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WO2025057172A1 - Procédés pour améliorer la tolérance de plantes au stress et plantes ainsi obtenues - Google Patents

Procédés pour améliorer la tolérance de plantes au stress et plantes ainsi obtenues Download PDF

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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
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stress
plant
plants
expression
tolerance
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Moshe Sagi
Zhadyrassyn NURBEKOVA
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BG Negev Technologies and Applications Ltd
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    • 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
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    • 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
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    • 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)
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    • 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
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    • 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
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    • 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)].

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Abstract

L'invention concerne un procédé permettant de conférer une tolérance au stress à une plante. Le procédé consiste à réguler à la baisse l'expression d'une aldéhyde oxydase 1 (AO1) et/ou d'une aldéhyde oxydase 2 (AO2), pour ainsi conférer une tolérance au stress à la plante. L'invention concerne également une plante ainsi obtenue.
PCT/IL2024/050929 2023-09-13 2024-09-13 Procédés pour améliorer la tolérance de plantes au stress et plantes ainsi obtenues Pending WO2025057172A1 (fr)

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