co-integration event.
  • the main advantage of co-integrate vectors is their high stability in Agrobacterium. However, the frequency of cointegration is low and their development is complex, requiring a detailed knowledge of the Ti plasmid and a high level of technical competence.
  • the helper plasmid is a Ti or Ri plasmid that has the vir genes with the T-DNA region deleted and acts in trans to effect T- DNA processing and transfer to plant cells of a T-DNA on a second plasmid (the binary vector).
  • Binary vectors have several main advantages: small size, ease of manipulation in Escherichia coli, high frequency of introduction into Agrobacterium, and independence of specific Ti and Ri plasmids. They have revolutionized the applications of AgroZzz c/erzMm-mediated gene transfer in plant science and are now used to the virtual exclusion of co-integrate vectors.
  • T-DNA vectors without extraneous DNA segments on the T-DNA.
  • These simple binary vectors consist of a very small T-DNA with a selectable marker gene tightly inserted between the left and right T-DNA borders and a short cloning region with a series of unique restriction sites for inserting genes- of-interest. As a consequence they are based on the minimum features necessary for efficient plant transformation by Agrobacterium.
  • the generation of plants with a single intact T-DNA is preferred.
  • the T-DNA is delineated by two 25 bp imperfect repeats, the so-called border sequences, which define target sites for the VirDl/VirD2 border specific endonucleases that initiate T-DNA processing.
  • the resulting single-stranded T-strand is transferred to plant cells rather than the double stranded T-DNA.
  • initiation of T-strand formation involves a single strand nick in the double- stranded T-DNA of the right border, predominantly between the third and fourth nucleotides.
  • the VirD protein after nicking the border, the VirD protein remains covalently linked to the 5' end of the resulting single-stranded T-strand. In some embodiments, the attachment of the VirD protein to the 5' right border end of the T- strand, rather than the border sequence, establishes the polarity between the borders. In some embodiments, this determines the initiation and termination sites for T-strand formation.
  • Vectors ior Agrobacterium-mediated transformation of plants generally contain two T-DNA border- like sequences in the correct orientation that ideally flank a series of restriction sites suitable for cloning genes intended for transfer.
  • efficient transformation is possible with, only a single border in the right border orientation. Deletion of the left border has minimal effect on T-DNA transfer, whereas deletion of the right border abolishes T-DNA transfer. Retaining two borders flanking the T-DNA helps to define both the initiation and end points of transfer, thereby facilitating the recovery of transformation events without vector backbone sequences.
  • T-strand initiation from the right border results, in most instances, in only 3 nucleotides of the right border being transferred upon plant transformation.
  • the end point of the T-DNA sequence is far less precise. It may occur at or about the left border, or even well beyond the left border. This is confirmed by DNA sequencing across the junctions of T-DNA integration events into plant genomes. The less precise end points at left border junctions results in the frequent integration of vector backbone sequences into plant genomes.
  • the modification of a CAX gene or a gene capable of synthesis of an activator of a CAX gene is carried out using one or more DNA-binding nucleic acids, such as alteration via an RNA-guided endonuclease (RGEN).
  • RGEN RNA-guided endonuclease
  • the alteration can be carried out using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins.
  • CRISPR system refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (transactivating CRISPR) sequence (e.g.
  • tracrRNA or an active partial tracrRNA a tracr- mate sequence (encompassing a "direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a "spacer” in the context of an endogenous CRISPR system), and/or other sequences and transcripts from a CRISPR locus.
  • the CRISPR/Cas nuclease or CRISPR/Cas nuclease system can include a noncoding RNA molecule (guide) RNA, which sequence-specifically binds to DNA, and a Cas protein (e.g., Cas9), with nuclease functionality (e.g., two nuclease domains).
  • a CRISPR system can derive from a type I, type II, or type III CRISPR system, e.g., derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes.
  • a Cas nuclease and gRNA are introduced into the cell.
  • target sites at the 5' end of the gRNA target the Cas nuclease to the target site, e.g., the gene, using complementary base pairing.
  • the target site may be selected based on its location immediately 5' of a protospacer adjacent motif (PAM) sequence, such as typically NGG, or NAG.
  • PAM protospacer adjacent motif
  • the gRNA is targeted to the desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of the guide RNA to correspond to the target DNA sequence.
  • a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence.
  • target sequence generally refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between the target sequence and a guide sequence promotes the formation of a CRISPR complex.
  • Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex.
  • a CRISPR system can induce double stranded breaks (DSBs) at one or more target sites of a CAX gene or a gene capable of synthesis of an activator of a CAX gene, followed by disruptions or alterations as discussed herein.
  • Cas9 variants deemed “nickases,” are used to nick a single strand at one or more target sites.
  • paired nickases can be used, e.g., to improve specificity, each directed by a pair of different gRNAs targeting sequences such that upon introduction of the nicks simultaneously, a 5' overhang is introduced.
  • catalytically inactive Cas9 is fused to a heterologous effector domain such as a transcriptional repressor or activator, to affect gene expression.
  • a target sequence for a CAX gene or a gene capable of synthesis of an activator of a CAX gene may comprise any polynucleotide, such as DNA or RNA polynucleotides.
  • a target sequence may be located in the nucleus or cytoplasm of the cell, such as within an organelle of the cell.
  • a sequence or template that may be used for recombination into the targeted locus comprising the target sequences is referred to as an "editing template” or "editing polynucleotide” or “editing sequence”.
  • an exogenous template polynucleotide may be referred to as an editing template.
  • the recombination is homologous recombination.
  • a tracr sequence which may comprise or consist of all or a portion of a wild-type tracr sequence (e.g.
  • a tracr sequence has sufficient complementarity to a tracr mate sequence to hybridize and participate in formation of the CRISPR complex, such as at least 50%, 60%, 70%, 80%, 90%, 95% or 99% of sequence complementarity along the length of the tracr mate sequence when optimally aligned.
  • one or more vectors driving expression of one or more elements of the CRISPR system can be introduced into the cell such that expression of the elements of the CRISPR system direct formation of the CRISPR complex at one or more target sites.
  • Components can also be delivered to cells as proteins and/or RNA.
  • a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors.
  • two or more of the elements expressed from the same or different regulatory elements may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector.
  • a vector may comprise one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a "cloning site").
  • one or more insertion sites are located upstream and/or downstream of one or more sequence elements of one or more vectors.
  • a single expression construct may be used to target CRISPR activity to multiple different, corresponding target sequences within a cell.
  • a vector may comprise a regulatory element operably linked to an enzyme-coding sequence encoding the CRISPR enzyme, such as a Cas protein.
  • Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, C
  • a CRISPR enzyme can be Cas9 (e.g., from S. pyogenes or S. pneumonia).
  • a CRISPR enzyme can direct cleavage of one or both strands at the location of a target sequence, such as within the target sequence and/or within the complement of the target sequence.
  • a vector can encode a CRISPR enzyme that is mutated with respect to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence.
  • an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand).
  • a Cas9 nickase may be used in combination with guide sequence(s), e.g., two guide sequences, which target respectively sense and antisense strands of the DNA target. This combination allows both strands to be nicked and used to induce NHEJ or HDR.
  • an enzyme coding sequence encoding the CRISPR enzyme is codon optimized for expression in particular cells, such as eukaryotic cells.
  • a eukaryotic cells may be those of or derived from a particular organism, such as a plant, fungi, bacteria, mammal, including but not limited to human, mouse, rat, rabbit, dog, or non-human primate.
  • codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence.
  • Codon bias differs in codon usage between organisms
  • mRNA messenger RNA
  • tRNA transfer RNA
  • the predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization.
  • a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence.
  • the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.
  • Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith- Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g. the Burrows Wheeler Aligner), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).
  • Burrows-Wheeler Transform e.g. the Burrows Wheeler Aligner
  • Clustal W Clustal W
  • Clustal X Clustal X
  • BLAT Novoalign
  • SOAP available at soap.genomics.org.cn
  • Maq available at maq.sourceforge.net
  • the CRISPR enzyme may be part of a fusion protein comprising one or more heterologous protein domains.
  • a CRISPR enzyme fusion protein may comprise any additional protein sequence, and optionally a linker sequence between any two domains.
  • protein domains that may be fused to a CRISPR enzyme include, without limitation, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity.
  • Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags.
  • reporter genes include, but are not limited to, glutathione-5- transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP).
  • GST glutathione-5- transferase
  • HRP horseradish peroxidase
  • CAT chloramphenicol acetyltransferase
  • beta galactosidase beta-glucuronidase
  • a CRISPR enzyme may be fused to a gene sequence encoding a protein or a fragment of a protein that bind DNA molecules or bind other cellular molecules, including but not limited to maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4A DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. Additional domains that may form part of a fusion protein comprising a CRISPR enzyme are described in US 20110059502, incorporated herein by reference.
  • the modification of a CAX gene or a gene capable of synthesis of an activator of a CAX gene is carried out using intragenic DNA transfer.
  • intragenic DNA transfer Despite the rapid global adoption of GM technology in agricultural crops, many concerns have been raised about the use of GM crops in agricultural production. These include ethical, religious and/or other concerns among the general public, with the main underlying issue often involving the transfer of genes across very wide taxonomic boundaries. Current advances in plant genomics are beginning to address some of these concerns. Many genes are now being identified from within the gene pools already used by plant breeders for transfer via plant transformation. More importantly, the design of vectors for plant transformation has recently progressed to the development of intragenic systems. This involves identifying plant-derived DNA sequences similar to important vector components.
  • a particularly useful approach involves adjoining two fragments from plant genomes to form sequences that have the functional equivalence of vectors elements such as: T-DNA borders for Agrobacterium- mediated transformation, bacterial origins of replication, and bacterial selectable elements.
  • vectors elements such as: T-DNA borders for Agrobacterium- mediated transformation, bacterial origins of replication, and bacterial selectable elements.
  • intragenic vectors have been identified from a wide range of plant species, suggesting that intragenic vectors can be constructed from the genome of any plant species.
  • Intragenic vectors provide a mechanism for the well-defined genetic improvement of plants with the entire DNA destined for transfer originating from within the gene pool already available to plant breeders.
  • the aim of such approaches is to design vectors capable of effecting gene transfer without the introduction of foreign DNA upon plant transformation. In this manner genes can be introgressed into elite cultivars in a single step without linkage drag and, most importantly, without the incorporation of foreign DNA, such that the resulting plants may be considered to be non-trans
  • the modification of a CAX gene or a gene capable of synthesis of an activator of a CAX gene is carried out by direct exogenous application of RNA molecules to a plant.
  • RNA molecules such a technique avoids production of either transgenic or genetically modified plants is through direct exogenous delivery of RNA molecules, rather than delivery of DNA which expresses RNA molecules.
  • exogenously applied RNA molecules spread through the plant, inducing RNA interference (RNAi) locally and systemically.
  • CAX1 confers tolerance to anoxia.
  • Multiple studies have shown that Ca-regulated transporters and proteins like calmodulin demonstrate altered expression during low O2 conditions (13, 24, 25). However, the exact identity and roles of the Ca transporters directly involved in the anoxia stress response are unknown.
  • the role of CAX genes in anoxia tolerance was clarified using a seedling survival assay where plants are exposed to anoxic conditions for a period of time and survival monitored upon return to normoxic conditions (ambient air, i.e., -21% O2). In an assay, 14-21-day-old plants were placed in a GasPak Anaerobic system that reduced O2 levels in the experimental chamber to -0% (FIG. 7A).
  • H2O2 burst has been shown to regulate the anoxia stress response, and intracellular H2O2 is known to be one of main sources of oxidative damage triggered upon reoxygenation after anoxic challenge (33).
  • H2O2 post-anoxia in caxl and caxl/3, consistent with the enhanced survival after low O2 stress in these mutants.
  • CAX genes in anoxia tolerance was further elucidated using a submergence survival assay where plants were exposed to anoxic conditions (e.g., submergence) for a period of time e.g., -38 hours) and survival was then monitored upon return to normoxic conditions (e.g., ambient air, i.e., -21% O2).
  • normoxic conditions e.g., ambient air, i.e., -21% O2
  • necrosis and/or leaf wilting phenotypes were readily apparent in otherwise healthy Col-0 (wildtype, aka “WT”) lines seven days after submergence treatment.
  • caxl deficient lines e.g., suppressed and/or eliminated expression, e.g., caxl mutant lines
  • FIG. 18C-18E lines caxl-1, caxl -2, and caxl -3
  • FIG. 18C-18E lines caxl-1, caxl -2, and caxl -3
  • caxl mutant lines transgenically expressed functional complement caxl (e.g., sCAXl, a functional truncation of CAX1) (FIG. 18G). It is noted that cax2 or cax3 lines had similar post- submergence phenotypes as Col-0 (wild-type) plants (FIG. 18H and 181). Additionally, anoxia resistance (e.g., as measured by submergence) of caxl plants was enhanced if cax3 was also mutated (FIG. 18F). caxl plants that underwent submergence treatment were followed throughout the course of their lifespan and found to flower and produce seeds in a manner similar to control wild-type plants that had not undergone submergence treatment. These results were markedly different when compared to wild-type plants that had undergone submergence treatment, such wild-type plants failed to continue developing and died before producing viable seeds.
  • functional complement caxl e.g., sCAXl, a functional truncation of CAX1
  • CAX genes in anoxia tolerance is further elucidated using a submergence survival assay where plants are exposed to anoxic conditions (e.g., submergence in salt water, e.g., salt water with ions and/or ionic concentrations that are relevant when considering natural environments) for a period of time e.g., -12 hours, -24 hours, -36 hours, -48 hours, or greater than -48 hours) and survival is then monitored upon return to normoxic conditions (e.g., ambient air, i.e., -21% O2).
  • anoxic conditions e.g., submergence in salt water, e.g., salt water with ions and/or ionic concentrations that are relevant when considering natural environments
  • normoxic conditions e.g., ambient air, i.e., -21% O2
  • caxl deficient lines e.g., mutant and/or otherwise deficient lines (e.g., suppressed and/or eliminated expression) comprising at least mutation and/or inhibition of caxl , e.g., caxl, caxl/2, caxl/3, caxl/4 mutant lines, etc.
  • caxl deficient lines e.g., mutant and/or otherwise deficient lines (e.g., suppressed and/or eliminated expression) comprising at least mutation and/or inhibition of caxl , e.g., caxl, caxl/2, caxl/3, caxl/4 mutant lines, etc.
  • Leaves were harvested from plants (samples: WT (wild-type), caxl, cax3, or caxl, cax3 double knock out mutant (caxDKO)) at the start of the treatment (0-h control) (FIG. 2A, FIG 8A), after being in the anoxia chamber for 4 hr (anoxia) (FIG. 2B, FIG. 8B) and 1 hr post 7- hour anoxia treatment (recovery) (FIGS. 2C; FIG. 8C).
  • RNAseq data for several of the genes was compared using RT-PCR (FIG. 10; Table 2), indicating that inferences drawn from the RNAseq were likely robust. Table 2. Primer Sequences for Genes selected for qRT-PCR Validation of RNAseq
  • caxl/3 mutant line was more tolerant than the caxl mutant line
  • an additional filter of caxl/3 > caxl was applied if the gene lists were large (n ⁇ 10). It was posited that enhanced expression or repression of specific genes prior to the stress could prime caxl and caxl/3 mutant lines for tolerance (FIG. 2B). It was thus assessed if any of the genes highly expressed in both caxl and caxl/3 mutant lines in control conditions were genes highly expressed in Col-0 and cax3 lines during anoxic or recovery conditions. This was proven to be true.
  • a monodehydroascorbate reductase 3 (AT3G09940.1), GRX480 (Atlg28480) and senescence regulator (AT5G45630.1), which are all involved in redox regulation, are highly expressed in caxl and caxl/3 lines prior to anoxia stress conditions but are also seen to be induced in WT and CAX3 upon anoxic challenge.
  • transgenic expression of the monodehydroascorbate reductase is known to enhance tolerance to temperature and oxidative stresses (38), hinting at a possible mechanism behind the anoxic tolerance in caxl and caxl/3.
  • the GRX480 transcription is salicylic acid (SA)-inducible and appears to have a role in SA/jasmonic acid (J A) cross-talk (39).
  • SA salicylic acid
  • J A jasmonic acid
  • JA metabolism relevant to anoxic tolerance may also be constitutively altered in the caxl and caxl/3 backgrounds is the repression of expression of ALLENE OXIDE CYCLASE 2 (AOC2, AT3G25770).
  • AOC2 catalyzes a crucial step in JA biosynthesis (40).
  • JA and ethylene signaling coordinate biosynthesis of the hydroxycinnamic acid amide defense compounds through modulation of the expression of Anthocyanin 5-aromatic acyltransferase 1 (AT5G61160) (41) and this transcript is downregulated in non-anoxic conditions in the tolerant lines.
  • a series of cold-regulated genes (At2g42530, At2g42540, At4g30650) and chloroplast and stomatai proteins (Atlg53801.1, Atlg53890.3, Atlg29170.1) were also down regulated in col-0 after anoxia while being constitutively repressed in caxl and caxl/3.
  • Some of these cold regulated genes are downregulated by calcium sensors which are highly expressed in the caxl and caxl/3 mutants (FIGS. 11A-11B) (42).
  • AT1G35140 AT1G36060; AT1G54100; AT1G54760; AT1G55810; AT1G56250;
  • AT1G58936 AT1G60750; AT1G62480; AT1G62510; AT1G63090; AT1G65970;
  • AT1G75750 AT1G76650; AT1G77120; AT1G79700; AT1G79910; AT1G80160;
  • AT2G17036 AT2G17850; AT2G19590; AT2G19800; AT2G20670; AT2G22880;
  • AT4G10250 AT4G10265; AT4G10270; AT4G15760; AT4G16160; AT4G16162;
  • AT4G26460 AT4G27450; AT4G28040; AT4G28811; AT4G30270; AT4G30370;
  • AT5G34830 AT5G35525; AT5G39160; AT5G39200; AT5G39580; AT5G39890;
  • AT5G47910 AT5G49360; AT5G54080; AT5G56100; AT5G56870; AT5G57220;
  • ATMG00400 ATMG00410; ATMG00510; ATMG00513; ATMG00516; ATMG00560;
  • ATMG01050 ; ATMG01120; ATMG01130; ATMG01170; ATMG01190; ATMG01200;
  • ATMG01360 [0140] GO analysis of these 313 genes identified similar categories as the sensitive lines: response to hypoxia, small molecule catabolism, anaerobic respiration, response to hydrogen peroxide and sucrose. Based on the improved tolerance of caxl/3 lines, we focused the list by imposing the condition of expression in caxl/3 being higher than in caxl. This generated a list of 81 transcripts; GO analysis for this list showed significant enrichment in genes related to changes in ethylene signaling and response to carbohydrates, classic plant- anoxic response components. If the 313 transcripts are analyzed to remove the transcripts also induced during anoxia in both Col-0 and cax3 there are 189 transcripts (FIG.
  • glyceraldehyde-3-phosphate dehydrogenase A subunit AT1G12900
  • fructose-bisphosphate aldolase 1 AT2G21330
  • fructose-bisphosphate aldolase 2 AT4G38970
  • phosphoglycerate kinase AT3G12780
  • Alcohol dehydrogenase 1 (AT1G77120) is upregulated in Arabidopsis thaliana in response to low oxygen(44); however, it is less up regulated during anoxia in both caxl and caxl/3 (FIG. 10).
  • This diminished induction may reflect the generally lower degree of O2 stress response induced in these mutants or possibly an element of metabolic reprogramming that underlies the enhanced tolerance of these lines.
  • arginase transcripts are upregulated, a response also seen in the mitochondria of rice experiencing anoxic stress (45). This increase flux through arginase could then possibly be producing more urea, again a characteristic of anoxia-stressed plant mitochondria (45).
  • RNAs were associated with chloroplast, mitochondrial and ER associated proteins (for example: AT1G28760.1; AT1G66510.1; AT2G01390.1; AT3G13310.1; AT5G15250.2; AT4G27657.1; AT5G15250.2) suggesting there could be some organelle cross-talk operating in this response.
  • 9 of the 41 transcripts are associated with mitochondrion function; it appears that disruption of the tonoplast H+/Ca transporter CAX1 impacts the anoxia dependent synthesis or stability of transcripts of mitochondrial origin. Previous work has shown that these mitochondrial RNAs are more stable than anticipated, and that heat stress enhances their detection (46).
  • Anoxia stress impacts the ER which results in the unfolded protein response (UPR) (1).
  • the caxl and caxl/3 lines demonstrated upregulation of GAAP3 (AT4G02690) that functions to mitigate the UPR and programed cell death (47, 48).
  • caxl and caxl/3 showed upregulation of AtPARK13 (AT5G27660.1) which targets misfolded proteins (49). Potentially, the caxl and caxl/3 tolerant lines perceive less ER stress during anoxia recovery than col-0 and cax3.
  • the list of 41 anoxia responsive transcripts in caxl and caxl/3 also includes several putative regulatory molecules that are interesting with respect to CAX function.
  • CBL-interacting protein kinase CBL15; AT5G01810
  • this Ca sensor has a role in carbohydrate metabolism, ABA and ethylene signaling (50).
  • this protein has domains that may interact with CAX1 (51).
  • CAX activity can also be modulated by pH changes (52) and a 14-3-3 protein (GRF11; AT1G34760) is upregulated in the caxl and caxl/3 anoxia- responsive transcriptome.
  • 14-3-3 proteins can bind to H+-ATPases to modulate cellular pH and ATP levels as well as modulate hormone levels, the heat stress response and mitochondrial function (53, 54), cellular elements closely linked to anoxic responses.
  • AT1G07500 AT1G08630; AT1G09950; AT1G10140; AT1G12610; AT1G14200;
  • AT1G1833O AT1G19530; AT1G19540; AT1G19620; AT1G21340; AT1G21940;
  • AT1G34575 AT1G35140; AT1G44414; AT1G50745; AT1G52560; AT1G53540;
  • AT1G66500 AT1G66510; AT1G67265; AT1G71000; AT1G71520; AT1G72060;
  • AT1G72416 AT1G72660; AT1G72760; AT1G73480; AT1G74310; AT1G74450;
  • AT2G07707 AT2G07724; AT2G07725; AT2G07727; AT2G07734; AT2G07771;
  • AT3G15500 AT3G16050; AT3G17400; AT3G19240; AT3G20340; AT3G20395;
  • AT3G22090 AT3G22100; AT3G23150; AT3G24500; AT3G25250; AT3G27220;
  • AT3G48240 AT3G49160; AT3G50310; AT3G50560; AT3G51910; AT3G53830;
  • AT4G26200 AT4G26460; AT4G27410; AT4G27450; AT4G27652; AT4G27654;
  • AT4G35770 AT4G36850; AT4G37710; AT4G38030; AT5G01740; AT5G02170;
  • AT5G05410 AT5G06980; AT5G07330; AT5G08150; AT5G09590; AT5G09930;
  • CCA1 organonitrogen compounds
  • AT2G46830 AT-HSFB2B (AT4G11660), AT4G18450, AT4G30370, ZAT6 (AT5G04340), HSFB2A (AT5G62020)
  • monooxygenase activity CYP81D7 (AT2G23190), MOI (AT4G15760.1), CYP707A1 (AT4G19230), CYP706A2 (AT4G22710), AT5G35320, CYP81F2 (AT5G57220), CYP71B10 (AT5G57260.1)).
  • the hypoxia/heat transcripts includes a well-characterized putative glucose-regulated protein that binds to the promoters of glucose-regulated heat shock responsive genes (AT1G66080).
  • the heat regulatory transcripts included heat shock transcription factors (At5g6202; At4gl l660), HSP20-like chaperones (AT5G37670; AT4G16555), and HSP70 (AT4G32208).
  • This enhancement of heat regulated genes in all probability causes the increased expression of PYRIDOXINE BIOSYNTHESIS 1.2 (PDX1.2, AT3G16050), which is under control of the HSFA1 family of heat shock transcriptional regulators (55).
  • PDX1.2 is a pseudoenzyme required for supplying adequate levels of vitamin B under deleterious conditions such as heat stress (55).
  • This list of the 500 most differentially expressed transcripts in caxl and caxl/3 during recovery also includes four other transcription factors: bHLH (At3g22100), AP2- EREBP (At4g 18450), NAC (At4g27410), and C2H2 (At5g04340) that provide indications about some of the hormonal system that these mutants may use to respond to the reoxygenation stress after anoxic treatment.
  • AP2-EREBP is an ethylene response factor and NAC is ABA responsive, suggesting that hormone signaling is altered in both caxl and caxl/3 (FIG. 13).
  • RNAs associated with a negative regulator of ABA signaling 15 (AT1G09950.1); a SAUR-like auxin-responsive protein (AT4G12410.1), and a JA-mediated signaling protein (JAS1/JAZ10; AT5G13220) are also highly expressed in the mutants.
  • JAZ10 is part of the JA-related defense response linked to slow wave potential signaling (56) and it is interesting to speculate that JAZ10 may be operating in responses to both electrical and Ca signals during anoxia recovery.
  • RNAs associated with altered redox regulation (AT3G44190.1, AT3G50560.1) were also highly expressed during recovery in the caxl and caxl/3 mutants. A molecular mechanism for altered redox regulation in these mutants could be caused in part by induction of C2H2, a zinc finger transcription factor that targets redox-related genes.
  • proline dehydrogenase (ERD5, AT3G30775) that has been previously shown to be hypoxia responsive (60). This enzyme may be important in respiration, with proline acting as an alternative energy substrate (61).
  • a transcript from an unknown protein response to proline was also upregulated, as was an amino acid transporter (AT5G02170).
  • a xyloglucan endotransglucosylase/hydrolase (At4g25810) is highly expressed in these lines as are transcripts involved in cell wall biogenesis (AT3G29810) and several RNAs that may impact cell wall and lipid biosynthesis are repressed during recovery (AT4G12490, AT4G12520, AT3G07970, AT5G12580).
  • FIG. 3B.1 and FIG. 3B.2 The top 40 enriched GO term categories are presented in FIG. 3B.1 and FIG. 3B.2.
  • FIG. 2D In support of the results from RNA-seq analysis and the data which showed lower levels of H2O2 in caxl and caxl/3 post anoxia (FIG. 2D), proteins shown to be increased in abundance in these genotypes were significantly enriched in GO terms related to oxidative stress including response to oxidative stress, cell redox homeostasis and oxidation reduction process (FIG. 3B.1 and FIG. 3B.2). Furthermore, protein changes were seen in various organelles (FIGS. 15A-15C).
  • Activated H2O2 removal may be related to the markedly increased abundance of hydrogen peroxide catabolic process proteins including APX1 (AT1G07890), GLO1 (AT3G14420), PRXIIF (AT3G06050), TL29 (AT4G09010) and CAT1- 3 (AT1G20630, AT4G35090, AT1G20620).
  • APX1 AT1G07890
  • GLO1 AT3G14420
  • PRXIIF AT3G06050
  • TL29 AT4G09010
  • CAT1- 3 AT1G20630, AT4G35090, AT1G20620.
  • proteomic analysis also indicated enrichment in proteins involved in glycolysis and captured significant increases in a number of proteins involved in the glycolytic pathway in caxl and caxl/3 post anoxia (GO enrichment, FIG. 3B.1; FIG. 3B.2; FIG. 16), including fructose 1,6 bisphosphatase, and phosphoglycerate kinase. With the latter showing similar responses when data from both approaches was overlaid onto KEGG pathways (FIG. 3C).
  • caxl -mediated anoxia tolerance likely involves signaling processes related to Ca, ROS and hormones and to pathways already linked to other abiotic and biotic stresses such as heat stress (FIGS. 10A-10B).
  • FIGS. 10A-10B To further investigate the roles of these processes in caxl-mediated anoxia tolerance, we applied various treatments to the plants and assessed changes in anoxia tolerance using the anoxia- reoxygenation recovery assay.
  • DPI Diphenyleneiodonium chloride
  • GSH glutathione
  • H2O2 (1 mM or lOOmM
  • H2O2 at ImM did not have any effect on anoxia tolerance in all lines, while lOOmM treatment 2 days prior to the anoxia partially rescued the anoxia sensitivity of Col-0 and cax3, and partially reduced the robustness of caxl and caxl/3.
  • ABA is a regulator of abiotic stress responses and exogenous application of 50 pM ABA 2 d prior to the anoxia treatment conferred anoxia tolerance to the Col-0 and cax3 lines.
  • This tolerance phenocopied the tolerance of caxl and caxl/3.
  • This phenotype was dependent on protein synthesis as pretreatment with cycloheximide (100 pM), a protein synthesis inhibitor, along with ABA rendered all the lines sensitive.
  • Various other treatments caused incremental changes in the anoxia tolerance phenotype: elicitors of plant defense responses and ethylene precursors and ethylene signaling inhibitors had modest effects; auxin, cytokinin and JA applications caused almost no change in anoxia stress phenotypes.
  • the second signal was slightly diminished (FIG. 5D).
  • the second Ca signal was observed earlier than water treated plants (peak at 140 sec compared to 195 sec in WT) and the peak intensity of the signal is increased to a level higher than the initial signal in WT.
  • the spatial distribution of the signal was also changed: in water control caxl-1 lines, the second Ca signal appeared to propagate from the base of the meristem of the rosette along the petiole and into the leaf blade of the leaves; while with SA, the Ca signal first appeared visible at the tip and edge of the leaf blade and migrated to the petiole (FIG. 5D). This result suggests caxl has a differential Ca response to SA compared to water controls.
  • a plant’ s oxygen supply can vary from normal (normoxia) to total depletion (anoxia). Tolerance to anoxia is relevant to wetland species, rice cultivation and transient waterlogging of crops. Decoding and transmitting calcium (Ca) signals may be an important component to anoxia tolerance; however, the contribution of intracellular Ca transporters in this process are poorly understood.
  • Four functional cation/proton exchangers (CAX1-4) in Arabidopsis thaliana help regulate Ca homeostasis around the vacuole. In specific embodiments of the present disclosure, any single or double CAX mutation that includes CAX1 are more tolerant to anoxic conditions.
  • CAX1 mediated anoxia changes have been identified that phenocopy changes present in anoxia tolerant crops: altered metabolic processes, diminished ROS production post anoxia, and altered hormone signaling. Comparing wild-type and CAX1 expressing genetically encoded Ca indicators (GECIs) demonstrated altered Ca signals in the CAX1 during anoxia recovery. This suggests that anoxia induced Ca signals around the plant vacuole are involved in organelle cross-talk, metabolic fluxes and ROS signaling.
  • the CAX1 anoxia response pathway will be engineered to circumvent the adverse effects of anoxic conditions that impair production agriculture.
  • CAX1 a gene that is neither highly expressed during oxygen stress conditions nor thought to be modified in plants adapted to oxygen limitations. Studies illustrated that caxl mutant lines are tolerant to anoxia conditions; this phenotype was specific to CAX1, but enhanced tolerance was found in caxl/3 mutant lines. It was established that post anoxia, caxl lines showed reduced ROS production; meanwhile, the caxl and caxl/3 lines demonstrated apparent changes in Ca signaling, metabolism and hormone signaling during and after the stress. Using various approaches it was explored how CAX1 mediated anoxia tolerance could be manipulated via changes in temperature, Ca levels, reactive oxygen species and phytohormones. It was also investigated how CAX1 changed the amplitude and duration of Ca signals during anoxia. This work highlights a central role of tonoplast localized H + /Ca transport in anoxia perception and recovery while further defining the molecular choreography of anoxia signaling.
  • Enzymes that regulate both sucrose breakdown and anaerobic fermentation are controlled in a temporal manner in response to oxygen deprivation (70).
  • the management of carbohydrate consumption and avoidance of oxidative stress have been proposed to be key determinants of anoxia tolerance (71).
  • Rice cultivars that vary in submergence tolerance display distinct transcript profiles for glycolytic and fermentation enzymes (72).
  • Our data point toward differences in both the mitochondria and plastid as well as glycolysis in caxl and caxl/3 impacting anoxia tolerance FIG. 3C; FIG. 15).
  • Increases in glycolysis post-anoxia may reflect an enhanced ability to return to cellular homeostasis by replenishing ATP stores in caxl which would have been significantly diminished during the anoxia treatment.
  • RNAseq revealed that genes involved in ABA biosynthesis (NCED9, AT1G78390) as well as enzymes involved in the conversion of active ABA to its storage form (UGT71B6) were upregulated in caxl and caxl/3.
  • RNA-seq data revealed a global change in the transcript abundances of Ca transporters and Ca sensors/effectors in both caxl and caxl/3 (FIG. 11).
  • CAX1 Loss of function mutations in Arabidopsis thaliana H+/Ca transporter CAX1 display a robust conditional phenotype, they gain tolerance to anoxia conditions (FIGS. 1A- 1E; FIG. 6). Disruption of CAX transport during this stress impacts a myriad of cellular processes: cell wall biosynthesis, metabolism, ROS, hormone and Ca signaling (FIG. 6). Furthermore, the mutants appear to be primed for the stress by having heightened expression of ROS related transcripts and diminished expression of cold tolerance genes during normal growth conditions. The caxl and caxl/3 lines also appear to be ‘warmed up” as they display heightened expression of heat responsive transcripts during the anoxia recovery phase (FIGS. 12C-12D).
  • Heat shock response involves the induction of a wide range of molecular chaperones to aid in protecting and refolding damaged proteins and so this constitutive production of e.g., heat shock proteins may equip caxl and caxl/3 plants with a ready-made machinery to cope with some of the cellular damage that anoxia and reoxygenation imposes.
  • caxl has altered cold- acclimation responses (14) and temperature perception may be an important factor in the observed anoxia tolerance phenotype.
  • CAX1 may be activated by the binding of a specific regulatory protein, such as SOS2 (Salt Overly Sensitive 2) protein kinase, CXIP1 (CAX interacting protein T), and CXIP4 (CAX interacting protein 4), to the autoinhibitory N-terminus of CAX1 (84).
  • SOS2 Salt Overly Sensitive 2 protein kinase
  • CXIP1 CAX interacting protein T
  • CXIP4 CAX interacting protein 4
  • a means of improving anoxia tolerance may include down-regulation of at least one gene capable of synthesis of at least one activator of CAX1 to suppress or eliminate CAX1 activation during anoxic conditions.
  • An alternative means of improving anoxia tolerance may include transient down-regulation of at least one gene capable of synthesis of at least one activator of at least one other CAX gene combined with down-regulation of at least one gene capable of synthesis of at least one activator of CAX1 to suppress or eliminate activation of multiple CAX genes during anoxic conditions.
  • a further alternate means of improving anoxia tolerance may include transiently suppressing and/or eliminating expression of an activated CAX1 or a combination of an activated CAX1 and at least one other activated CAX gene during anoxic conditions.
  • a further means of improving anoxia tolerance may include transient downregulation of a gene capable of synthesis of an activator of one or more CAX genes including CAX1 to suppress or eliminate CAX gene activation as well as transient down-regulation of expression of one or more activated CAX genes including CAX1 during anoxic conditions.
  • an activator of CAX1 is selected from the group consisting of SOS2 protein kinase, CXIP1, and CXIP4.
  • hpRNA hairpin RNA
  • An experimental limitation to this technology occurs when a gene is necessary for cell viability and reducing the levels below a certain threshold inhibits plant regeneration. This scenario requires that the gene be turned off and on in specific environmental conditions: CAX genes are necessary during specific growth conditions, thus a null mutation is not optimal for plant performance.
  • Inducible plant promoter systems to drive hpRNA CAX mediated gene silencing are available. Variations of this system can be used for tissue- specific hpRNA expression.
  • Chlorophyll fluorescence measurement Chlorophyll content was measured via spectroscopic absorbance of chlorophyll a and b, as previously described (23). Briefly chlorophyll was extracted from whole rosettes using 96% (vol/vol) DMSO and incubated at 65 °C water bath for 4 hours. Absorbance at 664, 647, and 750 nm was measured with a spectrophotometer (Cary 50) in ImL cuvettes. Chlorophyll a and b concentrations, and total chlorophyll content were calculated following the equations of reference and were normalized to tissue fresh weight.
  • RNAseq transcriptomic analysis Approximately 100 mg of full rosettes from anoxia-treated or untreated plants were frozen and ground in liquid nitrogen, and were subjected to Trizol total RNA extraction. Total RNA samples were sent to BGI Genomics (BGI Americas Corporation, Cambridge, MA, USA) for transcriptome sequencing on their DNBseq platform using 150 bp pair-end sequencing chemistry. Data processing and differential gene expression (DEG) analysis were also conducted by BGI. Sequences mapped to CAX1 and 3 were observed in caxl and cax3 and caxl/3 double mutants.
  • BGI Genomics BGI Americas Corporation, Cambridge, MA, USA
  • DEG differential gene expression
  • the homogenate was then filtered through Miracloth (Calbiochem, La Jolla, CA), and subsequently clarified by centrifugation to remove cellular debris.
  • the total proteins in supernatant 500 pL were then precipitated using 200 pL 10X TE, 200 pl of 0.3% sodium deoxycholate, and 200 pl of cold 72% TCA.
  • the protein pellets were washed in 90% room-temperature methanol and subsequently lyophilized.
  • Protein identification was carried out at the Institute for Molecular Biosciences proteomics facility at the University of Queensland, Brisbane. Proteins were analyzed using an Eksigent, Ekspert nano LC400 uHPLC coupled to a TripleTOF 6600+ System (SCIEX, Canada) equipped with a PicoView nanoflow ion source (New Objective, USA). Protein extract (up to 5 pl) was injected onto a ChromXP C18-CL column (3 pm, 75 pm x 150mm) (SCIEX, Canada). Mobile phase solvents consisted of, solvent A; 0.1% formic acid in water, and solvent B; 0.1% formic acid in acetonitrile.
  • the mass spectrometer was set to acquire 100ms of full scan TOF-MS data over the mass range 350-1500 m/z, followed by up to fifty 50ms full scan product ion data in IDA mode over the mass range 100-1500 m/z. Ions observed in the TOF-MS scan exceeding a threshold of 100 counts and a charge state of +2 to +5 were set to trigger the acquisition of product ion MS/MS spectra of the resultant 50 most intense ions.
  • Protein Identification and Quantification Protein Pilot 5.0.2 (SCIEX, Canada) was used to search spectra against the Uniprot Arabidopsis thaliana database (129652 proteins, May/ 12/2020) and encode the output mzIdentML file for the downstream analysis. Scaffold 4.8.6 (Proteome Software, Portland, OR, USA) was used to validate MS/MS-based protein identifications and quantification. Protein identifications were accepted if they could be established at greater than 99% probability and contained at least two unique peptides. Normalized spectral abundance factor (NSAF) was used for protein quantification (91, 92).
  • SAF Normalized spectral abundance factor
  • Bioinformatics Analysis for Gene Ontology Enrichment Analysis Proteins identified in at least 2 out of 3 biological replicates were considered as present in the corresponding genotypes. To evaluate the significance of comparative quantification by different genotypes, Student’s t-test was performed on the data, and the differences were assigned to be significant at a p-value less than 0.05. Differentially abundant proteins (DAPs, including exclusively present proteins) were submitted to David Bioinformatics Resources 6.8 for Gene Ontology enrichment analysis (93).
  • MDA Malondialdehyde Measurements. MDA was quantified using a colorimetric method described in Stewart & Bewley (94). Briefly, full rosette leaves were pulverized in liquid nitrogen and resuspended in 80% (vol/vol) ethanol, and the supernatant was mixed with a reactant mixture of 0.65% (wt/vol) thiobarbituric acid and 20% (wt/vol) trichloroacetic acid. After a 30-min incubation at 95 °C, absorbance was measured at 532 and 600 nm with a spectrophotometer (Cary 50) in ImL cuvettes.
  • a spectrophotometer Cary 50
  • ACC 50pM
  • MeJA 50pM
  • ABA 50pM
  • SA 50pM
  • GA 20pM
  • ethylene antagonists AVG (50pM), silver nitrate (200pM), RBOH inhibitor diphenyleneiodonium chloride (DPI) (8pM), Ca chelator EGTA (lOmM), ruthenium red (8pM), LaC13 (5mM), GdC13 (5mM), and W-7 (lOOpM).
  • ACC is 1 -Aminocyclopropane- 1- carboxylic acid
  • MeJA is methyl jasmonate
  • GA gibberellic acid
  • W-7 is W-7 hydrochloride.
  • RNA Extraction and Quantitative Real-Time qPCR Total RNA was extracted using trizol from 50mg full rosette leaves. For qRT-PCR, single- stranded cDNA was synthesized from 1 pg RNA using oligo dTis primers (Invitrogen). qRT-PCR was performed on a Biorad CFX96 Real-Time PCR System (Biorad) with iTAQ SYBR Green Master Mix (Bio-Rad). Primers used are listed in the table in Table 2. Relative transcript abundance was calculated using the comparative 2 -AACT method normalized to UBQ10 (95).
  • Col-0 or caxl-1 harboring the GCamP3 sensor constructs were grown on * strength MS media supplemented with 0.5% sucrose for 12 days before they were surface sprayed with either water control, lOmM EGTA, 50pM ABA or ImM SA, 4 hour prior to the 4h anoxia treatment.
  • water control lOmM EGTA, 50pM ABA or ImM SA
  • Ca-GCamP3 fluorescence was measured to assay cytosolic Ca concentrations immediately after the plants were re-exposed to atmospheric oxygen. The fluorescence in the first set of true leaves was analyzed using ImageJ. Experiments were run in duplicate each day and experiments repeated on different days. For example, assays with the water control were repeated at least 10 different times over the course of multiple months while the SA assay was repeated on two different days (two replicates each day).
  • RNA-Seq reveals novel genes and pathways associated with hypoxia duration and tolerance in tomato root. Sci. Rep. 10(1): 1692.
  • GENERAL REGULATORY FACTOR11 acts downstream of nitric oxide to regulate iron acquisition in Arabidopsis thaliana. New Phytol. 197(3):815-824.
  • Transgenic AEQUORIN reveals organ-specific cytosolic Ca2+ responses to anoxia and Arabidopsis thaliana seedlings. Plant Physiol. 11 l(l):243-257.

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Abstract

Compositions and methods for regulating calcium transporters in plants during anoxic conditions are used as a means of improving plant development and survival during periods of oxygen deprivation. In specific embodiments, such compositions and methods are useful as a means of improving crop performance during periods of anoxia. Embodiments of the disclosure include methods and compositions related to genetic modification for imparting anoxia tolerance to plants. In certain embodiments, the genetic modification may be applied to any kind of plant. In particular embodiments, expression of one or more CAX genes is genetically modified by the hand of man to impart anoxia tolerance to the modified plant as compared to a plant of the same kind that lacks the recombinantly introduced genetic modification. In specific embodiments, the one or more CAX genes that is modified is CAX1, and optionally, CAX2, CAX3 and/or CAX4.

Description

ALTERING PLANT CALCIUM TRANSPORT TO IMPROVE PLANT ANOXIA
TOLERANCE
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63/129,213 filed December 22, 2020, which is incorporated by reference herein in its entirety.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This disclosure was made with government support under 1557890 awarded by the National Science Foundation, and under 58-3092-5-001 awarded by the USDA. The government has certain rights in the invention.
TECHNICAL FIELD
[0003] In certain embodiments of the disclosure, the field of the disclosure includes plant biology, yeast biology, agriculture, agronomy, molecular biology, and/or cell biology, for example.
BACKGROUND
[0004] Plants must mitigate numerous stress conditions simultaneously (1). For example, flooding can impose limited oxygen (hypoxia) conditions, temperature changes, and altered oxidative conditions (2). The lack of oxygen (anoxia) reduces plant energy production (3). To survive and limit post- anoxic injury, plants regulate the flow of the little energy produced under these stressful conditions to essential processes (4-7). The perception and response to cellular oxygen (O2) levels is mediated by a subset of ERFs (Ethylene Response Factors) termed type VII ERFs which are indispensable in optimizing metabolic performance during low O2 conditions (8); Reactive Oxygen Species (ROS) signals are integrated with ethylene signaling and calcium (Ca) signaling may be one of the most important components of anoxia stress tolerance (9). Ca signaling differs among plants with marked differences between Arabidopsis thaliana and rice in the spatiotemporal parameters of the observed Ca signatures (10). Ca signaling differences appear to precede gene expression differences and metabolic fluxes, although precisely how Ca is triggering downstream events remains unclear (1). Conceptualizing signaling events involved in anoxia perception and tolerance is important to improve plant performance both during and after anoxia conditions.
[0005] Calcium is at the nexus between many facets of plant biology (11, 12). Calcium transporters on various membranes help to orchestrate responses to stresses such as changes in temperature and anoxia (11, 13-15). Calcium pumps and exchangers are found in multiple membrane systems, including the endoplasmic reticulum (ER), vacuole, and plasma membrane (PM). Calcium pumps (such as the Ca2+-ATPases) are considered low-capacity, high-affinity efflux systems, but within this broad group of transporters, H+/Ca2+ exchangers (CAXs) are distinct because they have high capacity and low affinity. While both Ca pumps and CAXs remove Ca from the cytoplasm, their individual contributions to the control of the magnitude or duration of different stimulus-specific Ca signals remains enigmatic.
[0006] There are six CAX genes in Arabidopsis thaliana, but only CAX1-4 are expressed and appear to be predominately localized to the vacuole (16). CAX2 and CAX4 are expressed at lower levels, and the majority of work on defining roles in Ca signaling and homeostasis has focused on the more highly expressed CAX1 and CAX3 (17). CAX1 is expressed in aerial tissue and is the dominant mechanism for Ca accumulation in the leaf (17); CAX3 is primarily expressed in the roots (18). Expression of CAX3 is increased in aerial tissue during stresses which may help remove increased Ca levels from the cytosol. In a caxl/cax3 double mutant there is reduced capacity for mesophyll Ca accumulation resulting in reduced cell wall extensibility, stomatai aperture, transpiration, CO2 assimilation, and leaf growth rate along with altered expression of cell wall-modifying proteins, and thicker cell walls (17). In yeast assays, CAX1 and CAX3 may function as heterodimers (18); furthermore, the two transporters are autoinhibited and co-expressed in guard cells where they may function singly or together in intracellular signaling (19). Absence of CAX1 induces ectopic expression of other CAXs, which is often sufficient to maintain mesophyll cellular uptake of Ca from the apoplast (20). There is also interplay between CAX transporters and other transporters; deletions in CAX1 reduce vacuolar H+/Ca2+ antiport activity by 50%, decrease vacuolar V- type proton ATPase activity by 40% and increase vacuolar Ca-ATPase activity by 36% (21). Given the functional connectivity among the various transporters, the prevailing wisdom suggests that CAXs are important for viability (22).
[0007] The present disclosure concerns methods and compositions that address the management of plant tolerance to anoxia and production of plants with an increased tolerance to anoxia. BRIEF SUMMARY
[0008] Anoxic conditions can be detrimental to plant development and yield. Recovery post-anoxia is also an important determinant of tolerance, yet mechanisms regulating this remain largely unknown. Anoxic responses have been extensively studied (4); however, less is known about the processes governing recovery from this stress and the signals that mediate these responses. Traditional approaches to study these events include differential gene expression studies and comparative genomic analysis (23).
[0009] In the present disclosure, an Arabidopsis thaliana mutant defective in endomembrane calcium transport that dramatically improves recovery from anoxic conditions has been identified and characterized. The present disclosure is directed to compositions and methods for regulating such calcium transporters during anoxic conditions as a means of improving plant development and survival during periods of oxygen deprivation. In specific embodiments, such compositions and methods are useful as a means of improving crop performance during periods of oxygen deprivation.
[0010] Embodiments of the disclosure include methods and compositions related to tolerance to anoxia, including tolerance that is acquired because of one or more genetic modifications by the hand of man. In embodiments, the genetic modification may be applied to any kind of plant. In particular embodiments, expression of one or more CAX genes is genetically modified by the hand of man to impart anoxia tolerance to the modified plant as compared to a plant of the same kind that lacks the recombinantly-introduced genetic modification. In particular embodiments, expression of one or more CAX genes is genetically modified by the hand of man to impart anoxia tolerance to the modified plant as compared to a plant of the same kind that lacks the genetic modification. In particular embodiments, expression of one or more CAX genes is genetically modified by the hand of man to impart anoxia tolerance to the modified plant as compared to a plant of the same kind that lacks the synthetic genetic modification. In specific embodiments, the one or more CAX genes that is modified is CAX1. In other aspects, the one or more CAX genes that is modified is CAX1 and one or more other genes, including one or more other CAX genes, such as CAX2, CAX3, and/or CAX4. In particular embodiments, expression of a gene capable of synthesis of one or more activators of one or more CAX genes is genetically modified by the hand of man to impart anoxia tolerance to the modified plant as compared to a plant of the same kind that lacks the recombinantly-introduced genetic modification. In specific embodiments, the gene capable of synthesis of one or more activators of one or more CAX genes that is modified is a gene capable of synthesis of one or more activators of CAX1. In other aspects, the gene capable of synthesis of one or more activators of one or more CAX genes that is modified is a gene capable of synthesis of one or more activators of CAX1 and one or more activators of one or more other genes, including one or more other CAX genes, such as CAX2, CAX3, and/or CAX4. In other aspects, the at least one activator of CAX1 is selected from the group consisting of SOS2 protein kinase, CXIP1, and CXIP4.
[0011] The disclosure encompasses anoxia tolerance for monocots and/or dicots and may encompass bryophytes (mosses), pteridophytes (ferns), gymnosperms (conifers), and angiosperms (flowering, seed-bearing plants), at least. In specific embodiments one or more crop plants are modified as disclosed herein so that they may be tolerant to anoxia as compared to crop plants without the recombinantly-introduced genetic modification or they may have enhanced tolerance compared to the level of tolerance they naturally had without the genetic modification.
[0012] In specific embodiments, a composition of the disclosure includes a genetically modified plant, part, and/or progeny thereof, excluding Arabidopsis thaliana, including a plurality of plant cells including: synthetically suppressed and/or eliminated expression of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4, in said plurality of plant cells. In specific embodiments, the plurality of plant cells include: one or more agents capable of driving gene silencing of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4, in said plurality of plant cells; and, optionally, a regulatory sequence capable of inducing said agent(s) to drive said gene silencing of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4, in said plurality of plant cells. In specific embodiments, the agent(s) include a nucleic acid sequence and/or a protein. In specific embodiments, the nucleic acid sequence includes exogenous DNA, intragenic DNA, and/or exogenous RNA. In specific embodiments, the protein includes a nuclease and/or a protease.
[0013] In specific embodiments, the agent(s) and, optionally, the regulatory sequence, are (a) naked; and/or (b) comprised in (i) a complex; (ii) a carrier system; (iii) a particle gun system; (iv) a viral vector; (v) an Agrobacterium vector; and/or (vi) a CRISPR vector. In specific embodiments, the agent(s) are capable of driving RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4.
[0014] In specific embodiments, the agent(s) are capable of expressing double stranded RNA (dsRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), small RNA (sRNA), microMRNA (mRNA), pre-miRNA, and/or pri-miRNA, capable of RNA interference (RNAi) of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4. In specific embodiments, the agent(s) include double stranded RNA (dsRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), small RNA (sRNA), microMRNA (mRNA), pre-miRNA, and/or pri-miRNA, capable of RNA interference (RNAi) of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4.
[0015] In specific embodiments, regulator sequence is capable of inducing said agent(s) to drive RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4. In specific embodiments, the regulatory sequence is capable of inducing said agent(s) to express double stranded RNA (dsRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), small RNA (sRNA), microMRNA (mRNA), pre-miRNA, and/or pri-miRNA, capable of RNA interference (RNAi) of CAX1 and, optionally, RNAi of at least one of CAX2, CAX3, or CAX4. In specific embodiments, the regulatory sequence is tissue specific and/or cell-type specific. In specific embodiments, the regulatory sequence is low oxygen-induced, chemical-induced, temperature-induced, and/or light-induced.
[0016] In specific embodiments, the plurality of plant cells includes suppressed or eliminated expression of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4 before, during, and/or after anoxic conditions. In specific embodiments, the agent(s) are capable of driving RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4 before, during, and/or after anoxic conditions. In specific embodiments, the plurality of plant cells include transient and/or stable suppressed or eliminated expression of CAX1 and, optionally, RNAi of at least one of CAX2, CAX3, or CAX4. In specific embodiments, the agent(s) are capable of driving transient and/or stable RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of CAX1 and, optionally, RNAi, TGS, and/or VIGS of at least one of CAX2, CAX3, or CAX4.
[0017] In specific embodiments, the plurality of plant cells have upregulated expression during anoxia of at least one gene selected from the group consisting of: AT 1G01355; AT1G02610; AT1G02620; AT1G03090; AT1G03610; AT1G06570; AT1G08630;
AT1G08930; AT1G10070; AT1G10140; AT1G10550; AT1G11260; AT1G12780;
AT1G14860; AT1G15040; AT1G15330; AT1G15670; AT1G17290; AT1G183OO;
AT1G18773; AT1G19396; AT1G19530; AT1G19540; AT1G19610; AT1G19620; AT1G24880; AT1G25560; AT1G27045; AT1G28330; AT1G30135; AT1G30720;
AT1G32910; AT1G33050; AT1G33055; AT1G34140; AT1G34760; AT1G35140;
AT1G36060; AT1G54100; AT1G54760; AT1G55810; AT1G56250; AT1G58936;
AT1G60750; AT1G62480; AT1G62510; AT1G63090; AT1G65970; AT1G66860;
AT1G67265; AT1G68935; AT1G71520; AT1G72060; AT1G72416; AT1G75750;
AT1G76650; AT1G77120; AT1G79700; AT1G79910; AT1G80160; AT1G80570;
AT1G80840; AT2G05400; AT2G07633; AT2G07638; AT2G07641; AT2G07648;
AT2G07658; AT2G07665; AT2G07669; AT2G07673; AT2G07674; AT2G07678;
AT2G07689; AT2G07696; AT2G07698; AT2G07714; AT2G07721; AT2G07724;
AT2G07725; AT2G07727; AT2G07734; AT2G07749; AT2G07771; AT2G07773;
AT2G07785; AT2G07798; AT2G07815; AT2G07825; AT2G07835; AT2G14210;
AT2G14900; AT2G15880; AT2G15960; AT2G 16060; AT2G16586; AT2G17036;
AT2G17850; AT2G19590; AT2G19800; AT2G20670; AT2G22880; AT2G22980;
AT2G25770; AT2G25780; AT2G26130; AT2G30600; AT2G30615; AT2G31810;
AT2G33160; AT2G33830; AT2G34390; AT2G34555; AT2G36110; AT2G36950;
AT2G37025; AT2G39400; AT2G39570; AT2G43520; AT2G44080; AT2G47520;
AT2G47950; AT3G02550; AT3G03270; AT3G04160; AT3G06435; AT3G10040;
AT3G1331O; AT3G13450; AT3G15440; AT3G15450; AT3G15620; AT3G16770;
AT3G17225; AT3G18530; AT3G19680; AT3G20395; AT3G21352; AT3G22640;
AT3G23150; AT3G23550; AT3G27220; AT3G28740; AT3G29370; AT3G29810;
AT3G29970; AT3G30775; AT3G43190; AT3G43850; AT3G44300; AT3G45300;
AT3G46230; AT3G47340; AT3G47675; AT3G47720; AT3G47870; AT3G48360;
AT3G48530; AT3G50560; AT3G51840; AT3G51910; AT3G55240; AT3G55970;
AT3G59060; AT3G61060; AT3G61260; AT3G62150; AT4G01120; AT4G01250;
AT4G02170; AT4G02380; AT4G02430; AT4G03205; AT4G04423; AT4G10250;
AT4G10265; AT4G10270; AT4G15760; AT4G16160; AT4G16162; AT4G 16563;
AT4G19230; AT4G19880; AT4G20953; AT4G22710; AT4G22780; AT4G24040;
AT4G24110; AT4G24230; AT4G25580; AT4G25707; AT4G25810; AT4G26460;
AT4G27450; AT4G28040; AT4G28811; AT4G30270; AT4G30370; AT4G30380;
AT4G32480; AT4G32630; AT4G32840; AT4G33070; AT4G33150; AT4G33560;
AT4G33970; AT4G34030; AT4G35770; AT4G36690; AT4G36730; AT4G36850;
AT4G37220; AT4G38470; AT4G39675; AT5G01740; AT5G01810; AT5G02200;
AT5G03380; AT5G03830; AT5G05530; AT5G06980; AT5G07440; AT5G07560; AT5G07570; AT5G08150; AT5G10040; AT5G11090; AT5G12020; AT5G12030;
AT5G14180; AT5G14470; AT5G15120; AT5G15250; AT5G18170; AT5G19120;
AT5G19550; AT5G20240; AT5G20250; AT5G20830; AT5G21170; AT5G22920;
AT5G26200; AT5G27893; AT5G28610; AT5G28630; AT5G28770; AT5G34830;
AT5G35525; AT5G39160; AT5G39200; AT5G39580; AT5G39890; AT5G40450;
AT5G41080; AT5G42825; AT5G43570; AT5G45340; AT5G47590; AT5G47910;
AT5G49360; AT5G54080; AT5G56100; AT5G56870; AT5G57220; AT5G57550;
AT5G57660; AT5G62520; AT5G63160; AT5G65207; AT5G66110; ATMG00010;
ATMG00060; ATMG00070; ATMG00080; ATMG00090; ATMG00110; ATMG00140;
ATMG00160; ATMG00260; ATMG00270; ATMG00285; ATMG00310; ATMG00400;
ATMG00410; ATMG00510; ATMG00513; ATMG00516; ATMG00560; ATMG00570;
ATMG00630; ATMG00640; ATMG00650; ATMG00680; ATMG00690; ATMG00730;
ATMG00900; ATMG00940; ATMG00960; ATMG00970; ATMG01000; ATMG01050;
ATMG01120; ATMG01130; ATMG01170; ATMG01190; ATMG01200; ATMG01210;
ATMG01220; ATMG01230; ATMG01260; ATMG01280; ATMG01320; and ATMG01360.
[0018] In specific embodiments, the plurality of plant cells have upregulated expression during recovery from anoxia of at least one gene selected from the group consisting of:
AT1G01720; AT1G03070; AT1G03090; AT1G03610; AT1G05575; AT1G06030;
AT1G07330; AT1G07350; AT1G07400; AT1G07500; AT1G08630; AT1G09950;
AT1G10140; AT1G12610; AT1G14200; AT1G15040; AT1G15330; AT1G15840;
AT1G16030; AT1G17870; AT1G183OO; AT1G1833O; AT1G19530; AT1G19540;
AT1G19620; AT1G21340; AT1G21940; AT1G22110; AT1G26800; AT1G27730;
AT1G28760; AT1G30070; AT1G30135; AT1G31370; AT1G32910; AT1G32928;
AT1G33055; AT1G33730; AT1G33760; AT1G34575; AT1G35140; AT1G44414;
AT1G50745; AT1G52560; AT1G53540; AT1G54050; AT1G55530; AT1G55810;
AT1G56170; AT1G56250; AT1G59860; AT1G59865; AT1G60190; AT1G61340;
AT1G66060; AT1G66080; AT1G66400; AT1G66500; AT1G66510; AT1G67265;
AT1G71000; AT1G71520; AT1G72060; AT1G72416; AT1G72660; AT1G72760;
AT1G73480; AT1G74310; AT1G74450; AT1G74930; AT1G75490; AT1G75750;
AT1G76600; AT1G76640; AT1G76650; AT1G77120; AT1G80840; AT2G01390;
AT2G07687; AT2G07696; AT2G07698; AT2G07707; AT2G07724; AT2G07725;
AT2G07727; AT2G07734; AT2G07771; AT2G07773; AT2G07785; AT2G07825;
AT2G07835; AT2G14900; AT2G15960; AT2G 16060; AT2G17850; AT2G20560; AT2G20670; AT2G22880; AT2G23110; AT2G23190; AT2G24100; AT2G25140;
AT2G26150; AT2G27580; AT2G29500; AT2G30615; AT2G32120; AT2G34390;
AT2G34600; AT2G36220; AT2G38240; AT2G38340; AT2G40340; AT2G40955;
AT2G44070; AT2G44080; AT2G44130; AT2G44840; AT2G46240; AT2G46790;
AT2G46830; AT2G47180; AT2G47520; AT3G02550; AT3G06435; AT3G07090;
AT3G07150; AT3G07350; AT3G08970; AT3G09350; AT3G09640; AT3G10020;
AT3G10040; AT3G11020; AT3G12190; AT3G12320; AT3G12580; AT3G1331O;
AT3G14200; AT3G15440; AT3G15450; AT3G15500; AT3G16050; AT3G17400;
AT3G19240; AT3G20340; AT3G20395; AT3G22090; AT3G22100; AT3G23150;
AT3G24500; AT3G25250; AT3G27220; AT3G28210; AT3G28740; AT3G29370;
AT3G29810; AT3G29970; AT3G30775; AT3G43850; AT3G44190; AT3G46070;
AT3G46230; AT3G47340; AT3G47720; AT3G48240; AT3G49160; AT3G50310;
AT3G50560; AT3G51910; AT3G53830; AT3G55580; AT3G55840; AT3G62260;
AT3G63350; AT4G01250; AT4G01435; AT4G02170; AT4G02425; AT4G02550;
AT4G02690; AT4G09150; AT4G10250; AT4G10265; AT4G10270; AT4G 11660;
AT4G12400; AT4G12410; AT4G13395; AT4G15280; AT4G 15420; AT4G15760;
AT4G16555; AT4G17250; AT4G18450; AT4G19570; AT4G21320; AT4G23493;
AT4G24110; AT4G24410; AT4G24570; AT4G25200; AT4G25380; AT4G25470;
AT4G25490; AT4G25580; AT4G25810; AT4G26200; AT4G26460; AT4G27410;
AT4G27450; AT4G27652; AT4G27654; AT4G27657; AT4G27670; AT4G28811;
AT4G29770; AT4G29780; AT4G30270; AT4G30370; AT4G32208; AT4G32480;
AT4G33070; AT4G34131; AT4G34410; AT4G35770; AT4G36850; AT4G37710;
AT4G38030; AT5G01740; AT5G02170; AT5G02810; AT5G03210; AT5G03720;
AT5G04340; AT5G04400; AT5G05220; AT5G05410; AT5G06980; AT5G07330;
AT5G08150; AT5G09590; AT5G09930; AT5G10040; AT5G10336; AT5G10695;
AT5G12020; AT5G12030; AT5G12110; AT5G13220; AT5G14470; AT5G15120;
AT5G15250; AT5G15450; AT5G17350; AT5G18065; AT5G18340; AT5G20250;
AT5G22680; AT5G22920; AT5G25450; AT5G27660; AT5G35320; AT5G37340;
AT5G37670; AT5G39580; AT5G39890; AT5G43620; AT5G43650; AT5G45340;
AT5G45640; AT5G47220; AT5G47590; AT5G47830; AT5G48570; AT5G49920;
AT5G51190; AT5G51440; AT5G51990; AT5G52050; AT5G52630; AT5G52640;
AT5G53680; AT5G54165; AT5G57260; AT5G57550; AT5G57560; AT5G57660;
AT5G58070; AT5G59720; AT5G59820; AT5G62020; AT5G62520; AT5G63130; AT5G63300; AT5G64170; AT5G64210; AT5G64510; AT5G66650; AT5G67080;
ATMG00060; ATMG00080; ATMG00090; ATMG00160; ATMG00270; ATMG00285;
ATMG00400; ATMG00410; ATMG00480; ATMG00510; ATMG00513; ATMG00516;
ATMG00560; ATMG00570; ATMG00640; ATMG00650; ATMG00690; ATMG00730;
ATMG00900; ATMG00940; ATMG00960; ATMG00990; ATMG01000; ATMG01050;
ATMG01120; ATMG01190; ATMG01200; ATMG01320; and ATMG01360.
[0019] In specific embodiments, the genetically modified plant, part, and/or progeny thereof is selected from the group consisting of whole plants, seedlings, leaves, stems, flowers, roots, fruits, seeds, spores, grafts comprising genetically modified scions and/or rootstocks, buddings comprising genetically modified buds and/or rootstocks, cuttings, bulbs, tubers, rhizomes, regenerable cells, tissue cultures of regenerable cells, regenerable protoplasts, tissue cultures of regenerable protoplasts, and other plant germplasms.
[0020] In specific embodiments, the genetically modified plant, part, and/or progeny thereof, is selected from the group consisting of the Viridiplantae, the Glaucophyta, and the Rhodophyta. In specific embodiments, the Viridiplantae are selected from the group consisting of green algae, hornworts, liverworts, mosses, ferns, lycophytes, gymnosperms, and angiosperms. In specific embodiments, the genetically modified plant, part, and/or progeny thereof, is monocotyledonous or dicotyledonous. In specific embodiments, the genetically modified plant, part, and/or progeny thereof is selected from the group consisting of herbs, shrubs, trees, and vines. In specific embodiments, the genetically modified plant, part, and/or progeny thereof, excludes research plants, parts, and/or progeny thereof.
[0021] In specific embodiments, the genetically modified plant, part, and/or progeny thereof, is a crop plant capable of producing a crop product. In specific embodiments, the crop plant is selected from the group consisting of food crop plants, forage crop plants, fodder crop plants, medicinal crop plants, industrial crop plants, energy crop plants, and ornamental crop plants. In specific embodiments, the crop product of said ornamental crop plant is selected from the group consisting of foliage-, flower-, and/or fruit-producing herbs, shrubs, trees, vines, and regenerable parts thereof. In specific embodiments, the crop product is selected from the group consisting of cereal grains, legumes, vegetables, nuts, seeds, roots, tubers, rhizomes, flowers, fruits, timber, plant leaves, plant oils, plant fats, plant fibers, plant juices, plant extracts, and combinations thereof. In specific embodiments, the crop plant is selected from the group consisting of rice plants, maize plants, barley plants, oat plants, rye plants, wheat plants, com plants, sorghum plants, soybean plants, pea plants, lentil plants, curcurbit plants, coffee plants, cocoa plants, rapeseed plants, sunflower plants, sugar cane plants, potato plants, palm plants, grape plants, apple plants, banana plants, plantain plants, cassava plants, sugar beet plants, tomato plants, sweet potato plants, yam plants, tobacco plants, cotton plants, rubber plants, tea plants, lettuce plants, pepper plants, onion plants, grape plants, pecan trees, timber trees, cannabis plants, poppy plants, and combinations thereof.
[0022] In specific embodiments, the genetically modified plant, part, and/or progeny thereof is not cultivated for harvest of crops. In specific embodiments, the genetically modified plant, part, and/or progeny thereof, not cultivated for harvest crops is selected from the group consisting of Arabidopsis halleri, Arabidopsis lyrata, Eutrema salsugineum, Cardamine hirsute, the Viridiplantae, the Glaucophytes, and the Rhodophytes.
[0023] In specific embodiments, a composition of the disclosure includes an inbred, hybrid, or varietal plant, part, or progeny thereof, of the genetically modified plant, part, or progeny thereof. In specific embodiments, a composition of the disclosure includes a genetically modified plant, part, and/or progeny thereof, propagated and/or regenerated from the seeds, spores, grafts comprising genetically modified scions and/or rootstocks, buddings comprising genetically modified buds and/or rootstocks, cuttings, bulbs, tubers, rhizomes, regenerable cells, tissue cultures of regenerable cells, regenerable protoplasts, tissue cultures of regenerable protoplasts, and/or other plant germplasms of the genetically modified plant, part, and/or progeny thereof. In specific embodiments, a composition of the disclosure includes a genetically modified seed produced by the genetically modified plant, part, and/or progeny thereof.
[0024] In specific embodiments, a composition of the disclosure includes a genetically modified plant, part, and/or progeny thereof, excluding Arabidopsis thaliana, including a plurality of plant cells including: synthetically suppressed and/or eliminated synthesis of an activator of CAX1 and, optionally, an activator of at least one of CAX2, CAX3, or CAX4, in said plurality of plant cells. In specific embodiments, the plurality of plant cells include: one or more agents capable of driving gene silencing of a gene capable of synthesis of said activator of CAX1 and, optionally, a gene capable of synthesis of said activator of at at least one of CAX2, CAX3, or CAX4, in said plurality of plant cells; and, optionally, a regulatory sequence capable of inducing said agent(s) to drive said gene silencing of said gene capable of synthesis of said activator of CAX1 and, optionally, said gene capable of synthesis of said activator of at at least one of CAX2, CAX3, or CAX4, in said plurality of plant cells. [0025] In specific embodiments, the agent(s) include a nucleic acid sequence and/or a protein. In specific embodiments, activator of CAX1 is capable of binding to the N-terminus of CAX1, and wherein said activator of at least one of CAX2, CAX3, or CAX4 is capable of binding to the N-terminus of at least one of CAX2, CAX3, or CAX4. In specific embodiments, activator of CAX1 is selected from the group consisting of SOS2 protein kinase, CXIP1, and CXIP4. In specific embodiments, the nucleic acid sequence includes exogenous DNA, intragenic DNA, and/or exogenous RNA. In specific embodiments, the protein includes a nuclease and/or a protease.
[0026] In specific embodiments, the agent(s) and, optionally, said regulatory sequence, are (a) naked; and/or (b) comprised in (i) a complex; (ii) a carrier system; (iii) a particle gun system; (iv) a viral vector; (v) an Agrobacterium vector; and/or (vi) a CRISPR vector. In specific embodiments, the agent(s) are capable of driving RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of said gene capable of synthesis of said activator of CAX1 and, optionally, said gene capable of synthesis of said activator of at at least one of CAX2, CAX3, or CAX4. In specific embodiments, the agent(s) are capable of expressing double stranded RNA (dsRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), small RNA (sRNA), microMRNA (mRNA), pre-miRNA, and/or pri-miRNA, capable of RNA interference (RNAi) of said gene capable of synthesis of said activator of CAX1 and, optionally, said gene capable of synthesis of said activator of at at least one of CAX2, CAX3, or CAX4. In specific embodiments, the agent(s) include double stranded RNA (dsRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), small RNA (sRNA), microMRNA (mRNA), pre-miRNA, and/or pri-miRNA capable of RNA interference (RNAi) of said gene capable of synthesis of said activator of CAX1 and, optionally, said gene capable of synthesis of said activator of at at least one of CAX2, CAX3, or CAX4.
[0027] In specific embodiments, the regulatory agent is capable of inducing said agent(s) to drive RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of said gene capable of synthesis of said activator of CAX1 and, optionally, said gene capable of synthesis of said activator of at at least one of CAX2, CAX3, or CAX4. In specific embodiments, the regulatory sequence is capable of inducing said agent(s) to express double stranded RNA (dsRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), small RNA (sRNA), microMRNA (mRNA), pre-miRNA, and/or pri-miRNA, capable of RNA interference (RNAi) of said gene capable of synthesis of said activator of CAX1 and, optionally, said gene capable of synthesis of said activator of at at least one of CAX2, CAX3, or CAX4. In specific embodiments, the said regulatory sequence is tissue specific and/or cell-type specific. In specific embodiments, the said regulatory sequence is low oxygen-induced, chemical-induced, temperature-induced, and/or light-induced.
[0028] In specific embodiments, the plurality of plant cells have suppressed and/or eliminated synthesis of said activator of CAX1 and, optionally, said activator of at least one of CAX2, CAX3, or CAX4, before, during, and/or after anoxic conditions. In specific embodiments, the agent(s) are capable of driving RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of said gene capable of synthesis of said activator of CAX1 and, optionally, said gene capable of synthesis of said activator of at least one of CAX2, CAX3, or CAX4, before, during, and/or after anoxic conditions. In specific embodiments, the plurality of plant cells have transient and/or stable suppressed or eliminated synthesis of said activator of CAX1 and, optionally, said activator of at least one of CAX2, CAX3, or CAX4. In specific embodiments, the agent(s) are capable of driving transient and/or stable RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of said gene capable of synthesis of said activator of CAX1 and, optionally, said activator of at least one of CAX2, CAX3, or CAX4.
[0029] In specific embodiments, the plurality of plant cells have upregulated expression during anoxia of at least one gene selected from the group consisting of: AT 1G01355; AT1G02610; AT1G02620; AT1G03090; AT1G03610; AT1G06570; AT1G08630;
AT1G08930; AT1G10070; AT1G10140; AT1G10550; AT1G11260; AT1G12780;
AT1G14860; AT1G15040; AT1G15330; AT1G15670; AT1G17290; AT1G183OO;
AT1G18773; AT1G19396; AT1G19530; AT1G19540; AT1G19610; AT1G19620;
AT1G24880; AT1G25560; AT1G27045; AT1G28330; AT1G30135; AT1G30720;
AT1G32910; AT1G33050; AT1G33055; AT1G34140; AT1G34760; AT1G35140;
AT1G36060; AT1G54100; AT1G54760; AT1G55810; AT1G56250; AT1G58936;
AT1G60750; AT1G62480; AT1G62510; AT1G63090; AT1G65970; AT1G66860;
AT1G67265; AT1G68935; AT1G71520; AT1G72060; AT1G72416; AT1G75750;
AT1G76650; AT1G77120; AT1G79700; AT1G79910; AT1G80160; AT1G80570;
AT1G80840; AT2G05400; AT2G07633; AT2G07638; AT2G07641; AT2G07648;
AT2G07658; AT2G07665; AT2G07669; AT2G07673; AT2G07674; AT2G07678;
AT2G07689; AT2G07696; AT2G07698; AT2G07714; AT2G07721; AT2G07724;
AT2G07725; AT2G07727; AT2G07734; AT2G07749; AT2G07771; AT2G07773;
AT2G07785; AT2G07798; AT2G07815; AT2G07825; AT2G07835; AT2G14210; AT2G14900; AT2G15880; AT2G15960; AT2G16060; AT2G16586; AT2G17036;
AT2G17850; AT2G19590; AT2G19800; AT2G20670; AT2G22880; AT2G22980;
AT2G25770; AT2G25780; AT2G26130; AT2G30600; AT2G30615; AT2G31810;
AT2G33160; AT2G33830; AT2G34390; AT2G34555; AT2G36110; AT2G36950;
AT2G37025; AT2G39400; AT2G39570; AT2G43520; AT2G44080; AT2G47520;
AT2G47950; AT3G02550; AT3G03270; AT3G04160; AT3G06435; AT3G10040;
AT3G1331O; AT3G13450; AT3G15440; AT3G15450; AT3G15620; AT3G16770;
AT3G17225; AT3G18530; AT3G19680; AT3G20395; AT3G21352; AT3G22640;
AT3G23150; AT3G23550; AT3G27220; AT3G28740; AT3G29370; AT3G29810;
AT3G29970; AT3G30775; AT3G43190; AT3G43850; AT3G44300; AT3G45300;
AT3G46230; AT3G47340; AT3G47675; AT3G47720; AT3G47870; AT3G48360;
AT3G48530; AT3G50560; AT3G51840; AT3G51910; AT3G55240; AT3G55970;
AT3G59060; AT3G61060; AT3G61260; AT3G62150; AT4G01120; AT4G01250;
AT4G02170; AT4G02380; AT4G02430; AT4G03205; AT4G04423; AT4G10250;
AT4G10265; AT4G10270; AT4G15760; AT4G16160; AT4G16162; AT4G16563;
AT4G19230; AT4G19880; AT4G20953; AT4G22710; AT4G22780; AT4G24040;
AT4G24110; AT4G24230; AT4G25580; AT4G25707; AT4G25810; AT4G26460;
AT4G27450; AT4G28040; AT4G28811; AT4G30270; AT4G30370; AT4G30380;
AT4G32480; AT4G32630; AT4G32840; AT4G33070; AT4G33150; AT4G33560;
AT4G33970; AT4G34030; AT4G35770; AT4G36690; AT4G36730; AT4G36850;
AT4G37220; AT4G38470; AT4G39675; AT5G01740; AT5G01810; AT5G02200;
AT5G03380; AT5G03830; AT5G05530; AT5G06980; AT5G07440; AT5G07560;
AT5G07570; AT5G08150; AT5G10040; AT5G11090; AT5G12020; AT5G12030;
AT5G14180; AT5G14470; AT5G15120; AT5G15250; AT5G18170; AT5G19120;
AT5G19550; AT5G20240; AT5G20250; AT5G20830; AT5G21170; AT5G22920;
AT5G26200; AT5G27893; AT5G28610; AT5G28630; AT5G28770; AT5G34830;
AT5G35525; AT5G39160; AT5G39200; AT5G39580; AT5G39890; AT5G40450;
AT5G41080; AT5G42825; AT5G43570; AT5G45340; AT5G47590; AT5G47910;
AT5G49360; AT5G54080; AT5G56100; AT5G56870; AT5G57220; AT5G57550;
AT5G57660; AT5G62520; AT5G63160; AT5G65207; AT5G66110; ATMG00010;
ATMG00060; ATMG00070; ATMG00080; ATMG00090; ATMG00110; ATMG00140;
ATMG00160; ATMG00260; ATMG00270; ATMG00285; ATMG00310; ATMG00400;
ATMG00410; ATMG00510; ATMG00513; ATMG00516; ATMG00560; ATMG00570; ATMG00630; ATMG00640; ATMG00650; ATMG00680; ATMG00690; ATMG00730;
ATMG00900; ATMG00940; ATMG00960; ATMG00970; ATMG01000; ATMG01050;
ATMG01120; ATMG01130; ATMG01170; ATMG01190; ATMG01200; ATMG01210;
ATMG01220; ATMG01230; ATMG01260; ATMG01280; ATMG01320; and ATMG01360.
[0030] In specific embodiments, the plurality of plant cells have upregulated expression during recovery from anoxia of at least one gene selected from the group consisting of:
AT1G01720; AT1G03070; AT1G03090; AT1G03610; AT1G05575; AT1G06030;
AT1G07330; AT1G07350; AT1G07400; AT1G07500; AT1G08630; AT1G09950;
AT1G10140; AT1G12610; AT1G14200; AT1G15040; AT1G15330; AT1G15840;
AT1G16030; AT1G17870; AT1G183OO; AT1G1833O; AT1G19530; AT1G19540;
AT1G19620; AT1G21340; AT1G21940; AT1G22110; AT1G26800; AT1G27730;
AT1G28760; AT1G30070; AT1G30135; AT1G31370; AT1G32910; AT1G32928;
AT1G33055; AT1G33730; AT1G33760; AT1G34575; AT1G35140; AT1G44414;
AT1G50745; AT1G52560; AT1G53540; AT1G54050; AT1G55530; AT1G55810;
AT1G56170; AT1G56250; AT1G59860; AT1G59865; AT1G60190; AT1G61340;
AT1G66060; AT1G66080; AT1G66400; AT1G66500; AT1G66510; AT1G67265;
AT1G71000; AT1G71520; AT1G72060; AT1G72416; AT1G72660; AT1G72760;
AT1G73480; AT1G74310; AT1G74450; AT1G74930; AT1G75490; AT1G75750;
AT1G76600; AT1G76640; AT1G76650; AT1G77120; AT1G80840; AT2G01390;
AT2G07687; AT2G07696; AT2G07698; AT2G07707; AT2G07724; AT2G07725;
AT2G07727; AT2G07734; AT2G07771; AT2G07773; AT2G07785; AT2G07825;
AT2G07835; AT2G14900; AT2G15960; AT2G 16060; AT2G17850; AT2G20560;
AT2G20670; AT2G22880; AT2G23110; AT2G23190; AT2G24100; AT2G25140;
AT2G26150; AT2G27580; AT2G29500; AT2G30615; AT2G32120; AT2G34390;
AT2G34600; AT2G36220; AT2G38240; AT2G38340; AT2G40340; AT2G40955;
AT2G44070; AT2G44080; AT2G44130; AT2G44840; AT2G46240; AT2G46790;
AT2G46830; AT2G47180; AT2G47520; AT3G02550; AT3G06435; AT3G07090;
AT3G07150; AT3G07350; AT3G08970; AT3G09350; AT3G09640; AT3G10020;
AT3G10040; AT3G11020; AT3G12190; AT3G12320; AT3G12580; AT3G1331O;
AT3G14200; AT3G15440; AT3G15450; AT3G15500; AT3G16050; AT3G17400;
AT3G19240; AT3G20340; AT3G20395; AT3G22090; AT3G22100; AT3G23150;
AT3G24500; AT3G25250; AT3G27220; AT3G28210; AT3G28740; AT3G29370;
AT3G29810; AT3G29970; AT3G30775; AT3G43850; AT3G44190; AT3G46070; AT3G46230; AT3G47340; AT3G47720; AT3G48240; AT3G49160; AT3G50310;
AT3G50560; AT3G51910; AT3G53830; AT3G55580; AT3G55840; AT3G62260;
AT3G63350; AT4G01250; AT4G01435; AT4G02170; AT4G02425; AT4G02550;
AT4G02690; AT4G09150; AT4G10250; AT4G10265; AT4G10270; AT4G11660;
AT4G12400; AT4G12410; AT4G13395; AT4G15280; AT4G15420; AT4G15760;
AT4G16555; AT4G17250; AT4G18450; AT4G19570; AT4G21320; AT4G23493;
AT4G24110; AT4G24410; AT4G24570; AT4G25200; AT4G25380; AT4G25470;
AT4G25490; AT4G25580; AT4G25810; AT4G26200; AT4G26460; AT4G27410;
AT4G27450; AT4G27652; AT4G27654; AT4G27657; AT4G27670; AT4G28811;
AT4G29770; AT4G29780; AT4G30270; AT4G30370; AT4G32208; AT4G32480;
AT4G33070; AT4G34131; AT4G34410; AT4G35770; AT4G36850; AT4G37710;
AT4G38030; AT5G01740; AT5G02170; AT5G02810; AT5G03210; AT5G03720;
AT5G04340; AT5G04400; AT5G05220; AT5G05410; AT5G06980; AT5G07330;
AT5G08150; AT5G09590; AT5G09930; AT5G10040; AT5G10336; AT5G10695;
AT5G12020; AT5G12030; AT5G12110; AT5G13220; AT5G14470; AT5G15120;
AT5G15250; AT5G15450; AT5G17350; AT5G18065; AT5G18340; AT5G20250;
AT5G22680; AT5G22920; AT5G25450; AT5G27660; AT5G35320; AT5G37340;
AT5G37670; AT5G39580; AT5G39890; AT5G43620; AT5G43650; AT5G45340;
AT5G45640; AT5G47220; AT5G47590; AT5G47830; AT5G48570; AT5G49920;
AT5G51190; AT5G51440; AT5G51990; AT5G52050; AT5G52630; AT5G52640;
AT5G53680; AT5G54165; AT5G57260; AT5G57550; AT5G57560; AT5G57660;
AT5G58070; AT5G59720; AT5G59820; AT5G62020; AT5G62520; AT5G63130;
AT5G63300; AT5G64170; AT5G64210; AT5G64510; AT5G66650; AT5G67080;
ATMG00060; ATMG00080; ATMG00090; ATMG00160; ATMG00270; ATMG00285;
ATMG00400; ATMG00410; ATMG00480; ATMG00510; ATMG00513; ATMG00516;
ATMG00560; ATMG00570; ATMG00640; ATMG00650; ATMG00690; ATMG00730;
ATMG00900; ATMG00940; ATMG00960; ATMG00990; ATMG01000; ATMG01050;
ATMG01120; ATMG01190; ATMG01200; ATMG01320; and ATMG01360.
[0031] In specific embodiments, the genetically modified plant, part, and/or progeny thereof is selected from the group consisting of whole plants, seedlings, leaves, stems, flowers, roots, fruits, seeds, spores, grafts comprising genetically modified scions and/or rootstocks, buddings comprising genetically modified buds and/or rootstocks, cuttings, bulbs, tubers, rhizomes, regenerable cells, tissue cultures of regenerable cells, regenerable protoplasts, tissue cultures of regenerable protoplasts, and other plant germplasms. In specific embodiments, the genetically modified plant, part, and/or progeny thereof, is selected from the group consisting of the Viridiplantae, the Glaucophyta, and the Rhodophyta. In specific embodiments, the Viridiplantae are selected from the group consisting of green algae, hornworts, liverworts, mosses, ferns, lycophytes, gymnosperms, and angiosperms. In specific embodiments, the genetically modified plant, part, and/or progeny thereof, is monocotyledonous or dicotyledonous. In specific embodiments, the genetically modified plant, part, and/or progeny thereof is selected from the group consisting of herbs, shrubs, trees, and vines. In specific embodiments, the genetically modified plant, part, and/or progeny thereof, excludes research plants, parts, and/or progeny thereof.
[0032] In specific embodiments, the genetically modified plant, part, and/or progeny thereof, is a crop plant capable of producing a crop product. In specific embodiments, the crop plant is selected from the group consisting of food crop plants, forage crop plants, fodder crop plants, medicinal crop plants, industrial crop plants, energy crop plants, and ornamental crop plants. In specific embodiments, the crop product of said ornamental crop plant is selected from the group consisting of foliage-, flower-, and/or fruit-producing herbs, shrubs, trees, vines, and regenerable parts thereof. In specific embodiments, the crop product is selected from the group consisting of cereal grains, legumes, vegetables, nuts, seeds, roots, tubers, rhizomes, flowers, fruits, timber, plant leaves, plant oils, plant fats, plant fibers, plant juices, plant extracts, and combinations thereof. In specific embodiments, the crop plant is selected from the group consisting of rice plants, maize plants, barley plants, oat plants, rye plants, wheat plants, com plants, sorghum plants, soybean plants, pea plants, lentil plants, curcurbit plants, coffee plants, cocoa plants, rapeseed plants, sunflower plants, sugar cane plants, potato plants, palm plants, grape plants, apple plants, banana plants, plantain plants, cassava plants, sugar beet plants, tomato plants, sweet potato plants, yam plants, tobacco plants, cotton plants, rubber plants, tea plants, lettuce plants, pepper plants, onion plants, grape plants, pecan trees, timber trees, cannabis plants, poppy plants, and combinations thereof.
[0033] In specific embodiments, the genetically modified plant, part, and/or progeny thereof is not cultivated for harvest of crops. In specific embodiments, the genetically modified plant, part, and/or progeny thereof, not cultivated for harvest crops is selected from the group consisting of Arabidopsis halleri, Arabidopsis lyrata, Eutrema salsugineum, Cardamine hirsute, the Viridiplantae, the Glaucophytes, and the Rhodophytes. [0034] In specific embodiments, a composition of the disclosure includes an inbred, hybrid, or varietal plant, part, or progeny thereof, of the genetically modified plant, part, or progeny thereof. In specific embodiments, a composition of the disclosure includes a genetically modified plant, part, and/or progeny thereof, propagated and/or regenerated from the seeds, spores, grafts comprising genetically modified scions and/or rootstocks, buddings comprising genetically modified buds and/or rootstocks, cuttings, bulbs, tubers, rhizomes, regenerable cells, tissue cultures of regenerable cells, regenerable protoplasts, tissue cultures of regenerable protoplasts, and/or other plant germplasms of the genetically modified plant, part, and/or progeny thereof. In specific embodiments, a composition of the disclosure includes a genetically modified seed produced by the genetically modified plant, part, and/or progeny thereof.
[0035] In specific embodiments, a method of the disclosure includes a method of producing a crop from the genetically modified crop plant, part, and/or progeny thereof, including the steps of: cultivating said genetically modified crop plant, part, and/or progeny thereof, to produce said crop; and harvesting said crop from said genetically modified crop plant, part, and/or progeny thereof.
[0036] In specific embodiments, a method of the disclosure includes a method of producing a genetically modified plant, part, and/or progeny thereof, excluding Arabidopsis thaliana, comprising a plurality of plant cells, wherein the method includes the step of: synthetically driving suppression and/or elimination of expression of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4, in said plurality of plant cells, wherein said synthetically driving step improves the anoxia tolerance of said genetically modified plant, part and/or progeny thereof.
[0037] In specific embodiments, the synthetically driving step further includes the steps of: introducing into said plurality of plant cells one or more agents, said agent(s) driving gene silencing of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4, in said plurality of plant cells; and, optionally, introducing into said plurality of plant cells a regulatory sequence, said regulatory sequence inducing said agent(s) to drive said gene silencing of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4, in said plurality of plant cells.
[0038] In specific embodiments, the one or more agents is a nucleic acid sequence and/or a protein. In specific embodiments, the nucleic acid sequence comprises exogenous DNA, intragenic DNA, and/or exogenous RNA. In specific embodiments, the protein comprises a nuclease and/or a protease. [0039] In specific embodiments, the introducing steps are accomplished by: direct transfer of exogenous DNA, intragenic DNA, exogenous RNA, or combinations, complexes, carrier systems, and/or particle gun systems thereof; and/or viral vector-mediated, Agrobacterium vector-mediated, and/or CRISPR vector-mediated gene transformation of exogenous and/or intragenic DNA. In specific embodiments, the direct transfer step is accomplished by: passive uptake; electroporation; polyethylene glycol treatment; electrophoresis; cell fusion with liposomes or spheroplasts; injection, silicon carbide whiskers, particle gun bombardment, spraying, soaking, pipetting, brushing, cationic nanoparticle carriers, clay nanosheet carriers, surfactant complexes, and/or peptide-based carriers.
[0040] In specific embodiments, the driving step further includes the step of driving RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) in expression of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4. In specific embodiments, the step of driving RNA interference (RNAi) further includes the step of expressing double stranded RNA (dsRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), small RNA (sRNA), microMRNA (mRNA), pre-miRNA, and/or pri-miRNA capable of RNA interference (RNAi) of expression of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4. In specific embodiments, the agent(s) comprise double stranded RNA (dsRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), small RNA (sRNA), microMRNA (mRNA), pre-miRNA, and/or pri-miRNA capable of RNA interference (RNAi) of expression of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4.
[0041] In specific embodiments, the inducing step further includes the step of inducing said agent(s) to drive RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) in expression of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4. In specific embodiments, the step of inducing said agent(s) to drive RNA interference (RNAi) further includes the step of inducing said agent(s) to express double stranded RNA (dsRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), small RNA (sRNA), microMRNA (mRNA), pre-miRNA, and/or pri-miRNA capable of RNA interference (RNAi) of expression of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4. In specific embodiments, the regulatory sequence is tissue specific and/or cell-type specific. In specific embodiments, the regulatory sequence is low oxygen-induced, chemical-induced, temperature-induced, and/or light-induced.
[0042] In specific embodiments, the said step of suppressing and/or eliminating expression of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4, occurs before, during, and/or after anoxic conditions. In specific embodiments, the step of driving RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of expression of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4, occurs before, during, and/or after anoxic conditions. In specific embodiments, the step of suppressing and/or eliminating expression of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4, is transient and/or stable. In specific embodiments, the step of driving RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of expression of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4, is transient and/or stable.
[0043] In specific embodiments, the plurality of plant cells upregulate expression during anoxia of at least one gene selected from the group consisting of: AT 1G01355;
AT1G02610; AT1G02620; AT1G03090; AT1G03610; AT1G06570; AT1G08630;
AT1G08930; AT1G10070; AT1G10140; AT1G10550; AT1G11260; AT1G12780;
AT1G14860; AT1G15040; AT1G15330; AT1G15670; AT1G17290; AT1G183OO;
AT1G18773; AT1G19396; AT1G19530; AT1G19540; AT1G19610; AT1G19620;
AT1G24880; AT1G25560; AT1G27045; AT1G28330; AT1G30135; AT1G30720;
AT1G32910; AT1G33050; AT1G33055; AT1G34140; AT1G34760; AT1G35140;
AT1G36060; AT1G54100; AT1G54760; AT1G55810; AT1G56250; AT1G58936;
AT1G60750; AT1G62480; AT1G62510; AT1G63090; AT1G65970; AT1G66860;
AT1G67265; AT1G68935; AT1G71520; AT1G72060; AT1G72416; AT1G75750;
AT1G76650; AT1G77120; AT1G79700; AT1G79910; AT1G80160; AT1G80570;
AT1G80840; AT2G05400; AT2G07633; AT2G07638; AT2G07641; AT2G07648;
AT2G07658; AT2G07665; AT2G07669; AT2G07673; AT2G07674; AT2G07678;
AT2G07689; AT2G07696; AT2G07698; AT2G07714; AT2G07721; AT2G07724;
AT2G07725; AT2G07727; AT2G07734; AT2G07749; AT2G07771; AT2G07773;
AT2G07785; AT2G07798; AT2G07815; AT2G07825; AT2G07835; AT2G14210;
AT2G14900; AT2G15880; AT2G15960; AT2G 16060; AT2G16586; AT2G17036;
AT2G17850; AT2G19590; AT2G19800; AT2G20670; AT2G22880; AT2G22980;
AT2G25770; AT2G25780; AT2G26130; AT2G30600; AT2G30615; AT2G31810;
AT2G33160; AT2G33830; AT2G34390; AT2G34555; AT2G36110; AT2G36950;
AT2G37025; AT2G39400; AT2G39570; AT2G43520; AT2G44080; AT2G47520;
AT2G47950; AT3G02550; AT3G03270; AT3G04160; AT3G06435; AT3G10040;
AT3G1331O; AT3G13450; AT3G15440; AT3G15450; AT3G15620; AT3G16770; AT3G17225; AT3G18530; AT3G19680; AT3G20395; AT3G21352; AT3G22640;
AT3G23150; AT3G23550; AT3G27220; AT3G28740; AT3G29370; AT3G29810;
AT3G29970; AT3G30775; AT3G43190; AT3G43850; AT3G44300; AT3G45300;
AT3G46230; AT3G47340; AT3G47675; AT3G47720; AT3G47870; AT3G48360;
AT3G48530; AT3G50560; AT3G51840; AT3G51910; AT3G55240; AT3G55970;
AT3G59060; AT3G61060; AT3G61260; AT3G62150; AT4G01120; AT4G01250;
AT4G02170; AT4G02380; AT4G02430; AT4G03205; AT4G04423; AT4G10250;
AT4G10265; AT4G10270; AT4G15760; AT4G16160; AT4G16162; AT4G16563;
AT4G19230; AT4G19880; AT4G20953; AT4G22710; AT4G22780; AT4G24040;
AT4G24110; AT4G24230; AT4G25580; AT4G25707; AT4G25810; AT4G26460;
AT4G27450; AT4G28040; AT4G28811; AT4G30270; AT4G30370; AT4G30380;
AT4G32480; AT4G32630; AT4G32840; AT4G33070; AT4G33150; AT4G33560;
AT4G33970; AT4G34030; AT4G35770; AT4G36690; AT4G36730; AT4G36850;
AT4G37220; AT4G38470; AT4G39675; AT5G01740; AT5G01810; AT5G02200;
AT5G03380; AT5G03830; AT5G05530; AT5G06980; AT5G07440; AT5G07560;
AT5G07570; AT5G08150; AT5G10040; AT5G11090; AT5G12020; AT5G12030;
AT5G14180; AT5G14470; AT5G15120; AT5G15250; AT5G18170; AT5G19120;
AT5G19550; AT5G20240; AT5G20250; AT5G20830; AT5G21170; AT5G22920;
AT5G26200; AT5G27893; AT5G28610; AT5G28630; AT5G28770; AT5G34830;
AT5G35525; AT5G39160; AT5G39200; AT5G39580; AT5G39890; AT5G40450;
AT5G41080; AT5G42825; AT5G43570; AT5G45340; AT5G47590; AT5G47910;
AT5G49360; AT5G54080; AT5G56100; AT5G56870; AT5G57220; AT5G57550;
AT5G57660; AT5G62520; AT5G63160; AT5G65207; AT5G66110; ATMG00010;
ATMG00060; ATMG00070; ATMG00080; ATMG00090; ATMG00110; ATMG00140;
ATMG00160; ATMG00260; ATMG00270; ATMG00285; ATMG00310; ATMG00400;
ATMG00410; ATMG00510; ATMG00513; ATMG00516; ATMG00560; ATMG00570;
ATMG00630; ATMG00640; ATMG00650; ATMG00680; ATMG00690; ATMG00730;
ATMG00900; ATMG00940; ATMG00960; ATMG00970; ATMG01000; ATMG01050;
ATMG01120; ATMG01130; ATMG01170; ATMG01190; ATMG01200; ATMG01210;
ATMG01220; ATMG01230; ATMG01260; ATMG01280; ATMG01320; and ATMG01360.
[0044] In specific embodiments, the plurality of plant cells upregulate expression during recovery from anoxia of at least one gene selected from the group consisting of: AT1G01720; AT1G03070; AT1G03090; AT1G03610; AT1G05575; AT1G06030; AT1G07330; AT1G07350; AT1G07400; AT1G07500; AT1G08630; AT1G09950;
AT1G10140; AT1G12610; AT1G14200; AT1G15040; AT1G15330; AT1G15840;
AT1G16030; AT1G17870; AT1G183OO; AT1G1833O; AT1G19530; AT1G19540;
AT1G19620; AT1G21340; AT1G21940; AT1G22110; AT1G26800; AT1G27730;
AT1G28760; AT1G30070; AT1G30135; AT1G31370; AT1G32910; AT1G32928;
AT1G33055; AT1G33730; AT1G33760; AT1G34575; AT1G35140; AT1G44414;
AT1G50745; AT1G52560; AT1G53540; AT1G54050; AT1G55530; AT1G55810;
AT1G56170; AT1G56250; AT1G59860; AT1G59865; AT1G60190; AT1G61340;
AT1G66060; AT1G66080; AT1G66400; AT1G66500; AT1G66510; AT1G67265;
AT1G71000; AT1G71520; AT1G72060; AT1G72416; AT1G72660; AT1G72760;
AT1G73480; AT1G74310; AT1G74450; AT1G74930; AT1G75490; AT1G75750;
AT1G76600; AT1G76640; AT1G76650; AT1G77120; AT1G80840; AT2G01390;
AT2G07687; AT2G07696; AT2G07698; AT2G07707; AT2G07724; AT2G07725;
AT2G07727; AT2G07734; AT2G07771; AT2G07773; AT2G07785; AT2G07825;
AT2G07835; AT2G14900; AT2G15960; AT2G 16060; AT2G17850; AT2G20560;
AT2G20670; AT2G22880; AT2G23110; AT2G23190; AT2G24100; AT2G25140;
AT2G26150; AT2G27580; AT2G29500; AT2G30615; AT2G32120; AT2G34390;
AT2G34600; AT2G36220; AT2G38240; AT2G38340; AT2G40340; AT2G40955;
AT2G44070; AT2G44080; AT2G44130; AT2G44840; AT2G46240; AT2G46790;
AT2G46830; AT2G47180; AT2G47520; AT3G02550; AT3G06435; AT3G07090;
AT3G07150; AT3G07350; AT3G08970; AT3G09350; AT3G09640; AT3G10020;
AT3G10040; AT3G11020; AT3G12190; AT3G12320; AT3G12580; AT3G1331O;
AT3G14200; AT3G15440; AT3G15450; AT3G15500; AT3G16050; AT3G17400;
AT3G19240; AT3G20340; AT3G20395; AT3G22090; AT3G22100; AT3G23150;
AT3G24500; AT3G25250; AT3G27220; AT3G28210; AT3G28740; AT3G29370;
AT3G29810; AT3G29970; AT3G30775; AT3G43850; AT3G44190; AT3G46070;
AT3G46230; AT3G47340; AT3G47720; AT3G48240; AT3G49160; AT3G50310;
AT3G50560; AT3G51910; AT3G53830; AT3G55580; AT3G55840; AT3G62260;
AT3G63350; AT4G01250; AT4G01435; AT4G02170; AT4G02425; AT4G02550;
AT4G02690; AT4G09150; AT4G10250; AT4G10265; AT4G10270; AT4G 11660;
AT4G12400; AT4G12410; AT4G13395; AT4G15280; AT4G 15420; AT4G15760;
AT4G16555; AT4G17250; AT4G18450; AT4G19570; AT4G21320; AT4G23493;
AT4G24110; AT4G24410; AT4G24570; AT4G25200; AT4G25380; AT4G25470; AT4G25490; AT4G25580; AT4G25810; AT4G26200; AT4G26460; AT4G27410;
AT4G27450; AT4G27652; AT4G27654; AT4G27657; AT4G27670; AT4G28811;
AT4G29770; AT4G29780; AT4G30270; AT4G30370; AT4G32208; AT4G32480;
AT4G33070; AT4G34131; AT4G34410; AT4G35770; AT4G36850; AT4G37710;
AT4G38030; AT5G01740; AT5G02170; AT5G02810; AT5G03210; AT5G03720;
AT5G04340; AT5G04400; AT5G05220; AT5G05410; AT5G06980; AT5G07330;
AT5G08150; AT5G09590; AT5G09930; AT5G10040; AT5G10336; AT5G10695;
AT5G12020; AT5G12030; AT5G12110; AT5G13220; AT5G14470; AT5G15120;
AT5G15250; AT5G15450; AT5G17350; AT5G18065; AT5G18340; AT5G20250;
AT5G22680; AT5G22920; AT5G25450; AT5G27660; AT5G35320; AT5G37340;
AT5G37670; AT5G39580; AT5G39890; AT5G43620; AT5G43650; AT5G45340;
AT5G45640; AT5G47220; AT5G47590; AT5G47830; AT5G48570; AT5G49920;
AT5G51190; AT5G51440; AT5G51990; AT5G52050; AT5G52630; AT5G52640;
AT5G53680; AT5G54165; AT5G57260; AT5G57550; AT5G57560; AT5G57660;
AT5G58070; AT5G59720; AT5G59820; AT5G62020; AT5G62520; AT5G63130;
AT5G63300; AT5G64170; AT5G64210; AT5G64510; AT5G66650; AT5G67080;
ATMG00060; ATMG00080; ATMG00090; ATMG00160; ATMG00270; ATMG00285;
ATMG00400; ATMG00410; ATMG00480; ATMG00510; ATMG00513; ATMG00516;
ATMG00560; ATMG00570; ATMG00640; ATMG00650; ATMG00690; ATMG00730;
ATMG00900; ATMG00940; ATMG00960; ATMG00990; ATMG01000; ATMG01050;
ATMG01120; ATMG01190; ATMG01200; ATMG01320; and ATMG01360.
[0045] In specific embodiments, a method of the disclosure includes a method of producing a genetically modified plant, part, and/or progeny thereof, excluding Arabidopsis thaliana, including a plurality of plant cells, wherein the method includes the step of: synthetically driving suppression and/or elimination of expression of a gene capable of synthesis of an activator of CAX1 and, optionally, a gene capable of synthesis of an activator of at least one of CAX2, CAX3, or CAX4, in said plurality of plant cells, wherein said synthetically driving step improves the anoxia tolerance of said genetically modified plant, part and/or progeny thereof.
[0046] In specific embodiments, the synthetically driving step further includes the steps of: introducing into said plurality of plant cells one or more agents, said agent(s) driving gene silencing of said gene capable of synthesis of an activator of CAX1 and, optionally, said gene capable of synthesis of an activator of at least one of CAX2, CAX3, or CAX4, in said plurality of plant cells; and, optionally, introducing into said plurality of plant cells a regulatory sequence, said regulatory sequence inducing said agent(s) to drive gene silencing of said gene capable of synthesis of an activator of CAX1 and, optionally, said gene capable of synthesis of an activator of at least one of CAX2, CAX3, or CAX4, in said plurality of plant cells.
[0047] In specific embodiments, the activator of CAX1 is capable of binding to the N- terminus of CAX1, and wherein said activator of at least one of CAX2, CAX3, or CAX4 is capable of binding to the N-terminus of at least one of CAX2, CAX3, or CAX4. In specific embodiments, the activator of CAX1 is selected from the group consisting of SOS2 protein kinase, CXIP1, and CXIP4.
[0048] In specific embodiments, the introducing steps are accomplished by: direct transfer of exogenous DNA, intragenic DNA, exogenous RNA, or combinations, complexes, carrier systems, and/or particle gun systems thereof; and/or viral vector-mediated, Agrobacterium vector-mediated, and/or CRISPR vector-mediated gene transformation of exogenous and/or intragenic DNA. In specific embodiments, the direct transfer step is accomplished by: passive uptake; electroporation; polyethylene glycol treatment; electrophoresis; cell fusion with liposomes or spheroplasts; injection, silicon carbide whiskers, particle gun bombardment, spraying, soaking, pipetting, brushing, cationic nanoparticle carriers, clay nanosheet carriers, surfactant complexes, and/or peptide-based carriers.
[0049] In specific embodiments, the driving step further includes the step of driving RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of a gene capable of synthesis of an activator of CAX1 and, optionally, a gene capable of synthesis of an activator of at least one of CAX2, CAX3, or CAX4. In specific embodiments, the step of driving RNA interference (RNAi) further includes the step of expressing double stranded RNA (dsRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), small RNA (sRNA), microMRNA (mRNA), pre-miRNA, and/or pri-miRNA capable of RNA interference (RNAi) of a gene capable of synthesis of at least one activator of CAX1 and, optionally, a gene capable of synthesis of an activator of at least one of CAX2, CAX3, or CAX. In specific embodiments, the agent(s) includes double stranded RNA (dsRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), small RNA (sRNA), microMRNA (mRNA), pre-miRNA, and/or pri-miRNA capable of RNA interference (RNAi) of a gene capable of synthesis of an activator of CAX1 and, optionally, a gene capable of synthesis of an activator of at least one of CAX2, CAX3, or CAX4. [0050] In specific embodiments, the inducing step further includes the step of inducing said agent(s) to drive RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of a gene capable of synthesis of an activator of CAX1 and, optionally, a gene capable of synthesis of an activator of at least one of CAX2, CAX3, or CAX4. In specific embodiments, the step of inducing said agent(s) to drive RNA interference (RNAi) further includes the step of inducing said agent to express double stranded RNA (dsRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), small RNA (sRNA), microMRNA (mRNA), pre-miRNA, and/or pri-miRNA capable of RNA interference (RNAi) of a gene capable of synthesis of an activator of CAX1 and, optionally, a gene capable of synthesis of an activator of at least one of CAX2, CAX3, or CAX4, wherein said inducing step is accomplished using said regulatory sequence. In specific embodiments, the regulatory sequence is tissue specific and/or cell-type specific. In specific embodiments, the regulatory sequence is low oxygen-induced, chemical-induced, temperature-induced, and/or light- induced.
[0051] In specific embodiments, the step of suppressing and/or eliminating expression of a gene capable of synthesis of an activator of CAX1 and, optionally, a gene capable of synthesis of an activator of at least one of CAX2, CAX3, or CAX4, occurs before, during, and/or after anoxic conditions. In specific embodiments, the step of driving RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of a gene capable of synthesis of an activator of CAX1 and, optionally, a gene capable of synthesis of an activator of at least one of CAX2, CAX3, or CAX4, occurs before, during, and/or after anoxic conditions. In specific embodiments, the step of suppressing and/or eliminating synthesis of at least one activator of CAX1 and, optionally, at least one activator of at least one of CAX2, CAX3, or CAX4, is transient and/or stable. In specific embodiments, the step of driving RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of a gene capable of synthesis of an activator of CAX1 and, optionally, a gene capable of synthesis of an activator of at least one of CAX2, CAX3, or CAX4, is transient and/or stable.
[0052] In specific embodiments, the plurality of plant cells upregulate expression during anoxia of at least one gene selected from the group consisting of: AT 1G01355; AT1G02610; AT1G02620; AT1G03090; AT1G03610; AT1G06570; AT1G08630;
AT1G08930; AT1G10070; AT1G10140; AT1G10550; AT1G11260; AT1G12780;
AT1G14860; AT1G15040; AT1G15330; AT1G15670; AT1G17290; AT1G183OO; AT1G18773; AT1G19396; AT1G19530; AT1G19540; AT1G19610; AT1G19620;
AT1G24880; AT1G25560; AT1G27045; AT1G28330; AT1G30135; AT1G30720;
AT1G32910; AT1G33050; AT1G33055; AT1G34140; AT1G34760; AT1G35140;
AT1G36060; AT1G54100; AT1G54760; AT1G55810; AT1G56250; AT1G58936;
AT1G60750; AT1G62480; AT1G62510; AT1G63090; AT1G65970; AT1G66860;
AT1G67265; AT1G68935; AT1G71520; AT1G72060; AT1G72416; AT1G75750;
AT1G76650; AT1G77120; AT1G79700; AT1G79910; AT1G80160; AT1G80570;
AT1G80840; AT2G05400; AT2G07633; AT2G07638; AT2G07641; AT2G07648;
AT2G07658; AT2G07665; AT2G07669; AT2G07673; AT2G07674; AT2G07678;
AT2G07689; AT2G07696; AT2G07698; AT2G07714; AT2G07721; AT2G07724;
AT2G07725; AT2G07727; AT2G07734; AT2G07749; AT2G07771; AT2G07773;
AT2G07785; AT2G07798; AT2G07815; AT2G07825; AT2G07835; AT2G14210;
AT2G14900; AT2G15880; AT2G15960; AT2G 16060; AT2G16586; AT2G17036;
AT2G17850; AT2G19590; AT2G19800; AT2G20670; AT2G22880; AT2G22980;
AT2G25770; AT2G25780; AT2G26130; AT2G30600; AT2G30615; AT2G31810;
AT2G33160; AT2G33830; AT2G34390; AT2G34555; AT2G36110; AT2G36950;
AT2G37025; AT2G39400; AT2G39570; AT2G43520; AT2G44080; AT2G47520;
AT2G47950; AT3G02550; AT3G03270; AT3G04160; AT3G06435; AT3G10040;
AT3G1331O; AT3G13450; AT3G15440; AT3G15450; AT3G15620; AT3G16770;
AT3G17225; AT3G18530; AT3G19680; AT3G20395; AT3G21352; AT3G22640;
AT3G23150; AT3G23550; AT3G27220; AT3G28740; AT3G29370; AT3G29810;
AT3G29970; AT3G30775; AT3G43190; AT3G43850; AT3G44300; AT3G45300;
AT3G46230; AT3G47340; AT3G47675; AT3G47720; AT3G47870; AT3G48360;
AT3G48530; AT3G50560; AT3G51840; AT3G51910; AT3G55240; AT3G55970;
AT3G59060; AT3G61060; AT3G61260; AT3G62150; AT4G01120; AT4G01250;
AT4G02170; AT4G02380; AT4G02430; AT4G03205; AT4G04423; AT4G10250;
AT4G10265; AT4G10270; AT4G15760; AT4G16160; AT4G16162; AT4G 16563;
AT4G19230; AT4G19880; AT4G20953; AT4G22710; AT4G22780; AT4G24040;
AT4G24110; AT4G24230; AT4G25580; AT4G25707; AT4G25810; AT4G26460;
AT4G27450; AT4G28040; AT4G28811; AT4G30270; AT4G30370; AT4G30380;
AT4G32480; AT4G32630; AT4G32840; AT4G33070; AT4G33150; AT4G33560;
AT4G33970; AT4G34030; AT4G35770; AT4G36690; AT4G36730; AT4G36850;
AT4G37220; AT4G38470; AT4G39675; AT5G01740; AT5G01810; AT5G02200; AT5G03380; AT5G03830; AT5G05530; AT5G06980; AT5G07440; AT5G07560;
AT5G07570; AT5G08150; AT5G10040; AT5G11090; AT5G12020; AT5G12030;
AT5G14180; AT5G14470; AT5G15120; AT5G15250; AT5G18170; AT5G19120;
AT5G19550; AT5G20240; AT5G20250; AT5G20830; AT5G21170; AT5G22920;
AT5G26200; AT5G27893; AT5G28610; AT5G28630; AT5G28770; AT5G34830;
AT5G35525; AT5G39160; AT5G39200; AT5G39580; AT5G39890; AT5G40450;
AT5G41080; AT5G42825; AT5G43570; AT5G45340; AT5G47590; AT5G47910;
AT5G49360; AT5G54080; AT5G56100; AT5G56870; AT5G57220; AT5G57550;
AT5G57660; AT5G62520; AT5G63160; AT5G65207; AT5G66110; ATMG00010;
ATMG00060; ATMG00070; ATMG00080; ATMG00090; ATMG00110; ATMG00140;
ATMG00160; ATMG00260; ATMG00270; ATMG00285; ATMG00310; ATMG00400;
ATMG00410; ATMG00510; ATMG00513; ATMG00516; ATMG00560; ATMG00570;
ATMG00630; ATMG00640; ATMG00650; ATMG00680; ATMG00690; ATMG00730;
ATMG00900; ATMG00940; ATMG00960; ATMG00970; ATMG01000; ATMG01050;
ATMG01120; ATMG01130; ATMG01170; ATMG01190; ATMG01200; ATMG01210;
ATMG01220; ATMG01230; ATMG01260; ATMG01280; ATMG01320; and ATMG01360.
[0053] In specific embodiments, the plurality of plant cells upregulate expression during recovery from anoxia of at least one gene selected from the group consisting of: AT1G01720; AT1G03070; AT1G03090; AT1G03610; AT1G05575; AT1G06030;
AT1G07330; AT1G07350; AT1G07400; AT1G07500; AT1G08630; AT1G09950;
AT1G10140; AT1G12610; AT1G14200; AT1G15040; AT1G15330; AT1G15840;
AT1G16030; AT1G17870; AT1G183OO; AT1G1833O; AT1G19530; AT1G19540;
AT1G19620; AT1G21340; AT1G21940; AT1G22110; AT1G26800; AT1G27730;
AT1G28760; AT1G30070; AT1G30135; AT1G31370; AT1G32910; AT1G32928;
AT1G33055; AT1G33730; AT1G33760; AT1G34575; AT1G35140; AT1G44414;
AT1G50745; AT1G52560; AT1G53540; AT1G54050; AT1G55530; AT1G55810;
AT1G56170; AT1G56250; AT1G59860; AT1G59865; AT1G60190; AT1G61340;
AT1G66060; AT1G66080; AT1G66400; AT1G66500; AT1G66510; AT1G67265;
AT1G71000; AT1G71520; AT1G72060; AT1G72416; AT1G72660; AT1G72760;
AT1G73480; AT1G74310; AT1G74450; AT1G74930; AT1G75490; AT1G75750;
AT1G76600; AT1G76640; AT1G76650; AT1G77120; AT1G80840; AT2G01390;
AT2G07687; AT2G07696; AT2G07698; AT2G07707; AT2G07724; AT2G07725;
AT2G07727; AT2G07734; AT2G07771; AT2G07773; AT2G07785; AT2G07825; AT2G07835; AT2G14900; AT2G15960; AT2G 16060; AT2G17850; AT2G20560;
AT2G20670; AT2G22880; AT2G23110; AT2G23190; AT2G24100; AT2G25140;
AT2G26150; AT2G27580; AT2G29500; AT2G30615; AT2G32120; AT2G34390;
AT2G34600; AT2G36220; AT2G38240; AT2G38340; AT2G40340; AT2G40955;
AT2G44070; AT2G44080; AT2G44130; AT2G44840; AT2G46240; AT2G46790;
AT2G46830; AT2G47180; AT2G47520; AT3G02550; AT3G06435; AT3G07090;
AT3G07150; AT3G07350; AT3G08970; AT3G09350; AT3G09640; AT3G10020;
AT3G10040; AT3G11020; AT3G12190; AT3G12320; AT3G12580; AT3G1331O;
AT3G14200; AT3G15440; AT3G15450; AT3G15500; AT3G16050; AT3G17400;
AT3G19240; AT3G20340; AT3G20395; AT3G22090; AT3G22100; AT3G23150;
AT3G24500; AT3G25250; AT3G27220; AT3G28210; AT3G28740; AT3G29370;
AT3G29810; AT3G29970; AT3G30775; AT3G43850; AT3G44190; AT3G46070;
AT3G46230; AT3G47340; AT3G47720; AT3G48240; AT3G49160; AT3G50310;
AT3G50560; AT3G51910; AT3G53830; AT3G55580; AT3G55840; AT3G62260;
AT3G63350; AT4G01250; AT4G01435; AT4G02170; AT4G02425; AT4G02550;
AT4G02690; AT4G09150; AT4G10250; AT4G10265; AT4G10270; AT4G 11660;
AT4G12400; AT4G12410; AT4G13395; AT4G15280; AT4G 15420; AT4G15760;
AT4G16555; AT4G17250; AT4G18450; AT4G19570; AT4G21320; AT4G23493;
AT4G24110; AT4G24410; AT4G24570; AT4G25200; AT4G25380; AT4G25470;
AT4G25490; AT4G25580; AT4G25810; AT4G26200; AT4G26460; AT4G27410;
AT4G27450; AT4G27652; AT4G27654; AT4G27657; AT4G27670; AT4G28811;
AT4G29770; AT4G29780; AT4G30270; AT4G30370; AT4G32208; AT4G32480;
AT4G33070; AT4G34131; AT4G34410; AT4G35770; AT4G36850; AT4G37710;
AT4G38030; AT5G01740; AT5G02170; AT5G02810; AT5G03210; AT5G03720;
AT5G04340; AT5G04400; AT5G05220; AT5G05410; AT5G06980; AT5G07330;
AT5G08150; AT5G09590; AT5G09930; AT5G10040; AT5G10336; AT5G10695;
AT5G12020; AT5G12030; AT5G12110; AT5G13220; AT5G14470; AT5G15120;
AT5G15250; AT5G15450; AT5G17350; AT5G18065; AT5G18340; AT5G20250;
AT5G22680; AT5G22920; AT5G25450; AT5G27660; AT5G35320; AT5G37340;
AT5G37670; AT5G39580; AT5G39890; AT5G43620; AT5G43650; AT5G45340;
AT5G45640; AT5G47220; AT5G47590; AT5G47830; AT5G48570; AT5G49920;
AT5G51190; AT5G51440; AT5G51990; AT5G52050; AT5G52630; AT5G52640;
AT5G53680; AT5G54165; AT5G57260; AT5G57550; AT5G57560; AT5G57660; AT5G58070; AT5G59720; AT5G59820; AT5G62020; AT5G62520; AT5G63130;
AT5G63300; AT5G64170; AT5G64210; AT5G64510; AT5G66650; AT5G67080;
ATMG00060; ATMG00080; ATMG00090; ATMG00160; ATMG00270; ATMG00285;
ATMG00400; ATMG00410; ATMG00480; ATMG00510; ATMG00513; ATMG00516;
ATMG00560; ATMG00570; ATMG00640; ATMG00650; ATMG00690; ATMG00730;
ATMG00900; ATMG00940; ATMG00960; ATMG00990; ATMG01000; ATMG01050;
ATMG01120; ATMG01190; ATMG01200; ATMG01320; and ATMG01360.
[0054] In specific embodiments, the progeny is produced by propagating said genetically modified plant or said part thereof, using asexual or sexual reproduction.
[0055] In specific embodiments, a composition of the disclosure includes a genetically modified seed produced by the genetically modified plant, part, and/or progeny thereof, prepared according to any one of the methods of the disclosure.
[0056] In specific embodiments, a composition of the disclosure includes a genetically modified plant, part, and/or progeny thereof, propagated and/or regenerated from the seeds, spores, grafts comprising genetically modified scions and/or rootstocks, buddings comprising genetically modified buds and/or rootstocks, cuttings, bulbs, tubers, rhizomes, regenerable cells, tissue cultures of regenerable cells, regenerable protoplasts, tissue cultures of regenerable protoplasts, and/or other plant germplasms of the genetically modified plant, part, and/or progeny thereof, produced according to any one of the methods of the disclosure.
[0057] In specific embodiments, a composition of the disclosure includes a crop produced by the genetically modified plant, part, and/or progeny thereof, prepared according to any one of the methods of the disclosure.
[0058] The foregoing has outlined rather broadly the features and technical advantages of the present disclosure in order that the detailed description that follows may be better understood. Additional features and advantages will be described hereinafter which form the subject of the claims herein. It should be appreciated by those skilled in the art that the conception and specific embodiments disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present designs. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope as set forth in the appended claims. The novel features which are believed to be characteristic of the designs disclosed herein, both as to the organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0059] For a more complete understanding of the present disclosure, reference is now made to the following descriptions taken in conjunction with the accompanying drawing, in which:
[0060] FIGS. 1A-1E illustrate particular embodiments of the disclosure showing the effect of CAX mutation on anoxia tolerance of Arabidopsis thaliana plants. FIG. 1A shows improved anoxia tolerance (reduced wilting and necrosis) of the caxl mutant line versus the Col-0 (wild-type, aka “WT”) line or the cax3 mutant line compared to normoxia. Anoxia tolerance of caxl mutant line was enhanced when CAX3 was also mutated, e.g., the caxl/3 (aka “double mutant knockout”, aka “caxl/3DKO”) line. Photographs are representative of more than 15 biological replicate plates for each condition. FIG. IB show improved postanoxia reoxygenation tolerance (reduced chlorophyll loss; reduced induction of lipid peroxidation, measured as malondialdehyde (MDA) level in leaves) of the caxl and caxl/3 mutant lines versus the Col-0 line or the cax3 mutant line. All results are Mean (M) ± Standard Error of the Mean (SEM), n = 3. FIG. 1C shows improved post-anoxia reoxygenation tolerance (reduced cell death, measured by Trypan blue staining) of the caxl and caxl/3 mutant lines versus the Col-0 line or the cax3 mutant line. The images are representative of three independent experiments. FIG. ID shows reduced plant stress (reduced chlorophyll loss, measured by fluorescence; reduced H2O2 levels, measured by H2DCFDA dye fluorescence) of the caxl and caxl/3 mutant lines versus the Col-0 line or the cax3 mutant line in post-anoxia reoxygenation conditions compared to normoxia. The images are representative of three independent experiments. FIG. IE shows reduced plant stress (reduced hydrogen peroxide (H2O2) levels, via staining with 3,3 '-diaminobenzidine (DAB)) of the caxl and caxl/3 mutant lines versus the Col-0 line or the cax3 mutant line in 1 hr and 8 hr post-anoxic reoxyganation conditions compared to normoxia. The images are representative of three independent experiments.
[0061] FIGS. 2A-2F illustrate particular embodiments of the disclosure showing RNAseq transcriptomic data for Arabidopsis thaliana plants. The caxl mutant line and the caxl/3 mutant line were compared with the cax3 mutant line and the Col-0 line. Plants were sampled prior to, during, and after exposure to anoxic conditions. FIG. 2A is a multi-dimension scaling (MDS) representation of Col-0, caxl, cax3, and caxl/3 transcriptomes before during and after anoxic treatment. Colors highlight the factor/time point and the shape defines the genotype. FIG. 2B shows Genome Ontology (GO) groups identified in caxl and caxl/3 preanoxia that coincide with genes differentially expressed in Col-0 and cax3 during anoxia and recovery (1-hour post 7-hour anoxia). FIG. 2C shows GO enrichment in caxl and caxl/3 (caxl/3>caxl) after 4 hours of anoxia. FIG. 2D shows GO enrichment in caxl and caxl/3 (caxl/3>caxl) during recovery. FIG. 2E shows a scheme depicting timing of leaf tissue harvesting for RNAseq analysis. Plants were taken for RNAseq analysis before anoxia (preconditions), after 4 hours of anoxia (anoxia) and 1 hour after a 7-hour anoxia treatment (recovery). FIG. 2F shows Metascape network visualization of the enriched genome ontology groups that were significantly different in both caxl and caxl/3 compared to cax3 and Col-0 under anoxia and recovery conditions. The nodes are color coded to delineate the similarities and differences among caxl and caxl/3 during anoxia and in the recovery phase. The size of the connecting dots represent the -loglO of the P value for that GO cluster.
[0062] FIGS. 3A-3C illustrate particular embodiments of the disclosure showing differentially abundant proteins (DAP) of WT, caxl mutant, and caxl/3 mutant genotypes of Arabidopsis thaliana plants based on normalized spectral abundance factor (NSAF). FIG. 3A shows a summary table of differentially abundant proteins (DAP) showing number of proteins which increased or decreased in abundance in the different genotypes. GO enrichment analysis was performed on the DAPs in caxl and caxl/3 post anoxia (recovery). Enrichment was performed using DAVID employing the hypergeometric test to determine significance. Rich factor is the ratio of identified protein numbers annotated in the given GO term pathway to the total number of input annotated proteins. The top 40 enriched GO term categories are presented in FIG. 3B.1, and FIG. 3B.2 for caxl and caxl/3 respectively. FIG. 3C shows a schematic diagram of the glycolytic pathway showing those proteins (P) and transcripts (T) regulated post anoxia. Red/Up arrow - increased; Blue/Down arrow - decreased. Similar responses for data from both transcriptomic analysis and protein abundance analysis are overlaid onto KEGG pathways.
[0063] FIGS 4A-4C illustrate particular embodiments of the disclosure showing the effect of Ca, ROS, temperature, and phytohormones on the anoxic tolerance. FIG. 4A shows the effect of exogenous application of chemicals or heat treatment on anoxia tolerance. EGTA is ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid, a chelating agent that binds calcium and thus lowers the levels found in the plant; ABA is abscisic acid, a plant hormone that is important in plant stress responses. Plants sprayed with water displayed phenotypes identical to untreated plants whereas 10 mM EDTA sprayed 8h prior to anoxia stress abolished the anoxia tolerance without impacting growth during normoxia; 50uM ABA sprayed 2d prior to anoxia increased anoxia tolerance in all lines as did a heat treatment applied 2d prior to the stress. FIG. 4B shows leaf death 4 days post anoxia for lines treated with water, EGTA, ABA, and heat. A leaf with > 50% necrosis was counted as an anoxia damaged leaf. FIG. 4C shows phytohormone measurements from 2.5-week-old Col-0 and caxl/3 plants. N=6. ABA is abscisic acid; BA is benzoic acid, a compound that elicits plant growth and development responses; CA is trans-cinnamic acid; COUMA is p-Coumaric acid which is a phenylpropaid in plants that can act as an antioxidant, IAA is indole-3-acetic acid, which has many effects including inducing cell elongation and cell division; SA is salicylic acid.
[0064] FIGS. 5A-5E illustrate particular embodiments of the disclosure showing Ca imaging studies of Col-0 and caxl mutant lines of Arabidopsis thaliana plants which express the Ca biosensor GCamP3. Col-0 and caxl plants expressing a stable genetically encoded Ca indicator GCamP3 were grown on * strength MS medium with 0.5% sucrose for 12 days before they were treated with anoxia stress for 4h. FIG. 5A shows fluorescence of GCamP3 recorded by streaming video immediately after the plants were re-exposed to oxygen. Images are representative of at least 6 independent experiments. White bar: 2 mm. Plants (12 days old) were surface sprayed with either water control (FIG. 5B), lOmM EGTA (FIG. 5C), 50uM ABA (FIG. 5D) or ImM SA (FIG. 5E), 4 hours prior to a 4h anoxia treatment. SA is salicylic acid and acts as a signal molecule in plant defense responses. Ca-GCamP3 fluorescence was measured to assay cytosolic Ca concentrations immediately after the plants were re-exposed to atmospheric conditions. Graphs are from a representative experiment. All experimental conditions were repeated at least twice each session on at least two different days. During postanoxia reoxygenation, the caxl mutant line displayed altered spatial and temporal Ca signaling versus the Col-0 line after returning to normoxia.
[0065] FIG. 6 illustrates a model comparing anoxia responses in CAX1 and caxl deficient lines (e.g., suppressed and/or eliminated expression). CAX1 plants appear to respond to anoxia with increased ROS production, and minor metabolic and hormone signaling changes. We speculate that Ca levels in the cytosol remain tightly regulated. In caxl there appears to be dampened ROS production, heightened hormone signaling and changes in metabolism. Calcium signaling is altered in the caxl lines, and we speculate that pH levels fluctuate less than in CAX1.
[0066] FIGS. 7A-7D illustrates a particular embodiment of the disclosure showing the effect of CAX mutation on anoxia tolerance of Arabidopsis thaliana plants. FIG. 7A is a diagrammatic representation of the GasPak system used for generating anoxic conditions. FIG. 7C, shows improved anoxia tolerance (reduced wilting and necrosis) compared to nomoxia for the caxl mutant line versus either the Col-0 (wild-type, aka “WT”) line or the cax3 mutant line (the experiment was repeated at least three times with 10-12 plants in each replicate). Phenotypes of the cax mutants were well-defined: the caxl mutant line was more tolerant to anoxic conditions and this phenotype was clearly visible three days after being removed from the GasPak. This tolerance was lost when transgenic CAX1 expressed sCAXl (FIG. 7B, the experiment was repeated at least three times with 10-12 plants in each replicate). The cax2, cax3 or cax4 had similar anoxia phenotypes as Col-0 (wild-type) plants (FIG. 7C). Meanwhile, the tolerance of caxl mutant line was enhanced if CAX3 was also mutated (FIG. 7C). Additionally, every double mutant analyzed was tolerant if one of the mutations was in CAX1 (FIG. 7C). Mutants for V-type ATPases like vha-a2 and vha-a3 and mutants regulating cation dependent proton transport activity such as nhxl-1 and nhx2-3 were susceptible to anoxia (FIG. 7D, two-week old plants grown on half-strength MS medium were used for the study. The experiment was repeated at least two times with 10-12 plants in each replicate).
[0067] FIGS. 8A-8C illustrate particular embodiments of the disclosure showing RNAseq transcriptomic data for Arabidopsis thaliana plants sampled after anoxia (FIG. 8A, A1-A7); recovery (FIG. 8B, B1-B12) and pre-conditions (FIG. 8C, C1-C7). The caxl mutant line and the cax 1/3 mutant line were compared with the cax3 mutant line and the Col-0 line. FIGS. 8A-8C illustrate genome ontology (GO) analysis which correlates the expressed transcripts in the samples with biologic function. Gene categories have been identified by Metascape and are numbered, e.g., groups A1-A7, groups B1-B13, and groups C1-C7. Heatmaps were created in R using pheatmap. Genes present in multiple categories are represented only once in the heat map. Red and blue represent up and downregulated gene expression. Color density indicating levels of fold change.
[0068] FIGS. 9A-9D illustrate particular embodiments of the disclosure showing enriched GO categories in Col-0 and cax3 for Arabidopsis thaliana plants sampled after anoxia and recovery. FIG. 9A shows GO categories among the genes commonly up regulated in Col- 0 and cax3 following anoxia. FIG. 9B shows GO categories among the genes commonly down regulated in Col-0 and cax3 following anoxia. FIG. 9C shows GO categories among the genes commonly up regulated in Col-0 and cax3 during recovery. FIG. 9D shows GO categories among the genes commonly down regulated in Col-0 and cax3 during recovery. Gene categories have been identified by Metascape.
[0069] FIG. 10 illustrates particular embodiments of the disclosure showing RNAseq data using real-time PCR (qRT-PCR) revealing a global change in the transcript abundances of Ca transporters, Ca sensor/effectors in both caxl and caxl/3 mutant lines. Eight genes were selected and analyzed in pre-conditions, anoxia and recovery in each genotypes (Col-0, caxl, cax3, caxl/3). Plots were generated from the ratio of transcripts levels obtained by qRT-PCR. ADH1 is Alcohol Dehydrogenase 1; CAT1 is Catalase 1; APX2 is Ascorbate peroxidase 2; GLR2.8 is Glutamate Receptor 2.8; CML41 is Calmodulin-like 41; HRE2 is Ethylene response factor 71; EBP is Ethylene-responsive element binding protein; HSFA2 is Heat shock factor A2.
[0070] FIGS. 11A-11B illustrate particular embodiments of the disclosure showing a hierarchically clustered heatmap showing the expression of approximately 30 different Ca sensors (FIG. 11A) and Ca transporters (FIG. 11B). Red and blue represent up and downregulated gene expression. Color density indicating levels of fold change. FIGS. 11A- 11B show that the caxl and caxl/3 specific DEGs here were enriched for GO categories associated with various stress and signaling pathways including calcium, heat, hydrogen peroxide, carbohydrates, ethylene, chitin, and wounding.
[0071] FIGS. 12A-12D illustrate particular embodiments of the disclosure showing enriched temperature-responsive genes in caxl and caxl/3 during pretreatment conditions (FIGS. 12A-12B) and during recovery (FIGS. 12C-12D). FIG. 12A shows Metascape output of genes which are highly down regulated in caxl and caxl/3 during the pre-treatment conditions. FIG. 12B shows expression analysis of the cold responsive genes highly down regulated in caxl and caxl/3 during the pre-treatment conditions. FIG. 12C shows Metascape output of genes which are highly up regulated in caxl and caxl/3 during recovery. FIG. 12D shows expression analysis of the heat responsive genes highly up regulated in caxl and caxl/3 during recovery. Red and blue represent up and downregulated gene expression. Color density indicating levels of fold change
[0072] FIG. 13 illustrates particular embodiments of the disclosure showing expression pattern of genes involved in ABA and SA pathways. Heatmap was created in R using pheatmap. Red and blue represent up and downregulated gene expression. Color density indicating levels of fold change.
[0073] FIG. 14 illustrates particular embodiments of the disclosure showing transcript analysis of CAX1 and CAX3 RNAs in caxl and cax3. Semi-quantitative RT-PCR was conducted in 2.5 week old Col-0, caxl, cax3 and caxl/3 to assess transcripts of CAX1 and CAX3. RNA fragments of CAX1 and CAX3 before, across and after the T-DNA insertion were investigated in the various genetic backgrounds. UBQ10 was used as an endogenous control. T-DNA-span primers anneal to regions that span across the T-DNA insertion site. Primers anneal to fragments before and after the T-DNA insertion were used to test the effect of T-DNA insertion on the transcription of these regions. This data shows that while CAX1 and CAX3 RNAs are present in the mutants, they are partial transcripts containing only the 5’ region before the T-DNA.
[0074] FIGS. 15A-15C illustrate particular embodiments of the disclosure showing pie charts of subcellular distributions of DAPs in Col-0 (FIG. 15A), caxl (FIG. 15B), and caxl/3 (FIG. 15C). Subcellular locations of proteins were suggested by SUBA consensus, “Mix” indicates the proteins with multiple predicted locations. Numbers in the figure represent the regulated protein counts for the corresponding subcellular compartments.
[0075] FIG. 16 illustrates particular embodiments of the disclosure showing a schematic overview of cellular H2O2 removal and oxidative stress relief in caxl and caxl/3 as evidenced by proteomic studies. SOD is Superoxide dismutase; CAT is Catalase; APX is L- ascorbate peroxidase 1; GPX is phospholipid hydroperoxide glutathione peroxidase; GLYR1 is Glyoxylate succinic semialdehyde reductase; GSTUJ is Glutathione S-transferase; PDX is Pyridoxal 5'-phosphate synthase; TRXM1 is Thioredoxin Ml; CDSP is Thioredoxin-like protein; PRX2E is Peroxiredoxin-2E; PRX2F is Peroxiredoxin-2F; and ACO3 is Aconitate hydratase 3.
[0076] FIG. 17 illustrates particular embodiments of the disclosure showing cytosolic calcium signals under dark conditions without anoxia in Col-0 and caxl. Col-0 and caxl expressing a stable genetically encoded Ca indicators GCamP3 were grown on half-strength MS with 0.5% sucrose for 12 day before they were wrapped in foil and placed in anaerobic bags without the GasPak for 4h. This was done as a negative control for anoxia. Fluorescence of GCamP3 was recorded by streaming video immediately after the plants were exposed to light. Images are representative of at least 4 independent experiments. White bar: 2 mm. [0077] FIGS. 18A-18I illustrates a particular embodiment of the disclosure showing the effect of CAX mutations on anoxia tolerance of Arabidopsis thaliana plants when assessed using submergence in water (e.g., fresh water) as an anoxic condition generating stimulus. FIG. 18A is a diagrammatic representation of the submergence tolerance assay used for generating anoxic conditions. FIG. 18B, shows Col-0 line (wild-type, aka “WT”) plant wilting and/or necrosis seven days after plants have been removed from 38 hours of submergence in water. FIG. 18C, 18D, and 18E represent results from various cax-1 mutant lines (caxl-1 (18C), caxl -2 (18D), and cax-13 (18E)) that exhibited reduced wilting and/or necrosis following submergence when compared to control lines. FIG. 18F represent results from cax-1/3 double mutant lines that showed reduced wilting and/or necrosis following submergence when compared to control lines. FIG. 18G show that complement based rescue of CAX1 expression in cax-1 mutant lines (“cax-1 complemented line”) restores wilting and/or necrosis phenotypes following submergence when compared to control cax-1 mutant lines that have not been rescued. FIG. 18H and 181 show that cax-3 or cax-2 single mutant lines show wilting and/or necrosis similar to WT lines following submergence. These experiments were repeated at least three times with 9-20 plants in each replicate. Phenotypes of the cax mutants were well-defined: the caxl mutant lines were more tolerant to anoxic conditions and this phenotype was clearly visible seven days after being removed from submergence. This tolerance to submergence mediated anoxia was lost when caxl mutant lines transgenically expressed functional CAX1 (FIG. 18G).
DETAILED DESCRIPTION
[0078] As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising", the words “a” or “an” may mean one or more than one. As used herein “another” may mean at least a second or more. Still further, the terms “having”, “including”, “containing” and “comprising” are interchangeable and one of skill in the art is cognizant that these terms are open ended terms. In specific embodiments, aspects of the disclosure may “consist essentially of’ or “consist of’ one or more sequences of the disclosure, for example. Some embodiments of the invention may consist of or consist essentially of one or more elements, method steps, and/or methods of the disclosure. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. The scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As used herein, the terms “or” and “and/or” are utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and/or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” It is specifically contemplated that x, y, or z may be specifically excluded from an embodiment.
[0079] The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” As used herein “another” may mean at least a second or more. The terms “about”, “substantially” and “approximately” mean, in general, the stated value plus or minus 5%.
[0080] Reference throughout this specification to “one embodiment,” “an embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment,” “an additional embodiment,” or “a further embodiment” or combinations thereof means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
[0081] Methods of Use
[0082] In another aspect, the present disclosure relates to methods of using the genetically modified plants described herein. The genetically modified plants may exhibit improved properties over a control plant. For example, the genetically modified plants may have improved tolerance to anoxia as compared to a control plant. As used herein, "anoxia” or “anoxic conditions" refer to conditions in which there is total oxygen depletion to plant cells, plants or parts of such genetically modified plants. In contrast, “hypoxia” or “hypoxic conditions” refer to conditions in which there is an oxygen deficiency but not total oxygen depletion.
[0083] Anoxia resistance may be measured using assays known in the art such as, for example, subjecting the plants to low oxygen-stress over a period of a certain number of days (e.g., via methods as described herein, e.g., via hypoxia generating apparatus, via submergence, via tight packing of soil, etc.,). In some embodiments, as used herein, a “control plant” is a plant that has not been modified as described herein. In some embodiments, exemplary control plants may include those from a natural plant species for a particular plant being modified. In specific embodiments, a control plant is a plant that has not been genetically modified by the hand of man to have improved anoxia tolerance, including a plant that has not been genetically modified with respect to one or more CAX genes, including at least CAX1 or a combination of CAX1 and one or more of CAX2, CAX3, and CAX4. In some embodiments, a control plant and/or condition is relative to a test plant and/or condition, one of skill in the art can determine if a certain genotype, assay condition, etc. is an appropriate control relative to the question(s) asked or comparisons being made. In certain embodiments, a control plant is one that has been genetically modified by the hand of man for the purposes of determining necessity and/or sufficiency of a particular biological phenomenon. In specific embodiments, a control plant is a plant that has not been genetically modified by the hand of man to have improved anoxia tolerance, including a plant that has not been genetically modified with respect to one or more genes capable of synthesis of one or more activators of one or more CAX genes, including at least one or more activators of CAX1 or a combination of one or more activators of CAX1 and one or more activators of one or more of CAX2, CAX3, and CAX4.
[0084] In some embodiments, methods described herein include planting any of the genetically modified plants described herein in a suitable area. In certain embodiments, the area may possess low oxygen levels, for example but not limited to, conditions such as areas of high elevation, extremely tight soil, submersion by water and/or ice, etc. In certain embodiments, the area may generally have normal oxygen levels but is at risk of acute anoxia. In certain embodiments, the area may have normal oxygen levels but is at risk of submersion. For example, in some embodiments, the area may be an area that is subject to flooding, such as near any body of water and including a flood plain (e.g., a river basin, near a reservoir and/or other standing body of fresh water, near an estuary and/or swamp, near a tidal flood zone, etc.). In some embodiments, genetically modified plants described herein have increased tolerance to anoxic stress and may be useful for planting in areas where low oxygen levels are possible, predictable, and/or expected.
[0085] In some embodiments, following production of genetically modified plants as described herein, the genetically modified plants may be suitably stored and/or their seeds may be suitably stored. The genetically modified plants and/or their progeny including seeds may be used for commercial purposes and/or research purposes. In particular embodiments, the genetically modified plants and/or their progeny including seeds are planted for the purpose of being used as a crop or to produce crops and harvesting thereof. I. Types of Plants
[0086] In another aspect of the present disclosure, plants are provided for genetic modification. The plants may include any one of the plant cells, plant parts, or entire plants encompassed herein. The plants may include plants in which every cell of the plant is a plant cell modified as described herein. Alternatively, the plants may include plants in which only certain tissues within the plant include the plant cells described herein. For example, with respect to genetic engineering techniques, it is contemplated that the plants may only have plant cells including modified expression of CAX genes in certain tissues of the plant. For example, with respect to genetic engineering techniques, it is contemplated that the plants may only have plant cells including modified synthesis of activators of CAX genes in certain tissues of the plant.
[0087] As used herein, a “plant” includes any multicellular organism of the Kingdom Plantae. Clades of Kingdom Plantae include the Viridiplantae, the Glaucophytes, and the Rhodophytes. The Viridiplantae includes green algae and embryophytes (hornworts, liverworts, mosses, ferns, lycophytes, gymnosperms, and angiosperms). The Glaucophytes include fresh water algae. The Rhodophytes include red algae.
[0088] As used herein, a “plant, part, or progeny thereof’ includes any portion of the plant including, without limitation, a whole plant or a portion of a plant such as a part of a root, leaf, stem, seed, pod, flower, tissue, plant germplasm, asexual propagate, or any progeny thereof. For example, a corn plant refers to the whole com plant or portions thereof including, without limitation, the leaves, flowers, fruits, stems, roots, or otherwise. As used herein, a “plant germplasm” is any living tissue from which new plants can be grown, such as a seed or another plant part - a leaf, a piece of stem, pollen, spores, or even just a few cells that can be turned into a whole plant. A plant germplasm contains the genetic information for the plant. Plant germplasms may include whole plants, seedlings, leaves, stems, flowers, roots, fruits, seeds, grafts, buddings, cuttings, bulbs, tubers, rhizomes, regenerable cells, tissue cultures of regenerable cells, regenerable protoplasts, and tissue cultures of regenerable protoplasts.
[0089] Suitable plants may include, without limitation, a rice plant, a maize plant, a barley plant, an oat plant, a rye plant, a wheat plant, a corn plant, a sorghum plant, a soybean plant, a pea plant, a lentil plant, a curcurbit plant, coffee plant, a cocoa plant, a rapeseed plant, sunflower plant, a sugar cane plant, a potato plant, a palm plant, a grape plant, an apple plant, a banana plant, a plantain plant, a cassava plant, a sugar beet plant, a tomato plant, a sweet potato plant, a yam plant, a tobacco plant, a cotton plant, a natural rubber plant, a tea plant, a lettuce plant, a pepper plant, an onion plant, a grape plant, a pecan tree, a timber tree, a cannabis plant, or a poppy plant.
[0090] Plants may be sexually or asexually propagated to produce progeny. Plants may be cultivated from seeds, seedlings, grafts, cuttings, spores, rhizomes, tubers, or whole plants regenerated from cultured plant tissues or cells. Plants may be cultivated in natural or artificial culture medium. Cultivation may be performed in hydroponic systems or in soil media. Cultivation may occur in a container or in an open area. Indoor cultivation may occur in greenhouses while cultivation on open land may occur on farmland, orchards, vineyards, gardens, and/or forests, for example.
II. Modification of Plants
[0091] Genetic modification as encompassed herein includes any modification made to the genetic material of an organism. As described herein, genetic modifications are generally synthetic genetic modifications. A synthetic genetic modification is a genetic modification performed by the hand of man and does not encompass naturally produced mutations.
[0092] Genetic modification may be accomplished using genetic engineering techniques or by plant breeding (selection, hybridization, and induced mutation). Genetic modification as encompassed herein includes altered levels of expression of one or more genes normally present in an organism’s genome. Genetic modification may include removal of one or more genes normally present in an organism’s genome. Genetic modification may involve one or more mutations of one or more genes normally present in an organism’s genome. Genetic modification may involve insertion of genes into an organism’ s genome which are not normally present. Genetic modification may involve insertion into the cells of an organism of agents capable of gene silencing of one or more genes normally present in an organism’s genome. Genetic modification may involve insertion into and/or deletion out of the cells of an organism of nucleic acid sequences (DNA and/or RNA), nucleases (DNAses and/or RNAses), and/or proteases.
[0093] Genetic modifications are generally obtained through genetic engineering. Genetic engineering generally refers to the intentional introduction of a targeted genetic change to achieve a specific result. The resultant organism is generally referred to as a genetically modified organism (GMO). In specific embodiments, a whole GMO plant may be regenerated from genetically modified regenerable plant parts (e.g. tissue cultures of plant cells or seeds). [0094] Plants with exogenous DNA incorporated within are generally referred to as transgenic plants. In some embodiments, intragenic DNA may be used to achieve the same specific result. Intragenic DNA may be produced by identifying functional equivalents of vector components from within the genome of a specific target plant species and using such DNA sequences to assemble vectors for transformation of that species. In some embodiments, plants with intragenic DNA incorporated within are not considered transgenic plants. As used herein, the terms “genetically modified plants” and “GMO plants” encompass both transgenic and non-transgenic plants produced according to the methods disclosed herein. Techniques that may be used to produce GMO plants with heritable genetic modifications include but are not limited to, physical (direct) gene transfer, Agrobacterium-mediated gene transfer, and/or CRISPR systems.
[0095] Viral vectors and exogenous application of RNA molecules may also be used to achieve the same specific result as transgenic or intragenic gene modification. However, viral vectors and exogenous application of RNA molecules generally achieve non-heritable modifications in gene expression which are present only during the lifetime of the plant, as the underlying genetic code remains unaffected. In certain circumstance, physical (direct) and/or Agrobacterium-mediated gene transformation techniques may be used to achieve nonintegrative genetic modification. As used herein, the terms “genetically modified plants” and “GMO plants” refer to plants possessing transgenic or intragenic gene modification as well as plants with modified gene expression based on insertion of nonintegrative nucleic acid sequences such as viral vectors and/or exogenous RNA into plant cells.
III. Gene Silencing of Plants
[0096] New methods for plant trait improvement through specific gene alteration are highly desirable. These include methods for over-expression of genes or gene silencing. Gene silencing is the regulation of gene expression in a cell to prevent the expression of a certain gene and encompasses gene knock down (suppression of gene expression) and/or gene knockout (elimination of gene expression). A powerful technique for sequence-specific gene silencing is through RNA interference (RNAi). RNAi is a mechanism in which expression of an individual gene can be specifically silenced by introducing a double- stranded RNA (dsRNA) that is homologous to the selected gene, into cells. Such double stranded RNA (dsRNA) is also referred to as hairpin RNA (hpRNA). Inside the cell, dsRNA molecules are cut into shorter double stranded RNA fragments or single-stranded hairpin RNA fragments of 21-27 nucleotides by an RNase Ill-related enzyme (Dicer). These fragments, called small interfering RNAs (siRNAs), get incorporated into the RNA-induced silencing complex (RISC). After additional processing, the siRNAs are transformed into single-stranded RNAs that act as guide sequences to eventually cleave target messenger RNAs. By using RNAi to specifically silence relevant target genes, one can alter basic traits of an organism. Specifically for plants, RNAi holds incredible potential for modifications that may lead to increased stress resistance and better crop yield.
[0097] In plants, RNAi is typically performed by producing transgenic plants that overexpress an exogenous DNA fragment that is transcribed to produce a dsRNA. However, intragenic DNA fragments may also be used to avoid the controversy surrounding transgenic plants. In some embodiments, dsRNA may be directly introduced into plant cells rather than being indirectly expressed from DNA. In some embodiments, this dsRNA is then processed into siRNAs that mediate the cleavage and silencing of target genes, typically by targeting cleavage of the target gene by an RNA Induced Silencing Complex (RISC) or by translational repression. In some embodiments, dsRNA are utilized to silence genes of interest as described herein (e.g., CAX1, CAX2, CAX3, CAX4, regulators of CAX gene activity, etc.).
[0098] MicroRNA (miRNA) regulation is a specialized branch of the RNA silencing pathway that evolved towards gene regulation, diverging from conventional RNAi. miRNAs are a specific class of small RNAs that are encoded in gene-like elements organized in a characteristic partial inverted repeat. When transcribed, microRNA genes give rise to partially basepaired stem-looped precursor RNAs (pri-miRNAs and pre-miRNAs) from which the microRNAs are subsequently processed. miRNAs are typically about 21 nucleotides in length. The released miRNAs are incorporated into RISC-like complexes containing a particular subset of Argonaute proteins that exert sequence- specific gene repression. In some embodiments, miRNAs are utilized to silence genes of interest as described herein e.g., CAX1, CAX2, CAX3, CAX4, regulators of CAX gene activity, etc.).
IV. Genetic Engineering of Plants
[0099] There are numerous genetic engineering techniques that may be used to achieve the specific result of gene silencing in plants. Certain techniques employ foreign (exogenous) DNA transfer (physical, viral, bacterial, or CRISPR systems) while other techniques avoid exogenous DNA (intragenic DNA transfer, exogenous RNA application). Genetic engineering techniques may result in stable modification involving integration of DNA into the host genome (physical, bacterial, CRISPR systems) and/or transient modification which is nonintegrative (physical, bacterial, viral, exogenous RNA application). In some embodiments, genetic engineering techniques such as, but not limited to, those described herein are utilized to silence genes of interest as described herein (e.g., CAX1, CAX2, CAX3, CAX4, regulators of CAX gene activity, etc.).
I. Physical (Direct) DNA Transfer
[0100] In some embodiments, the modification of a CAX gene or a gene capable of synthesis of an activator of a CAX gene is carried out using physical (direct) gene transfer. Direct gene transfer involves the uptake of naked DNA by plant cells and its subsequent integration into the genome. The target cells can include: isolated protoplasts or cells; cultured tissues, organs or plants; intact pollen, seeds, and plants. Direct DNA transfer methods are entirely physical processes with no biological interactions to introduce the DNA into plant cells and therefore no “host range” limitations associated with Agrobacterium-mediated transformation. Methods to effect direct DNA transfer can involve a wide range of approaches, including: passive uptake; the use of electroporation; treatments with polyethylene glycol; electrophoresis; cell fusion with liposomes or spheroplasts; microinjection, silicon carbide whiskers, and particle bombardment. Of the various approaches, particle bombardment is almost exclusively used because there are no limitations to the target tissue. However, one limitation of particle bombardment is the overall length of the DNA. Longer DNA molecules are likely to shear either upon particle acceleration or impact.
[0101] Vectors for direct DNA uptake only need to be standard bacterial plasmids to allow propagation of the vector. It is usual for such vectors to be small, high-copy plasmids capable of propagation in Escherichia coli. This allows convenient construction of plasmids using well-established molecular biology protocols and ensures high yields of vector upon plasmid isolation and purification for subsequent use in transformation. Various authors claim a preference to use DNA of a specific form (circular or linear, double- or single-stranded). However, comparisons of all four combinations of DNA conformation in parallel experiments resulted in similar transformation frequencies and integration patterns.
II. Viral-Mediated Gene Transformation
[0102] In some embodiments, the modification of a CAX gene or a gene capable of synthesis of an activator of a CAX gene is carried out using a viral-mediated gene transformation. The use of plant viruses as vehicles to introduce and express nonviral genes in plants is well known. When a DNA virus is utilized, suitable modifications can be made to the virus itself. Alternatively, the virus DNA can first be cloned into a bacterial plasmid for ease of constructing the desired viral vector with the foreign DNA. The virus can then be excised from the plasmid. A bacterial origin of replication can be attached to the viral DNA, which is then replicated by the bacteria. Transcription and translation of this DNA will produce the coat protein which will encapsulate the viral DNA. If an RNA virus is utilized, the virus is generally cloned as a cDNA and inserted into a plasmid. The DNA of the plasmid is then used to make all of the constructions. The RNA virus is then produced by transcribing the viral sequence of the plasmid and translation of the viral genes to produce the coat protein(s) which encapsulate the viral RNA.
[0103] Infection of plants with modified viruses is simpler and quicker than the regeneration of stably transformed plants since plant viruses are often small in size (between 3000 and 10,000 nucleotides), are easy to manipulate, have the inherent ability to enter the plant cell, lead to the immediate expression of the heterologous gene and will multiply to produce a high copy number of the gene of interest. Viral vectors have been engineered for delivery of genetic material and expression of recombinant proteins in plants. Viral expression systems are considered transient expression systems as the viral vectors are not integrated into the genome of the host, however, depending on which virus is used, virus multiplication and gene expression can persist for long periods (up to several weeks or months).
[0104] Plant virus vectors provide advantages in quick and high level of foreign gene expression in plants due to the infection nature of plant viruses. The full length of the plant viral genome can be used as a vector, but often a viral component is deleted, for example the coat protein, and transgenic ORFs are subcloned in that place. The ICK motif protein expression ORF can be subcloned into such a site to create a viral vector. These viral vectors can be introduced into plants mechanically since they are infectious themselves, for example through plant wound, spray-on etc. They can also be transformed into plants by agroinfection by cloning the virus vector into the T-DNA of the crown gall bacterium, Agrobacterium tumefaciens, or the hairy root bacterium, Agrobacterium rhizogenes. The expression of the ICK motif protein in this vector is controlled by the replication of the RNA virus, and the virus translation to mRNA for replication is controlled by a strong viral promoter, for example, 35S promoter from Cauliflower mosaic virus. Viral vectors with ICK motif protein expression ORF are usually cloned into T-DNA region in a binary vector that can replicate itself in both E. coli strains and Agrobacterium strains. The transient transformation of a plant can be done by infiltration of the plant leaves with the Agrobacterium cells which contain the viral vector for ICK motif protein expression. In the transient transformed plant, it is common for the foreign protein expression to be ceased in a short period of time due to the post-transcriptional gene silencing (PTGS). Sometimes a PTGS suppressing protein gene is necessary to be cotransformed into the plant transiently with the same type of viral vector that drives the expression of with the ICK motif protein expression ORF. This improves and extends the expression of the ICK motif protein in the plant. The most commonly used PTGS suppressing protein is P19 protein discovered from tomato bushy stunt virus (TBSV).
III. Agrobacterium-Mediated Gene Transfer
[0105] In some embodiments, the modification of a CAX gene or a gene capable of synthesis of an activator of a CAX gene is carried out using Agrobacterium-mediated gene transfer. Agrobacterium strains induce crown galls or hairy roots on plants by the natural transfer of a discrete segment of DNA (T-DNA) to plant cells. The T-DNA region contains genes that induce tumor or hairy root formation and opine biosynthesis in plant cells. In Agrobacterium the T-DNA resides on the Ti or Ri plasmids along with several virulence loci with key vir genes responsible for the transfer process. The action of these vir genes, combined with several other chromosomal-based genes in Agrobacterium, and specific plant proteins effect the transfer and integration of the T-DNA into the nuclear genome of plant cells. Short imperfect direct repeats of about 25 bp, known as the right and left border (RB and LB respectively), define the outer limits of the T-DNA region.
[0106] The genes on the T-DNA of Ti and Ri plasmids responsible for tumor or hairy root formation are well known to result in plants with an abnormal phenotype or prevent the regeneration of plants. The development of “disarmed” Agrobacterium strains with either the deletion of the genes responsible for tumor formation or the complete removal of the T-DNA was crucial for Agrobacterium-mediated gene transfer to plants. These approaches lead to the development of co-integrate vectors and binary vectors respectively.
[0107] With co-integrate vectors the foreign DNA is integrated into the resident Ti plasmid. The tumor-inducing genes of the T-DNA are first removed leaving the right border and left border sequences. The foreign DNA is then inserted into a vector that cannot replicate in Agrobacterium cells, but can recombine with the Ti plasmids through a single or double recombination event at a homologous site previously introduced between the right border and left border sequences. This results in a co-integration event between the two plasmids. A later refinement resulted in the split-end vector system in which only the left border is retained on the Ti plasmid and the right border is restored by the co-integration event. The main advantage of co-integrate vectors is their high stability in Agrobacterium. However, the frequency of cointegration is low and their development is complex, requiring a detailed knowledge of the Ti plasmid and a high level of technical competence.
[0108] The demonstration that the T-DNA and the vir region of Ti plasmids could be separated onto two different plasmids contributed to the development of binary vectors, a key step to greatly simplify AgroZzz c/erzMm-mediated gene transfer. The helper plasmid is a Ti or Ri plasmid that has the vir genes with the T-DNA region deleted and acts in trans to effect T- DNA processing and transfer to plant cells of a T-DNA on a second plasmid (the binary vector). Binary vectors have several main advantages: small size, ease of manipulation in Escherichia coli, high frequency of introduction into Agrobacterium, and independence of specific Ti and Ri plasmids. They have revolutionized the applications of AgroZzz c/erzMm-mediated gene transfer in plant science and are now used to the virtual exclusion of co-integrate vectors.
[0109] To facilitate the development of transgenic plants a wide range of binary vectors with versatile T-DNA regions have been constructed. These often contain alternative cloning regions with a different series of unique restriction endonuclease sites for insertion of genes for transfer to plants and/or alternative selectable marker genes. However, many binary vectors also contain extraneous DNA elements on the T-DNA region that are present as a matter of convenience rather than of necessity for the development of a desired transgenic plant. Examples include the lacZ' region coding for P-galactosidase reporter genes, origins of plasmid replication, and bacterial marker genes.
[0110] For the general release of transgenic plants into agricultural production, such extraneous DNA regions either necessitate additional risk assessment or may be unacceptable to regulatory authorities. This led to the development of minimal T-DNA vectors, without extraneous DNA segments on the T-DNA. These simple binary vectors consist of a very small T-DNA with a selectable marker gene tightly inserted between the left and right T-DNA borders and a short cloning region with a series of unique restriction sites for inserting genes- of-interest. As a consequence they are based on the minimum features necessary for efficient plant transformation by Agrobacterium.
[0111] In some embodiments, for optimal transgene function, the generation of plants with a single intact T-DNA is preferred. In some embodiments, the T-DNA is delineated by two 25 bp imperfect repeats, the so-called border sequences, which define target sites for the VirDl/VirD2 border specific endonucleases that initiate T-DNA processing. In some embodiments, the resulting single-stranded T-strand is transferred to plant cells rather than the double stranded T-DNA. In some embodiments, initiation of T-strand formation involves a single strand nick in the double- stranded T-DNA of the right border, predominantly between the third and fourth nucleotides. In some embodiments, after nicking the border, the VirD protein remains covalently linked to the 5' end of the resulting single-stranded T-strand. In some embodiments, the attachment of the VirD protein to the 5' right border end of the T- strand, rather than the border sequence, establishes the polarity between the borders. In some embodiments, this determines the initiation and termination sites for T-strand formation.
[0112] Vectors ior Agrobacterium-mediated transformation of plants generally contain two T-DNA border- like sequences in the correct orientation that ideally flank a series of restriction sites suitable for cloning genes intended for transfer. However, efficient transformation is possible with, only a single border in the right border orientation. Deletion of the left border has minimal effect on T-DNA transfer, whereas deletion of the right border abolishes T-DNA transfer. Retaining two borders flanking the T-DNA helps to define both the initiation and end points of transfer, thereby facilitating the recovery of transformation events without vector backbone sequences.
[0113] The well-defined nature of T-strand initiation from the right border results, in most instances, in only 3 nucleotides of the right border being transferred upon plant transformation. However, at the left border, the end point of the T-DNA sequence is far less precise. It may occur at or about the left border, or even well beyond the left border. This is confirmed by DNA sequencing across the junctions of T-DNA integration events into plant genomes. The less precise end points at left border junctions results in the frequent integration of vector backbone sequences into plant genomes.
IV. CRISPR
[0114] In some embodiments, the modification of a CAX gene or a gene capable of synthesis of an activator of a CAX gene is carried out using one or more DNA-binding nucleic acids, such as alteration via an RNA-guided endonuclease (RGEN). For example, the alteration can be carried out using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins. In general, "CRISPR system" refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated ("Cas") genes, including sequences encoding a Cas gene, a tracr (transactivating CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr- mate sequence (encompassing a "direct repeat" and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a "spacer" in the context of an endogenous CRISPR system), and/or other sequences and transcripts from a CRISPR locus.
[0115] The CRISPR/Cas nuclease or CRISPR/Cas nuclease system can include a noncoding RNA molecule (guide) RNA, which sequence-specifically binds to DNA, and a Cas protein (e.g., Cas9), with nuclease functionality (e.g., two nuclease domains). In some embodiments, one or more elements of a CRISPR system can derive from a type I, type II, or type III CRISPR system, e.g., derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes.
[0116] In some aspects, a Cas nuclease and gRNA (including a fusion of crRNA specific for the target sequence and fixed tracrRNA) are introduced into the cell. In general, target sites at the 5' end of the gRNA target the Cas nuclease to the target site, e.g., the gene, using complementary base pairing. The target site may be selected based on its location immediately 5' of a protospacer adjacent motif (PAM) sequence, such as typically NGG, or NAG. In this respect, the gRNA is targeted to the desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of the guide RNA to correspond to the target DNA sequence. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence. Typically, "target sequence" generally refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between the target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex.
[0117] In some embodiments, a CRISPR system can induce double stranded breaks (DSBs) at one or more target sites of a CAX gene or a gene capable of synthesis of an activator of a CAX gene, followed by disruptions or alterations as discussed herein. In some embodiments, Cas9 variants, deemed "nickases," are used to nick a single strand at one or more target sites. In some embodiments, paired nickases can be used, e.g., to improve specificity, each directed by a pair of different gRNAs targeting sequences such that upon introduction of the nicks simultaneously, a 5' overhang is introduced. In other embodiments, catalytically inactive Cas9 is fused to a heterologous effector domain such as a transcriptional repressor or activator, to affect gene expression.
[0118] In some embodiments, a target sequence for a CAX gene or a gene capable of synthesis of an activator of a CAX gene may comprise any polynucleotide, such as DNA or RNA polynucleotides. In some embodiments, a target sequence may be located in the nucleus or cytoplasm of the cell, such as within an organelle of the cell. Generally, a sequence or template that may be used for recombination into the targeted locus comprising the target sequences is referred to as an "editing template" or "editing polynucleotide" or "editing sequence". In some aspects, an exogenous template polynucleotide may be referred to as an editing template. In some aspects, the recombination is homologous recombination.
[0119] Typically, in the context of an endogenous CRISPR system, formation of the CRISPR complex (comprising the guide sequence hybridized to the target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence. In some embodiments, a tracr sequence, which may comprise or consist of all or a portion of a wild-type tracr sequence (e.g. about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a wild-type tracr sequence), may also form part of the CRISPR complex, such as by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence that is operably linked to the guide sequence. In some embodiments, a tracr sequence has sufficient complementarity to a tracr mate sequence to hybridize and participate in formation of the CRISPR complex, such as at least 50%, 60%, 70%, 80%, 90%, 95% or 99% of sequence complementarity along the length of the tracr mate sequence when optimally aligned.
[0120] In some embodiments, one or more vectors driving expression of one or more elements of the CRISPR system can be introduced into the cell such that expression of the elements of the CRISPR system direct formation of the CRISPR complex at one or more target sites. Components can also be delivered to cells as proteins and/or RNA. For example, in some embodiments, a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors. Alternatively, in some embodiments, two or more of the elements expressed from the same or different regulatory elements, may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector. In some embodiments, a vector may comprise one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a "cloning site"). In some embodiments, one or more insertion sites are located upstream and/or downstream of one or more sequence elements of one or more vectors. In some embodiments, when multiple different guide sequences are used, a single expression construct may be used to target CRISPR activity to multiple different, corresponding target sequences within a cell.
[0121] In some embodiments, a vector may comprise a regulatory element operably linked to an enzyme-coding sequence encoding the CRISPR enzyme, such as a Cas protein. Non-limiting examples of Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof. These enzymes are known; for example, the amino acid sequence of S. pyogenes Cas9 protein may be found in the SwissProt database under accession number Q99ZW2.
[0122] In some embodiments, a CRISPR enzyme can be Cas9 (e.g., from S. pyogenes or S. pneumonia). In some embodiments, a CRISPR enzyme can direct cleavage of one or both strands at the location of a target sequence, such as within the target sequence and/or within the complement of the target sequence. In some embodiments, a vector can encode a CRISPR enzyme that is mutated with respect to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence. For example, an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand). In some embodiments, a Cas9 nickase may be used in combination with guide sequence(s), e.g., two guide sequences, which target respectively sense and antisense strands of the DNA target. This combination allows both strands to be nicked and used to induce NHEJ or HDR.
[0123] In some embodiments, an enzyme coding sequence encoding the CRISPR enzyme is codon optimized for expression in particular cells, such as eukaryotic cells. In some embodiments, a eukaryotic cells may be those of or derived from a particular organism, such as a plant, fungi, bacteria, mammal, including but not limited to human, mouse, rat, rabbit, dog, or non-human primate. In general, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization.
[0124] In general, a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.
[0125] Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith- Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g. the Burrows Wheeler Aligner), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).
[0126] In some embodiments, the CRISPR enzyme may be part of a fusion protein comprising one or more heterologous protein domains. In some embodiments, a CRISPR enzyme fusion protein may comprise any additional protein sequence, and optionally a linker sequence between any two domains. Examples of protein domains that may be fused to a CRISPR enzyme include, without limitation, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione-5- transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP). A CRISPR enzyme may be fused to a gene sequence encoding a protein or a fragment of a protein that bind DNA molecules or bind other cellular molecules, including but not limited to maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4A DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. Additional domains that may form part of a fusion protein comprising a CRISPR enzyme are described in US 20110059502, incorporated herein by reference.
V. Intragenic DNA Transfers
[0127] In some embodiments, the modification of a CAX gene or a gene capable of synthesis of an activator of a CAX gene is carried out using intragenic DNA transfer. Despite the rapid global adoption of GM technology in agricultural crops, many concerns have been raised about the use of GM crops in agricultural production. These include ethical, religious and/or other concerns among the general public, with the main underlying issue often involving the transfer of genes across very wide taxonomic boundaries. Current advances in plant genomics are beginning to address some of these concerns. Many genes are now being identified from within the gene pools already used by plant breeders for transfer via plant transformation. More importantly, the design of vectors for plant transformation has recently progressed to the development of intragenic systems. This involves identifying plant-derived DNA sequences similar to important vector components. A particularly useful approach involves adjoining two fragments from plant genomes to form sequences that have the functional equivalence of vectors elements such as: T-DNA borders for Agrobacterium- mediated transformation, bacterial origins of replication, and bacterial selectable elements. Such DNA fragments have been identified from a wide range of plant species, suggesting that intragenic vectors can be constructed from the genome of any plant species. Intragenic vectors provide a mechanism for the well-defined genetic improvement of plants with the entire DNA destined for transfer originating from within the gene pool already available to plant breeders. The aim of such approaches is to design vectors capable of effecting gene transfer without the introduction of foreign DNA upon plant transformation. In this manner genes can be introgressed into elite cultivars in a single step without linkage drag and, most importantly, without the incorporation of foreign DNA, such that the resulting plants may be considered to be non-transgenic. VI. Direct Exogenous RNA Application
[0128] In some embodiments, the modification of a CAX gene or a gene capable of synthesis of an activator of a CAX gene is carried out by direct exogenous application of RNA molecules to a plant. Such a technique avoids production of either transgenic or genetically modified plants is through direct exogenous delivery of RNA molecules, rather than delivery of DNA which expresses RNA molecules. Such exogenously applied RNA molecules spread through the plant, inducing RNA interference (RNAi) locally and systemically. Success has been achieved after direct application through spraying, mechanical inoculation, loading onto clay nanosheets, foliar pipetting or brushing, and/or soaking of seeds/roots, trunk injections, soil/root drench, petiole adsorption, cationic nanoparticle carriers, surfactant complexes, and/or peptide-based carriers. Plants have been treated with in vitro-synthesized RNA preparations as well as crude nucleic acid extracts purified from dsRNA/hpRNA-expressing bacterial strains and treated with DNAse and RNAse. RNA molecules other than dsRNA have also been used, including siRNA and single- stranded RNA (ssRNAs).
EXAMPLES
I. Results
[0129] Loss of CAX1 confers tolerance to anoxia. Multiple studies have shown that Ca-regulated transporters and proteins like calmodulin demonstrate altered expression during low O2 conditions (13, 24, 25). However, the exact identity and roles of the Ca transporters directly involved in the anoxia stress response are unknown. The role of CAX genes in anoxia tolerance was clarified using a seedling survival assay where plants are exposed to anoxic conditions for a period of time and survival monitored upon return to normoxic conditions (ambient air, i.e., -21% O2). In an assay, 14-21-day-old plants were placed in a GasPak Anaerobic system that reduced O2 levels in the experimental chamber to -0% (FIG. 7A). If the plants were placed in this chamber for only 5 h, they emerged with no obvious deleterious effect on subsequent survival. After between 6-9 h in the anaerobic chamber, phenotypes of the cax mutants relative to wild type plants were well-defined: the caxl line was more tolerant to anoxic conditions and this phenotype was clearly visible three days after being removed from the GasPak chamber (FIG. 1A, FIG. 7A). This tolerance was lost when transgenic caxl expressed sCAXl (a functional truncation of CAX1; FIG. 7B). The cax2, cax3 or cax4 had similar anoxia phenotypes as Col-0 (wild-type) plants (FIG. 7C). Meanwhile, the tolerance of caxl was enhanced if CAX3 was also mutated (FIG. 1A, FIG. 7C). Additionally, every double mutant analyzed was tolerant if one of the mutations was in CAX1 (FIG. 7C).
[0130] Given that CAX transporters have been shown to play various roles in altering plant signaling, pH and membrane transport, a study was performed to assess the specificity of this anoxia tolerance. Using the same assay, lines previously identified as altering Ca transport, pH, and vacuolar transport were phenotyped (FIG. 7D; Table 1). These included the autoinhibited Ca-ATPase [AC As] localized on the vacuolar membrane (ACA4 and ACA11) (26). Also, two independent V-type ATPases loss-of-function mutant lines vha-a2 and vha-a3 (27, 28) and mutants with reduced Na+/H+ dependent proton transport namely nhxl-1 and nhx2-3 (29, 30). Each of these lines showed the same anoxia sensitivity as col-0 (FIG. 7D; Table 1).
Table 1. Anoxia Response of Various Mutants Involved in Signaling Pathways (Hormone,
Ca, ROS and pH), Phosphorylation and Immune Responses.
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
[0131] Additional measurements were made to assess the damage experienced by these plants during reoxygenation. At 8 h post-anoxia, all the lines still maintained leaf turgor and only showed slight chlorophyll loss. At 24 post-anoxia, WT and cax3 displayed more chlorophyll loss while caxl and caxl/3 maintained their leaf chlorophyll content (FIG. IB) (31). Oxidative-stress-induced lipid peroxidation (by malondialdehyde measurement in leaves) was elevated in WT and cax3 both immediately after and 8 h post-anoxia, but was less elevated in caxl and caxl/3 (FIG. IB) (32).
[0132] Trypan blue staining of seedlings was performed to investigate cell death during the anoxia stress response. A mild degree of cell death was observed in pre-treatment conditions. At 7h post anoxia, WT and cax3 mutants displayed significantly more cell death throughout the leaves, compared to caxl and caxl/3 (FIG. 1C), which also showed greater survival at the whole plant level (FIG. ID). This observation suggests that the difference in anoxia tolerance between caxl, caxl/3 and both Col-0 and cax3 lies in the difference in the severity of initiation of cell death.
[0133] Since ROS are important signal molecules mediating plant stress responses and have been closely linked to hypoxic response systems (33), hydrogen peroxide levels were measured using the cell permeable dye H2DCFDA in plants exposed to anoxic conditions or to normoxia (34) (FIG. ID). The col-0 and cax3 plants displayed elevated H2DCFDA fluorescence post- anoxia while caxl and caxl/3 displayed lower levels. Staining with 3,3'- diaminobenzidine (DAB) was also performed as another independent measure hydrogen peroxide levels (35). Again, caxl and caxl/3 showed lower H2O2 accumulation (Figure IE). A H2O2 burst has been shown to regulate the anoxia stress response, and intracellular H2O2 is known to be one of main sources of oxidative damage triggered upon reoxygenation after anoxic challenge (33). Here we observed a reduction in H2O2 post-anoxia in caxl and caxl/3, consistent with the enhanced survival after low O2 stress in these mutants.
[0134] Additionally, the role of CAX genes in anoxia tolerance was further elucidated using a submergence survival assay where plants were exposed to anoxic conditions (e.g., submergence) for a period of time e.g., -38 hours) and survival was then monitored upon return to normoxic conditions (e.g., ambient air, i.e., -21% O2). As described in FIG. 18A, plants were raised for 21 days in normal growth conditions, 21 -day-old plants were then submerged for 38 hours in fresh water to generate anaerobic conditions. As shown in FIG. 18B, necrosis and/or leaf wilting phenotypes were readily apparent in otherwise healthy Col-0 (wildtype, aka “WT”) lines seven days after submergence treatment. However, in line with the results described herein, caxl deficient lines (e.g., suppressed and/or eliminated expression, e.g., caxl mutant lines) (FIG. 18C-18E; lines caxl-1, caxl -2, and caxl -3) each exhibited decreased necrosis and/or leaf wilting phenotypes relative to controls seven days after submergence treatment. This tolerance was lost when caxl mutant lines transgenically expressed functional complement caxl (e.g., sCAXl, a functional truncation of CAX1) (FIG. 18G). It is noted that cax2 or cax3 lines had similar post- submergence phenotypes as Col-0 (wild-type) plants (FIG. 18H and 181). Additionally, anoxia resistance (e.g., as measured by submergence) of caxl plants was enhanced if cax3 was also mutated (FIG. 18F). caxl plants that underwent submergence treatment were followed throughout the course of their lifespan and found to flower and produce seeds in a manner similar to control wild-type plants that had not undergone submergence treatment. These results were markedly different when compared to wild-type plants that had undergone submergence treatment, such wild-type plants failed to continue developing and died before producing viable seeds.
[0135] The role of CAX genes in anoxia tolerance is further elucidated using a submergence survival assay where plants are exposed to anoxic conditions (e.g., submergence in salt water, e.g., salt water with ions and/or ionic concentrations that are relevant when considering natural environments) for a period of time e.g., -12 hours, -24 hours, -36 hours, -48 hours, or greater than -48 hours) and survival is then monitored upon return to normoxic conditions (e.g., ambient air, i.e., -21% O2). caxl deficient lines (e.g., mutant and/or otherwise deficient lines (e.g., suppressed and/or eliminated expression) comprising at least mutation and/or inhibition of caxl , e.g., caxl, caxl/2, caxl/3, caxl/4 mutant lines, etc. ) exhibit decreased necrosis and/or leaf wilting phenotypes relative to controls.
[0136] RNAseq Reveals CAX1 and CAX1/3 Changes in Transcripts During Anoxia and Recovery. To identify molecular processes contributing to the observed difference between the sensitive lines (Col-0, cax3) and tolerant lines (caxl, caxl/3), the leaves of 21 old plants were subject to RNAseq analysis before, during and after anoxic conditions (36). Leaves were harvested from plants (samples: WT (wild-type), caxl, cax3, or caxl, cax3 double knock out mutant (caxDKO)) at the start of the treatment (0-h control) (FIG. 2A, FIG 8A), after being in the anoxia chamber for 4 hr (anoxia) (FIG. 2B, FIG. 8B) and 1 hr post 7- hour anoxia treatment (recovery) (FIGS. 2C; FIG. 8C).
[0137] Treatments and mutants clearly clustered separately in a multidimensional scaling plot, suggesting the RNAseq was likely revealing differences between responses to the different treatments in the various genotypes (FIG. 2A). A large number of genes responded significantly to the treatments, and their responses in the Col-0 and cax3 lines were similar to previous studies (FIGS. 9A-9D) (23). Thus, enriched gene ontology (GO) categories for the genes up and down regulated reveals widespread enrichment in ‘hypoxia-’, ‘anoxia’-and oxidative stress-related ontology categories demonstrating that the anoxia conditions produced by the GasPak are likely similar to low O2 conditions used in previous studies (FIGS. 9A-9B) (23, 37). Similarly, the enriched gene ontology categories for the recovery period demonstrate that the up and down regulated genes represent similar gene expression patterns to previous studies using different experimental conditions (FIG. 9C-9D) (37). RNAseq data for several of the genes was compared using RT-PCR (FIG. 10; Table 2), indicating that inferences drawn from the RNAseq were likely robust. Table 2. Primer Sequences for Genes selected for qRT-PCR Validation of RNAseq
Transcriptomic Analysis.
Figure imgf000061_0001
[0138] Given that caxl/3 mutant line was more tolerant than the caxl mutant line, an additional filter of caxl/3 > caxl (and caxl > caxl/3 for repressed genes) was applied if the gene lists were large (n<10). It was posited that enhanced expression or repression of specific genes prior to the stress could prime caxl and caxl/3 mutant lines for tolerance (FIG. 2B). It was thus assessed if any of the genes highly expressed in both caxl and caxl/3 mutant lines in control conditions were genes highly expressed in Col-0 and cax3 lines during anoxic or recovery conditions. This was proven to be true. For example, a monodehydroascorbate reductase 3 (AT3G09940.1), GRX480 (Atlg28480) and senescence regulator (AT5G45630.1), which are all involved in redox regulation, are highly expressed in caxl and caxl/3 lines prior to anoxia stress conditions but are also seen to be induced in WT and CAX3 upon anoxic challenge. Further, transgenic expression of the monodehydroascorbate reductase is known to enhance tolerance to temperature and oxidative stresses (38), hinting at a possible mechanism behind the anoxic tolerance in caxl and caxl/3. These transcriptional patterns also suggest a role for hormonal responses linked to defense. For example, the GRX480 transcription is salicylic acid (SA)-inducible and appears to have a role in SA/jasmonic acid (J A) cross-talk (39). Further evidence that JA metabolism relevant to anoxic tolerance may also be constitutively altered in the caxl and caxl/3 backgrounds is the repression of expression of ALLENE OXIDE CYCLASE 2 (AOC2, AT3G25770). AOC2 catalyzes a crucial step in JA biosynthesis (40). Similarly, JA and ethylene signaling coordinate biosynthesis of the hydroxycinnamic acid amide defense compounds through modulation of the expression of Anthocyanin 5-aromatic acyltransferase 1 (AT5G61160) (41) and this transcript is downregulated in non-anoxic conditions in the tolerant lines. A series of cold-regulated genes (At2g42530, At2g42540, At4g30650) and chloroplast and stomatai proteins (Atlg53801.1, Atlg53890.3, Atlg29170.1) were also down regulated in col-0 after anoxia while being constitutively repressed in caxl and caxl/3. Some of these cold regulated genes are downregulated by calcium sensors which are highly expressed in the caxl and caxl/3 mutants (FIGS. 11A-11B) (42).
[0139] Analysis of the 500 most differentially expressed transcripts in caxl and caxl/3 during anoxia identifies the following 313 transcripts as being upregulated in both caxl and caxl/3: AT1G01355; AT1G02610; AT1G02620; AT1G03090; AT1G03610; AT1G06570;
AT1G08630; AT1G08930; AT1G10070; AT1G10140; AT1G10550; AT1G11260;
AT1G12780; AT1G14860; AT1G15040; AT1G15330; AT1G15670; AT1G17290;
AT1G183OO; AT1G18773; AT1G19396; AT1G19530; AT1G19540; AT1G19610;
AT1G19620; AT1G24880; AT1G25560; AT1G27045; AT1G28330; AT1G30135;
AT1G30720; AT1G32910; AT1G33050; AT1G33055; AT1G34140; AT1G34760;
AT1G35140; AT1G36060; AT1G54100; AT1G54760; AT1G55810; AT1G56250;
AT1G58936; AT1G60750; AT1G62480; AT1G62510; AT1G63090; AT1G65970;
AT1G66860; AT1G67265; AT1G68935; AT1G71520; AT1G72060; AT1G72416;
AT1G75750; AT1G76650; AT1G77120; AT1G79700; AT1G79910; AT1G80160;
AT1G80570; AT1G80840; AT2G05400; AT2G07633; AT2G07638; AT2G07641;
AT2G07648; AT2G07658; AT2G07665; AT2G07669; AT2G07673; AT2G07674;
AT2G07678; AT2G07689; AT2G07696; AT2G07698; AT2G07714; AT2G07721;
AT2G07724; AT2G07725; AT2G07727; AT2G07734; AT2G07749; AT2G07771;
AT2G07773; AT2G07785; AT2G07798; AT2G07815; AT2G07825; AT2G07835;
AT2G14210; AT2G14900; AT2G15880; AT2G15960; AT2G 16060; AT2G16586;
AT2G17036; AT2G17850; AT2G19590; AT2G19800; AT2G20670; AT2G22880;
AT2G22980; AT2G25770; AT2G25780; AT2G26130; AT2G30600; AT2G30615;
AT2G31810; AT2G33160; AT2G33830; AT2G34390; AT2G34555; AT2G36110;
AT2G36950; AT2G37025; AT2G39400; AT2G39570; AT2G43520; AT2G44080; AT2G47520; AT2G47950; AT3G02550; AT3G03270; AT3G04160; AT3G06435;
AT3G10040; AT3G1331O; AT3G13450; AT3G15440; AT3G15450; AT3G15620;
AT3G16770; AT3G17225; AT3G18530; AT3G19680; AT3G20395; AT3G21352;
AT3G22640; AT3G23150; AT3G23550; AT3G27220; AT3G28740; AT3G29370;
AT3G29810; AT3G29970; AT3G30775; AT3G43190; AT3G43850; AT3G44300;
AT3G45300; AT3G46230; AT3G47340; AT3G47675; AT3G47720; AT3G47870;
AT3G48360; AT3G48530; AT3G50560; AT3G51840; AT3G51910; AT3G55240;
AT3G55970; AT3G59060; AT3G61060; AT3G61260; AT3G62150; AT4G01120;
AT4G01250; AT4G02170; AT4G02380; AT4G02430; AT4G03205; AT4G04423;
AT4G10250; AT4G10265; AT4G10270; AT4G15760; AT4G16160; AT4G16162;
AT4G16563; AT4G19230; AT4G19880; AT4G20953; AT4G22710; AT4G22780;
AT4G24040; AT4G24110; AT4G24230; AT4G25580; AT4G25707; AT4G25810;
AT4G26460; AT4G27450; AT4G28040; AT4G28811; AT4G30270; AT4G30370;
AT4G30380; AT4G32480; AT4G32630; AT4G32840; AT4G33070; AT4G33150;
AT4G33560; AT4G33970; AT4G34030; AT4G35770; AT4G36690; AT4G36730;
AT4G36850; AT4G37220; AT4G38470; AT4G39675; AT5G01740; AT5G01810;
AT5G02200; AT5G03380; AT5G03830; AT5G05530; AT5G06980; AT5G07440;
AT5G07560; AT5G07570; AT5G08150; AT5G10040; AT5G11090; AT5G12020;
AT5G12030; AT5G14180; AT5G14470; AT5G15120; AT5G15250; AT5G18170;
AT5G19120; AT5G19550; AT5G20240; AT5G20250; AT5G20830; AT5G21170;
AT5G22920; AT5G26200; AT5G27893; AT5G28610; AT5G28630; AT5G28770;
AT5G34830; AT5G35525; AT5G39160; AT5G39200; AT5G39580; AT5G39890;
AT5G40450; AT5G41080; AT5G42825; AT5G43570; AT5G45340; AT5G47590;
AT5G47910; AT5G49360; AT5G54080; AT5G56100; AT5G56870; AT5G57220;
AT5G57550; AT5G57660; AT5G62520; AT5G63160; AT5G65207; AT5G66110;
ATMG00010; ATMG00060; ATMG00070; ATMG00080; ATMG00090; ATMG00110;
ATMG00140; ATMG00160; ATMG00260; ATMG00270; ATMG00285; ATMG00310;
ATMG00400; ATMG00410; ATMG00510; ATMG00513; ATMG00516; ATMG00560;
ATMG00570; ATMG00630; ATMG00640; ATMG00650; ATMG00680; ATMG00690;
ATMG00730; ATMG00900; ATMG00940; ATMG00960; ATMG00970; ATMG01000;
ATMG01050; ATMG01120; ATMG01130; ATMG01170; ATMG01190; ATMG01200;
ATMG01210; ATMG01220; ATMG01230; ? FMG01260; ATMG01280; ATMG01320; and
ATMG01360. [0140] GO analysis of these 313 genes identified similar categories as the sensitive lines: response to hypoxia, small molecule catabolism, anaerobic respiration, response to hydrogen peroxide and sucrose. Based on the improved tolerance of caxl/3 lines, we focused the list by imposing the condition of expression in caxl/3 being higher than in caxl. This generated a list of 81 transcripts; GO analysis for this list showed significant enrichment in genes related to changes in ethylene signaling and response to carbohydrates, classic plant- anoxic response components. If the 313 transcripts are analyzed to remove the transcripts also induced during anoxia in both Col-0 and cax3 there are 189 transcripts (FIG. 2A-2F); in this analysis a GO category related to cell wall metabolism emerges. If these 189 transcripts are filtered for caxl/3 expression being higher than caxl, 41 transcripts emerge; Enhanced expression of ATP-dependent 6-phosphofructokinase 6 (PFK; AT4G32840) was observed among these 41 transcripts. This enzyme is a key regulator of glycolytic flux and performs the second phosphorylation step in the glycolytic pathway (43). Indeed, a closer look at the glycolytic pathway shows many of the enzymes in the glycolytic pathway are upregulated in caxl and caxl/3: glyceraldehyde-3-phosphate dehydrogenase A subunit (AT1G12900), fructose-bisphosphate aldolase 1 (AT2G21330), fructose-bisphosphate aldolase 2 (AT4G38970), and phosphoglycerate kinase (AT3G12780). Alcohol dehydrogenase 1 (AT1G77120) is upregulated in Arabidopsis thaliana in response to low oxygen(44); however, it is less up regulated during anoxia in both caxl and caxl/3 (FIG. 10). This diminished induction may reflect the generally lower degree of O2 stress response induced in these mutants or possibly an element of metabolic reprogramming that underlies the enhanced tolerance of these lines. In caxl and caxl/3 arginase transcripts are upregulated, a response also seen in the mitochondria of rice experiencing anoxic stress (45). This increase flux through arginase could then possibly be producing more urea, again a characteristic of anoxia-stressed plant mitochondria (45).
[0141] Among the 41 transcripts a large number of RNAs were associated with chloroplast, mitochondrial and ER associated proteins (for example: AT1G28760.1; AT1G66510.1; AT2G01390.1; AT3G13310.1; AT5G15250.2; AT4G27657.1; AT5G15250.2) suggesting there could be some organelle cross-talk operating in this response. In fact, 9 of the 41 transcripts are associated with mitochondrion function; it appears that disruption of the tonoplast H+/Ca transporter CAX1 impacts the anoxia dependent synthesis or stability of transcripts of mitochondrial origin. Previous work has shown that these mitochondrial RNAs are more stable than anticipated, and that heat stress enhances their detection (46). Anoxia stress impacts the ER which results in the unfolded protein response (UPR) (1). The caxl and caxl/3 lines demonstrated upregulation of GAAP3 (AT4G02690) that functions to mitigate the UPR and programed cell death (47, 48). Furthermore, caxl and caxl/3 showed upregulation of AtPARK13 (AT5G27660.1) which targets misfolded proteins (49). Potentially, the caxl and caxl/3 tolerant lines perceive less ER stress during anoxia recovery than col-0 and cax3.
[0142] The list of 41 anoxia responsive transcripts in caxl and caxl/3 also includes several putative regulatory molecules that are interesting with respect to CAX function. For example, CBL-interacting protein kinase (CBL15; AT5G01810); this Ca sensor has a role in carbohydrate metabolism, ABA and ethylene signaling (50). Furthermore, this protein has domains that may interact with CAX1 (51). CAX activity can also be modulated by pH changes (52) and a 14-3-3 protein (GRF11; AT1G34760) is upregulated in the caxl and caxl/3 anoxia- responsive transcriptome. 14-3-3 proteins can bind to H+-ATPases to modulate cellular pH and ATP levels as well as modulate hormone levels, the heat stress response and mitochondrial function (53, 54), cellular elements closely linked to anoxic responses.
[0143] Evaluation of the 500 most differentially expressed transcripts in caxl and caxl/3 during recovery identifies the following 156 genes also found in Col-0 and cax3, but identifies the following more than 170 transcripts as specifically upregulated only in both caxl and cax 1/3 (FIGS. 2A-2F; FIGS. 8A-8C): AT1G01720; AT1G03070; AT1G03090; AT1G03610; AT1G05575; AT1G06030; AT1G07330; AT1G07350; AT1G07400;
AT1G07500; AT1G08630; AT1G09950; AT1G10140; AT1G12610; AT1G14200;
AT1G15040; AT1G15330; AT1G15840; AT1G16030; AT1G17870; AT1G183OO;
AT1G1833O; AT1G19530; AT1G19540; AT1G19620; AT1G21340; AT1G21940;
AT1G22110; AT1G26800; AT1G27730; AT1G28760; AT1G30070; AT1G30135;
AT1G31370; AT1G32910; AT1G32928; AT1G33055; AT1G33730; AT1G33760;
AT1G34575; AT1G35140; AT1G44414; AT1G50745; AT1G52560; AT1G53540;
AT1G54050; AT1G55530; AT1G55810; AT1G56170; AT1G56250; AT1G59860;
AT1G59865; AT1G60190; AT1G61340; AT1G66060; AT1G66080; AT1G66400;
AT1G66500; AT1G66510; AT1G67265; AT1G71000; AT1G71520; AT1G72060;
AT1G72416; AT1G72660; AT1G72760; AT1G73480; AT1G74310; AT1G74450;
AT1G74930; AT1G75490; AT1G75750; AT1G76600; AT1G76640; AT1G76650;
AT1G77120; AT1G80840; AT2G01390; AT2G07687; AT2G07696; AT2G07698;
AT2G07707; AT2G07724; AT2G07725; AT2G07727; AT2G07734; AT2G07771;
AT2G07773; AT2G07785; AT2G07825; AT2G07835; AT2G14900; AT2G15960; AT2G 16060; AT2G17850; AT2G20560; AT2G20670; AT2G22880; AT2G23110;
AT2G23190; AT2G24100; AT2G25140; AT2G26150; AT2G27580; AT2G29500;
AT2G30615; AT2G32120; AT2G34390; AT2G34600; AT2G36220; AT2G38240;
AT2G38340; AT2G40340; AT2G40955; AT2G44070; AT2G44080; AT2G44130;
AT2G44840; AT2G46240; AT2G46790; AT2G46830; AT2G47180; AT2G47520;
AT3G02550; AT3G06435; AT3G07090; AT3G07150; AT3G07350; AT3G08970;
AT3G09350; AT3G09640; AT3G10020; AT3G10040; AT3G11020; AT3G12190;
AT3G12320; AT3G12580; AT3G1331O; AT3G14200; AT3G15440; AT3G15450;
AT3G15500; AT3G16050; AT3G17400; AT3G19240; AT3G20340; AT3G20395;
AT3G22090; AT3G22100; AT3G23150; AT3G24500; AT3G25250; AT3G27220;
AT3G28210; AT3G28740; AT3G29370; AT3G29810; AT3G29970; AT3G30775;
AT3G43850; AT3G44190; AT3G46070; AT3G46230; AT3G47340; AT3G47720;
AT3G48240; AT3G49160; AT3G50310; AT3G50560; AT3G51910; AT3G53830;
AT3G55580; AT3G55840; AT3G62260; AT3G63350; AT4G01250; AT4G01435;
AT4G02170; AT4G02425; AT4G02550; AT4G02690; AT4G09150; AT4G10250;
AT4G10265; AT4G10270; AT4G 11660; AT4G12400; AT4G12410; AT4G13395;
AT4G15280; AT4G 15420; AT4G15760; AT4G16555; AT4G17250; AT4G18450;
AT4G19570; AT4G21320; AT4G23493; AT4G24110; AT4G24410; AT4G24570;
AT4G25200; AT4G25380; AT4G25470; AT4G25490; AT4G25580; AT4G25810;
AT4G26200; AT4G26460; AT4G27410; AT4G27450; AT4G27652; AT4G27654;
AT4G27657; AT4G27670; AT4G28811; AT4G29770; AT4G29780; AT4G30270;
AT4G30370; AT4G32208; AT4G32480; AT4G33070; AT4G34131; AT4G34410;
AT4G35770; AT4G36850; AT4G37710; AT4G38030; AT5G01740; AT5G02170;
AT5G02810; AT5G03210; AT5G03720; AT5G04340; AT5G04400; AT5G05220;
AT5G05410; AT5G06980; AT5G07330; AT5G08150; AT5G09590; AT5G09930;
AT5G10040; AT5G10336; AT5G10695; AT5G12020; AT5G12030; AT5G12110;
AT5G13220; AT5G14470; AT5G15120; AT5G15250; AT5G15450; AT5G17350;
AT5G 18065; AT5G18340; AT5G20250; AT5G22680; AT5G22920; AT5G25450;
AT5G27660; AT5G35320; AT5G37340; AT5G37670; AT5G39580; AT5G39890;
AT5G43620; AT5G43650; AT5G45340; AT5G45640; AT5G47220; AT5G47590;
AT5G47830; AT5G48570; AT5G49920; AT5G51190; AT5G51440; AT5G51990;
AT5G52050; AT5G52630; AT5G52640; AT5G53680; AT5G54165; AT5G57260;
AT5G57550; AT5G57560; AT5G57660; AT5G58070; AT5G59720; AT5G59820; AT5G62020; AT5G62520; AT5G63130; AT5G63300; AT5G64170; AT5G64210; AT5G64510; AT5G66650; AT5G67080; ATMG00060; ATMG00080; ATMG00090; ATMG00160; ATMG00270; ATMG00285; ATMG00400; ATMG00410; ATMG00480; ATMG00510; ATMG00513; ATMG00516; ATMG00560; ATMG00570; ATMG00640; ATMG00650; ATMG00690; ATMG00730; ATMG00900; ATMG00940; ATMG00960; ATMG00990; ATMG01000; ATMG01050; ATMG01120; ATMG01190; ATMG01200; ATMG01320; and ATMG01360.
[0144] More than 200 transcripts were uniquely repressed in these backgrounds. Imposing the condition that the expression in caxl/3 being greater than caxl identified 70 transcripts as uniquely upregulated and 9 down regulated in caxl and caxl/3. The enriched GO category for these 70 genes were predominately cellular response to hypoxia and heat. Many of these genes have been previously identified in hypoxia/anoxia stress they were just expressed at 14 enhanced levels in caxl/3 and caxl. This list differed from that found for the caxl and caxl/3 anoxia phase in many genes especially those related to response to organonitrogen compounds (CCA1 (AT2G46830), AT-HSFB2B (AT4G11660), AT4G18450, AT4G30370, ZAT6 (AT5G04340), HSFB2A (AT5G62020) ) and monooxygenase activity (CYP81D7 (AT2G23190), MOI (AT4G15760.1), CYP707A1 (AT4G19230), CYP706A2 (AT4G22710), AT5G35320, CYP81F2 (AT5G57220), CYP71B10 (AT5G57260.1)). The hypoxia/heat transcripts includes a well-characterized putative glucose-regulated protein that binds to the promoters of glucose-regulated heat shock responsive genes (AT1G66080). The heat regulatory transcripts included heat shock transcription factors (At5g6202; At4gl l660), HSP20-like chaperones (AT5G37670; AT4G16555), and HSP70 (AT4G32208). This enhancement of heat regulated genes in all probability causes the increased expression of PYRIDOXINE BIOSYNTHESIS 1.2 (PDX1.2, AT3G16050), which is under control of the HSFA1 family of heat shock transcriptional regulators (55). PDX1.2 is a pseudoenzyme required for supplying adequate levels of vitamin B under deleterious conditions such as heat stress (55).
[0145] This list of the 500 most differentially expressed transcripts in caxl and caxl/3 during recovery also includes four other transcription factors: bHLH (At3g22100), AP2- EREBP (At4g 18450), NAC (At4g27410), and C2H2 (At5g04340) that provide indications about some of the hormonal system that these mutants may use to respond to the reoxygenation stress after anoxic treatment. Thus, the transcription factor AP2-EREBP is an ethylene response factor and NAC is ABA responsive, suggesting that hormone signaling is altered in both caxl and caxl/3 (FIG. 13). Consistent with this model, RNAs associated with a negative regulator of ABA signaling 15 (AT1G09950.1); a SAUR-like auxin-responsive protein (AT4G12410.1), and a JA-mediated signaling protein (JAS1/JAZ10; AT5G13220) are also highly expressed in the mutants. JAZ10 is part of the JA-related defense response linked to slow wave potential signaling (56) and it is interesting to speculate that JAZ10 may be operating in responses to both electrical and Ca signals during anoxia recovery. RNAs associated with altered redox regulation (AT3G44190.1, AT3G50560.1) were also highly expressed during recovery in the caxl and caxl/3 mutants. A molecular mechanism for altered redox regulation in these mutants could be caused in part by induction of C2H2, a zinc finger transcription factor that targets redox-related genes.
[0146] Metabolic changes in caxl and caxl/3 recovery period were also apparent in the transcriptomic data with several RNAs related to carbon regulation showing differential expression (AT1G06030; AT1G15330.1; AT1G66080.1). One of these transcripts encodes for a fructokinase (FRK) which in rice has been previously shown to be responsive to low oxygen levels (57). In addition, the mitochondrial alternative oxidase (AOX) pathway appears to be upregulated in caxl and caxl/3 during recovery (NAD7, ATMG00510; NAD4L, ATMG00650; NAD3, ATMG00990). Multiple studies have indicated that AOX is upregulated in hypoxia with this induction preventing overproduction of superoxide and other ROS (58, 59). AOX is thought to facilitate nitrite-dependent NO production; this reaction protects the mitochondria during stress conditions.
[0147] Within the mitochondria there may also be changes in proline metabolism. A proline dehydrogenase (ERD5, AT3G30775) that has been previously shown to be hypoxia responsive (60). This enzyme may be important in respiration, with proline acting as an alternative energy substrate (61). A transcript from an unknown protein response to proline (AT2g27580) was also upregulated, as was an amino acid transporter (AT5G02170).
[0148] Cell wall and membrane modifications may also be altered in caxl and caxl/3 during recovery. A xyloglucan endotransglucosylase/hydrolase (At4g25810) is highly expressed in these lines as are transcripts involved in cell wall biogenesis (AT3G29810) and several RNAs that may impact cell wall and lipid biosynthesis are repressed during recovery (AT4G12490, AT4G12520, AT3G07970, AT5G12580).
[0149] Protein Changes in CAX1 and CAX1/3 post-Anoxia. To provide additional insight into how the CAX proteins are involved in anoxia, global changes in protein abundance between WT, caxl, and caxl/cax3, pre and post anoxia were analyzed. In total, amongst all genotypes, 473 unique proteins were identified from up to a total of 17339 spectra, total number of 473 differentially abundant proteins (DAP) were identified across the 3 genotypes based on their normalized spectral abundance factor (NSAF) (62), with numbers of increased and decreased proteins in the genotypes listed in the Table in FIG. 3A.
[0150] DAP in caxl and caxl/3 were subjected to enrichment analysis based on functional annotation of GO terms. The top 40 enriched GO term categories are presented in FIG. 3B.1 and FIG. 3B.2. In support of the results from RNA-seq analysis and the data which showed lower levels of H2O2 in caxl and caxl/3 post anoxia (FIG. 2D), proteins shown to be increased in abundance in these genotypes were significantly enriched in GO terms related to oxidative stress including response to oxidative stress, cell redox homeostasis and oxidation reduction process (FIG. 3B.1 and FIG. 3B.2). Furthermore, protein changes were seen in various organelles (FIGS. 15A-15C). Activated H2O2 removal may be related to the markedly increased abundance of hydrogen peroxide catabolic process proteins including APX1 (AT1G07890), GLO1 (AT3G14420), PRXIIF (AT3G06050), TL29 (AT4G09010) and CAT1- 3 (AT1G20630, AT4G35090, AT1G20620).
[0151] In agreement with tran scrip tomic data, proteomic analysis also indicated enrichment in proteins involved in glycolysis and captured significant increases in a number of proteins involved in the glycolytic pathway in caxl and caxl/3 post anoxia (GO enrichment, FIG. 3B.1; FIG. 3B.2; FIG. 16), including fructose 1,6 bisphosphatase, and phosphoglycerate kinase. With the latter showing similar responses when data from both approaches was overlaid onto KEGG pathways (FIG. 3C).
[0152] Accessing the Roles of Ca, ROS, Heat and Phytohormones on CAX1 and CAX1/3 mediated Anoxia tolerance. Previous studies on anoxia stress responses (17) and the caxl and caxl/3 specific DEGs here suggested to us that caxl -mediated anoxia tolerance likely involves signaling processes related to Ca, ROS and hormones and to pathways already linked to other abiotic and biotic stresses such as heat stress (FIGS. 10A-10B). To further investigate the roles of these processes in caxl-mediated anoxia tolerance, we applied various treatments to the plants and assessed changes in anoxia tolerance using the anoxia- reoxygenation recovery assay. Critically, in all cases, applying the treatments described below to Col-0, cax3, caxl, and caxl/3 caused none, or minimal phenotypic alterations to lines grown in normoxia conditions (FIGS. 4A-4B). Thus, to analyze the role of Ca, EGTA (10 mM), and LaC13 (5 mM), were sprayed onto the plants 8 h prior to the anoxia stress and compared to lines sprayed with water. Both EGTA, which chelates Ca, and LaC13, a nonselective Ca channel blocker, abolished the anoxia tolerance of both caxl and caxl/3. Meanwhile, the effect of nonselective Ca channel blockers and CaM antagonist W7 were not as severe: caxl and caxl/3 were slightly less robust after the stress but largely retained their tolerance.
[0153] Previous studies have shown that caxl has altered sensitivity to low temperatures; furthermore, acclimation of plants to higher temperatures has been shown to enhance anoxia tolerance (14, 63). The RNA-seq data also revealed altered expression of temperature regulated transcripts (FIGS. 2A-2E; FIGS. 12A-12D). We thus tested the effects of both cold and heat acclimation on anoxia tolerance; lines grown at 4°C for two days prior to the anoxia stress displayed no changes in their anoxia phenotypes; however, when the lines were adapted to 28°C for two days and then stressed, Col-0 and cax3 became as tolerant to the anoxia conditions as caxl and caxl/3 (FIGS. 4A-4B).
[0154] To assess the role of ROS and oxidative stress in anoxia tolerance, Diphenyleneiodonium chloride (DPI) (8 pM), glutathione (GSH) (3 mM), and H2O2 (1 mM or lOOmM) were sprayed on the plants 8 h before anoxia. DPI, an inhibitor of the superoxide producing enzyme NADPH oxidase, slightly enhanced the tolerance of Col-0 and cax3 and marginally reduced the tolerance of caxl and caxl/3; however, caxl and caxl/3 were still more tolerant. H2O2 at ImM did not have any effect on anoxia tolerance in all lines, while lOOmM treatment 2 days prior to the anoxia partially rescued the anoxia sensitivity of Col-0 and cax3, and partially reduced the robustness of caxl and caxl/3.
[0155] ABA is a regulator of abiotic stress responses and exogenous application of 50 pM ABA 2 d prior to the anoxia treatment conferred anoxia tolerance to the Col-0 and cax3 lines. This tolerance phenocopied the tolerance of caxl and caxl/3. This phenotype was dependent on protein synthesis as pretreatment with cycloheximide (100 pM), a protein synthesis inhibitor, along with ABA rendered all the lines sensitive. Various other treatments caused incremental changes in the anoxia tolerance phenotype: elicitors of plant defense responses and ethylene precursors and ethylene signaling inhibitors had modest effects; auxin, cytokinin and JA applications caused almost no change in anoxia stress phenotypes.
[0156] To address if caxl and caxl/3 have altered hormone levels which could have primed the plants for the anoxia stress direct measurement of hormone levels using LC-MS/MS was employed (64). The profiling from unstressed leaves suggested that under these growth conditions there was a reduction in ABA levels in caxl/3 compared to Col-0 (FIG. 4C).
[0157] As an alternative approach to measure the impact of Ca2+ signaling, hormone levels and ROS response on anoxia tolerance in our assay conditions, various genetic mutations in hormone, Ca and ROS signaling were tested for anoxia tolerance (Table 1). Lines with altered levels and sensitivity to ABA displayed no changes in anoxia tolerance (Table 1). Similarly, mutants perturbed in SA, ethylene, GA and JA levels were comparable to the water controls in their anoxia sensitivity (Table 1). Furthermore, mutations involved in ROS and defense signaling also had phenotypes that closely resembled Col-0 (Table 1).
[0158] Changes in caxl Cytosolic Ca Levels during reoxygenation. In order to investigate the relationship between CAX1 function and changes in cytosolic Ca and anoxia tolerance, stable Col-0 and caxl lines were generated expressing the Ca biosensor GCamP3, a GFP based fluorescent protein with a calmodulin domain (65). Without anoxia treatment but keeping all other growth conditions identical, these Col-0 and caxl lines showed very modest changes in cytosolic Ca levels (FIG. 17). After 4 h of anoxia treatment, these lines were reoxygenated and immediately imaged for their cytosolic Ca signature. In Col-0 there was a prominent initial Ca signal which dissipated within 90 sec (FIG. 5A); a second less prominent Ca signal peaked around 200 sec post reoxygenation. In caxl, the initial cytosolic Ca signal after reoxygenation was greatly diminished, while a larger Ca signal peaked at around 200 sec (FIG. 5A). To assess the potential role of apoplastic Ca in the generation of the Ca signature during anoxia response, the lines were treated with Ca chelator EGTA prior to the anoxia stress. In Col-0, the initial cytosolic Ca signal was similar to control conditions; however, the second Ca signal at around 200 sec was diminished (FIG. 5B). In contrast, the caxl Ca signals during reoxygenation were no longer apparent. Given that EGTA treatment confers anoxia sensitivity to the caxl lines (FIGS. 4A-4B), we posit that these EGTA-sensitive caxl-mediated Ca signals may be components of the tolerance phenotype.
[0159] The RNA-seq and phenotyping experiments suggested that hormones could impact Ca signaling during anoxia recovery (FIG. 13). ABA pretreatment of the GCamP3- expressing lines was used to assess if this hormone influenced these Ca signals post-anoxia. In Col-0, the initial Ca signal was similar to those in water-control treated plants and dissipated quickly (FIG. 5C), however, the second Ca signal was increased in magnitude and prolonged compared to the water control. In caxl, ABA treatment led to diminished Ca signals (FIG. 5C). Similarly, experiments using SA pretreatment in Col-0 demonstrated the initial strong Ca signal post-anoxia was maintained and dissipated in a manner similar to water-treated controls. The second signal was slightly diminished (FIG. 5D). In caxl, the second Ca signal was observed earlier than water treated plants (peak at 140 sec compared to 195 sec in WT) and the peak intensity of the signal is increased to a level higher than the initial signal in WT. The spatial distribution of the signal was also changed: in water control caxl-1 lines, the second Ca signal appeared to propagate from the base of the meristem of the rosette along the petiole and into the leaf blade of the leaves; while with SA, the Ca signal first appeared visible at the tip and edge of the leaf blade and migrated to the petiole (FIG. 5D). This result suggests caxl has a differential Ca response to SA compared to water controls.
[0160] Discussion. A plant’ s oxygen supply can vary from normal (normoxia) to total depletion (anoxia). Tolerance to anoxia is relevant to wetland species, rice cultivation and transient waterlogging of crops. Decoding and transmitting calcium (Ca) signals may be an important component to anoxia tolerance; however, the contribution of intracellular Ca transporters in this process are poorly understood. Four functional cation/proton exchangers (CAX1-4) in Arabidopsis thaliana help regulate Ca homeostasis around the vacuole. In specific embodiments of the present disclosure, any single or double CAX mutation that includes CAX1 are more tolerant to anoxic conditions. Using phenotypic measurements, RNAseq and proteomic approaches, CAX1 mediated anoxia changes have been identified that phenocopy changes present in anoxia tolerant crops: altered metabolic processes, diminished ROS production post anoxia, and altered hormone signaling. Comparing wild-type and CAX1 expressing genetically encoded Ca indicators (GECIs) demonstrated altered Ca signals in the CAX1 during anoxia recovery. This suggests that anoxia induced Ca signals around the plant vacuole are involved in organelle cross-talk, metabolic fluxes and ROS signaling. In specific embodiments, the CAX1 anoxia response pathway will be engineered to circumvent the adverse effects of anoxic conditions that impair production agriculture.
[0161] Stress recovery is vital for plant survival. Anoxia reduces plant energy production (4); this lack of oxygen can be viewed as leading to sequential stresses, where both the lack of oxygen and the subsequent reoxygenation periods pose distinct stressors; tolerance being determined by the ability to acclimate to both phases (23). Plants may use spatial and temporal dynamics in ROS, ethylene, and Ca to convey information about anoxic conditions (13, 66). Here caxl Arabidopsis thaliana lines were identified as producing a robust anoxia/reoxygenation tolerance phenotype; additionally, caxl/3 was demonstrated as more tolerant than caxl. Using these mutants, molecular and physiological processes and regulatory components influencing anoxia tolerance were identified.
[0162] Plant roots and shoots have different mechanisms for hypoxia/anoxia stress tolerance (67). Our work has concentrated on aerial portions of the stress response. Previous work in Arabidopsis thaliana has used tissue- specific root ion profiling to show roles of CAX and Ca-ATPase transporters in hypoxia (13). This root-focused approach suggests that caxl does not have a hypoxia phenotype; furthermore, these studies demonstrate a gene termed CAX11 (CCX5) is involved in hypoxia responses; however, this transporter is not a bonafide CAX (68) and is more closely related to mammalian K+-dependent Na+/Ca2+ antiporters (69).
[0163] Here CAX1, a gene that is neither highly expressed during oxygen stress conditions nor thought to be modified in plants adapted to oxygen limitations, was analyzed. Studies illustrated that caxl mutant lines are tolerant to anoxia conditions; this phenotype was specific to CAX1, but enhanced tolerance was found in caxl/3 mutant lines. It was established that post anoxia, caxl lines showed reduced ROS production; meanwhile, the caxl and caxl/3 lines demonstrated apparent changes in Ca signaling, metabolism and hormone signaling during and after the stress. Using various approaches it was explored how CAX1 mediated anoxia tolerance could be manipulated via changes in temperature, Ca levels, reactive oxygen species and phytohormones. It was also investigated how CAX1 changed the amplitude and duration of Ca signals during anoxia. This work highlights a central role of tonoplast localized H+/Ca transport in anoxia perception and recovery while further defining the molecular choreography of anoxia signaling.
[0164] Enzymes that regulate both sucrose breakdown and anaerobic fermentation are controlled in a temporal manner in response to oxygen deprivation (70). The management of carbohydrate consumption and avoidance of oxidative stress have been proposed to be key determinants of anoxia tolerance (71). Rice cultivars that vary in submergence tolerance display distinct transcript profiles for glycolytic and fermentation enzymes (72). Our data point toward differences in both the mitochondria and plastid as well as glycolysis in caxl and caxl/3 impacting anoxia tolerance (FIG. 3C; FIG. 15). Increases in glycolysis post-anoxia may reflect an enhanced ability to return to cellular homeostasis by replenishing ATP stores in caxl which would have been significantly diminished during the anoxia treatment. During anoxia it is likely that glycolytic flux would have been diverted into the oxidative arm of the pentose phosphate cycle to enhance NADPH production which would fuel the cellular antioxidant systems (63). This is supported by the transcriptomic data at 4h anoxia in caxl and caxl/3 showing increased transcription of glucose 6-phosphate dehydrogenase, 6- phosphogluconolactonase and 6-phosphogluconate dehydrogenase.
[0165] During O2 depletion, studies suggests ethylene accumulation down-regulates ABA levels (73). Conversely, in both crops and Arabidopsis thaliana, ABA limits ethylene production (74) suggesting there may be a complex interplay of hormonal regulation behind the low oxygen responses mediated by CAX-dependent pathways. Here how exogenous application of various hormones and chemicals altered the anoxic tolerance among cax mutants was analyzed. Two-day treatment of Arabidopsis thaliana lines with ABA improved anoxia tolerance in every line analyzed (FIGS. 4A-4B) ABA and Ca signals convergently modulate many plant stress responses (75) and it is speculated that the caxl mutant may bypass ABA and directly modulate downstream signaling. Meanwhile, various ethylene and ethylene inhibitor treatments did not dramatically alter anoxia tolerance. We posited that caxl and cax 1/3 may have constitutively heightened ABA levels that alter stomatai conductance to improve anoxia tolerance. However, our hormone measurements did not reveal dramatically altered levels of ABA in caxl or cax 1/3 lines in non-stressed conditions (FIG. 4C). Furthermore, previous work has suggested that caxl and cax 1/3 stomatai conductance is not differentially sensitive to ABA (76). RNAseq revealed that genes involved in ABA biosynthesis (NCED9, AT1G78390) as well as enzymes involved in the conversion of active ABA to its storage form (UGT71B6) were upregulated in caxl and caxl/3. Multiple ABA responsive genes were also upregulated, in addition to MYB2 which is a transcription factor involved in ABA regulation (FIG. 13). Proteomics data due to its limited coverage depth, revealed less differentially regulated proteins. However, a stress-induced protein kinase (Kinl At5gl5960) related to ABA signaling was induced in caxl, and caxl/3 prior to the anoxia stress. Taken together the data infer altered ABA responses in caxl and caxl/3 that are suggestive of elevated ABA production; however, our direct ABA measurements do not support this as the mechanism behind differential ABA response in caxl and caxl/3. One caveat is that the elevation of ABA synthesis could trigger rapid conversion into its inactive storage forms, which may lead to lower detection via LC-MS (77).
[0166] The abrupt reoxygenation post anoxia is thought to result in a ROS burst in recovering tissues due to the reactivation of photosynthetic and mitochondrial electron transport promoting excessive electron and proton leakage (78, 79). Reoxygenation led to increased ROS production in Col-0 and cax3, whereas caxl and caxl/3 showed dampened ROS production (FIGS. 1D-1E). Exogenous application of H2O2 at lOOmM enhanced the anoxia tolerance in Col-0 and cax3. Oxidative stress induces endogenous ABA production, which might explain how exogenous H2O2 was also able to improve anoxia tolerance.
[0167] Calcium is involved in multiple signal transduction pathways, and the involvement of Ca in low O2 responses has been observed in many plants (80). Using a transgenic luminescence Ca indicator, previous work has shown the cotyledons and leaves of Arabidopsis thaliana seedlings developed a biphasic Ca response to anoxia (81). caxl/3 has been shown to exhibit a reduced capacity for mesophyll Ca accumulation which results in reduced cell wall extensibility, stomatai aperture, transpiration, and CO2 assimilation (17); it is tempting to speculate that these changes impact anoxia tolerance; however, none of these phenotypes are caxl specific. Here exogenous application of Ca chelators, and Ca channel blockers were used to impair anoxia tolerance in all lines, demonstrating the general importance of Ca signals in anoxia stress responses. Using Ca imaging, upon returning to normoxia, caxl lines were observed to display altered spatial and temporal Ca signaling (FIG. 5A-5D). A working model is that differences in Ca signaling during the anoxia and recovery stages directly impact the changes in transcripts and protein abundance seen in the various CAX mutants (FIGS. 2C-2E; FIGS. 3A-3C, FIG. 6). RNA-seq data revealed a global change in the transcript abundances of Ca transporters and Ca sensors/effectors in both caxl and caxl/3 (FIG. 11). Many of these changes were apparent prior to the anoxia stress and have been reported previously (17). A working model is that impaired H+/Ca2+ transport resulting from loss of CAX1, that can be exacerbated by a simultaneous reduction in CAX3 activity, elevates cytosolic Ca levels causing enhanced expression of a multitude of Ca regulated proteins (FIG. 6; FIG. 11).
[0168] Oxygen deprivation affects many organelles especially the mitochondria and ER (82); organelles emit stress signals whose processing and integration then initiate the adaptive responses (83): this work highlights the additional importance of vacuolar stress signals in anoxia responses and the ability of Ca changes around the tonoplast to impart changes in various organelles (FIG. 15; FIG. 16). This work suggests that caxl and caxl/3 have altered UPR (AT4G02690; AT5G27660.1) at the ER both during anoxia and recovery. Additionally, there are changes in AOX and numerous mitochondrial transcripts.
[0169] Loss of function mutations in Arabidopsis thaliana H+/Ca transporter CAX1 display a robust conditional phenotype, they gain tolerance to anoxia conditions (FIGS. 1A- 1E; FIG. 6). Disruption of CAX transport during this stress impacts a myriad of cellular processes: cell wall biosynthesis, metabolism, ROS, hormone and Ca signaling (FIG. 6). Furthermore, the mutants appear to be primed for the stress by having heightened expression of ROS related transcripts and diminished expression of cold tolerance genes during normal growth conditions. The caxl and caxl/3 lines also appear to be ‘warmed up” as they display heightened expression of heat responsive transcripts during the anoxia recovery phase (FIGS. 12C-12D). Heat shock response involves the induction of a wide range of molecular chaperones to aid in protecting and refolding damaged proteins and so this constitutive production of e.g., heat shock proteins may equip caxl and caxl/3 plants with a ready-made machinery to cope with some of the cellular damage that anoxia and reoxygenation imposes. Previous work has also shown that caxl has altered cold- acclimation responses (14) and temperature perception may be an important factor in the observed anoxia tolerance phenotype.
[0170] Conclusions. This disclosure adds granularity to the role of tonoplast localized Ca transport in plant stress responses. The caxl mediated anoxia tolerance is caused by extensive changes in both metabolism and signaling. Indeed, one of the most important findings of this study is the breath and amplitude of changes caused by loss of a single tonoplast H+/Ca transporter (FIG. 6). To harness this knowledge for translational applications, judicious application is advised: CAX transporters impact a variety of plant signal transduction pathways and loss of CAX1 could be deleterious in specific environments (22). However, the CAX transporters appear to be autoinhibited, requiring a protein partner for activation (84). During anoxic conditions, CAX1 may be activated by the binding of a specific regulatory protein, such as SOS2 (Salt Overly Sensitive 2) protein kinase, CXIP1 (CAX interacting protein T), and CXIP4 (CAX interacting protein 4), to the autoinhibitory N-terminus of CAX1 (84).
[0171] A means of improving anoxia tolerance may include down-regulation of at least one gene capable of synthesis of at least one activator of CAX1 to suppress or eliminate CAX1 activation during anoxic conditions. An alternative means of improving anoxia tolerance may include transient down-regulation of at least one gene capable of synthesis of at least one activator of at least one other CAX gene combined with down-regulation of at least one gene capable of synthesis of at least one activator of CAX1 to suppress or eliminate activation of multiple CAX genes during anoxic conditions. A further alternate means of improving anoxia tolerance may include transiently suppressing and/or eliminating expression of an activated CAX1 or a combination of an activated CAX1 and at least one other activated CAX gene during anoxic conditions. A further means of improving anoxia tolerance may include transient downregulation of a gene capable of synthesis of an activator of one or more CAX genes including CAX1 to suppress or eliminate CAX gene activation as well as transient down-regulation of expression of one or more activated CAX genes including CAX1 during anoxic conditions. In specific embodiments, an activator of CAX1 is selected from the group consisting of SOS2 protein kinase, CXIP1, and CXIP4.
[0172] Understanding plant gene function can be facilitated by altering transcript levels by known hairpin RNA (hpRNA) mediated silencing techniques. (85-87). An experimental limitation to this technology occurs when a gene is necessary for cell viability and reducing the levels below a certain threshold inhibits plant regeneration. This scenario requires that the gene be turned off and on in specific environmental conditions: CAX genes are necessary during specific growth conditions, thus a null mutation is not optimal for plant performance. Inducible plant promoter systems to drive hpRNA CAX mediated gene silencing are available. Variations of this system can be used for tissue- specific hpRNA expression.
II. Materials and Methods
[0173] Plant materials. The Arabidopsis thaliana wild-type (Col-0) and CAX mutant seeds (caxl-1, cax3-l and caxl-l/cax3-l double mutant (caxl/3)) and sCAXl construct used in the study have been previously characterized (20). The CAX mutants were genotyped to confirm the presence of the T-DNA insertions. The binary construct harboring p35S:sCAXl has been previously described (88). To complement the loss of CAX1 in caxl-1, the sCAXl construct was transformed into Agrobacterium tumefaciens GV3101 (Invitrogen, CA, USA) and subsequently transformed into caxl-l(89).
[0174] Plant growth, anoxia, and flooding treatment. Arabidopsis thaliana seeds were sterilized in 20% bleach for 15 min before they were planted on 1/2X Mirashige and Skoog (MS) media containing 0.8% agar (wt/vol) and 0.5% sucrose (wt/vol), and were grown under equal day/night light conditions [12 h light, 22 °C, 180 pmol-m-2 s-l, and 12 h night 20 °C]. For anoxia treatments, 2.5 to 3-week-old plants were foil covered and then placed in a GasPak using anaerobic atmosphere generation bags (Sigma) for 7h or an anoxia chamber (Anaerobe Systems, AS-580, Morgan Hill, CA) for 10 h. After the treatment the plants are returned to the growth chamber. Images are taken 4 days after the anoxia treatment to assess plant tolerance.
[0175] Chlorophyll fluorescence measurement. Chlorophyll content was measured via spectroscopic absorbance of chlorophyll a and b, as previously described (23). Briefly chlorophyll was extracted from whole rosettes using 96% (vol/vol) DMSO and incubated at 65 °C water bath for 4 hours. Absorbance at 664, 647, and 750 nm was measured with a spectrophotometer (Cary 50) in ImL cuvettes. Chlorophyll a and b concentrations, and total chlorophyll content were calculated following the equations of reference and were normalized to tissue fresh weight.
[0176] RNAseq transcriptomic analysis. Approximately 100 mg of full rosettes from anoxia-treated or untreated plants were frozen and ground in liquid nitrogen, and were subjected to Trizol total RNA extraction. Total RNA samples were sent to BGI Genomics (BGI Americas Corporation, Cambridge, MA, USA) for transcriptome sequencing on their DNBseq platform using 150 bp pair-end sequencing chemistry. Data processing and differential gene expression (DEG) analysis were also conducted by BGI. Sequences mapped to CAX1 and 3 were observed in caxl and cax3 and caxl/3 double mutants. Semi-quantitative RT-PCR analysis of the CAX1 and 3 transcripts in these mutants suggested that these RNA fragments detected by RNA-seq were results of partial mRNA fragments transcribed before the T-DNA insertions (FIG. 14). These partial mRNA fragments is informative regarding the transcriptional regulation of CAX1 and 3 by anoxia stress.
[0177] Mass Spectrometry and Proteomic Analysis. Arabidopsis whole rosette leaves were harvested from 2.5-week-old plants and proteins were extracted from leaves (90). In brief, 3g of tissue were ground in liquid N2 and were then subjected to extraction using prechilled buffer containing: 100 mm Tris-MES, pH 8.0, 1 mm EGTA, 5 mm dithiothreitol, 4 mm MgSO4, 5% [w/v] insoluble PVP, and plant protease inhibitor cocktails (Sigma Aldrich, St. Louis, MO) to the recommended concentration by manufacturer. The homogenate was then filtered through Miracloth (Calbiochem, La Jolla, CA), and subsequently clarified by centrifugation to remove cellular debris. The total proteins in supernatant (500 pL) were then precipitated using 200 pL 10X TE, 200 pl of 0.3% sodium deoxycholate, and 200 pl of cold 72% TCA. The protein pellets were washed in 90% room-temperature methanol and subsequently lyophilized.
[0178] Protein identification was carried out at the Institute for Molecular Biosciences proteomics facility at the University of Queensland, Brisbane. Proteins were analyzed using an Eksigent, Ekspert nano LC400 uHPLC coupled to a TripleTOF 6600+ System (SCIEX, Canada) equipped with a PicoView nanoflow ion source (New Objective, USA). Protein extract (up to 5 pl) was injected onto a ChromXP C18-CL column (3 pm, 75 pm x 150mm) (SCIEX, Canada). Mobile phase solvents consisted of, solvent A; 0.1% formic acid in water, and solvent B; 0.1% formic acid in acetonitrile. Linear gradients of 5-30% solvent B were run over 120 min at 400nL/minute flow rate, followed by 30-90% solvent B for 3 min, then 90% solvent B for 17 min, for peptide elution. The gradient was then returned to 5% solvent B for equilibration prior to the next sample injection. Column temperature was maintained at 45°C throughout. The ion spray voltage was set to 2600V, declustering potential at 80V, curtain gas flow 25 psi, nebuliser gas 30 psi, and interface heater at 150°C. The mass spectrometer was set to acquire 100ms of full scan TOF-MS data over the mass range 350-1500 m/z, followed by up to fifty 50ms full scan product ion data in IDA mode over the mass range 100-1500 m/z. Ions observed in the TOF-MS scan exceeding a threshold of 100 counts and a charge state of +2 to +5 were set to trigger the acquisition of product ion MS/MS spectra of the resultant 50 most intense ions.
[0179] Protein Identification and Quantification. Protein Pilot 5.0.2 (SCIEX, Canada) was used to search spectra against the Uniprot Arabidopsis thaliana database (129652 proteins, May/ 12/2020) and encode the output mzIdentML file for the downstream analysis. Scaffold 4.8.6 (Proteome Software, Portland, OR, USA) was used to validate MS/MS-based protein identifications and quantification. Protein identifications were accepted if they could be established at greater than 99% probability and contained at least two unique peptides. Normalized spectral abundance factor (NSAF) was used for protein quantification (91, 92).
[0180] Bioinformatics Analysis for Gene Ontology Enrichment Analysis. Proteins identified in at least 2 out of 3 biological replicates were considered as present in the corresponding genotypes. To evaluate the significance of comparative quantification by different genotypes, Student’s t-test was performed on the data, and the differences were assigned to be significant at a p-value less than 0.05. Differentially abundant proteins (DAPs, including exclusively present proteins) were submitted to David Bioinformatics Resources 6.8 for Gene Ontology enrichment analysis (93).
[0181] Malondialdehyde Measurements. MDA was quantified using a colorimetric method described in Stewart & Bewley (94). Briefly, full rosette leaves were pulverized in liquid nitrogen and resuspended in 80% (vol/vol) ethanol, and the supernatant was mixed with a reactant mixture of 0.65% (wt/vol) thiobarbituric acid and 20% (wt/vol) trichloroacetic acid. After a 30-min incubation at 95 °C, absorbance was measured at 532 and 600 nm with a spectrophotometer (Cary 50) in ImL cuvettes.
[0182] Hormone and pharmacological treatments of plants. Plants were treated with the following reagents: ACC (50pM), MeJA (50pM), ABA (50pM), SA (50pM), GA (20pM), and ethylene antagonists AVG (50pM), silver nitrate (200pM), RBOH inhibitor diphenyleneiodonium chloride (DPI) (8pM), Ca chelator EGTA (lOmM), ruthenium red (8pM), LaC13 (5mM), GdC13 (5mM), and W-7 (lOOpM). ACC is 1 -Aminocyclopropane- 1- carboxylic acid; MeJA is methyl jasmonate; GA is gibberellic acid; and W-7 is W-7 hydrochloride. These treatments were dissolved in sterile water and sprayed topically onto the plants in petri-dishes 8 hours or 2 days before they were treated with anoxia stress. As controls a set of plants were sprayed with water and subject to anoxia; a separate groups of plants were sprayed with the reagents and not subject to the anoxia.
[0183] RNA Extraction and Quantitative Real-Time qPCR. Total RNA was extracted using trizol from 50mg full rosette leaves. For qRT-PCR, single- stranded cDNA was synthesized from 1 pg RNA using oligo dTis primers (Invitrogen). qRT-PCR was performed on a Biorad CFX96 Real-Time PCR System (Biorad) with iTAQ SYBR Green Master Mix (Bio-Rad). Primers used are listed in the table in Table 2. Relative transcript abundance was calculated using the comparative 2-AACT method normalized to UBQ10 (95).
[0184] In situ staining of hydrogen peroxide, superoxide radical and cell death in Arabidopsis thaliana leaves. The in situ staining of hydrogen peroxide in Arabidopsis thaliana leaves was performed according to a previous study (35). Briefly, whole rosettes were stained in 1 mg/mL 3,3 '-diaminobenzidine (DAB) dissolved in 20mM Na2HPO4 (pH 5.6) and 0.05% Tween-20, for 6h. The stained rosette was then destained with 80% ethanol at 80 C. Arabidopsis thaliana leaves were assayed for cell death using trypan blue (96). Whole rosettes were harvested and stained with lactophenol-trypan blue solution (10 ml of lactic acid, 10 ml of glycerol, 10 g of phenol, 10 mg of trypan blue, dissolved in 10 ml of distilled water). Leaves were stained at room temperature without vacuum infiltration in the solution for Ih, and then decolorized overnight in 100% ethanol solution. The blue precipitation indicates location of cell death.
[0185] Generation of cytosolic Ca indicator expressing plants. Col-0 harboring a stable p35S::GCaMP3 Ca biosensor construct (65) (GCamP3/Col-0) was crossed with caxl to obtain GCamP3/caxl. F3 plants that were verified as caxl-1 were then assayed for expression of the GCamP3.
[0186] Real-time measurements of cytosolic Ca in plant leaves post anoxia. Arabidopsis thaliana plants were imaged with a motorized fluorescence stereo microscope (Zeiss Axio Zoom V16) equipped with a PlanApo Z l.Ox objective lens and a Zeiss AxioCam HRm sCMOS camera. The GFP-based Ca indicator, GCaMP3 was excited using a mercury lamp (Zeiss HXP 200c Illuminator), a 470/40 nm excitation filter, and a 500 nm dichroic mirror. The green fluorescent signal passing through a 535/50 nm filter was acquired every 1 s using Zeiss Zen pro imaging software. Col-0 or caxl-1 harboring the GCamP3 sensor constructs were grown on * strength MS media supplemented with 0.5% sucrose for 12 days before they were surface sprayed with either water control, lOmM EGTA, 50pM ABA or ImM SA, 4 hour prior to the 4h anoxia treatment. For anoxia treatment, the surgical tape around the plates was removed to allow air exchange in and out of the plates. The plates were then foil covered and placed in heat sealed AnaeroPouch (Thermo Fisher, Waltham, MA). Ca-GCamP3 fluorescence was measured to assay cytosolic Ca concentrations immediately after the plants were re-exposed to atmospheric oxygen. The fluorescence in the first set of true leaves was analyzed using ImageJ. Experiments were run in duplicate each day and experiments repeated on different days. For example, assays with the water control were repeated at least 10 different times over the course of multiple months while the SA assay was repeated on two different days (two replicates each day).
[0187] Statistical Analysis. Statistical analyses were performed with ANOVA or the student T-Test formula in Microsoft Excel. Significance was set at P < 0.05. Data were presented as means ± SEMs.
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Claims

What is claimed is: A genetically modified plant, part, and/or progeny thereof, excluding Arabidopsis thaliana, comprising a plurality of plant cells comprising: synthetically suppressed and/or eliminated expression of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4, in said plurality of plant cells. The genetically modified plant, part, and/or progeny thereof, according to claim 1, wherein said plurality of plant cells comprise: one or more agents capable of driving gene silencing of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4, in said plurality of plant cells; and optionally, a regulatory sequence capable of inducing said agent(s) to drive said gene silencing of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4, in said plurality of plant cells. The genetically modified plant, part, and/or progeny thereof, according to claim 1 or 2, wherein said agent(s) comprise a nucleic acid sequence and/or a protein. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 1-3, wherein said nucleic acid sequence comprises exogenous DNA, intragenic DNA, and/or exogenous RNA. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 1-4, wherein said protein comprises a nuclease and/or a protease. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 1-5 wherein said agent(s) and, optionally, said regulatory sequence, are (a) naked; and/or (b) comprised in (i) a complex; (ii) a carrier system; (iii) a particle gun system; (iv) a viral vector; (v) an Agrobacterium vector; and/or (vi) a CRISPR vector. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 1-6, wherein said agent(s) are capable of driving RNA interference (RNAi),
85 transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 1-7, wherein said agent(s) are capable of expressing double stranded RNA (dsRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), small RNA (sRNA), microMRNA (mRNA), pre-miRNA, and/or pri-miRNA, capable of RNA interference (RNAi) of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 1-8, wherein said agent(s) comprises double stranded RNA (dsRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), small RNA (sRNA), microMRNA (mRNA), pre-miRNA, and/or pri-miRNA, capable of RNA interference (RNAi) of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 1-9, wherein said regulator sequence is capable of inducing said agent(s) to drive RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 1-10, wherein said regulatory sequence is capable of inducing said agent(s) to express double stranded RNA (dsRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), small RNA (sRNA), microMRNA (mRNA), pre-miRNA, and/or pri-miRNA, capable of RNA interference (RNAi) of CAX1 and, optionally, at at least one of CAX2, CAX3, or CAX4. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 1-11, wherein said regulatory sequence is tissue specific and/or cell-type specific. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 1-12, wherein said regulatory sequence is low oxygen-induced, chemical- induced, temperature-induced, and/or light-induced.
86 The genetically modified plant, part, and/or progeny thereof, according to any one of claims 1-13, wherein said plurality of plant cells comprise suppressed or eliminated expression of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4 before, during, and/or after anoxic conditions. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 1-14, wherein said agent(s) are capable of driving RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4 before, during, and/or after anoxic conditions. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 1-15, wherein said plurality of plant cells comprise transient and/or stable suppressed or eliminated expression of CAX1 and, optionally, at at least one of CAX2, CAX3, or CAX4. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 1-16, wherein said agent(s) are capable of driving transient and/or stable RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of CAX1 and, optionally, at at least one of CAX2, CAX3, or CAX4. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 1-17, wherein said plurality of plant cells comprise upregulated expression during anoxia of at least one gene selected from the group consisting of: AT1G01355; AT1G02610; AT1G02620; AT1G03090; AT1G03610; AT1G06570; AT1G08630; AT1G08930; AT1G10070; AT1G10140; AT1G10550; AT1G11260; AT1G12780; AT1G14860; AT1G15040; AT1G15330; AT1G15670; AT1G17290; AT1G183OO; AT1G18773; AT1G19396; AT1G19530; AT1G19540; AT1G19610; AT1G19620; AT1G24880; AT1G25560; AT1G27045; AT1G28330; AT1G30135; AT1G30720; AT1G32910; AT1G33050; AT1G33055; AT1G34140; AT1G34760; AT1G35140; AT1G36060; AT1G54100; AT1G54760; AT1G55810; AT1G56250; AT1G58936; AT1G60750; AT1G62480; AT1G62510; AT1G63090; AT1G65970; AT1G66860; AT1G67265; AT1G68935; AT1G71520; AT1G72060; AT1G72416; AT1G75750; AT1G76650; AT1G77120; AT1G79700; AT1G79910; AT1G80160; AT1G80570; AT1G80840; AT2G05400; AT2G07633; AT2G07638; AT2G07641; AT2G07648;
87 AT2G07658; AT2G07665; AT2G07669; AT2G07673; AT2G07674; AT2G07678;
AT2G07689; AT2G07696; AT2G07698; AT2G07714; AT2G07721; AT2G07724;
AT2G07725; AT2G07727; AT2G07734; AT2G07749; AT2G07771; AT2G07773;
AT2G07785; AT2G07798; AT2G07815; AT2G07825; AT2G07835; AT2G14210;
AT2G14900; AT2G15880; AT2G15960; AT2G16060; AT2G16586; AT2G17036;
AT2G17850; AT2G19590; AT2G19800; AT2G20670; AT2G22880; AT2G22980;
AT2G25770; AT2G25780; AT2G26130; AT2G30600; AT2G30615; AT2G31810;
AT2G33160; AT2G33830; AT2G34390; AT2G34555; AT2G36110; AT2G36950;
AT2G37025; AT2G39400; AT2G39570; AT2G43520; AT2G44080; AT2G47520;
AT2G47950; AT3G02550; AT3G03270; AT3G04160; AT3G06435; AT3G10040;
AT3G1331O; AT3G13450; AT3G15440; AT3G15450; AT3G15620; AT3G16770;
AT3G17225; AT3G18530; AT3G19680; AT3G20395; AT3G21352; AT3G22640;
AT3G23150; AT3G23550; AT3G27220; AT3G28740; AT3G29370; AT3G29810;
AT3G29970; AT3G30775; AT3G43190; AT3G43850; AT3G44300; AT3G45300;
AT3G46230; AT3G47340; AT3G47675; AT3G47720; AT3G47870; AT3G48360;
AT3G48530; AT3G50560; AT3G51840; AT3G51910; AT3G55240; AT3G55970;
AT3G59060; AT3G61060; AT3G61260; AT3G62150; AT4G01120; AT4G01250;
AT4G02170; AT4G02380; AT4G02430; AT4G03205; AT4G04423; AT4G10250;
AT4G10265; AT4G10270; AT4G15760; AT4G16160; AT4G16162; AT4G16563;
AT4G19230; AT4G19880; AT4G20953; AT4G22710; AT4G22780; AT4G24040;
AT4G24110; AT4G24230; AT4G25580; AT4G25707; AT4G25810; AT4G26460;
AT4G27450; AT4G28040; AT4G28811; AT4G30270; AT4G30370; AT4G30380;
AT4G32480; AT4G32630; AT4G32840; AT4G33070; AT4G33150; AT4G33560;
AT4G33970; AT4G34030; AT4G35770; AT4G36690; AT4G36730; AT4G36850;
AT4G37220; AT4G38470; AT4G39675; AT5G01740; AT5G01810; AT5G02200;
AT5G03380; AT5G03830; AT5G05530; AT5G06980; AT5G07440; AT5G07560;
AT5G07570; AT5G08150; AT5G10040; AT5G11090; AT5G12020; AT5G12030;
AT5G14180; AT5G14470; AT5G15120; AT5G15250; AT5G18170; AT5G19120;
AT5G19550; AT5G20240; AT5G20250; AT5G20830; AT5G21170; AT5G22920;
AT5G26200; AT5G27893; AT5G28610; AT5G28630; AT5G28770; AT5G34830;
AT5G35525; AT5G39160; AT5G39200; AT5G39580; AT5G39890; AT5G40450;
AT5G41080; AT5G42825; AT5G43570; AT5G45340; AT5G47590; AT5G47910;
AT5G49360; AT5G54080; AT5G56100; AT5G56870; AT5G57220; AT5G57550;
88 AT5G57660; AT5G62520; AT5G63160; AT5G65207; AT5G66110; ATMG00010;
ATMG00060; ATMG00070; ATMG00080; ATMG00090; ATMG00110;
ATMG00140; ATMG00160; ATMG00260; ATMG00270; ATMG00285;
ATMG00310; ATMG00400; ATMG00410; ATMG00510; ATMG00513;
ATMG00516; ATMG00560; ATMG00570; ATMG00630; ATMG00640;
ATMG00650; ATMG00680; ATMG00690; ATMG00730; ATMG00900;
ATMG00940; ATMG00960; ATMG00970; ATMG01000; ATMG01050;
ATMG01120; ATMG01130; ATMG01170; ATMG01190; ATMG01200;
ATMG01210; ATMG01220; ATMG01230; ATMG01260; ATMG01280;
ATMG01320; and ATMG01360. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 1-18, wherein said plurality of plant cells comprise upregulated expression during recovery from anoxia of at least one gene selected from the group consisting of:
AT1G01720; AT1G03070; AT1G03090; AT1G03610; AT1G05575; AT1G06030;
AT1G07330; AT1G07350; AT1G07400; AT1G07500; AT1G08630; AT1G09950;
AT1G10140; AT1G12610; AT1G14200; AT1G15040; AT1G15330; AT1G15840;
AT1G16030; AT1G17870; AT1G183OO; AT1G1833O; AT1G19530; AT1G19540;
AT1G19620; AT1G21340; AT1G21940; AT1G22110; AT1G26800; AT1G27730;
AT1G28760; AT1G30070; AT1G30135; AT1G31370; AT1G32910; AT1G32928;
AT1G33055; AT1G33730; AT1G33760; AT1G34575; AT1G35140; AT1G44414;
AT1G50745; AT1G52560; AT1G53540; AT1G54050; AT1G55530; AT1G55810;
AT1G56170; AT1G56250; AT1G59860; AT1G59865; AT1G60190; AT1G61340;
AT1G66060; AT1G66080; AT1G66400; AT1G66500; AT1G66510; AT1G67265;
AT1G71000; AT1G71520; AT1G72060; AT1G72416; AT1G72660; AT1G72760;
AT1G73480; AT1G74310; AT1G74450; AT1G74930; AT1G75490; AT1G75750;
AT1G76600; AT1G76640; AT1G76650; AT1G77120; AT1G80840; AT2G01390;
AT2G07687; AT2G07696; AT2G07698; AT2G07707; AT2G07724; AT2G07725;
AT2G07727; AT2G07734; AT2G07771; AT2G07773; AT2G07785; AT2G07825;
AT2G07835; AT2G14900; AT2G15960; AT2G16060; AT2G17850; AT2G20560;
AT2G20670; AT2G22880; AT2G23110; AT2G23190; AT2G24100; AT2G25140;
AT2G26150; AT2G27580; AT2G29500; AT2G30615; AT2G32120; AT2G34390;
AT2G34600; AT2G36220; AT2G38240; AT2G38340; AT2G40340; AT2G40955;
AT2G44070; AT2G44080; AT2G44130; AT2G44840; AT2G46240; AT2G46790;
89 AT2G46830; AT2G47180; AT2G47520; AT3G02550; AT3G06435; AT3G07090;
AT3G07150; AT3G07350; AT3G08970; AT3G09350; AT3G09640; AT3G10020;
AT3G10040; AT3G11020; AT3G12190; AT3G12320; AT3G12580; AT3G1331O;
AT3G14200; AT3G15440; AT3G15450; AT3G15500; AT3G16050; AT3G17400;
AT3G19240; AT3G20340; AT3G20395; AT3G22090; AT3G22100; AT3G23150;
AT3G24500; AT3G25250; AT3G27220; AT3G28210; AT3G28740; AT3G29370;
AT3G29810; AT3G29970; AT3G30775; AT3G43850; AT3G44190; AT3G46070;
AT3G46230; AT3G47340; AT3G47720; AT3G48240; AT3G49160; AT3G50310;
AT3G50560; AT3G51910; AT3G53830; AT3G55580; AT3G55840; AT3G62260;
AT3G63350; AT4G01250; AT4G01435; AT4G02170; AT4G02425; AT4G02550;
AT4G02690; AT4G09150; AT4G10250; AT4G10265; AT4G10270; AT4G11660;
AT4G12400; AT4G12410; AT4G13395; AT4G15280; AT4G15420; AT4G15760;
AT4G16555; AT4G17250; AT4G18450; AT4G19570; AT4G21320; AT4G23493;
AT4G24110; AT4G24410; AT4G24570; AT4G25200; AT4G25380; AT4G25470;
AT4G25490; AT4G25580; AT4G25810; AT4G26200; AT4G26460; AT4G27410;
AT4G27450; AT4G27652; AT4G27654; AT4G27657; AT4G27670; AT4G28811;
AT4G29770; AT4G29780; AT4G30270; AT4G30370; AT4G32208; AT4G32480;
AT4G33070; AT4G34131; AT4G34410; AT4G35770; AT4G36850; AT4G37710;
AT4G38030; AT5G01740; AT5G02170; AT5G02810; AT5G03210; AT5G03720;
AT5G04340; AT5G04400; AT5G05220; AT5G05410; AT5G06980; AT5G07330;
AT5G08150; AT5G09590; AT5G09930; AT5G10040; AT5G10336; AT5G10695;
AT5G12020; AT5G12030; AT5G12110; AT5G13220; AT5G14470; AT5G15120;
AT5G15250; AT5G15450; AT5G17350; AT5G18065; AT5G18340; AT5G20250;
AT5G22680; AT5G22920; AT5G25450; AT5G27660; AT5G35320; AT5G37340;
AT5G37670; AT5G39580; AT5G39890; AT5G43620; AT5G43650; AT5G45340;
AT5G45640; AT5G47220; AT5G47590; AT5G47830; AT5G48570; AT5G49920;
AT5G51190; AT5G51440; AT5G51990; AT5G52050; AT5G52630; AT5G52640;
AT5G53680; AT5G54165; AT5G57260; AT5G57550; AT5G57560; AT5G57660;
AT5G58070; AT5G59720; AT5G59820; AT5G62020; AT5G62520; AT5G63130;
AT5G63300; AT5G64170; AT5G64210; AT5G64510; AT5G66650; AT5G67080;
ATMG00060; ATMG00080; ATMG00090; ATMG00160; ATMG00270;
ATMG00285; ATMG00400; ATMG00410; ATMG00480; ATMG00510;
ATMG00513; ATMG00516; ATMG00560; ATMG00570; ATMG00640;
90 ATMG00650; ATMG00690; ATMG00730; ATMG00900; ATMG00940;
ATMG00960; ATMG00990; ATMG01000; ATMG01050; ATMG01120;
ATMG01190; ATMG01200; ATMG01320; and ATMG01360. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 1-19, wherein said genetically modified plant, part, and/or progeny thereof is selected from the group consisting of whole plants, seedlings, leaves, stems, flowers, roots, fruits, seeds, spores, grafts comprising genetically modified scions and/or rootstocks, buddings comprising genetically modified buds and/or rootstocks, cuttings, bulbs, tubers, rhizomes, regenerable cells, tissue cultures of regenerable cells, regenerable protoplasts, tissue cultures of regenerable protoplasts, and other plant germplasms. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 1-20, wherein said genetically modified plant, part, and/or progeny thereof, is selected from the group consisting of the Viridiplantae, the Glaucophyta, and the Rhodophyta. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 1-21, wherein said Viridiplantae are selected from the group consisting of green algae, homworts, liverworts, mosses, ferns, lycophytes, gymnosperms, and angiosperms. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 1-22, wherein said genetically modified plant, part, and/or progeny thereof, is monocotyledonous or dicotyledonous. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 1-23, wherein said genetically modified plant, part, and/or progeny thereof is selected from the group consisting of herbs, shrubs, trees, and vines. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 1-24, wherein said genetically modified plant, part, and/or progeny thereof, excludes research plants, parts, and/or progeny thereof.
91 The genetically modified plant, part, and/or progeny thereof, according to any one of claims 1-25, wherein said genetically modified plant, part, and/or progeny thereof, is a crop plant capable of producing a crop product. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 1-26, wherein said crop plant is selected from the group consisting of food crop plants, forage crop plants, fodder crop plants, medicinal crop plants, industrial crop plants, energy crop plants, and ornamental crop plants. The genetically modified plant, part, and/or progeny thereof according to any one of claims 1-27, wherein said crop product of said ornamental crop plant is selected from the group consisting of foliage-, flower-, and/or fruit-producing herbs, shrubs, trees, vines, and regenerable parts thereof. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 1-28, wherein said crop product is selected from the group consisting of cereal grains, legumes, vegetables, nuts, seeds, roots, tubers, rhizomes, flowers, fruits, timber, plant leaves, plant oils, plant fats, plant fibers, plant juices, plant extracts, and combinations thereof. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 1-29, wherein said crop plant is selected from the group consisting of rice plants, maize plants, barley plants, oat plants, rye plants, wheat plants, com plants, sorghum plants, soybean plants, pea plants, lentil plants, curcurbit plants, coffee plants, cocoa plants, rapeseed plants, sunflower plants, sugar cane plants, potato plants, palm plants, grape plants, apple plants, banana plants, plantain plants, cassava plants, sugar beet plants, tomato plants, sweet potato plants, yam plants, tobacco plants, cotton plants, rubber plants, tea plants, lettuce plants, pepper plants, onion plants, grape plants, pecan trees, timber trees, cannabis plants, poppy plants, and combinations thereof. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 1-30, wherein said genetically modified plant, part, and/or progeny thereof is not cultivated for harvest of crops. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 1-31, wherein said genetically modified plant, part, and/or progeny thereof, not
92 cultivated for harvest crops is selected from the group consisting of Arabidopsis halleri, Arabidopsis lyrata, Eutrema salsugineum, Cardamine hirsute, the Viridiplantae, the Glaucophytes, and the Rhodophytes. An inbred, hybrid, or varietal plant, part, or progeny thereof, of said genetically modified plant, part, or progeny thereof, of any one of claims 1-32. A genetically modified plant, part, and/or progeny thereof, propagated and/or regenerated from said seeds, said spores, said grafts comprising genetically modified scions and/or rootstocks, said buddings comprising genetically modified buds and/or rootstocks, said cuttings, said bulbs, said tubers, said rhizomes, said regenerable cells, said tissue cultures of regenerable cells, said regenerable protoplasts, said tissue cultures of regenerable protoplasts, and/or said other plant germplasms of said genetically modified plant, part, and/or progeny thereof, according to any one of claims 1-33. A genetically modified seed produced by said genetically modified plant, part, and/or progeny thereof, according to any one of claims 1-34. A genetically modified plant, part, and/or progeny thereof, excluding Arabidopsis thaliana, comprising a plurality of plant cells comprising: synthetically suppressed and/or eliminated synthesis of an activator of CAX1 and, optionally, an activator of at least one of CAX2, CAX3, or CAX4, in said plurality of plant cells. The genetically modified plant, part, and/or progeny thereof, according to claim 36, wherein said plurality of plant cells comprise: one or more agents capable of driving gene silencing of a gene capable of synthesis of said activator of CAX1 and, optionally, a gene capable of synthesis of said activator of at at least one of CAX2, CAX3, or CAX4, in said plurality of plant cells; and optionally, a regulatory sequence capable of inducing said agent(s) to drive said gene silencing of said gene capable of synthesis of said activator of CAX1 and, optionally, said gene capable of synthesis of said activator of at at least one of CAX2, CAX3, or CAX4, in said plurality of plant cells. The genetically modified plant, part, and/or progeny thereof, according to claim 36 or 37, wherein said agent(s) comprise a nucleic acid sequence and/or a protein. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 36-38, wherein said activator of CAX1 is capable of binding to the N-terminus of CAX1, and wherein said activator of at least one of CAX2, CAX3, or CAX4 is capable of binding to the N-terminus of at least one of CAX2, CAX3, or CAX4. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 36-39, wherein said activator of CAX1 is selected from the group consisting of SOS2 protein kinase, CXIP1, and CXIP4. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 36-40, wherein said nucleic acid sequence comprises exogenous DNA, intragenic DNA, and/or exogenous RNA. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 36-41, wherein said protein comprises a nuclease and/or a protease. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 36-42, wherein said agent(s) and, optionally, said regulatory sequence, are (a) naked; and/or (b) comprised in (i) a complex; (ii) a carrier system; (iii) a particle gun system; (iv) a viral vector; (v) an Agrobacterium vector; and/or (vi) a CRISPR vector. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 36-43, wherein said agent(s) are capable of driving RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of said gene capable of synthesis of said activator of CAX1 and, optionally, said gene capable of synthesis of said activator of at at least one of CAX2, CAX3, or CAX4. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 36-44, wherein said agent(s) are capable of expressing double stranded RNA (dsRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), small RNA (sRNA), microMRNA (mRNA), pre-miRNA, and/or pri-miRNA, capable of RNA interference (RNAi) of said gene capable of synthesis of said activator of CAX1 and, optionally, said gene capable of synthesis of said activator of at at least one of CAX2, CAX3, or CAX4. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 36-45, wherein said agent(s) comprise double stranded RNA (dsRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), small RNA (sRNA), microMRNA (mRNA), pre-miRNA, and/or pri-miRNA capable of RNA interference (RNAi) of said gene capable of synthesis of said activator of CAX1 and, optionally, said gene capable of synthesis of said activator of at at least one of CAX2, CAX3, or CAX4. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 36-46, wherein said regulatory agent is capable of inducing said agent(s) to drive RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of said gene capable of synthesis of said activator of CAX1 and, optionally, said gene capable of synthesis of said activator of at at least one of CAX2, CAX3, or CAX4. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 36-47, wherein said regulatory sequence is capable of inducing said agent(s) to express double stranded RNA (dsRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), small RNA (sRNA), microMRNA (mRNA), pre-miRNA, and/or pri-miRNA, capable of RNA interference (RNAi) of said gene capable of synthesis of said activator of CAX1 and, optionally, said gene capable of synthesis of said activator of at at least one of CAX2, CAX3, or CAX4. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 36-48, wherein said regulatory sequence is tissue specific and/or cell-type specific. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 36-49, wherein said regulatory sequence is low oxygen-induced, chemical- induced, temperature-induced, and/or light-induced. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 36-50, wherein said plurality of plant cells comprise suppressed and/or
95 eliminated synthesis of said activator of CAX1 and, optionally, said activator of at least one of CAX2, CAX3, or CAX4, before, during, and/or after anoxic conditions. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 36-51, wherein said agent(s) are capable of driving RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of said gene capable of synthesis of said activator of CAX1 and, optionally, said gene capable of synthesis of said activator of at least one of CAX2, CAX3, or CAX4, before, during, and/or after anoxic conditions. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 36-52, wherein said plurality of plant cells comprise transient and/or stable suppressed or eliminated synthesis of said activator of CAX1 and, optionally, said activator of at least one of CAX2, CAX3, or CAX4. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 36-53, wherein said agent(s) are capable of driving transient and/or stable RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of said gene capable of synthesis of said activator of CAX1 and, optionally, said activator of at least one of CAX2, CAX3, or CAX4. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 36-54, wherein said plurality of plant cells comprise upregulated expression during anoxia of at least one gene selected from the group consisting of: AT1G01355; AT1G02610; AT1G02620; AT1G03090; AT1G03610; AT1G06570; AT1G08630; AT1G08930; AT1G10070; AT1G10140; AT1G10550; AT1G11260; AT1G12780; AT1G14860; AT1G15040; AT1G15330; AT1G15670; AT1G17290; AT1G183OO; AT1G18773; AT1G19396; AT1G19530; AT1G19540; AT1G19610; AT1G19620; AT1G24880; AT1G25560; AT1G27045; AT1G28330; AT1G30135; AT1G30720; AT1G32910; AT1G33050; AT1G33055; AT1G34140; AT1G34760; AT1G35140; AT1G36060; AT1G54100; AT1G54760; AT1G55810; AT1G56250; AT1G58936; AT1G60750; AT1G62480; AT1G62510; AT1G63090; AT1G65970; AT1G66860; AT1G67265; AT1G68935; AT1G71520; AT1G72060; AT1G72416; AT1G75750; AT1G76650; AT1G77120; AT1G79700; AT1G79910; AT1G80160; AT1G80570; AT1G80840; AT2G05400; AT2G07633; AT2G07638; AT2G07641; AT2G07648;
96 AT2G07658; AT2G07665; AT2G07669; AT2G07673; AT2G07674; AT2G07678;
AT2G07689; AT2G07696; AT2G07698; AT2G07714; AT2G07721; AT2G07724;
AT2G07725; AT2G07727; AT2G07734; AT2G07749; AT2G07771; AT2G07773;
AT2G07785; AT2G07798; AT2G07815; AT2G07825; AT2G07835; AT2G14210;
AT2G14900; AT2G15880; AT2G15960; AT2G16060; AT2G16586; AT2G17036;
AT2G17850; AT2G19590; AT2G19800; AT2G20670; AT2G22880; AT2G22980;
AT2G25770; AT2G25780; AT2G26130; AT2G30600; AT2G30615; AT2G31810;
AT2G33160; AT2G33830; AT2G34390; AT2G34555; AT2G36110; AT2G36950;
AT2G37025; AT2G39400; AT2G39570; AT2G43520; AT2G44080; AT2G47520;
AT2G47950; AT3G02550; AT3G03270; AT3G04160; AT3G06435; AT3G10040;
AT3G1331O; AT3G13450; AT3G15440; AT3G15450; AT3G15620; AT3G16770;
AT3G17225; AT3G18530; AT3G19680; AT3G20395; AT3G21352; AT3G22640;
AT3G23150; AT3G23550; AT3G27220; AT3G28740; AT3G29370; AT3G29810;
AT3G29970; AT3G30775; AT3G43190; AT3G43850; AT3G44300; AT3G45300;
AT3G46230; AT3G47340; AT3G47675; AT3G47720; AT3G47870; AT3G48360;
AT3G48530; AT3G50560; AT3G51840; AT3G51910; AT3G55240; AT3G55970;
AT3G59060; AT3G61060; AT3G61260; AT3G62150; AT4G01120; AT4G01250;
AT4G02170; AT4G02380; AT4G02430; AT4G03205; AT4G04423; AT4G10250;
AT4G10265; AT4G10270; AT4G15760; AT4G16160; AT4G16162; AT4G16563;
AT4G19230; AT4G19880; AT4G20953; AT4G22710; AT4G22780; AT4G24040;
AT4G24110; AT4G24230; AT4G25580; AT4G25707; AT4G25810; AT4G26460;
AT4G27450; AT4G28040; AT4G28811; AT4G30270; AT4G30370; AT4G30380;
AT4G32480; AT4G32630; AT4G32840; AT4G33070; AT4G33150; AT4G33560;
AT4G33970; AT4G34030; AT4G35770; AT4G36690; AT4G36730; AT4G36850;
AT4G37220; AT4G38470; AT4G39675; AT5G01740; AT5G01810; AT5G02200;
AT5G03380; AT5G03830; AT5G05530; AT5G06980; AT5G07440; AT5G07560;
AT5G07570; AT5G08150; AT5G10040; AT5G11090; AT5G12020; AT5G12030;
AT5G14180; AT5G14470; AT5G15120; AT5G15250; AT5G18170; AT5G19120;
AT5G19550; AT5G20240; AT5G20250; AT5G20830; AT5G21170; AT5G22920;
AT5G26200; AT5G27893; AT5G28610; AT5G28630; AT5G28770; AT5G34830;
AT5G35525; AT5G39160; AT5G39200; AT5G39580; AT5G39890; AT5G40450;
AT5G41080; AT5G42825; AT5G43570; AT5G45340; AT5G47590; AT5G47910;
AT5G49360; AT5G54080; AT5G56100; AT5G56870; AT5G57220; AT5G57550;
97 AT5G57660; AT5G62520; AT5G63160; AT5G65207; AT5G66110; ATMG00010;
ATMG00060; ATMG00070; ATMG00080; ATMG00090; ATMG00110;
ATMG00140; ATMG00160; ATMG00260; ATMG00270; ATMG00285;
ATMG00310; ATMG00400; ATMG00410; ATMG00510; ATMG00513;
ATMG00516; ATMG00560; ATMG00570; ATMG00630; ATMG00640;
ATMG00650; ATMG00680; ATMG00690; ATMG00730; ATMG00900;
ATMG00940; ATMG00960; ATMG00970; ATMG01000; ATMG01050;
ATMG01120; ATMG01130; ATMG01170; ATMG01190; ATMG01200;
ATMG01210; ATMG01220; ATMG01230; ATMG01260; ATMG01280;
ATMG01320; and ATMG01360. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 36-55, wherein said plurality of plant cells comprise upregulated expression during recovery from anoxia of at least one gene selected from the group consisting of: AT1G01720; AT1G03070; AT1G03090; AT1G03610; AT1G05575; AT1G06030; AT1G07330; AT1G07350; AT1G07400; AT1G07500; AT1G08630; AT1G09950; AT1G10140; AT1G12610; AT1G14200; AT1G15040; AT1G15330; AT1G15840; AT1G16030; AT1G17870; AT1G183OO; AT1G1833O; AT1G19530; AT1G19540; AT1G19620; AT1G21340; AT1G21940; AT1G22110; AT1G26800; AT1G27730; AT1G28760; AT1G30070; AT1G30135; AT1G31370; AT1G32910; AT1G32928; AT1G33055; AT1G33730; AT1G33760; AT1G34575; AT1G35140; AT1G44414; AT1G50745; AT1G52560; AT1G53540; AT1G54050; AT1G55530; AT1G55810; AT1G56170; AT1G56250; AT1G59860; AT1G59865; AT1G60190; AT1G61340; AT1G66060; AT1G66080; AT1G66400; AT1G66500; AT1G66510; AT1G67265; AT1G71000; AT1G71520; AT1G72060; AT1G72416; AT1G72660; AT1G72760; AT1G73480; AT1G74310; AT1G74450; AT1G74930; AT1G75490; AT1G75750; AT1G76600; AT1G76640; AT1G76650; AT1G77120; AT1G80840; AT2G01390; AT2G07687; AT2G07696; AT2G07698; AT2G07707; AT2G07724; AT2G07725; AT2G07727; AT2G07734; AT2G07771; AT2G07773; AT2G07785; AT2G07825; AT2G07835; AT2G14900; AT2G15960; AT2G16060; AT2G17850; AT2G20560; AT2G20670; AT2G22880; AT2G23110; AT2G23190; AT2G24100; AT2G25140; AT2G26150; AT2G27580; AT2G29500; AT2G30615; AT2G32120; AT2G34390; AT2G34600; AT2G36220; AT2G38240; AT2G38340; AT2G40340; AT2G40955; AT2G44070; AT2G44080; AT2G44130; AT2G44840; AT2G46240; AT2G46790;
98 AT2G46830; AT2G47180; AT2G47520; AT3G02550; AT3G06435; AT3G07090;
AT3G07150; AT3G07350; AT3G08970; AT3G09350; AT3G09640; AT3G10020;
AT3G10040; AT3G11020; AT3G12190; AT3G12320; AT3G12580; AT3G1331O;
AT3G14200; AT3G15440; AT3G15450; AT3G15500; AT3G16050; AT3G17400;
AT3G19240; AT3G20340; AT3G20395; AT3G22090; AT3G22100; AT3G23150;
AT3G24500; AT3G25250; AT3G27220; AT3G28210; AT3G28740; AT3G29370;
AT3G29810; AT3G29970; AT3G30775; AT3G43850; AT3G44190; AT3G46070;
AT3G46230; AT3G47340; AT3G47720; AT3G48240; AT3G49160; AT3G50310;
AT3G50560; AT3G51910; AT3G53830; AT3G55580; AT3G55840; AT3G62260;
AT3G63350; AT4G01250; AT4G01435; AT4G02170; AT4G02425; AT4G02550;
AT4G02690; AT4G09150; AT4G10250; AT4G10265; AT4G10270; AT4G11660;
AT4G12400; AT4G12410; AT4G13395; AT4G15280; AT4G15420; AT4G15760;
AT4G16555; AT4G17250; AT4G18450; AT4G19570; AT4G21320; AT4G23493;
AT4G24110; AT4G24410; AT4G24570; AT4G25200; AT4G25380; AT4G25470;
AT4G25490; AT4G25580; AT4G25810; AT4G26200; AT4G26460; AT4G27410;
AT4G27450; AT4G27652; AT4G27654; AT4G27657; AT4G27670; AT4G28811;
AT4G29770; AT4G29780; AT4G30270; AT4G30370; AT4G32208; AT4G32480;
AT4G33070; AT4G34131; AT4G34410; AT4G35770; AT4G36850; AT4G37710;
AT4G38030; AT5G01740; AT5G02170; AT5G02810; AT5G03210; AT5G03720;
AT5G04340; AT5G04400; AT5G05220; AT5G05410; AT5G06980; AT5G07330;
AT5G08150; AT5G09590; AT5G09930; AT5G10040; AT5G10336; AT5G10695;
AT5G12020; AT5G12030; AT5G12110; AT5G13220; AT5G14470; AT5G15120;
AT5G15250; AT5G15450; AT5G17350; AT5G18065; AT5G18340; AT5G20250;
AT5G22680; AT5G22920; AT5G25450; AT5G27660; AT5G35320; AT5G37340;
AT5G37670; AT5G39580; AT5G39890; AT5G43620; AT5G43650; AT5G45340;
AT5G45640; AT5G47220; AT5G47590; AT5G47830; AT5G48570; AT5G49920;
AT5G51190; AT5G51440; AT5G51990; AT5G52050; AT5G52630; AT5G52640;
AT5G53680; AT5G54165; AT5G57260; AT5G57550; AT5G57560; AT5G57660;
AT5G58070; AT5G59720; AT5G59820; AT5G62020; AT5G62520; AT5G63130;
AT5G63300; AT5G64170; AT5G64210; AT5G64510; AT5G66650; AT5G67080;
ATMG00060; ATMG00080; ATMG00090; ATMG00160; ATMG00270;
ATMG00285; ATMG00400; ATMG00410; ATMG00480; ATMG00510;
ATMG00513; ATMG00516; ATMG00560; ATMG00570; ATMG00640;
99 ATMG00650; ATMG00690; ATMG00730; ATMG00900; ATMG00940;
ATMG00960; ATMG00990; ATMG01000; ATMG01050; ATMG01120;
ATMG01190; ATMG01200; ATMG01320; and ATMG01360. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 36-56, wherein said genetically modified plant, part, and/or progeny thereof is selected from the group consisting of whole plants, seedlings, leaves, stems, flowers, roots, fruits, seeds, spores, grafts comprising genetically modified scions and/or rootstocks, buddings comprising genetically modified buds and/or rootstocks, cuttings, bulbs, tubers, rhizomes, regenerable cells, tissue cultures of regenerable cells, regenerable protoplasts, tissue cultures of regenerable protoplasts, and other plant germplasms. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 36-57, wherein said genetically modified plant, part, and/or progeny thereof, is selected from the group consisting of the Viridiplantae, the Glaucophyta, and the Rhodophyta. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 36-58, wherein said Viridiplantae are selected from the group consisting of green algae, homworts, liverworts, mosses, ferns, lycophytes, gymnosperms, and angiosperms. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 36-59, wherein said genetically modified plant, part, and/or progeny thereof, is monocotyledonous or dicotyledonous. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 36-60, wherein said genetically modified plant, part, and/or progeny thereof is selected from the group consisting of herbs, shrubs, trees, and vines. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 36-61, wherein said genetically modified plant, part, and/or progeny thereof, excludes research plants, parts, and/or progeny thereof.
100 The genetically modified plant, part, and/or progeny thereof, according to any one of claims 36-62, wherein said genetically modified plant, part, and/or progeny thereof, is a crop plant capable of producing a crop product. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 36-63, wherein said crop plant is selected from the group consisting of food crop plants, forage crop plants, fodder crop plants, medicinal crop plants, industrial crop plants, energy crop plants, and ornamental crop plants. The genetically modified plant, part, and/or progeny thereof according to any one of claims 36-64, wherein said crop product of said ornamental crop plant is selected from the group consisting of foliage-, flower-, and/or fruit-producing herbs, shrubs, trees, vines, and regenerable parts thereof. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 36-65, wherein said crop product is selected from the group consisting of cereal grains, legumes, vegetables, nuts, seeds, roots, tubers, rhizomes, flowers, fruits, timber, plant leaves, plant oils, plant fats, plant fibers, plant juices, plant extracts, and combinations thereof. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 36-66, wherein said crop plant is selected from the group consisting of rice plants, maize plants, barley plants, oat plants, rye plants, wheat plants, corn plants, sorghum plants, soybean plants, pea plants, lentil plants, curcurbit plants, coffee plants, cocoa plants, rapeseed plants, sunflower plants, sugar cane plants, potato plants, palm plants, grape plants, apple plants, banana plants, plantain plants, cassava plants, sugar beet plants, tomato plants, sweet potato plants, yam plants, tobacco plants, cotton plants, rubber plants, tea plants, lettuce plants, pepper plants, onion plants, grape plants, pecan trees, timber trees, cannabis plants, poppy plants, and combinations thereof. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 36-67, wherein said genetically modified plant, part, and/or progeny thereof is not cultivated for harvest of crops. The genetically modified plant, part, and/or progeny thereof, according to any one of claims 36-68, wherein said genetically modified plant, part, and/or progeny thereof, not
101 cultivated for harvest crops is selected from the group consisting of Arabidopsis halleri, Arabidopsis lyrata, Eutrema salsugineum, Cardamine hirsute, the Viridiplantae, the Glaucophytes, and the Rhodophytes. An inbred, hybrid, or varietal plant, part, or progeny thereof, of said genetically modified plant, part, or progeny thereof, of any one of claims 36-69. A genetically modified plant, part, and/or progeny thereof, propagated and/or regenerated from said seeds, said spores, said grafts comprising genetically modified scions and/or rootstocks, said buddings comprising genetically modified buds and/or rootstocks, said cuttings, said bulbs, said tubers, said rhizomes, said regenerable cells, said tissue cultures of regenerable cells, said regenerable protoplasts, said tissue cultures of regenerable protoplasts, and/or said other plant germplasms of said genetically modified plant, part, and/or progeny thereof, according to any one of claims 36-70. A genetically modified seed produced by said genetically modified plant, part, and/or progeny thereof, according to any one of claims 36-71. A method of producing a crop from said genetically modified crop plant, part, and/or progeny thereof, of any one of claims 1-72, comprising the steps of: cultivating said genetically modified crop plant, part, and/or progeny thereof, to produce said crop; and harvesting said crop from said genetically modified crop plant, part, and/or progeny thereof. A method of producing a genetically modified plant, part, and/or progeny thereof, excluding Arabidopsis thaliana, comprising a plurality of plant cells, wherein the method comprises the step of: synthetically driving suppression and/or elimination of expression of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4, in said plurality of plant cells, wherein said synthetically driving step improves the anoxia tolerance of said genetically modified plant, part and/or progeny thereof.
102 The method of claim 74, wherein the synthetically driving step further comprises the steps of: introducing into said plurality of plant cells one or more agents, said agent(s) driving gene silencing of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4, in said plurality of plant cells; and optionally, introducing into said plurality of plant cells a regulatory sequence, said regulatory sequence inducing said agent(s) to drive said gene silencing of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4, in said plurality of plant cells. The method of claim 74 or 75, wherein said one or more agents is a nucleic acid sequence and/or a protein. The method of any one of claims 74-76, wherein said nucleic acid sequence comprises exogenous DNA, intragenic DNA, and/or exogenous RNA. The method of any one of claims 74-77, wherein said protein comprises a nuclease and/or a protease. The method of any one of claims 74-78, wherein said introducing steps are accomplished by: direct transfer of exogenous DNA, intragenic DNA, exogenous RNA, or combinations, complexes, carrier systems, and/or particle gun systems thereof; and/or viral vector-mediated, Agrobacterium vector-mediated, and/or CRISPR vector- mediated gene transformation of exogenous and/or intragenic DNA. The method of any one of claims 74-79, wherein said direct transfer step is accomplished by: passive uptake; electroporation; polyethylene glycol treatment; electrophoresis; cell fusion with liposomes or spheroplasts; injection, silicon carbide whiskers, particle gun bombardment, spraying, soaking, pipetting, brushing, cationic nanoparticle carriers, clay nanosheet carriers, surfactant complexes, and/or peptide- based carriers. The method of any one of claims 74-80, wherein said driving step further comprises the step of driving RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) in expression of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4.
103 The method of any one of claims 74-81, wherein said step of driving RNA interference (RNAi) further comprises the step of expressing double stranded RNA (dsRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), small RNA (sRNA), microMRNA (mRNA), pre-miRNA, and/or pri-miRNA capable of RNA interference (RNAi) of expression of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4. The method of any one of claims 74-82, wherein said agent(s) comprise double stranded RNA (dsRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), small RNA (sRNA), microMRNA (mRNA), pre-miRNA, and/or pri-miRNA capable of RNA interference (RNAi) of expression of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4. The method of any one of claims 74-83, wherein said inducing step further comprises the step of inducing said agent(s) to drive RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) in expression of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4. The method of any one of claims 74-84, wherein said step of inducing said agent(s) to drive RNA interference (RNAi) further comprises the step of inducing said agent(s) to express double stranded RNA (dsRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), small RNA (sRNA), microMRNA (mRNA), pre-miRNA, and/or pri-miRNA capable of RNA interference (RNAi) of expression of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4. The method of any one of claims 74-85, wherein said regulatory sequence is tissue specific and/or cell-type specific. The method of any one of claims 74-86, wherein said regulatory sequence is low oxygen-induced, chemical-induced, temperature-induced, and/or light-induced. The method of any one of claims 74-87, wherein said step of suppressing and/or eliminating expression of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4, occurs before, during, and/or after anoxic conditions. The method of any one of claims 74-88, wherein said step of driving RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing
104 (VIGS) of expression of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4, occurs before, during, and/or after anoxic conditions. The method of any one of claims 74-89, wherein said step of suppressing and/or eliminating expression of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4, is transient and/or stable. The method of any one of claims 74-90, wherein said step of driving RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of expression of CAX1 and, optionally, at least one of CAX2, CAX3, or CAX4, is transient and/or stable. The method of any one of claims 74-91, wherein said plurality of plant cells upregulate expression during anoxia of at least one gene selected from the group consisting of: AT1G01355; AT1G02610; AT1G02620; AT1G03090; AT1G03610; AT1G06570; AT1G08630; AT1G08930; AT1G10070; AT1G10140; AT1G10550; AT1G11260; AT1G12780; AT1G14860; AT1G15040; AT1G15330; AT1G15670; AT1G17290; AT1G183OO; AT1G18773; AT1G19396; AT1G19530; AT1G19540; AT1G19610; AT1G19620; AT1G24880; AT1G25560; AT1G27045; AT1G28330; AT1G30135; AT1G30720; AT1G32910; AT1G33050; AT1G33055; AT1G34140; AT1G34760; AT1G35140; AT1G36060; AT1G54100; AT1G54760; AT1G55810; AT1G56250; AT1G58936; AT1G60750; AT1G62480; AT1G62510; AT1G63090; AT1G65970; AT1G66860; AT1G67265; AT1G68935; AT1G71520; AT1G72060; AT1G72416; AT1G75750; AT1G76650; AT1G77120; AT1G79700; AT1G79910; AT1G80160; AT1G80570; AT1G80840; AT2G05400; AT2G07633; AT2G07638; AT2G07641; AT2G07648; AT2G07658; AT2G07665; AT2G07669; AT2G07673; AT2G07674; AT2G07678; AT2G07689; AT2G07696; AT2G07698; AT2G07714; AT2G07721; AT2G07724; AT2G07725; AT2G07727; AT2G07734; AT2G07749; AT2G07771; AT2G07773; AT2G07785; AT2G07798; AT2G07815; AT2G07825; AT2G07835; AT2G14210; AT2G14900; AT2G15880; AT2G15960; AT2G16060; AT2G16586; AT2G17036; AT2G17850; AT2G19590; AT2G19800; AT2G20670; AT2G22880; AT2G22980; AT2G25770; AT2G25780; AT2G26130; AT2G30600; AT2G30615; AT2G31810; AT2G33160; AT2G33830; AT2G34390; AT2G34555; AT2G36110; AT2G36950; AT2G37025; AT2G39400; AT2G39570; AT2G43520; AT2G44080; AT2G47520; AT2G47950; AT3G02550; AT3G03270; AT3G04160; AT3G06435;
105 AT3G10040; AT3G1331O; AT3G13450; AT3G15440; AT3G15450; AT3G15620;
AT3G16770; AT3G17225; AT3G18530; AT3G19680; AT3G20395; AT3G21352;
AT3G22640; AT3G23150; AT3G23550; AT3G27220; AT3G28740; AT3G29370;
AT3G29810; AT3G29970; AT3G30775; AT3G43190; AT3G43850; AT3G44300;
AT3G45300; AT3G46230; AT3G47340; AT3G47675; AT3G47720; AT3G47870;
AT3G48360; AT3G48530; AT3G50560; AT3G51840; AT3G51910; AT3G55240;
AT3G55970; AT3G59060; AT3G61060; AT3G61260; AT3G62150; AT4G01120;
AT4G01250; AT4G02170; AT4G02380; AT4G02430; AT4G03205; AT4G04423;
AT4G10250; AT4G10265; AT4G10270; AT4G15760; AT4G16160; AT4G16162;
AT4G16563; AT4G19230; AT4G19880; AT4G20953; AT4G22710; AT4G22780;
AT4G24040; AT4G24110; AT4G24230; AT4G25580; AT4G25707; AT4G25810;
AT4G26460; AT4G27450; AT4G28040; AT4G28811; AT4G30270; AT4G30370;
AT4G30380; AT4G32480; AT4G32630; AT4G32840; AT4G33070; AT4G33150;
AT4G33560; AT4G33970; AT4G34030; AT4G35770; AT4G36690; AT4G36730;
AT4G36850; AT4G37220; AT4G38470; AT4G39675; AT5G01740; AT5G01810;
AT5G02200; AT5G03380; AT5G03830; AT5G05530; AT5G06980; AT5G07440;
AT5G07560; AT5G07570; AT5G08150; AT5G10040; AT5G11090; AT5G12020;
AT5G12030; AT5G14180; AT5G14470; AT5G15120; AT5G15250; AT5G18170;
AT5G19120; AT5G19550; AT5G20240; AT5G20250; AT5G20830; AT5G21170;
AT5G22920; AT5G26200; AT5G27893; AT5G28610; AT5G28630; AT5G28770;
AT5G34830; AT5G35525; AT5G39160; AT5G39200; AT5G39580; AT5G39890;
AT5G40450; AT5G41080; AT5G42825; AT5G43570; AT5G45340; AT5G47590;
AT5G47910; AT5G49360; AT5G54080; AT5G56100; AT5G56870; AT5G57220;
AT5G57550; AT5G57660; AT5G62520; AT5G63160; AT5G65207; AT5G66110;
ATMG00010; ATMG00060; ATMG00070; ATMG00080; ATMG00090;
ATMG00110; ATMG00140; ATMG00160; ATMG00260; ATMG00270;
ATMG00285; ATMG00310; ATMG00400; ATMG00410; ATMG00510;
ATMG00513; ATMG00516; ATMG00560; ATMG00570; ATMG00630;
ATMG00640; ATMG00650; ATMG00680; ATMG00690; ATMG00730;
ATMG00900; ATMG00940; ATMG00960; ATMG00970; ATMG01000;
ATMG01050; ATMG01120; ATMG01130; ATMG01170; ATMG01190;
ATMG01200; ATMG01210; ATMG01220; ATMG01230; ATMG01260;
ATMG01280; ATMG01320; and ATMG01360.
106 The method of any one of claims 74-92, wherein said plurality of plant cells upregulate expression during recovery from anoxia of at least one gene selected from the group consisting of: AT1G01720; AT1G03070; AT1G03090; AT1G03610; AT1G05575; AT1G06030; AT1G07330; AT1G07350; AT1G07400; AT1G07500; AT1G08630; AT1G09950; AT1G10140; AT1G12610; AT1G14200; AT1G15040; AT1G15330; AT1G15840; AT1G16030; AT1G17870; AT1G183OO; AT1G1833O; AT1G19530; AT1G19540; AT1G19620; AT1G21340; AT1G21940; AT1G22110; AT1G26800; AT1G27730; AT1G28760; AT1G30070; AT1G30135; AT1G31370; AT1G32910; AT1G32928; AT1G33055; AT1G33730; AT1G33760; AT1G34575; AT1G35140; AT1G44414; AT1G50745; AT1G52560; AT1G53540; AT1G54050; AT1G55530; AT1G55810; AT1G56170; AT1G56250; AT1G59860; AT1G59865; AT1G60190; AT1G61340; AT1G66060; AT1G66080; AT1G66400; AT1G66500; AT1G66510; AT1G67265; AT1G71000; AT1G71520; AT1G72060; AT1G72416; AT1G72660; AT1G72760; AT1G73480; AT1G74310; AT1G74450; AT1G74930; AT1G75490; AT1G75750; AT1G76600; AT1G76640; AT1G76650; AT1G77120; AT1G80840; AT2G01390; AT2G07687; AT2G07696; AT2G07698; AT2G07707; AT2G07724; AT2G07725; AT2G07727; AT2G07734; AT2G07771; AT2G07773; AT2G07785; AT2G07825; AT2G07835; AT2G14900; AT2G15960; AT2G16060; AT2G17850; AT2G20560; AT2G20670; AT2G22880; AT2G23110; AT2G23190; AT2G24100; AT2G25140; AT2G26150; AT2G27580; AT2G29500; AT2G30615; AT2G32120; AT2G34390; AT2G34600; AT2G36220; AT2G38240; AT2G38340; AT2G40340; AT2G40955; AT2G44070; AT2G44080; AT2G44130; AT2G44840; AT2G46240; AT2G46790; AT2G46830; AT2G47180; AT2G47520; AT3G02550; AT3G06435; AT3G07090; AT3G07150; AT3G07350; AT3G08970; AT3G09350; AT3G09640; AT3G10020; AT3G10040; AT3G11020; AT3G12190; AT3G12320; AT3G12580; AT3G1331O; AT3G14200; AT3G15440; AT3G15450; AT3G15500; AT3G16050; AT3G17400; AT3G19240; AT3G20340; AT3G20395; AT3G22090; AT3G22100; AT3G23150; AT3G24500; AT3G25250; AT3G27220; AT3G28210; AT3G28740; AT3G29370; AT3G29810; AT3G29970; AT3G30775; AT3G43850; AT3G44190; AT3G46070; AT3G46230; AT3G47340; AT3G47720; AT3G48240; AT3G49160; AT3G50310; AT3G50560; AT3G51910; AT3G53830; AT3G55580; AT3G55840; AT3G62260; AT3G63350; AT4G01250; AT4G01435; AT4G02170; AT4G02425; AT4G02550; AT4G02690; AT4G09150; AT4G10250; AT4G10265; AT4G10270;
107 AT4G11660; AT4G12400; AT4G12410; AT4G13395; AT4G15280; AT4G15420; AT4G15760; AT4G16555; AT4G17250; AT4G18450; AT4G19570; AT4G21320; AT4G23493; AT4G24110; AT4G24410; AT4G24570; AT4G25200; AT4G25380; AT4G25470; AT4G25490; AT4G25580; AT4G25810; AT4G26200; AT4G26460; AT4G27410; AT4G27450; AT4G27652; AT4G27654; AT4G27657; AT4G27670; AT4G28811; AT4G29770; AT4G29780; AT4G30270; AT4G30370; AT4G32208; AT4G32480; AT4G33070; AT4G34131; AT4G34410; AT4G35770; AT4G36850; AT4G37710; AT4G38030; AT5G01740; AT5G02170; AT5G02810; AT5G03210; AT5G03720; AT5G04340; AT5G04400; AT5G05220; AT5G05410; AT5G06980; AT5G07330; AT5G08150; AT5G09590; AT5G09930; AT5G10040; AT5G10336; AT5G10695; AT5G12020; AT5G12030; AT5G12110; AT5G13220; AT5G14470; AT5G15120; AT5G15250; AT5G15450; AT5G17350; AT5G18065; AT5G18340; AT5G20250; AT5G22680; AT5G22920; AT5G25450; AT5G27660; AT5G35320; AT5G37340; AT5G37670; AT5G39580; AT5G39890; AT5G43620; AT5G43650; AT5G45340; AT5G45640; AT5G47220; AT5G47590; AT5G47830; AT5G48570; AT5G49920; AT5G51190; AT5G51440; AT5G51990; AT5G52050; AT5G52630; AT5G52640; AT5G53680; AT5G54165; AT5G57260; AT5G57550; AT5G57560; AT5G57660; AT5G58070; AT5G59720; AT5G59820; AT5G62020; AT5G62520; AT5G63130; AT5G63300; AT5G64170; AT5G64210; AT5G64510; AT5G66650; AT5G67080; ATMG00060; ATMG00080; ATMG00090; ATMG00160;
ATMG00270; ATMG00285; ATMG00400; ATMG00410; ATMG00480;
ATMG00510; ATMG00513; ATMG00516; ATMG00560; ATMG00570;
ATMG00640; ATMG00650; ATMG00690; ATMG00730; ATMG00900;
ATMG00940; ATMG00960; ATMG00990; ATMG01000; ATMG01050;
ATMG01120; ATMG01190; ATMG01200; ATMG01320; and ATMG01360. A method of producing a genetically modified plant, part, and/or progeny thereof, excluding Arabidopsis thaliana, comprising a plurality of plant cells, wherein the method comprises the step of: synthetically driving suppression and/or elimination of expression of a gene capable of synthesis of an activator of CAX1 and, optionally, a gene capable of synthesis of an activator of at least one of CAX2, CAX3, or CAX4, in said plurality of plant cells,
108 wherein said synthetically driving step improves the anoxia tolerance of said genetically modified plant, part and/or progeny thereof. The method of claim 94, wherein the synthetically driving step further comprises the steps of: introducing into said plurality of plant cells one or more agents, said agent(s) driving gene silencing of said gene capable of synthesis of an activator of CAX1 and, optionally, said gene capable of synthesis of an activator of at least one of CAX2, CAX3, or CAX4, in said plurality of plant cells; and optionally, introducing into said plurality of plant cells a regulatory sequence, said regulatory sequence inducing said agent(s) to drive gene silencing of said gene capable of synthesis of an activator of CAX1 and, optionally, said gene capable of synthesis of an activator of at least one of CAX2, CAX3, or CAX4, in said plurality of plant cells. The method of claim 94 or 95, wherein said activator of CAX1 is capable of binding to the N-terminus of CAX1, and wherein said activator of at least one of CAX2, CAX3, or CAX4 is capable of binding to the N-terminus of at least one of CAX2, CAX3, or CAX4. The method of any one of claims 94-96, wherein said activator of CAX1 is selected from the group consisting of SOS2 protein kinase, CXIP1, and CXIP4. The method of any one of claims 94-97, wherein said introducing steps are accomplished by: direct transfer of exogenous DNA, intragenic DNA, exogenous RNA, or combinations, complexes, carrier systems, and/or particle gun systems thereof; and/or viral vector-mediated, Agrobacterium vector-mediated, and/or CRISPR vector- mediated gene transformation of exogenous and/or intragenic DNA. The method of any one of claims 94-98, wherein said direct transfer step is accomplished by: passive uptake; electroporation; polyethylene glycol treatment; electrophoresis; cell fusion with liposomes or spheroplasts; injection, silicon carbide whiskers, particle gun bombardment, spraying, soaking, pipetting, brushing, cationic nanoparticle carriers, clay nanosheet carriers, surfactant complexes, and/or peptide- based carriers.
109 The method of any one of claims 94-99, wherein said driving step further comprises the step of driving RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of a gene capable of synthesis of an activator of CAX1 and, optionally, a gene capable of synthesis of an activator of at least one of CAX2, CAX3, or CAX4. The method of any one of claims 94-100, wherein said step of driving RNA interference (RNAi) further comprises the step of expressing double stranded RNA (dsRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), small RNA (sRNA), microMRNA (mRNA), pre-miRNA, and/or pri-miRNA capable of RNA interference (RNAi) of a gene capable of synthesis of at least one activator of CAX1 and, optionally, a gene capable of synthesis of an activator of at least one of CAX2, CAX3, or CAX. The method of any one of claims 94-101, wherein said agent(s) comprise double stranded RNA (dsRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), small RNA (sRNA), microMRNA (mRNA), pre-miRNA, and/or pri-miRNA capable of RNA interference (RNAi) of a gene capable of synthesis of an activator of CAX1 and, optionally, a gene capable of synthesis of an activator of at least one of CAX2, CAX3, or CAX4. The method of any one of claims 94-99, wherein said inducing step further comprises the step of inducing said agent(s) to drive RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of a gene capable of synthesis of an activator of CAX1 and, optionally, a gene capable of synthesis of an activator of at least one of CAX2, CAX3, or CAX4. The method of any one of claims 94-100, wherein said step of inducing said agent(s) to drive RNA interference (RNAi) further comprises the step of inducing said agent to express double stranded RNA (dsRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), small RNA (sRNA), microMRNA (mRNA), pre-miRNA, and/or pri-miRNA capable of RNA interference (RNAi) of a gene capable of synthesis of an activator of CAX1 and, optionally, a gene capable of synthesis of an activator of at least one of CAX2, CAX3, or CAX4, wherein said inducing step is accomplished using said regulatory sequence.
110 The method of any one of claims 94-104, wherein said regulatory sequence is tissue specific and/or cell-type specific. The method of any one of claims 94-105, wherein said regulatory sequence is low oxygen-induced, chemical-induced, temperature-induced, and/or light-induced. The method of any one of claims 94-106, wherein said step of suppressing and/or eliminating expression of a gene capable of synthesis of an activator of CAX1 and, optionally, a gene capable of synthesis of an activator of at least one of CAX2, CAX3, or CAX4, occurs before, during, and/or after anoxic conditions. The method of any one of claims 94-107, wherein said step of driving RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of a gene capable of synthesis of an activator of CAX1 and, optionally, a gene capable of synthesis of an activator of at least one of CAX2, CAX3, or CAX4, occurs before, during, and/or after anoxic conditions. The method of any one of claims 94-108, wherein said step of suppressing and/or eliminating synthesis of at least one activator of CAX1 and, optionally, at least one activator of at least one of CAX2, CAX3, or CAX4, is transient and/or stable. The method of any one of claims 94-109, wherein said step of driving RNA interference (RNAi), transcriptional gene silencing (TGS), and/or virus induced gene silencing (VIGS) of a gene capable of synthesis of an activator of CAX1 and, optionally, a gene capable of synthesis of an activator of at least one of CAX2, CAX3, or CAX4, is transient and/or stable. The method of any one of claims 94- 110, wherein said plurality of plant cells upregulate expression during anoxia of at least one gene selected from the group consisting of: AT1G01355; AT1G02610; AT1G02620; AT1G03090; AT1G03610; AT1G06570; AT1G08630; AT1G08930; AT1G10070; AT1G10140; AT1G10550; AT1G11260; AT1G12780; AT1G14860; AT1G15040; AT1G15330; AT1G15670; AT1G17290; AT1G183OO; AT1G18773; AT1G19396; AT1G19530; AT1G19540; AT1G19610; AT1G19620; AT1G24880; AT1G25560; AT1G27045; AT1G28330; AT1G30135; AT1G30720; AT1G32910; AT1G33050; AT1G33055; AT1G34140; AT1G34760; AT1G35140; AT1G36060; AT1G54100; AT1G54760; AT1G55810; AT1G56250;
111 AT1G58936; AT1G60750; AT1G62480; AT1G62510; AT1G63090; AT1G65970;
AT1G66860; AT1G67265; AT1G68935; AT1G71520; AT1G72060; AT1G72416;
AT1G75750; AT1G76650; AT1G77120; AT1G79700; AT1G79910; AT1G80160;
AT1G80570; AT1G80840; AT2G05400; AT2G07633; AT2G07638; AT2G07641;
AT2G07648; AT2G07658; AT2G07665; AT2G07669; AT2G07673; AT2G07674;
AT2G07678; AT2G07689; AT2G07696; AT2G07698; AT2G07714; AT2G07721;
AT2G07724; AT2G07725; AT2G07727; AT2G07734; AT2G07749; AT2G07771;
AT2G07773; AT2G07785; AT2G07798; AT2G07815; AT2G07825; AT2G07835;
AT2G14210; AT2G14900; AT2G15880; AT2G15960; AT2G16060; AT2G16586;
AT2G17036; AT2G17850; AT2G19590; AT2G19800; AT2G20670; AT2G22880;
AT2G22980; AT2G25770; AT2G25780; AT2G26130; AT2G30600; AT2G30615;
AT2G31810; AT2G33160; AT2G33830; AT2G34390; AT2G34555; AT2G36110;
AT2G36950; AT2G37025; AT2G39400; AT2G39570; AT2G43520; AT2G44080;
AT2G47520; AT2G47950; AT3G02550; AT3G03270; AT3G04160; AT3G06435;
AT3G10040; AT3G1331O; AT3G13450; AT3G15440; AT3G15450; AT3G15620;
AT3G16770; AT3G17225; AT3G18530; AT3G19680; AT3G20395; AT3G21352;
AT3G22640; AT3G23150; AT3G23550; AT3G27220; AT3G28740; AT3G29370;
AT3G29810; AT3G29970; AT3G30775; AT3G43190; AT3G43850; AT3G44300;
AT3G45300; AT3G46230; AT3G47340; AT3G47675; AT3G47720; AT3G47870;
AT3G48360; AT3G48530; AT3G50560; AT3G51840; AT3G51910; AT3G55240;
AT3G55970; AT3G59060; AT3G61060; AT3G61260; AT3G62150; AT4G01120;
AT4G01250; AT4G02170; AT4G02380; AT4G02430; AT4G03205; AT4G04423;
AT4G10250; AT4G10265; AT4G10270; AT4G15760; AT4G16160; AT4G16162;
AT4G 16563; AT4G19230; AT4G19880; AT4G20953; AT4G22710; AT4G22780;
AT4G24040; AT4G24110; AT4G24230; AT4G25580; AT4G25707; AT4G25810;
AT4G26460; AT4G27450; AT4G28040; AT4G28811; AT4G30270; AT4G30370;
AT4G30380; AT4G32480; AT4G32630; AT4G32840; AT4G33070; AT4G33150;
AT4G33560; AT4G33970; AT4G34030; AT4G35770; AT4G36690; AT4G36730;
AT4G36850; AT4G37220; AT4G38470; AT4G39675; AT5G01740; AT5G01810;
AT5G02200; AT5G03380; AT5G03830; AT5G05530; AT5G06980; AT5G07440;
AT5G07560; AT5G07570; AT5G08150; AT5G10040; AT5G11090; AT5G12020;
AT5G12030; AT5G14180; AT5G14470; AT5G15120; AT5G15250; AT5G18170;
AT5G19120; AT5G19550; AT5G20240; AT5G20250; AT5G20830; AT5G21170;
112 AT5G22920; AT5G26200; AT5G27893; AT5G28610; AT5G28630; AT5G28770;
AT5G34830; AT5G35525; AT5G39160; AT5G39200; AT5G39580; AT5G39890;
AT5G40450; AT5G41080; AT5G42825; AT5G43570; AT5G45340; AT5G47590;
AT5G47910; AT5G49360; AT5G54080; AT5G56100; AT5G56870; AT5G57220;
AT5G57550; AT5G57660; AT5G62520; AT5G63160; AT5G65207; AT5G66110;
ATMG00010; ATMG00060; ATMG00070; ATMG00080; ATMG00090;
ATMG00110; ATMG00140; ATMG00160; ATMG00260; ATMG00270;
ATMG00285; ATMG00310; ATMG00400; ATMG00410; ATMG00510;
ATMG00513; ATMG00516; ATMG00560; ATMG00570; ATMG00630;
ATMG00640; ATMG00650; ATMG00680; ATMG00690; ATMG00730;
ATMG00900; ATMG00940; ATMG00960; ATMG00970; ATMG01000;
ATMG01050; ATMG01120; ATMG01130; ATMG01170; ATMG01190;
ATMG01200; ATMG01210; ATMG01220; ATMG01230; ATMG01260;
ATMG01280; ATMG01320; and ATMG01360. The method of any one of claims 94- 111, wherein said plurality of plant cells upregulate expression during recovery from anoxia of at least one gene selected from the group consisting of: AT1G01720; AT1G03070; AT1G03090; AT1G03610; AT1G05575; AT1G06030; AT1G07330; AT1G07350; AT1G07400; AT1G07500; AT1G08630; AT1G09950; AT1G10140; AT1G12610; AT1G14200; AT1G15040; AT1G15330; AT1G15840; AT1G16030; AT1G17870; AT1G183OO; AT1G1833O; AT1G19530; AT1G19540; AT1G19620; AT1G21340; AT1G21940; AT1G22110; AT1G26800; AT1G27730; AT1G28760; AT1G30070; AT1G30135; AT1G31370; AT1G32910; AT1G32928; AT1G33055; AT1G33730; AT1G33760; AT1G34575; AT1G35140; AT1G44414; AT1G50745; AT1G52560; AT1G53540; AT1G54050; AT1G55530; AT1G55810; AT1G56170; AT1G56250; AT1G59860; AT1G59865; AT1G60190; AT1G61340; AT1G66060; AT1G66080; AT1G66400; AT1G66500; AT1G66510; AT1G67265; AT1G71000; AT1G71520; AT1G72060; AT1G72416; AT1G72660; AT1G72760; AT1G73480; AT1G74310; AT1G74450; AT1G74930; AT1G75490; AT1G75750; AT1G76600; AT1G76640; AT1G76650; AT1G77120; AT1G80840; AT2G01390; AT2G07687; AT2G07696; AT2G07698; AT2G07707; AT2G07724; AT2G07725; AT2G07727; AT2G07734; AT2G07771; AT2G07773; AT2G07785; AT2G07825; AT2G07835; AT2G14900; AT2G15960; AT2G16060; AT2G17850; AT2G20560; AT2G20670; AT2G22880; AT2G23110; AT2G23190; AT2G24100;
113 AT2G25140; AT2G26150; AT2G27580; AT2G29500; AT2G30615; AT2G32120;
AT2G34390; AT2G34600; AT2G36220; AT2G38240; AT2G38340; AT2G40340;
AT2G40955; AT2G44070; AT2G44080; AT2G44130; AT2G44840; AT2G46240;
AT2G46790; AT2G46830; AT2G47180; AT2G47520; AT3G02550; AT3G06435;
AT3G07090; AT3G07150; AT3G07350; AT3G08970; AT3G09350; AT3G09640;
AT3G10020; AT3G10040; AT3G11020; AT3G12190; AT3G12320; AT3G12580;
AT3G1331O; AT3G14200; AT3G15440; AT3G15450; AT3G15500; AT3G16050;
AT3G17400; AT3G19240; AT3G20340; AT3G20395; AT3G22090; AT3G22100;
AT3G23150; AT3G24500; AT3G25250; AT3G27220; AT3G28210; AT3G28740;
AT3G29370; AT3G29810; AT3G29970; AT3G30775; AT3G43850; AT3G44190;
AT3G46070; AT3G46230; AT3G47340; AT3G47720; AT3G48240; AT3G49160;
AT3G50310; AT3G50560; AT3G51910; AT3G53830; AT3G55580; AT3G55840;
AT3G62260; AT3G63350; AT4G01250; AT4G01435; AT4G02170; AT4G02425;
AT4G02550; AT4G02690; AT4G09150; AT4G10250; AT4G10265; AT4G10270;
AT4G 11660; AT4G12400; AT4G12410; AT4G13395; AT4G15280; AT4G15420;
AT4G15760; AT4G16555; AT4G17250; AT4G18450; AT4G19570; AT4G21320;
AT4G23493; AT4G24110; AT4G24410; AT4G24570; AT4G25200; AT4G25380;
AT4G25470; AT4G25490; AT4G25580; AT4G25810; AT4G26200; AT4G26460;
AT4G27410; AT4G27450; AT4G27652; AT4G27654; AT4G27657; AT4G27670;
AT4G28811; AT4G29770; AT4G29780; AT4G30270; AT4G30370; AT4G32208;
AT4G32480; AT4G33070; AT4G34131; AT4G34410; AT4G35770; AT4G36850;
AT4G37710; AT4G38030; AT5G01740; AT5G02170; AT5G02810; AT5G03210;
AT5G03720; AT5G04340; AT5G04400; AT5G05220; AT5G05410; AT5G06980;
AT5G07330; AT5G08150; AT5G09590; AT5G09930; AT5G10040; AT5G10336;
AT5G10695; AT5G12020; AT5G12030; AT5G12110; AT5G13220; AT5G14470;
AT5G15120; AT5G15250; AT5G15450; AT5G17350; AT5G18065; AT5G18340;
AT5G20250; AT5G22680; AT5G22920; AT5G25450; AT5G27660; AT5G35320;
AT5G37340; AT5G37670; AT5G39580; AT5G39890; AT5G43620; AT5G43650;
AT5G45340; AT5G45640; AT5G47220; AT5G47590; AT5G47830; AT5G48570;
AT5G49920; AT5G51190; AT5G51440; AT5G51990; AT5G52050; AT5G52630;
AT5G52640; AT5G53680; AT5G54165; AT5G57260; AT5G57550; AT5G57560;
AT5G57660; AT5G58070; AT5G59720; AT5G59820; AT5G62020; AT5G62520;
AT5G63130; AT5G63300; AT5G64170; AT5G64210; AT5G64510; AT5G66650;
114 AT5G67080; ATMG00060; ATMG00080; ATMG00090; ATMG00160;
ATMG00270; ATMG00285; ATMG00400; ATMG00410; ATMG00480;
ATMG00510; ATMG00513; ATMG00516; ATMG00560; ATMG00570;
ATMG00640; ATMG00650; ATMG00690; ATMG00730; ATMG00900;
ATMG00940; ATMG00960; ATMG00990; ATMG01000; ATMG01050;
ATMG01120; ATMG01190; ATMG01200; ATMG01320; and ATMG01360. The method of any one of claims 74-112, wherein said progeny is produced by propagating said genetically modified plant or said part thereof, using asexual or sexual reproduction. A genetically modified seed produced by said genetically modified plant, part, and/or progeny thereof, prepared according to any one of the methods of claims 74-113. A genetically modified plant, part, and/or progeny thereof, propagated and/or regenerated from said seeds, said spores, said grafts comprising genetically modified scions and/or rootstocks, said buddings comprising genetically modified buds and/or rootstocks, said cuttings, said bulbs, said tubers, said rhizomes, said regenerable cells, said tissue cultures of regenerable cells, said regenerable protoplasts, said tissue cultures of regenerable protoplasts, and/or said other plant germplasms of said genetically modified plant, part, and/or progeny thereof, produced according to any one of the methods of claims 74-114. A crop produced by said genetically modified plant, part, and/or progeny thereof, prepared according to any one of the methods of claims 74-115.
115
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CN117904187A (en) * 2024-01-04 2024-04-19 南京农业大学 Application of calcium hydrogen transporter OsCAX a in regulation and control of rice stomata development and/or photosynthesis capacity
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CN115595331A (en) * 2022-10-13 2023-01-13 浙江农林大学（Cn） Infection liquid and cinnamomum camphora instantaneous transformation method
JP2024110396A (en) * 2023-02-02 2024-08-15 国立研究開発法人農業・食品産業技術総合研究機構 Agrobacterium bacteria, Agrobacterium bacterial strains, Rhizobium bacterial strains, soybean cultivation treatment agents, soybean cultivation soil production method, soybean cultivation seed production method, and soybean cultivation method
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CN116536336A (en) * 2023-03-15 2023-08-04 重庆文理学院 Longan fruit size and number regulating gene DlCYP71B10-like and application thereof
CN116536336B (en) * 2023-03-15 2023-12-22 重庆文理学院 Longan fruit size and number regulating gene DlCYP71B10-like and application thereof
CN117904187A (en) * 2024-01-04 2024-04-19 南京农业大学 Application of calcium hydrogen transporter OsCAX a in regulation and control of rice stomata development and/or photosynthesis capacity
CN118063577A (en) * 2024-03-15 2024-05-24 中国热带农业科学院南亚热带作物研究所 Pineapple Ca2+/H+ antiporter AcCAX3, isolated nucleic acid molecules and their applications
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