[go: up one dir, main page]

WO2025057172A1 - Methods of improving tolerance of plants to stress and plants generated thereby - Google Patents

Methods of improving tolerance of plants to stress and plants generated thereby Download PDF

Info

Publication number
WO2025057172A1
WO2025057172A1 PCT/IL2024/050929 IL2024050929W WO2025057172A1 WO 2025057172 A1 WO2025057172 A1 WO 2025057172A1 IL 2024050929 W IL2024050929 W IL 2024050929W WO 2025057172 A1 WO2025057172 A1 WO 2025057172A1
Authority
WO
WIPO (PCT)
Prior art keywords
stress
plant
plants
expression
tolerance
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/IL2024/050929
Other languages
French (fr)
Inventor
Moshe Sagi
Zhadyrassyn NURBEKOVA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BG Negev Technologies and Applications Ltd
Original Assignee
BG Negev Technologies and Applications Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by BG Negev Technologies and Applications Ltd filed Critical BG Negev Technologies and Applications Ltd
Publication of WO2025057172A1 publication Critical patent/WO2025057172A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • 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
    • C12N15/8273—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 for drought, cold, salt 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C12N9/222—Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes
    • C12N9/226—Class 2 CAS enzyme complex, e.g. single CAS protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C12Y102/03—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with oxygen as acceptor (1.2.3)
    • C12Y102/03001—Aldehyde oxidase (1.2.3.1), i.e. retinal oxidase
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

  • the present invention in some embodiments thereof, relates to methods of improving tolerance of plants to stress and plants generated.
  • Aldehyde oxidases are a multigene family that oxidizes a variety of aldehydes, including the oxidation of abscisic aldehyde (ABAld) to the phytohormone abscisic acid (ABA).
  • ABAld abscisic aldehyde
  • the protein architecture of the Arabidopsis AO (AAO) multigene family comprises FAD, Fe-S and molybdenum cofactor (Moco) domains as its prosthetic groups (Koshiba et al., 1996 Plant Physiology Volume 110, Issue 3, March 1996, Pages 781-789).
  • the approximate molecular mass of the AO monomer is ca.
  • AOs carry their catalytic function by forming homodimers as well as heterodimers in plants (Akaba et al., 1999 The Journal of Biochemistry, Volume 126, Issue 2, August 1999, Pages 395-401).
  • Arabidopsis four AO genes encode AAO1, AAO2, AAO3 and AAO4, and their expression patterns have been shown to be tissue specific; AAO1 is predominantly expressed in seedlings, roots, stem and seeds, but shows a significant expression in rosette leaves as well.
  • AAO2 is mainly expressed in seedlings and root and in rosette leaves
  • AAO3 is expressed in seedlings (at lower levels than AAO1 and AAO2), roots, stem flowers and rosette leaves
  • AAO4 is abundant in siliques but is expressed to a certain level in flower, root and stem.
  • the AAOs are characterized by differential substrate specificities that play a key role in identifying and assigning their biological roles.
  • AAO1 and AAO2 homodimers catalyze the oxidation of indole-3 -acetaldehyde and 1 -naphthaldehyde, respectively, with very high efficiency, whereas their heterodimer (AAO1 ::AAO2) exhibits intermediate substrate specificities, oxidizing both aldehydes with intermediate specificity.
  • the homodimer of AAO3 and its heterodimer with AA02 oxidize ABald to ABA.
  • AA03 has received special attention owing to its involvement in ABA biosynthesis and its importance in normal and stress conditions.
  • AA01 was shown to be implicated in the biosynthesis of indole-3 -carboxylic acid, yet further roles of AA01 as well as the role of AA02 is not known (Nurbekova et al., 2021 5(108) December 2021 Pages 1439-1455, 2024, Seo et al., 2000 The Plant J. 23(4):481-488; Seo et al. 2000 PNAS 97 23 12908-12913).
  • Aldehydes can be extremely toxic when produced in excess because of their inherent chemical reactivity and under normal physiological conditions aldehydes are formed constitutively and need to be detoxified. Yet, there is an increasing body of evidence for the generation of toxic levels of aldehydes in response to environmental stresses, especially lipid peroxidation-derived reactive carbonyl species such as malondialdehyde (MDA), acrolein and 4-hydroxyl-2-nonenal (HNE).
  • MDA malondialdehyde
  • HNE 4-hydroxyl-2-nonenal
  • reactive carbonyl species RCS
  • ROS reactive oxygen species
  • Detoxification by oxidation of toxic aldehydes was attributed to several enzymes but rarely to aldehyde oxidases. Recently demonstrated was the importance of active AA03 and AA04 in delaying rosette leaves and siliques senescence respectively, by oxidizing toxic aldehydes accumulated in siliques or leaves exposed to toxic aldehyde, dark stress UV-C irradiation or natural senescence (Srivastava et al., 2017; Nurbekova et al., 2021).
  • Nurbekova et al. 2021 states: that AA01 and AA02 activities do not play a role in UV-C sensitivity in AO3 knockout plants.
  • a method of conferring stress tolerance to a plant comprising down-regulating expression of an aldehyde oxidase 1 (AO1) and/or aldehyde oxidase 2 (AO2), thereby conferring stress tolerance to the plant.
  • AO1 aldehyde oxidase 1
  • AO2 aldehyde oxidase 2
  • a plant having been treated with an agent down-regulating expression of aldehyde oxidase 1 (AO1) and/or aldehyde oxidase 2 (AO2) such that the plant or plant cell exhibits reduced expression of AO1 and/or AO2, as compared to a control plant.
  • a plant cell having been treated with an agent down-regulating expression of aldehyde oxidase 1 (AO1) and/or aldehyde oxidase 2 (AO2) such that the plant cell exhibits reduced expression of AO1 and/or AO2, as compared to a control plant cell.
  • AO1 aldehyde oxidase 1
  • AO2 aldehyde oxidase 2
  • a method of producing a plant exhibiting tolerance to stress comprising growing the plant as described herein or regenerating the cell as described herein.
  • a method of selecting a plant exhibiting tolerance to stress comprising:
  • the down-regulating expression is of AO2.
  • the down-regulating expression is of AO1.
  • the down-regulating expression is of AO2 and AO1.
  • the stress is abiotic stress.
  • the abiotic stress is selected from the group consisting of drought stress, oxidative stress, radiation stress, temperature stress, light stress, nutrient stress, heavy metal stress, salinity stress wounding stress and flooding stress.
  • the abiotic stress is selected from the group consisting of drought stress, oxidative stress and radiation stress.
  • the abiotic stress is drought stress.
  • the drought stress comprises extreme drought stress, as defined by fast water loss (8 to 16% within 3 to 7 hours).
  • the abiotic stress is radiation stress.
  • the stress is not UV-C stress.
  • the stress is biotic stress.
  • the growing is under stress conditions.
  • the plant is a crop plant.
  • the down-regulating expression is by a nucleic acid agent.
  • the nucleic acid agent is a genome editing agent or an RNA editing agent.
  • the nucleic acid agent is an RNA silencing agent.
  • the down-regulating expression is in a constitutive manner.
  • the down-regulating expression is in a tissue specific manner.
  • the down-regulating is in a leaf tissue.
  • FIGs. 1A-C show Arabidopsis aldehyde oxidases activity assessment with different aldehydes.
  • AO enzyme activity was determined in a reaction solution containing 100 mM Tris-HCl (pH 7.5), 1 mM 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 0.1 mM phenazine methosulfate (PMS) and 1 mM aldehydes (except for HNE and abscisic aldehyde loaded with 0.25mM and 0.2 mM, respectively).
  • reaction was stopped after 4h with aaolKO mutant and 3 h with WT and aao2 KO mutant [SALK l 04895 (KO-95)] by immersing the gel in 5% acetic acid and band images were captured and analyzed for relative intensity (RI) of AAO1/AAO3 using ImageJ software (www(dot)/imagej (dot)nih(dot)gov/ij/).
  • FIGs. 2A-D show the effects of Rose Bengal on chlorophyll and aldehyde level in leaves of Arabidopsis wild-type (WT) and aao2-KO plants.
  • A. Appearance of control (water treated) and Rose Bengal treated WT and aao2 plants. Representative photograph of wild-type and aao2 [SALK l 04895 (KO-95) and SAIL 563 G09 (KO-563)] appearance in response to Rose Bengal. 23-day old plants were sprayed with 0.05 mM Rose Bengal and photographed 3 d later and
  • B Remaining chlorophyll in rosette leaves (left to right are the oldest to younger leaves) was determined.
  • FIGs. 3A-D show in gel aldehyde oxidase activity of wild-type and the Arabidopsis aldehyde oxidases (AAOs) double mutant (in which there is a single functioning AAO I (.suu ⁇ >/).
  • AAOs Arabidopsis aldehyde oxidases
  • 150 pg crude protein extract from rosette leaves of WT and saaol, saao2 and saao3 were fractionated by NATIVE PAGE for the activity assay using A. abscisic aldehyde, B. Indole-3 carbaldehyde (I3CA), C. Zrans-2-nonenal, D. Benzaldehyde.
  • Relative intensity (RI) was calculated for each band by using ImageJ software (https://imagei.nih.gov/ii/). The intensities of the activity bands were compared with those obtained with WT control (as 100%).
  • FIG. 4 show the relative transcript expression of AA01 (At5g20960), AA02 (At3g43600) and AA03 (At2g27150) in rosette leaves of 23-days post germination Arabidopsis WT, aaolSingle (aaolS) and independent aao3Singles (aao3Ss) mutant plants.
  • aaolS was generated by silencing AA03 in aao2 [SALK 104895 (KO-95)], and aao3Ss was generated by silencing AA01 in aao2( KO-95) plants or silencing AA02 in aaol [SALK 018100 (al -100)] plants.
  • FIGs. 5A-E show the determination of UV-C-irradiation-induced senescence and senescence-related factors in rosette leaves of Arabidopsis aldehyde oxidases single mutants [aaolSingle (aaolS) and aao3Singles (aao3Ss)], aao2KO [SALK 104895 (KO-95) and wild-type (WT) plants.
  • A Representative photograph of WT, aao2, aaolS and aao3Ss (aao3S-l, aao3S-12, aao3S-18) rosette leaves in untreated (control) and UV-C irradiation treated plants.
  • Aldehyde oxidase 3 (AA03) in gel activity in control and UV-C treated WT, aao2 [KO-95), SAIL 563 G09 (KO-563)] and aao3Ss (aao3S-l, aao3S-12, aao3S-18) using abscisic aldehyde as the specific substrate for AA03.
  • aao2 (KO-95 and KO-563)
  • aao3Ss (aao3S-l, aao3S-12, aao3S-18) rosette leaves were fractionated by NATIVE PAGE and were used for activity.
  • FIGs. 6A-E show the determination of Rose-Bengal -induced senescence and senescence- related factors in rosette leaves of Arabidopsis aldehyde oxidases single mutants [aaol Single (aaolS) and aao3Singles (aao3Ss)], aao2KO [SALK104895 (KO-95) and wild-type (WT) plants.
  • Aldehyde oxidases 3 (AAO3) in gel activity in control and Rose-Bengal treated WT, aao2 [KO- 95), SAIL 563 G09 (KO-563)] and aao3Ss using abscisic aldehyde as the specific substrate for AAO3.
  • AAO3 Aldehyde oxidases 3
  • FIGs. 7A-C show that. Determination of UV-C-irradiation-induced senescence in rosette leaves of Arabidopsis aldehyde oxidases al -100 [(SALK 018100 (aaol KO)] as well as AA01 OE (AAO1-OE was described in Nurbekova et al., 2021).
  • 21-days post germination plants were exposed to UV-C irradiation (100 mJ) and were kept in a growth room for 96 hours and thereafter documented together with rosette leaves of plants not exposed to UV-C (control), (a) Representative photograph of WT, al -100 andAAOl-OE rosette leaves in untreated (control) and UV-C irradiation treated plants.
  • FIGs. 8A-B show water loss in detached rosette leaves of 24 d old Arabidopsis WT, aao2 mutant (KO-95), aaol single mutant [al-11-10-(95)] and aao3 single mutants [(a3-l-95) and (a3- 18-7-(l 00)] grown in soil.
  • A. Plants were detached and kept in covered 20x20 cm Petri dishes for 3 and 7 hours. Error bars represent ⁇ SE (n 6 similar positioned rosette leaves from 6 different plants).
  • RWC Relative Water Content
  • aao3S is the average of 2 independent single aao3 (a3-l-95) and (a3-18-7-100) and aao2KO is the average of 2 independent KO of aao2 (KO-95 and KO-563). Values denoted with different letters above the bars are significantly different according to the Turkey-Kramer HSD mean-separation test (P ⁇ 0.05).
  • the present invention in some embodiments thereof, relates to methods of improving tolerance of plants to stress and plants generated.
  • AOs Aldehyde oxidases
  • Four AO genes encode AO1, AO2, AO3 and AO4, and their expression patterns have been shown to be tissue specific. Recently demonstrated was the importance of active AO3 in delaying rosette leaves senescence, by oxidizing toxic aldehydes accumulated in leaves exposed to toxic aldehyde, dark stress, UV-C irradiation or natural senescence.
  • stress tolerance can be achieved by down-regulating the expression of aldehyde oxidase 1 (AO1, AAO1 in Arabidopsis) and aldehyde oxidase 2 (AO2, AAO2 in Arabidopsis), thereby augmenting the stress tolerance activity of aldehyde oxidase 3 (AO3, AAO3 in Arabidopsis).
  • aldehyde modifying enzymes such as aldehyde dehydrogenase and aldehyde oxidases e.g., AO3 increases detoxification of aldehydes, hence it is unexpected that reducing the levels of such enzymes would be beneficial.
  • aldehyde modifying enzymes such as aldehyde dehydrogenase and aldehyde oxidases e.g., AO3 increases detoxification of aldehydes, hence it is unexpected that reducing the levels of such enzymes would be beneficial.
  • the present inventors demonstrated enhancement of AA01 and/or AA03 oxidizing activity on a variety of aldehydes by knocking out AA02 in aao2 mutant (Example 1).
  • AA02 leading to reduced protein expression level affected AA03 and AAOl’s capacity to oxidize specific aldehydes under oxidation stress induced by Rose-Bengal (Example 2).
  • Down-regulation of AA01 and AA02 indicated that AA02 protein expression level affects AA03 capacity to oxidize toxic aldehydes in rosette leaves of plants exposed to irradiation (UV-C) or oxidation stress induced by Rose-Bengal application (Example 3).
  • Example 4 showed that mutant impaired in AA01 and AA02 expression causes significant improvement in chlorophyll levels as compared to wild type (WT) in response to stress.
  • a method of conferring stress tolerance to a plant comprising down-regulating expression of an aldehyde oxidase 1 (AO1) and/or aldehyde oxidase 2 (AO2), thereby conferring stress tolerance to the plant.
  • AO1 aldehyde oxidase 1
  • AO2 aldehyde oxidase 2
  • plant encompasses a whole plant, a grafted plant, ancestor(s) and progeny of the plants and plant parts, including seeds, shoots, stems, roots (including tubers), rootstock, scion, and plant cells, tissues and organs.
  • the plant may be in any form including suspension cultures, embryos, meristematic regions, callus tissue, leaves, gametophytes, sporophytes, pollen, and microspores.
  • Plants that are particularly useful in the methods of the invention include all plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants including a fodder or forage legume, ornamental plant, food crop, tree, or shrub selected from the list comprising Acacia spp., Acer spp., Actinidia spp., Aesculus spp., Agathis australis, Albizia amara, Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadaba farinosa, Calliandra spp, Camellia sinensis, Canna indica, Capsicum spp., Cassia spp., Centroe
  • the plant used by the method of the invention is a crop plant such as rice, maize, wheat, barley, peanut, potato, sesame, olive tree, palm oil, banana, soybean, sunflower, canola, sugarcane, alfalfa, millet, leguminosae (bean, pea), flax, lupinus, rapeseed, tobacco, poplar and cotton.
  • a crop plant such as rice, maize, wheat, barley, peanut, potato, sesame, olive tree, palm oil, banana, soybean, sunflower, canola, sugarcane, alfalfa, millet, leguminosae (bean, pea), flax, lupinus, rapeseed, tobacco, poplar and cotton.
  • the plant is a dicotyledonous plant.
  • the plant is a monocotyledonous plant.
  • tolerance refers to the ability of a plant to withstand or cope with adverse environmental conditions that would normally negatively impact growth, development, or yield of a plant of a given species at a given developmental stage.
  • tolerance is generally interchangeably used with resistance, though in some cases they have different meanings. For instance, in the case of biotic stress tolerance or resistance:
  • resistance is as defined by the ISF (International Seed Federation) Vegetable and Ornamental Crops Section for describing the reaction of plants to pests or pathogens, and abiotic stresses for the Vegetable Seed Industry. Specifically, by resistance, it is meant the ability of a plant variety to restrict at least to some degree the multiplication of the virus. Symptoms, even if present, are mild as compared to susceptible plants.
  • tolerant plants are therefore resistant to symptom expression or are symptomless carriers of the virus.
  • conferring refers to increasing tolerance or resistance of a plant to stress conditions.
  • increasing refers to a statistically significant increase in tolerance compared to the level of tolerance obtained in plants of the same species and developmental stage, as typically determined quantitatively.
  • the increase can be by at least, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 2 fold, 3 fold, 5 fold or 10 fold.
  • the level of oxidized aldehydes can be a proxy for tolerance to stress, it can be used for following or for predicting the level of tolerance.
  • plant aldehyde refers to organic compounds characterized by the presence of a carbonyl group bonded to a hydrogen atom. They are common in plants and play significant roles in various physiological processes, including growth, defense, and aroma.
  • Aldehyde oxidation is a measure of stress, and measurement is typically effected in the leaf tissue or siliques.
  • plant aldehydes for which oxidized state tissue level is determined include, but are not limited to, cinnamaldehyde, vanillin, benzaldehyde, hexanal, hexenal, nonanal, citral (neral and geranial), formaldehyde, acetaldehyde, salicylaldehyde, furfural, anisaldehyde, trans-2- hexenal, octanal, 2,4-decadienal, abscisic aldehyde, acrolein, propionaldehyde, butyraldehyde, crotonaldehyde, glyoxal, phenylacetaldehyde, 3 -methylbutanal, 2-methylpropanal, decanal, dodecanal, heptanal, dodecenal, sinapaldehyde, coniferyl aldehyde, 4-hydroxynonenal (HNE)
  • the measured aldehydes for which oxidized state tissue level is determined are selected from the group consisting of cinnamaldehyde, benzaldehyde, hexanal, citral (neral and geranial), abscisic acid, acrolein, crotonaldehyde, decanal, dodecanal, heptanal, dodecenal, sinapaldehyde, coniferyl aldehyde, and 4-hydroxynonenal (HNE) and ) and indole-3 -carbaldehy de (ICHO).
  • cinnamaldehyde benzaldehyde
  • hexanal citral (neral and geranial)
  • abscisic acid acrolein, crotonaldehyde, decanal, dodecanal, heptanal, dodecenal, sinapaldehyde, coniferyl aldehyde, and 4-hydroxy
  • the measured oxidized aldehyde is abscisic acid which is specific to AO3.
  • TBARS Thiobarbituric Acid Reactive Substances
  • MDA malondialdehyde
  • TAA thiobarbituric acid
  • HPLC High-Performance Liquid Chromatography
  • MDA and 4-hydroxynonenal HNE
  • Derivatization agents such as 2,4-dinitrophenylhydrazine (DNPH) can be used to form hydrazones for easier detection.
  • DNPH 2,4-dinitrophenylhydrazine
  • GC-MS Gas Chromatography-Mass Spectrometry
  • LC-MS Liquid Chromatography-Mass Spectrometry
  • Spectrophotometric and fluorometric assays can quantify aldehydes based on their ability to form colored or fluorescent products upon reaction with specific reagents like DNPH or Nash reagent. This method is not specific to a given aldehyde.
  • Enzyme-Linked Immunosorbent Assay uses specific antibodies against aldehy demodified proteins or aldehydes to quantify them in a sample.
  • Nuclear Magnetic Resonance (NMR) Spectroscopy detects aldehydes based on their unique magnetic resonance signals in a magnetic field. This method provides non-destructive analysis and detailed information about molecular structures.
  • the Thiobarbituric Acid Reactive Substances (TBARS) assay is used for tor MDA.
  • aldehyde oxidation is determined using HPLC and/or Mass Spectrometry detection.
  • phenotypic appearance can be used to measure tolerance such as leaf color as in Figure 2A.
  • Other measures can be used to measure tolerance to stress, such as biomass, dry weight, growth rate, vigor, yield, seed set, oil content, fiber yield, fiber quality, fiber length, plant height, , photosynthetic capacity, fertilizer use efficiency (e.g., nitrogen use efficiency), and more.
  • plant vigor refers to the amount (measured by weight) of tissue produced by the plant in a given time. Hence increased vigor could determine or affect the plant yield or the yield per growing time or growing area. In addition, early vigor (seed and/or seedling) results in improved field stand.
  • plant yield refers to the amount (e.g., as determined by weight or size) or quantity (numbers) of tissues or organs produced per plant or per growing season. Hence increased yield could affect the economic benefit one can obtain from the plant in a certain growing area and/or growing time.
  • a plant yield can be affected by various parameters including, but not limited to, plant biomass; plant vigor; growth rate; seed yield; seed or grain quantity; seed or grain quality; oil yield; content of oil, starch and/or protein in harvested organs (e.g., seeds or vegetative parts of the plant); number of flowers (florets) per panicle (expressed as a ratio of number of filled seeds over number of primary panicles); harvest index; number of plants grown per area; number and size of harvested organs per plant and per area; number of plants per growing area (density); number of harvested organs in field; total leaf area; carbon assimilation and carbon partitioning (the distribution/allocation of carbon within the plant); resistance to shade; number of harvestable organs (e.g.
  • seed yield refers to the number or weight of the seeds per plant, pod or spike weight, seeds per pod, or per growing area or to the weight of a single seed, or to the oil extracted per seed. Hence seed yield can be affected by seed dimensions (e.g., length, width, perimeter, area and/or volume), number of (filled) seeds and seed filling rate and by seed oil content.
  • increase seed yield per plant could affect the economic benefit one can obtain from the plant in a certain growing area and/or growing time; and increase seed yield per growing area could be achieved by increasing seed yield per plant, and/or by increasing number of plants grown on the same given area or by increase harvest index (seed yield per the total biomass).
  • seed also referred to as “grain” or “kernel” as used herein refers to a small embryonic plant enclosed in a covering called the seed coat (usually with some stored food), the product of the ripened ovule of gymnosperm and angiosperm plants which occurs after fertilization and some growth within the mother plant.
  • oil content refers to the amount of lipids in a given plant organ, either the seeds (seed oil content) or the vegetative portion of the plant (vegetative oil content) and is typically expressed as percentage of dry weight (10 % humidity of seeds) or wet weight (for vegetative portion).
  • oil content is affected by intrinsic oil production of a tissue (e.g., seed, vegetative portion), as well as the mass or size of the oil-producing tissue per plant or per growth period.
  • increase in oil content of the plant can be achieved by increasing the size/mass of a plant's tissue(s) which comprise oil per growth period.
  • increased oil content of a plant can be achieved by increasing the yield, growth rate, biomass and vigor of the plant.
  • plant biomass refers to the amount (e.g., measured in grams of air-dry tissue) of a tissue produced from the plant in a growing season, which could also determine or affect the plant yield or the yield per growing area.
  • An increase in plant biomass can be in the whole plant or in parts thereof such as aboveground (harvestable) parts, vegetative biomass, leaf size or area, leaf thickness, roots and seeds.
  • root biomass refers to the total weight of the plant’ s root(s). Root biomass can be determined directly by weighing the total root material (fresh and/or dry weight) of a plant.
  • the root biomass can be indirectly determined by measuring root coverage, root density and/or root length of a plant.
  • plants having a larger root coverage exhibit higher fertilizer (e.g., nitrogen) use efficiency and/or higher water use efficiency as compared to plants with a smaller root coverage.
  • fertilizer e.g., nitrogen
  • root coverage refers to the total area or volume of soil or of any plant-growing medium encompassed by the roots of a plant.
  • the root coverage is the minimal convex volume encompassed by the roots of the plant.
  • each plant has a characteristic root system, e.g., some plants exhibit a shallow root system (e.g., only a few centimeters below ground level), while others have a deep in soil root system (e.g., a few tens of centimeters or a few meters deep in soil below ground level), measuring the root coverage of a plant can be performed in any depth of the soil or of the plant-growing medium, and comparison of root coverage between plants of the same species (e.g., the plant in which there is down-regulation of A01/A02 and control plants as described herein) should be performed by measuring the root coverage in the same depth.
  • a characteristic root system e.g., some plants exhibit a shallow root system (e.g., only a few centimeters below ground level), while others have a deep in soil root system (e.g., a few tens of centimeters or a few meters deep in soil below ground level)
  • measuring the root coverage of a plant can be performed in any depth of the
  • the root coverage is the minimal convex area encompassed by the roots of a plant in a specific depth.
  • root density refers to the density of roots in a given area (e.g., area of soil or any plant growing medium).
  • the root density can be determined by counting the root number per a predetermined area at a predetermined depth (in units of root number per area, e.g., mm , cm or m ).
  • root length refers to the total length of the longest root of a single plant.
  • root length growth rate refers to the change in total root length per plant per time unit (e.g., per day).
  • growth rate refers to the increase in plant organ/tissue size per time (can be measured in cm 2 per day or cm/day).
  • photosynthetic capacity is a measure of the maximum rate at which leaves are able to fix carbon during photosynthesis. It is typically measured as the amount of carbon dioxide that is fixed per square meter per second, for example as pmol m' 2 sec' 1 . Plants are able to increase their photosynthetic capacity by several modes of action, such as by increasing the total leaves area (e.g., by increase of leaves area, increase in the number of leaves, and increase in plant’s vigor, e.g., the ability of the plant to grow new leaves along time course) as well as by increasing the ability of the plant to efficiently execute carbon fixation in the leaves. Hence, the increase in total leaves area can be used as a reliable measurement parameter for photosynthetic capacity increment.
  • plant height refers to measuring plant height as indication for plant growth status, assimilates allocation and yield potential. In addition, plant height is an important trait to prevent lodging (collapse of plants with high biomass and height) under high density agronomical practice.
  • Plant height is measured in various ways depending on the plant species but it is usually measured as the length between the ground level and the top of the plant, e.g., the head or the reproductive tissue.
  • abiotic stress refers to any adverse effect on metabolism, growth, reproduction and/or viability of a plant. Accordingly, abiotic stress can be induced by suboptimal environmental growth conditions such as, for example, salinity, osmotic stress, water deprivation, drought, flooding, freezing, low or high temperature, heavy metal toxicity, anaerobiosis, nutrient deficiency (e.g., nitrogen deficiency or limited nitrogen), atmospheric pollution or UV irradiation.
  • suboptimal environmental growth conditions such as, for example, salinity, osmotic stress, water deprivation, drought, flooding, freezing, low or high temperature, heavy metal toxicity, anaerobiosis, nutrient deficiency (e.g., nitrogen deficiency or limited nitrogen), atmospheric pollution or UV irradiation.
  • the abiotic stress is selected from the group consisting of drought stress, oxidative stress, radiation stress, temperature stress, light stress, nutrient stress, heavy metal stress, salinity stress wound stress and flooding stress.
  • the abiotic stress is selected from the group consisting of drought stress, oxidative stress and radiation stress.
  • the abiotic stress is drought stress.
  • the drought stress comprises extreme drought stress, as defined by fast water loss (8 to 16% within 3 to 7 hours).
  • the abiotic stress is oxidative stress.
  • the abiotic stress is radiation stress.
  • the radiation is UV-C.
  • the stress is not radiation stress.
  • the stress is not UV-C stress.
  • drought conditions refers to growth conditions with limited water availability.
  • extreme drought conditions relates to water deprivation (as represented by root detachment model) where there is fast water loss e.g., 8-16 % within 3-7 hours.
  • oxidative stress refers to a condition where there is an imbalance between the production of reactive oxygen species (ROS) and the plant’s ability to detoxify or neutralize them.
  • ROS reactive oxygen species
  • ROS levels are highly reactive molecules generated during various metabolic processes, particularly under stress conditions such as drought, high light intensity, or pathogen infection.
  • antioxidant defenses comprised of enzymes like catalase and superoxide dismutase, and non-enzymatic antioxidants like ascorbic acid and glutathione — oxidative damage occurs.
  • Oxidative stress thus represents a critical challenge that plants must manage to maintain cellular homeostasis and overall health.
  • reactive carbonyl species can act as agents to mediate reactive oxygen species (ROS) signals to target proteins such as heat shock-responsive gene regulation, ABA signaling for stomatai closure, and auxin signaling for lateral root formation in plants.
  • ROS reactive oxygen species
  • RCS can mediate ROS-induced programmed cell death as well as senescence.
  • Plant aldehyde oxidases were shown to generate H2O2 and 02", while oxidizing various aldehydes (Yesbergenova et al., 2005, Srivastava et al., 2017). Rose Bengal presents a compound, which elicits oxidative stress.
  • UV radiation stress refers to the adverse effects caused by excessive or intense radiation, primarily from sunlight or other artificial sources, on plant health and development. This type of stress often results from high levels of ultraviolet (UV) radiation or excessive visible light, which can lead to the generation of reactive oxygen species (ROS) and cause damage to cellular components like proteins, lipids, and DNA. High-intensity radiation can impair photosynthesis by damaging chlorophyll and disrupting the photosynthetic machinery, reducing the plant’s ability to synthesize essential nutrients and energy. Additionally, radiation stress can trigger protective responses such as the production of UV-ab sorbing compounds and the activation of stress-related genes, but prolonged exposure can overwhelm these defenses, leading to reduced growth, yield loss, and compromised plant health.
  • UV radiation ultraviolet
  • ROS reactive oxygen species
  • temperature stress refers to the physiological and biochemical challenges that arise from exposure to temperatures outside the optimal range for growth and development.
  • Cold stress or low-temperature stress, occurs when plants are exposed to freezing or near-freezing conditions, leading to issues such as ice formation within cells, which can damage cellular structures, disrupt metabolic processes, and impair water uptake. This stress often results in reduced growth, delayed flowering, and diminished yield.
  • heat stress or high- temperature stress, happens when plants experience temperatures significantly above their optimal range, leading to overheating of cellular components, increased water loss through transpiration, and disruption of photosynthesis. Heat stress can cause protein denaturation, enzyme inactivation, and reduced reproductive success, ultimately leading to decreased plant productivity and health.
  • Both cold and heat stress require plants to activate various stress response mechanisms, such as the synthesis of heat shock proteins or cold-responsive proteins, to mitigate damage and maintain homeostasis.
  • nutrient stress refers to a state when there is an inadequate supply or imbalance of essential nutrients required for optimal growth and development. This stress can result from deficiencies or excesses of key nutrients such as nitrogen, phosphorus, potassium, calcium, magnesium, and micronutrients like iron, zinc, and manganese. Nutrient deficiencies can impair various physiological processes, including photosynthesis, protein synthesis, and enzyme function, leading to symptoms such as chlorosis, stunted growth, poor root development, and reduced crop yields. Conversely, nutrient excesses can cause toxicity, alter nutrient uptake dynamics, and disrupt plant metabolism. Effective nutrient management is crucial to prevent nutrient stress and ensure that plants receive the appropriate balance of nutrients for healthy growth and productivity. Nutrient stress can be caused under fertilizer-limiting conditions or nutrientlimiting conditions.
  • fertilizer-limiting conditions refers to growth conditions which include a level (e.g., concentration) of a fertilizer applied which is below the level needed for normal plant metabolism, growth, reproduction and/or viability.
  • nitrogen-limiting conditions refers to growth conditions which include a level (e.g., concentration) of nitrogen (e.g., ammonium or nitrate) applied which is below the level needed for normal plant metabolism, growth, reproduction and/or viability.
  • a level e.g., concentration
  • nitrogen e.g., ammonium or nitrate
  • light stress occurs when plants are exposed to light conditions that are suboptimal or excessively intense, impacting their growth and development. Light stress can result from either too much light, leading to overexposure that causes damage to photosynthetic machinery, chlorophyll degradation, and the formation of reactive oxygen species, or too little light, which can reduce photosynthesis, hinder growth, and limit energy production. Excessive light can cause photoinhibition and damage to cellular structures, while insufficient light can lead to etiolation and poor plant health.
  • wound stress refers to the physiological and biochemical responses triggered by physical damage or injury to plant tissues. This type of stress can result from mechanical wounds such as cuts, bruises, or abrasions, as well as from damage caused by insects, herbivores, or pathogens. When a plant experiences wound stress, it activates a range of defensive mechanisms to repair the damage and mitigate further injury. These responses include the production of wound-induced proteins, such as proteinase inhibitors and defensive enzymes, the synthesis of secondary metabolites like jasmonic acid and phenolic compounds, and the reinforcement of cell walls. Additionally, plants may initiate localized and systemic signaling pathways to coordinate responses across the tissue and to neighboring areas.
  • “flooding stress” refers to when roots are submerged in excess water (not optimal to the plant species), leading to a lack of oxygen in the soil and disrupting normal plant function. This condition impairs root respiration and nutrient uptake, resulting in reduced growth, weakened plant structures, and potential root rot. The lack of oxygen can also trigger the production of toxic metabolites and affect cellular energy processes.
  • heavy metal stress refers to the adverse effects caused by the accumulation of toxic levels of heavy metals, such as lead (Pb), cadmium (Cd), mercury (Hg), arsenic (As), and copper (Cu), in the soil and plant tissues. These metals can enter plants through contaminated soil or water and disrupt various physiological processes. Heavy metal stress leads to oxidative damage, interfering with photosynthesis, respiration, and nutrient uptake, and can cause growth inhibition, chlorosis, and reduced biomass. Toxic metals can also displace essential nutrients, impair enzyme function, and lead to the production of reactive oxygen species (ROS), further exacerbating stress.
  • ROS reactive oxygen species
  • biotic stress refers to the adverse effects caused by living organisms that negatively impact plant health and growth. This type of stress is caused by various biotic factors, including pathogens such as bacteria, fungi, and viruses, as well as pests like insects and nematodes. Biotic stress can lead to disease, damage to plant tissues, and reduced nutrient availability. Plants respond to biotic stress through a range of defensive mechanisms, including the production of antimicrobial compounds, activation of immune responses, and physical barriers like thickened cell walls.
  • Drought tolerance assay/Osmoticum assay - To analyze whether the plants (modified to down-regulate A01/A02, also may be referred to as “test plants”) are more tolerant to drought, an osmotic stress produced by the non-ionic osmolyte sorbitol in the medium can be performed. Control and the treated plants are germinated and grown in plant-agar plates for 4 days, after which they are transferred to plates containing 500 mM sorbitol. The treatment causes growth retardation, then both control and test plants are compared, by measuring plant weight (wet and dry), yield, and by growth rates measured as time to flowering.
  • soil-based drought screens are performed with test plants. Seeds from control Arabidopsis plants and test plants germinated and transferred to pots. Drought stress is obtained after irrigation is ceased accompanied by placing the pots on absorbent paper to enhance the soildrying rate. Test and control plants are compared to each other when the majority of the control plants develop severe wilting. Plants are re-watered after obtaining a significant fraction of the control plants displaying a severe wilting. Plants are ranked comparing to controls for each of two criteria: tolerance to the drought conditions and recovery (survival) following re-watering.
  • Cold stress tolerance To analyze cold stress, mature (25 day old) plants are transferred to 4 °C chambers for 1 or 2 weeks, with constitutive light. Later on plants are moved back to greenhouse. Two weeks later damages from chilling period, resulting in growth retardation and other phenotypes, are compared between both control and test plants, by measuring plant weight (wet and dry), and by comparing growth rates measured as time to flowering, plant size, yield, and the like.
  • Heat stress tolerance is achieved by exposing the plants to temperatures above 34 °C for a certain period. Plant tolerance is examined after transferring the plants back to 22 °C for recovery and evaluation after 5 days relative to internal controls or plants not exposed to neither cold or heat stress.
  • Water use efficiency - can be determined as the biomass produced per unit transpiration. To analyze WUE, leaf relative water content can be measured in control and test plants. Fresh weight (FW) is immediately recorded; then leaves are soaked for 8 hours in distilled water at room temperature in the dark, and the turgid weight (TW) is recorded. Total dry weight (DW) is recorded after drying the leaves at 60 °C to a constant weight.
  • Fertilizer use efficiency To analyze whether the test plants are more responsive to fertilizers, plants are grown in agar plates or pots with a limited amount of fertilizer, as described, for example, in Yanagisawa et al (Proc Natl Acad Sci U S A. 2004; 101 :7833-8). The plants are analyzed for their overall size, time to flowering, yield, protein content of shoot and/or grain. The parameters checked are the overall size of the mature plant, its wet and dry weight, the weight of the seeds yielded, the average seed size and the number of seeds produced per plant.
  • NUE nitrogen use efficiency
  • PUE phosphate use efficiency
  • KUE potassium use efficiency
  • Nitrogen use efficiency To analyze whether the test plants (e.g., Arabidopsis plants) are more responsive to nitrogen, plant are grown in 0.75-3 mM (nitrogen deficient conditions) or 6- 10 mM (optimal nitrogen concentration). Plants are allowed to grow for additional 25 days or until seed production. The plants are then analyzed for their overall size, time to flowering, yield, protein content of shoot and/or grain/ seed production. The parameters checked can be the overall size of the plant, wet and dry weight, the weight of the seeds yielded, the average seed size and the number of seeds produced per plant.
  • Nitrogen Use efficiency assay using plantlets - The assay is done according to Yanagisawa-S. et al. with minor modifications (“Metabolic engineering with Dofl transcription factor in plants: Improved nitrogen assimilation and growth under low-nitrogen conditions” Proc. Natl. Acad. Sci. USA 101, 7833-7838). Briefly, test plants which are grown for 7-10 days in 0.5 x MS [Murashige-Skoog] supplemented with a selection agent are transferred to two nitrogenlimiting conditions: MS media in which the combined nitrogen concentration (NEENCh and KNCh) was 0.75 mM (nitrogen deficient conditions) or 6-15 mM (optimal nitrogen concentration).
  • Plants are allowed to grow for additional 30-40 days and then photographed, individually removed from the Agar (the shoot without the roots) and immediately weighed (fresh weight) for later statistical analysis. Constructs for which only T1 seeds are available are sown on selective media and at least 20 seedlings (each one representing an independent transformation event) are carefully transferred to the nitrogen-limiting media. For constructs for which T2 seeds are available, different transformation events are analyzed. Usually, 20 randomly selected plants from each event are transferred to the nitrogen-limiting media allowed to grow for 3-4 additional weeks and individually weighed at the end of that period. Test plants are compared to control plants grown in parallel under the same conditions.
  • N (nitrogen) concentration determination in the structural parts of the plants involves the potassium persulfate digestion method to convert organic N to NCh’ (Purcell and King 1996 Argon. J. 88: 111-113, the modified Cd" mediated reduction of NCh’ to NCh” (Vodovotz 1996 Biotechniques 20:390-394) and the measurement of nitrite by the Griess assay (Vodovotz 1996, supra). The absorbance values are measured at 550 nm against a standard curve of NaNCh. The procedure is described in details in Samonte et al. 2006 Agron. J. 98: 168-176.
  • Germination tests compare the percentage of seeds from test plants that could complete the germination process to the percentage of seeds from control plants that are treated in the same manner. Normal conditions are considered for example, incubations at 22 °C under 22-hour light 2-hour dark daily cycles. Evaluation of germination and seedling vigor is conducted between 4 and 14 days after planting. The basal media is 50 % MS medium (Murashige and Skoog, 1962 Plant Physiology 15, 473-497).
  • Germination is checked also at unfavorable conditions such as cold (incubating at temperatures lower than 10 °C instead of 22 °C) or using seed inhibition solutions that contain high concentrations of an osmolyte such as sorbitol (at concentrations of 50 mM, 100 mM, 200 mM, 300 mM, 500 mM, and up to 1000 mM) or applying increasing concentrations of salt (of 50 mM, 100 mM, 200 mM, 300 mM, 500 mM NaCl).
  • an osmolyte such as sorbitol
  • salt of 50 mM, 100 mM, 200 mM, 300 mM, 500 mM NaCl
  • the effect of down-regulating AO1/AO2 on plant’s vigor, growth rate, biomass, yield and/or oil content can be determined using known methods.
  • Plant vigor The plant vigor can be calculated by the increase in growth parameters such as leaf area, fiber length, rosette diameter, plant fresh weight and the like per time.
  • the growth rate can be measured using digital analysis of growing plants. For example, images of plants growing in greenhouse on plot basis can be captured every 3 days and the rosette area can be calculated by digital analysis. Rosette area growth is calculated using the difference of rosette area between days of sampling divided by the difference in days between samples.
  • rosette parameters such as rosette area, rosette diameter and/or rosette growth rate in a plant model such as Arabidopsis predicts an increase in canopy coverage and/or plot coverage in a target plant such as Brassica sp., soy, com, wheat, Barley, oat, cotton, rice, tomato, sugar beet, and vegetables such as cucumber.
  • conferring tolerance to stress is achieved by down-regulating expression of AO1 and/or AO2 (aldehyde oxidase 1 and/or aldehyde oxidase 2).
  • aldehyde oxidase 1 refers to / T5G20960 ( AAO1) or natural orthologs thereof.
  • aldehyde oxidase 2 refers to AT3G43600 (AAO2) or natural orthologs thereof.
  • aldehyde oxidase 3 refers to AT2G27150 (AA03) or natural orthologs thereof.
  • aldehyde oxidase 4 refers to AT1G04580 (AA04) or natural orthologs thereof.
  • Table 1 Aldehyde oxidases (and their gene accession numbers) present in crops to be improved by genes modification according to some embodiments of the invention.
  • downregulates expression refers to downregulating the expression of a protein (e.g. AO1 and/or AO2) at the genomic (e.g. homologous recombination and site specific endonucleases) and/or the transcript level using a variety of molecules which interfere with transcription and/or translation (e.g., RNA silencing agents) or on the protein level (e.g., aptamers, small molecules and inhibitory peptides and the like).
  • a protein e.g. AO1 and/or AO2
  • genomic e.g. homologous recombination and site specific endonucleases
  • transcript level e.g., RNA silencing agents
  • Down regulation of expression may be either transient or stable. According to specific embodiments, down regulating expression refers to the absence of mRNA and/or protein, as detected by RT-PCR or Western blot, respectively.
  • down regulating expression refers to a decrease in the level of mRNA and/or protein, as detected by RT-PCR or Western blot, respectively.
  • the reduction may be by at least a 10 %, at least 20 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 %, at least 95 % or at least 99 % reduction as compared to that in a plant of the same species in which down-regulation has not been performed, yet of the same genetic background and developmental stage.
  • Non-limiting examples of agents capable of down regulating AO1 and/or AO2 expression are described in details hereinbelow.
  • Down-regulation at the nucleic acid level is typically effected using a nucleic acid agent, having a nucleic acid backbone, DNA, RNA, mimetics thereof or a combination of same.
  • the nucleic acid agent may be encoded from a DNA molecule or provided to the cell per se.
  • the downregulating agent is a polynucleotide.
  • the downregulating agent is a polynucleotide capable of hybridizing to a gene or mRNA encoding the protein e.g., AO1 and/or AO2.
  • the downregulating agent directly interacts with the gene of e.g., AO1 and/or AO2.
  • the agent directly binds the gene.
  • the downregulating agent is an RNA silencing agent or a genome or RNA editing agent.
  • RNA silencing refers to a group of regulatory mechanisms [e.g. RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), quelling, co-suppression, and translational repression] mediated by RNA molecules which result in the inhibition or "silencing" of the expression of a corresponding protein-coding gene.
  • RNAi RNA interference
  • TGS transcriptional gene silencing
  • PTGS post-transcriptional gene silencing
  • quelling co-suppression
  • co-suppression co-suppression
  • translational repression mediated by RNA molecules which result in the inhibition or "silencing" of the expression of a corresponding protein-coding gene.
  • RNA silencing agent refers to an RNA which is capable of specifically inhibiting or “silencing" the expression of a target gene.
  • the RNA silencing agent is capable of preventing complete processing (e.g, the full translation and/or expression) of an mRNA molecule through a post-transcriptional silencing mechanism.
  • RNA silencing agents include non-coding RNA molecules, for example RNA duplexes comprising paired strands, as well as precursor RNAs from which such small non-coding RNAs can be generated.
  • Exemplary RNA silencing agents include dsRNAs such as siRNAs, miRNAs and shRNAs.
  • the RNA silencing agent is capable of inducing RNA interference. In another embodiment, the RNA silencing agent is capable of mediating translational repression.
  • the RNA silencing agent is specific to the target RNA (e.g., AO1 and/or AO2 and does not cross inhibit or silence other targets AO3, AO4 and AO5 or other non-specific off-targets; as determined by PCR, Western blot, Immunohistochemistry and/or flow cytometry.
  • target RNA e.g., AO1 and/or AO2 and does not cross inhibit or silence other targets AO3, AO4 and AO5 or other non-specific off-targets; as determined by PCR, Western blot, Immunohistochemistry and/or flow cytometry.
  • RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs).
  • Downregulation of AO1 and/or AO2 can also be achieved by inactivating the gene via introducing targeted mutations involving loss-of function alterations (e.g. point mutations, deletions and insertions) in the gene structure.
  • targeted mutations involving loss-of function alterations e.g. point mutations, deletions and insertions
  • null mutation or a “null allele” is a mutation that leads to a non- transcribable RNA and/or non-translatable protein product or a protein product which is nonfunctional.
  • loss-of-function alterations refers to any mutation in the DNA sequence of a gene (AO1 and/or AO2) which results in downregulation of the expression level and/or activity of the expressed product, i.e., the mRNA transcript and/or the translated protein.
  • Non-limiting examples of such loss-of-function alterations include a missense mutation, z.e., a mutation which changes an amino acid residue in the protein with another amino acid residue and thereby abolishes the enzymatic activity of the protein; a nonsense mutation, /. ⁇ ?., a mutation which introduces a stop codon in a protein, e.g., an early stop codon which results in a shorter protein devoid of the enzymatic activity; a frame-shift mutation, z.e., a mutation, usually, deletion or insertion of nucleic acid(s) which changes the reading frame of the protein, and may result in an early termination by introducing a stop codon into a reading frame (e.g., a truncated protein, devoid of the enzymatic activity), or in a longer amino acid sequence (e.g., a readthrough protein) which affects the secondary or tertiary structure of the protein and results in a non-functional protein, devoid of the enzymatic
  • the mutation is a null mutation.
  • a null mutation is a gene mutation that leads to its not being transcribed into RNA and/or translated into a functional protein. According to a specific embodiment, the mutation causes the protein not being translated at all or completely degraded (e.g., as determined by Western blot).
  • loss-of-function alteration of a gene may comprise at least one allele of the gene.
  • allele refers to any of one or more alternative forms of a gene locus, all of which alleles relate to a trait or characteristic. In a diploid cell or organism, the two alleles of a given gene occupy corresponding loci on a pair of homologous chromosomes.
  • loss-of-function alteration of a gene comprises both alleles of the gene.
  • the genes of AO1 and/or AO2 may be in a homozygous form.
  • homozygosity is a condition where both alleles at the locus are characterized by the same nucleotide sequence. Heterozygosity refers to different mutations in the gene at the locus.
  • loss-of-function alterations claimed herein are non-naturally occurring, i.e., not found in nature, and a result of man-made activities.
  • nucleic acid alterations to a gene of interest can be designed publically available sources or obtained commercially from Transposagen, Addgene and Sangamo Biosciences. Following is a description of various exemplary methods used to introduce nucleic acid alterations to a gene of interest and agents for implementing same that can be used according to specific embodiments of the present invention.
  • Genome Editing using engineered endonucleases - this approach refers to a reverse genetics method using artificially engineered nucleases to cut and create specific double-stranded breaks at a desired location(s) in the genome, which are then repaired by cellular endogenous processes such as, homology directed repair (HDR) and non-homologous end-joining (NFfEJ).
  • HDR homology directed repair
  • NFfEJ non-homologous end-joining
  • HDR utilizes a homologous sequence as a template for regenerating the missing DNA sequence at the break point.
  • a DNA repair template containing the desired sequence must be present during HDR.
  • Genome editing cannot be performed using traditional restriction endonucleases since most restriction enzymes recognize a few base pairs on the DNA as their target and the probability is very high that the recognized base pair combination will be found in many locations across the genome resulting in multiple cuts not limited to a desired location.
  • restriction enzymes recognize a few base pairs on the DNA as their target and the probability is very high that the recognized base pair combination will be found in many locations across the genome resulting in multiple cuts not limited to a desired location.
  • ZFNs Zinc finger nucleases
  • TALENs transcription-activator like effector nucleases
  • CRISPR/Cas system CRISPR/Cas system.
  • Meganucleases are commonly grouped into four families: the LAGLID ADG family, the GIY-YIG family, the His-Cys box family and the HNH family. These families are characterized by structural motifs, which affect catalytic activity and recognition sequence. For instance, members of the LAGLID ADG family are characterized by having either one or two copies of the conserved LAGLID ADG motif. The four families of meganucleases are widely separated from one another with respect to conserved structural elements and, consequently, DNA recognition sequence specificity and catalytic activity. Meganucleases are found commonly in microbial species and have the unique property of having very long recognition sequences (>14bp) thus making them naturally very specific for cutting at a desired location.
  • meganucleases can be designed using the methods described in e.g., Certo, MT et al.
  • ZFNs and TALENs Two distinct classes of engineered nucleases, zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), have both proven to be effective at producing targeted double-stranded breaks (Christian et al., 2010; Kim et al., 1996; Li et al., 2011; Mahfouz et al., 2011; Miller et al., 2010).
  • ZFNs and TALENs restriction endonuclease technology utilizes a non-specific DNA cutting enzyme which is linked to a specific DNA binding domain (either a series of zinc finger domains or TALE repeats, respectively).
  • a restriction enzyme whose DNA recognition site and cleaving site are separate from each other is selected. The cleaving portion is separated and then linked to a DNA binding domain, thereby yielding an endonuclease with very high specificity for a desired sequence.
  • An exemplary restriction enzyme with such properties is Fokl. Additionally Fokl has the advantage of requiring dimerization to have nuclease activity and this means the specificity increases dramatically as each nuclease partner recognizes a unique DNA sequence.
  • Fokl nucleases have been engineered that can only function as heterodimers and have increased catalytic activity.
  • the heterodimer functioning nucleases avoid the possibility of unwanted homodimer activity and thus increase specificity of the doublestranded break.
  • ZFNs and TALENs are constructed as nuclease pairs, with each member of the pair designed to bind adjacent sequences at the targeted site.
  • the nucleases bind to their target sites and the Fokl domains heterodimerize to create a double-stranded break. Repair of these double-stranded breaks through the nonhomologous end-joining (NHEJ) pathway most often results in small deletions or small sequence insertions. Since each repair made by NHEJ is unique, the use of a single nuclease pair can produce an allelic series with a range of different deletions at the target site.
  • NHEJ nonhomologous end-joining
  • deletions typically range anywhere from a few base pairs to a few hundred base pairs in length, but larger deletions have successfully been generated in cell culture by using two pairs of nucleases simultaneously (Carlson et al., 2012; Lee et al., 2010).
  • the double- stranded break can be repaired via homology directed repair to generate specific modifications (Li et al., 2011; Miller et al., 2010; Urnov et al., 2005).
  • ZFNs rely on Cys2- His2 zinc fingers and TALENs on TALEs. Both of these DNA recognizing peptide domains have the characteristic that they are naturally found in combinations in their proteins. Cys2-His2 Zinc fingers typically found in repeats that are 3 bp apart and are found in diverse combinations in a variety of nucleic acid interacting proteins. TALEs on the other hand are found in repeats with a one-to-one recognition ratio between the amino acids and the recognized nucleotide pairs.
  • Zinc fingers correlated with a triplet sequence are attached in a row to cover the required sequence
  • OPEN low-stringency selection of peptide domains vs. triplet nucleotides followed by high-stringency selections of peptide combination vs. the final target in bacterial systems
  • ZFNs can also be designed and obtained commercially from e.g., Sangamo BiosciencesTM (Richmond, CA).
  • TALEN Method for designing and obtaining TALENs are described in e.g. Reyon et al. Nature Biotechnology 2012 May;30(5):460-5; Miller et al. Nat Biotechnol. (2011) 29: 143-148; Cermak et al. Nucleic Acids Research (2011) 39 (12): e82 and Zhang et al. Nature Biotechnology (2011) 29 (2): 149-53.
  • a recently developed web-based program named Mojo Hand was introduced by Mayo Clinic for designing TAL and TALEN constructs for genome editing applications (can be accessed through www(dot)talendesign(dot)org).
  • TALEN can also be designed and obtained commercially from e.g., Sangamo BiosciencesTM (Richmond, CA).
  • CRISPR-Cas system - has been exemplified in the examples section which follows.
  • Many bacteria and archea contain endogenous RNA-based adaptive immune systems that can degrade nucleic acids of invading phages and plasmids. These systems consist of clustered regularly interspaced short palindromic repeat (CRISPR) genes that produce RNA components and CRISPR associated (Cas) genes that encode protein components.
  • CRISPR RNAs crRNAs
  • crRNAs contain short stretches of homology to specific viruses and plasmids and act as guides to direct Cas nucleases to degrade the complementary nucleic acids of the corresponding pathogen.
  • RNA/protein complex RNA/protein complex and together are sufficient for sequence-specific nuclease activity: the Cas9 nuclease, a crRNA containing 20 base pairs of homology to the target sequence, and a trans-activating crRNA (tracrRNA) (Jinek et al. Science (2012) 337: 816-821). It was further demonstrated that a synthetic chimeric guide RNA (gRNA) composed of a fusion between crRNA and tracrRNA could direct Cas9 to cleave DNA targets that are complementary to the crRNA in vitro.
  • gRNA synthetic chimeric guide RNA
  • transient expression of Cas9 in conjunction with synthetic gRNAs can be used to produce targeted double-stranded brakes in a variety of different species (Cho et al., 2013; Cong et al., 2013; DiCarlo etal., 2013; Hwang etal., 2013a, b; Jinek etal., 2013; Mali et al., 2013).
  • the CRIPSR/Cas system for genome editing contains two distinct components: a gRNA and an endonuclease e.g. Cas9.
  • the gRNA is typically a 20 nucleotide sequence encoding a combination of the target homologous sequence (crRNA) and the endogenous bacterial RNA that links the crRNA to the Cas9 nuclease (tracrRNA) in a single chimeric transcript.
  • the gRNA/Cas9 complex is recruited to the target sequence by the base-pairing between the gRNA sequence and the complement genomic DNA.
  • the genomic target sequence must also contain the correct Protospacer Adjacent Motif (PAM) sequence immediately following the target sequence.
  • PAM Protospacer Adjacent Motif
  • the binding of the gRNA/Cas9 complex localizes the Cas9 to the genomic target sequence so that the Cas9 can cut both strands of the DNA causing a double-strand break.
  • the double-stranded brakes produced by CRISPR/Cas can undergo homologous recombination or NHEJ.
  • the Cas9 nuclease has two functional domains: RuvC and HNH, each cutting a different DNA strand. When both of these domains are active, the Cas9 causes double strand breaks in the genomic DNA.
  • CRISPR/Cas A significant advantage of CRISPR/Cas is that the high efficiency of this system coupled with the ability to easily create synthetic gRNAs enables multiple genes to be targeted simultaneously. In addition, the majority of cells carrying the mutation present biallelic mutations in the targeted genes.
  • nickases Modified versions of the Cas9 enzyme containing a single inactive catalytic domain, either RuvC- or HNH-, are called ‘nickases’. With only one active nuclease domain, the Cas9 nickase cuts only one strand of the target DNA, creating a single-strand break or 'nick'. A single-strand break, or nick, is normally quickly repaired through the HDR pathway, using the intact complementary DNA strand as the template. However, two proximal, opposite strand nicks introduced by a Cas9 nickase are treated as a double-strand break, in what is often referred to as a 'double nick' CRISPR system.
  • a double-nick can be repaired by either NHEJ or HDR depending on the desired effect on the gene target.
  • using the Cas9 nickase to create a double-nick by designing two gRNAs with target sequences in close proximity and on opposite strands of the genomic DNA would decrease off- target effect as either gRNA alone will result in nicks that will not change the genomic DNA.
  • dCas9 Modified versions of the Cas9 enzyme containing two inactive catalytic domains
  • dCas9 can be utilized as a platform for DNA transcriptional regulators to activate or repress gene expression by fusing the inactive enzyme to known regulatory domains.
  • the binding of dCas9 alone to a target sequence in genomic DNA can interfere with gene transcription.
  • Non-limiting examples of a gRNA that can be used in the present invention are shown in the Examples section which follows.
  • the introduced variation confers a non- naturally occurring variation.
  • both gRNA and Cas9 should be expressed in a target cell.
  • Cas9 can also be provided as mRNA or protein to the cell.
  • the insertion vector can contain both cassettes on a single plasmid or the cassettes are expressed from two separate plasmids.
  • CRISPR plasmids are commercially available such as the px330 plasmid from Addgene.
  • “Hit and run” or “in-out” - involves a two-step recombination procedure.
  • an insertion-type vector containing a dual positive/negative selectable marker cassette is used to introduce the desired sequence alteration.
  • the insertion vector contains a single continuous region of homology to the targeted locus and is modified to carry the mutation of interest.
  • This targeting construct is linearized with a restriction enzyme at a one site within the region of homology, electroporated into the cells, and positive selection is performed to isolate homologous recombinants. These homologous recombinants contain a local duplication that is separated by intervening vector sequence, including the selection cassette.
  • targeted clones are subjected to negative selection to identify cells that have lost the selection cassette via intrachromosomal recombination between the duplicated sequences.
  • the local recombination event removes the duplication and, depending on the site of recombination, the allele either retains the introduced mutation or reverts to wild type. The end result is the introduction of the desired modification without the retention of any exogenous sequences.
  • the “double-replacement” or “tag and exchange” strategy - involves a two-step selection procedure similar to the hit and run approach, but requires the use of two different targeting constructs.
  • a standard targeting vector with 3' and 5' homology arms is used to insert a dual positive/negative selectable cassette near the location where the mutation is to be introduced.
  • homologous targeted clones are identified.
  • a second targeting vector that contains a region of homology with the desired mutation is electroporated into targeted clones, and negative selection is applied to remove the selection cassette and introduce the mutation.
  • the final allele contains the desired mutation while eliminating unwanted exogenous sequences.
  • Site-Specific Recombinases The Cre recombinase derived from the Pl bacteriophage and Flp recombinase derived from the yeast Saccharomyces cerevisiae are site-specific DNA recombinases each recognizing a unique 34 base pair DNA sequence (termed “Lox” and “FRT”, respectively) and sequences that are flanked with either Lox sites or FRT sites can be readily removed via site-specific recombination upon expression of Cre or Flp recombinase, respectively.
  • the Lox sequence is composed of an asymmetric eight base pair spacer region flanked by 13 base pair inverted repeats.
  • Cre recombines the 34 base pair lox DNA sequence by binding to the 13 base pair inverted repeats and catalyzing strand cleavage and religation within the spacer region.
  • the staggered DNA cuts made by Cre in the spacer region are separated by 6 base pairs to give an overlap region that acts as a homology sensor to ensure that only recombination sites having the same overlap region recombine.
  • the site specific recombinase system offers means for the removal of selection cassettes after homologous recombination. This system also allows for the generation of conditional altered alleles that can be inactivated or activated in a temporal or tissue-specific manner.
  • the Cre and Flp recombinases leave behind a Lox or FRT “scar” of 34 base pairs. The Lox or FRT sites that remain are typically left behind in an intron or 3' UTR of the modified locus, and current evidence suggests that these sites usually do not interfere significantly with gene function.
  • Cre/Lox and Flp/FRT recombination involves introduction of a targeting vector with 3' and 5' homology arms containing the mutation of interest, two Lox or FRT sequences and typically a selectable cassette placed between the two Lox or FRT sequences. Positive selection is applied and homologous recombinants that contain targeted mutation are identified. Transient expression of Cre or Flp in conjunction with negative selection results in the excision of the selection cassette and selects for cells where the cassette has been lost. The final targeted allele contains the Lox or FRT scar of exogenous sequences.
  • Transposases refers to an enzyme that binds to the ends of a transposon and catalyzes the movement of the transposon to another part of the genome.
  • transposon refers to a mobile genetic element comprising a nucleotide sequence which can move around to different positions within the genome of a single cell. In the process the transposon can cause mutations and/or change the amount of a DNA in the genome of the cell.
  • transposon systems that are able to also transpose in cells e.g. vertebrates have been isolated or designed, such as Sleeping Beauty [Izsvak and Ivies Molecular Therapy (2004) 9, 147-156], piggyBac [Wilson et al. Molecular Therapy (2007) 15, 139-145], Tol2 [Kawakami et al. PNAS (2000) 97 (21): 11403-11408] or Frog Prince [Miskey et al. Nucleic Acids Res. Dec 1, (2003) 31(23): 6873-6881], Generally, DNA transposons translocate from one DNA site to another in a simple, cut-and-paste manner.
  • PB is a 2.5 kb insect transposon originally isolated from the cabbage looper moth, Trichoplusia ni.
  • the PB transposon consists of asymmetric terminal repeat sequences that flank a transposase, PBase.
  • PBase recognizes the terminal repeats and induces transposition via a “cut- and-paste” based mechanism, and preferentially transposes into the host genome at the tetranucleotide sequence TTAA.
  • the TTAA target site is duplicated such that the PB transposon is flanked by this tetranucleotide sequence.
  • the transposase system When mobilized, PB typically excises itself precisely to reestablish a single TTAA site, thereby restoring the host sequence to its pretransposon state. After excision, PB can transpose into a new location or be permanently lost from the genome.
  • the transposase system offers an alternative means for the removal of selection cassettes after homologous recombination quit similar to the use Cre/Lox or Flp/FRT.
  • the PB transposase system involves introduction of a targeting vector with 3' and 5' homology arms containing the mutation of interest, two PB terminal repeat sequences at the site of an endogenous TTAA sequence and a selection cassette placed between PB terminal repeat sequences.
  • Genome editing using recombinant adeno-associated virus (rAAV) platform - this genomeediting platform is based on rAAV vectors which enable insertion, deletion or substitution of DNA sequences in the genomes of live mammalian cells.
  • the rAAV genome is a single-stranded deoxyribonucleic acid (ssDNA) molecule, either positive- or negative-sensed, which is about 4.7 kb long.
  • ssDNA deoxyribonucleic acid
  • These single-stranded DNA viral vectors have high transduction rates and have a unique property of stimulating endogenous homologous recombination in the absence of double-strand DNA breaks in the genome.
  • rAAV genome editing has the advantage in that it targets a single allele and does not result in any off- target genomic alterations.
  • rAAV genome editing technology is commercially available, for example, the rAAV GENESISTM system from HorizonTM (Cambridge, UK).
  • Constructs useful in the methods according to the present invention may be constructed using recombinant DNA technology well known to persons skilled in the art.
  • the coding sequence constructs may be inserted into vectors, which may be commercially available, suitable for transforming into plants and suitable for expression of the gene of interest in the transformed cells.
  • the genetic construct can be an expression vector wherein the nucleic acid sequence is operably linked to one or more regulatory sequences allowing expression in the plant cells.
  • Plant cells may be transformed stably or transiently with the nucleic acid constructs or with naked DNA or RNA of the present invention.
  • stable transformation the nucleic acid molecule of the present invention is integrated into the plant genome and as such it represents a stable and inherited trait.
  • transient transformation the nucleic acid molecule is expressed by the cell transformed but it is not integrated into the genome and as such it represents a transient trait. According to a specific embodiment, down-regulating expression is in a constitutive manner.
  • down-regulating expression is in a tissue specific manner.
  • down-regulating is in a leaf tissue.
  • the Agrobacterium system includes the use of plasmid vectors that contain defined DNA segments that integrate into the plant genomic DNA. Methods of inoculation of the plant tissue vary depending upon the plant species and the Agrobacterium delivery system. A widely used approach is the leaf disc procedure which can be performed with any tissue explant that provides a good source for initiation of whole plant differentiation. Horsch et al. in Plant Molecular Biology Manual A5, Kluwer Academic Publishers, Dordrecht (1988) p. 1-9. A supplementary approach employs the Agrobacterium delivery system in combination with vacuum infiltration. The Agrobacterium system is especially viable in the creation of transgenic dicotyledonous plants.
  • DNA transfer into plant cells There are various methods of direct DNA transfer into plant cells.
  • electroporation the protoplasts are briefly exposed to a strong electric field.
  • microinjection the DNA is mechanically injected directly into the cells using very small micropipettes.
  • microparticle bombardment the DNA is adsorbed on microprojectiles such as magnesium sulfate crystals or tungsten particles, and the microprojectiles are physically accelerated into cells or plant tissues.
  • Micropropagation is a process of growing new generation plants from a single piece of tissue that has been excised from a selected parent plant or cultivar. This process permits the mass reproduction of plants having the preferred tissue expressing the fusion protein.
  • the new generated plants which are produced are genetically identical to, and have all of the characteristics of, the original plant.
  • Micropropagation allows mass production of quality plant material in a short period of time and offers a rapid multiplication of selected cultivars in the preservation of the characteristics of the original transgenic or transformed plant.
  • the advantages of cloning plants are the speed of plant multiplication and the quality and uniformity of plants produced.
  • Micropropagation is a multi-stage procedure that requires alteration of culture medium or growth conditions between stages.
  • the micropropagation process involves four basic stages: Stage one, initial tissue culturing; stage two, tissue culture multiplication; stage three, differentiation and plant formation; and stage four, greenhouse culturing and hardening.
  • Stage one initial tissue culturing
  • stage two initial tissue culturing
  • stage two the initial tissue culture is multiplied until a sufficient number of tissue samples are produced to meet gradually increased so that it can be grown in the natural environment.
  • Viruses that have been shown to be useful for the transformation of plant hosts include CaMV, TMV, TRV and BV. Transformation of plants using plant viruses is described in U.S. Pat. No. 4,855,237 (BGV), EP-A 67,553 (TMV), Japanese Published Application No. 63-14693 (TMV), EPA 194,809 (BV), EPA 278,667 (BV); and Gluzman, Y. et al., Communications in Molecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, New York, pp. 172-189 (1988). Pseudovirus particles for use in expressing foreign DNA in many hosts, including plants, is described in WO 87/06261.
  • plants of the invention can be obtained by producing one parental line with down-regulated AO1 and another with down-regulated AO2.
  • a hybrid can be obtained by crossing, where the selected progeny has down -regulated expression of both AO1 and AO2, preferably by genome editing.
  • Such a progeny is also selected to exclude presence of foreign DNA (such as coding for the endonuclease, e.g., Cas9) according to some embodiments of the invention.
  • Desirable inbred or parent lines are developed by continuous self-pollinations and/or backcrosses and selection of the best breeding lines, sometimes utilizing molecular markers to speed up the selection process.
  • the hybrid seed can be produced indefinitely, as long as the homozygosity of the parents are maintained.
  • stable parental lines refers to open pollinated, inbred lines, stable for the desired plants over cycles of self-pollination and planting. According to a specific embodiment, 95% of the genome is in a homozygous form in the parental lines of the present invention.
  • a common practice in plant breeding is using the method of backcrossing to develop new varieties by single trait conversion.
  • single trait conversion refers to the incorporation of new single gene into a parent line wherein essentially all of the desired morphological and physiological characteristics of the parent lines are recovered in addition to the single gene transferred.
  • backcrossing refers to the repeated crossing of a hybrid progeny back to one of the parental plant.
  • the parental plant which contributes the gene for the desired characteristic is termed the non-recurrent or donor parent, as mentioned hereinabove. This terminology refers to the fact that the non-recurrent parent is used one time in the backcross protocol and therefore does not recur.
  • the parental plant to which the gene from the non-recurrent parent are transferred is known as the recurrent parent as it is used for several rounds in the backcrossing protocol.
  • a plant from the original varieties of interest (recurrent parent) is crossed to a plant selected from second varieties (non-recurrent parent) that carries the gene, introgression or hamplotype of interest to be transferred.
  • the resulting progeny from this cross are then crossed again to the recurrent parent and the process is repeated until a plant is obtained wherein essentially all of the desired morphological and physiological characteristics of the recurrent parent are recovered in the converted plant, in addition to the transferred gene from the non-recurrent parent.
  • NIL near-isogenic lines
  • Backcrossing methods can be used with the present invention to improve or introduce a characteristic into the parent lines.
  • a method of selecting a plant exhibiting tolerance to stress comprising:
  • Marker assisted breeding as described above can be used in this method, looking for genetic variation in AO1 and/or AO2.
  • selection can be done by measuring levels of oxidized aldehydes. This method is effective since it is possible to measure this early marker already upon leaf emergence.
  • selection can be done by measuring tolerance to stress.
  • present teachings provide for plants and progeny which are characterized by increased tolerance to stress and have a genetic variation in AO1 and/or AO2 gene.
  • a plant having been treated with an agent down-regulating expression of aldehyde oxidase 1 (AO1) and/or aldehyde oxidase 2 (AO2) such that said plant or plant cell exhibits reduced expression of AO1 and/or AO2, as compared to a control plant.
  • AO1 aldehyde oxidase 1
  • AO2 aldehyde oxidase 2
  • a plant cell having been treated with an agent down-regulating expression of aldehyde oxidase 1 (AO1) and/or aldehyde oxidase 2 (AO2) such that said plant cell exhibits reduced expression of AO1 and/or AO2, as compared to a control plant cell.
  • AO1 aldehyde oxidase 1
  • AO2 aldehyde oxidase 2
  • the plant or the plant seed is an inbred.
  • the plant is a hybrid plant or the seed is a hybrid seed.
  • the invention also relates to progeny of the plant (having down-regulated expression of AO1 and/or AO2) of the invention.
  • progeny can be produced by sexual or vegetative reproduction of a plant of the invention or a progeny plant thereof.
  • the progeny plant may be modified in one or more other characteristics. Such additional modifications are for example effected by mutagenesis or by transformation with a transgene.
  • progeny is intended to mean the offspring or the first and all further descendants from a cross with a plant of the invention that shows tolerance to stress as described herein.
  • Progeny of the invention are descendants of any cross with a plant of the invention that carries the mutation (in a homozygous form) trait that leads to tolerance.
  • Progeny also encompasses plants that carry the trait of the invention which are obtained from other plants of the invention by vegetative propagation or multiplication.
  • embodiments described herein furthermore, relate to hybrid seed and to a method of producing hybrid seed comprising crossing a first parent plant with a second parent plant and harvesting the resultant hybrid seed.
  • the trait is recessive, therefore both parent plants need to be homozygous for the trait in order for all of the hybrid seed to carry the trait of the invention. They need not necessarily be uniform for other traits.
  • Embodiments described herein also relate to the germplasm of the plants.
  • the germplasm is constituted by all inherited characteristics of an organism and according to the invention encompasses at least the trait of the invention.
  • growing plants or selecting plants is effected under stress or in a region known to be at risk of stress (e.g., drought).
  • stress e.g., drought
  • processed products of the plants which comprise DNA showing genetic variation in AO1 and AO2 which elicits down-regulation of these genes.
  • compositions, methods or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range.
  • the phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.
  • any Sequence Identification Number can refer to either a DNA sequence or a RNA sequence, depending on the context where that SEQ ID NO is mentioned, even if that SEQ ID NO is expressed only in a DNA sequence format or a RNA sequence format.
  • the aldehyde oxidase (AO1; AO2 and AO3) mutant of Solarium lycopersicum was generated using CRISPR/Cas9 system.
  • Two sgRNA were designed for each of the aldehyde oxidase (listed in Table 2a, below) using CRISPR-P(Liu et al 2017, doi: 10.1016/j.molp.2017.01.003) and the tomato genome assembly (SL2.50).
  • a PCR reaction was carried out with a primer containing the gRNA sequence and a universal primer (TGTGGTCTCAAGCGTAATGCCAACTTTGTAC, SEQ ID NO 7, UP1SG), using the plasmid pICH86966:: AtU6p::gRNA_PDS (Addgene plasmid 46966) as a template (Table 2b).
  • the PCR products were then cloned into level 1 vectors pICH47751 (gRNAl) and pICH47761 (gRNA2).
  • level 1 vectors containing the gRNAs were then assembled together with the plasmids pICH47732-NOSpro::NPTII, pICH47742-35Spro:Cas9, pICH41780 End-link (Addgene plasmid 48019), into the binary level 2 vector pAGM4723 using Bpil enzyme.
  • Agrobacterium tumefaciens GV3101, and the cotyledon transformation method (McCormick, 1997) were used to transform all constructs into M82 (sp).
  • Specific primers for the CAS9 sequence [Forward- CGCTAATCTTGCAGGTAGCC, SEQ ID NO: 8 CAS9PF and Reverse-
  • Genomic DNA of CRISPR/cas9 generated mutant is isolated using Qiagen DNeasy® Plant Pro Kit to visualize pattern of targeted mutagenesis using PCR amplification and sequencing.
  • the DNA fragments spanning the Cas9/gRNA target sequences are amplified by PCR (primer sequences listed in Table 3, below) using PlatinumTM SuperFiTM PCR Master Mix (invitrogen).
  • the PCR is run with a final volume of 20 pl, containing; 40 ng of gDNA, 1 pl of forward and reverse primer (10 pm) each and 10 pl of 2X PCR master mix.
  • the thermocycler is set at 95°C for 5 min, 35 cycles at 95°C for 30s, specific annealing temperature (55-60°C) for 15s for and 72°C for 30s followed by 72°C for 5min.
  • the PCR product is used for Sanger sequencing to recognize the mutation. Based on sequence, the marker is desgined. If there is mutation inside the restriction enzyme sequence, then a PCR-based marker with a possible restriction enzyme is designed. In case there is no enzyme then dCAPS (Derived Cleaved Amplified Polymorphic Sequences) primers are designed to amplify a region of DNA containing the mutation of interest. One of the primers is designed to introduce a mismatched nucleotide at the mutation site, to introduce or destroy a restriction enzyme recognition site when the PCR product is generated.
  • dCAPS Deived Cleaved Amplified Polymorphic Sequences
  • Table 2a Sequence of sgRNA used for CRISPR/Cas9 mediated mutation
  • Table 2b Sequence of primers used for golden gate assembly and transformation confirmation
  • Table 3 Details of primers to amplify genomic DNA PCR analysis
  • Table 4 -Nomenclature of mutants used
  • AAO2 protein expression level affects AAO3 and AAOl’s capacity to oxidize specific aldehydes under stresses such as Rose-Bengal
  • RNA interference (RNAi) technique was used using S ALK l 04895 (aao2) and S ALK O 18100 (aaol ) KO mutant plants.
  • Flowering aao2 were dipped with Agrobacterium GV3101 strain containing AAO1 RNAi or AAO3 RNAi constructs to generate saao3 or saaol, respectively.
  • the homozygous AAO-compromised lines were exposed to UV-C irradiation or Rose- Bengal spray after verification of the mutations by detection of the transcript's expression of the targeted genes as compared to the expression in WT leaves ( Figure 4).
  • rosette leaves of the aaolS (aaolS-11) mutants impaired in AAO3 and AAO2 expressions exhibited significantly lower remaining chlorophyll level than WT leaves 3 days after exposing to 250 mJ of UV-C irradiation or 0.05 mM of Rose-Bengal application.
  • aao3Ss mutants impaired in AA01 and AAO2 expression, as well as the aao2 (KO-95) mutant exhibited significantly higher remaining chlorophyll level than WT in response to the applied stresses ( Figures 5A-B and Figures 6A-B).
  • aldehydes level in rosette leaves was carried out 3 days after the UV-C irradiation, and revealed significantly higher level of benzaldehyde, crotonaldehyde, propionaldehyde and HNE in WT leaves compared to aao2KO and the three aao3Ss (aao3S-l, aao3S-12, aao3S-18) mutants.
  • Impairment in AAO1 improves plant resistance to UV-C irradiation., while its overexpression responds as WT
  • aao3S the average of the two independents single aao3 (a3-l-95) and (a3-18-7-10G) and aao2KO (KO-95) exhibited lower water loss than WT and single functioning aaol ⁇ al-ll-10 (95)].

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Virology (AREA)
  • Cell Biology (AREA)
  • Botany (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

A method of conferring stress tolerance to a plant is provided. The method comprising down-regulating expression of an aldehyde oxidase 1 (AO1) and/or aldehyde oxidase 2 (AO2), thereby conferring stress tolerance to the plant. Also provided are plant generated thereby.

Description

METHODS OF IMPROVING TOLERANCE OF PLANTS TO STRESS AND PLANTS GENERATED THEREBY
RELATED APPLICATIONS:
This Application claims the benefit of priority from U.S. Provisional Patent Application Nos. and 63/538,085 filed September 13, 2023 and 63/683,249 filed August 15, 2024, each of which is incorporated herein by reference in its entirety.
SEQUENCE LISTING STATEMENT
The XML file, entitled 101393. xml, created on September 13, 2024, comprising 20,480 bytes, submitted concurrently with the filing of this application is incorporated herein by reference.
FIELD AND BACKGROUND OF THE INVENTION
The present invention, in some embodiments thereof, relates to methods of improving tolerance of plants to stress and plants generated.
Aldehyde oxidases (AOs, EC 1.2.3.1) are a multigene family that oxidizes a variety of aldehydes, including the oxidation of abscisic aldehyde (ABAld) to the phytohormone abscisic acid (ABA). The protein architecture of the Arabidopsis AO (AAO) multigene family comprises FAD, Fe-S and molybdenum cofactor (Moco) domains as its prosthetic groups (Koshiba et al., 1996 Plant Physiology Volume 110, Issue 3, March 1996, Pages 781-789). The approximate molecular mass of the AO monomer is ca. 145 kDa, and the AOs carry their catalytic function by forming homodimers as well as heterodimers in plants (Akaba et al., 1999 The Journal of Biochemistry, Volume 126, Issue 2, August 1999, Pages 395-401). In Arabidopsis, four AO genes encode AAO1, AAO2, AAO3 and AAO4, and their expression patterns have been shown to be tissue specific; AAO1 is predominantly expressed in seedlings, roots, stem and seeds, but shows a significant expression in rosette leaves as well. AAO2 is mainly expressed in seedlings and root and in rosette leaves, whereas AAO3 is expressed in seedlings (at lower levels than AAO1 and AAO2), roots, stem flowers and rosette leaves, while AAO4 is abundant in siliques but is expressed to a certain level in flower, root and stem.
The AAOs are characterized by differential substrate specificities that play a key role in identifying and assigning their biological roles. AAO1 and AAO2 homodimers catalyze the oxidation of indole-3 -acetaldehyde and 1 -naphthaldehyde, respectively, with very high efficiency, whereas their heterodimer (AAO1 ::AAO2) exhibits intermediate substrate specificities, oxidizing both aldehydes with intermediate specificity. The homodimer of AAO3 and its heterodimer with AA02 oxidize ABald to ABA. Among the four AAOs, AA03 has received special attention owing to its involvement in ABA biosynthesis and its importance in normal and stress conditions. AA01 was shown to be implicated in the biosynthesis of indole-3 -carboxylic acid, yet further roles of AA01 as well as the role of AA02 is not known (Nurbekova et al., 2021 5(108) December 2021 Pages 1439-1455, 2024, Seo et al., 2000 The Plant J. 23(4):481-488; Seo et al. 2000 PNAS 97 23 12908-12913).
Aldehydes can be extremely toxic when produced in excess because of their inherent chemical reactivity and under normal physiological conditions aldehydes are formed constitutively and need to be detoxified. Yet, there is an increasing body of evidence for the generation of toxic levels of aldehydes in response to environmental stresses, especially lipid peroxidation-derived reactive carbonyl species such as malondialdehyde (MDA), acrolein and 4-hydroxyl-2-nonenal (HNE). Notably, reactive carbonyl species (RCS) can act as agents to mediate reactive oxygen species (ROS) signals to target proteins such as heat shock-responsive gene regulation, ABA signaling for stomatai closure, and auxin signaling for lateral root formation in plants. Yet, RCS can mediate ROS-induced programmed cell death as well as senescence. Plant aldehyde oxidases were shown to generate H2O2 and 02", while oxidizing various aldehydes (Yesbergenova et al., 2005 The Plant J. 42(6):862-876, Srivastava et al., 2017 Plant Physiology, Volume 173, Issue 4, April 2017, Pages 1977-1997). Yet, above a certain level, aldehydes increase may result in enhancement of reactive oxygen species generation and oxidative stress as was shown before (see Fig. S8 in Nurbekova et al., 2021).
Detoxification by oxidation of toxic aldehydes was attributed to several enzymes but rarely to aldehyde oxidases. Recently demonstrated was the importance of active AA03 and AA04 in delaying rosette leaves and siliques senescence respectively, by oxidizing toxic aldehydes accumulated in siliques or leaves exposed to toxic aldehyde, dark stress UV-C irradiation or natural senescence (Srivastava et al., 2017; Nurbekova et al., 2021).
Nurbekova et al. 2021 states: that AA01 and AA02 activities do not play a role in UV-C sensitivity in AO3 knockout plants.
SUMMARY OF THE INVENTION
According to an aspect of some embodiments of the present invention there is provided a method of conferring stress tolerance to a plant, the method comprising down-regulating expression of an aldehyde oxidase 1 (AO1) and/or aldehyde oxidase 2 (AO2), thereby conferring stress tolerance to the plant. According to an aspect of some embodiments of the present invention there is provided a plant having been treated with an agent down-regulating expression of aldehyde oxidase 1 (AO1) and/or aldehyde oxidase 2 (AO2) such that the plant or plant cell exhibits reduced expression of AO1 and/or AO2, as compared to a control plant.
According to an aspect of some embodiments of the present invention there is provided a plant cell having been treated with an agent down-regulating expression of aldehyde oxidase 1 (AO1) and/or aldehyde oxidase 2 (AO2) such that the plant cell exhibits reduced expression of AO1 and/or AO2, as compared to a control plant cell.
According to an aspect of some embodiments of the present invention there is provided a method of producing a plant exhibiting tolerance to stress, the method comprising growing the plant as described herein or regenerating the cell as described herein.
According to an aspect of some embodiments of the present invention there is provided a method of selecting a plant exhibiting tolerance to stress, the method comprising:
(a) providing plants having been treated with an agent down-regulating expression of aldehyde oxidase 1 (AO1) and/or aldehyde oxidase 2 (AO2);
(b) selecting from the plants, a plant which exhibits tolerance to stress.
According to some embodiments of the invention, the down-regulating expression is of AO2.
According to some embodiments of the invention, the down-regulating expression is of AO1.
According to some embodiments of the invention, the down-regulating expression is of AO2 and AO1.
According to some embodiments of the invention, the stress is abiotic stress.
According to some embodiments of the invention, the abiotic stress is selected from the group consisting of drought stress, oxidative stress, radiation stress, temperature stress, light stress, nutrient stress, heavy metal stress, salinity stress wounding stress and flooding stress.
According to some embodiments of the invention, the abiotic stress is selected from the group consisting of drought stress, oxidative stress and radiation stress.
According to some embodiments of the invention, the abiotic stress is drought stress.
According to some embodiments of the invention, the drought stress comprises extreme drought stress, as defined by fast water loss (8 to 16% within 3 to 7 hours).
According to some embodiments of the invention, the abiotic stress is oxidative stress.
According to some embodiments of the invention, the abiotic stress is radiation stress.
According to some embodiments of the invention, the radiation is UV-C. According to some embodiments of the invention, the stress is not radiation stress.
According to some embodiments of the invention, the stress is not UV-C stress.
According to some embodiments of the invention, the stress is biotic stress.
According to some embodiments of the invention, the growing is under stress conditions.
According to some embodiments of the invention, the plant is a crop plant.
According to some embodiments of the invention, the down-regulating expression is by a nucleic acid agent.
According to some embodiments of the invention, the nucleic acid agent is a genome editing agent or an RNA editing agent.
According to some embodiments of the invention, the nucleic acid agent is an RNA silencing agent.
According to some embodiments of the invention, the down-regulating expression is in a constitutive manner.
According to some embodiments of the invention, the down-regulating expression is in a tissue specific manner.
According to some embodiments of the invention, the down-regulating is in a leaf tissue.
Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.
In the drawings:
FIGs. 1A-C show Arabidopsis aldehyde oxidases activity assessment with different aldehydes. A. AA03 in-gel activity (most upper activity band) in rosette leaves of aaol knock out (ko) (SALK 018100) four- week-old plants. B. AA01 and AA03 activity (most upper activity band) as well as AA02 activity (lowest activity band) in wild type (WT) and aao2 KO mutant [SALK l 04895 (KO-95)] in 12-days old seedling and C. rosette leaves of four- week-old plants.100 pg protein extract from rosette leaves or seedling were fractionated on native gel and AO activity detection with indicated aldehyde were carried out. AO enzyme activity was determined in a reaction solution containing 100 mM Tris-HCl (pH 7.5), 1 mM 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 0.1 mM phenazine methosulfate (PMS) and 1 mM aldehydes (except for HNE and abscisic aldehyde loaded with 0.25mM and 0.2 mM, respectively). The reaction was stopped after 4h with aaolKO mutant and 3 h with WT and aao2 KO mutant [SALK l 04895 (KO-95)] by immersing the gel in 5% acetic acid and band images were captured and analyzed for relative intensity (RI) of AAO1/AAO3 using ImageJ software (www(dot)/imagej (dot)nih(dot)gov/ij/).
FIGs. 2A-D show the effects of Rose Bengal on chlorophyll and aldehyde level in leaves of Arabidopsis wild-type (WT) and aao2-KO plants. A. Appearance of control (water treated) and Rose Bengal treated WT and aao2 plants. Representative photograph of wild-type and aao2 [SALK l 04895 (KO-95) and SAIL 563 G09 (KO-563)] appearance in response to Rose Bengal. 23-day old plants were sprayed with 0.05 mM Rose Bengal and photographed 3 d later and B. Remaining chlorophyll in rosette leaves (left to right are the oldest to younger leaves) was determined. C. Aldehyde profiling (nmole gFW'1) of control (blue bars) and Rose Bengal treated (red bars) plants (results were adjusted according to the fresh weight of control) 17h after the application. Leaves from 3 different plants were taken as one replica and the bars represent the average of at least 4 replicas. Different letters above the bar indicate significant difference (Tukey- Kramer HSD test, P<0.05). Asterisk shows a significant difference within genotype. D. In gel activity of control and Rose Bengal treated WT, aao2, aaol [SALK 018100 (al-100)] and aao3 [SAIL 78 H09 (KO9)] mutants. 150 pg crude protein extract from rosette leaves of WT and aao2, aaol and aao3 were fractionated by NATIVE PAGE for the activity assay using the indicated aldehydes. Relative intensity was calculated using ImageJ software ((https://imagei.nih.gov/ii/).) for each band. The intensity of the upper activity bands was compared with that obtained with WT control (as 100%). RI indicates Relative Intensity. KO -95 and KO-563 are SALK l 04895 and SAIL 563 G09 respectively. Al-100 is SALK 018100 (aaol KO).
FIGs. 3A-D show in gel aldehyde oxidase activity of wild-type and the Arabidopsis aldehyde oxidases (AAOs) double mutant (in which there is a single functioning AAO I (.suu<>/). 150 pg crude protein extract from rosette leaves of WT and saaol, saao2 and saao3 were fractionated by NATIVE PAGE for the activity assay using A. abscisic aldehyde, B. Indole-3 carbaldehyde (I3CA), C. Zrans-2-nonenal, D. Benzaldehyde. Relative intensity (RI) was calculated for each band by using ImageJ software (https://imagei.nih.gov/ii/). The intensities of the activity bands were compared with those obtained with WT control (as 100%).
FIG. 4 show the relative transcript expression of AA01 (At5g20960), AA02 (At3g43600) and AA03 (At2g27150) in rosette leaves of 23-days post germination Arabidopsis WT, aaolSingle (aaolS) and independent aao3Singles (aao3Ss) mutant plants. The expression level of each of the transcripts in WT, aaolS (aaolS-11) and aao3Ss (aao3S-l, aao3S-7, aao3S-ll, aao3S-12, aao3S-18) mutants was compared with the corresponding transcript in WT after normalization to the transcript of EF-la (At5g60390), as the housekeeping gene, and presented as relative expression. Different letters above the bar show significant differences (Tukey-Kramer HSD test, P<0.05). aaolS was generated by silencing AA03 in aao2 [SALK 104895 (KO-95)], and aao3Ss was generated by silencing AA01 in aao2( KO-95) plants or silencing AA02 in aaol [SALK 018100 (al -100)] plants.
FIGs. 5A-E show the determination of UV-C-irradiation-induced senescence and senescence-related factors in rosette leaves of Arabidopsis aldehyde oxidases single mutants [aaolSingle (aaolS) and aao3Singles (aao3Ss)], aao2KO [SALK 104895 (KO-95) and wild-type (WT) plants. A. Representative photograph of WT, aao2, aaolS and aao3Ss (aao3S-l, aao3S-12, aao3S-18) rosette leaves in untreated (control) and UV-C irradiation treated plants. 21-days post germination (DPG) plants exposed to 250 mJ of UV-C irradiation were kept in a growth room for 72 hours and thereafter documented together with rosette leaves of plants not exposed to UV-C (control). Scale bar=2 cm. B. The level of remaining chlorophyll in the first seven leaves (oldest to youngest from left to right) after exposing WT and the various mutant plants to UV-C treatment. C. Indicated aldehyde profiling in control (blue bars) and UV-C treated (red bars) plants. Leaves from 3 different plants were taken as one replica and the bars show the average of at least 4 replicas. aao3Ss represents the average of the 3 independent aao3 single mutants. D. Aldehyde oxidase 3 (AA03) in gel activity in control and UV-C treated WT, aao2 [KO-95), SAIL 563 G09 (KO-563)] and aao3Ss (aao3S-l, aao3S-12, aao3S-18) using abscisic aldehyde as the specific substrate for AA03. 150 pg crude protein extracted from WT, aao2 (KO-95 and KO-563) and aao3Ss (aao3S-l, aao3S-12, aao3S-18) rosette leaves were fractionated by NATIVE PAGE and were used for activity. The gels were scanned after 30 min, and intensity of the activity bands was estimated using ImageJ software (http://imagei .nih.gov/ii/) and compared with that obtained with UV-C untreated (control) WT (employed as 100%) and presented as relative intensity (RI). E. Abscisic acid (ABA) level in rosette leaves of WT, aao2KO (KO-95, KO-563), aaolS (aaolS-11) and aao3Ss (aao3S-l, aao3S-12, aao3S-18) untreated control (blue bars) and UV-C treated (red bars) plants. Different letters above the bar indicate significant difference (Tukey-Kramer HSD test, P<0.05). Asterisk shows significant differences between treatments within the same genotype (Student’s t test, P < 0.05).
FIGs. 6A-E show the determination of Rose-Bengal -induced senescence and senescence- related factors in rosette leaves of Arabidopsis aldehyde oxidases single mutants [aaol Single (aaolS) and aao3Singles (aao3Ss)], aao2KO [SALK104895 (KO-95) and wild-type (WT) plants. A. Representative photograph of WT, aao2, aaolS and aao3Ss (aao3S-7, aao3S-12, aao3S-18) rosette leaves in untreated (control) and Rose-Bengal treated plants. 21-DPG plants treated with 0.05 mM of Rose-Bengal were kept in a growth room for 72 hours and thereafter documented. Scale bar=2 cm. B. Damage level in leaves as shown in (A) was calculated as described in the materials and methods. Means +-SEM (n = 6). C. Indicated aldehyde profiling in control (blue bars) and Rose-Bengal treated (red bars) plants. Seventeen hours after Rose-Bengal treatment leaves from 3 different untreated (control) or treated plants were taken as one replica and the bars show the average of at least 4 replicas. aao3Ss is average of the 3 independent aao3 singles plants. D. Aldehyde oxidases 3 (AAO3) in gel activity in control and Rose-Bengal treated WT, aao2 [KO- 95), SAIL 563 G09 (KO-563)] and aao3Ss using abscisic aldehyde as the specific substrate for AAO3. 150 pg crude protein extract from WT, aao2 (KO-95, KO-563) and aao3Ss rosette leaves was fractionated by NATIVE PAGE and were used for the in-gel activity. The gels were scanned after Ih, and the intensity of the activity bands was estimated using Imaged software (http://imagei .nih.gov/ii/) and compared with that obtained with Rose-Bengal untreated (control) WT (employed as 100%) and presented as relative intensity (RI). E. ABA content in rosette leaves of WT, aao2KO (KO-95, KO-563), aaolS (aaolS-11) and aao3Ss (aao3S-7, aao3S-12, aao3S-18) untreated control (blue bars) and Rose-Bengal treated (red bars). Different letters above the bar indicate significant differences (Tukey-Kramer HSD test, P<0.05). Asterisk shows significant differences between treatments within the same genotype (Student’s t test, P < 0.05).
FIGs. 7A-C show that. Determination of UV-C-irradiation-induced senescence in rosette leaves of Arabidopsis aldehyde oxidases al -100 [(SALK 018100 (aaol KO)] as well as AA01 OE (AAO1-OE was described in Nurbekova et al., 2021). 21-days post germination plants were exposed to UV-C irradiation (100 mJ) and were kept in a growth room for 96 hours and thereafter documented together with rosette leaves of plants not exposed to UV-C (control), (a) Representative photograph of WT, al -100 andAAOl-OE rosette leaves in untreated (control) and UV-C irradiation treated plants. 21-days post germination (DPG) plants exposed to 100 mJ of UV- C irradiation were kept in a growth room for 96 hours and thereafter documented together with rosette leaves of plants not exposed to UV-C (control). Scale bar=2 cm. (b and c) The level of remaining chlorophyll in the first six leaves (oldest to youngest from left to right) after exposing WT and the various mutant plants to UV-C treatment in two independent experiments. Different letters above the bar indicate significant difference (Tukey-Kramer HSD test, P<0.05).
FIGs. 8A-B show water loss in detached rosette leaves of 24 d old Arabidopsis WT, aao2 mutant (KO-95), aaol single mutant [al-11-10-(95)] and aao3 single mutants [(a3-l-95) and (a3- 18-7-(l 00)] grown in soil. A. Plants were detached and kept in covered 20x20 cm Petri dishes for 3 and 7 hours. Error bars represent ±SE (n=6 similar positioned rosette leaves from 6 different plants). B. Relative Water Content (RWC) in rosette leaves of plants kept in the covered Petri dishes for 7 hours as described in A. Error bars represent ±SE (n=5). aao3S is the average of 2 independent single aao3 (a3-l-95) and (a3-18-7-100) and aao2KO is the average of 2 independent KO of aao2 (KO-95 and KO-563). Values denoted with different letters above the bars are significantly different according to the Turkey-Kramer HSD mean-separation test (P < 0.05).
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
The present invention, in some embodiments thereof, relates to methods of improving tolerance of plants to stress and plants generated.
Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.
The involvement of reactive aldehydes in stress-induced damage to plants was previously reported. Aldehyde oxidases (AOs) are a multigene family that oxidizes a variety of aldehydes rendering them less toxic. Four AO genes encode AO1, AO2, AO3 and AO4, and their expression patterns have been shown to be tissue specific. Recently demonstrated was the importance of active AO3 in delaying rosette leaves senescence, by oxidizing toxic aldehydes accumulated in leaves exposed to toxic aldehyde, dark stress, UV-C irradiation or natural senescence.
Whilst conceiving embodiments of the invention and reducing them to practice, the present inventors have uncovered that stress tolerance can be achieved by down-regulating the expression of aldehyde oxidase 1 (AO1, AAO1 in Arabidopsis) and aldehyde oxidase 2 (AO2, AAO2 in Arabidopsis), thereby augmenting the stress tolerance activity of aldehyde oxidase 3 (AO3, AAO3 in Arabidopsis). This is surprising since it is generally known that the activity of aldehyde modifying enzymes such as aldehyde dehydrogenase and aldehyde oxidases e.g., AO3 increases detoxification of aldehydes, hence it is unexpected that reducing the levels of such enzymes would be beneficial. As is illustrated hereinbelow and in the Examples section which follows, the present inventors demonstrated enhancement of AA01 and/or AA03 oxidizing activity on a variety of aldehydes by knocking out AA02 in aao2 mutant (Example 1). Down-regulation of AA02 leading to reduced protein expression level affected AA03 and AAOl’s capacity to oxidize specific aldehydes under oxidation stress induced by Rose-Bengal (Example 2). Down-regulation of AA01 and AA02 indicated that AA02 protein expression level affects AA03 capacity to oxidize toxic aldehydes in rosette leaves of plants exposed to irradiation (UV-C) or oxidation stress induced by Rose-Bengal application (Example 3). Example 4 showed that mutant impaired in AA01 and AA02 expression causes significant improvement in chlorophyll levels as compared to wild type (WT) in response to stress. Focusing on AO1, the present inventors showed that impairment in AA01 elicited plant resistance to UV-C irradiation, while its overexpression responds as WT. Absence of AA02 expression not only enhances plant resistance to irradiation or oxidative stress but also improves plant resistance to harsh drought (Example 6).
Collectively, these results show that down-regulation of AO1 and/or AO2 can be used to improve the tolerance of plants to stress.
Thus, according to an aspect of the invention there is provided a method of conferring stress tolerance to a plant, the method comprising down-regulating expression of an aldehyde oxidase 1 (AO1) and/or aldehyde oxidase 2 (AO2), thereby conferring stress tolerance to the plant.
The term '"plant" as used herein encompasses a whole plant, a grafted plant, ancestor(s) and progeny of the plants and plant parts, including seeds, shoots, stems, roots (including tubers), rootstock, scion, and plant cells, tissues and organs. The plant may be in any form including suspension cultures, embryos, meristematic regions, callus tissue, leaves, gametophytes, sporophytes, pollen, and microspores. Plants that are particularly useful in the methods of the invention include all plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants including a fodder or forage legume, ornamental plant, food crop, tree, or shrub selected from the list comprising Acacia spp., Acer spp., Actinidia spp., Aesculus spp., Agathis australis, Albizia amara, Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadaba farinosa, Calliandra spp, Camellia sinensis, Canna indica, Capsicum spp., Cassia spp., Centroema pubescens, Chacoomeles spp., Cinnamomum cassia, Coffea arabica, Colophospermum mopane, Coronillia varia, Cotoneaster serotina, Crataegus spp., Cucumis spp., Cupressus spp., Cyathea dealbata, Cydonia oblonga, Cryptomeria japonica, Cymbopogon spp., Cynthea dealbata, Cydonia oblonga, Dalbergia monetaria, Davallia divaricata, Desmodium spp., Dicksonia squarosa, Dibeteropogon amplectens, Dioclea spp, Dolichos spp., Dorycnium rectum, Echinochloa pyramidalis, Ehraffia spp., Eleusine coracana, Eragrestis spp., Erythrina spp., Eucalypfus spp., Euclea schimperi, Eulalia vi/losa, Pagopyrum spp., Feijoa sellowlana, Fragaria spp., Flemingia spp, Freycinetia banksli, Geranium thunbergii, GinAgo biloba, Glycine javanica, Gliricidia spp, Gossypium hirsutum, Grevillea spp., Guibourtia coleosperma, Hedysarum spp., Hemaffhia altissima, Heteropogon contoffus, Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hypeffhelia dissolute, Indigo incamata, Iris spp., Leptarrhena pyrolifolia, Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetia simplex, Lotonus bainesli, Lotus spp., Macrotyloma axillare, Malus spp., Manihot esculenta, Medicago saliva, Metasequoia glyptostroboides, Musa sapientum, Nicotianum spp., Onobrychis spp., Ornithopus spp., Oryza spp., Peltophorum africanum, Pennisetum spp., Persea gratissima, Petunia spp., Phaseolus spp., Phoenix canariensis, Phormium cookianum, Photinia spp., Picea glauca, Pinus spp., Pisum sativam, Podocarpus totara, Pogonarthria fleckii, Pogonaffhria squarrosa, Populus spp., Prosopis cineraria, Pseudotsuga menziesii, Pterolobium stellatum, Pyrus communis, Quercus spp., Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhus natalensis, Ribes grossularia, Ribes spp., Robinia pseudoacacia, Rosa spp., Rubus spp., Salix spp., Schyzachyrium sanguineum, Sciadopitys vefficillata, Sequoia sempervirens, Sequoiadendron giganteum, Sorghum bicolor, Spinacia spp., Sporobolus fimbriatus, Stiburus alopecuroides, Stylosanthos humilis, Tadehagi spp, Taxodium distichum, Themeda triandra, Trifolium spp., Triticum spp., Tsuga heterophylla, Vaccinium spp., Vicia spp., Vitis vinifera, Watsonia pyramidata, Zantedeschia aethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli, Brussels sprouts, cabbage, canola, carrot, cauliflower, celery, collard greens, flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean, straw, sugar beet, sugar cane, sunflower, tomato, squash tea, maize, wheat, barley, rye, oat, peanut, pea, lentil and alfalfa, cotton, rapeseed, canola, pepper, sunflower, tobacco, eggplant, eucalyptus, a tree, an ornamental plant, a perennial grass and a forage crop. Alternatively algae and other non-Viridiplantae can be used for the methods of the present invention.
According to some embodiments of the invention, the plant used by the method of the invention is a crop plant such as rice, maize, wheat, barley, peanut, potato, sesame, olive tree, palm oil, banana, soybean, sunflower, canola, sugarcane, alfalfa, millet, leguminosae (bean, pea), flax, lupinus, rapeseed, tobacco, poplar and cotton.
According to some embodiments of the invention the plant is a dicotyledonous plant.
According to some embodiments of the invention the plant is a monocotyledonous plant. As used herein “tolerance” refers to the ability of a plant to withstand or cope with adverse environmental conditions that would normally negatively impact growth, development, or yield of a plant of a given species at a given developmental stage.
The term “tolerance” is generally interchangeably used with resistance, though in some cases they have different meanings. For instance, in the case of biotic stress tolerance or resistance:
The term “resistance” is as defined by the ISF (International Seed Federation) Vegetable and Ornamental Crops Section for describing the reaction of plants to pests or pathogens, and abiotic stresses for the Vegetable Seed Industry. Specifically, by resistance, it is meant the ability of a plant variety to restrict at least to some degree the multiplication of the virus. Symptoms, even if present, are mild as compared to susceptible plants.
The term “Tolerance” is used herein to indicate a phenotype of a plant wherein at least some of the disease-symptoms remain absent upon exposure of said plant to an infective dose of virus, but virus multiplication remains unaffected as in susceptible plants. According to some embodiments, tolerant plants are therefore resistant to symptom expression or are symptomless carriers of the virus.
As used herein “conferring” refers to increasing tolerance or resistance of a plant to stress conditions.
As used herein “increasing” refers to a statistically significant increase in tolerance compared to the level of tolerance obtained in plants of the same species and developmental stage, as typically determined quantitatively.
Thus, the increase can be by at least, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 2 fold, 3 fold, 5 fold or 10 fold.
Since the level of oxidized aldehydes can be a proxy for tolerance to stress, it can be used for following or for predicting the level of tolerance.
As used herein “plant aldehyde” refers to organic compounds characterized by the presence of a carbonyl group bonded to a hydrogen atom. They are common in plants and play significant roles in various physiological processes, including growth, defense, and aroma.
Aldehyde oxidation is a measure of stress, and measurement is typically effected in the leaf tissue or siliques.
Examples of plant aldehydes for which oxidized state tissue level is determined include, but are not limited to, cinnamaldehyde, vanillin, benzaldehyde, hexanal, hexenal, nonanal, citral (neral and geranial), formaldehyde, acetaldehyde, salicylaldehyde, furfural, anisaldehyde, trans-2- hexenal, octanal, 2,4-decadienal, abscisic aldehyde, acrolein, propionaldehyde, butyraldehyde, crotonaldehyde, glyoxal, phenylacetaldehyde, 3 -methylbutanal, 2-methylpropanal, decanal, dodecanal, heptanal, dodecenal, sinapaldehyde, coniferyl aldehyde, 4-hydroxynonenal (HNE) and indole-3 -carbaldehy de (ICHO).
According to a specific embodiment, the measured aldehydes for which oxidized state tissue level is determined are selected from the group consisting of cinnamaldehyde, benzaldehyde, hexanal, citral (neral and geranial), abscisic acid, acrolein, crotonaldehyde, decanal, dodecanal, heptanal, dodecenal, sinapaldehyde, coniferyl aldehyde, and 4-hydroxynonenal (HNE) and ) and indole-3 -carbaldehy de (ICHO).
According to a specific embodiment, the measured oxidized aldehyde is abscisic acid which is specific to AO3.
To determine the level of oxidized aldehydes in plant tissue, several analytical methods can be employed. One common method is the Thiobarbituric Acid Reactive Substances (TBARS) assay, which measures malondialdehyde (MDA), a byproduct of lipid peroxidation. In this assay, plant tissue is homogenized and reacted with thiobarbituric acid (TBA) under acidic and heated conditions, and the MDA- TBA adduct is measured spectrophotometrically at 532 nm. This method is not specific to a specific aldehyde, and thus measures general tissue level.
High-Performance Liquid Chromatography (HPLC) is another method where specific aldehydes like MDA and 4-hydroxynonenal (HNE) are separated and quantified based on their retention time and absorption spectra. Derivatization agents such as 2,4-dinitrophenylhydrazine (DNPH) can be used to form hydrazones for easier detection. HPLC offers high specificity and sensitivity.
Gas Chromatography-Mass Spectrometry (GC-MS) separates and identifies aldehydes based on their mass-to-charge ratio after volatilization and ionization. Plant samples are often derivatized to improve volatility, and the analysis provides high sensitivity and precision in quantifying volatile and semi-volatile aldehydes.
Liquid Chromatography-Mass Spectrometry (LC-MS) combines liquid chromatography with mass spectrometry for enhanced detection and identification of aldehydes. This method provides high sensitivity and precise quantification.
Spectrophotometric and fluorometric assays can quantify aldehydes based on their ability to form colored or fluorescent products upon reaction with specific reagents like DNPH or Nash reagent. This method is not specific to a given aldehyde.
Enzyme-Linked Immunosorbent Assay (ELISA) uses specific antibodies against aldehy demodified proteins or aldehydes to quantify them in a sample. Nuclear Magnetic Resonance (NMR) Spectroscopy detects aldehydes based on their unique magnetic resonance signals in a magnetic field. This method provides non-destructive analysis and detailed information about molecular structures.
These methods can be chosen based on the specific aldehydes of interest, the required sensitivity and specificity, and the nature of the plant tissue being analyzed. Combining methods, such as using HPLC with MS detection, can offer a more comprehensive and accurate determination of oxidized aldehydes in plant tissues.
According to a specific embodiment, the Thiobarbituric Acid Reactive Substances (TBARS) assay is used for tor MDA.
According to a specific embodiment, aldehyde oxidation is determined using HPLC and/or Mass Spectrometry detection.
Alternatively or additionally, phenotypic appearance can be used to measure tolerance such as leaf color as in Figure 2A. Other measures can be used to measure tolerance to stress, such as biomass, dry weight, growth rate, vigor, yield, seed set, oil content, fiber yield, fiber quality, fiber length, plant height, , photosynthetic capacity, fertilizer use efficiency (e.g., nitrogen use efficiency), and more.
As used herein the phrase "plant vigor" refers to the amount (measured by weight) of tissue produced by the plant in a given time. Hence increased vigor could determine or affect the plant yield or the yield per growing time or growing area. In addition, early vigor (seed and/or seedling) results in improved field stand.
As used herein the phrase "plant yield" refers to the amount (e.g., as determined by weight or size) or quantity (numbers) of tissues or organs produced per plant or per growing season. Hence increased yield could affect the economic benefit one can obtain from the plant in a certain growing area and/or growing time.
It should be noted that a plant yield can be affected by various parameters including, but not limited to, plant biomass; plant vigor; growth rate; seed yield; seed or grain quantity; seed or grain quality; oil yield; content of oil, starch and/or protein in harvested organs (e.g., seeds or vegetative parts of the plant); number of flowers (florets) per panicle (expressed as a ratio of number of filled seeds over number of primary panicles); harvest index; number of plants grown per area; number and size of harvested organs per plant and per area; number of plants per growing area (density); number of harvested organs in field; total leaf area; carbon assimilation and carbon partitioning (the distribution/allocation of carbon within the plant); resistance to shade; number of harvestable organs (e.g. seeds), seeds per pod, weight per seed; and modified architecture [such as increase stalk diameter, thickness or improvement of physical properties (e.g. elasticity)]. As used herein the phrase “seed yield” refers to the number or weight of the seeds per plant, pod or spike weight, seeds per pod, or per growing area or to the weight of a single seed, or to the oil extracted per seed. Hence seed yield can be affected by seed dimensions (e.g., length, width, perimeter, area and/or volume), number of (filled) seeds and seed filling rate and by seed oil content. Hence increase seed yield per plant could affect the economic benefit one can obtain from the plant in a certain growing area and/or growing time; and increase seed yield per growing area could be achieved by increasing seed yield per plant, and/or by increasing number of plants grown on the same given area or by increase harvest index (seed yield per the total biomass).
The term "seed" (also referred to as "grain" or "kernel") as used herein refers to a small embryonic plant enclosed in a covering called the seed coat (usually with some stored food), the product of the ripened ovule of gymnosperm and angiosperm plants which occurs after fertilization and some growth within the mother plant.
The phrase “oil content” as used herein refers to the amount of lipids in a given plant organ, either the seeds (seed oil content) or the vegetative portion of the plant (vegetative oil content) and is typically expressed as percentage of dry weight (10 % humidity of seeds) or wet weight (for vegetative portion).
It should be noted that oil content is affected by intrinsic oil production of a tissue (e.g., seed, vegetative portion), as well as the mass or size of the oil-producing tissue per plant or per growth period.
In one embodiment, increase in oil content of the plant can be achieved by increasing the size/mass of a plant's tissue(s) which comprise oil per growth period. Thus, increased oil content of a plant can be achieved by increasing the yield, growth rate, biomass and vigor of the plant.
As used herein the phrase "plant biomass" refers to the amount (e.g., measured in grams of air-dry tissue) of a tissue produced from the plant in a growing season, which could also determine or affect the plant yield or the yield per growing area. An increase in plant biomass can be in the whole plant or in parts thereof such as aboveground (harvestable) parts, vegetative biomass, leaf size or area, leaf thickness, roots and seeds.
It should be noted that an increase in plant’s dry weight, rosette area, leaf blade area, leaf petiole length, leaf thickness, shoot dry weight, shoot fresh weight, vegetative dry weight, and/or total dry matter per plant indicates an increased biomass as compared to a matching control plant under the same growth conditions (control plant=being of the same species, and developmental stage but not having been modified for reduced expression of AO1 and/or AO2). As used herein the term “root biomass” refers to the total weight of the plant’ s root(s). Root biomass can be determined directly by weighing the total root material (fresh and/or dry weight) of a plant.
Additional or alternatively, the root biomass can be indirectly determined by measuring root coverage, root density and/or root length of a plant.
It should be noted that plants having a larger root coverage exhibit higher fertilizer (e.g., nitrogen) use efficiency and/or higher water use efficiency as compared to plants with a smaller root coverage.
As used herein the phrase “root coverage” refers to the total area or volume of soil or of any plant-growing medium encompassed by the roots of a plant.
According to some embodiments of the invention, the root coverage is the minimal convex volume encompassed by the roots of the plant.
It should be noted that since each plant has a characteristic root system, e.g., some plants exhibit a shallow root system (e.g., only a few centimeters below ground level), while others have a deep in soil root system (e.g., a few tens of centimeters or a few meters deep in soil below ground level), measuring the root coverage of a plant can be performed in any depth of the soil or of the plant-growing medium, and comparison of root coverage between plants of the same species (e.g., the plant in which there is down-regulation of A01/A02 and control plants as described herein) should be performed by measuring the root coverage in the same depth.
According to some embodiments of the invention, the root coverage is the minimal convex area encompassed by the roots of a plant in a specific depth.
As used herein the term “root density” refers to the density of roots in a given area (e.g., area of soil or any plant growing medium). The root density can be determined by counting the root number per a predetermined area at a predetermined depth (in units of root number per area, e.g., mm , cm or m ).
As used herein the phrase “root length” refers to the total length of the longest root of a single plant.
As used herein the phrase “root length growth rate” refers to the change in total root length per plant per time unit (e.g., per day).
As used herein the phrase “growth rate” refers to the increase in plant organ/tissue size per time (can be measured in cm2 per day or cm/day).
As used herein the phrase “photosynthetic capacity” (also known as “Amax”) is a measure of the maximum rate at which leaves are able to fix carbon during photosynthesis. It is typically measured as the amount of carbon dioxide that is fixed per square meter per second, for example as pmol m'2 sec'1. Plants are able to increase their photosynthetic capacity by several modes of action, such as by increasing the total leaves area (e.g., by increase of leaves area, increase in the number of leaves, and increase in plant’s vigor, e.g., the ability of the plant to grow new leaves along time course) as well as by increasing the ability of the plant to efficiently execute carbon fixation in the leaves. Hence, the increase in total leaves area can be used as a reliable measurement parameter for photosynthetic capacity increment.
As used herein the phrase “plant height” refers to measuring plant height as indication for plant growth status, assimilates allocation and yield potential. In addition, plant height is an important trait to prevent lodging (collapse of plants with high biomass and height) under high density agronomical practice.
Plant height is measured in various ways depending on the plant species but it is usually measured as the length between the ground level and the top of the plant, e.g., the head or the reproductive tissue.
The phrase "abiotic stress" as used herein refers to any adverse effect on metabolism, growth, reproduction and/or viability of a plant. Accordingly, abiotic stress can be induced by suboptimal environmental growth conditions such as, for example, salinity, osmotic stress, water deprivation, drought, flooding, freezing, low or high temperature, heavy metal toxicity, anaerobiosis, nutrient deficiency (e.g., nitrogen deficiency or limited nitrogen), atmospheric pollution or UV irradiation.
According to a specific embodiment, the abiotic stress is selected from the group consisting of drought stress, oxidative stress, radiation stress, temperature stress, light stress, nutrient stress, heavy metal stress, salinity stress wound stress and flooding stress.
According to a specific embodiment, the abiotic stress is selected from the group consisting of drought stress, oxidative stress and radiation stress.
According to a specific embodiment, the abiotic stress is drought stress.
According to a specific embodiment, the drought stress comprises extreme drought stress, as defined by fast water loss (8 to 16% within 3 to 7 hours).
According to a specific embodiment, the abiotic stress is oxidative stress.
According to a specific embodiment, the abiotic stress is radiation stress.
According to a specific embodiment, the radiation is UV-C.
According to a specific embodiment, the stress is not radiation stress.
According to a specific embodiment, the stress is not UV-C stress.
As used herein, the phrase “drought conditions” refers to growth conditions with limited water availability. As used herein “extreme drought conditions” relates to water deprivation (as represented by root detachment model) where there is fast water loss e.g., 8-16 % within 3-7 hours.
As used herein, the phrase “oxidative stress” refers to a condition where there is an imbalance between the production of reactive oxygen species (ROS) and the plant’s ability to detoxify or neutralize them. ROS, including superoxide radicals, hydrogen peroxide, and hydroxyl radicals, are highly reactive molecules generated during various metabolic processes, particularly under stress conditions such as drought, high light intensity, or pathogen infection. When ROS levels exceed the plant’s antioxidant defenses — comprised of enzymes like catalase and superoxide dismutase, and non-enzymatic antioxidants like ascorbic acid and glutathione — oxidative damage occurs. This damage can affect cellular components such as lipids, proteins, and nucleic acids, leading to impaired function and structure, ultimately impacting plant growth, development, and productivity. Oxidative stress thus represents a critical challenge that plants must manage to maintain cellular homeostasis and overall health.
Notably, reactive carbonyl species (RCS) can act as agents to mediate reactive oxygen species (ROS) signals to target proteins such as heat shock-responsive gene regulation, ABA signaling for stomatai closure, and auxin signaling for lateral root formation in plants. Yet, RCS can mediate ROS-induced programmed cell death as well as senescence. Plant aldehyde oxidases were shown to generate H2O2 and 02", while oxidizing various aldehydes (Yesbergenova et al., 2005, Srivastava et al., 2017). Rose Bengal presents a compound, which elicits oxidative stress.
As used herein “radiation stress” refers to the adverse effects caused by excessive or intense radiation, primarily from sunlight or other artificial sources, on plant health and development. This type of stress often results from high levels of ultraviolet (UV) radiation or excessive visible light, which can lead to the generation of reactive oxygen species (ROS) and cause damage to cellular components like proteins, lipids, and DNA. High-intensity radiation can impair photosynthesis by damaging chlorophyll and disrupting the photosynthetic machinery, reducing the plant’s ability to synthesize essential nutrients and energy. Additionally, radiation stress can trigger protective responses such as the production of UV-ab sorbing compounds and the activation of stress-related genes, but prolonged exposure can overwhelm these defenses, leading to reduced growth, yield loss, and compromised plant health.
As used herein “temperature stress” refers to the physiological and biochemical challenges that arise from exposure to temperatures outside the optimal range for growth and development. Cold stress, or low-temperature stress, occurs when plants are exposed to freezing or near-freezing conditions, leading to issues such as ice formation within cells, which can damage cellular structures, disrupt metabolic processes, and impair water uptake. This stress often results in reduced growth, delayed flowering, and diminished yield. Conversely, heat stress, or high- temperature stress, happens when plants experience temperatures significantly above their optimal range, leading to overheating of cellular components, increased water loss through transpiration, and disruption of photosynthesis. Heat stress can cause protein denaturation, enzyme inactivation, and reduced reproductive success, ultimately leading to decreased plant productivity and health. Both cold and heat stress require plants to activate various stress response mechanisms, such as the synthesis of heat shock proteins or cold-responsive proteins, to mitigate damage and maintain homeostasis.
As used herein “nutrient stress” refers to a state when there is an inadequate supply or imbalance of essential nutrients required for optimal growth and development. This stress can result from deficiencies or excesses of key nutrients such as nitrogen, phosphorus, potassium, calcium, magnesium, and micronutrients like iron, zinc, and manganese. Nutrient deficiencies can impair various physiological processes, including photosynthesis, protein synthesis, and enzyme function, leading to symptoms such as chlorosis, stunted growth, poor root development, and reduced crop yields. Conversely, nutrient excesses can cause toxicity, alter nutrient uptake dynamics, and disrupt plant metabolism. Effective nutrient management is crucial to prevent nutrient stress and ensure that plants receive the appropriate balance of nutrients for healthy growth and productivity. Nutrient stress can be caused under fertilizer-limiting conditions or nutrientlimiting conditions.
As used herein the phrase “fertilizer-limiting conditions” refers to growth conditions which include a level (e.g., concentration) of a fertilizer applied which is below the level needed for normal plant metabolism, growth, reproduction and/or viability.
As used herein the phrase “nitrogen-limiting conditions” refers to growth conditions which include a level (e.g., concentration) of nitrogen (e.g., ammonium or nitrate) applied which is below the level needed for normal plant metabolism, growth, reproduction and/or viability.
As used herein “light stress”, occurs when plants are exposed to light conditions that are suboptimal or excessively intense, impacting their growth and development. Light stress can result from either too much light, leading to overexposure that causes damage to photosynthetic machinery, chlorophyll degradation, and the formation of reactive oxygen species, or too little light, which can reduce photosynthesis, hinder growth, and limit energy production. Excessive light can cause photoinhibition and damage to cellular structures, while insufficient light can lead to etiolation and poor plant health.
As used herein “wound stress” refers to the physiological and biochemical responses triggered by physical damage or injury to plant tissues. This type of stress can result from mechanical wounds such as cuts, bruises, or abrasions, as well as from damage caused by insects, herbivores, or pathogens. When a plant experiences wound stress, it activates a range of defensive mechanisms to repair the damage and mitigate further injury. These responses include the production of wound-induced proteins, such as proteinase inhibitors and defensive enzymes, the synthesis of secondary metabolites like jasmonic acid and phenolic compounds, and the reinforcement of cell walls. Additionally, plants may initiate localized and systemic signaling pathways to coordinate responses across the tissue and to neighboring areas.
As used herein “flooding stress” refers to when roots are submerged in excess water (not optimal to the plant species), leading to a lack of oxygen in the soil and disrupting normal plant function. This condition impairs root respiration and nutrient uptake, resulting in reduced growth, weakened plant structures, and potential root rot. The lack of oxygen can also trigger the production of toxic metabolites and affect cellular energy processes.
As used herein “heavy metal stress” refers to the adverse effects caused by the accumulation of toxic levels of heavy metals, such as lead (Pb), cadmium (Cd), mercury (Hg), arsenic (As), and copper (Cu), in the soil and plant tissues. These metals can enter plants through contaminated soil or water and disrupt various physiological processes. Heavy metal stress leads to oxidative damage, interfering with photosynthesis, respiration, and nutrient uptake, and can cause growth inhibition, chlorosis, and reduced biomass. Toxic metals can also displace essential nutrients, impair enzyme function, and lead to the production of reactive oxygen species (ROS), further exacerbating stress.
As used herein “biotic stress” refers to the adverse effects caused by living organisms that negatively impact plant health and growth. This type of stress is caused by various biotic factors, including pathogens such as bacteria, fungi, and viruses, as well as pests like insects and nematodes. Biotic stress can lead to disease, damage to plant tissues, and reduced nutrient availability. Plants respond to biotic stress through a range of defensive mechanisms, including the production of antimicrobial compounds, activation of immune responses, and physical barriers like thickened cell walls.
It is well accepted that during stress, toxic metabiolites are formed such as toxic aldehydes.
Methods of determining tolerance to stress are well known in the art, some are described infra.
Drought tolerance assay/Osmoticum assay - To analyze whether the plants (modified to down-regulate A01/A02, also may be referred to as “test plants”) are more tolerant to drought, an osmotic stress produced by the non-ionic osmolyte sorbitol in the medium can be performed. Control and the treated plants are germinated and grown in plant-agar plates for 4 days, after which they are transferred to plates containing 500 mM sorbitol. The treatment causes growth retardation, then both control and test plants are compared, by measuring plant weight (wet and dry), yield, and by growth rates measured as time to flowering.
Conversely, soil-based drought screens are performed with test plants. Seeds from control Arabidopsis plants and test plants germinated and transferred to pots. Drought stress is obtained after irrigation is ceased accompanied by placing the pots on absorbent paper to enhance the soildrying rate. Test and control plants are compared to each other when the majority of the control plants develop severe wilting. Plants are re-watered after obtaining a significant fraction of the control plants displaying a severe wilting. Plants are ranked comparing to controls for each of two criteria: tolerance to the drought conditions and recovery (survival) following re-watering.
Cold stress tolerance - To analyze cold stress, mature (25 day old) plants are transferred to 4 °C chambers for 1 or 2 weeks, with constitutive light. Later on plants are moved back to greenhouse. Two weeks later damages from chilling period, resulting in growth retardation and other phenotypes, are compared between both control and test plants, by measuring plant weight (wet and dry), and by comparing growth rates measured as time to flowering, plant size, yield, and the like.
Heat stress tolerance - Heat stress tolerance is achieved by exposing the plants to temperatures above 34 °C for a certain period. Plant tolerance is examined after transferring the plants back to 22 °C for recovery and evaluation after 5 days relative to internal controls or plants not exposed to neither cold or heat stress.
Water use efficiency - can be determined as the biomass produced per unit transpiration. To analyze WUE, leaf relative water content can be measured in control and test plants. Fresh weight (FW) is immediately recorded; then leaves are soaked for 8 hours in distilled water at room temperature in the dark, and the turgid weight (TW) is recorded. Total dry weight (DW) is recorded after drying the leaves at 60 °C to a constant weight.
Fertilizer use efficiency - To analyze whether the test plants are more responsive to fertilizers, plants are grown in agar plates or pots with a limited amount of fertilizer, as described, for example, in Yanagisawa et al (Proc Natl Acad Sci U S A. 2004; 101 :7833-8). The plants are analyzed for their overall size, time to flowering, yield, protein content of shoot and/or grain. The parameters checked are the overall size of the mature plant, its wet and dry weight, the weight of the seeds yielded, the average seed size and the number of seeds produced per plant. Other parameters that may be tested are: the chlorophyll content of leaves (as nitrogen plant status and the degree of leaf verdure is highly correlated), amino acid and the total protein content of the seeds or other plant parts such as leaves or shoots, oil content, etc. Similarly, instead of providing nitrogen at limiting amounts, phosphate or potassium can be added at increasing concentrations. Again, the same parameters measured are the same as listed above. In this way, nitrogen use efficiency (NUE), phosphate use efficiency (PUE) and potassium use efficiency (KUE) are assessed, checking the ability of the test plants to thrive under nutrient restraining conditions.
Nitrogen use efficiency - To analyze whether the test plants (e.g., Arabidopsis plants) are more responsive to nitrogen, plant are grown in 0.75-3 mM (nitrogen deficient conditions) or 6- 10 mM (optimal nitrogen concentration). Plants are allowed to grow for additional 25 days or until seed production. The plants are then analyzed for their overall size, time to flowering, yield, protein content of shoot and/or grain/ seed production. The parameters checked can be the overall size of the plant, wet and dry weight, the weight of the seeds yielded, the average seed size and the number of seeds produced per plant. Other parameters that may be tested are: the chlorophyll content of leaves (as nitrogen plant status and the degree of leaf greenness is highly correlated), amino acid and the total protein content of the seeds or other plant parts such as leaves or shoots and oil content. Transformed plants not exhibiting substantial physiological and/or morphological effects, or exhibiting higher measured parameters levels than wild-type plants, are identified as nitrogen use efficient plants.
Nitrogen Use efficiency assay using plantlets - The assay is done according to Yanagisawa-S. et al. with minor modifications (“Metabolic engineering with Dofl transcription factor in plants: Improved nitrogen assimilation and growth under low-nitrogen conditions” Proc. Natl. Acad. Sci. USA 101, 7833-7838). Briefly, test plants which are grown for 7-10 days in 0.5 x MS [Murashige-Skoog] supplemented with a selection agent are transferred to two nitrogenlimiting conditions: MS media in which the combined nitrogen concentration (NEENCh and KNCh) was 0.75 mM (nitrogen deficient conditions) or 6-15 mM (optimal nitrogen concentration). Plants are allowed to grow for additional 30-40 days and then photographed, individually removed from the Agar (the shoot without the roots) and immediately weighed (fresh weight) for later statistical analysis. Constructs for which only T1 seeds are available are sown on selective media and at least 20 seedlings (each one representing an independent transformation event) are carefully transferred to the nitrogen-limiting media. For constructs for which T2 seeds are available, different transformation events are analyzed. Usually, 20 randomly selected plants from each event are transferred to the nitrogen-limiting media allowed to grow for 3-4 additional weeks and individually weighed at the end of that period. Test plants are compared to control plants grown in parallel under the same conditions.
Nitrogen determination - The procedure for N (nitrogen) concentration determination in the structural parts of the plants involves the potassium persulfate digestion method to convert organic N to NCh’ (Purcell and King 1996 Argon. J. 88: 111-113, the modified Cd" mediated reduction of NCh’ to NCh" (Vodovotz 1996 Biotechniques 20:390-394) and the measurement of nitrite by the Griess assay (Vodovotz 1996, supra). The absorbance values are measured at 550 nm against a standard curve of NaNCh. The procedure is described in details in Samonte et al. 2006 Agron. J. 98: 168-176.
Germination tests - Germination tests compare the percentage of seeds from test plants that could complete the germination process to the percentage of seeds from control plants that are treated in the same manner. Normal conditions are considered for example, incubations at 22 °C under 22-hour light 2-hour dark daily cycles. Evaluation of germination and seedling vigor is conducted between 4 and 14 days after planting. The basal media is 50 % MS medium (Murashige and Skoog, 1962 Plant Physiology 15, 473-497).
Germination is checked also at unfavorable conditions such as cold (incubating at temperatures lower than 10 °C instead of 22 °C) or using seed inhibition solutions that contain high concentrations of an osmolyte such as sorbitol (at concentrations of 50 mM, 100 mM, 200 mM, 300 mM, 500 mM, and up to 1000 mM) or applying increasing concentrations of salt (of 50 mM, 100 mM, 200 mM, 300 mM, 500 mM NaCl).
The effect of down-regulating AO1/AO2 on plant’s vigor, growth rate, biomass, yield and/or oil content can be determined using known methods.
Plant vigor - The plant vigor can be calculated by the increase in growth parameters such as leaf area, fiber length, rosette diameter, plant fresh weight and the like per time.
Growth rate - The growth rate can be measured using digital analysis of growing plants. For example, images of plants growing in greenhouse on plot basis can be captured every 3 days and the rosette area can be calculated by digital analysis. Rosette area growth is calculated using the difference of rosette area between days of sampling divided by the difference in days between samples.
It should be noted that an increase in rosette parameters such as rosette area, rosette diameter and/or rosette growth rate in a plant model such as Arabidopsis predicts an increase in canopy coverage and/or plot coverage in a target plant such as Brassica sp., soy, com, wheat, Barley, oat, cotton, rice, tomato, sugar beet, and vegetables such as cucumber.
Thus, as mentioned, conferring tolerance to stress is achieved by down-regulating expression of AO1 and/or AO2 (aldehyde oxidase 1 and/or aldehyde oxidase 2).
As used herein “aldehyde oxidase 1” refers to / T5G20960 ( AAO1) or natural orthologs thereof.
As used herein “aldehyde oxidase 2” refers to AT3G43600 (AAO2) or natural orthologs thereof. As used herein “aldehyde oxidase 3” refers to AT2G27150 (AA03) or natural orthologs thereof.
As used herein “aldehyde oxidase 4” refers to AT1G04580 (AA04) or natural orthologs thereof.
Table 1: Aldehyde oxidases (and their gene accession numbers) present in crops to be improved by genes modification according to some embodiments of the invention.
Figure imgf000024_0001
Figure imgf000025_0001
As used herein the phrase “downregulates expression” refers to downregulating the expression of a protein (e.g. AO1 and/or AO2) at the genomic (e.g. homologous recombination and site specific endonucleases) and/or the transcript level using a variety of molecules which interfere with transcription and/or translation (e.g., RNA silencing agents) or on the protein level (e.g., aptamers, small molecules and inhibitory peptides and the like).
Down regulation of expression may be either transient or stable. According to specific embodiments, down regulating expression refers to the absence of mRNA and/or protein, as detected by RT-PCR or Western blot, respectively.
According to other specific embodiments down regulating expression refers to a decrease in the level of mRNA and/or protein, as detected by RT-PCR or Western blot, respectively. The reduction may be by at least a 10 %, at least 20 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 %, at least 95 % or at least 99 % reduction as compared to that in a plant of the same species in which down-regulation has not been performed, yet of the same genetic background and developmental stage.
Non-limiting examples of agents capable of down regulating AO1 and/or AO2 expression are described in details hereinbelow.
Down-regulation at the nucleic acid level
Down-regulation at the nucleic acid level is typically effected using a nucleic acid agent, having a nucleic acid backbone, DNA, RNA, mimetics thereof or a combination of same. The nucleic acid agent may be encoded from a DNA molecule or provided to the cell per se.
According to specific embodiments, the downregulating agent is a polynucleotide.
According to specific embodiments, the downregulating agent is a polynucleotide capable of hybridizing to a gene or mRNA encoding the protein e.g., AO1 and/or AO2.
According to specific embodiments, the downregulating agent directly interacts with the gene of e.g., AO1 and/or AO2.
According to specific embodiments, the agent directly binds the gene.
According to specific embodiments the downregulating agent is an RNA silencing agent or a genome or RNA editing agent.
Thus, downregulation of gene expression can be achieved by RNA silencing. This is exemplified in the double mutants of the Examples section which follows. As used herein, the phrase "RNA silencing" refers to a group of regulatory mechanisms [e.g. RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), quelling, co-suppression, and translational repression] mediated by RNA molecules which result in the inhibition or "silencing" of the expression of a corresponding protein-coding gene.
As used herein, the term "RNA silencing agent" refers to an RNA which is capable of specifically inhibiting or "silencing" the expression of a target gene. In certain embodiments, the RNA silencing agent is capable of preventing complete processing (e.g, the full translation and/or expression) of an mRNA molecule through a post-transcriptional silencing mechanism. RNA silencing agents include non-coding RNA molecules, for example RNA duplexes comprising paired strands, as well as precursor RNAs from which such small non-coding RNAs can be generated. Exemplary RNA silencing agents include dsRNAs such as siRNAs, miRNAs and shRNAs.
In one embodiment, the RNA silencing agent is capable of inducing RNA interference. In another embodiment, the RNA silencing agent is capable of mediating translational repression.
According to an embodiment of the invention, the RNA silencing agent is specific to the target RNA (e.g., AO1 and/or AO2 and does not cross inhibit or silence other targets AO3, AO4 and AO5 or other non-specific off-targets; as determined by PCR, Western blot, Immunohistochemistry and/or flow cytometry.
RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs).
Downregulation of AO1 and/or AO2 can also be achieved by inactivating the gene via introducing targeted mutations involving loss-of function alterations (e.g. point mutations, deletions and insertions) in the gene structure.
As used herein “a null mutation” or a “null allele” is a mutation that leads to a non- transcribable RNA and/or non-translatable protein product or a protein product which is nonfunctional.
Also provided is a cultivated plant containing the modified gene as described herein.
As used herein, the phrase “loss-of-function alterations” refers to any mutation in the DNA sequence of a gene (AO1 and/or AO2) which results in downregulation of the expression level and/or activity of the expressed product, i.e., the mRNA transcript and/or the translated protein. Non-limiting examples of such loss-of-function alterations include a missense mutation, z.e., a mutation which changes an amino acid residue in the protein with another amino acid residue and thereby abolishes the enzymatic activity of the protein; a nonsense mutation, /.<?., a mutation which introduces a stop codon in a protein, e.g., an early stop codon which results in a shorter protein devoid of the enzymatic activity; a frame-shift mutation, z.e., a mutation, usually, deletion or insertion of nucleic acid(s) which changes the reading frame of the protein, and may result in an early termination by introducing a stop codon into a reading frame (e.g., a truncated protein, devoid of the enzymatic activity), or in a longer amino acid sequence (e.g., a readthrough protein) which affects the secondary or tertiary structure of the protein and results in a non-functional protein, devoid of the enzymatic activity of the non-mutated polypeptide; a readthrough mutation due to a frame-shift mutation or a modified stop codon mutation (i.e., when the stop codon is mutated into an amino acid codon), with an abolished enzymatic activity; a promoter mutation, i.e., a mutation in a promoter sequence, usually 5' to the transcription start site of a gene, which results in downregulation of a specific gene product; a regulatory mutation, i.e., a mutation in a region upstream or downstream, or within a gene, which affects the expression of the gene product; a deletion mutation, i.e., a mutation which deletes coding nucleic acids in a gene sequence and which may result in a frame-shift mutation or an in-frame mutation (within the coding sequence, deletion of one or more amino acid codons); an insertion mutation, z.e., a mutation which inserts coding or non-coding nucleic acids into a gene sequence, and which may result in a frame-shift mutation or an in-frame insertion of one or more amino acid codons; an inversion, z.e., a mutation which results in an inverted coding or non-coding sequence; a splice mutation z.e., a mutation which results in abnormal splicing or poor splicing; and a duplication mutation, z.e., a mutation which results in a duplicated coding or non-coding sequence, which can be in-frame or can cause a frame-shift.
According to a specific embodiment, the mutation is a null mutation.
As used herein “a null mutation” is a gene mutation that leads to its not being transcribed into RNA and/or translated into a functional protein. According to a specific embodiment, the mutation causes the protein not being translated at all or completely degraded (e.g., as determined by Western blot).
According to specific embodiments, loss-of-function alteration of a gene may comprise at least one allele of the gene.
The term "allele" as used herein, refers to any of one or more alternative forms of a gene locus, all of which alleles relate to a trait or characteristic. In a diploid cell or organism, the two alleles of a given gene occupy corresponding loci on a pair of homologous chromosomes.
According to other specific embodiments loss-of-function alteration of a gene comprises both alleles of the gene. In such instances the genes of AO1 and/or AO2 may be in a homozygous form. According to this embodiment, homozygosity is a condition where both alleles at the locus are characterized by the same nucleotide sequence. Heterozygosity refers to different mutations in the gene at the locus.
According to other specific embodiments loss-of-function alterations claimed herein are non-naturally occurring, i.e., not found in nature, and a result of man-made activities.
Methods of introducing nucleic acid alterations to a gene of interest are well known in the art [see for example Menke D. Genesis (2013) 51 : - 618; Capecchi, Science (1989) 244: 1288- 1292; Santiago et al. Proc Natl Acad Sci USA (2008) 105:5809-5814; International Patent Application Nos. WO 2014085593, WO 2009071334 and WO 2011146121; US Patent Nos. 8771945, 8586526, 6774279 and UP Patent Application Publication Nos. 20030232410, 20050026157, US20060014264; the contents of which are incorporated by reference in their entireties] and include targeted homologous recombination, site specific recombinases, PB transposases and genome editing by engineered nucleases. Agents for introducing nucleic acid alterations to a gene of interest can be designed publically available sources or obtained commercially from Transposagen, Addgene and Sangamo Biosciences. Following is a description of various exemplary methods used to introduce nucleic acid alterations to a gene of interest and agents for implementing same that can be used according to specific embodiments of the present invention.
Genome Editing using engineered endonucleases - this approach refers to a reverse genetics method using artificially engineered nucleases to cut and create specific double-stranded breaks at a desired location(s) in the genome, which are then repaired by cellular endogenous processes such as, homology directed repair (HDR) and non-homologous end-joining (NFfEJ). NFfEJ directly joins the DNA ends in a double-stranded break, while HDR utilizes a homologous sequence as a template for regenerating the missing DNA sequence at the break point. In order to introduce specific nucleotide modifications to the genomic DNA, a DNA repair template containing the desired sequence must be present during HDR. Genome editing cannot be performed using traditional restriction endonucleases since most restriction enzymes recognize a few base pairs on the DNA as their target and the probability is very high that the recognized base pair combination will be found in many locations across the genome resulting in multiple cuts not limited to a desired location. To overcome this challenge and create site-specific single- or doublestranded breaks, several distinct classes of nucleases have been discovered and bioengineered to date. These include the meganucleases, Zinc finger nucleases (ZFNs), transcription-activator like effector nucleases (TALENs) and CRISPR/Cas system.
Meganucleases - Meganucleases are commonly grouped into four families: the LAGLID ADG family, the GIY-YIG family, the His-Cys box family and the HNH family. These families are characterized by structural motifs, which affect catalytic activity and recognition sequence. For instance, members of the LAGLID ADG family are characterized by having either one or two copies of the conserved LAGLID ADG motif. The four families of meganucleases are widely separated from one another with respect to conserved structural elements and, consequently, DNA recognition sequence specificity and catalytic activity. Meganucleases are found commonly in microbial species and have the unique property of having very long recognition sequences (>14bp) thus making them naturally very specific for cutting at a desired location. This can be exploited to make site-specific double-stranded breaks in genome editing. One of skill in the art can use these naturally occurring meganucleases, however the number of such naturally occurring meganucleases is limited. To overcome this challenge, mutagenesis and high throughput screening methods have been used to create meganuclease variants that recognize unique sequences. For example, various meganucleases have been fused to create hybrid enzymes that recognize a new sequence. Alternatively, DNA interacting amino acids of the meganuclease can be altered to design sequence specific meganucleases (see e.g., US Patent 8,021,867). Meganucleases can be designed using the methods described in e.g., Certo, MT et al. Nature Methods (2012) 9:073-975; U.S. Patent Nos. 8,304,222; 8,021,867; 8, 119,381; 8, 124,369; 8, 129,134; 8,133,697; 8,143,015; 8,143,016; 8, 148,098; or 8, 163,514, the contents of each are incorporated herein by reference in their entirety. Alternatively, meganucleases with site specific cutting characteristics can be obtained using commercially available technologies e.g., Precision Biosciences' Directed Nuclease Editor™ genome editing technology.
ZFNs and TALENs - Two distinct classes of engineered nucleases, zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), have both proven to be effective at producing targeted double-stranded breaks (Christian et al., 2010; Kim et al., 1996; Li et al., 2011; Mahfouz et al., 2011; Miller et al., 2010).
Basically, ZFNs and TALENs restriction endonuclease technology utilizes a non-specific DNA cutting enzyme which is linked to a specific DNA binding domain (either a series of zinc finger domains or TALE repeats, respectively). Typically a restriction enzyme whose DNA recognition site and cleaving site are separate from each other is selected. The cleaving portion is separated and then linked to a DNA binding domain, thereby yielding an endonuclease with very high specificity for a desired sequence. An exemplary restriction enzyme with such properties is Fokl. Additionally Fokl has the advantage of requiring dimerization to have nuclease activity and this means the specificity increases dramatically as each nuclease partner recognizes a unique DNA sequence. To enhance this effect, Fokl nucleases have been engineered that can only function as heterodimers and have increased catalytic activity. The heterodimer functioning nucleases avoid the possibility of unwanted homodimer activity and thus increase specificity of the doublestranded break.
Thus, for example to target a specific site, ZFNs and TALENs are constructed as nuclease pairs, with each member of the pair designed to bind adjacent sequences at the targeted site. Upon transient expression in cells, the nucleases bind to their target sites and the Fokl domains heterodimerize to create a double-stranded break. Repair of these double-stranded breaks through the nonhomologous end-joining (NHEJ) pathway most often results in small deletions or small sequence insertions. Since each repair made by NHEJ is unique, the use of a single nuclease pair can produce an allelic series with a range of different deletions at the target site. The deletions typically range anywhere from a few base pairs to a few hundred base pairs in length, but larger deletions have successfully been generated in cell culture by using two pairs of nucleases simultaneously (Carlson et al., 2012; Lee et al., 2010). In addition, when a fragment of DNA with homology to the targeted region is introduced in conjunction with the nuclease pair, the double- stranded break can be repaired via homology directed repair to generate specific modifications (Li et al., 2011; Miller et al., 2010; Urnov et al., 2005).
Although the nuclease portions of both ZFNs and TALENs have similar properties, the difference between these engineered nucleases is in their DNA recognition peptide. ZFNs rely on Cys2- His2 zinc fingers and TALENs on TALEs. Both of these DNA recognizing peptide domains have the characteristic that they are naturally found in combinations in their proteins. Cys2-His2 Zinc fingers typically found in repeats that are 3 bp apart and are found in diverse combinations in a variety of nucleic acid interacting proteins. TALEs on the other hand are found in repeats with a one-to-one recognition ratio between the amino acids and the recognized nucleotide pairs. Because both zinc fingers and TALEs happen in repeated patterns, different combinations can be tried to create a wide variety of sequence specificities. Approaches for making site-specific zinc finger endonucleases include, e.g., modular assembly (where Zinc fingers correlated with a triplet sequence are attached in a row to cover the required sequence), OPEN (low-stringency selection of peptide domains vs. triplet nucleotides followed by high-stringency selections of peptide combination vs. the final target in bacterial systems), and bacterial one-hybrid screening of zinc finger libraries, among others. ZFNs can also be designed and obtained commercially from e.g., Sangamo Biosciences™ (Richmond, CA).
Method for designing and obtaining TALENs are described in e.g. Reyon et al. Nature Biotechnology 2012 May;30(5):460-5; Miller et al. Nat Biotechnol. (2011) 29: 143-148; Cermak et al. Nucleic Acids Research (2011) 39 (12): e82 and Zhang et al. Nature Biotechnology (2011) 29 (2): 149-53. A recently developed web-based program named Mojo Hand was introduced by Mayo Clinic for designing TAL and TALEN constructs for genome editing applications (can be accessed through www(dot)talendesign(dot)org). TALEN can also be designed and obtained commercially from e.g., Sangamo Biosciences™ (Richmond, CA).
CRISPR-Cas system - Has been exemplified in the examples section which follows. Many bacteria and archea contain endogenous RNA-based adaptive immune systems that can degrade nucleic acids of invading phages and plasmids. These systems consist of clustered regularly interspaced short palindromic repeat (CRISPR) genes that produce RNA components and CRISPR associated (Cas) genes that encode protein components. The CRISPR RNAs (crRNAs) contain short stretches of homology to specific viruses and plasmids and act as guides to direct Cas nucleases to degrade the complementary nucleic acids of the corresponding pathogen. Studies of the type II CRISPR/Cas system of Streptococcus pyogenes have shown that three components form an RNA/protein complex and together are sufficient for sequence-specific nuclease activity: the Cas9 nuclease, a crRNA containing 20 base pairs of homology to the target sequence, and a trans-activating crRNA (tracrRNA) (Jinek et al. Science (2012) 337: 816-821). It was further demonstrated that a synthetic chimeric guide RNA (gRNA) composed of a fusion between crRNA and tracrRNA could direct Cas9 to cleave DNA targets that are complementary to the crRNA in vitro. It was also demonstrated that transient expression of Cas9 in conjunction with synthetic gRNAs can be used to produce targeted double-stranded brakes in a variety of different species (Cho et al., 2013; Cong et al., 2013; DiCarlo etal., 2013; Hwang etal., 2013a, b; Jinek etal., 2013; Mali et al., 2013).
The CRIPSR/Cas system for genome editing contains two distinct components: a gRNA and an endonuclease e.g. Cas9.
The gRNA is typically a 20 nucleotide sequence encoding a combination of the target homologous sequence (crRNA) and the endogenous bacterial RNA that links the crRNA to the Cas9 nuclease (tracrRNA) in a single chimeric transcript. The gRNA/Cas9 complex is recruited to the target sequence by the base-pairing between the gRNA sequence and the complement genomic DNA. For successful binding of Cas9, the genomic target sequence must also contain the correct Protospacer Adjacent Motif (PAM) sequence immediately following the target sequence. The binding of the gRNA/Cas9 complex localizes the Cas9 to the genomic target sequence so that the Cas9 can cut both strands of the DNA causing a double-strand break. Just as with ZFNs and TALENs, the double-stranded brakes produced by CRISPR/Cas can undergo homologous recombination or NHEJ.
The Cas9 nuclease has two functional domains: RuvC and HNH, each cutting a different DNA strand. When both of these domains are active, the Cas9 causes double strand breaks in the genomic DNA.
A significant advantage of CRISPR/Cas is that the high efficiency of this system coupled with the ability to easily create synthetic gRNAs enables multiple genes to be targeted simultaneously. In addition, the majority of cells carrying the mutation present biallelic mutations in the targeted genes.
However, apparent flexibility in the base-pairing interactions between the gRNA sequence and the genomic DNA target sequence allows imperfect matches to the target sequence to be cut by Cas9.
Modified versions of the Cas9 enzyme containing a single inactive catalytic domain, either RuvC- or HNH-, are called ‘nickases’. With only one active nuclease domain, the Cas9 nickase cuts only one strand of the target DNA, creating a single-strand break or 'nick'. A single-strand break, or nick, is normally quickly repaired through the HDR pathway, using the intact complementary DNA strand as the template. However, two proximal, opposite strand nicks introduced by a Cas9 nickase are treated as a double-strand break, in what is often referred to as a 'double nick' CRISPR system. A double-nick can be repaired by either NHEJ or HDR depending on the desired effect on the gene target. Thus, if specificity and reduced off-target effects are crucial, using the Cas9 nickase to create a double-nick by designing two gRNAs with target sequences in close proximity and on opposite strands of the genomic DNA would decrease off- target effect as either gRNA alone will result in nicks that will not change the genomic DNA.
Modified versions of the Cas9 enzyme containing two inactive catalytic domains (dead Cas9, or dCas9) have no nuclease activity while still able to bind to DNA based on gRNA specificity. The dCas9 can be utilized as a platform for DNA transcriptional regulators to activate or repress gene expression by fusing the inactive enzyme to known regulatory domains. For example, the binding of dCas9 alone to a target sequence in genomic DNA can interfere with gene transcription.
There are a number of publically available tools available to help choose and/or design target sequences as well as lists of bioinformatically determined unique gRNAs for different genes in different species such as the Feng Zhang lab's Target Finder, the Michael Boutros lab's Target Finder (E-CRISP), the RGEN Tools: Cas-OFFinder, the CasFinder: Flexible algorithm for identifying specific Cas9 targets in genomes and the CRISPR Optimal Target Finder.
Non-limiting examples of a gRNA that can be used in the present invention are shown in the Examples section which follows.
According to another specific embodiment, the introduced variation confers a non- naturally occurring variation.
In order to use the CRISPR system, both gRNA and Cas9 should be expressed in a target cell. Cas9 can also be provided as mRNA or protein to the cell. The insertion vector can contain both cassettes on a single plasmid or the cassettes are expressed from two separate plasmids. CRISPR plasmids are commercially available such as the px330 plasmid from Addgene.
“Hit and run” or “in-out” - involves a two-step recombination procedure. In the first step, an insertion-type vector containing a dual positive/negative selectable marker cassette is used to introduce the desired sequence alteration. The insertion vector contains a single continuous region of homology to the targeted locus and is modified to carry the mutation of interest. This targeting construct is linearized with a restriction enzyme at a one site within the region of homology, electroporated into the cells, and positive selection is performed to isolate homologous recombinants. These homologous recombinants contain a local duplication that is separated by intervening vector sequence, including the selection cassette. In the second step, targeted clones are subjected to negative selection to identify cells that have lost the selection cassette via intrachromosomal recombination between the duplicated sequences. The local recombination event removes the duplication and, depending on the site of recombination, the allele either retains the introduced mutation or reverts to wild type. The end result is the introduction of the desired modification without the retention of any exogenous sequences.
The “double-replacement” or “tag and exchange” strategy - involves a two-step selection procedure similar to the hit and run approach, but requires the use of two different targeting constructs. In the first step, a standard targeting vector with 3' and 5' homology arms is used to insert a dual positive/negative selectable cassette near the location where the mutation is to be introduced. After electroporation and positive selection, homologous targeted clones are identified. Next, a second targeting vector that contains a region of homology with the desired mutation is electroporated into targeted clones, and negative selection is applied to remove the selection cassette and introduce the mutation. The final allele contains the desired mutation while eliminating unwanted exogenous sequences.
Site-Specific Recombinases - The Cre recombinase derived from the Pl bacteriophage and Flp recombinase derived from the yeast Saccharomyces cerevisiae are site-specific DNA recombinases each recognizing a unique 34 base pair DNA sequence (termed “Lox” and “FRT”, respectively) and sequences that are flanked with either Lox sites or FRT sites can be readily removed via site-specific recombination upon expression of Cre or Flp recombinase, respectively. For example, the Lox sequence is composed of an asymmetric eight base pair spacer region flanked by 13 base pair inverted repeats. Cre recombines the 34 base pair lox DNA sequence by binding to the 13 base pair inverted repeats and catalyzing strand cleavage and religation within the spacer region. The staggered DNA cuts made by Cre in the spacer region are separated by 6 base pairs to give an overlap region that acts as a homology sensor to ensure that only recombination sites having the same overlap region recombine.
Basically, the site specific recombinase system offers means for the removal of selection cassettes after homologous recombination. This system also allows for the generation of conditional altered alleles that can be inactivated or activated in a temporal or tissue-specific manner. Of note, the Cre and Flp recombinases leave behind a Lox or FRT “scar” of 34 base pairs. The Lox or FRT sites that remain are typically left behind in an intron or 3' UTR of the modified locus, and current evidence suggests that these sites usually do not interfere significantly with gene function.
Thus, Cre/Lox and Flp/FRT recombination involves introduction of a targeting vector with 3' and 5' homology arms containing the mutation of interest, two Lox or FRT sequences and typically a selectable cassette placed between the two Lox or FRT sequences. Positive selection is applied and homologous recombinants that contain targeted mutation are identified. Transient expression of Cre or Flp in conjunction with negative selection results in the excision of the selection cassette and selects for cells where the cassette has been lost. The final targeted allele contains the Lox or FRT scar of exogenous sequences.
Transposases - As used herein, the term “transposase” refers to an enzyme that binds to the ends of a transposon and catalyzes the movement of the transposon to another part of the genome.
As used herein the term “transposon” refers to a mobile genetic element comprising a nucleotide sequence which can move around to different positions within the genome of a single cell. In the process the transposon can cause mutations and/or change the amount of a DNA in the genome of the cell.
A number of transposon systems that are able to also transpose in cells e.g. vertebrates have been isolated or designed, such as Sleeping Beauty [Izsvak and Ivies Molecular Therapy (2004) 9, 147-156], piggyBac [Wilson et al. Molecular Therapy (2007) 15, 139-145], Tol2 [Kawakami et al. PNAS (2000) 97 (21): 11403-11408] or Frog Prince [Miskey et al. Nucleic Acids Res. Dec 1, (2003) 31(23): 6873-6881], Generally, DNA transposons translocate from one DNA site to another in a simple, cut-and-paste manner. Each of these elements has their own advantages, for example, Sleeping Beauty is particularly useful in region-specific mutagenesis, whereas Tol2 has the highest tendency to integrate into expressed genes. Hyperactive systems are available for Sleeping Beauty and piggyBac. Most importantly, these transposons have distinct target site preferences, and can therefore introduce sequence alterations in overlapping, but distinct sets of genes. Therefore, to achieve the best possible coverage of genes, the use of more than one element is particularly preferred. The basic mechanism is shared between the different transposases, therefore we will describe piggyBac (PB) as an example.
PB is a 2.5 kb insect transposon originally isolated from the cabbage looper moth, Trichoplusia ni. The PB transposon consists of asymmetric terminal repeat sequences that flank a transposase, PBase. PBase recognizes the terminal repeats and induces transposition via a “cut- and-paste” based mechanism, and preferentially transposes into the host genome at the tetranucleotide sequence TTAA. Upon insertion, the TTAA target site is duplicated such that the PB transposon is flanked by this tetranucleotide sequence. When mobilized, PB typically excises itself precisely to reestablish a single TTAA site, thereby restoring the host sequence to its pretransposon state. After excision, PB can transpose into a new location or be permanently lost from the genome. Typically, the transposase system offers an alternative means for the removal of selection cassettes after homologous recombination quit similar to the use Cre/Lox or Flp/FRT. Thus, for example, the PB transposase system involves introduction of a targeting vector with 3' and 5' homology arms containing the mutation of interest, two PB terminal repeat sequences at the site of an endogenous TTAA sequence and a selection cassette placed between PB terminal repeat sequences. Positive selection is applied and homologous recombinants that contain targeted mutation are identified. Transient expression of PBase removes in conjunction with negative selection results in the excision of the selection cassette and selects for cells where the cassette has been lost. The final targeted allele contains the introduced mutation with no exogenous sequences.
For PB to be useful for the introduction of sequence alterations, there must be a native TTAA site in relatively close proximity to the location where a particular mutation is to be inserted.
Genome editing using recombinant adeno-associated virus (rAAV) platform - this genomeediting platform is based on rAAV vectors which enable insertion, deletion or substitution of DNA sequences in the genomes of live mammalian cells. The rAAV genome is a single-stranded deoxyribonucleic acid (ssDNA) molecule, either positive- or negative-sensed, which is about 4.7 kb long. These single-stranded DNA viral vectors have high transduction rates and have a unique property of stimulating endogenous homologous recombination in the absence of double-strand DNA breaks in the genome. One of skill in the art can design a rAAV vector to target a desired genomic locus and perform both gross and/or subtle endogenous gene alterations in a cell. rAAV genome editing has the advantage in that it targets a single allele and does not result in any off- target genomic alterations. rAAV genome editing technology is commercially available, for example, the rAAV GENESIS™ system from Horizon™ (Cambridge, UK).
Constructs useful in the methods according to the present invention, such as for downregulating expression of AO 1 and/or AO2 may be constructed using recombinant DNA technology well known to persons skilled in the art. The coding sequence constructs may be inserted into vectors, which may be commercially available, suitable for transforming into plants and suitable for expression of the gene of interest in the transformed cells. The genetic construct can be an expression vector wherein the nucleic acid sequence is operably linked to one or more regulatory sequences allowing expression in the plant cells.
Plant cells may be transformed stably or transiently with the nucleic acid constructs or with naked DNA or RNA of the present invention. In stable transformation, the nucleic acid molecule of the present invention is integrated into the plant genome and as such it represents a stable and inherited trait. In transient transformation, the nucleic acid molecule is expressed by the cell transformed but it is not integrated into the genome and as such it represents a transient trait. According to a specific embodiment, down-regulating expression is in a constitutive manner.
According to a specific embodiment, down-regulating expression is in a tissue specific manner.
According to a specific embodiment, down-regulating is in a leaf tissue.
There are various methods of introducing foreign genes into both monocotyledonous and dicotyledonous plants (Potrykus, I., Annu. Rev. Plant. Physiol., Plant. Mol. Biol. (1991) 42:205- 225; Shimamoto et al., Nature (1989) 338:274-276).
The principle methods of causing stable integration of exogenous DNA into plant genomic DNA include two main approaches:
(i) Agrobacterium-mediated gene transfer: Klee et al. (1987) Annu. Rev. Plant Physiol. 38:467-486; Klee and Rogers in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes, eds. Schell, J., and Vasil, L.K., Academic Publishers, San Diego, Calif. (1989) p. 2-25; Gatenby, in Plant Biotechnology, eds. Kung, S. and Amtzen, C. J., Butterworth Publishers, Boston, Mass. (1989) p. 93-112.
(ii) direct DNA uptake: Paszkowski et al., in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego, Calif. (1989) p. 52-68; including methods for direct uptake of DNA into protoplasts, Toriyama, K. et al. (1988) Bio/Technology 6: 1072-1074. DNA uptake induced by brief electric shock of plant cells: Zhang et al. Plant Cell Rep. (1988) 7:379-384. Fromm et al. Nature (1986) 319:791-793. DNA injection into plant cells or tissues by particle bombardment, Klein et al. Bio/Technology (1988) 6:559-563; McCabe et al. Bio/Technology (1988) 6:923-926; Sanford, Physiol. Plant. (1990) 79:206-209; by the use of micropipette systems: Neuhaus et al., Theor. Appl. Genet. (1987) 75:30-36; Neuhaus and Spangenberg, Physiol. Plant. (1990) 79:213-217;
Glass fibers or silicon carbide whisker transformation of cell cultures, embryos or callus tissue, U.S. Pat. No. 5,464,765 or by the direct incubation of DNA with germinating pollen, DeWet et al. in Experimental Manipulation of Ovule Tissue, eds. Chapman, G. P. and Mantell, S. H. and Daniels, W. Longman, London, (1985) p. 197-209; and Ohta, Proc. Natl. Acad. Sci. USA (1986) 83:715-719.
The Agrobacterium system includes the use of plasmid vectors that contain defined DNA segments that integrate into the plant genomic DNA. Methods of inoculation of the plant tissue vary depending upon the plant species and the Agrobacterium delivery system. A widely used approach is the leaf disc procedure which can be performed with any tissue explant that provides a good source for initiation of whole plant differentiation. Horsch et al. in Plant Molecular Biology Manual A5, Kluwer Academic Publishers, Dordrecht (1988) p. 1-9. A supplementary approach employs the Agrobacterium delivery system in combination with vacuum infiltration. The Agrobacterium system is especially viable in the creation of transgenic dicotyledonous plants.
There are various methods of direct DNA transfer into plant cells. In electroporation, the protoplasts are briefly exposed to a strong electric field. In microinjection, the DNA is mechanically injected directly into the cells using very small micropipettes. In microparticle bombardment, the DNA is adsorbed on microprojectiles such as magnesium sulfate crystals or tungsten particles, and the microprojectiles are physically accelerated into cells or plant tissues.
Following stable transformation plant propagation is exercised. The most common method of plant propagation is by seed. Regeneration by seed propagation, however, has the deficiency that due to heterozygosity there is a lack of uniformity in the crop, since seeds are produced by plants according to the genetic variances governed by Mendelian rules. Basically, each seed is genetically different and each will grow with its own specific traits. Therefore, it is preferred that the transformed plant be produced such that the regenerated plant has the identical traits and characteristics of the parent transgenic plant. Therefore, it is preferred that the transformed plant be regenerated by micropropagation which provides a rapid, consistent reproduction of the transformed plants.
However other methods of production are also contemplated including sexual reproduction (and selection for the phenotype whether morphologically or using molecular markers as described herein), tissue culture and more.
Micropropagation is a process of growing new generation plants from a single piece of tissue that has been excised from a selected parent plant or cultivar. This process permits the mass reproduction of plants having the preferred tissue expressing the fusion protein. The new generated plants which are produced are genetically identical to, and have all of the characteristics of, the original plant. Micropropagation allows mass production of quality plant material in a short period of time and offers a rapid multiplication of selected cultivars in the preservation of the characteristics of the original transgenic or transformed plant. The advantages of cloning plants are the speed of plant multiplication and the quality and uniformity of plants produced.
Micropropagation is a multi-stage procedure that requires alteration of culture medium or growth conditions between stages. Thus, the micropropagation process involves four basic stages: Stage one, initial tissue culturing; stage two, tissue culture multiplication; stage three, differentiation and plant formation; and stage four, greenhouse culturing and hardening. During stage one, initial tissue culturing, the tissue culture is established and certified contaminant-free. During stage two, the initial tissue culture is multiplied until a sufficient number of tissue samples are produced to meet gradually increased so that it can be grown in the natural environment.
Viruses that have been shown to be useful for the transformation of plant hosts include CaMV, TMV, TRV and BV. Transformation of plants using plant viruses is described in U.S. Pat. No. 4,855,237 (BGV), EP-A 67,553 (TMV), Japanese Published Application No. 63-14693 (TMV), EPA 194,809 (BV), EPA 278,667 (BV); and Gluzman, Y. et al., Communications in Molecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, New York, pp. 172-189 (1988). Pseudovirus particles for use in expressing foreign DNA in many hosts, including plants, is described in WO 87/06261.
According to a specific embodiment, plants of the invention can be obtained by producing one parental line with down-regulated AO1 and another with down-regulated AO2. A hybrid can be obtained by crossing, where the selected progeny has down -regulated expression of both AO1 and AO2, preferably by genome editing. Such a progeny (hybrid, or further selected to obtain an inbred) is also selected to exclude presence of foreign DNA (such as coding for the endonuclease, e.g., Cas9) according to some embodiments of the invention.
In breeding programs desirable traits from two or more germplasm sources or gene pools are combined to develop superior breeding varieties. Desirable inbred or parent lines are developed by continuous self-pollinations and/or backcrosses and selection of the best breeding lines, sometimes utilizing molecular markers to speed up the selection process.
Once the parental lines that give the best hybrid performance have been identified e.g., both carrying the loss of function mutation as described above, the hybrid seed can be produced indefinitely, as long as the homozygosity of the parents are maintained.
As defined herein, the phrase "stable parental lines" refers to open pollinated, inbred lines, stable for the desired plants over cycles of self-pollination and planting. According to a specific embodiment, 95% of the genome is in a homozygous form in the parental lines of the present invention.
A common practice in plant breeding is using the method of backcrossing to develop new varieties by single trait conversion.
The phrase "single trait conversion" as used herein refers to the incorporation of new single gene into a parent line wherein essentially all of the desired morphological and physiological characteristics of the parent lines are recovered in addition to the single gene transferred.
The term "backcrossing" as used herein refers to the repeated crossing of a hybrid progeny back to one of the parental plant. The parental plant which contributes the gene for the desired characteristic is termed the non-recurrent or donor parent, as mentioned hereinabove. This terminology refers to the fact that the non-recurrent parent is used one time in the backcross protocol and therefore does not recur. The parental plant to which the gene from the non-recurrent parent are transferred is known as the recurrent parent as it is used for several rounds in the backcrossing protocol.
In a typical backcross protocol, a plant from the original varieties of interest (recurrent parent) is crossed to a plant selected from second varieties (non-recurrent parent) that carries the gene, introgression or hamplotype of interest to be transferred. The resulting progeny from this cross are then crossed again to the recurrent parent and the process is repeated until a plant is obtained wherein essentially all of the desired morphological and physiological characteristics of the recurrent parent are recovered in the converted plant, in addition to the transferred gene from the non-recurrent parent.
Thus, near-isogenic lines (NIL) may be created by many backcrosses to produce an array of individuals that are nearly identical in genetic composition except for the trait or genomic region under interrogation in this case loss of function genetic alteration.
Backcrossing methods can be used with the present invention to improve or introduce a characteristic into the parent lines.
Thus, according to an aspect of the invention there is provided a method of selecting a plant exhibiting tolerance to stress, the method comprising:
(a) providing plants having been treated with an agent down-regulating expression of aldehyde oxidase 1 (AO1) and/or aldehyde oxidase 2 (AO2), as described herein; and
(b) selecting from the plants, a plant which exhibits tolerance to stress.
Marker assisted breeding (selection) as described above can be used in this method, looking for genetic variation in AO1 and/or AO2.
Alternatively or additionally, selection can be done by measuring levels of oxidized aldehydes. This method is effective since it is possible to measure this early marker already upon leaf emergence.
Alternatively or additionally, selection can be done by measuring tolerance to stress.
Thus the present teachings provide for plants and progeny which are characterized by increased tolerance to stress and have a genetic variation in AO1 and/or AO2 gene.
Thus, there is provided a plant having been treated with an agent down-regulating expression of aldehyde oxidase 1 (AO1) and/or aldehyde oxidase 2 (AO2) such that said plant or plant cell exhibits reduced expression of AO1 and/or AO2, as compared to a control plant.
Alternatively or additionally, there is provided a plant cell having been treated with an agent down-regulating expression of aldehyde oxidase 1 (AO1) and/or aldehyde oxidase 2 (AO2) such that said plant cell exhibits reduced expression of AO1 and/or AO2, as compared to a control plant cell.
According to a specific embodiment, the plant or the plant seed is an inbred.
According to a specific embodiment, the plant is a hybrid plant or the seed is a hybrid seed.
The invention also relates to progeny of the plant (having down-regulated expression of AO1 and/or AO2) of the invention. Such progeny can be produced by sexual or vegetative reproduction of a plant of the invention or a progeny plant thereof. In addition to this, the progeny plant may be modified in one or more other characteristics. Such additional modifications are for example effected by mutagenesis or by transformation with a transgene.
As used herein the word "progeny" is intended to mean the offspring or the first and all further descendants from a cross with a plant of the invention that shows tolerance to stress as described herein. Progeny of the invention are descendants of any cross with a plant of the invention that carries the mutation (in a homozygous form) trait that leads to tolerance. Progeny also encompasses plants that carry the trait of the invention which are obtained from other plants of the invention by vegetative propagation or multiplication.
As mentioned, embodiments described herein, furthermore, relate to hybrid seed and to a method of producing hybrid seed comprising crossing a first parent plant with a second parent plant and harvesting the resultant hybrid seed. In this case the trait is recessive, therefore both parent plants need to be homozygous for the trait in order for all of the hybrid seed to carry the trait of the invention. They need not necessarily be uniform for other traits.
Embodiments described herein also relate to the germplasm of the plants. The germplasm is constituted by all inherited characteristics of an organism and according to the invention encompasses at least the trait of the invention.
Also provided is a method of producing a plant exhibiting tolerance to stress, the method comprising growing the plant as described herein or regenerating the plant cell as described herein.
According to a specific embodiment, growing plants or selecting plants is effected under stress or in a region known to be at risk of stress (e.g., drought).
Also contemplated are processed products of the plants which comprise DNA showing genetic variation in AO1 and AO2 which elicits down-regulation of these genes.
As used herein the term “about” refers to ± 10 %.
The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".
The term “consisting of’ means “including and limited to”. The term "consisting essentially of' means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.
Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides. It is understood that any Sequence Identification Number (SEQ ID NO) disclosed in the instant application can refer to either a DNA sequence or a RNA sequence, depending on the context where that SEQ ID NO is mentioned, even if that SEQ ID NO is expressed only in a DNA sequence format or a RNA sequence format.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.
Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.
EXAMPLES
Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.
MATERIAL AND METHODS
Identification of mutation generated by CRISPR/Cas9 in Tomato
The aldehyde oxidase (AO1; AO2 and AO3) mutant of Solarium lycopersicum was generated using CRISPR/Cas9 system. Two sgRNA were designed for each of the aldehyde oxidase (listed in Table 2a, below) using CRISPR-P(Liu et al 2017, doi: 10.1016/j.molp.2017.01.003) and the tomato genome assembly (SL2.50). To generate the binary vectors containing a CRISPR/Cas9 cassette with 35Spro:Cas9 and the two gRNA’s the Golden Gate assembly method was used (Werner et al., 2012, doi: 10.4161/bbug.3.1.18223; Engler et al., 2014, DOI: 10(dot)1021/sb4001504). For each of the gRNAs, a PCR reaction was carried out with a primer containing the gRNA sequence and a universal primer (TGTGGTCTCAAGCGTAATGCCAACTTTGTAC, SEQ ID NO 7, UP1SG), using the plasmid pICH86966:: AtU6p::gRNA_PDS (Addgene plasmid 46966) as a template (Table 2b). The PCR products were then cloned into level 1 vectors pICH47751 (gRNAl) and pICH47761 (gRNA2). Finally, the level 1 vectors containing the gRNAs were then assembled together with the plasmids pICH47732-NOSpro::NPTII, pICH47742-35Spro:Cas9, pICH41780 End-link (Addgene plasmid 48019), into the binary level 2 vector pAGM4723 using Bpil enzyme. Agrobacterium tumefaciens GV3101, and the cotyledon transformation method (McCormick, 1997) were used to transform all constructs into M82 (sp). Specific primers for the CAS9 sequence [Forward- CGCTAATCTTGCAGGTAGCC, SEQ ID NO: 8 CAS9PF and Reverse-
TGCCAGCTCGTTACCTTTCT, SEQ ID NO: 9) CAS9PR], were used to isolate mutants free of the insertion cassette (Table 2b).
Genomic DNA of CRISPR/cas9 generated mutant is isolated using Qiagen DNeasy® Plant Pro Kit to visualize pattern of targeted mutagenesis using PCR amplification and sequencing. The DNA fragments spanning the Cas9/gRNA target sequences are amplified by PCR (primer sequences listed in Table 3, below) using Platinum™ SuperFi™ PCR Master Mix (invitrogen). The PCR is run with a final volume of 20 pl, containing; 40 ng of gDNA, 1 pl of forward and reverse primer (10 pm) each and 10 pl of 2X PCR master mix. The thermocycler is set at 95°C for 5 min, 35 cycles at 95°C for 30s, specific annealing temperature (55-60°C) for 15s for and 72°C for 30s followed by 72°C for 5min. The PCR product is used for Sanger sequencing to recognize the mutation. Based on sequence, the marker is desgined. If there is mutation inside the restriction enzyme sequence, then a PCR-based marker with a possible restriction enzyme is designed. In case there is no enzyme then dCAPS (Derived Cleaved Amplified Polymorphic Sequences) primers are designed to amplify a region of DNA containing the mutation of interest. One of the primers is designed to introduce a mismatched nucleotide at the mutation site, to introduce or destroy a restriction enzyme recognition site when the PCR product is generated.
Table 2a: Sequence of sgRNA used for CRISPR/Cas9 mediated mutation
Figure imgf000044_0001
Table 2b: Sequence of primers used for golden gate assembly and transformation confirmation
Figure imgf000045_0001
Table 3: Details of primers to amplify genomic DNA PCR analysis
Figure imgf000045_0002
Table 4: -Nomenclature of mutants used
Figure imgf000046_0001
Figure imgf000046_0002
EXAMPLE 1 Enhancement of AAO1 and/or AAO3 in the absence of active AAO2 in aao2 mutant
To gain an insight into the role of AA03 in detoxifying aldehydes, we examined a range of aldehydes including reactive aldehydes. ABal was used for in-gel activity to mark the position of AA03 mobile activity band. The presence of upper band in WT and aaol and its absence in aao3 KOs indicated that the upper activity band in aaol is solely catalyzed by AA03. The application of various aldehydes at the level of 1 mM [except for HNE (0.25 m M) andABal (0.2 mM)] exhibited that AA03 catalyzed the oxidation of aromatic and aliphatic aldehydes. It showed a high activity for cinnamaldehyde, and moderate activity for acrolein, benzaldehyde HNE (Figure 1 A). The capacity of aao2 mutant to oxidize a range of aldehydes including reactive aldehydes was compared to WT to get more insight into the function of AA02. The crude protein extracted from the rosette leaves of aao2KO (KO95) and WT was fractionated by NATIVE PAGE. Significantly, knockout of aao2KO (KO-95) mutant exhibited only the upper activity band, whereas WT exhibited additional middle and the lowest activity bands (Figure IB). The application of various aldehydes at the level of 1 mM [except for 4-hydroxyl-2-nonenal (HNE) (0.25 mM) and ABal (0.1 mM)] exhibited that upper in-gel activity band generated by aao2 (KO-95) extracted proteins had a more intense activity band (higher activity level) with almost all aldehydes employed as substrates as compared with WT. These results indicate that the absence of AA02 enhances the oxidation capacity of aldehydes by AA03 and/or AAO1 (Figure IB and C), suggesting a role for AAO2 in the homeostasis of aldehydes level by modulating another/other aldehyde oxidase/s activity in 12 days old seedling leaves (Figure IB) as well as rosette leaves of 21 days old plants (Figure 1C).
EXAMPLE 2
AAO2 protein expression level affects AAO3 and AAOl’s capacity to oxidize specific aldehydes under stresses such as Rose-Bengal
The enhancement of AAO1 and/or AAO3 in the absence of active AAO2 in aao2 mutant under control conditions (FigureslB and 1C), led the present inventors to study the effect of Rose- Bengal, a singlet oxygen generator that participates in lipid peroxidation and resulting toxic aldehydes. Spraying 0.05 mM Rose-Bengal on plants grown in plates containing 0.5 MS induced early senescence symptoms in rosette leaves of WT, three days after treatment, whereas aao2 leaves showed much less visible senescence symptoms and significantly higher levels of remaining chlorophyll (Figures 2A and 2B). Detection of aldehydes 17 h after Rose-Bengal application revealed stronger enhancement of acrolein, acetaldehyde, benzaldehyde, crotonaldehyde, propionaldehyde and HNE in WT than in aao2 mutant leaves (Figure 2C). The in-gel assay carried out to examine the AAO3 and AAO1 activity levels in mock and Rose-Bengal treated WT, aao2 (SAZL 563 G09; KO-563), aao3 (SAIL 78 H09; KO9) and aaol (SALK 018100; al-100) mutants using trans-3-nonenal or I3CA as substrate, exhibited higher band intensity of AAO1 and/or AAO3 [the most upper activity band as described in Nurbekova et al., 2021, Figure 1C and Table S3 therein] in aao2 mutant compared to WT in the mock (control) treated plants in both aldehydes applied (Figure 2D). For the trans-2-nonenal the activity by AAO3 under control conditions was higher than AAO1 as indicated by the stronger intensity in the most upper band in aaol compared to the aao3 mutant, whereas I3CA was a preferable substrate for AAO1 activity, as indicated by the stronger intensity of the most upper band in aao3 compared to aaol mutant (Figure 2D). The application of Rose-Bengal resulted in enhancement in the most upper band, indicating the activation of AA01 and/or AA03 at the absence and presence of AA02 activity. Examination of AA01 in aao3 (KO9) and AA03 activity in aaol (al-100) mutant revealed that both were enhanced as the result of Rose-Bengal application as shown in either aldehyde substrate used, showing higher activity level of AA01 than AA03 (Figure 2D). Parallel to AA01 and AA03 increase, AA02 exhibited decreased activity level when treated with Rose- Bengal. As for the heterodimer activity bands [middle bands], AA0EAA02 in WT and aao3 mutant exhibited an increase in response to the applied stress, whereas AA03 : AA02 heterodimer band in aaol tended to show a declined intensity of the activity band evident in aaol mutant (Figure 2D). The results indicate that AA02 protein expression level affects AA03 and AAOl’s capacity to oxidize specific aldehydes under stresses such as Rose-Bengal.
EXAMPLE 3
Employing Single functioning mutants (double mutants) indicates that AAO2 protein expression level affects AAO3 capacity to oxidize toxic aldehydes in rosette leaves of plants exposed to UV-C irradiation or Rose-Bengal application
The present inventors have constructed (heArabidopsis independent double mutants that are single functioning AA01 (saaol), saao2 and saao3. For doing so, the RNA interference (RNAi) technique was used using S ALK l 04895 (aao2) and S ALK O 18100 (aaol ) KO mutant plants. Flowering aao2 were dipped with Agrobacterium GV3101 strain containing AAO1 RNAi or AAO3 RNAi constructs to generate saao3 or saaol, respectively. Flowering aaol were dipped with GV3101 containing AAO2 RNAi or AAO3 RNAi constructs to generate another saao3 as well as saao2, respectively. For the generation of AAO1 and AAO3 RNAi constructs, amplified AAO1 and AAO3 fragments were employed for sense and antisense orientation. The in-gel activities of various single active mutants employing Abscisic aldehyde or other aldehydes such as Indole -carboxy aldehyde (I3CA), transe-2-nonenal or benzaldehyde can be seen in Figures 3 A-C.
The homozygous AAO-compromised lines were exposed to UV-C irradiation or Rose- Bengal spray after verification of the mutations by detection of the transcript's expression of the targeted genes as compared to the expression in WT leaves (Figure 4).
EXAMPLE 4 aao2KO mutant and aao3Ss mutants impaired in AAO1 and AAO2 expression show significant improvement in chlorophyll levels as compared to WT in response to stress
Notably, rosette leaves of the aaolS (aaolS-11) mutants impaired in AAO3 and AAO2 expressions exhibited significantly lower remaining chlorophyll level than WT leaves 3 days after exposing to 250 mJ of UV-C irradiation or 0.05 mM of Rose-Bengal application. In contrast, aao3Ss mutants (aao3S-l, aao3S-7, aao3S-12, aao3S-18) impaired in AA01 and AAO2 expression, as well as the aao2 (KO-95) mutant exhibited significantly higher remaining chlorophyll level than WT in response to the applied stresses (Figures 5A-B and Figures 6A-B). Further, detection of aldehydes level in rosette leaves was carried out 3 days after the UV-C irradiation, and revealed significantly higher level of benzaldehyde, crotonaldehyde, propionaldehyde and HNE in WT leaves compared to aao2KO and the three aao3Ss (aao3S-l, aao3S-12, aao3S-18) mutants.
EXAMPLE 5
Impairment in AAO1 improves plant resistance to UV-C irradiation., while its overexpression responds as WT
Since UV-C irradiation and Rose-Bengal application enhanced AA03 activity in al-100 mutant as compared to the control untreated plants (see Figure 2D), the present inventors wanted to investigate if the absence of active AA01 indeed can improve plant resistance to UV-C irradiation similar to the absence of AA02, Al-100 [SALK 018100 (aaol KO)]. Additionally, the present inventors also examined this effect in AA01 overexpression (OE) (AA01-0E was described in Nurbekova et al., 2021). Accordingly, 21-days post germination plants were exposed to UV-C irradiation (100 mJ) and were kept in a growth room for 96 hours and thereafter documented together with rosette leaves of plants not exposed to UV-C (control). Notably, rosette leaves of the Al-100 mutant exhibited significantly higher remaining chlorophyll level than WT leaves, whereas AAO1-OE exhibited similar remaining chlorophyll as WT in response to the applied stress (Figures 7A-C). These results indicate that impairment in AA01 improves plant resistance to UV-C irradiation, likely by enhancing AAO3 activity. Interestingly, AAO1 OE responded as WT.
EXAMPLE 6
Enhanced expression of AAO3 in the absence of AAO2, or impairment of AAO2 and AAO1 results in decreased water loss in plants exposed to root detached stress
To further examine a possible biological relevance, detached plants of single KO mutants of AAO2 (KO 95) as well as two single functioning mutants (double mutants) of aao3, the a3-l - 95 and a3-18-7-100, and single functioning mutant of aaol, the al-ll-10-(95) together with WT were exposed to harsh drought stress. Water loss in detached rosette leaves of 24 d old Arabidopsis WT, aao2 mutant (KO-95), aaol single mutant (al-ll-10-(95)) and aao3 single mutants (a.3-1- 95) and (a3-18-7-(100)) mutants grew in soil is shown in Figure 8A, as well as the relative water content (RWC) in rosette leaves of plants kept in the covered Petri dishes for 7 hours as shown in Figure 8A. aao3S the average of the two independents single aao3 (a3-l-95) and (a3-18-7-10G) and aao2KO (KO-95) exhibited lower water loss than WT and single functioning aaol \al-ll-10 (95)].
The results indicate that the absence of AA02 expression not only enhances plant resistance to abiotic stresses such as UV-C irradiation or Rose-Bengal spray, by AA03 activity enhanced aldehyde detoxification activity, but also improves plant resistance to harsh drought.
Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.
It is the intent of the Applicant(s) that all publications, patents and patent applications referred to in this specification are to be incorporated in their entirety by reference into the specification, as if each individual publication, patent or patent application was specifically and individually noted when referenced that it is to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is/are hereby incorporated herein by reference in its/their entirety.

Claims

WHAT IS CLAIMED IS:
1. A method of conferring stress tolerance to a plant, the method comprising downregulating expression of an aldehyde oxidase 1 (AO1) and/or aldehyde oxidase 2 (AO2), thereby conferring stress tolerance to the plant.
2. A plant having been treated with an agent down-regulating expression of aldehyde oxidase 1 (AO1) and/or aldehyde oxidase 2 (AO2) such that said plant or plant cell exhibits reduced expression of AO1 and/or AO2, as compared to a control plant.
3. A plant cell having been treated with an agent down-regulating expression of aldehyde oxidase 1 (AO1) and/or aldehyde oxidase 2 (AO2) such that said plant cell exhibits reduced expression of AO1 and/or AO2, as compared to a control plant cell.
4. A method of producing a plant exhibiting tolerance to stress, the method comprising growing the plant of claim 2 or regenerating the cell of claim 3.
5. A method of selecting a plant exhibiting tolerance to stress, the method comprising:
(a) providing plants having been treated with an agent down-regulating expression of aldehyde oxidase 1 (AO1) and/or aldehyde oxidase 2 (AO2);
(b) selecting from said plants, a plant which exhibits tolerance to stress.
6. The method of claim 1, wherein said down-regulating expression is of AO2.
7. The method of claim 1, wherein said down-regulating expression is of AO1.
8. The method of claim 1, wherein said down-regulating expression is of AO2 and
AO1.
9. The method of claim 1, wherein said stress is abiotic stress.
10. The method of claim 9, wherein said abiotic stress is selected from the group consisting of drought stress, oxidative stress, radiation stress, temperature stress, light stress, nutrient stress, heavy metal stress, salinity stress wounding stress and flooding stress.
11. The method of claim 1, wherein said abiotic stress is selected from the group consisting of drought stress, oxidative stress and radiation stress.
12. The method of claim 1, wherein said abiotic stress is drought stress.
13. The method of claim 12, wherein said drought stress comprises extreme drought stress, as defined by fast water loss (8 to 16% within 3 to 7 hours).
14. The method of claim 1, wherein said abiotic stress is oxidative stress.
15. The method of claim 1, wherein said abiotic stress is radiation stress.
16. The method of claim 1, wherein said radiation is UV-C.
17. The method of claim 1, wherein said stress is not radiation stress.
18. The method of claim 1, wherein said stress is not UV-C stress.
19. The method of claim 1, wherein said stress is biotic stress.
20. The method of claim 4, wherein said growing is under stress conditions.
21. The method of claim 1, wherein said plant is a crop plant.
22. The method of claim 1, wherein said down-regulating expression is by a nucleic acid agent.
23. The method of claim 22, wherein said nucleic acid agent is a genome editing agent or an RNA editing agent.
24. The method of claim 1, wherein said nucleic acid agent is an RNA silencing agent.
25. The method of claim 1 , wherein said down-regulating expression is in a constitutive manner.
26. The method of claim 1, wherein said down-regulating expression is in a tissue specific manner.
27. The method of claim 26, wherein said down-regulating is in a leaf tissue.
PCT/IL2024/050929 2023-09-13 2024-09-13 Methods of improving tolerance of plants to stress and plants generated thereby Pending WO2025057172A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US202363538085P 2023-09-13 2023-09-13
US63/538,085 2023-09-13
US202463683249P 2024-08-15 2024-08-15
US63/683,249 2024-08-15

Publications (1)

Publication Number Publication Date
WO2025057172A1 true WO2025057172A1 (en) 2025-03-20

Family

ID=95021794

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IL2024/050929 Pending WO2025057172A1 (en) 2023-09-13 2024-09-13 Methods of improving tolerance of plants to stress and plants generated thereby

Country Status (1)

Country Link
WO (1) WO2025057172A1 (en)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090158465A1 (en) * 2006-08-31 2009-06-18 D-Helix Transgenic plants with enhanced drought-resistance and method for producing the plants
US20090307796A1 (en) * 2006-06-16 2009-12-10 Annie Marion-Poll Plants with increased tolerance to water deficit
WO2010127969A1 (en) * 2009-05-06 2010-11-11 Basf Plant Science Company Gmbh Plants having enhanced yield-related traits and/or enhanced abiotic stress tolerance and a method for making the same
US20140090102A1 (en) * 2011-01-24 2014-03-27 Stéphane Pien Use of the rd29 promoter or fragments thereof for stress-inducible expression of transgenes in cotton
US20180310557A1 (en) * 2015-04-24 2018-11-01 Asilomar Bio, Inc. Methods for hydraulic enhancement of crops
US20200377900A1 (en) * 2019-05-29 2020-12-03 Monsanto Technology Llc Methods and compositions for generating dominant alleles using genome editing
IN202111038068A (en) * 2021-08-23 2023-03-03

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090307796A1 (en) * 2006-06-16 2009-12-10 Annie Marion-Poll Plants with increased tolerance to water deficit
US20090158465A1 (en) * 2006-08-31 2009-06-18 D-Helix Transgenic plants with enhanced drought-resistance and method for producing the plants
WO2010127969A1 (en) * 2009-05-06 2010-11-11 Basf Plant Science Company Gmbh Plants having enhanced yield-related traits and/or enhanced abiotic stress tolerance and a method for making the same
US20140090102A1 (en) * 2011-01-24 2014-03-27 Stéphane Pien Use of the rd29 promoter or fragments thereof for stress-inducible expression of transgenes in cotton
US20180310557A1 (en) * 2015-04-24 2018-11-01 Asilomar Bio, Inc. Methods for hydraulic enhancement of crops
US20200377900A1 (en) * 2019-05-29 2020-12-03 Monsanto Technology Llc Methods and compositions for generating dominant alleles using genome editing
IN202111038068A (en) * 2021-08-23 2023-03-03

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ĐURIĆ MARIJA, SUBOTIĆ ANGELINA, PROKIĆ LJILJANA, TRIFUNOVIĆ-MOMČILOV MILANA, CINGEL ALEKSANDAR, VUJIČIĆ MILORAD, MILOŠEVIĆ SNEŽANA: "Morpho-Physiological and Molecular Evaluation of Drought and Recovery in Impatiens walleriana Grown Ex Vitro", PLANTS, MDPI AG, vol. 9, no. 11, 1 November 2020 (2020-11-01), pages 1559 - 22, XP093294776, ISSN: 2223-7747, DOI: 10.3390/plants9111559 *
HAMISCH DOMENICA, KAUFHOLDT DAVID, KUCHERNIG JENNIFER-CHRISTIN, BITTNER FLORIAN, MENDEL RALF R, HÄNSCH ROBERT, POPKO JENNIFER: "Transgenic Poplar Plants for the Investigation of ABA-Dependent Salt and Drought Stress Adaptation in Trees", AMERICAN JOURNAL OF PLANR SCIENCES, vol. 7, 18 July 2016 (2016-07-18), pages 1337 - 1356, XP093294707, DOI: 10.4236/ajps.2016.79128 *
NURBEKOVA ZHADYRASSYN, SRIVASTAVA SUDHAKAR, DU NJA ZAI, TUREČKOVÁ VERONICA, STRAND MIROSLAV, OMAROV RUSTEM, SAGI MOSHE: "AAO2 impairment improves aldehyde detoxification by AAO3 in Arabidopsis leaves exposed to UVC or Rose Bengal", BIORXIV, COLD SPRING HARBOR LABORATORY, 1 September 2023 (2023-09-01), pages 1 - 34, XP093294787, Retrieved from the Internet <URL:https://www.biorxiv.org/content/10.1101/2023.09.22.559040v2.full.pdf> DOI: 10.1101/2023.09.22.559040 *
NURBEKOVA ZHADYRASSYN, SRIVASTAVA SUDHAKAR, STANDING DOMINIC, KURMANBAYEVA ASSYLAY, BEKTUROVA AIZAT, SOLTABAYEVA AIGERIM, OSHANOVA: "Arabidopsis aldehyde oxidase 3, known to oxidize abscisic aldehyde to abscisic acid, protects leaves from aldehyde toxicity", THE PLANT JOURNAL, BLACKWELL SCIENTIFIC PUBLICATIONS, OXFORD., GB, vol. 108, no. 5, 1 December 2021 (2021-12-01), GB , pages 1439 - 1455, XP093294726, ISSN: 0960-7412, DOI: 10.1111/tpj.15521 *
SHAKIROV ZAIR S, KHAKIMOV SARDOR A, SHOMURODOV KHABIBULLO F: "Effect of salinity and drought on symbiotical and biochemical properties of Onobrychis and alfalfa", AGRICULLTURE SCIENCES, vol. 3, no. 3, 19 January 2012 (2012-01-19), pages 444 - 454, XP093294763, DOI: 10.4236/as.2012.33052 *

Similar Documents

Publication Publication Date Title
US12275939B2 (en) Modifying the specificity of plant non-coding RNA molecules for silencing gene expression
US9562235B2 (en) MicroRNA compositions and methods for enhancing plant resistance to abiotic stress
US20140013469A1 (en) ISOLATED POLYNUCLEOTIDES EXPRESSING OR MODULATING microRNAs OR TARGETS OF SAME, TRANSGENIC PLANTS COMPRISING SAME AND USES THEREOF IN IMPROVING NITROGEN USE EFFICIENCY, ABIOTIC STRESS TOLERANCE, BIOMASS, VIGOR OR YIELD OF A PLANT
US20220090121A1 (en) Isolated polynucleotides and polypeptides associated with plants resistance to pathogenic fungi
US9902956B2 (en) Nucleic acid agents for overexpressing or downregulating RNA interference targets and uses of same in improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of a plant
AU2019252985A1 (en) Polynucleotides and polypeptides of plant and bacterial origin for protecting plants from pathogenic fungi
WO2018015957A1 (en) Compositions and methods for generating a haploid of a target plant
WO2018015956A1 (en) Compositions and methods for generating a haploid of a target plant
US20170218388A1 (en) Method of improving abiotic stress tolerance of plants and plants generated thereby
US20150299724A1 (en) Transgenic Plants Exhibiting Improved Resistance to Abiotic Stress
WO2025057172A1 (en) Methods of improving tolerance of plants to stress and plants generated thereby
WO2025199008A1 (en) Loss of function in gdsl lipases increases plant root biomass
HK40028645B (en) Modifying the specificity of plant non-coding rna molecules for silencing gene expression

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24864879

Country of ref document: EP

Kind code of ref document: A1