WO2022148710A1 - Agents for improving water use efficiency - Google Patents

Abstract

The invention provides use of quercetin to reduce the water requirement or enhance water use efficiency of a plant. The quercetin can promote stomatal closing in a plant, thereby reducing the rate of transpiration and water loss from the plant. The invention also provides a method for increasing tolerance to water scarcity and/or for reducing the consequence of water scarcity in a plant, comprising contacting the plant with quercetin.

Description

Aqents for improvinq water use efficiency

Technical field

The present invention relates generally to methods and materials for improving water utilisation efficiency in crop plants while mitigating any concomitant reduction in photosynthetic activity.

Background art

Water use efficiency (WUE) is defined as the amount of carbon assimilated as biomass or grain produced per unit of water used by the crop.

Human pressures on global water availability mean that future agricultural sustainability is going to require increased crop water-use efficiency.

Crop yield is primarily water-limited in areas of West Asia and North Africa with a Mediterranean climate (Zhang H, Oweis T. Water-yield relations and optimal irrigation scheduling of wheat in the Mediterranean region. Agricultural Water Management. 1999 Jan 1 ; 38(3) : 195-211).

Water shortage is recognized as the most critical constraint that limits China’s capacity for food security. Although China’s water resources are large in absolute terms, the average water resources per capita are less than one-third of the global average (Huang, Jing, et al. "Water-scarcity footprints and water productivities indicate unsustainable wheat production in China." Agricultural Water Management 224 (2019): 105744.).

Some of the challenges facing water-limited crop yield in Europe are discussed in the report of the European Environment Agency - see e.g. https://www.eea.europa.eu/data-and-maps/indicators/crop-yield-variability- 2/assessment discusses the effect of water limitation to winter wheat in Europe based on multiple climate models. The report notes that there is a clear indication of deteriorating agroclimatic conditions in terms of increased drought stress and a shortening of the active growing season in central and southern Europe.

For example, bread wheat is the staple food for a substantial proportion of the world’s population. Given that one of the leading constraints on wheat grain productivity is soil moisture availability ( 1 ), improved WUE is an important priority for programs improving wheat or other crops (2-5).

Improvements in grain yield and crop water productivity arise from breeding for superior varieties and from better agronomic and water management practices.

Because plants lose water via transpiration through the same leaf epidermal stomatal pores through which they also gain the carbon dioxide (CO2) used in photosynthesis, carbon assimilation and water loss are coupled with one another (6). A key question is whether improvements in WUE through reduced water loss can be achieved without a incurring a collateral penalty from reduced CO2 uptake and consequent damaged photosynthetic performance (6-8).

Whilst the phytohormone abscisic acid (ABA) enhances stomatal closing by promoting the activity of OPEN STOMATA 1 (OST1), a sucrose non-fermenting 1 (SNFI)-related protein kinase (9-11), attempts to manipulate the ABA signaling system to enhance crop WUE without compromising photosynthetic performance have had limited success (12-15).

Thus it can be seen that providing methods of improving water utilisation efficiency while mitigating any concomitant reduction in photosynthetic activity would provide a useful contribution to the art.

Disclosure of the invention

The present inventors have shown that application of quercetin to crop plants can reduce the water requirement of the crop without incurring penalties with respect to carbon fixation, nitrogen assimilation and plant growth.

More specifically, as described hereinafter, the invention concerns the trade-off between carbon assimilation and water loss which arises from the dual function of stomatal pores to both control carbon dioxide influx and transpirational water efflux

The inventors show that quercetin promotes stomatal closing while boosting photosynthetic activity, thus negating the effects of reduced stomatal conductance on carbon fixation. By way of demonstration, a quercetin-spray treatment improved water use efficiency and grain yield of moisture-deficient field-grown bread wheat, and also showed beneficial effects in rice, tomato and tobacco.

Interestingly another flavonoid accumulated in lines showing high water use efficiency (kaempferol) did not demonstrate the same technical benefits.

Without wishing to be bound by theory, it appears that quercetin promotes stomatal closing by binding directly with OPEN STOMATA 1 (OST1), an abscisic acid (ABA)- activated sucrose non-fermenting 1 (SNFI)-related protein kinase, and amplifies OST1 kinase activity by interfering with the inhibitory interaction between OST1 and type 2C protein phosphatases.

Quercetin is an aglycone, but naturally occurs mainly as glycosides (and other derivatives). As explained below, the inventors have shown that examples of such derivatives also bind OST 1 and have other favourable properties. Thus the use of quercetin in these derived forms is embraced by the present invention. Where the term “quercetin” is used herein in relation to the invention, it will be understood that, unless context demands otherwise, the use of quercetin-derivative applies mutatis mutandis.

Thus application of a quercetin-based chemical intervention strategy will enable enhanced water use efficiency and yield, for example for wheat and other crop plants, thus contributing to future food security and agricultural sustainability. Flavonoids have previously been mentioned (amongst other agents) in relation to plant growth. For example US2016050921 A1 utilises mixtures of Genistein and Daidzein, and Hesperetin and Naringenin to apparently improve plant growth. Although quercetin is referred to, there is no demonstration of any technical effect for that compound, and no mention of any flavonoid reducing water requirements or boosting photosynthesis. Furthermore, as noted above in relation to kaempferol, the effects of one flavonoid in crop plants cannot be extrapolated to quite different ones.

US2016106110A1 concerns compositions comprising one or more microorganisms and one or more germinants (for example lactate, lactose, bicarbonate, fructose, glucose, mannose, galactose, alanine, asparagine, cysteine, glutamine, norvatine, serine, threonine, valine, glycine, inosine, taurocholate, and combinations thereof) for enhancing germination. Optional further ingredients include flavonols. The present invention is not for inducing germination, and is not based on the use of microorganisms or any of these germinants.

WO2019152632A1 relates to compositions and methods for increasing tolerance to abiotic stress and/or for reducing the consequence of abiotic stress in a plant and/or part thereof. The compositions all include at least one dicarboxylic acid and/or a salt thereof, in combination with another composition selected from a variety agents, including kaempferol or quercetin. Quercetin is not used in any of the Examples, and the present invention is not based on the use of compositions further comprising dicarboxylic acid and/or a salt thereof.

US2007265166A1 concerns methods of treating specific crop plants when those plants have reached specific developmental stages, and a method for improving the yield of a crop produced by a plurality of plants which comprises contacting said plants with at least one composition that comprises at least one cyclopropene.

Certain non-cyclopropene agents are also mentioned in relation plant growth regulation, including, in relation to IAA oxidase, “quercitin” (sic). The present invention is not based on the use of cyclopropene, or providing quercitrin, or of targeting IAA oxidase with co — factors or inhibitors of IAA oxidase.

In one aspect of the invention there is provided use of quercetin to reduce the water requirement or enhance water use efficiency (WUE) of a plant.

In a further aspect of the invention there is provided use of quercetin to promote stomatal closing in a plant, thereby reducing the rate of transpiration and water loss from the plant.

In a further aspect of the invention there is provided a method for increasing tolerance to water scarcity and/or for reducing the consequence of water scarcity in a plant, comprising contacting the plant with quercetin, or applying quercetin to the plant.

Some further aspects and embodiments of the invention will now be discussed in more detail:

The present invention thus provides for the use of quercetin to reduce the water requirement or water loss of a plant while mitigating or avoiding penalties with respect to carbon fixation, nitrogen assimilation or plant growth, as compared to an otherwise comparable (in terms of type and stage of plant and environment) untreated control plant not treated with quercetin. This can be used, for example, to improve drought resistance, or tolerance to other causes of water scarcity.

As described herein, it appears that quercetin can bind directly with OPEN STOMATA 1 (OST1), an abscisic acid (ABA)-activated sucrose non-fermenting 1 (SNFI)-related protein kinase, and amplify OST1 kinase activity by interfering with the inhibitory interaction between OST1 and type 2C protein phosphatases. Furthermore quercetin treatment can boost chlorophyll content and increase the protein abundances of multiple genes involved in photosynthesis, sucrose metabolism and sucrose transport resulting in increased photosynthetic capacity and biomass production. The invention provides use of quercetin to do or achieve any one or more of these effects in a crop plant.

The practice of the invention may increase WUE by at least 2, 3, 4, 5%, 10%, or 20% or more compared to a comparable untreated control plant not treated with quercetin.

Water use efficiency (WUE) is a concept introduced 100 years ago by Briggs and Shantz (1913) showing a relationship between plant productivity and water use. They introduced the term, WUE, as a measure of the amount of biomass produced per unit of water used by a plant. Since that time, there have been countless original papers and reviews written on the topic. Briefly, WUE is defined as the amount of carbon assimilated as biomass or grain produced per unit of water used by the crop (for example biomass kg ha-1 mm-1) (see e.g. Hatfield JL, Dold C. Water-Use Efficiency: Advances and Challenges in a Changing Climate. Front Plant Sci. 2019 (10:103. doi: 10.3389)

Quercetin has the following formula as an aglycone:

In one embodiment the quercetin used in the invention is the aglycone. However it is well known in the art that quercetin naturally occurs mainly as glycosides (and other derivatives) and the use of quercetin in these derived forms is embraced by the present invention.

Common quercetin glycosides and other derivatives are described, for example, by Materska, Malgorzata. "Quercetin and its derivatives: chemical structure and bioactivity-a review." Polish journal of food and nutrition sciences 58.4 (2008). Such derivatives often share the bioactive properties of the aglycones. Examples described therein include (with reference to the following formula):

Glc: glucose; Rha: rhamnose; Ara: arabinose; X: rhamnosylglucose; M: -CH ; Sul: - SO Na; Y: 2-acetylgalactose; Z: prenyl.

The inventors have shown that glycosylation does not affect the binding of quercetin to OST1, and indeed that glycosides such as quercetin-3-O- glucoside have a higher binding affinity than quercetin (see Figure 15). Further such glycosides are highly soluble.

Preferred quercetin derivatives which may be employed in the present invention are glycosides, which include quercetin-3-O-glycosides e.g. glucoside, -rhamnoside, or - galactoside.

Increased drought tolerance can be measured as described herein e.g. based on size, number or yield (e.g. in relation to grain or fruit, or yield of other commodity) of the plants, for example per unit of cultivated or covered area. A preferred measure is the total carbon and total nitrogen content in above-ground parts. As described herein it appears that quercetin can promote plant growth and biomass accumulation by simultaneous enhancement of carbon and nitrogen assimilation.

Thus in some embodiments the treatment increases total carbon and total nitrogen content in above-ground parts of the plant. In some embodiments the quercetin treatment does not modify the carbon-to-nitrogen ratio of the plant.

By “not modifying the carbon-to-nitrogen ratio of the plant” is meant that following the application (e.g. in the ensuing 1, 2, 3, 4, 5, 6, 7, 14 days) of the quercetin the carbon-to-nitrogen ratio of the plant is substantially unaffected as compared to a comparable ‘untreated’ control plant (not treated with quercetin).

The present invention enhances the ability of a plant and/or part thereof exposed to water scarcity conditions and contacted with a quercetin-containing composition to withstand those conditions better than a control plant and/or part thereof (i.e. , a plant and/or part thereof that has been exposed to the same stress but has not been contacted with the composition.

The invention may be applied to plants or crops, whether cultivated or otherwise, both indoors and outdoors. Preferably the plant is crop plant, preferably being cultivated in an open air environment.

The plant treated according to the invention will generally be one which is, or is believed to be, or believed to be at risk of, being moisture deficient or water limited.

By “moisture deficient or water limited” is meant that the growing conditions (environment) of the plant are such that water availability is a rate limiting step for growth or yield.

For example the invention may be utilised with a plant being grown in an irrigated environment with an Irrigation Water Productivity (IWP) for the plant of < 5 kg/m-3.

IWP, defined as the yield produced per unit of irrigation water use, is a widely accepted comprehensive indicator for analysis of and management of the irrigation and crop interaction. For example an IWP<4.5 was calculated for the high-yielding wheat plants grown in Shijiazhuang, Hebei province, China (Table S1, Fig. 1B,

Fig4J).

Alternatively or additionally, the invention may be utilised with a plant being grown in an environment with a Water-Scarcity Footprint (WSF) of < 0.05 m3 H₂0_e kg-1.

IWP and WSF may be calculated by the methods known in the art, and in particular as described by Huang, Jing, et al. "Water-scarcity footprints and water productivities indicate unsustainable wheat production in China." Agricultural Water Management 224 (2019): 105744 in sections 2.1 - 2.3, which are herein incorporated by cross- reference.

The plants treated according to the invention may be in any moisture deficient region. Examples include dry or arid regions in North America, West Asia, North Africa,

China, or Europe, for example Southern Europe. In one embodiment the plant is wheat grown in one of these regions.

Methods known in the art that allow quercetin-containing compositions to contact the plant include spraying, foaming, fogging or misting, pouring, brushing, dipping, dusting, sprinkling, scattering, atomizing, broadcasting, or soaking

The quercetin-containing composition of the present invention may be contacted with the entire plant or may be contacted with one or more plant parts. Plant parts include any part of a plant, including, for example, leaves, flowers, buds, blooms etc..

In some embodiments, the quercetin-containing composition of the present invention is a liquid, and the liquid is sprayed onto crop plants growing in a field. In other embodiments, the liquid is sprayed onto plants growing inside, for example in a greenhouse or indoors, or in other controlled environments.

In some embodiments, the amount of quercetin used in one spraying operation is 0.1 gram per hectare (g/ha) or more; or 0.5 g/ha or more; or 1 g/ha or more; or 5 g/ha or more; or 25 g/ha or more; or 50 g/ha or more; or 100 g/ha or more.

Independently, in some embodiments, the amount of quercetin used in one spraying operation is 6000 g/ha or less; or 3000 g/ha or less; or 1500 g/ha or less.

The timing of the treatment may be judged according to need and convenience - typically it may be seasonal, or repeated several times during a dry season or period. The present inventors have shown that the effect of quercetin can be detected in guard cells and other parts of the leaf after around 1 day from treatment, and its effects are still detectable after 10 days.

Where desired quercetin may be applied to plants as a slow-release composition, for example to prolong its effect of during periods of low water availability.

Technologies (e.g. nanoparticles, hydrogels etc,) for the slow release of agriculturally relevant molecules are well known in the art, and may be applied mutatis mutandis to the use of quercetin in the present invention see e.g. Remya V.R., George J.S., Thomas S. (2020) Manufacturing of Slow- and Controlled-Release Pesticides. In: K. R. R., Thomas S., Volova T., K. J. (eds) Controlled Release of Pesticides for Sustainable Agriculture. Springer, Cham https://doi.org/10.1007/978-3-030-23396- 9_5; Ramli, Ros Azlinawati. "Slow release fertilizer hydrogels: a review." Polymer Chemistry 10.45 (2019): 6073-6090; Guertal, E. A. "Slow-release nitrogen fertilizers in vegetable production: a review." HortTechnology 19.1 (2009): 16-19;

US4435383A ("Slow release pesticide formulations") the disclosures of which are incorporated herein by cross reference.

Slow release formulations have the benefit of prolonging the beneficial effects of quercetin, and reduce the rate at which applied quercetin is metabolized in plants. Reduced frequency of application can increase the efficiency of absorption as well as reducing the cost.

Quercetin administration may be performed one time or more than one time on a particular group of plants during a single growing season.

For example the composition may be contacted with a plant and/or part thereof 1 to 10 times per season, 1 to 11 times per season, 1 to 12 times per season, 1 to 13 times per season, 1 to 14 times per season, 1 to 15 times per season, and the like.

In some embodiments, the number of days between applications of (i.e., contacting the plant and/or part thereof with) quercetin may be 1 day to 100 days, 1 day to 95 days, 1 day to 90 days, 1 day to 85 days, 1 day to 80 days, 1 day to 75 days, 1 day to 70 days, 1 day to 65 days, 1 day to 60 days, 1 day to 55 days, 1 day to 50 days, 1 day to 45 days, 1 day to 40 days, and the like, and any combination thereof. In still other embodiments of the present invention, the number of days between applications of any of the compositions of the invention may be 1 day, 4 days, 7 days, 10 days, 13 days, 15 days, 18 days, 20 days, 25, days, 28, days, 30 days, 32, days, 35 days, 38 days, 40 days, 45 days, and the like, and any combination thereof.

The administration may be carried out immediately following application of water to the plants by irrigation or rainfall e.g. within 1, 2, 3, 4, 5, 6 or 7 days.

The administration may be carried out around or during anthesis, and following anthesis e.g. between 7 and 21 days after anthesis.

The precise timing of the application of quercetin may not be critical. Where the plant is wheat, the may be contacted with quercetin during one, or two or more of F8.0,

F9.0, F10.0, and F10.5 stages. In some embodiments, wheat plants are contacted during each of F8.0, F9.0, F10.0, and F10.5 (referring to the stages of the Feekes scale).

As an example, an application to a maize or soybean plant may be made between the v2 (2 leaf) and V6 (six leaf) stage of growth. In a tomato plant, applications may begin pretransplant and continue at two to three week intervals through early harvest. In citrus, applications may begin prior to flowering and continue at three to four week intervals for six or seven applications.

The quercetin when used in the present invention is exogenous to the plant. It may be applied as a composition which consists essentially of quercetin. However a composition may further comprise e.g. an agriculturally acceptable carrier such as a carrier, support, filler, dispersant, emulsifier, wetter, adjuvant, solubilizer, colorant, tackifier, binder, anti-foaming agent and/or surfactant, such as are well known in the art (see e.g. WO2019/152632).

In addition to adjuvants, the quercetin may be applied as part of a tank-mix with one or more other active compatible plant protection or improvement products (e.g. herbicides, fungicides, insecticides) subject to normal safety guidelines.

Agriculturally acceptable carriers can be solid or liquid. Solid carriers include, but are not limited to, silicas, silica gels, silicates, talc, kaolin, limestone, lime, chalk, bole, loess, clay, dolomite, attapulgite clay, bentonite, acid clay, pyrophillite, talc, calcite, corn starch powder, dolomite, diatomaceous earth, calcium sulfate, magnesium sulfate, magnesium oxide, ground synthetic materials, resins, waxes, polysaccharides, e.g. cellulose, starch, fertilizers such as ammonium sulfate, ammonium phosphate, ammonium nitrate and ureas, products of vegetable origin such as cereal meal, tree bark meal, wood meal and nutshell meal (e.g., walnut shell powder), cellulose powders and the like; and combinations thereof. Non-limiting examples of liquid carriers include water, alcohols, ketones, petroleum fractions, aromatic or paraffinic hydrocarbons, chlorinated hydrocarbons, liquefied gases and the like, and combinations thereof. Thus, liquid carriers can include, but are not limited to, xylene, methylnaphthalene and the like, isopropanol, ethylene glycol, cellosolve and the like, acetone, cyclohexanone, isophorone and the like, vegetable oils such as soybean oil, cottonseed oil, corn oil and the like, dimethyl sulfoxide, acetonitrile, and combinations thereof.

In some embodiments the composition does not include a microorganism and/or a germinant.

In some embodiments the composition does not include a dicarboxylic acid and/or a salt thereof

In some embodiments does not include a cyclopropane.

In some embodiments the composition contains 0.05 to 1 , 5 or 10 mM of the quercetin, more preferably about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 mM.

A composition as described above e.g. containing 0.05 to 1, 5 or 10 mM quercetin or quercetin derivative (e.g. quercetin-3-O-glycoside) , more preferably about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 mM, in combination with an agriculturally acceptable carrier as described above, forms a further aspect of the invention. The composition may be supplied in a container suitable for application to plants, and may be supplied with a written description of use in accordance with the uses or methods of the invention (i.e. to reduce the water requirement or enhance WUE of a plant; to increase tolerance to water scarcity and/or for reduce the consequence of water scarcity in a plant) and optionally describing one or more of suitable plants for use; suitable regions for use; suitable timing or regularity of application of the quercetin or derivative.

Plants to which the present invention may be applied include, but are not limited to, gymnosperms, angiosperms (monocots and dicots), ferns, fern allies, bryophytes, and combinations thereof.

Plants to which the present invention may be applied includes those defined in the “Definition and Classification of Commodities,” published by the Food and Agriculture Organization of the United Nations (“FAO”) statisticians and available here: http://www.fao.org/economic/ess/ess-standards/en/

These are grouped as follows:

1. Cereals: wheat, rice barley, maize, popcorn, rye, oats, millets, sorghum, buckwheat, quinoa, fonio, triticale, canary seed, mixed grain, other cereals (including inter alia: canagua or coaihua (Chenopodium pallidicaule); quihuicha or Inca wheat (Amaranthus caudatus); adlay or Job's tears (Coix lacryma-jobi); wild rice (Zizania aquatica ))

2. Roots and tubers: potatoes, sweet potatoes, cassava, yautia (cocoyam), taro (cocoyam), yams, other roots and tubers (including inter alia: arracacha (Arracacoa xanthorrhiza); arrowroot (Maranta arundinacea); chufa (Cyperus esculentus); sago palm (Metroxylon spp.); oca and ullucu (Oxalis tuberosa and Ullucus tuberosus); yam bean, jicama (Pachyrxhizus erosus, P. angulatus); mashua (Tropaeolum tuberosum); Jerusalem artichoke, topinambur (Helianthus tuberosus))

3. Sugar crops and sweeteners and derived products: Sugar cane, Sugar beet, Other sugar crops (including inter alia: sugar maple (Acer saccharum); sweet sorghum (Sorghum saccharatum); sugar palm (Arenga saccharifera)

4. Pulses: beans, broad beans, peas, chick-peas, cow peas, pigeon peas, lentils, bambara beans, vetches, lupins, other pulses (including inter alia: lablab or hyacinth bean (Dolichos spp.); jack or sword bean (Canavalia spp.); winged bean (Psophocarpus tetragonolobus); guar bean (Cyamopsis tetragonoloba); velvet bean (Stizolobium spp.); yam bean (Pachyrrhizus erosus))

5. Nuts: brazil nuts, cashew nuts, chestnuts, almonds, walnuts, pistachios, kola nuts, hazelnuts (filberts), areca nuts, other nuts (including inter alia: pecan nut (Carya illinoensis); butter or swarri nut (Caryocar nuciferum); pili nut, Java almond, Chinese olives (Canarium spp.); paradise or sapucaia nut (Lecythis zabucajo); Queensland, macadamia nut (Macadamia ternifolia); pignolia nut (Pinus pinea ))

6. Oil-bearing crops: soybeans, groundnuts, coconuts, oil palm fruit, olives, karite nuts, castor beans, sunflower seed, rapeseed, tung nuts, safflower seed, sesame seed, mustard seed, poppy seed, melonseed, tallowtree seeds, kapok fruit, seed cotton, linseed, hempseed, other oil seeds (including inter alia: beech nut (Fagus sylvatica);( Aleurites moluccana);(Carapa guineensis);(Croton tiglium);(Bassia latifolia);(Guizotia abyssinica);(Licania rigida);(Perilla frutescens);(Jatropha curcas);(Shorea robusta);(Pongamia glabra);(Astrocaryum spp.))

7. Vegetables: cabbages, artichokes, asparagus, lettuce, spinach, cassava leaves, tomato, cauliflower, pumpkins, cucumbers and gherkins, eggplants, chillies and peppers, green onions, dry onions, garlic, leeks and other alliaceous vegetables, green beans, green peas , green broad beans, string beans, carrots, okra, green corn, mushrooms, watermelons, cantaloupes melons, other vegetables (including inter alia: bamboo shoots (Bambusa spp.); beets, chards (Beta vulgaris); capers (Capparis spinosa); cardoons (Cynara cardunculus); celery (Apium graveolens); chervil (Anthriscus cerefolium); cress (Lepidium sativum); fennel (Foeniculum vulgare); horseradish (Cochlearia armoracia); marjoram, sweet (Majorana hortensis); oyster plant (Tragopogon porrifolius); parsley (Petroselinum crispum); parsnips (Pastinaca sativa); radish (Raphanus sativus); rhubarb (Rheum spp.); rutabagas, swedes (Brassica napus); savory (Satureja hortensis); scorzonera (Scorzonera hispanica); sorrel (Rumex acetosa); soybean sprouts tarragon (Artemisia dracunculus); watercress (Nasturtium officinale))

8. Fruits: bananas, plantains, oranges, tangerines and mandarins and clementines and satsumas, lemons and limes, grapefruit and pomelo, citrus fruit, apples, pears, quinces, spome fruit, apricots, sour cherries, cherries, peaches and nectarines, plums, stone fruit, strawberries, raspberries, gooseberries, currants, blueberries, cranberries, berries, grapes, figs, persimmons, kiwi fruit, mangoes, avocados, pineapples, dates, cashewapple, papayas, other fruit (including inter alia: breadfruit (Artocarpus incisa); carambola (Averrhoa carambola); cherimoya, custard apple (Annona spp.); durian (Durio zibethinus); feijoa (Feijoa sellowiana); guava (Psidium guajava); hog plum, mombin (Spondias spp.); jackfruit (Artocarpus integrifolia); longan (Nephelium longan); mammee (Mammea americana); mangosteen (Garcinia mangostana); naranjillo (Solanum quitoense); passion fruit (Passiflora edulis); rambutan (Nephelium lappaceum); sapote, mamey Colorado (Calocarpum mammosum); sapodilla (Achras sapota); star apple, cainito (Chrysophyllum spp.)] azarole (Crataegus azarolus); babaco (Carica pentagona); elderberry (Sambucus nigra); jujube (Zizyphus jujuba); litchi (Nephelium litchi); loquat (Eriobotrya japonica); medlar (Mespilus germanica); pawpaw (Asimina triloba); pomegranate (Punica granatum); prickly pear (Opuntia ficus-indica); rose hips (Rosa spp.); rowanberry (Sorbus aucuparia); service-apple (Sorbus domestica); tamarind (Tamarindus indica); tree-strawberry (Arbutus unedo)

9. Fibres of vegetal origin: cotton, flax, hemp, kapok, jute, ramie, sisal, and other fibers, from plants.

10. Spices: pepper, pimento, vanilla, cinnamon (canella), nutmeg and mace and cardamons, cloves, anise and badian and fennel, ginger, other spices including bay leaves (Laurus nobilis); dill seed (Anethum graveolens); fenugreek seed (Trigonella foenum-graecum); saffron (Crocus sativus); thyme (Thymus vulgaris); turmeric (Curcuma long a)

11. Fodder crops and products: Fodder crops are crops that are cultivated primarily for animal feed. By extension, natural grasslands and pastures are included whether they are cultivated or not. Examples include maize for forage, sorghum for forage, rye grass for forage, clover for forage, alfalfa for forage, green oilseeds for silage, legumes for silage, grasses for forage, cabbage for fodder, pumpkins for fodder, turnips for fodder, beets for fodder, carrots for fodder, swedes for fodder.

12. Stimulant crops and derived products: green coffee, cocoa beans, tea, mate

13. tobacco and rubber and other crops: chicory roots, carobs, hops, citronella, peppermint & spearmint, plants providing essential oils, pyrethrum plants (Chrysanthemum), tobacco, rubber, natural gums (including inter alia: balata (Manilkara bidentata); ceara (Manihot glaziovii)] chicle gum (Achras zapota)] guayule (Parthenium argentatum)] gutta-percha ( Palachium gutta ); jelutong ( Dieva costulana )), arabic gum, other resins (including inter alia: copaiba, copal ( Copaifera spp.)] gum tragacanth ( some Astragalus spp.)] incense ( Boswellia spp.)] myrrh, opopanax, Mecca balsam (Commiphora spp.); tolu balsam, peru balsam ( Myroxilon balsamum; M. Pereira)) vegetable waxes (including inter alia: candelilla ( Euphorbia antisyphilitica; Pedilanthus pavonis)] carnauba ( Copernicia cerifera)] urucury ( Attaiea excelsa)] palm wax ( Ceroxylon andicolum))

In some embodiments, plants are treated that are not members of the genus Nicotiana.

In some embodiments, plants are treated that are not Arabidopsis.

In some embodiments the plant is selected from apple, tomato, cherry, pear, pepper, cucumber, honeydew melon, watermelon, cantaloupe, papaya, mango, pineapple, avocado, plum, bean, squash, peach, apricot, grape, strawberry, raspberry, blueberry, mango, cranberry, gooseberry, banana, fig, clementine, kumquat, orange, grapefruit, tangerine, lemon, lime, hazelnut, pistachio, walnut, macadamia, almond, pecan, Litchi, soybeans, corn, sugar cane, camelina, peanut, cotton, canola, alfalfa, timothy, tobacco, tomato, sugarbeet, potato, pea, carrot, wheat, rice, barley, rye, triticale, turf, lettuce, rose, tulip, violet, basil, oil palm, elm, ash, oak, maple, fir, spruce, cedar, pine, birch, cypress, coffee, miscanthus, switchgrass, or arundo, or any combination thereof.

On some embodiments the plant is a selected from the list consisting of wheat, tomato, tobacco, barley, oats, rye, triticale, and rice.

On some embodiments the plant is a selected from the list consisting of wheat, tomato, and tobacco.

On some embodiments the plant is a wheat.

In one embodiment the invention improves plant yield by at least 3, 4, 5, 6, 7, 8, 9, 10%.

In one embodiment the invention improves grain yield by at least 3, 4, 5, 6, 7, 8, 9, 10%.

Grain yield may be compared to an otherwise comparable untreated control plant not treated with quercetin, which is likewise present in a moisture deprived environment. Quercetin treatment may be used to boost grain yield in moisture-stress exposed plants, for example to a level similar to that of non-treated fully irrigated plants.

On one embodiment the quercetin increases WUE(grain) (kg ha-1 mm-1) by at least 5%, 10%, or 20%.

WUEg may be a calculated by the methods known in the art, and in particular as described by Nagore, Maria Lujan, et al. "Water use efficiency for grain yield in an old and two more recent maize hybrids." Field Crops Research 214 (2017): 185-193, in sections 2.3 - 2.4, which are herein incorporated by cross-reference.

As described herein, for plants grown in moisture-stress conditions, quercetin treatment had a positive effect on tiller numbers, grain length, grain width and thousand-grain weight, and total grain yield.

Definitions

Unless context demands otherwise, by “substantially” is meant that the criterion in question is met in at least at least 90%, more preferably at least 95% of the relevant instances.

When it is stated herein that the invention “is not based on” the use of a reagent or entity, this indicates that such a reagent or entity may be specifically and deliberately excluded from compositions of the invention, for example the compositions do not contain the reagent or entity as an active ingredient, or contain only very low levels of such reagent or entity.

In some embodiments, it is contemplated to contact a group of crop plants with quercetin at a certain desired "Stage of development”. In such cases, it is contemplated that such contacting may be performed when the ratio of the number of plants that have reached the desired stage of development to the total number of plants in the group is at least 0.5, or at least 0.75, or at least 0.9 (i.e., when the portion of plants that have reached the desired stage of development is at least 50%, or 75%, or 90%).

As used herein, "a," "an" or "the" can mean one or more than one (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, etc.). For example, a plant can mean a plurality of plants.

Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted.

As used in the description of the invention and the appended claims, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

Also as used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

The term “about,” as used herein when referring to a measurable value such as an amount or concentration and the like, is meant to encompass variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified value as well as the specified value. For example, “about X” where X is the measurable value, is meant to include X as well as variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of X.

A range provided herein for a measureable value may include any other range and/or individual value therein.

As used herein, phrases such as "between X and Y" and "between about X and Y" should be interpreted to include X and Y. As used herein, phrases such as "between about X and Y" mean "between about X and about Y" and phrases such as "from about X to Y" mean "from about X to about Y."

The term “comprise,” “comprises” and “comprising” as used herein, specify the presence of the stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

As used herein, the phrase “consisting essentially of means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, the term “consisting essentially of when used in a claim of this invention is not intended to be interpreted to be equivalent to “comprising.”

Any sub-titles herein are included for convenience only, and are not to be construed as limiting the disclosure in any way.

The invention will now be further described with reference to the following non limiting Figures and Examples. Other embodiments of the invention will occur to those skilled in the art in the light of these.

The disclosure of all references cited herein, inasmuch as it may be used by those skilled in the art to carry out the invention, is hereby specifically incorporated herein by cross-reference.

Figure Legends

Figure 1. Flavonols enhance carbon assimilation and water use. (A) Appearance of mature plants. Scale bar, 10 cm. (B) Grain yield. (C) Transpiration rate. (D) CO2 assimilation rate. (E) WUEi. (F) Stomatal sensitivity to ABA. (G) Time-course of water loss from detached leaves. (H) SR3 plants exhibit a superior level of drought tolerance. Water was withheld from 2-week-old plants for 14 days. Scale bar, 5 cm.

(I) DPBA stained leaves. Scale bar, 10 pm. (J) Flavonols accumulation. (K, L) Relative transcript abundance of F3’H (K) and FLS1 (L) in SR3 plants relative to that in moisture-stressed JN177 plants. Data shown as mean ± SEM (panels B-G, K and L, n = 3; panel J, n = 50). Statistical analyses used Duncan's multiple range tests, the same lowercase letter denotes a non-significant difference between means ( P > 0.05). Figure 2. Quercetin-binding properties of OST1. (A-F) Photosynthetic performance and WUE of 5-week-old plants sprayed with 5 ml_ 0.5 mM quercetin or 5 mL O.1 mM ABA: (A) stomatal conductance, (B) WUEi, (C) chlorophyll content, (D) CO2 assimilation rate, (E) appearance of plants at 7 days after the treatment (scale bar, 10 cm), and (F) biomass. (G) In vitro pull-down assays. (H) In vivo pull-down assays. Total proteins are extracted from wheat plants pre-treated with or without ABA. (I) Six amino acid residues in the Dl domain define a putative binding site for quercetin. (J-L) MST assays. OST1 binds to quercetin (J), quercetin interferes with the interaction between OST1 and ABI1 (K), phosphorylation of the Dll domain enhances its binding affinity to quercetin (L). Q, quercetin; K, Kaempferol. Data shown as mean ± SEM (panels A-D and F, n = 6; panels J-L, n = 3). Statistical analyses used Duncan's multiple range tests, the same lowercase letter denotes a non-significant difference between means (P> 0.05).

Figure 3. Quercetin activation of SLAC1 anion channels. (A) Quercetin inhibits the ABI1 -mediated dephosphorylation of GST-tagged OST1 and SLAC^NT proteins. The upper panel shows ³²P-labeled phosphorylated OST1 and SLAC^NT, while lower one shows the quantification of OST 1 , AB11 and SLAC^NT. (B-G) Effects of quercetin on ABA-mediated activation of SLAC1 channels in oocytes. Whole cell currents (B, D and F) and current-voltage (C, E and G) relationships imply that quercetin reverses the inhibition of OST1 -mediated SLAC1 activation by ABI1 (B, C); PYL4 does not activate SLAC1 currents in the absence of ABA, but does so in its presence (D, E); ABA-activated SLAC1 anion currents are enhanced in the presence of quercetin (F, G). Q, quercetin; K, Kaempferol. Data (panels C, E and G) shown as mean ± SEM ( n = 5).

Figure 4. Quercetin-spray treatment boosts wheat yield and WUE. (A)

Transpiration rate. (B) CO2 assimilation rate. (C) WUEi. (D) Distribution of total carbon (C) within the plant. (E) Distribution of total nitrogen (N) within the plant. (F) The carbon-to-nitrogen ratio. (G) Grain numbers. (H) Tiller numbers. (I) Grain weight. (J) Grain yield. Data shown as mean ± SEM (panels A-C, n = 5; panels F and J, n = 3; panels G-l, n = 30). Statistical analyses used Duncan's multiple range tests, the same lowercase letter denotes a non-significant difference between means ( P > 0.05).

Fig. 5. The up-regulation of flavonoid metabolisms is associated with drought tolerance. (A) The appearance of mature plants. Scale bar, 10 cm. (B) WUEi. Data shown as mean ± SEM ( n = 3). Statistical analyses used Duncan's multiple range tests, the same lowercase letter denotes a non-significant difference between means (P> 0.05). (C) Transcriptome response to moisture stress. (D) The flavonoid biosynthesis pathway. (E) The differential transcriptional response to moisture stress of JN177 and SR3. Data shown as mean ± SEM ( n = 3). Transcript abundances are expressed relative to that of F3’H in JN177 (set to one). A Student’s f-test was used to generate the P values.

Fig. 6. Moisture stress-induced accumulation of flavonols. (A-D) The content of quercetin and kaempferol in the leaves of 2-week-old wheat plants either kept well- watered or deprived of water for 7 days. Data (panels B and D) shown as mean ± SEM (n = 3). Statistical analyses used Duncan's multiple range tests, the same lowercase letter denotes a non-significant difference between means ( P > 0.05).

Fig. 7. The down-regulation of FLS1 confers increased susceptibility to drought-induced mortality in SR3 plants. (A) Abundance of FLS1 transcript in transgenic relative to non-transgenic plants. Data shown as mean ± SEM ( n = 3). A Student’s f-test was used to generate the P values. (B) DPBA stained leaves. Scale bar, 10 pm. (C) The accumulation of flavonols in the stomatal guard cells shown in B. Data shown as mean ± SEM ( n = 30). Statistical analyses used Duncan's multiple range tests, the same lowercase letter denotes a non-significant difference between means ( P > 0.05). (D) The performance of 2-week-old SR3 FLS1 knock-down plants in response to the withholding of water for 14 days.

Fig. 8. The up-regulation of FLS1 improves water use efficiency and drought tolerance. (A) Abundance of FLS1 transcript in transgenic relative to non-transgenic plants. (B) The content of quercetin and kaempferol in the leaves of 3-week-old wheat plants. (C) The accumulation of flavonols shown in B. (D) DPBA- staining shows that the major site of flavonols deposition was in and around the stomatal guard cells. (E) The accumulation of flavonols in the stomatal guard cells shown in D. Data shown as mean ± SEM ( n = 30). Statistical analyses used Duncan's multiple range tests, the same lowercase letter denotes a non-significant difference between means (P > 0.05). (F) The stomatal sensitivity to ABA. (G) Transpiration rate. (H) CO2 assimilation rate. (I) WUEi. (J) The relative rates of water loss from detached leaves. Data (panels A, C and F-J) shown as mean ± SEM ( n = 3). A Student’s f-test was used to generate the P values. (K) FLS1- overexpressors shown an enhanced level of moisture stress tolerance. 2 week-old plants were deprived of water for 14 days.

Fig. 9. Quercetin-spray treatment enhances the ability of plants to tolerate moisture stress. (A) Transpiration rate. (B) The rates of water loss from detached leaves. (C) CO2 assimilation rate. (D) WUEi. (E) Stomatal sensitivity to ABA. Data shown as mean ± SEM (panels A-D, n = 4; panel E, n = 3). Student’s f-test was used to generate the P values. (F) Application of quercetin improves the drought resistance of wheat. 2-week-old plants were deprived of water for 14 days and sprayed with 5 ml_ 0.5 mM quercetin on the eighteenth day. Scale bar, 5 cm.

Fig. 10. Application of quercetin enhances photosynthetic capacity. (A) Relative shoot abundances of transcripts of multiple genes regulating photosynthesis, sucrose metabolism and transport in 3-week-old wheat plants treated with 0.5 mM quercetin, relative to abundance in non-treated controls (set to one). Data shown as mean ± SEM (n = 3). (B) Immunoblot detection of Lhcal, Lhcb2, PsaD, PsbE, PsbO and PsbP proteins in wheat plants shown in A. HSP90 serves as loading control.

Fig. 11. Flavonols accumulation in the region of the stomatal guard cells. 3- week-old wheat leaves sprayed with either 0.5 mM quercetin or 0.5 mM kaempferol are stained with DPBA, and the resulting fluorescent signal is captured by confocal microscopy (Zeiss LSM710). Scale bar, 50 pm.

Fig. 12. Kaempferol-spray treatment had no impact on the plants’ ability to tolerate moisture stress. (A) Transpiration rate. (B) The relative rates of water loss from detached leaves. (C) CO2 assimilation rate. (D) WUEi. (E) Sensitivity of the stomata to exogenously supplied ABA. Data shown as mean ± SEM (panels A-D, n = 4; panel E, n = 3). Student’s f-test was used to generate the P values. (F) The appearance of 2-week-old wheat plants deprived of water for 14 days and then treated with 0.5 mM kaempferol on the eighteenth day the sixth day. Scale bar, 5 cm.

Fig. 13. Quercetin treatment enhances transcription and protein abundances of OST1. (A) The relative abundance of OST1 transcript. 3-week-old plants were sprayed with either 0.5 mM quercetin or kaempferol. Data shown as mean ± SEM ( n = 3). Abundance was measured relative to that of non-treated control plants (set to one). (B) The abundance of OST1. HSP90 was used as the loading control.

Fig. 14. Quercetin-binding properties of OST1. (A) Schematic representation of the two conserved domains of OST 1 and alignment of sequences from different plant species. OST 1 , a wheat OST 1 homolog (MK203873); AtOST 1 , an A. thaliana OST 1 homolog (AT4G33950); OsSAPK8, a rice OST1 homolog (0s03g0764800). Asterisks indicate consensus sequences for CK2 activity that are mutagenized. (B) A putative quercetin-binding pocket is present in the OST1 catalytic kinase domain. (C) Mutations engineered by NAAIRS substitutions within those putative binding sites do not compromise the affinity of OST 1 for quercetin. Data shown as mean ± SEM ( n = 3).

Fig. 15. Binding affinities of quercetin and its derivatives with OST1. (A)

Quercetin. (B) Quercetin 3 b-D-glucoside. (C) Quercetin 3-rhamnoside. (D) Quercetin 3-D-galactoside. Data shown as mean ± SEM ( n = 3).

Fig. 16. The effects of flavonols on OST1 -dependent activation of SLAC1 anion channels. Whole cell currents (A) and current-voltage (B) relationships imply that neither quercetin nor kaempferol affects the activation by OST 1 kinase of a SLAC1 anion channel. Data shown as mean ± SEM ( n = 3).

Fig. 17. Quercetin-spray treatment increases grain yield. (A) The appearance of mature wheat plants. 0 m³/ha, non-irrigated plants; 750 m³/ha, partially irrigated plants; 1,500 m³/ha, fully irrigated plants. Scale bar: 10 cm. (B) Grain width. Scale bar: 1 cm. (C) Grain length. Scale bar: 1 cm.

Fig. 18. Quercetin-spray treatment improves drought-tolerance in both wheat and rice. (A) The appearance of 5-week-old rice plants deprived of water for 15 days, and plants treated with 0.2 mM quercetin on the first day after drought treatment. (B) The appearance of 5-week-old wheat plants deprived of water for 10 days, and plants treated with 0.2 mM quercetin on the first day after drought treatment. (C) The appearance of 5-week-old rice and wheat plants deprived of water for 16 days, and plants treated with 0.5 mM quercetin on the eighth day after drought treatment.

Fig. 19. Quercetin-spray treatment improves drought-tolerance in tobacco.

The appearance of 10-day-old tobacco plants deprived of water for 16 days, and plants treated with 0.5 mM quercetin on the eighth day after drought treatment. Scale bar, 2cm.

Fig. 20. Quercetin-spray treatment improves drought-tolerance in tomato.

The appearance of 3-week-old tomato plants deprived of water for 14 days, and plants treated with 0.5 mM quercetin on the eighth day after drought treatment. Scale bar, 2cm.

Fig. 21. Effect of quercetin spray on transpiration rate, C02 assimilation efficiency and water use efficiency of Wheat.

Fig. 22. Field trials of quercetin on wheat.

Examples

Example 1 - Field-grown experiment to investigate water use efficiency in winter bread wheat germplasm.

We found that the high-yielding wheat varieties exhibit genetic variation with respect to plant height, tiller number and grain yield when grown under limited soil moisture conditions (table S1). For example, the wheat cultivar Shanrong3 (SR3, 16) out- yielded its parental wheat variety Jinan177 (JN177) when the plants were exposed to moisture stress, increasing yield to a level similar to that of fully irrigated JN177 plants (Fig. 1A, B). Gas exchange measurements showed that whilst SR3 plants have higher photosynthetic activities than JN177 plants, but their transpiration rates are lower (Fig. 1C, D). As a result, the ratio of the instantaneous rates of CO2 assimilation over transpiration (WUEi, 3) was found to be higher in SR3 than in JN177 (Fig. 1E). Furthermore, we found that SR3 stomatal closing is more responsive to exogenous ABA than in JN177 (Fig. 1F), SR3 rate of water loss is lower than in JN177 (Fig. 1G), and that SR3 leaves recover more readily from moisture stress than those of JN177 (Fig. 1H). These results suggest that the superior WUE of SR3 is due to combined reduced rates of transpiration and increased rates of CO2 fixation.

Example 2 - Analysis of cultivars and CSSLs derived from JN177 x SR3

From 240 chromosome segment substitution lines (CSSLs) derived from a cross between JN177 and SR3, we identified one (CSSL15) that accumulates greater biomass than the JN177 recurrent parent, and also displays a WUEi superior to that of JN177 (and similar to that of SR3; fig. 5A, B). In contrast, another line (CSSL21) behaves similarly to JN177 (fig. 5A, B). Transcriptome-wide RNA sequencing following a 7 day exposure to moisture stress revealed the mRNA abundances of the genes involved in flavonoid biosynthesis to be increased in both SR3 and CSSL15 (versus JN177 and CSSL21; fig. 5C). Quantitative real-time PCR assays confirmed moisture stress induced increases in relative abundances of mRNAs from genes F3’H and FLS1 {17), whose products catalyze specific steps in flavonoid biosynthesis (fig. 5D), to be higher in SR3 than in JN177 (fig. 5E). Furthermore, the flavonols quercetin and kaempferol also accumulate to higher levels in SR3 than in JN177 leaves, and these levels are further increased by moisture stress (fig. 6), in accord with previous observations that biotic or abiotic stress exposure elicits accumulation of these flavonoids (18). Staining of wheat leaf tissue preparations with DPBA (diphenylboric acid 2-aminoethyl ester, a flavonol-indicating fluorescent dye) indicated the major site of flavonol accumulation to be in and around the stomatal guard cells (Fig. 11), as previously observed in Arabidopsis thaliana (19) and tomato (20). The intensity of the DPBA signal was greater in moisture-stressed plants than in well-watered controls, and this effect was particularly prominent in SR3 (Fig. 1J). Finally, whilst the abundance of both FLS1 and F’3H mRNAs increases during prolonged moisture stress, FLS1 mRNA abundance rises more steeply than that of F’3H , and this effect is more pronounced in SR3 than in JN177 leaves (Fig. 1K, L).

Example 3 - Analysis of transgenic plants with modified flavonoid biosynthesis

To determine if the superior WUE of SR3 is dependent upon FLS1 function, we next generated transgenic SR3 pUbi::RNAi-FLS1 wheat plants in which FLS1 expression was suppressed (fig. 7A), showing that this suppression reduced the stomatal DPBA signal (fig. 7B, C). We found that moisture stress causes SR3 pUbi::RNAi-FLS1 plants to lose water more rapidly, to wilt earlier and to die sooner than SR3 controls, while also under-performing with respect to CO2 fixation and WUEi (Fig. 1C-G). In contrast, transgenic FLS1 over-expressor plants accumulate increased amounts of both quercetin and kaempferol (fig. 8A-C), and display an enhanced moisture stress- induced flavonol DPBA signal in and around their stomatal guard cells (fig. 8D, E). The key effect of pUbi::FLS1 -dependent constitutive FLS1 expression is to reduce transpirational water loss, enhance CO2 fixation and WUEi, increase stomatal ABA sensitivity, and improve the tolerance of moisture stress (fig. 8F-K). The phenotypes conferred by pUbi::RNAi-FLS1 and pUbi::FLS1 suggest that the manipulation of cellular flavonol content is an effective way of reducing transpiration without compromising photosynthesis, and that SR3 exhibits increased WUE because of increased FLS 7-dependent flavonol content.

Example 4 - Effect of flavonol application to plants

We next determined the effects of direct flavonol application, by spraying plants with solutions containing quercetin or kaempferol, first finding that quercetin treatment substantially reduces the rates of transpiration and water loss (fig. 9A, B), increases CO2 assimilation, WUEi and the stomatal sensitivity to ABA (fig. 9C-E), and enhances post-stress recovery and survival (fig. 9F). To some extent, these effects mimicked those elicited by spraying with ABA (Fig. 2A, B), except that whilst quercetin treatment boosted chlorophyll content (Fig. 2C), and enhanced the mRNA (fig. 10A) and protein (fig. 10B) abundances of multiple genes involved in photosynthesis (for example, Lhcal and PsaD), sucrose metabolism (for example, TPS1 and TPP1) and sucrose transport (for example, STU1A and SWEET2A), resulting in increased photosynthetic capacity (Fig. 2D) and biomass production (Fig. 2E, F). In contrast, ABA treatment had the opposite effects on photosynthesis and biomass accumulation (Fig. 2D-F), and was less promotive of chlorophyll content (Fig. 2C). Whilst treatment with either quercetin or kaempferol enhanced DPBA-detectable stomatal guard cell flavonol content (fig. 11), kaempferol treatment does not detectably alter rates of water loss, transpiration and carbon assimilation, WUEi and stomatal ABA sensitivity, or viability in moisture-stress conditions (fig. 12). We conclude that, of the two flavonols displaying FLSI-dependent increase in stomatal guard cell content, it is quercetin, but not kaempferol, that confers the increased WUE of SR3.

In further experiments, after 10 days of water withholding, quercetin was sprayed on the leaves of wheat. The quercetin in the leaves was visualized by DPBA staining. In comparison with the leaves before spraying, those sprayed with quercetin for one and three days had higher flavonol content (results not shown).

Example 5 - Relationship between quercetin- and ABA-mediated regulation of stomatal closure.

In canonical ABA signaling (9,21), PP2C phosphatases interact with and dephosphorylate OST1 , thus inhibiting its activity (11, 12). In contrast, ABA activates OST1 by binding to pyrabactin resistance1/PYR1-like/regulatory components of ABA receptors (PYR/PYL/RCAR) ABA receptors, thus capturing PP2Cs, releasing OST1 from PP2C-dependent inhibition, and promoting the activities of stomatal guard cell slow-type anion channel SLAC1 (SLOWANION CHANNEL-ASSOCIATED1; 22,23) and other targets (11). We first found that increased quercetin cellular content (due either to spray treatment or to pUbi::FLS1 expression) increased the abundance of OST1 mRNA and OST1 protein (fig. 13). Moreover, pull-down assays demonstrated that quercetin was able to bind directly with OST 1 both in vitro and in vivo (Fig. 2G, H), and the binding affinity of quercetin with OST1 could be enhanced by exogenous ABA treatment (Fig. 2H). From a three-dimensional (3-D) structural model of the wheat OST 1 constructed on the basis of the crystal structure of the homologous A. thaliana OST1 (24), we discerned two putative quercetin-binding pockets, one located at the area within the protein’s Dl domain and the other in its catalytic kinase domain (Fig. 2I; fig. 14A, B). Microscale thermophoresis (MST) binding assays revealed that quercetin and its derivatives, such as quercetin-3^-D-glucoside, does indeed bind OST1 (Fig. 2J; fig. 15), but not ABA-INSENSITIVE1 (a wheat ABI1 PP2C; 25) or PYRABACTIN RESISTANCE-LIKE4 (PYL4, a wheat ABA receptor; 1) (Fig. 2J). Further MST binding assays using mutant OST1 proteins with NAAIRS (Asp-Ala-Ala-lle-Arg-Ser hexapeptide; 26) substitutions in the two putative quercetin binding sites showed that whilst mutating the Dl domain completely abolishes quercetin binding (Fig. 2G, J), but mutating the catalytic kinase domain has no detectable effect on quercetin binding (fig. 14C).

Whilst OST 1 interacts with ABI1 , we found this affinity interaction to be reduced in the presence of quercetin (but not of kaempferol) (Fig. 2K). A previous study showed that the conserved serine residues present in the Dll (fig. 14A) domain of OST1 are phosphorylated by casein kinase 2 (CK2; 27), thus enhancing the ability of OST1 to bind to PP2Cs. We next found that an OST1^4A variant (27), in which all four of the Dll domain serine residues have been mutated to alanine, and is more stable than OST1 on account of reduced PP2C binding (27), exhibits a much reduced affinity for quercetin (Fig. 2L). Conversely, an OST1^4E variant (27), in which the serine residues of the Dll domain have been mutated to phosphorylation-mimicking glutamate residues, binds quercetin more readily than wild-type OST1 (Fig. 2L). These results suggest that CK2-mediated conformational change at the Dll domain can influence binding affinity of quercetin with Dl domain, and that this influence links the physiological consequences of quercetin and ABA signaling.

We next found that recombinant GST (Glutathione S-transferase)-OST 1 protein is able to phosphorylate the N terminal domain of wheat homolog of rice SLAC (SLAC^NT; 28), but this effect is inhibited in the presence of the ABI1 PP2C (Fig. 3A). Furthermore, we also found that ABI1 -mediated dephosphorylation of both OST1 and SLAC^NT is repressed by quercetin but not by kaempferol (Fig. 3A). To determine if quercetin regulates OST1-mediated activation of SLAC1 anion channels, we next used oocytes co-expressing OST1 and SLAC1 (23, 28). Voltage clamp experiments revealed OST1 activates SLAC1 -mediated currents in these oocytes, and that this anion channel current activity is not detectably affected by the presence of either quercetin or kaempferol (fig. 16). However, whilst ABI1 almost completely suppresses the OST1 -activated anion channel current, this suppression is reduced by quercetin (but not by kaempferol; Fig. 3B, C). In addition, anion current enhancement is when ABA is injected into the cytoplasm of oocytes co-expressing SLAC1, ABI1, OST1 and PYL4 (Fig. 3D, E), an effect which becomes even stronger in the presence of quercetin (Fig. 3F, G). This latter result is consistent with the previous observation that quercetin treatment increases the stomatal sensitivity to exogenously supplied ABA (fig. 9E). We conclude that the enhanced WUE of SR3 is conferred by quercetin-promoted amplification of OST1 -activated SLAC anion channel-dependent stomatal aperture reduction, which in turn reduces water loss through transpiration.

Example 6 - Effect of exogenous quercetin on grain yield winter wheat

Having shown that exogenous quercetin promotes stomatal closing, we next determined the effect on grain yield of spraying field-grown winter wheat (cultivar Jimai22) with 185 mL/m²0.5 mM quercetin. The plants were sprayed twice: firstly when close to anthesis, and secondly at 15 days post-anthesis.

We found that sprayed plants stay green longer than water-sprayed controls, consistent with our previous observation of the positive effect of quercetin on leaf chlorophyll content (Fig. 2C). In addition, quercetin-sprayed plants exhibit decreased transpiration rates, increased photosynthesis and superior WUEi, in both moisture- stress exposed plants and fully irrigated controls (Fig. 4A-C). Furthermore, quercetin- sprayed plants exhibit increases in total carbon and total nitrogen content in above ground parts, particularly in grains (Fig. 4D, E). However, there is no detectable effect of the quercetin-spray treatment on the carbon-to-nitrogen ratio (Fig. 4F), suggesting that quercetin promotes plant growth and biomass accumulation by simultaneous enhancement of carbon and nitrogen assimilation.

With respect to plants grown in moisture-stress conditions, quercetin treatment has no detectable impact on grain number per ear (Fig. 4G), but a positive effect on tiller numbers, grain length, grain width and thousand-grain weight, and total grain yield (Fig. 4H-J; Fig. 17). Finally, quercetin treatment boosts grain yield by -7.2% in moisture-stress exposed plants, thus increasing yield to a level similar to that of non- treated fully irrigated plants (Fig. 4J).

Figs. 18 to 20 show that the invention is likewise applicable to a wide variety of monocot and dicot plant types including rice, tobacco, and tomato.

Fig. 21 further illustrates the effect on transpiration rate, C02 assimilation efficiency and water use efficiency of quercetin. Wheat was sprayed with a control and 200 mM quercetin for seven days. The results show a lower transpiration rate but higher C02 assimilation efficiency and water use efficiency in leaves sprayed with 200 pM quercetin for seven days.

Fig. 22 further illustrates field trials of quercetin on wheat. Wheat plants with limited irrigation were sprayed with quercetin at the grain filling stage. After two weeks, the leaves quercetin became yellow, but those sprayed with 200 pM quercetin spray were still green.

Materials and Methods for Examples 1 to 6 Plant materials and field experiments

The chromosome segment substitution lines (CSSLs) were derived from a cross between SR3 and JN177 (the recurrent parent). Two selected CSSL lines, CSSL15 and CSSL21, each carrying a different spectrum of genomic segments introgressed from the variety SR3. The winter wheat plants grown in the field were the varieties Jimai22 (JM22), Jinnan177 (JN177), Shanrong3 (SR3) and various transgenic derivatives of SR3; the materials were planted at a commercial density at the Experimental Station of the Shijiazhuang Academy of Agriculture and Forestry Sciences (Shijiazhuang, Hebei province). The 1.2 m ^c 10 m plots were arranged as a randomized block design with nine replicates: three plots, designated “0 m³/ha”, were not given any irrigation, three (“750 m³/ha”) were irrigated a single time at the point when the plants had reached the jointing stage (defined as when the internodal tissue begins to elongate, forming a stem), while the final three (“1,500 m³/ha”) were irrigated both at the jointing stage and at anthesis stage. The methods used to quantify plant performance and to determine tissue carbon and nitrogen contents have been described elsewhere (29).

Transgene constructs

The full length FLS1 (TraesCS6A02G331400) cDNA sequence was amplified from total RNA extracted from 2-week-old SR3 seedling and then inserted into pMD18-T (TaKaRa Bio, 6011) for sequencing-based validation. The sequence was introduced into pUbi::nos (30) to generate the construct pUbi::FLS1. A 583 bp fragment of FLS1 cDNA was used to generate the construct pActin::RNAi-FLS1. The transgenic plants were generated by Agrobacterium-medi atedi transformation, using methods described elsewhere (31,32). Relevant primer sequences are given in table S2.

Quantitative real-time PCR (qRT-PCR)

RNA was extracted from 2-week-old plants which had been either well-watered or moisture-stressed (water withheld for 7 or 14 days) using the TRIzol® Reagent (Invitrogen, 15596-018), and treated with RNase-free DNase I (Roche LifeScience, 11284932001) according to the manufacturer’s protocol. The full-length cDNA was reverse-transcribed using M-MLV Reverse Transcriptase (Invitrogen, 28025-013).

The resulting cDNAs were used as a template for qRT-PCRs performed as described elsewhere (29). The wheat Actin gene (TraesCS5B02G124100) was used as the reference gene. Each genotype/treatment combination was represented by three biological replicates. All relevant primer sequences are given in table S3.

Transcriptome analysis

Two-week-old wheat seedlings were raised under well-watered conditions, after which water was withheld for seven days. RNA was extracted from leaves using the TRIzol® Reagent (Invitrogen, 15596-018), from which RNA-seq libraries were constructed, and then high-throughput transcriptome sequencing was undertaken using an lllumina HiSeq XTen sequencer (Biomarker Technologies Corporation, Beijing, China). The differentially expressed genes (DEGs) were assigned to sequences for which the FKPM parameter’s absolute log2 ratio exceeded 2, and the DEGs identified using this criterion were functionally assigned using the Gene Ontology enrichment analysis method (geneontology.org/docs/go-enrichment- analysis/). The negative Iog10 enrichment p values (-logioP) associated with enriched pathways were used to perform a cluster analysis based on Genesis1.8.1 software (33).

Gas exchange measurements

Leaf gas exchange rates were measured using a LI-COR LI-6800 infrared gas analyzer (LI-COR Biosciences, Lincoln, United States). Measurements were taken in the morning between 9:00 a.m. and 11:00 a.m. The CO2 flow rate and leaf temperature were kept constant at, respectively, 1000 mM s ¹ and 25°C.

Measurements of stomatal conductance and transpiration rate were conducted at a CO2 concentration of 400 ppm. After an initial light adaptation of 10 min, the photon flux density of red and blue light (9:1 ratio) was incrementally increased up to 50, 100, 200, 400, 600, 800, 1,200, 1,400, 1,600, 1,800 and 2,000 mol rrr² s ¹.

Stomatal aperture experiments

The wheat leaves were harvested from 3-week-old plants and floated in 20 mM KCI,

1 mM CaCh, 5 mM MES-KOH (pH6.2). Stomatal aperture was measured as described elsewhere (34). The ratio between the percentage of closed stomata in ABA-treated leaves and that of untreated control leaves was used to indicate the sensitivity of stomatal movement to the ABA treatment.

Moisture stress treatments

Surface-sterilized grains (20% w/v sodium hypochlorite for 30 min, followed by a rinse in deionized water) were planted in soil and the emerging plants were raised for two weeks under a constant temperature of 25°C, a 16 h photoperiod and a relative humidity of 60%. Water was then withheld for 14 days. To analyze the effect of providing stressed plants with flavonol treatment, 5 mL of a 0.5 mM solution of either quercetin or kaempferol was sprayed on the leaves of plants which had not been watered for 11 days. At the end of the stress treatment, the plants were watered and their recovery from the stress was monitored. Leaf water loss measurements

Leaves of flavonol treated plants were detached 12 h after spraying, weighted (fresh weight, FW) and laid abaxial side up at room temperature to dessicate, after which they were reweighed. At least 15 leaves per genotype were monitored. The desiccated leaves were then baked for 24 h at 80°C, and weighed (dry weight, DW). The water loss rate parameter was calculated from the expression [(FW-DesW)/(FW- DW)]x 100%.

Determination of quercetin and kaempferol

The method used to quantify wheat leaf flavonol content was adapted from procedures described elsewhere (35). A ~ 0.5 g aliquot of leaf tissue harvested from 3-week-old plants was freeze-dried for 48 h, milled to a powder and extracted in 50% v/v methanol (50 pL per mg leaf tissue). Following a centrifugation (13,000 ^c g, 15 min, 4°C) to remove particulate matter, an equal volume of 2 M HCI was added to the supernatant, and the mixture held at 70°C for 40 min. An equal volume of methanol was then added, the centrifugation step was repeated, and the resulting supernatants were used for flavonol quantification. The content of quercetin and kaempferol was obtained following a separation step using an HPLC-MS device: a 20 pL aliquot of sample was injected into a Diamonsil C18 column (4.6 ^c 150 mm, 5 pm, Dikma), the compounds were eluted by using 5% formic acid in acetonitrile-water (60:40, v/v), supplied at a flow rate of 0.8 mL/min, the eluted compounds were detected spectrophotometrically at 365 nm by the liquid chromatography system (Dionex, UltiMate3000, UHPLC) coupled with an ESI-Q-TOF mass spectrometer (Bruker Daltonics, Impact HD). Based on the peak molecular weight of the positive charged forms of either quercetin or kaempferol, the ratio of the peak area was calculated to obtain the relative contents of quercetin and kaempferol.

DPBA staining

DPBA staining was used to identify the sub-cellular localization of flavonol deposition. The epidermal strips were peeled from 3-week-old plants, incubated for 15 min in aqueous 0.25% w/v DPBA (Sigma-Aldrich, CAS number:524-95-8) containing 0.05% v/v Triton X-100, and subsequently cleared by steeping for 10 min in boiling 96% ethanol. Fluorescent signals were captured by confocal microscopy (Zeiss LSM710), and their intensity quantified by an analysis of the resulting micrographs, using ImageJ software (rsb.info.nih.gov/ij/).

Expression of recombinant proteins

Each of the full-length cDNAs of PYL4 (MG273654), OST1 (TraesCS2A02G303900) and ABI1 (TraesCS3A02G209200) was amplified from 3-week-old SR3 seedlings and then inserted into the pEASY-T1 cloning vector (TransGene Biotech, CB101) for validation by sequencing. The cDNAs of PYL4, ABI1 and OST1 and its NAAIRS (Asp-Ala-Ala-lle-Arg-Ser hexapeptide; 25) variants were sub-cloned into a modified pET28a plasmid using a pEASY-Uni Seamless Cloning and Assembly Kit (TransGene Biotech, CU101-01) and the recombined plasmids inserted into E. coli Transetta (DE3). Once the Oϋboo of the resulting E. coli cultures had reached 0.6, 0.5 mM isopropyl^-d-thiogalactopyranoside (IPTG) was added to induce the expression of the recombinant protein. After an 8 h incubation at 28°C, the cells were disrupted by sonication and the soluble protein fraction assayed by SDS-PAGE. Ni-NTA affinity chromatography (Qiagen, 30210) was used to purify the recombinant proteins. All relevant primer sequences are given in table S2.

Microscale thermophoresis (MST) binding assays

Purified OST1 and its mutant variants were histidine-tagged using a Monolith™ His- Tag Labeling Kit RED-tris-NTA (NanoTemper Technologies, MO-L008) following the manufacturer’s recommended procedure. The labeled proteins were diluted to 0.2 mM in 50 mM NaH₂P0₄/Na2HP0₄ (pH7.5), 0.2 M NaCI, 5% v/v glycerol, 0.01% v/v Tween 20. The concentration of quercetin or kaempferol chosen was in the range 0.3 nM to 10 mM. The mixture of labelled recombinant protein and flavonol was incubated for 5 min in 50 mM Tris-HCI (pH7.4), 0.15 M NaCI, 10 mM MgCI₂, 0.05% v/v Tween-20 before being loaded into a Monolith silica capillary (Monolith NT.115 Standard Treated Capillaries, MO-K002; Monolith™ NT.115 MST Premium Coated Capillaries, MO-K005). The assay output was recorded by a Monolish NT.115 device (Nano Temper Technologies), the recorded data was further analyzed by using MO Affinity Analysis v2.2.4 software.

Two-electrode voltage clamp recordings

Each of the full-length cDNAs of SLAC1 (TraesCS2A02G398000), OST1, ABU and PYL4 was amplified from 3-week-old SR3 seedlings and the amplicons were inserted into a Xenopus laevis oocyte expression vectors (36). The methods used to prepare oocytes, inject cRNAs and to carry out voltage clamp measurements have been described elsewhere (37-39). Data recording analysis were performed by using pCIampIO (Axon Instrument, CA, USA) and Origin 2015 software.

Pull-down assays

Biotin was linked to the 7-OH of the quercetin A ring to create biotin-linked quercetin (Bio-Q) with a short chain between biotin and quercetin (Q-bio; 40), using the services of AbMART (www.ab-mart.com/). Total proteins are extracted from wheat plants pre-treated with or without ABA. A 10 pL aliquot of 5mM Bio-Q in DMSO combined with ~ 2 pg purified His-OST1 was diluted by the addition of 100 pL 20 mM Tris-HCI (pH7.0) containing a protease inhibition cocktail (Roche LifeScience, 11873580001). After holding for 30 min at 25°C, a 20 pL aliquot of streptavidin beads was added and the mixture left at room temperature for 2 h. The beads were rinsed three times in 120 mM Tris-HCI (pH7.2) containing 100 mM NaCI and 0.2% v/v Triton X-100. The beads were suspended in 5 pL 5* SDS loading buffer (250 mM Tris-HCI (pH6.8), 500 mM DTT, 10% SDS, 0.5% bromophenol blue, 50% glycerol) and boiled for 10 min, the supernatant was subsequently separated by SDS-PAGE and the fragments transferred to an lmmobilon®-P Transfer Membrane (Millipore,

IPVH00010). Proteins were detected by immunoblot using anti-OST1 antibodies. All relevant primer sequences are given in table S2.

In vitro protein kinase activity assays

The cDNAs of OST1, ABU and SLAC1^NT (encoding the N terminal domain of SLAC1, 1-200 amino acids) were amplified from 3-week-old SR3 seedlings and then inserted into the pdonor vector (Invitrogen, 12536017) for validation by sequencing. Then, the donors were sub-cloned into a modified pGEX4T-1 plasmid and the recombined plasmids inserted into E. coli Transetta (DE3). Once the OD600 of the resulting E. coli cultures had reached 0.6, 0.5 mM isopropyl^-d-thiogalactopyranoside (IPTG) was added to induce the expression of the recombinant protein. After an 8 h incubation at 28°C, the cells were disrupted by sonication and the soluble protein fraction assayed by SDS-PAGE. Glutathione Sepharose 4B (GE Healthcare, 17075605) was used to purify the GST recombinant proteins. A 9.5 pl_ solution of 0.2 g/L purified recombinant OST1 or ABI1 protein in 20 mM Tris-HCI (pH7.5), 75 mM NaCI, 1 mM DTT, 10 mM MgCI₂, 100 mM ATP, 0.4 m Ci [y-³²P] ATP (Perkin-Elmer, NEG502A) was combined with 0.5 mI_ 100 mM quercetin (or kaempferol). The reactions were held at 30°C for 60 minutes. The reactions were terminated by the addition of 5* SDS loading buffer and boiled at 95°C for 5 minutes. The reaction products were separated by SDS-PAGE and stained with Coomassie Brilliant Blue R- 250 (Sigma-Aldrich, 6104-59-2). Radioactively labeled products were visualized by exposing the gels to a storage phosphor screen (GE Healthcare, 28-9564-75), and the data were captured using a Typhoon scanner FLA 9500 (GE Healthcare, Bio- Sciences AB, Uppsala, Sweden).

Statistical analysis

Data are presented as means ± s.e.m. The number used in each experiment is indicated in the figure legends. Statistical analysis was performed using either Student’s f-test or Duncan’s multiple range test as described elsewhere (29).

Statistical significance was called when the P-values lay below 0.05.

Supplementary T able 1. The agronomic performance of the set of investigated wheat varieties grown under either well-watered or moisture- deficient conditions

pplementary Table 2. The primer sequences used for transgene constructs.

Supplementary Table 3. The primer sequences used for qRT-PCR assays.

REFERENCES AND NOTES

1, S. Y Park, F. C. Peterson, A. Mosquna, J. Yao, B. F. Volkman, S. R. Cutler, Agrochemical control of plant water use using engineered abscisic acid receptors. Nature 520, 545-548 (2015).

2, R. Mega, etai, Tuning water-use efficiency and drought tolerance in wheat using abscisic acid receptors. Nat Plants 5, 153-159 (2019).

3, A. M. Hetherington, F. I. Woodward, The role of stomata in sensing and driving environmental change. Nature 424, 901-908 (2003).

4, S. Jasechko, Z. D. Sharp, J. J. Gibson, S. J. Birks, Y. Yi, P. J. Fawcett, Terrestrial water fluxes dominated by transpiration. Nature 496, 347-350 (2013).

5, M. Papanatsiou, J. Petersen, L. Henderson, Y. Wang, J. M. Christie, M. R. Blatt, Optogenetic manipulation of stomatal kinetics improves carbon assimilation, water use, and growth. Science 363, 1456-1459 (2019).

6, Y. Ma, etai, Regulators of PP2C phosphatase activity function as abscisic acid sensors. Science 324, 1064-1068 (2009).

7, S. Y. Park, etai., Abscisic acid inhibits type 2C protein phosphatases via the PYR/PYL family of START proteins. Science 324, 1068-1071 (2009).

8, T. H. Kim, M. Bohmer, H. Hu, N. Nishimura, J. I. Schroeder, Guard cell signal transduction network: advances in understanding abscisic acid, CO2, and Ca²⁺ signaling. Annu. Rev. Plant Biol. 61, 561-591 (2010).

9, Y. N. Chang, C. Zhu, J. Jiang, H. Zhang, J. K. Zhu, C. G. Duan. Epigenetic regulation in plant abiotic stress responses. J Integr Plant Biol doi: 10.1111/jipb.12899 (2019).

10, Z. Yang, et ai, Abscisic acid receptors and coreceptors modulate plant water use efficiency and water productivity. Plant Physiol. 180, 1066-1080 (2019).

11, A. C. Mustilli, S. Merlot, A. Vavasseur, F. Fenzi, J. Giraudat, Arabidopsis OST1 protein kinase mediates the regulation of stomatal aperture by abscisic acid and acts upstream of reactive oxygen species production. Plant Cell 14, 3089-3099 (2002).

12, H. Fujii, etai, In vitro reconstitution of an abscisic acid signalling pathway. Nature 462, 660-664 (2009).

13, E. Pennisi, The blue revolution, drop by drop, gene by gene. Science 320, 171-173 (2008). , N. D. Mueller, J. S. Gerber, M. Johnston, D. K. Ray, N. Ramankutty, J. A. Foley, Closing yield gaps through nutrient and water management. Nature 490, 254-257 (2012). , A. S. Vaidya, et ai, Dynamic control of plant water use using designed ABA receptor agonists. Science 366, eaaw8848 (2019). , S. Liu, et al., A wheat SIMILAR TO RCD-ONE gene enhances seedling growth and abiotic stress resistance by modulating redox homeostasis and maintaining genomic integrity. Plant Cell 26, 164-180 (2014). , B. Winkel-Shirley, Flavonoid biosynthesis: a colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiol. 126, 485-493 (2001). , E. Grotewold, The genetics and biochemistry of floral pigments. Annu Rev Plant Biol. 57, 761-780 (2006). , J. M. Watkins, R J. Hechler, G. K. Mudav, Ethylene-induced flavonol accumulation in guard cells suppresses reactive oxygen species and moderates stomatal aperture. Plant Physiol. 164, 1707-1717 (2014). , J. M. Watkins, J. M. Chapman, G. K. Muday, Abscisic acid-induced reactive oxygen species are modulated by flavonols to control stomata aperture. Plant Physiol. 175, 1807-1825 (2017). , K. Miyazono, etai, Structural basis of abscisic acid signalling. Nature 462, 609-614 (2009). , S. R. Cutler, P. L. Rodriguez, R. R. Finkelstein, S. R. Abrams, Abscisic acid: emergence of a core signaling network. Annu. Rev. Plant Biol. 61, 651-679 (2010)., B. Brandt, etai, Reconstitution of abscisic acid activation of SLAC1 anion channel by CPK6 and OST1 kinases and branched ABI1 PP2C phosphatase action. Proc. Natl Acad. Sci. USA 109, 10593-10598 (2012). , C. Yunta, M. Martinez-Ripoll, J. K. Zhu, A. Albert, The structure of Arabidopsis thaliana OST1 provides insights into the kinase regulation mechanism in response to osmotic stress. J Mol Biol. 414, 135-144 (2011). , J. Leung, M. Bouvier-Durand, P C. Morris, D. Guerrier, F. Chefdor, J. Giraudat, Arabidopsis ABA response gene ABI1: features of a calcium-modulated protein phosphatase. Science 264, 1448-1452 (1994). , R. A. Mosher, W. E. Durrant, D. Wang, J. Song, X. Dong, A comprehensive structure- function analysis of Arabidopsis SNI1 defines essential regions and transcriptional repressor activity. Plant Cell 18, 1750-1765 (2006). , B. Vilela, E. Najar, V. Lumbreras, J. Leung, M. Pages, Casein Kinase 2 negatively regulates abscisic acid-activated SnRK2s in the core abscisic acid-signaling module. Mol Plant 8, 709-721 (2015). , S. Sun, et ai, Protein kinase OsSAPK8 functions as an essential activator of S-type anion channel OsSLACI, which is nitrate-selective in rice. Planta 243, 489-500 (2016)., S. Li, etai, Modulating plant growth-metabolism coordination for sustainable agriculture. Nature 560, 595-600 (2018). , H. Sun, etai, Heterotrim eric G proteins regulate nitrogen-use efficiency in rice. Nat Genet. 46, 652-656 (2014). , T. Zhao, et ai, Transgenic wheat progeny resistant to powdery mildew generated by Agrobacterium inoculum to the basal portion of wheat seedling. Plant Cell Rep. 25, 1199-1204 (2006). , X. Huang, etai, Natural variation in the DEP1 locus enhances grain yield in rice. Nat. Genet. 41, 494-497 (2009). , A. Sturn, etai, Genesis: Cluster analysis of microarray data. Bioinformatics. 18, 207- 208 (2002). , Z. M. Pei, K. Kuchitsu, J. M. Ward, M. Schwarz, J. I. Schroeder, Differential abscisic acid regulation of guard cell slow anion channels in Arabidopsis wild-type and abi1 and abi2 mutants. Plant Cell 9, 409-423 (1997). , D. K. Owens, et ai, Functional analysis of a predicted flavonol synthase gene family in Arabidopsis. Plant Physiol. 147, 1046-1061 (2008). , H. H. Nour-Eldin, B. G. Hansen, M. H. Norholm, J. K. Jensen, B. A. Halkier,

Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments. Nucleic Acids Res 34, e122 (2006). , B. Brandt, etai, Reconstitution of abscisic acid activation of SLAC1 anion channel by CPK6 and OST 1 kinases and branched ABI1 PP2C phosphatase action. Proc Natl Acad Sci USA 109, 10593-10598 (2012). , S. Sun, et ai, Protein kinase OsSAPK8 functions as an essential activator of S-type anion channel OsSLACI, which is nitrate-selective in rice. Planta 243, 489-500 (2016). , Y. Pan, etai, Dynamic interactions of plant CNGC subunits and calmodulins drive oscillatory Ca²⁺ channel activities. Dev Ce/I 48, 710-725 e715 (2019). , R. E. Wang, etai, Biotinylated quercetin as an intrinsic photoaffinity proteomics probe for the identification of quercetin target proteins. Bioorg Med Chem. 19, 4710-4720 Ĩ2011).

Claims

Claims

1 Use of quercetin or derivative thereof to reduce the water requirement or enhance water use efficiency (WUE) of a plant.

2 Use of quercetin or derivative thereof to promote stomatal closing in a plant, thereby reducing the rate of transpiration and water loss from the plant.

3 A method for increasing tolerance to water scarcity and/or for reducing the consequence of water scarcity in a plant, comprising contacting the plant with quercetin or derivative thereof.

4 The use or method as claimed in any one of claims 1 to 3 wherein the quercetin derivative is a quercetin glycoside, more preferably a quercetin 3-O-glycoside, more preferably quercetin 3-O-glucoside

5 The use or method as claimed in any one of claims 1 to 4 wherein the quercetin or derivative thereof treatment does not modify the carbon-to-nitrogen ratio of the plant.

6 The use or method as claimed in any one of claims 1 to 5 wherein the plant is crop plant, optionally being cultivated in an open air environment.

7 The use or method as claimed in any one of claims 1 to 6 wherein the plant is moisture deficient or water limited when the quercetin or derivative thereof is used or contacted with the plant.

8 The use or method as claimed in any one of claims 1 to 7 wherein the plant is being grown in an irrigated environment with an Irrigation Water Productivity (IWP) for the plant of < 5 kg/m-3.

9 The use or method as claimed in any one of claims 1 to 8 wherein the plant is being grown in an environment with a Water-Scarcity Footprint (WSF) of <0.05 m3 FhO_e kg-1

10 The use or method as claimed in any one of claims 1 to 9 wherein the quercetin or derivative thereof is used or contacted with the plant by spraying, foaming, fogging or misting, pouring, brushing, dipping, dusting, sprinkling, scattering, atomizing, broadcasting, or soaking, most preferably by spraying. 11 The use or method as claimed in claim 10 wherein the quercetin or derivative thereof is used or contacted with the leaves of the plant.

12 The use or method as claimed in any one of claims 1 to 11 wherein the quercetin or derivative thereof is used or contacted with the plant by spraying between 50 and 500 ml_/m²of a composition comprising quercetin.

13 The use or method as claimed in any one of claims 1 to 12 wherein the quercetin or derivative thereof is used or contacted with the plant as a composition consisting essentially of quercetin or derivative thereof.

14 The use or method as claimed in any one of claims 1 to 13 wherein the quercetin or derivative thereof is used or contacted with the plant as a composition comprising quercetin or derivative thereof, which is optionally part of a tank spray and/or which is optionally a controlled release or slow release composition.

15 The use or method as claimed in claim 14 wherein the composition further comprise(s) an agriculturally acceptable carrier, support, filler, dispersant, emulsifier, wetter, adjuvant, solubilizer, colorant, tackifier, binder, anti-foaming agent, slow release particle or gel and/or surfactant.

16 The use or method as claimed in claim 14 or claim 15 wherein the composition does not include a microorganism and/or a germinant.

17 The use or method as claimed in any one of claims 14 to 16 wherein the composition does not include a dicarboxylic acid and/or a salt thereof.

18 The use or method as claimed in any one of claims 14 to 17 wherein the composition does not include a cyclopropane.

19 The use or method as claimed in any one of claims 13 to 18 claim wherein the composition contains 0.1 to 1 mM, more preferably about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 mM, quercetin or derivative thereof and is optionally an aqueous composition.

20 The use or method as claimed in any one of claims 1 to 19 wherein the plant is selected from the list consisting of: apple, tomato, cherry, pear, pepper, cucumber, honeydew melon, watermelon, cantaloupe, papaya, mango, pineapple, avocado, plum, bean, squash, peach, apricot, grape, strawberry, raspberry, blueberry, mango, cranberry, gooseberry, banana, fig, clementine, kumquat, orange, grapefruit, tangerine, lemon, lime, hazelnut, pistachio, walnut, macadamia, almond, pecan, Litchi, soybeans, corn, sugar cane, camelina, peanut, cotton, canola, alfalfa, timothy, tobacco, tomato, sugarbeet, potato, pea, carrot, wheat, rice, barley, oats, rye, triticale, turf, lettuce.

21 The use or method as claimed in any one of claims 1 to 20 wherein the plant is a selected from the list consisting of wheat, tomato, rice, tobacco, barley, oats, rye, triticale.

22 The use or method as claimed in claim 21 wherein the plant is a selected from the list consisting of wheat, tomato, rice, tobacco.

23 The use or method as claimed in claim 22 wherein the plant is wheat.

24 The use or method as claimed in any one of claims 1 to 23 wherein the quercetin or derivative thereof improves yield by at least 3, 4, 5, 6, 7, 8, 9, or 10%.

25 The use or method as claimed in claim 24 wherein the yield is grain yield

26 The use or method as claimed in any one of claims 1 to 25 wherein the quercetin or derivative thereof increases WUE(grain) (kg ha-1 mm-1) by at least 5%, 10%, or 20%

27 A composition comprising quercetin or a derivative thereof as defined in any one of claims 13 to 19, for use in a method as described in any one of claims 3 to 12.