WO2016113727A2 - Use of a superabsorbent polymer for improving plant health by changing the gene expression in a plant - Google Patents

Use of a superabsorbent polymer for improving plant health by changing the gene expression in a plant Download PDF

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Publication number
WO2016113727A2
WO2016113727A2 PCT/IB2016/053466 IB2016053466W WO2016113727A2 WO 2016113727 A2 WO2016113727 A2 WO 2016113727A2 IB 2016053466 W IB2016053466 W IB 2016053466W WO 2016113727 A2 WO2016113727 A2 WO 2016113727A2
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WO
WIPO (PCT)
Prior art keywords
plant
protein
improved
superabsorbent polymer
increased
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PCT/IB2016/053466
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French (fr)
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WO2016113727A3 (en
Inventor
Jorge SANZ-GOMEZ
Harald Köhle
Alexander Wissemeier
Pilar Puente
Michael Seufert
Markus Schmid
Alban Glaser
Stephan Saum
Angel Rodriguez
Ramon NAVARRA-MESTRE
Ana ALONSO RAMÍREZ
Nicolas Rodriguez CARLOS
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Basf Se
Basf (China) Company Limited
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Priority to PCT/IB2016/053466 priority Critical patent/WO2016113727A2/en
Publication of WO2016113727A2 publication Critical patent/WO2016113727A2/en
Publication of WO2016113727A3 publication Critical patent/WO2016113727A3/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H3/00—Processes for modifying phenotypes, e.g. symbiosis with bacteria
    • A01H3/04—Processes for modifying phenotypes, e.g. symbiosis with bacteria by treatment with chemicals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/02—Saturated carboxylic acids or thio analogues thereof; Derivatives thereof
    • A01N37/04—Saturated carboxylic acids or thio analogues thereof; Derivatives thereof polybasic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
    • A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
    • A01N43/14—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings
    • A01N43/16—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings with oxygen as the ring hetero atom

Definitions

  • the present invention relates to the treatment of agricultural plants with polymers in order to improve plant performance.
  • WO 2007/104720 A1 describes the use of superabsorbent polymers in the form of water-absor- bent granular material which is used as carrier for pesticide compositions suitable for combating arthropod pests, snails and nematodes in agricultural production of plants and products derived therefrom.
  • WO 2010/037630 A1 discloses the treatment of plants with superabsorbers in order to promote the aboveground root growth of plants. It is described that such effect is achieved by the addition of superabsorbent polymer to the growing medium. It is further suggested that such process is particularly useful for cultivating plants under arid or semi-arid conditions or comparable water-deficient environments.
  • WO 2013/060848 A1 discloses the preparation of water-swellable polymers that are suitable for absorbing and storing aqueous fluids. The polymers are suggested for improving soil quality, the preparation of agricultural soil and the re-cultivation of wasteland. It is further described that these water-swellable polymers can be used for improving the water storage ability of culture soil thereby improving the quality of agricultural soil.
  • superabsorbent polymers can be used in the cultivation of plants for changing gene expression including activating, in- creasing, inhibiting or reducing the activity of selected plant genes known for their high physiological relevance.
  • the first specific finding of the present invention is the use of a superabsorbent polymer for improving plant health by changing the gene expression in a plant, characterized in that the plant is treated with the superabsorbent polymer, the plant treated with the superabsorbent polymer is cultivated, and at least one gene of the plant is up-regulated by the addition of the superabsorbent polymer, and/or at least one gene is down-regulated by the addition of the superabsorbent polymer, wherein improved plant health is determined by increased yield of plant product, improved plant vigor, enhanced quality of the plant and/or improved tolerance or resistance of the plant to abiotic and/or biotic stress factors.
  • the increased yield of a plant product is determined by grains, fruits, vegetables, nuts, grains, seeds, wood and/or flowers and combinations thereof.
  • the improved plant vigor is determined by improved vitality of the plant, improved plant growth, improved plant development, im- proved visual appearance, improved plant stand, less plant verse/lodging, improved emergence, enhanced root growth and/or more developed root system, enhanced nodulation, in particular rhizobial nodulation, bigger leaf blade, bigger size, increased plant weight, increased plant height, increased tiller number, increased number of side shoots, increased number of flowers per plant, increased shoot growth, increased root growth like extensive root system, in- creased yield when grown on poor soils or unfavorable climate, enhanced photosynthetic activity, preferably based on increased stomatal conductance and/or increased CO2 assimilation rate, increased stomatal conductance, increased C0 2 assimilation rate, enhanced pigment content like chlorophyll content, flowering, earlier fruiting, earlier and improved germination, earlier grain maturity, improved self-defense mechanisms, less non-productive tillers, less dead basal leaves, less input needed such as fertilizers or
  • the enhanced quality of the plant is determined by increased nutrient content, increased protein content, increased content of fatty acids, increased metabolite content, increased carotenoid content, increased sugar content, increased content of amino acids, including essential amino acids, improved nutrient composition, improved protein composition, improved composition of fatty acids, improved metabolite composition, improved carotenoid composition, improved sugar composition, improved amino acids composition, improved or optimal fruit color, improved leaf color, higher storage capacity, and/or higher processability of the harvested products and combinations thereof.
  • the improved tolerance or resistance of the plant to abiotic stress factors is determined by tolerance and/or resistance to heat stress including temperatures higher than 30°C, temperature conditions causing heat damage to a plant like heat damaged foliage or burnt leaves, cold stress like temperature conditions below 10°C, periods of thawing and freezing, frost, variations in temperature like temperatures conditions that lead to the freezing of water either for extended periods of time or only temporary periods, temperature unusual for the season, drought stress, exposure to cold water, flood, water- logging, wind, sun light, particularly sun light causing signs of scorch, sun burn or similar signs of irradiation and heat stress to the plant, acid or alkaline pH conditions in the soil with pH values lower than pH 5 and/or pH values higher than 9, salt stress like soil salinity, soil erosion, inorganic pollution, soil contamination or soil pollution with chemicals, particularly with heavy metals, preferably chromium, lead, cadmium, arsenic, antimony, mercury, iron, thallium, barium, be- ryll
  • the improved tolerance or resistance of the plant to biotic stress factors is determined by tolerance and/or resistance to the attack of living organisms including pests like insects, arachnides, nematodes, competing plants like weeds, microorganisms like fungi, bacteria and/or viruses and combinations thereof.
  • the up-regulated plant gene is selected from pathogenesis related protein P4, xyloglucan-specific fungal endoglucanase inhibitor protein, sucrose synthase, beta-1 ,2-glucanase, chitinase, serine acetyltransferase and combi- nations thereof.
  • the down-regulated plant gene is selected from Jerusalem 1 , lipoxygenase, verticillium wilt disease resistance protein Ve2, AVr9/CF-9 rapidly elicited protein 1 , ripening regulated protein DDTFR10/A, salt responsive protein 2 and combinations thereof.
  • the plant is an agricultural plant like wheat, rye, barley, triticale, oats, sorghum or rice, beet, sugar beet or fodder beet, fruits like pomes, apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries, blackberries or gooseberries, leguminous plants, such as lentils, peas, alfalfa or soybeans, oil plants, such as rape, oil-seed rape, canola, juncea, linseed, mustard, olives, sunflowers, coconut, cocoa beans, castor oil plants, oil palms, ground nuts or soybeans, cucurbits, such as squashes, cucumber or melons, fiber plants, such as cotton, flax, hemp or jute, citrus fruit, such as oranges, lemons, grapefruits or mandarins, vegetables, such as spinach, lettuce, asparagus, cabbages, carrots, onions, tomatoes, potatoes, cucurbits or pap
  • the superabsorbent polymer is selected from polyacrylic acid and cellulose-modified polyacrylic acid or combinations thereof.
  • the superabsorbent polymer is in the form of granules, having average particle size in the range of 0.1 to 5 mm, preferably from 0.2 to 4 mm.
  • the second specific finding of the present invention is a process for improving the plant health by changing the gene expression in a plant comprising the steps of treating the plant with a superabsorbent polymer, cultivating the plant treated with the superabsorbent polymer, and up- regulating at least one gene of the plant by the addition of the superabsorbent polymer, and/or down-regulating at least one gene of the plant by the addition of the superabsorbent polymer, wherein improved plant health is determined by increased yield of plant product, improved plant vigor, enhanced quality of the plant and/or improved tolerance or resistance of the plant to abiotic and/or biotic stress factors.
  • the third specific finding of the present invention is a process of determining the change in gene expression in a plant upon treatment of the plant with a superabsorbent polymer, comprising the steps of treating the plant with a superabsorbent polymer, cultivating the plant treated with a superabsorbent polymer, and measuring the up-regulation and/or down-regulation of at least one gene of the plant during and/or after cultivation of the plant.
  • measuring the up-regulation and/or down-regulation of at least one gene of the plant is carried out by use of a transcriptomic assay, preferably by use of a microarray.
  • the present invention is defined by the surprising use of superabsorbent polymers in the cultivation of plants allowing improving the plant's health by systematically changing the gene expression of the plant, wherein the plant health can be for instance determined by increased yield of plant product, improved plant vigor, enhanced quality of the plant and/or improved tolerance or resistance of the plant to abiotic and/or biotic stress factors.
  • An increase in yield of a plant product can be determined with respect to grains, fruits, vegetables, nuts, grains, seeds, wood and/or flowers and combinations thereof. That is, the amount of plant product obtained after practice of the present invention relative to the amount of plant product obtained when the plant is not treated according to the present invention must be determined based on the above plant products.
  • An improvement in plant vigor can be determined with respect to improved vitality of the plant, improved plant growth, improved plant development, improved visual appearance, improved plant stand, less plant verse/lodging, improved emergence, enhanced root growth and/or more developed root system, enhanced nodulation, in particular rhizobial nodulation, bigger leaf blade, bigger size, increased plant weight, increased plant height, increased tiller number, increased number of side shoots, increased number of flowers per plant, increased shoot growth, increased root growth like extensive root system, increased yield when grown on poor soils or unfavorable climate, enhanced photosynthetic activity, preferably based on increased stomatal conductance and/or increased CO2 assimilation rate, increased stomatal conductance, increased CO2 assimilation rate, enhanced pigment content like chlorophyll content, flowering, earlier fruiting, earlier and improved germination, earlier grain maturity, improved self-defense response, less non-productive tillers, less dead basal leaves, less input needed such as fertiliz- ers or water, greener leaves, complete maturation
  • An enhancement in quality of the plant can be determined by increased nutrient content, increased protein content, increased content of fatty acids, increased metabolite content, increased carotenoid content, increased sugar content, increased content of amino acids, includ- ing essential amino acids, improved nutrient composition, improved protein composition, improved composition of fatty acids, improved metabolite composition, improved carotenoid composition, improved sugar composition, improved amino acids composition, improved or optimal fruit color, improved leaf color, higher storage capacity, and/or higher processability of the harvested products and combinations thereof.
  • An improvement in tolerance or resistance of the plant to abiotic stress factors can be determined by heat stress including temperatures higher than 30°C, temperature conditions causing heat damage to a plant like heat damaged foliage or burnt leaves, cold stress like temperature conditions below 10°C, periods of thawing and freezing, frost, variations in temperature like temperatures conditions that lead to the freezing of water either for extended periods of time or only temporary periods, temperature unusual for the season, drought stress, exposure to cold water, flood, water-logging, wind, sun light, particularly sun light causing signs of scorch, sun burn or similar signs of irradiation and heat stress to the plant, acid or alkaline pH conditions in the soil with pH values lower than pH 5 and/or pH values higher than 9, salt stress like soil salinity, soil erosion, inorganic pollution, soil contamination or soil pollution with chemicals, particularly with heavy metals, preferably chromium, lead, cadmium, arsenic, antimony, mercury, iron, thallium, barium, beryllium, polonium, uranium, toxic waste,
  • An improvement in tolerance or resistance of the plant to biotic stress factors can be determined by the attack of living organisms including pests like insects, arachnides, nematodes, competing plants like weeds, microorganisms like fungi, bacteria and/or viruses and combinations thereof.
  • the plant's overall health is generally regulated, altered, re-stored and defended by the plant's cellular metabolism which includes the plant's physiological response on the molecular level. Metabolic responses in the plant also include the activation, the increase, the inhibition or decrease of enzymatic activities. Such enzymatic activities are very complex. Clearly, such mechanisms of altered plant metabolism will be largely effected and regulated by changes in the gene expression in the plant.
  • the plant's ability to respond by selective gene activity to defend, alter, improve or restore its health represents an attractive goal in the agricultural industry for improved cultivation of plants, particularly in view of maximizing harvest and crop production as well as the yield of any other product obtainable from the plant.
  • the present invention refers to such changes in gene expression with relevance to the plant's health.
  • the present inventors were able to demonstrate that the treatment and/or cultivation of plants in the presence of superabsorbent polymers allows to selectively regulate, alter, re-store and defend plant's health because it is possible to alter, i.e. up-regulate and/or down- regulate plant genes with relevance for the plant's health by the treatment and/or cultivation of plants with superabsorbent polymers. Accordingly, the present invention effectively provides a cultivation approach for plants with superabsorbent polymers that allows artificially influencing the plant's health.
  • the increase of gene expression i.e. a gene which is already actively expressed at a certain level in the absence of a superabsorbent polymer shows an increase in gene expression upon treatment and/or cultivation with superabsorbent polymer
  • the levels of gene expression of each gene can be activated, increased, inhibited or decreased due to the treatment of the plant with the superabsorbent polymer.
  • Activated or increased levels of gene expression are preferably measured by an up-regulation of a gene of the plant on the level of transcription while inhibited or decreased levels of gene expression are preferably measured by a down-regulation of a gene of the plant on the level of transcription.
  • the up-regulation of a gene on the transcriptional level as defined by the present invention defines any increase of the level of transcription of DNA to RNA of a particular DNA segment, which is preferably a segment encoding for a gene.
  • the down-regulation of a gene on the tran- scriptional level as defined by the present invention defines any decrease of the level of transcription of DNA to RNA of a particular DNA segment, which is preferably a segment encoding for a gene.
  • Up-regulation or down-regulation of gene expression in a certain plant treated with superabsor- bent polymer is defined by any increase or decrease in gene expression relative to the level of gene expression in the same plant when being not treated with superabsorbent polymer under otherwise identical growth conditions.
  • any gene in a given plant can be up-regulated and/or down-regulated with respect to its level of transcription. Therefore, the present invention includes any gene that is up-regulated or down-regulated to a measurable extent upon treatment and/or cultivation of the plant with the superabsorbent polymer.
  • the genes identified in the present invention are for instance classified and defined in the present invention following the system of the Gene Ontology (GO) project which is a well-accepted collaborative effort in this technical field to address the need for consistent descriptions of gene products in different databases.
  • the GO Consortium has meanwhile grown to include many da- tabases, including several of the world's major repositories for plant, animal and microbial genomes. The GO project can be assessed by the following link http://www.geneontology.org/.
  • the GO project is commonly used in the art based on its three systematic vocabularies (ontologies) to unambiguously define gene products in terms of their associated biological processes, cellular components and molecular functions in a species-independent manner.
  • the Gene Ontology project is herein used as an ontology of defined terms representing gene product properties including three domains: cellular component, the parts of a cell or its extracellular environment; molecular function, the elemental activities of a gene product at the molecular level, such as binding or catalysis; and biological process, operations or sets of molecular events with a defined beginning and end, pertinent to the functioning of integrated living units: cells, tissues, organs, and organisms.
  • gene families as defined below according to the present invention rely on the classification of gene families as defined by the Gene Ontology project and as broadly ac- cepted in the present field.
  • the present invention refers to the following first list of genes which are up-regulated in response to treatment and/or cultivation with super- absorbent polymers, preferably by treatment and/or cultivation with a superabsorbent polymer comprising polyacrylic acid:
  • RNA-dependent DNA replication GO:0006278
  • bHLH transcriptional factor Mo- lus x domestica
  • ICE1 INDUCER OF CBF EXPRESSION 1
  • DNA binding / transcription activator/ transcription factor 3*E-89 sinomatal lineage progression
  • GO:0010440 positive regulation of transcription GO:0045941 , guard mother cell differentiation GO:0010444, response to cold GO:0009409, multicellular organismal development GO:0007275, response to stress GO:0006950, response to freezing GO:0050826);
  • P osp oenolpyruvate carboxylase kinase (LOC543633) (signal transduction GO:0007165, protein amino acid phosphorylation GO:0006468); * Ribonuclease (male) (response to wounding GO:000961 1 , jasmonic acid mediated signaling pathway GO:0009867, response to stress GO:0006950, anthocyanin biosynthetic process GO:0009718); Aspartic proteinase nepenthe- sin-1 precursor, putative (Ricinus communis) (proteolysis GO:0006508); Polyphenol oxidase E, chloroplastic; (PPO); Catechol oxidase (metabolic process GO:0008152, oxidation reduction GO:00551 14); Endo-1 ,4-betaglucanase precursor (celluloselase) (ripening GO:0009835, metabolic process GO:0008152
  • RNA processing GO:0007017 Transducin family protein / WD-40 repeat family protein (Arabidopsis thaliana) (rRNA processing GO:0006364); Tobacco fibrillarin homolog (Nicotiana tabacum)6*E-31 (RNA processing GO:0006396, RNA methylation GO:0001510, snoRNA metabolic process
  • MAR-binding protein [Nicotiana tabacum] (ribosome biogenesis GO:0042254, cell growth GO:0016049, rRNA processing, GO:0006364, snRN P protein import into nucleus GO:0006608); EF 1-alpha (AA 1 -448) (LOC544055) (translation GO:0006412, translational elongation GO:0006414); 6-deoxocastasterone oxidase (Dwarf) (steroid biosynthetic process GO:0006694, lipid biosynthetic process GO:0008610, brassinosteroid biosynthetic process GO:0016132, oxidation reduction GO:00551 14); Wound-induced proteinase inhibitor 2 * E-17 (response to wounding GO:000961 1 ); Fasciclin-like arabinogalactan protein 13 (Gossypium hirsu-
  • the present invention further refers to the following second list of genes which are up-regulated in response to treatment and/or cultivation with superabsorbent polymers, preferably by treatment and/or cultivation with a superabsorbent polymer comprising lignocellulose-modified polyacrylic acid:
  • Pathogenesis-related protein P4 (defense response to fungus GO:0050832, xenobiotic metabolic process GO:0006805, response to biotic stimulus GO:0009607); Xyloglucan-specific fungal endoglucanase inhibitor protein precursor (Lycopersicon esculentum) (ACI25 /// Xegip) (proteolysis GO:0006508); PR protein (PR1 b1 ) (defense response to fungus GO:0050832, xe- nobiotic metabolic process GO:0006805, response to biotic stimulus GO:0009607); Pathogenesis-related protein P2 (PR-P2) (defense response to fungus GO:0050832, chitin catabolic process GO:0006032, cell wall catabolic process GO:0016998, defense response to bacterium GO:0042742, response to biotic stimulus GO:0009607); Putative copia-like polyprotein (Lyco- persicon
  • the present invention further refers to the fol- lowing third list of genes which are up-regulated in response to treatment and/or cultivation with superabsorbent polymers, preferably by treatment and/or cultivation with a superabsorbent polymer comprising polyacrylic acid and lignocellulose-modified polyacrylic acid:
  • Beta-1 ,3-glucanase (LOC543987) (defense response GO:0006952, carbohydrate metabolic process GO:0005975); NP24 protein precursor (LOC543979) (defense response to fungus GO:0050832, response to stress GO:0006950, response to biotic stimulus GO:0009607);
  • Chitinase (LOC544148) (defense response GO:0006952, polysaccharide catabolic process GO:0000272, carbohydrate metabolic process GO:0005975, response to biotic stimulus GO:0009607); Pathogenesis-related protein PR (PR23) (pr p23) (defense response to fungus GO:0050832, response to stress GO:0006950, xenobiotic metabolic process GO:0006805, response to biotic stimulus GO:0009607); Psi14B protein (psi14B) (metabolic process
  • the present invention further refers to the following fourth list of genes which are down-regulated in response to treatment and/or cultivation with superabsorbent polymers, preferably by treatment and/or cultivation with a superabsorbent polymer comprising polyacrylic acid:
  • Hexose transporter protein (LOC543728) (Transmembrane transport GO:0055085);
  • Glutamate dehydrogenase (legdhl ) (cellular amino acid metabolic process GO:0006520); ADP- glucose pyrophosphorylase large subunit (AGP-S1) (glycogen biosynthetic process
  • GO:0008152 response to stress GO:0006950, phospholipid biosynthetic process GO:0008654, post-embryonic root development GO:0048528, phosphatidylcholine biosynthetic process GO:0006656, lipid biosynthetic process GO:0008610); Late embryogenesis (Lea)-like protein (LOC544157) (response to desiccation GO:0009269); Dihydroflavonol-reductase(LOC544150) (metabolic process GO:0008152, flavonoid biosynthetic process, cellular metabolic process GO:0044237, oxidation reduction GO:00551 14); Jasmonic acid 3 (LEJA3) (regulation of transcription GO:0045449); 20G-Fe(ll) oxidoreductase (Populus trichocarpa) (short-day photoperi- odism, flowering GO:004857
  • GO:0030308 protein folding GO:0006457, G2 phase of mitotic cell cycle GO:0000085, DNA replication GO:0006260, regulation of transcription GO:0045449); Cell Division Protein AAA ATPase family, putative, expressed (Oryza sativa (japonica cultivargroup) (mitochondrial respiratory chain complex IV assembly GO:0033617, mitochondrial respiratory chain complex I assembly GO:0032981 , mitochondrial cytochrome bc(1 ) complex assembly GO:0034551 , cell division GO:0051301 , sensory perception of sound GO:0007605, mitochondrion organization GO:0007005); Transporter-like protein (Solanum tuberosum) (response to drug GO:0042493, sterol biosynthetic process GO:0016126, phospholipid transport GO:0015914); Putative allan- toinase (Solanum tuberosum) (purine base metabolic process
  • the present invention further refers to the fol- lowing fifth list of genes which are down-regulated in response to treatment and/or cultivation with superabsorbent polymers, preferably by treatment and/or cultivation with a superabsorbent polymer comprising lignocellulose-modified polyacrylic acid:
  • Photoperiod responsive protein Solanum tuberosum subsp. Andigena AT3G 18710.1 PUB29 (PLANT U-BOX 29) (ubiquitin-protein ligase response to chitin GO:0010200, protein ubiquitina- tion GO:0016567, modification-dependent protein catabolic process GO:0019941 ); Avr9/Cf-9 rapidly elicited protein 231 (Nicotiana tabacum) AT3G28340.1 , GATL10 Galacturonosyltransfer- ase-like 10), polygalacturonate 4-alphagalacturonosyltransferase/ transferase (transferring hex- osyl groups, transferase activity GO:0016757);
  • the present invention further refers to the fol- lowing sixth list of genes which are down-regulated in response to treatment and/or cultivation with superabsorbent polymers, preferably by treatment and/or cultivation with a superabsorbent polymer comprising polyacrylic acid and lignocellulose-modified polyacrylic acid:
  • GRAS1 protein (GRAS1 ), (response to chitin GO:0010200, regulation of DNA-dependent tran- scription GO:0006355, regulation of transcription GO:0045449, photomorphogenesis
  • GO:0009640 protein similar to UBC9 (UBIQUITIN CONJUGATING ENZYM E 9), ubiquitin-pro- tein ligase isoform 2 (Vitis vinifera), (post-translational protein modification GO:0043687, ubiqui- tin-dependent protein catabolic processGO:000651 1 , modification- dependent protein catabolic process GO:0019941 , regulation of protein metabolic process GO:0051246); ABA 8 -hydrox- ylase CYP707A1 (Solanum tuberosum) (response to water deprivation GO:0009414, response to stress GO:0006950, abscisic acid catabolic process GO:0046345, abscisic acid metabolic process GO:0009687 oxidation reduction GO:00551 14, response to red light GO:00101 14, release of seed from dormancy GO:0048838, response to red or far red light GO:
  • GO:0045449 NAK-type protein kinase (Nicotiana tabacum) (defense response to fungus GO:0050832, protein amino acid autophosphorylation GO:0046777); Lipoxygenase (loxD) (fatty acid biosynthetic process GO:0006633, oxidation reduction GO:0055114, oxylipin biosynthetic process GO:0031408, lipid biosynthetic process GO:0008610); WRKY-like transcription factor (Solanum peruvianum) (defense response to fungus GO:0050832, camalexin biosynthetic process GO:0010120, response to water deprivation GO:0009414, response to cold GO:0009409, response to osmotic stress GO:0006970, regulation of DNA-dependent transcription
  • GO:0006355 defense response to bacterium GO:0042742, response to heat GO:0009408, response to chitin GO:0010200, transcription GO:0006350, response to salt stressGO: 0009651 , regulation of transcription GO:0045449); WRKY transcription factor lld-1 splice variant 1/WRKY transcription factor lld-1 splice variant 2 (LOC100125891/WR KYIId-1) (response to chitin GO:0010200, regulation of DNA-dependent transcription GO:0006355, transcription
  • GO:0006952 modification-dependent protein catabolic process GO:0019941 , protein complex assembly GO:0006461 ); Unnamed protein product [Vitis vinifera] AT1 G70740.1 protein kinase family protein (plant-type hypersensitive response GO:0009626, protein amino acid phosphorylation GO:0006468, response to molecule of bacterial origin GO:0002237); Ripening regulated protein DDTFR10/A DDTFR10/A (Lycopersicon esculentum) AT5G61600.1 ethylene-responsive element-binding family protein (response to chitin GO:0010200, ethylene mediated signaling pathway GO:0009873, response to cold GO:0009409, defense response GO:0006952, regulation of DNA-dependent transcription GO:0006355); RNA polymerase beta subunit (Solanum lycopersicum) (DNA-dependent transcription GO:0006351); N-hydroxycinnamoyl-
  • the following genes are preferred target genes for the up-regulation and/or down-regulation upon treatment of the corresponding plants with superabsorbent polymers and during cultivation of the plants.
  • Genes according to the present invention that are particularly preferred with respect to the up- regulation of gene activity caused by treatment of the plant with superabsorbent polymer, espe- cially by the treatment with polyacrylic acid and/or lignocellulose-modified polyacrylic acid, and cultivation of the plant are as follows:
  • Beta-1 ,3-glucanase (LOC543987), NP24 protein precursor (LOC543979), Chitinase
  • cdkB2 Enolase; 2-phospho-D-glycerate hydro-lyase, Iy200-like protein (Solanum tuberosum), Osmotin-like protein (LOC543971 ), 34 kDa outer mitochondrial membrane protein porin-like protein (Solanum tuberosum)4*E-28 VDAC3 (VOLTAGE DEPENDENT ANION CHANNEL 3); voltage-gated anion channel, Putative delta TIP (Nicotiana glauca) DELTATIP; ammonia trans- porter, methylammonium transmembrane transporter/ water Channel, 40S ribosomal protein S4, Hypothetical protein (Vitis vinifera) PTAC12 (PLASTID TRANSCRIPTIONALLY ACTIVE12), Pathogenesisrelated protein P4 (P4), Xyloglucan-specific fungal endoglucanase inhibitor protein precursor (Lycopersicon esculentum)(ACI25 /// Xegip), PR protein (PR1 b
  • Genes according to the present invention that are particularly preferred with respect to the down-regulation of gene activity caused by treatment of the plant with superabsorbent polymer, especially by the treatment with polyacrylic acid and/or lignocellulose-modified polyacrylic acid, and cultivation of the plant are as follows:
  • Photoperiod responsive protein Solanum tuberosum subsp. Andigena
  • PUB29 PLAY U-BOX 29
  • ubiquitin-protein ligase Avr9/Cf-9 rapidly elicited protein 231 (Nicotiana tabacum)
  • GATL10 Galacturonosyltransferase-like 10
  • ubiquitin-protein ligase isoform 2 (Vitis vinifera), ABA 8'-hydroxylase CYP707A1 (Solanum tuberosum), Hypothetical protein (Vitis vinifera) aminoacid transporter family protein, Protein SSM 1 , putative (Ricinus communis), Putative protein kinase (Solanum demissum), Whitefly-induced gp91-phox (Wfi1 ), WRKY transcription factor lld-2 (WRKYIId-2), NAK-type protein kinase (Nicotiana tabacum), Lipoxygenase (loxD), WRKY-like transcription factor (Solanum peruvianum), WRKY transcription factor lld-1 splice variant 1/WRKY transcrip- tion factor lld
  • Avr9/Cf-9 rapidly elicited protein 1 (Nicotiana tabacum)ERF5 (ETHYLENE RESPONSIVE ELEMENT BINDING FACTOR 5); DNA binding / transcription activator/ transcription factor, Double WRKY type transfactor (Solanum tuberosum), Predicted protein (Populus trichocarpa) ATL2; protein binding / zinc ion binding, Unnamed protein product [Vitis vinifera] protein kinase family Protein, Ripening regulated protein DDTFR10/A DDTFR10/A (Lycopersicon esculentum ethylene-responsive element-binding family protein, * RNA polymerase beta subunit (Solanum lycopersicum), Nhydroxycinnamoyl- CoA:tyramine Nhydroxycinnamoyl transferase THT1-3 THT1-3 (Lycopersicon esculentum), Nam-like protein 1 [Petunia x hybrida] ana
  • Especially preferred genes according to the present invention that are up-regulated are selected from pathogenesis related protein P4 (especially relevant for the plant's defense/self-defense response), xyloglucan-specific fungal endoglucanase inhibitor protein (especially relevant for the plant's anti-fungal response), sucrose synthase (especially relevant for the biosynthesis of sugars, improved sugar content and/or sugar composition and/or the plant's general health), beta-1 ,3-glucanase (especially relevant for improved plant defense/self-defense response), chi- tinase (especially relevant for the plant's anti-fungal response), serine acetyltransferase (especially relevant for improved amino acid and protein content, particularly cysteine, and/or the plant's health in general), and combinations thereof.
  • pathogenesis related protein P4 especially relevant for the plant's defense/self-defense response
  • xyloglucan-specific fungal endoglucanase inhibitor protein especially relevant for the plant's anti-fungal response
  • Especially preferred genes according to the present invention that are down-regulated are selected from Jerusalem 1 (especially preferred for growth repression), lipoxygenase (especially pre- ferred for improved and/or increased content of fatty acids and/or plant's general health), verti- cillium wilt disease resistance protein Ve2 (especially relevant for the plant's self-defense response), ACS6 (especially relevant for altered, especially reduced, production of ethylene and/or inhibition of its reception by the plant), cell wall peroxidase (especially relevant for the plant's response to oxidative stress).
  • AVr9/CF-9 rapidly elicited protein 1 (especially relevant for altered, especially reduced, production of ethylene and/or inhibition of its reception by the plant, ripening and/or flowering), ripening regulated protein DDTFR10/A (especially relevant for the plant's response to fungi, altered, especially reduced, production of ethylene and/or inhibition of its reception by the plant, ripening, flowering and the plant's general health), salt responsive protein 2 (especially relevant for the plant's resistance and/or tolerance to salt stress like soil sa- linity and/or plant's general health) and combinations thereof.
  • Superabsorbent polymers according to the present invention are well-known synthetic particulate organic polymers which are solid and hydrophilic, which are insoluble in water and which are capable of absorbing a multiple of their weight of water or aqueous solutions, thereby form- ing a water containing polymer gel, but which on drying again form particles.
  • Superabsorbent polymers according to the present invention are generally capable of absorbing at least 100 parts by weight of water per one part by weight of superabsorbent polymer (deionised water at 25°C, pH 7.5, 1 bar). The amount of water or aqueous solution a superabsorbent polymer is capable of absorbing, is also termed as absorption capacity or maximal absorption.
  • superabsorbent polymers are preferred which have an absorption capacity for deionised water (pH 7.5, 25°C, 1 bar) of at least 150 g/g, e.g. 150 to 500 g/g, in particular 200 to 500 g/g, more preferably 300 to 500 g/g of superabsorbent polymers.
  • superabsorbent polymers are preferred which have an absorption capacity for a 0.1 % by weight aqueous solution of sodium chloride of at least 100 g/g, in particular 100 to 300 g/g of superabsorbent polymer (pH 7.5, 25°C, 1 bar).
  • the maximal absorption or absorption capacity can be determined by routine methods known e.g. from F.
  • the superabsorbent polymer material is preferably in the form of granules.
  • Preferred super- absorbent polymer granules are those which have a moderate swelling rate, i.e. superabsor- bents, wherein the time required to achieve 60% of the maximal absorption is at least 10 minutes, in particular from 10 to 100 minutes. These values can be determined according to standard methods as described in F. L. Buchholz et al., loc. cit, p. 154 (swelling kinetics meth- ods).
  • the superabsorbent polymers may be nonionic or ionic crosslinked polymers.
  • the superabsorbent polymer is preferably selected from crosslinked anionic su- perabsorbent polymers, in particular from covalently crosslinked anionic superabsorbent polymers.
  • a survey of suitable superabsorbent polymers is e.g. given in F. L. Buchholz et al., loc. cit., p. 11-14.
  • Crosslinked anionic superabsorbent polymers are crosslinked polymers which comprise anionic functional groups or acidic groups, which can be neutralized in water, e.g.
  • sulfonic acid groups SO3H or SO3
  • phosphonate groups PO3H2 or PO3
  • carboxylate groups CO2H or CO2
  • These polymers are in principle obtainable by a process which comprises co- polymerizing a monoethylenically unsaturated acidic monomer and a crosslinking monomer optionally in the presence of a grafting base and optionally in the presence of one or more further neutral monoethylenically unsaturated monomers.
  • the carboxylate groups make up at least 80 mol-%, in particular at least 95 mol-%, of the acidic groups.
  • Suitable acidic monomers include monoethylenically unsaturated mono- and dicarboxylic acids having preferably from 3 to 8 carbon atoms such as acrylic acid, methacrylic acid, ethacrylic acid, [alpha]-chloroacrylic acid, crotonic acid, maleic acid, maleic anhydride, itaconic acid, citra- conic acid, mesaconic acid, glutaconic acid, aconitic acid and fumaric acid; monoesters of monoethylenically unsaturated dicarboxylic acids having from 4 to 10 and preferably from 4 to 6 carbon atoms, for example monoesters of maleic acid such as monomethyl maleate; monoeth- ylenically unsaturated sulfonic acids and phosphonic acids, for example vinylsulfonic acid, al- lylsulfonic acid, sulfoethyl acrylate, sulfoethyl methacrylate, sulfopropyl
  • the acidic monomers usually make up at least 15 %, by weight, preferably at least 20 % by weight, of the superabsorbent polymer, e.g. 15 to 99.9 % by weight, in particular from 20 to 99.8 % by weight, based on the acidic form of the anionic superabsorbent polymer.
  • the monoethylenically unsaturated carboxylic acid CA or the salt thereof accounts for at least 80 mol-%, in particular at least 95 mol-% of the total amount of polymerized acidic monomeres.
  • crosslinking monomers include compounds having at least two, for example 2, 3, 4 or 5, ethylenically unsaturated double bonds in the molecule. These compounds are also referred to as crosslinker monomers.
  • crosslinker monomers are N,N'-methylenebisacrylamide, polyethylene glycol diacrylates and polyethylene glycol dimethacrylates, each derived from poly- ethylene glycols having a molecular weight from 106 to 8500 and preferably from 400 to 2000, trimethylolpropane triacrylate, trimethylol propane trimethacrylate, ethylene glycol diacrylate, ethylene glycol dimethacrylate, propylene glycol diacrylate, propylene glycol dimethacrylate, butanediol diacrylate, butanediol dimethacrylate, hexanediol diacrylate, hexanediol dimethacrylate, diethylene glycol diacrylate, diethylene glycol dimethacrylate, triethylene glycol
  • allyl acrylate and allyl methacrylate also triallylamine, dialkyldiallylammonium halides such as dimethyldiallylammonium chloride and diethyldiallylammonium chloride, tetraallyleth- ylenediamine, divinylbenzene, diallyl phthalate, polyethylene glycol divinyl ethers of polyethylene glycols having a molecular weight from 106 to 4000, trimethylolpropane diallyl ether, bu- tanediol divinyl ether, pentaerythritol triallyl ether, reaction products of 1 mol of ethylene glycol diglycidyl ether or polyethylene glycol diglycidyl ether with 2 mol of pentaerythritol triallyl ether or allyl alcohol, and divinylethyleneurea.
  • dialkyldiallylammonium halides such as dimethyldiallylammonium chloride and diethy
  • the amount of crosslinker monomer is generally in the range from 0.05 to 20% by weight, preferably in the range from 0.1 to 10% by weight and especially in the range from 0.2 to 5% by weight, based on the weight of the superabsorbent polymer in the acidic form.
  • Suitable grafting bases can be of natural or synthetic origin. They include oligo- and polysac- charides such as starches, i.e. native starches from the group consisting of corn (maize) starch, potato starch, wheat starch, rice starch, tapioca starch, sorghum starch, manioca starch, pea starch or mixtures thereof, modified starches, starch degradation products, for example oxidative ⁇ , enzymatically or hydrolytically degraded starches, dextrins, for example roast dextrins, and also lower oligo- and polysaccharides, for example cyclodextrins having from 4 to 8 ring members.
  • starches i.e. native starches from the group consisting of corn (maize) starch, potato starch, wheat starch, rice starch, tapioca starch, sorghum starch, manioca starch, pea starch or mixtures thereof, modified starches, starch degradation
  • Useful oligo- and polysaccharides further include cellulose and also starch and cellulose derivatives. It is also possible to use polyvinyl alcohols, homo- and copolymers of N-vi- nylpyrrolidone, polyamines, polyamides, hydrophilic polyesters or polyalkylene oxides, especially polyethylene oxide and polypropylene oxide as a grafting base.
  • the amount of grafting base may be up to 50 % by weight of the weight of the superabsorbent polymer in the acidic form, e.g. from 1 to 50 % by weight.
  • the monomers forming the superabsorbent polymer may also contain neutral monoethylenicaily unsaturated monomers which do not have a polymerizable group or an acidic group.
  • Examples are monoethylenicaily unsaturated hydrophilic monomers, i.e. monomers having a water solubil- ity of at least 80 g/l at 25°C, 1 bar, including hydroxyalkyl esters of monoethylenicaily unsaturated monocarboxylic acids, e.g.
  • hydroxyalkyl acrylates and methacrylates such as hydrox- yethyl acrylate and hydroxyethylmethacrylate, amides of monoethylenicaily unsaturated monocarboxylic acids such as acrylamide and methacrylamide, monomers having a polyether group, such as vinyl, allyl and methallyl ethers of polyethylene glycols and esters of monoethylenicaily unsaturated monocarboxylic acids and polyethers, such as polyethylenglykol acrylate and poly- ethyleneglycol methacrylate.
  • amides of monoethylenicaily unsaturated monocarboxylic acids such as acrylamide and methacrylamide
  • monomers having a polyether group such as vinyl, allyl and methallyl ethers of polyethylene glycols and esters of monoethylenicaily unsaturated monocarboxylic acids and polyethers, such as polyethylenglykol acrylate and poly-
  • the neutral monomers make up from 10 to 84.9 % by weight, in particular from 20 to 79.9 % by weight of the super- absorbent polymer in the acidic form.
  • Preferred anionic superabsorbent polymers have a moderate charge density, i.e. the amount of acidic groups in the superabsorbent polymer is preferably from 0.1 to 1.1 mol per 100 g of superabsorbent polymer, in particular form 0.2 to 1 mol per 100 g of superabsorbent polymer, based on the weight of the superabsorbent polymer in the acidic form.
  • the water absorbent polymer is a crosslinked copolymer or graft copolymer of ethylenically unsaturated monomers M which comprise at least one monoethylenically unsaturated carboxylic acid CA or a salt thereof at least one amide of a monoethylenically unsaturated acid (monomer AM), and a crosslinking monomer in polymerized form.
  • Suitable monoethylenically unsaturated carboxylic acids CA comprise monoethylenically unsaturated mono-carboxylic acids having 3 to 8 carbon atoms, such as acrylic acid and meth- acrylic acid, and monoethylenically unsaturated dicarboxylic acids having from 4 to 8 carbon atoms, such as maleic acid, fumaric acid, itaconic acid and citraconic acid.
  • Suitable salts of monoethylenically unsaturated carboxylic acids CA comprise the alkali metal salts and the ammonium salts, in particular the potassium or sodium salts.
  • Preferred monoethylenically unsaturated carboxylic acids CA include mono-carboxylic acids having 3 to 8 carbon atoms, in particular acrylic acid and methacrylic acid and the salts thereof, in particular the alkalimetal salts thereof, and more preferably the alkali metal salts of acrylic acid, especially the sodium salt and the potassium salt of acrylic acid.
  • Suitable amides of monoethylenically unsaturated acids are the amides of monoethylenically unsaturated mono-carboxylic acids having 3 to 8 carbon atoms, in particular acrylamide and methacrylamide.
  • the water absorbent polymer is preferably a covalently crosslinked copolymer, i.e. it contains a crosslinking monomer as defined above.
  • the carboxylic acid CA and the amide AM make up at least 80% by weight, e.g. from 80 to 99.95% by weight, and more preferably at least 90% by weight, e.g. from 90 to 99.9% by weight, of the ethylenically unsaturated monomers M forming the superabsorbent polymer.
  • the crosslinking monomer will generally make up from 0.05 to 20% by weight, in particular from 0.1 to 10% by weight of the monomers M.
  • the monomers M comprise at least 90% by weight, e.g. from 90 to 99.9% by weight, based on the total weight of monomers M, of a mixture of acrylic acid or a salt thereof, in particular an alkali metal salt thereof, more preferably the potassium salt of acrylic acid, and acrylamide.
  • the superabsorbent polymer comprises in polymerized form:
  • amide AM 10 to 84.9 % in particular 20 to 79.8 % by weight of at least one amide AM, preferably an amide of a monoethylenically unsaturated mono-carboxylic acid having 3 to 8 carbon atoms, in particular acrylamide; and
  • % by weight are based on the superabsorbent polymer in the acidic form, the amount of mono- mers AM and CA making up at least 90 %, e.g. 90 to 99.9 % of the monomers forming the superabsorbent polymer.
  • Suitable superabsorbent polymers of this type are known in the art, e.g. from US 4,417,992, US 3,669,103 and WO 01/25493. They are also commercially available, e.g. from SNF SA., France, under the trademark Aquasorb(R), e.g. Aquasorb(R) 3005 KL, 3005 KM, 3005 L and 3005 M.
  • Aquasorb(R) e.g. Aquasorb(R) 3005 KL, 3005 KM, 3005 L and 3005 M.
  • the water absorbent polymer is a cross- linked copolymer or graft copolymer of ethylenically unsaturated monomers M which comprise at least 80 % by weight, e.g. from 80 to 99.95% by weight, preferably at least 90 % by weight, e.g. from 90 to 99.9% by weight, based on the total amount of monomers M, of a mixture of at least one monoethylenically unsaturated carboxylic acid CA, preferably acrylic and at least one alkali metal salt of a monoethylenically unsaturated carboxylic acid CA, preferably a potassium salt or sodium salt thereof, more preferably the potassium salt or sodium salt of acrylic acid.
  • the water absorbent polymer is preferably a covalently crosslinked copolymer.
  • the crosslinking monomer will generally make up from 0.05 to 20% by weight, in particular from 0.1 to 10% by weight of the monomers M.
  • the superabsorbent polymer of this embodiment comprises in polymerized form: 15 to 89.9 %, in particular 20 to 79.8 % by weight of at least one carboxylic acid CA, preferably acrylic acid;
  • % by weight are based on the superabsorbent polymer in the acidic form, the amount of carboxylic acid CA and the salt of CA making up at least 90 %, e.g. 90 to 99.9 % of the monomers forming the superabsorbent polymer.
  • Very preferred superabsorbent polymers according to the present invention include polyacrylic acid and lignocellulose-modified polyacrylic acid, especially the alkali metal salts and the ammonium salts, in particular the potassium or sodium salts thereof.
  • Such type of polymers are commercially available, e.g. from BASF AG under the trade names Luquasorb(R), e.g.
  • Luquasorb(R) 1280 Luquasorb(R) 1060, Luquasorb(R) 1 160, Luquasorb(R) 1061 and Hy- Sorb(R) and Phytogel and Vitola® of BASF, respectively.
  • the average particle size of the superabsorbent polymer granules ranges from 0.1 to 5 mm, preferably from 0.2 to 5 mm, in particular from 0.5 to 4 mm.
  • the average particle size is the weight average of the diameter which may be determined by microscopy or by sieving anal- ysis, preferably sieving analysis.
  • the superabsorbent polymer granules are surface crosslinked (see F. L. Buchholz, loc. cit. pp. 97 to 103, and the literature cited therein).
  • the surface crosslinked polymer granules some of the functional groups in the surface region of the superabsorbent polymer granules have been crosslinked by reaction with polyfunctional compounds.
  • Surface crosslinking can be a covalent or ionic crosslinking.
  • the surface of superabsorbent polymer granules which are used for preparing the pesticide composition, may have been treated with additives to reduce their dustiness and/or to ease their flow, including treatment with anti-caking additives such as particulate silica, in particular fumed silica, optionally in combination with polyols, or quaternary surfactants.
  • additives such as particulate silica, in particular fumed silica, optionally in combination with polyols, or quaternary surfactants.
  • the superabsorbent polymer material is dried using the established technology in the art includ- ing spray drying and the like. Drying will be carried out until a water content of not more than 20 % wt, preferably not more than 10 wt% and even more preferably until a water content of 0.5 to 20 wt%, most preferably 1 to 15 wt% is achieved.
  • the dried superabsorbent polymer material is preferably classified and milled using the estab- lished technology of this field to obtain the superabsorbent polymer in the form of granules.
  • the plant is treated with the superabsorbent polymer by applying superabsorbent polymer in an amount of about 1 to 1000 kg/ha culture soil, preferably 50 to 800 kg/ha culture soil and most preferably 100 to 500 kg/ha culture soil.
  • the usual agricultural methods can be used for applying the superabsorbent polymer to the culture soil either by simply spreading the superabsorbent over the soil or mixing it with the soil before applying a layer of superabsorbent polymer and soil to the ground or working the super- absorbent polymer into the soil using conventional agricultural techniques.
  • the latter operations can be optionally combined with the application of fertilizer.
  • the superabsorbent polymer is applied to the soil either before the seed is brought to the soil, or before the seedling is planted into the ground or during the cultivation of the already growing plant.
  • Cultivation of the plant in the presence of the superabsorbent polymer includes leaving the su- perabsorbent polymer on the soil where the plant is grown for extended periods of time, prefera- bly for the whole growth cycle until harvesting the desired crop or fruit or plant product.
  • plant generally comprises all plants of economic importance and/or men- grown plants. They are preferably selected from agricultural, silvicultural and ornamental plants, more preferably agricultural plants and silvicultural plants, utmost preferably agricultural plants.
  • plant (or plants) is a synonym of the term “crop” which is to be understood as a plant of economic importance and/or a men-grown plant.
  • plant as used herein includes all parts of a plant such as germinating seeds, emerging seedlings, herbaceous vegetation as well as established woody plants including all belowground portions (such as the roots) and above- ground portions.
  • the plants to be treated according to the invention are selected from the group consisting of agricultural, silvicultural, ornamental and horticultural plants, each in its natural or genetically modified form, more preferably from agricultural plants.
  • the aforementioned methods for increasing the health of a plant and/or increasing the control of undesirable vegetation and/or increasing the control of phytopathogenic fungi comprises treating the plant propagules, preferably the seeds of an agricultural, horticultural, ornamental or silvicultural plant selected from the group consisting of transgenic or non- transgenic plants with a mixture according to the present invention.
  • the plant to be treated according to the method of the invention is an agricultural plant.
  • Agricultural plants are plants of which a part or all is harvested or cultivated on a commercial scale or which serve as an important source of feed, food, fibres (e.g. cotton, linen), combustibles (e.g. wood, bioethanol, biodiesel, biomass) or other chemical compounds.
  • Agricul- tural plants also horticultural plants, i.e. plants grown in gardens (and not on fields), such as certain fruits and vegetables.
  • Preferred agricultural plants are for example cereals, e.g. wheat, rye, barley, triticale, oats, sorghum or rice; beet, e.g.
  • sugar beet or fodder beet fruits, such as pomes, stone fruits or soft fruits, e.g. apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries, blackberries or gooseberries; leguminous plants, such as lentils, peas, al- falfa or soybeans; oil plants, such as rape, oil-seed rape, canola, juncea (Brassica juncea), linseed, mustard, olives, sunflowers, coconut, cocoa beans, castor oil plants, oil palms, ground nuts or soybeans; cucurbits, such as squashes, cucumber or melons; fiber plants, such as cotton, flax, hemp or jute; citrus fruit, such as oranges, lemons, grapefruits or mandarins; vegetables, such as spinach, lettuce, asparagus, cabbages, carrots, onions, tomatoes, potatoes, cu- curbits or paprika; lauraceous plants, such as avocados, cinnamon or camphor; energy and raw material plants
  • More preferred agricultural plants are field crops, such as potatoes, sugar beets, cereals such as wheat, rye, barley, triticale, oats, sorghum, rice, corn, cotton, rape, sunflowers, oilseed rape, juncea and canola, legumes such as soybeans, peas and beans (fieldbeans), lentil, sugar cane, turf; ornamentals; or vegetables, such as cucumbers, tomatoes, or onions, leeks, lettuce, squashes, alfalfa, clover most preferred agricultural plants are potatoes, beans (fieldbeans), alfalfa, sugar cane, turf, sugar beets, cereals such as wheat, rye, triticale, barley, oats, sorghum, rice, corn, cotton, soybeans, oilseed rape, canola, juncea, sunflower, sugar cane, peas, lentils and alfalfa and utmost preferred plants are selected from soybean, wheat, sunflowers, canola, juncea,
  • the plants to be treated are selected from soybean, wheat, sunflower, canola, oilseed rape, corn, cotton, sugar cane, juncea, peas, lentils and alfalfa.
  • the utmost preferred plant is soybean.
  • the plants to be treated are selected from wheat, barley, corn, soybean, rice, canola and sunflower.
  • the plant to be treated according to the method of the invention is a horticultural plant.
  • the term "horticultural plants” are to be understood as plants which are commonly used in horticulture - e. g. the cultivation of ornamentals, vegetables and/or fruits. Examples for ornamentals are turf, geranium, pelargonia, petunia, begonia and fuchsia.
  • vegetables are potatoes, tomatoes, peppers, cucurbits, cucumbers, melons, watermelons, garlic, onions, carrots, cabbage, beans, peas and lettuce and more preferably from tomatoes, onions, peas and lettuce. Tomatoes are especially preferred.
  • fruits are apples, pears, cherries, strawberry, citrus, peaches, apricots and blue- berries.
  • the plant to be treated according to the method of the invention is an ornamental plant.
  • Ornamental plants are plants which are commonly used in gardening, e.g. in parks, gardens and on balconies. Examples are turf, geranium, pelargonia, petunia, begonia and fuchsia.
  • the plant to be treated according to the method of the invention is a silvicultural plants.
  • the term "silvicultural plant” is to be understood as trees, more specifically trees used in reforestation or industrial plantations.
  • Industrial plantations generally serve for the com- worthal production of forest products, such as wood, pulp, paper, rubber tree, Christmas trees, or young trees for gardening purposes.
  • silvicultural plants are conifers, like pines, in particular Pinus spec, fir and spruce, eucalyptus, tropical trees like teak, rubber tree, oil palm, willow (Salix), in particular SaNx spec, poplar (cottonwood), in particular Populus spec, beech, in particular Fagus spec, birch, oil palm and oak.
  • the present invention also includes genetically modified plants.
  • Genetically modified plants are plants, which genetic material has been so modified by the use of recombinant DNA techniques that under natural circumstances cannot readily be obtained by cross breeding, mutations or natural recombination.
  • one or more genes have been integrated into the genetic material of a genetically modified plant in order to improve certain properties of the plant.
  • Such genetic modifications also include but are not limited to targeted post-transtional modification of protein(s), oligo- or polypeptides e. g. by glycosylation or polymer additions such as prenylated, acetylated or farnesylated moieties or PEG moieties.
  • EP-A 242 236, EP-A 242 246) or oxynil herbicides see e. g. US 5,559,024) as a result of conventional methods of breeding or genetic engineering.
  • Several cultivated plants have been rendered tolerant to herbicides by conventional methods of breeding (mutagenesis), e. g. Clearfield ® summer rape (Canola, BASF SE, Germany) being tolerant to imidazolinones, e. g. imazamox.
  • plants are also covered that are by the use of recombinant DNA techniques capable to synthesize one or more insecticidal proteins, especially those known from the bacterial genus Bacillus, particularly from Bacillus thuringiensis, such as ⁇ -endotoxins, e. g. CrylA(b), CrylA(c), CrylF, CrylF(a2), CryllA(b), CrylllA, CrylllB(bl) or Cry9c; vegetative insecticidal pro- teins (VIP), e. g. VIP1 , VIP2, VIP3 or VIP3A; insecticidal proteins of bacteria colonizing nematodes, e. g. Photorhabdus spp.
  • VIP vegetative insecticidal pro- teins
  • toxins produced by animals such as scorpion toxins, arachnid toxins, wasp toxins, or other insect-specific neurotoxins
  • toxins produced by fungi such Streptomycetes toxins, plant lectins, such as pea or barley lectins; agglutinins
  • proteinase inhibitors such as trypsin inhibitors, serine protease inhibitors, patatin, cystatin or papain inhibitors
  • ribosome-inactivating proteins (RIP) such as ricin, maize-RIP, abrin, luffin, saporin or bryodin
  • steroid metabolism enzymes such as 3-hydroxysteroid oxidase, ecdyster- oid-IDP-glycosyl-transferase, cholesterol oxidases, ecdysone inhibitors or HMG-CoA-reductase
  • ion channel blockers such as block
  • insecticidal proteins or toxins are to be understood expressly also as pre-toxins, hybrid proteins, truncated or otherwise modified proteins.
  • Hybrid proteins are characterized by a new combination of protein domains, (see, e. g. WO 02/015701 ).
  • Further examples of such toxins or genetically modified plants capable of synthesizing such toxins are disclosed, e. g., in EP-A 374 753, WO 93/007278,
  • WO 95/34656 EP-A 427 529, EP-A 451 878, WO 03/18810 und WO 03/52073.
  • the methods for producing such genetically modified plants are generally known to the person skilled in the art and are described, e. g. in the publications mentioned above.
  • These insecticidal proteins contained in the genetically modified plants impart to the plants producing these proteins tolerance to harmful pests from all taxonomic groups of athropods, especially to beetles (Coelop- tera), two-winged insects (Diptera), and moths (Lepidoptera) and to nematodes (Nematoda).
  • Genetically modified plants capable to synthesize one or more insecticidal proteins are, e.
  • WO 03/018810 MON 863 from Monsanto Europe S.A., Belgium (corn cultivars producing the Cry3Bb1 toxin), IPC 531 from Monsanto Europe S.A., Belgium (cotton cultivars producing a modified version of the CrylAc toxin) and 1507 from Pioneer Overseas Corporation, Belgium (corn cultivars producing the Cry1 F toxin and PAT enzyme).
  • plants are also covered that are by the use of recombinant DNA techniques capable to synthesize one or more proteins to increase the resistance or tolerance of those plants to bacterial, viral or fungal pathogens.
  • proteins are the so-called "pathogenesis- related proteins" (PR proteins, see, e. g. EP-A 392 225), plant disease resistance genes (e. g. potato cultivars, which express resistance genes acting against Phytophthora infestans derived from the mexican wild potato Solanum bulbocastanum) or T4-lysozym (e. g. potato cultivars capable of synthesizing these proteins with increased resistance against bacteria such as Erwinia amylvora).
  • PR proteins pathogenesis- related proteins
  • plant disease resistance genes e. g. potato cultivars, which express resistance genes acting against Phytophthora infestans derived from the mexican wild potato Solanum bulbocastanum
  • T4-lysozym e. g. potato cultiv
  • plants are also covered that are by the use of recombinant DNA techniques capable to synthesize one or more proteins to increase the productivity (e. g. bio mass production, grain yield, starch content, oil content or protein content), tolerance to drought, salinity or other growth-limiting environmental factors or tolerance to pests and fungal, bacterial or viral pathogens of those plants.
  • productivity e. g. bio mass production, grain yield, starch content, oil content or protein content
  • plants are also covered that contain by the use of recombinant DNA techniques a modified amount of substances of content or new substances of content, specifically to improve human or animal nutrition, e. g. oil crops that produce health-promoting long-chain omega-3 fatty acids or unsaturated omega-9 fatty acids (e. g. Nexera ® rape, DOW Agro Sciences, Canada).
  • plants are also covered that contain by the use of recombinant DNA techniques a modified amount of substances of content or new substances of content, specifically to improve raw material production, e. g. potatoes that produce increased amounts of amylopectin (e. g. Amflora ® potato, BASF SE, Germany).
  • the up-regulation and down-regulation of the genes in the plant following treatment and cultivation of the plant with the superabsorbent polymer is measured by a differential gene expression approach.
  • RNA preferably mRNA
  • the relative increase or decrease in amount of RNA, preferably mRNA is the parameter indicating the increase or decrease of gene activity.
  • a conventional differential (gene) display particularly for mRNA, can be used to identify genes which are up-regulated or down-regulated in activity while quantitative real time PCR allows the quantitative measurement of up-regulation and down-regulation of genes.
  • Quantitative real time PCR and related techniques can be carried out by the skilled person based on the established standard protocols.
  • DNA microarray technology as known in the art and commercially available, is used for measuring the up-regulation and down-regulation of genes of interest.
  • One preferred approach is based on the GeneChip® tomato genome array as available from Affymetrix.
  • the present invention is directed to a process of changing the gene expression in a plant comprising the steps of treating the plant with a superabsorbent polymer, cultivating the plant treated with the superabsorbent polymer, and up-regulating at least one gene of the plant by the addition of the superabsorbent polymer, and/or down-regulating at least one gene of the plant by the addition of the superabsorbent polymer.
  • the plant is cultivated in the presence of an external stress factor.
  • the present invention is directed to a process of determining the change in gene expression in a plant upon treatment of the plant with a superabsorbent polymer, comprising the steps of treating the plant with a superabsorbent polymer, cultivating the plant treated with a superabsorbent polymer, optionally in the presence of an external stress factor, and measuring the up-regulation and/or down-regulation of at least one gene of the plant during and/or after cultivation of the plant.
  • measuring the up-regulation and/or down- regulation of at least one gene of the plant is carried out by use of a transcriptomic assay, preferably by use of a microarray.
  • Embodiment 1 Further embodiments according to the present invention include the following embodiments: Embodiment 1 :
  • Process for improving plant health by changing the gene expression in a plant characterized in that the plant is treated and/or cultivated with a superabsorbent polymer and at least one gene of the plant is up-regulated by the addition of the superabsorbent polymer, and/or at least one gene is down-regulated by the addition of the superabsorbent polymer, wherein improved plant health is determined by increased yield of plant product, improved plant vigor, enhanced quality of the plant and/or improved tolerance or resistance of the plant to abiotic and/or biotic stress factors.
  • Embodiment 2 is a diagrammatic representation of Embodiment 1:
  • Embodiment 3 is a diagrammatic representation of Embodiment 3
  • improved plant vigor is determined by improved vitality of the plant, improved plant growth, improved plant development, improved visual ap- pearance, improved plant stand, less plant verse/lodging, improved emergence, enhanced root growth and/or more developed root system, enhanced nodulation, in particular rhizobial nodula- tion, bigger leaf blade, bigger size, increased plant weight, increased plant height, increased tiller number, increased number of side shoots, increased number of flowers per plant, increased shoot growth, increased root growth like extensive root system, increased yield when grown on poor soils or unfavorable climate, enhanced photosynthetic activity, preferably based on increased stomatal conductance and/or increased CO2 assimilation rate, increased stomatal conductance, increased CO2 assimilation rate, enhanced pigment content like chlorophyll content, flowering, earlier fruiting, earlier and improved germination, earlier grain maturity, improved self-defense mechanisms, less non-productive tillers, less dead basal leaves, less input needed such as fertiliz
  • improved tolerance or resistance of the plant to abiotic stress factors is determined by improved tolerance or resistance to heat stress including temperatures higher than 30°C, temperature conditions causing heat damage to a plant like heat damaged foliage or burnt leaves, cold stress like temperature conditions below 10°C, peri- ods of thawing and freezing, frost, variations in temperature like temperatures conditions that lead to the freezing of water either for extended periods of time or only temporary periods, temperature unusual for the season, drought stress, exposure to cold water, flood, water-logging, wind, sun light, particularly sun light causing signs of scorch, sun burn or similar signs of irradiation and heat stress to the plant, acid or alkaline pH conditions in the soil with pH values lower than pH 5 and/or pH values higher than 9, salt stress like soil salinity, soil erosion, inorganic pollution, soil contamination or soil pollution with chemicals, particularly with heavy metals, preferably chromium, lead, cadmium, arsenic, antimony, mercury, iron, thallium, barium, bery
  • Embodiment 7 is a diagrammatic representation of Embodiment 7:
  • up-regulated plant gene is selected from pathogenesis related protein P4, xyloglucan-specific fungal endoglucanase inhibitor protein, sucrose synthase, beta-1 ,2-glucanase, chitinase, serine acetyltransferase and combinations thereof.
  • Embodiment 8 is a diagrammatic representation of Embodiment 8
  • the plant is an agricultural plant like wheat, rye, barley, triticale, oats, sorghum or rice, beet, sugar beet or fodder beet, fruits like pomes, apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries, blackberries or gooseberries, leguminous plants, such as lentils, peas, alfalfa or soybeans, oil plants, such as rape, oil-seed rape, canola, juncea, linseed, mustard, olives, sunflowers, coconut, cocoa beans, castor oil plants, oil palms, ground nuts or soybeans, cucurbits, such as squashes, cucumber or melons, fiber plants, such as cotton, flax, hemp or jute, citrus fruit, such as oranges, lemons, grapefruits or mandarins, vegetables, such as spinach, lettuce, asparagus, cabbages, carrots, onions, tomatoes, potatoes, cucurbits or
  • Embodiment 1 1 is a diagrammatic representation of Embodiment 1 1 :
  • Process for improving the plant health by changing the gene expression in a plant comprising the steps of treating and/or cultivating the plant with a superabsorbent polymer, and up-regulating at least one gene of the plant by the addition of the superabsorbent polymer, and/or down- regulating at least one gene of the plant by the addition of the superabsorbent polymer, wherein improved plant health is determined by increased yield of plant product, improved plant vigor, enhanced quality of the plant and/or improved tolerance or resistance of the plant to abiotic and/or biotic stress factors.
  • Embodiment 13 Embodiment 13:
  • Process of determining the change in gene expression in a plant upon treatment of the plant with a superabsorbent polymer comprising the steps of treating and/or cultivating the plant with a superabsorbent polymer and measuring the up-regulation and/or down-regulation of at least one gene of the plant during and/or after cultivation of the plant.
  • Embodiment 14 is a diagrammatic representation of Embodiment 14:
  • the plant material was collected and immediately frozen in liquid nitrogen.
  • RN A extraction the commercially available kit NucleoSpin RNA plant (Macherey-Nagel) was used, following the manufacturer's instructions.
  • RNA from plants Three technical replicates for each sample were used for biological hybridization of the samples. Isolation of RNA from plants:
  • "TRIZOL® Reagent” Invitrogen was used following the supplier's instructions. The plant material was maintained at -80°C until use. 100 mg of leaves or root of the plant frozen in liquid nitrogen were homogenized in a mortar and placed in liquid nitrogen for storage of the frozen plant tissue.
  • RNA Quantification of the amount of RNA for each sample was carried out using a commercial NanoDrop spectrophotometer ND 1000.
  • cDNA was prepared by reverse transcription and subsequently amplified by quantitative real time PCR of the selected marker genes of the plant samples collected at 10, 20, 30, 40, 50 and 60 days after application of the superabsorbent polymer.
  • RMA multi array average
  • the seeds were sterilized with 70 % ethanol for 10 minutes, a commercial bleach (5 % sodium hypochlorite) was used for 10 minutes, followed by repeated washing of the seeds with sterile, distilled water. The seeds were planted same day. After 18 days the seedlings were transplanted into sockets of 0.1 I to have a good root development, 8 days later the seedlings were transferred into 16 liters pots (two seedlings for each pot).
  • a conventional NPK fertilizer (20-20-20) was used for cultivation of the plants.
  • the NPK fertilizer was applied in doses of 1 grain / pot to each plant of the pots 50 days after application of the superabsorbent polymer.
  • As the substrate sterile peat and sterile or non-sterile soil brought from Utrera (Sevilla) was used as cultivation medium. Sterilization of the soil before use was carried out by two sterilization cycles for 1 h at 121 °C in autoclave. The same amount of water was added to each pot.
  • the plants were kept in the greenhouse CIALE throughout cultivation, under controlled conditions of 25-27°C during the day and 21 °C at night, with a photoperiod of 15 h and relative hu- midity of 40-60 %.
  • the tomato plants were examined at 10, 20, 30, 40, 50 and 60 days after adding the different treatments.
  • plant material was collected from the leaves and the roots of at least 3 plants of each treatment.
  • Leaf samples collected after 10 days of the application of the superabsorbent polymer were used for microarray analysis, while the rest was used for verifying the data.
  • Fig. 1 and Fig. 2 show the Volcano plots of p-values of the t-test of H 1 ("t-test-H 1 ") vs. the control experiment, the parameter on the horizontal x axis is "log2(fold change)", the parameter on the vertical y axis is "-logl O(p-value)", “FC” stands for "fold change”, “P-val” stands for "p-value”.
  • the differential gene expression found in the present experiment for the cultivation of the plants in the presence of superabsorbent polymer can be summarized as follows:
  • Fig. 3 shows this Venn diagram, wherein the left oval refers to the t-test of H1 at FC2 ("t-test- H 1 -FC2"), the right oval refers to the t-test of H2 at FC2 ("t-test-H2-FC2").
  • the Venn diagram shows that 53 genes were differentially expressed during cultivation of the plants in the presence of both superabsorbent polymers H 1 and H2, while 46 genes were up- regulated and 7 genes were down-regulated in the presence of both superabsorbent polymers.

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Abstract

The invention relates to the use of superabsorbent polymers for altering the gene expression in plants and to a method for determining changes in gene expression upon treatment of plants with superabsorbent polymers.

Description

Use of a superabsorbent polymer for improving plant health by changing the gene expression in a plant
Description
The present invention relates to the treatment of agricultural plants with polymers in order to improve plant performance.
Technical background
The treatment of plants with polymers during cultivation has been only sporadically described in the art.
WO 2007/104720 A1 describes the use of superabsorbent polymers in the form of water-absor- bent granular material which is used as carrier for pesticide compositions suitable for combating arthropod pests, snails and nematodes in agricultural production of plants and products derived therefrom.
WO 2010/037630 A1 discloses the treatment of plants with superabsorbers in order to promote the aboveground root growth of plants. It is described that such effect is achieved by the addition of superabsorbent polymer to the growing medium. It is further suggested that such process is particularly useful for cultivating plants under arid or semi-arid conditions or comparable water-deficient environments. Similarly, WO 2013/060848 A1 discloses the preparation of water-swellable polymers that are suitable for absorbing and storing aqueous fluids. The polymers are suggested for improving soil quality, the preparation of agricultural soil and the re-cultivation of wasteland. It is further described that these water-swellable polymers can be used for improving the water storage ability of culture soil thereby improving the quality of agricultural soil.
Consequently, the application of polymers, particularly superabsorbent polymers, as previously suggested in the art for the cultivation of plants, are limited to the use as carrier materials for pesticidal compounds or as supportive water-binding mediums improving water supply to the plant. On the other hand, it has not been described in the art that superabsorbent polymers have the ability to directly act on the plants to be cultivated.
In contrast to the above-mentioned previous teachings suggesting superabsorbent materials as mere auxiliary agents in plant cultivation carrying pesticidal compounds or assisting culture soil in more sustained water supply, the present inventors have surprisingly found that superabsor- bent polymers can be directly used in the cultivation of plants for manipulating the plant's physiologic response in a variety of different ways.
This has been unexpectedly demonstrated by the present inventors by studying the plant's response during and after the cultivation of the plant in the presence of superabsorbent polymer. It is shown herein that the cultivation of plants in the presence of superabsorbent polymers alters the transcriptional level of many different genes which are important for the plant's response to various biotic and abiotic factors, particularly the plants response to abiotic stress, especially derived from chemical stimuli and its defense response to external biotic stress factors
By the technical effects described for the first time in the present invention, it has become possible to systematically alter the plant's physiology on the molecular level by the use of super- absorbent polymers. It is demonstrated in the present invention that superabsorbent polymers can be used in the cultivation of plants for changing gene expression including activating, in- creasing, inhibiting or reducing the activity of selected plant genes known for their high physiological relevance.
The foregoing and other objectives are solved by the subject-matter of the present invention. The first specific finding of the present invention is the use of a superabsorbent polymer for improving plant health by changing the gene expression in a plant, characterized in that the plant is treated with the superabsorbent polymer, the plant treated with the superabsorbent polymer is cultivated, and at least one gene of the plant is up-regulated by the addition of the superabsorbent polymer, and/or at least one gene is down-regulated by the addition of the superabsorbent polymer, wherein improved plant health is determined by increased yield of plant product, improved plant vigor, enhanced quality of the plant and/or improved tolerance or resistance of the plant to abiotic and/or biotic stress factors.
In another preferred embodiment of the present invention, the increased yield of a plant product is determined by grains, fruits, vegetables, nuts, grains, seeds, wood and/or flowers and combinations thereof.
In another preferred embodiment of the present invention, the improved plant vigor is determined by improved vitality of the plant, improved plant growth, improved plant development, im- proved visual appearance, improved plant stand, less plant verse/lodging, improved emergence, enhanced root growth and/or more developed root system, enhanced nodulation, in particular rhizobial nodulation, bigger leaf blade, bigger size, increased plant weight, increased plant height, increased tiller number, increased number of side shoots, increased number of flowers per plant, increased shoot growth, increased root growth like extensive root system, in- creased yield when grown on poor soils or unfavorable climate, enhanced photosynthetic activity, preferably based on increased stomatal conductance and/or increased CO2 assimilation rate, increased stomatal conductance, increased C02 assimilation rate, enhanced pigment content like chlorophyll content, flowering, earlier fruiting, earlier and improved germination, earlier grain maturity, improved self-defense mechanisms, less non-productive tillers, less dead basal leaves, less input needed such as fertilizers or water, greener leaves, complete maturation under shortened vegetation periods, less fertilizers needed, less seeds needed, easier harvesting, ripening, longer shelf-life, longer panicles, delay of senescence, stronger and/or more productive tillers, better extractability of ingredients, improved quality of seeds for being seeded in the following seasons for seed production, reduced production of ethylene and/or the inhibition of its reception by the plant, growth repression, and combinations thereof.
In another preferred embodiment of the present invention, the enhanced quality of the plant is determined by increased nutrient content, increased protein content, increased content of fatty acids, increased metabolite content, increased carotenoid content, increased sugar content, increased content of amino acids, including essential amino acids, improved nutrient composition, improved protein composition, improved composition of fatty acids, improved metabolite composition, improved carotenoid composition, improved sugar composition, improved amino acids composition, improved or optimal fruit color, improved leaf color, higher storage capacity, and/or higher processability of the harvested products and combinations thereof.
In another preferred embodiment of the present invention, the improved tolerance or resistance of the plant to abiotic stress factors is determined by tolerance and/or resistance to heat stress including temperatures higher than 30°C, temperature conditions causing heat damage to a plant like heat damaged foliage or burnt leaves, cold stress like temperature conditions below 10°C, periods of thawing and freezing, frost, variations in temperature like temperatures conditions that lead to the freezing of water either for extended periods of time or only temporary periods, temperature unusual for the season, drought stress, exposure to cold water, flood, water- logging, wind, sun light, particularly sun light causing signs of scorch, sun burn or similar signs of irradiation and heat stress to the plant, acid or alkaline pH conditions in the soil with pH values lower than pH 5 and/or pH values higher than 9, salt stress like soil salinity, soil erosion, inorganic pollution, soil contamination or soil pollution with chemicals, particularly with heavy metals, preferably chromium, lead, cadmium, arsenic, antimony, mercury, iron, thallium, barium, be- ryllium, polonium, uranium, toxic waste, nuclear waste, acid rain, air pollution, preferably radiation like high UV radiation due to the exposure to the decreasing ozone layer, increased ozone levels, nitrogen oxides and/or sulfur oxides, oxidative stress, organic pollution, oil and/or fuel dumping or spilling, nuclear radiation, contact with sewage, over-fertilization, nutrient deficiencies, herbicide injuries, plant wounding, compaction, natural disasters, preferably tornadoes, hurricanes, wildfires, flooding and combinations thereof.
In another preferred embodiment of the present invention, the improved tolerance or resistance of the plant to biotic stress factors is determined by tolerance and/or resistance to the attack of living organisms including pests like insects, arachnides, nematodes, competing plants like weeds, microorganisms like fungi, bacteria and/or viruses and combinations thereof.
In another preferred embodiment of the present invention, the up-regulated plant gene is selected from pathogenesis related protein P4, xyloglucan-specific fungal endoglucanase inhibitor protein, sucrose synthase, beta-1 ,2-glucanase, chitinase, serine acetyltransferase and combi- nations thereof.
In another preferred embodiment of the present invention, the down-regulated plant gene is selected from gras 1 , lipoxygenase, verticillium wilt disease resistance protein Ve2, AVr9/CF-9 rapidly elicited protein 1 , ripening regulated protein DDTFR10/A, salt responsive protein 2 and combinations thereof.
In another preferred embodiment of the present invention, the plant is an agricultural plant like wheat, rye, barley, triticale, oats, sorghum or rice, beet, sugar beet or fodder beet, fruits like pomes, apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries, blackberries or gooseberries, leguminous plants, such as lentils, peas, alfalfa or soybeans, oil plants, such as rape, oil-seed rape, canola, juncea, linseed, mustard, olives, sunflowers, coconut, cocoa beans, castor oil plants, oil palms, ground nuts or soybeans, cucurbits, such as squashes, cucumber or melons, fiber plants, such as cotton, flax, hemp or jute, citrus fruit, such as oranges, lemons, grapefruits or mandarins, vegetables, such as spinach, lettuce, asparagus, cabbages, carrots, onions, tomatoes, potatoes, cucurbits or paprika, lauraceous plants, such as avocados, cinnamon or camphor, energy and raw material plants, such as corn, soybean, rape, canola, sugar cane or oil palm, corn, tobacco, nuts, coffee, tea, bananas, vines, hop, turf, natu- ral rubber plants or ornamental and forestry plants, such as flowers, shrubs, broad-leaved trees or evergreens, like conifers.
In another preferred embodiment of the present invention, the superabsorbent polymer is selected from polyacrylic acid and cellulose-modified polyacrylic acid or combinations thereof.
In another preferred embodiment of the present invention, the superabsorbent polymer is in the form of granules, having average particle size in the range of 0.1 to 5 mm, preferably from 0.2 to 4 mm. The second specific finding of the present invention is a process for improving the plant health by changing the gene expression in a plant comprising the steps of treating the plant with a superabsorbent polymer, cultivating the plant treated with the superabsorbent polymer, and up- regulating at least one gene of the plant by the addition of the superabsorbent polymer, and/or down-regulating at least one gene of the plant by the addition of the superabsorbent polymer, wherein improved plant health is determined by increased yield of plant product, improved plant vigor, enhanced quality of the plant and/or improved tolerance or resistance of the plant to abiotic and/or biotic stress factors.
The third specific finding of the present invention is a process of determining the change in gene expression in a plant upon treatment of the plant with a superabsorbent polymer, comprising the steps of treating the plant with a superabsorbent polymer, cultivating the plant treated with a superabsorbent polymer, and measuring the up-regulation and/or down-regulation of at least one gene of the plant during and/or after cultivation of the plant. In another preferred embodiment of the present invention, measuring the up-regulation and/or down-regulation of at least one gene of the plant is carried out by use of a transcriptomic assay, preferably by use of a microarray. The present invention is defined by the surprising use of superabsorbent polymers in the cultivation of plants allowing improving the plant's health by systematically changing the gene expression of the plant, wherein the plant health can be for instance determined by increased yield of plant product, improved plant vigor, enhanced quality of the plant and/or improved tolerance or resistance of the plant to abiotic and/or biotic stress factors.
An increase in yield of a plant product can be determined with respect to grains, fruits, vegetables, nuts, grains, seeds, wood and/or flowers and combinations thereof. That is, the amount of plant product obtained after practice of the present invention relative to the amount of plant product obtained when the plant is not treated according to the present invention must be determined based on the above plant products.
An improvement in plant vigor can be determined with respect to improved vitality of the plant, improved plant growth, improved plant development, improved visual appearance, improved plant stand, less plant verse/lodging, improved emergence, enhanced root growth and/or more developed root system, enhanced nodulation, in particular rhizobial nodulation, bigger leaf blade, bigger size, increased plant weight, increased plant height, increased tiller number, increased number of side shoots, increased number of flowers per plant, increased shoot growth, increased root growth like extensive root system, increased yield when grown on poor soils or unfavorable climate, enhanced photosynthetic activity, preferably based on increased stomatal conductance and/or increased CO2 assimilation rate, increased stomatal conductance, increased CO2 assimilation rate, enhanced pigment content like chlorophyll content, flowering, earlier fruiting, earlier and improved germination, earlier grain maturity, improved self-defense response, less non-productive tillers, less dead basal leaves, less input needed such as fertiliz- ers or water, greener leaves, complete maturation under shortened vegetation periods, less fertilizers needed, less seeds needed, easier harvesting, ripening, longer shelf-life, longer panicles, delay of senescence, stronger and/or more productive tillers, better extractability of ingredients, improved quality of seeds for being seeded in the following seasons for seed production, altered or reduced production of ethylene and/or the inhibition of its reception by the plant, growth repression, and combinations thereof.
An enhancement in quality of the plant can be determined by increased nutrient content, increased protein content, increased content of fatty acids, increased metabolite content, increased carotenoid content, increased sugar content, increased content of amino acids, includ- ing essential amino acids, improved nutrient composition, improved protein composition, improved composition of fatty acids, improved metabolite composition, improved carotenoid composition, improved sugar composition, improved amino acids composition, improved or optimal fruit color, improved leaf color, higher storage capacity, and/or higher processability of the harvested products and combinations thereof.
An improvement in tolerance or resistance of the plant to abiotic stress factors can be determined by heat stress including temperatures higher than 30°C, temperature conditions causing heat damage to a plant like heat damaged foliage or burnt leaves, cold stress like temperature conditions below 10°C, periods of thawing and freezing, frost, variations in temperature like temperatures conditions that lead to the freezing of water either for extended periods of time or only temporary periods, temperature unusual for the season, drought stress, exposure to cold water, flood, water-logging, wind, sun light, particularly sun light causing signs of scorch, sun burn or similar signs of irradiation and heat stress to the plant, acid or alkaline pH conditions in the soil with pH values lower than pH 5 and/or pH values higher than 9, salt stress like soil salinity, soil erosion, inorganic pollution, soil contamination or soil pollution with chemicals, particularly with heavy metals, preferably chromium, lead, cadmium, arsenic, antimony, mercury, iron, thallium, barium, beryllium, polonium, uranium, toxic waste, nuclear waste, acid rain, air pollution, prefer- ably radiation like high UV radiation due to the exposure to the decreasing ozone layer, increased ozone levels, nitrogen oxides and/or sulfur oxides, oxidative stress, organic pollution, oil and/or fuel dumping or spilling, nuclear radiation, contact with sewage, over-fertilization, nutrient deficiencies, herbicide injuries, plant wounding, compaction, natural disasters, preferably tornadoes, hurricanes, wildfires, flooding and combinations thereof.
An improvement in tolerance or resistance of the plant to biotic stress factors can be determined by the attack of living organisms including pests like insects, arachnides, nematodes, competing plants like weeds, microorganisms like fungi, bacteria and/or viruses and combinations thereof. The plant's overall health is generally regulated, altered, re-stored and defended by the plant's cellular metabolism which includes the plant's physiological response on the molecular level. Metabolic responses in the plant also include the activation, the increase, the inhibition or decrease of enzymatic activities. Such enzymatic activities are very complex. Clearly, such mechanisms of altered plant metabolism will be largely effected and regulated by changes in the gene expression in the plant.
Therefore, the plant's ability to respond by selective gene activity to defend, alter, improve or restore its health represents an attractive goal in the agricultural industry for improved cultivation of plants, particularly in view of maximizing harvest and crop production as well as the yield of any other product obtainable from the plant.
Consequently, it is of high interest to establish new ways of systematically manipulating the expression of genes involved in the plant's health. The present invention refers to such changes in gene expression with relevance to the plant's health.
Unexpectedly, the present inventors were able to demonstrate that the treatment and/or cultivation of plants in the presence of superabsorbent polymers allows to selectively regulate, alter, re-store and defend plant's health because it is possible to alter, i.e. up-regulate and/or down- regulate plant genes with relevance for the plant's health by the treatment and/or cultivation of plants with superabsorbent polymers. Accordingly, the present invention effectively provides a cultivation approach for plants with superabsorbent polymers that allows artificially influencing the plant's health.
This is a very important technical finding since it proves that the treatment or cultivation of plants with superabsorbent polymers allows manipulating the plant's physiological response and activity already on the genetic level.
Changes in gene expression in the plant include ...
the activation of gene expression of a gene by treatment/cultivation of the plant with a superabsorbent polymer while this gene was not expressed in the absence of the super- absorbent polymer,
- the increase of gene expression, i.e. a gene which is already actively expressed at a certain level in the absence of a superabsorbent polymer shows an increase in gene expression upon treatment and/or cultivation with superabsorbent polymer,
- the inhibition of gene expression upon treatment and/or cultivation of the plant with a superabsorbent polymer, and/or
- the decrease of the level of gene expression upon treatment and/or cultivation with superabsorbent polymer relative to the gene expression level present in the absence of the superabsorbent polymer.
Accordingly, the levels of gene expression of each gene can be activated, increased, inhibited or decreased due to the treatment of the plant with the superabsorbent polymer. Activated or increased levels of gene expression are preferably measured by an up-regulation of a gene of the plant on the level of transcription while inhibited or decreased levels of gene expression are preferably measured by a down-regulation of a gene of the plant on the level of transcription.
The up-regulation of a gene on the transcriptional level as defined by the present invention defines any increase of the level of transcription of DNA to RNA of a particular DNA segment, which is preferably a segment encoding for a gene. The down-regulation of a gene on the tran- scriptional level as defined by the present invention defines any decrease of the level of transcription of DNA to RNA of a particular DNA segment, which is preferably a segment encoding for a gene.
Up-regulation or down-regulation of gene expression in a certain plant treated with superabsor- bent polymer is defined by any increase or decrease in gene expression relative to the level of gene expression in the same plant when being not treated with superabsorbent polymer under otherwise identical growth conditions.
Basically any gene in a given plant can be up-regulated and/or down-regulated with respect to its level of transcription. Therefore, the present invention includes any gene that is up-regulated or down-regulated to a measurable extent upon treatment and/or cultivation of the plant with the superabsorbent polymer. The genes identified in the present invention are for instance classified and defined in the present invention following the system of the Gene Ontology (GO) project which is a well-accepted collaborative effort in this technical field to address the need for consistent descriptions of gene products in different databases. The GO Consortium has meanwhile grown to include many da- tabases, including several of the world's major repositories for plant, animal and microbial genomes. The GO project can be assessed by the following link http://www.geneontology.org/.
The GO project is commonly used in the art based on its three systematic vocabularies (ontologies) to unambiguously define gene products in terms of their associated biological processes, cellular components and molecular functions in a species-independent manner. Accordingly, the Gene Ontology project is herein used as an ontology of defined terms representing gene product properties including three domains: cellular component, the parts of a cell or its extracellular environment; molecular function, the elemental activities of a gene product at the molecular level, such as binding or catalysis; and biological process, operations or sets of molecular events with a defined beginning and end, pertinent to the functioning of integrated living units: cells, tissues, organs, and organisms.
In the following, the gene families as defined below according to the present invention rely on the classification of gene families as defined by the Gene Ontology project and as broadly ac- cepted in the present field.
According to the definitions of the Gene Ontology, the present invention refers to the following first list of genes which are up-regulated in response to treatment and/or cultivation with super- absorbent polymers, preferably by treatment and/or cultivation with a superabsorbent polymer comprising polyacrylic acid:
*GAST1 5*E-26 AT3G02885.1 GASA5 (GAST1 PROTEIN HOMOLOG 5) 5*E-23 (defense response GO:0006952, response to gibberellins stimulus GO:0009739, gibberellic acid mediated signaling GO:0009740); Hypothetical protein (Vitis vinifera) 3*E-51 AT2G03500.1 myb family transcription factor 1*E-28 (ethylene mediated signaling pathway GO:0009873, leaf senescence GO:0010150, regulation of DNA-dependent transcription GO:0006355, response to ethylene stimulus GO:0009723), primary root development GO:0080022, regulation of anthocyanin metabolic process GO:0031537, cytokinin mediated signaling GO:0009736, regulation of chlorophyll biosynthetic process GO:0010380, two-component signal transduction system (phosphorelay) GO:0000160, response to cytokinin stimulus GO:0009735, regulation of stomatal movement GO:00101 19, shoot development GO:0048367); Unnamed protein product (Vitis vinifera) 4*E- 52 AT1G02205.2 CER1 (ECERIFERUM 1 ); Octadecanal decarbonylase 2*E-48 (DNA integration GO:0015074, fatty acid biosynthetic process GO:0006633, oxidation reduction
GO:00551 14, RNA-dependent DNA replication GO:0006278); bHLH transcriptional factor (Ma- lus x domestica) 2*E-1 19 AT3G26744.1 ICE1 (INDUCER OF CBF EXPRESSION 1 ); DNA binding / transcription activator/ transcription factor 3*E-89 (stomatal lineage progression
GO:0010440, positive regulation of transcription GO:0045941 , guard mother cell differentiation GO:0010444, response to cold GO:0009409, multicellular organismal development GO:0007275, response to stress GO:0006950, response to freezing GO:0050826);
P osp oenolpyruvate carboxylase kinase (LOC543633) (signal transduction GO:0007165, protein amino acid phosphorylation GO:0006468); *Ribonuclease (male) (response to wounding GO:000961 1 , jasmonic acid mediated signaling pathway GO:0009867, response to stress GO:0006950, anthocyanin biosynthetic process GO:0009718); Aspartic proteinase nepenthe- sin-1 precursor, putative (Ricinus communis) (proteolysis GO:0006508); Polyphenol oxidase E, chloroplastic; (PPO); Catechol oxidase (metabolic process GO:0008152, oxidation reduction GO:00551 14); Endo-1 ,4-betaglucanase precursor (celullase) (ripening GO:0009835, metabolic process GO:0008152, response to nematode GO:0009624, cell wall organization GO:0007047, pattern specification process GO:0007389, polysaccharide catabolic process GO:0000272, cellulose catabolic process GO:0030245); Ribosomal protein L7 1*E-17 (translation GO:0006412); Arginine decarboxylase (add ) (arginine catabolic process GO:0006527, spermidine biosynthetic process GO:0008295); LOB domain containing protein, putative (Ricinus communis) 9*E- 28 (response to gibberellins stimulus GO:0009739); Uroporphyrin-lll methyltransferase, putative (Ricinus communis) (metabolic process GO:0008152, siroheme biosynthetic process
GO:0019354, cobalamin biosynthetic process GO:0009236, oxidation reduction GO:00551 14, porphyrin biosynthetic process GO:0006779); 40S ribosomal protein S5 (Capsicum annuum) (translation GO:0006412); Chaperonin-60kD, ch60, putative (Ricinus communis) (cellular protein metabolic process GO:0044267, protein folding GO:0006457, response to cadmium ion GO:0046686, response to stress GO:0006950, response to heat GO:0009408, mitochondrion organization GO:0007005); P40-like protein (Solanum tuberosum) 1*E-30 (response to osmotic stress GO:0006970; response to salt stress GO:0009651 , translation GO:0006412), Alpha-tubu- lin (Ceratopteris richardii) (protein polymerization GO:0051258, response to cadmium ion GO:0046686, microtubule-based movement GO:0007018, microtubule-based process
GO:0007017); Transducin family protein / WD-40 repeat family protein (Arabidopsis thaliana) (rRNA processing GO:0006364); Tobacco fibrillarin homolog (Nicotiana tabacum)6*E-31 (RNA processing GO:0006396, RNA methylation GO:0001510, snoRNA metabolic process
GO:0016074, rRNA processing GO:0006364); MYB transcription factor [Camellia sinensis]1*E- 30 (regulation of DNA-dependent transcription GO:0006355, transcription GO:0006350); Homo- cysteine S-methyltransferase (Populus trichocarpa) (amino acid biosynthetic process
GO:0008652, S-methylmethionine cycle GO:0033528, methionine biosynthetic process
GO:0009086); MAR-binding protein [Nicotiana tabacum] (ribosome biogenesis GO:0042254, cell growth GO:0016049, rRNA processing, GO:0006364, snRN P protein import into nucleus GO:0006608); EF 1-alpha (AA 1 -448) (LOC544055) (translation GO:0006412, translational elongation GO:0006414); 6-deoxocastasterone oxidase (Dwarf) (steroid biosynthetic process GO:0006694, lipid biosynthetic process GO:0008610, brassinosteroid biosynthetic process GO:0016132, oxidation reduction GO:00551 14); Wound-induced proteinase inhibitor 2*E-17 (response to wounding GO:000961 1 ); Fasciclin-like arabinogalactan protein 13 (Gossypium hirsu- tum) (threonine biosynthetic process GO:0009088); TRANSPARENT TESTA 12 protein, puta- tive (Ricinus communis) AT1G71 140.1 MATE (efflux family protein transport GO:0006810, pro- anthocyanidin biosynthetic process GO:0010023, flavonoid biosynthetic process GO:0009813, maintenance of seed dormancy GO:0010231 ); Putative ketol-acid reductoisomerase (Capsicum annuum) (valine biosynthetic process GO:0009099, response to cadmium ion GO:0046686, amino acid biosynthetic process GO:0008652, metabolic process GO:0008152, isoleucine biosynthetic process GO:0009097, oxidation reduction GO:0055114, branched chain family amino acid biosynthetic process GO:0009082); Histone H3.2 (nucleosome assembly GO:0006334); Phosphoenolpyruvate carboxylase (Suaeda aralocaspica) (tricarboxylic acid cycle GO:0006099, carbon utilization by fixation of carbon dioxide GO:0015977, oxidation reduction GO:00551 14, photosynthesis GO:0015979); Mitogen-activated protein kinase (Solanum tuberosum) (phosphorylation GO:0016310, response to fungus GO:0009620, systemic acquired resistance, salicylic acid mediated signaling pathway GO:0009862, jasmonic acid and ethylene-dependent systemic resistance GO:0009861 , response to cold GO:0009409, protein amino acid phosphoryla- tion GO:0006468, response to abscisic acid stimulus GO:0009737, hyperosmotic response GO:0006972, defense response GO:0006952, response to salt stress GO:0009651 , jasmonic acid and ethylene-dependent systemic resistance, jasmonic acid mediated signaling pathway GO:0009868, hypotonic salinity response GO:0042539, response to biotic stimulus
GO:0009607); 6-deoxocastasterone oxidase (Dwarf) (steroid biosynthetic process GO:0006694, lipid biosynthetic process GO:0008610, brassinosteroid biosynthetic process, oxidation reduction GO:00551 14); B2-type cyclin dependent kinase (cdkB2) (protein phosphorylation
GO:0006468); Enolase; 2-phospho-D-glycerate hydro-lyase (response to cadmium ion
GO:0046686, response to cold GO:0009409, response to salt stress GO:0009651 , response to light stimulus GO:0009416, glycolysis GO:0006096, response to abscisic acid stimulus
GO:0009737); Iy200-like protein (Solanum tuberosum) (translational elongation GO:0006414); 34 kDa outer mitochondrial membrane protein porin-like protein (Solanum tuberosum)4*E-28 AT5G15090.1 VDAC3 (VOLTAGE DEPENDENT ANION CHANNEL 3); voltage-gated anion Channel (anion transport GO:0006820, transport GO:0006810, plant-type hypersensitive response GO:0009626, defense response GO:0006952, defense response to bacterium
GO:0042742); Putative delta TIP (Nicotiana glauca) AT3G16240.1 DELTATIP (ammonia transporter/ methylammonium transmembrane transporter/ water channel transport GO:0006810); 40S ribosomal protein S4 (translation GO:0006412); Hypothetical protein (Vitis vinifera) AT2G34640.1 PTAC12 (PLASTID TRANSCRIPTIONALLY ACTIVE 12) (transcription from plas- tid promoter GO:0042793, positive regulation of DNA-dependent transcription GO:0045893);
According to the definitions of the Gene Ontology, the present invention further refers to the following second list of genes which are up-regulated in response to treatment and/or cultivation with superabsorbent polymers, preferably by treatment and/or cultivation with a superabsorbent polymer comprising lignocellulose-modified polyacrylic acid:
Pathogenesis-related protein P4 (P4) (defense response to fungus GO:0050832, xenobiotic metabolic process GO:0006805, response to biotic stimulus GO:0009607); Xyloglucan-specific fungal endoglucanase inhibitor protein precursor (Lycopersicon esculentum) (ACI25 /// Xegip) (proteolysis GO:0006508); PR protein (PR1 b1 ) (defense response to fungus GO:0050832, xe- nobiotic metabolic process GO:0006805, response to biotic stimulus GO:0009607); Pathogenesis-related protein P2 (PR-P2) (defense response to fungus GO:0050832, chitin catabolic process GO:0006032, cell wall catabolic process GO:0016998, defense response to bacterium GO:0042742, response to biotic stimulus GO:0009607); Putative copia-like polyprotein (Lyco- persicon esculentum) 7*E-28 (DNA integration GO:0015074); CBF/DREB-like transcription factor 1 (Poncirus trifoliate)9*E-17 AT1G12610.1 DDF1 (DWARF AND DELAYED FLOWERING 1 ); DNA binding / sequence-specific DNA binding / transcription factor 3*E-17 (abscisic acid mediated signaling GO:0009738, response to water deprivation GO:0009414, response to cold GO:0009409, regulation of DNA-dependent transcription GO:0006950, cell growth
GO:0016049, regulation of timing of transition from vegetative to reproductive phase
GO:0048510, response to salt stress GO:0009651 , cold acclimation GO:0009631); Aldo/keto reductase AKR (Manihot esculenta) AT1 G60710.1 ATB2; Oxidoreductase (cellular response to stress GO:0033554, auxin mediated signaling pathway GO:0009734, oxidation reduction GO:00551 14); Proteinase inhibitor I (ER1 ) (response to wounding GO:000961 1); Putative transcriptional activator CBF1 (Lycopersicon esculentum) (LeCBFI protein) AT4G25480.1 DREB1A (DEHYDRATION RESPONSE ELEM ENT B1A); DNA binding / transcription activator/ transcription factor (abscisic acid mediated signaling GO:0009738, response to water deprivation GO:0009414, response to cold GO:0009409, regulation of transcription, DNA-dependent GO:0006355, cell growth GO:0016049, regulation of timing of transition from vegetative to reproductive phase GO:0048510, cold acclimation GO:0009631 ); Wound-induced protein (Lycopersicon esculentum) (pi 1 protein) (defense response to fungus GO:0050832, cell wall catabolic process GO:0016998, defense response to bacterium GO:0042742, systemic acquired resistance GO:0009627, response to ethylene stimulus GO:0009723, chitin catabolic process GO:0006032, response to salt stress GO:0009651 , response to virus GO:0009615); Sucrose synthase(Solanum lycopersicum) (sus3) (biosynthetic process GO:0009058, sucrose metabolic process GO:0005985);
According to the definitions of the Gene Ontology, the present invention further refers to the fol- lowing third list of genes which are up-regulated in response to treatment and/or cultivation with superabsorbent polymers, preferably by treatment and/or cultivation with a superabsorbent polymer comprising polyacrylic acid and lignocellulose-modified polyacrylic acid:
Beta-1 ,3-glucanase (LOC543987) (defense response GO:0006952, carbohydrate metabolic process GO:0005975); NP24 protein precursor (LOC543979) (defense response to fungus GO:0050832, response to stress GO:0006950, response to biotic stimulus GO:0009607);
Chitinase (LOC544148) (defense response GO:0006952, polysaccharide catabolic process GO:0000272, carbohydrate metabolic process GO:0005975, response to biotic stimulus GO:0009607); Pathogenesis-related protein PR (PR23) (pr p23) (defense response to fungus GO:0050832, response to stress GO:0006950, xenobiotic metabolic process GO:0006805, response to biotic stimulus GO:0009607); Psi14B protein (psi14B) (metabolic process
GO:0008152); Serine acetyltransferase [Nicotiana plumbaginifolia) (cysteine biosynthetic process from serine GO:0006535); Serine acetyltransferase [Nicotiana plumbaginifolia) (response to cadmium ion GO:0046686, cellular response to sulfate starvation GO:0009970, amino acid biosynthetic process GO:0008652, response to cold GO:0009409, cysteine biosynthetic process from serine GO:0006535); According to the definitions of the Gene Ontology, the present invention further refers to the following fourth list of genes which are down-regulated in response to treatment and/or cultivation with superabsorbent polymers, preferably by treatment and/or cultivation with a superabsorbent polymer comprising polyacrylic acid:
Hexose transporter protein (LOC543728) (Transmembrane transport GO:0055085);
Glutamate dehydrogenase (legdhl ) (cellular amino acid metabolic process GO:0006520); ADP- glucose pyrophosphorylase large subunit (AGP-S1) (glycogen biosynthetic process
GO:0005978); Pre-pro-cysteine proteinase (LOC544144) (proteolysis GO:0006508); GRAS6 protein (GRAS6) (regulation of transcription GO:0045449); GRAS2 (response to chitin
GO:0010200, regulation of DNA-dependent transcription GO:0006355, photomorphogenesis GO:0009640); Protein of unknown function (LOC544143) (exocytosis GO:0006887); Glutathione S-transferase (Petunia x hybrida) 3*E-26 (response to oxidative stress GO:0006979, toxin catabolic process GO:0009407, response to stress GO:0006950); DNAJ-like protein (DNAJ) (protein folding GO:0006457); Zinc-finger protein (Solanum chacoense) AT1G27730.1 STZ (salt tolerance zinc finger) (nucleic acid binding, transcription factor, regulation of DNA-dependent transcription, transcription repressor, zinc ion binding GO:0006355); Chalcone synthase (LOC778294) (metabolic process GO:0008152, biosynthetic process GO:0009058, flavonoid biosynthetic process GO:0009813); Subtilisin-like protease (Nicotiana tabacum) AT5G67360.1 ARA12; serine-type endopeptidase (seed coat development GO:0010214, mucilage extrusion from seed coat GO:0080001 , proteolysis GO:0006508, mucilage metabolic process during seed coat development GO:0048359, negative regulation of catalytic activity GO:0043086); Phos- phoethanolamine N-methyltransferase (unidimensional cell growth GO:0009826, choline biosynthetic process GO:0042425, pollen development GO:0009555, metabolic process
GO:0008152, response to stress GO:0006950, phospholipid biosynthetic process GO:0008654, post-embryonic root development GO:0048528, phosphatidylcholine biosynthetic process GO:0006656, lipid biosynthetic process GO:0008610); Late embryogenesis (Lea)-like protein (LOC544157) (response to desiccation GO:0009269); Dihydroflavonol-reductase(LOC544150) (metabolic process GO:0008152, flavonoid biosynthetic process, cellular metabolic process GO:0044237, oxidation reduction GO:00551 14); Jasmonic acid 3 (LEJA3) (regulation of transcription GO:0045449); 20G-Fe(ll) oxidoreductase (Populus trichocarpa) (short-day photoperi- odism, flowering GO:0048575, flower development GO:0009908, unidimensional cell growth GO:0009826, gibberellin metabolic process GO:0009685, response to gibberellin stimulus GO:0009739, oxidation reduction GO:00551 14, gibberellic acid mediated signaling
GO:0009740, gibberellin biosynthetic process GO:0009686); Ethylene-responsive late embryo- genesis-like protein (ER5) (response to desiccation GO:0009269); RSH-like protein (Capsicum annuum) (guanosine tetraphosphate metabolic process GO:0015969); CYP72A58 (Nicotiana tabacum) (oxidation reduction GO:0055114, alkaloid metabolic process GO:0009820); Putative DnaJ protein (Camellia sinensis) AT1 G80920.1 J8; heat shock protein binding / unfolded protein binding (protein folding GO:0006457, response to unfolded protein GO:0006986, DNA replication GO:0006260, response to stress GO:0006950, response to heat GO:0009408); Chitinase (LOC544149) (polysaccharide catabolic process GO:0000272, carbohydrate metabolic process GO:0005975, chitin catabolic process GO:0006032, defense response GO:0006952, response to biotic stimulus, cell wall macromolecule catabolic process GO:0016998); SBT1 protein (SBT1) (proteolysis GO:0006508, negative regulation of catalytic activity GO:0043086); Wound induced protein (LOC544072); l-box binding factor (negative regulation of cell growth
GO:0030308, protein folding GO:0006457, G2 phase of mitotic cell cycle GO:0000085, DNA replication GO:0006260, regulation of transcription GO:0045449); Cell Division Protein AAA ATPase family, putative, expressed (Oryza sativa (japonica cultivargroup) (mitochondrial respiratory chain complex IV assembly GO:0033617, mitochondrial respiratory chain complex I assembly GO:0032981 , mitochondrial cytochrome bc(1 ) complex assembly GO:0034551 , cell division GO:0051301 , sensory perception of sound GO:0007605, mitochondrion organization GO:0007005); Transporter-like protein (Solanum tuberosum) (response to drug GO:0042493, sterol biosynthetic process GO:0016126, phospholipid transport GO:0015914); Putative allan- toinase (Solanum tuberosum) (purine base metabolic process GO:0006144, allantoin catabolic process GO:0000256); Putative allantoinase (Solanum tuberosum) (allantoin assimilation pathway GO:0009442, purine base metabolic process GO:0006144, allantoin catabolic process GO:0000256); EIL3 protein (EIL3); Tonoplast intrinsic protein 1 ;2 (Mimosa pudica) (water homeostasis GO:0030104, response to stress GO:0006950, defense response to bacterium
GO:0042742, urea transport GO:0015840); Senescence associated protein (Nicotiana taba- cum) AT4G35770.1 SEN1 (SENESCENCE 1 ); RUB1 conjugating enzyme (RCE1 ) (protein modification process GO:0006464, post-translational protein modification GO:0043687, regulation of protein metabolic process GO:0051246); Glutaredoxin (Solanum tuberosum) (electron transport chain GO:0022900, cell redox homeostasis, GO:0045454); Proline dehydrogenase (PDH) (gluta- mate biosynthetic process GO:0006537, proline catabolic process GO:0006562, oxidation reduction GO:0055114); Ammonium transporter 1 member 1 (LeAMTI) (protein polymerization GO:0051258, ammonium transport GO:0015696, response to nematode GO:0009624, me- thylammonium transport GO:0015843); Xyloglycan endotransglycosylase (tXET-B2) (carbohydrate metabolic process GO:0005975, cellular glucan metabolic process GO:0006073); Phy- tophthorainhibited protease 1 (pipl) (proteolysis GO:0006508); Cytosolic ascorbate peroxidase 2 (APX2) (response to oxidative stress GO:0006979, oxidation reduction GO:0055114); Beta- 1 ,3-glucanase (LOC543986) (carbohydrate metabolic process GO:0005975, defense response GO:0006952); PR-5x (PR-5) (defense response to fungus GO:0050832, defense response GO:0006952, response to stress GO:0006950, xenobiotic metabolic process GO:0006805, response to biotic stimulus GO:0009607);
According to the definitions of the Gene Ontology, the present invention further refers to the fol- lowing fifth list of genes which are down-regulated in response to treatment and/or cultivation with superabsorbent polymers, preferably by treatment and/or cultivation with a superabsorbent polymer comprising lignocellulose-modified polyacrylic acid:
Photoperiod responsive protein (Solanum tuberosum subsp. Andigena) AT3G 18710.1 PUB29 (PLANT U-BOX 29) (ubiquitin-protein ligase response to chitin GO:0010200, protein ubiquitina- tion GO:0016567, modification-dependent protein catabolic process GO:0019941 ); Avr9/Cf-9 rapidly elicited protein 231 (Nicotiana tabacum) AT3G28340.1 , GATL10 Galacturonosyltransfer- ase-like 10), polygalacturonate 4-alphagalacturonosyltransferase/ transferase (transferring hex- osyl groups, transferase activity GO:0016757);
According to the definitions of the Gene Ontology, the present invention further refers to the fol- lowing sixth list of genes which are down-regulated in response to treatment and/or cultivation with superabsorbent polymers, preferably by treatment and/or cultivation with a superabsorbent polymer comprising polyacrylic acid and lignocellulose-modified polyacrylic acid:
GRAS1 protein (GRAS1 ), (response to chitin GO:0010200, regulation of DNA-dependent tran- scription GO:0006355, regulation of transcription GO:0045449, photomorphogenesis
GO:0009640); protein similar to UBC9 (UBIQUITIN CONJUGATING ENZYM E 9), ubiquitin-pro- tein ligase isoform 2 (Vitis vinifera), (post-translational protein modification GO:0043687, ubiqui- tin-dependent protein catabolic processGO:000651 1 , modification- dependent protein catabolic process GO:0019941 , regulation of protein metabolic process GO:0051246); ABA 8 -hydrox- ylase CYP707A1 (Solanum tuberosum) (response to water deprivation GO:0009414, response to stress GO:0006950, abscisic acid catabolic process GO:0046345, abscisic acid metabolic process GO:0009687 oxidation reduction GO:00551 14, response to red light GO:00101 14, release of seed from dormancy GO:0048838, response to red or far red light GO:0009639); Hypothetical protein (Vitis vinifera) AT1 G47670.1 amino acid transporter family protein (membrane GO:0016020, integral to membrane GO:0016021 ); Protein SSM 1 , putative (Ricinus communis) (metabolic process GO:0008152); Putative protein kinase (Solanum demissum) (defense response to fungus GO:0050832, protein amino acid autophosphorylation GO:0046777); Whitefly- induced gp91-phox (Wfi1 ) (oxidation reduction GO:0055114); WRKY transcription factor lld-2 (WRKYIId-2) (response to chitin GO:0010200, regulation of DNA-dependent transcription GO:0006355, defense response to bacterium GO:0042742, regulation of transcription
GO:0045449); NAK-type protein kinase (Nicotiana tabacum) (defense response to fungus GO:0050832, protein amino acid autophosphorylation GO:0046777); Lipoxygenase (loxD) (fatty acid biosynthetic process GO:0006633, oxidation reduction GO:0055114, oxylipin biosynthetic process GO:0031408, lipid biosynthetic process GO:0008610); WRKY-like transcription factor (Solanum peruvianum) (defense response to fungus GO:0050832, camalexin biosynthetic process GO:0010120, response to water deprivation GO:0009414, response to cold GO:0009409, response to osmotic stress GO:0006970, regulation of DNA-dependent transcription
GO:0006355, defense response to bacterium GO:0042742, response to heat GO:0009408, response to chitin GO:0010200, transcription GO:0006350, response to salt stressGO: 0009651 , regulation of transcription GO:0045449); WRKY transcription factor lld-1 splice variant 1/WRKY transcription factor lld-1 splice variant 2 (LOC100125891/WR KYIId-1) (response to chitin GO:0010200, regulation of DNA-dependent transcription GO:0006355, transcription
GO:0006350, defense response to bacterium GO:0042742, regulation of transcription
GO:0045449); Nine-cisepoxycarotenoid dioxygenase (LOC544163) (response to water depriva- tion GO:0009414, response to osmotic stress GO:0006970, hyperosmotic salinity response GO:0042538, oxidation reduction GO:00551 14, abscisic acid biosynthetic process); ZPT2-13 (Petunia x hybrida) AT3G46070.1 zinc finger (C2H2 type) family protein (regulation of transcrip- tion GO:0045449); Verticillium wilt disease resistance protein Ve2 (Ve2) (Brassinosteroid homeostasis GO:0010268, xylem and phloem pattern formation GO:0010051 , leaf vascular tissue pattern formation GO:0010305, auxin mediated signaling pathway GO:0009734, protein amino acid phosphorylation GO:0006468, brassinosteroid mediated signaling GO:0009742, response to UV-B GO:0010224, positive regulation of flower development GO:0009911 , detection of brassinosteroid stimulus GO:0009729, phloem transport GO:0010233); Myb-related transcription factor (THM16) (Phenylpropanoid biosynthetic process GO:0009699, response to UV GO:000941 1 , regulation of DNA-dependent transcription GO:0006355); 1-aminocyclopropane- 1-carboxylate synthase ACS6 (Solanum lycopersicum) (response to wounding GO:000961 1 , ripening GO:0009835, response to auxin stimulus GO:0009733, response to mechanical stimulus GO:0009612, induction of apoptosis by oxidative stress GO:0008631 , response to jasmonic acid stimulus GO:0009753, ethylene biosynthetic process GO:0009693, biosynthetic process GO:0009058, defense response GO:0006952); Cell wall peroxidase (Capsicum annuum) AT1G14550.1 anionic peroxidase, putative (response to oxidative stress GO:0006979, hydro- gen peroxide catabolic process GO:0042744, oxidation reduction GO:0055114); NAC domain protein, IPR003441 (Populus trichocarpa) (response to wounding GO:0009611 , response to auxin stimulus GO:0009733, somatic stem cell division GO:0048103, regulation of DNA-dependent transcription GO:0006355, response to chitin GO:0010200, positive regulation of asymmetric cell division GO:0045770, root cap development GO:0048829, negative regulation of ab- scisic acid mediated signaling GO:0009788); Auxin-regulated protein (Lycopersicon esculentum) (LOC543701); Photoperiod responsive protein (Solanum tuberosum subsp. Andigena) (response to chitin GO:0010200, protein ubiquitination GO:0016567, modification-dependent protein catabolic process GO:0019941 ); Avr9/Cf-9 rapidly elicited protein 1 (Nicotiana tabacum) AT5G47230.1 ERF5 (ETHYLENE RESPONSIVE ELEMENT BINDING FACTOR 5), DNA-bind- ing /transcription activator (response to chitin GO:0010200, ethylene mediated signaling pathway GO:0009873, response to cold GO:0009409, defense response GO:0006952, regulation of DNA-dependent transcription GO:0006355); Double WRKY type transfactor (Solanum tuberosum) (defense response to fungus GO:0050832, camalexin biosynthetic process
GO:0010120, response to water deprivation GO:0009414, response to cold GO:0009409, re- sponse to osmotic stress GO:0006970, regulation of DNA-dependent transcription
GO:0006355, defense response to bacterium GO:0042742, response to heat GO:0009408, response to chitin GO:0010200, response to salt stress GO:0009651 , regulation of transcription GO:0045449); Les.5654.1.S1_at Predicted protein (Populus trichocarpa) AT3G16720.1 ATL2; protein binding / zinc-ion binding (response to chitin GO:0010200, defense response
GO:0006952, modification-dependent protein catabolic process GO:0019941 , protein complex assembly GO:0006461 ); Unnamed protein product [Vitis vinifera] AT1 G70740.1 protein kinase family protein (plant-type hypersensitive response GO:0009626, protein amino acid phosphorylation GO:0006468, response to molecule of bacterial origin GO:0002237); Ripening regulated protein DDTFR10/A DDTFR10/A (Lycopersicon esculentum) AT5G61600.1 ethylene-responsive element-binding family protein (response to chitin GO:0010200, ethylene mediated signaling pathway GO:0009873, response to cold GO:0009409, defense response GO:0006952, regulation of DNA-dependent transcription GO:0006355); RNA polymerase beta subunit (Solanum lycopersicum) (DNA-dependent transcription GO:0006351); N-hydroxycinnamoyl-CoA:tyramine N-hydroxycinnamoyl transferase THT1-3 THT1-3 (Lycopersicon esculentum) (metabolic process GO:0008152); Nam-like protein 1 [Petunia x hybrida] AT3G49530.1 anac062 (Arabidopsis NAC domain containing protein 62), transcription factor (response to chitin GO:0010200, regulation of DNA-dependent transcription GO:0006355, defense response to virus GO:0051607); NAD(P)H-quinone oxidoreductase subunit 1 , chloroplastic; NAD(P)H dehydrogenase subunit 1 ; NDH subunit 1 ; NADH plastoquinone oxidoreductase subunit 1 1*E-25 (oxidation reduction GO:00551 14); Ring domain containing protein (Capsicum annuum) AT4G03510.1 RMA1 ; protein binding / ubiquitin-protein ligase/ zinc ion binding (integral to membrane GO:0016021 , salt responsive protein 2 (Solanum lycopersicum); WRKY transcription factor-30 (Capsicum an- nuum) (positive regulation of transcription GO:0045941 , response to chitin GO:0010200, regulation of DNA-dependent transcription GO:0006355, regulation of defense response GO:0031347, defense response to bacterium, incompatible interaction GO:0009816);
As a general finding, the following genes are preferred target genes for the up-regulation and/or down-regulation upon treatment of the corresponding plants with superabsorbent polymers and during cultivation of the plants.
Genes according to the present invention that are particularly preferred with respect to the up- regulation of gene activity caused by treatment of the plant with superabsorbent polymer, espe- cially by the treatment with polyacrylic acid and/or lignocellulose-modified polyacrylic acid, and cultivation of the plant are as follows:
Beta-1 ,3-glucanase (LOC543987), NP24 protein precursor (LOC543979), Chitinase
LOC544148), Pathogenesis-related protein PR (PR23), Psi14B protein (psi14B), Serine Acetyltransferase (Nicotiana plumbaginifolia), Serine acetyltransferase (Nicotiana plumbagini- folia), TPSI1 protein (TPSI 1 ), GAST1 5*E-26 GASA5 (GAST1 PROTEIN HOMOLOG 5) 5*E-23, Anthocyanin acyltransferase 2*E-10, Hypothetical protein (Vitis vinifera) 3*E-51 myb family transcription factor 1*E-28, Unnamed protein product (Vitis vinifera) 4*E-52 CER1 (ECERIFERUM 1 ); Octadecanal decarbonylase 2*E-48, bHLH transcriptional factor (Malus x domestica) 2*E- 1 19 ICE1 (INDUCER OF CBF EXPRESSION 1 ); DNA binding / transcription activator/ transcription factor 3*E-89, Phosphoenolpyruvate carboxylase kinase (LOC543633), Neryl diphosphate synthase 1 (NDPS1 ), Ribonuclease (male), Aspartic proteinase nepenthesin-1 precursor, putative (Ricinus ommunis), Polyphenol oxidase E, chloroplastic; (PPO); Catechol oxidase, Endo- 1 ,4-betaglucanase Precursor (celullase), Ribosomal protein L7 1*E-17, Arginine decarboxylase (add ), LOB domain containing protein, putative (Ricinus communis) 9*E-28, Uroporphyrin-lll methyltransferase, putative (Ricinus communis), 40S ribosomal protein S5 (Capsicum annuum), Chaperonin-60kD, ch60, putative (Ricinus communis), P40-like protein (Solanum tuberosum) 1*E-30, Alpha-tubulin (Ceratopteris richardii), Transducin family protein / WD-40 repeat family protein, Tobacco fibrillarin homolog (Nicotiana t tabacum) 6*E-31 , MYB transcription factor [Ca- mellia sinensis]1*E-30, Homocysteine smethyltransferase
(Populus trichocarpa), LeArcAI protein, MAR-binding protein [Nicotiana tabacum], EF 1 -alpha (AA 1 -448) (LOC544055), 6-deoxocastasterone oxidase (Dwarf), Extensin (LOC544158), Wound-induced proteinase inhibitor 2*E-17, Fasciclin-like arabinogalactan protein 13 (Gossypi- umhirsutum), TRANSPARENT TESTA 12 protein, putative (Ricinus communis) MATE efflux family protein, Putative ketol-acid reductoisomerase (Capsicum annuum), Histone H3.2, Phos- phoenolpyruvate carboxylase (Suaeda aralocaspica), Mitogen-activated protein kinase (Sola- num tuberosum), 6-deoxocastasterone oxidase (Dwarf), B2-type cyclin dependent kinase
(cdkB2), Enolase; 2-phospho-D-glycerate hydro-lyase, Iy200-like protein (Solanum tuberosum), Osmotin-like protein (LOC543971 ), 34 kDa outer mitochondrial membrane protein porin-like protein (Solanum tuberosum)4*E-28 VDAC3 (VOLTAGE DEPENDENT ANION CHANNEL 3); voltage-gated anion channel, Putative delta TIP (Nicotiana glauca) DELTATIP; ammonia trans- porter, methylammonium transmembrane transporter/ water Channel, 40S ribosomal protein S4, Hypothetical protein (Vitis vinifera) PTAC12 (PLASTID TRANSCRIPTIONALLY ACTIVE12), Pathogenesisrelated protein P4 (P4), Xyloglucan-specific fungal endoglucanase inhibitor protein precursor (Lycopersicon esculentum)(ACI25 /// Xegip), PR protein (PR1 b1 ), Pathogenesis related protein P2 (PR-P2), Putative copia-like polyprotein (Lycopersicon esculentum) 7*E-28, CBF/DREB-like transcription factor 1 (Poncirus trifoliate)9*E-17 DDF1 (DWARF AND DELAYED FLOWERING 1 ); DNA binding / sequence specific DN A binding / transcription factor 3*E-17, Aldo/keto reductase AKR (Manihot esculenta) ATB2; oxidoreductase, Proteinase inhibitor I (ER1 ), Putative transcriptional activator CBF1 (Lycopersicon esculentum) (LeCBFI protein) DREB1 A (DEHYDRATION RESPONSE ELEMENT B1A); DNA binding / transcription activator/ transcription factor, Wound-induced protein (Lycopersicon esculentum) (pi 1 protein), Sucrose synthase(Solanum lycopersicum) (sus3).
Genes according to the present invention that are particularly preferred with respect to the down-regulation of gene activity caused by treatment of the plant with superabsorbent polymer, especially by the treatment with polyacrylic acid and/or lignocellulose-modified polyacrylic acid, and cultivation of the plant are as follows:
Photoperiod responsive protein (Solanum tuberosum subsp. Andigena) PUB29 (PLANT U-BOX 29); ubiquitin-protein ligase, Avr9/Cf-9 rapidly elicited protein 231 (Nicotiana tabacum) GATL10 (Galacturonosyltransferase-like 10); polygalacturonate 4-alphagalacturonosyltransferase/ transferase, transferring hexosyl groups, Hexose transporter protein (LOC543728), Glutamate dehydrogenase (legdhl ), ADP-glucose pyrophosphorylase large subunit (AGP-S1 ), Pre-pro-cysteine proteinase (LOC544144), GRAS6 protein (GRAS6), GRAS2, Protein of unknown function (LOC544143), Glutathione Stransferase (Petunia x hybrida) 3*E-26, DNAJ-like protein (DNAJ), zinc-finger protein (Solanum chacoense) STZ (salt tolerance zinc finger); nucleic acid binding; transcription factor; transcription repressor; zinc ion binding, Chalcone synthase(LOC778294), protease (Nicotiana tabacum) ARA12; serine-type endopeptidase, Phosphoethanolamine N-me- thyltransferase, *Late embryogenesis (Lea)-like protein (LOC544157), Dihydroflavonol 4-reduc- tase (LOC544150), Jasmonic acid 3 (LEJA3), 20G-Fe(ll) oxidoreductase (Populus trichocarpa), Ethyleneresponsive late embryogenesis-like protein(ER5), RSH-like protein (Capsicum annuum), CYP72A58 (Nicotiana tabacum), Putative DnaJ protein (Camellia sinensis) J8; heat shock protein, binding / unfolded protein binding, Chitinase (LOC544149), SBT1 protein (SBT1 ), Wound induced protein (LOC544072), l-box binding factor, Cell Division Protein AAA ATPase family, putative, expressed (Oryza sativa (japonica cultivargroup)), Transporter-like protein (So- lanum tuberosum), Putative allantoinase (Solanum tuberosum), Putative allantoinase (Solanum tuberosum), EIL3 protein (EIL3), Tonoplast intrinsic protein 1 ;2 (Mimosa pudica), Senescence- associated protein (Nicotiana tabacum) SEN1 (SENESCENCE 1), RUB1 conjugating enzyme (RCE1 ), RUB1 conjugating enzyme (RCE1 ), Proline Dehydrogenase (PDH), Ammonium transporter 1 member 1 (LeAMTI ), Xyloglycan endotransglycosylase (tXET-B2), Phytophthorainhib- ited protease 1 (pipl ), Cytosolic ascorbate peroxidase 2 (APX2), Beta-1 ,3-glucanase
(LOC543986), PR-5x (PR-5), GRAS1 protein (GRAS1 ), Similar to UBC9 (UBIQUITIN CONJUGATING ENZYME 9); ubiquitin-protein ligase isoform 2 (Vitis vinifera), ABA 8'-hydroxylase CYP707A1 (Solanum tuberosum), Hypothetical protein (Vitis vinifera) aminoacid transporter family protein, Protein SSM 1 , putative (Ricinus communis), Putative protein kinase (Solanum demissum), Whitefly-induced gp91-phox (Wfi1 ), WRKY transcription factor lld-2 (WRKYIId-2), NAK-type protein kinase (Nicotiana tabacum), Lipoxygenase (loxD), WRKY-like transcription factor (Solanum peruvianum), WRKY transcription factor lld-1 splice variant 1/WRKY transcrip- tion factor lld-1 splice variant 2 (LOC100125891/WR KYIId-1), Nine-cisepoxycarotenoid dioxy- genase (LOC544163), ZPT2-13 (Petunia xhybrida)zinc finger (C2H2 type)family protein, Verti- cillium wilt disease resistance protein Ve2 (Ve2), Myb-related transcription factor (THM 16), 1- aminocyclopropane-1-carboxylate synthase ACS6 (Solanum lycopersicum), Cell wall peroxidase (Capsicum annuum) anionic peroxidase, putative, NAC domain protein, I PR003441 (Popu- lus trichocarpa), Auxin-regulated protein (Lycopersicon esculentum) (LOC543701), Photoperiod responsive protein (Solanum tuberosum subsp. Andigena), Avr9/Cf-9 rapidly elicited protein 1 (Nicotiana tabacum)ERF5 (ETHYLENE RESPONSIVE ELEMENT BINDING FACTOR 5); DNA binding / transcription activator/ transcription factor, Double WRKY type transfactor (Solanum tuberosum), Predicted protein (Populus trichocarpa) ATL2; protein binding / zinc ion binding, Unnamed protein product [Vitis vinifera] protein kinase family Protein, Ripening regulated protein DDTFR10/A DDTFR10/A (Lycopersicon esculentum ethylene-responsive element-binding family protein, *RNA polymerase beta subunit (Solanum lycopersicum), Nhydroxycinnamoyl- CoA:tyramine Nhydroxycinnamoyl transferase THT1-3 THT1-3 (Lycopersicon esculentum), Nam-like protein 1 [Petunia x hybrida] anac062 (Arabidopsis NAC, domain containing protein 62); transcription factor, *NAD(P)H-quinone oxidoreductase subunit 1 , chloroplastic; NAD(P)H dehydrogenase subunit 1 ; NDH subunit 1 ; NADHplastoquinone oxidoreductase subunit 1 *E- 25, Ring domain containing protein (Capsicum annuum) RMA1 ; protein binding / ubiquitin-protein ligase/ zinc ion binding, Salt responsive protein 2 (Solanum lycopersicum), WRKY transcription factor-30 (Capsicum annuum).
Especially preferred genes according to the present invention that are up-regulated are selected from pathogenesis related protein P4 (especially relevant for the plant's defense/self-defense response), xyloglucan-specific fungal endoglucanase inhibitor protein (especially relevant for the plant's anti-fungal response), sucrose synthase (especially relevant for the biosynthesis of sugars, improved sugar content and/or sugar composition and/or the plant's general health), beta-1 ,3-glucanase (especially relevant for improved plant defense/self-defense response), chi- tinase (especially relevant for the plant's anti-fungal response), serine acetyltransferase (especially relevant for improved amino acid and protein content, particularly cysteine, and/or the plant's health in general), and combinations thereof.
Especially preferred genes according to the present invention that are down-regulated are selected from gras 1 (especially preferred for growth repression), lipoxygenase (especially pre- ferred for improved and/or increased content of fatty acids and/or plant's general health), verti- cillium wilt disease resistance protein Ve2 (especially relevant for the plant's self-defense response), ACS6 (especially relevant for altered, especially reduced, production of ethylene and/or inhibition of its reception by the plant), cell wall peroxidase (especially relevant for the plant's response to oxidative stress). AVr9/CF-9 rapidly elicited protein 1 (especially relevant for altered, especially reduced, production of ethylene and/or inhibition of its reception by the plant, ripening and/or flowering), ripening regulated protein DDTFR10/A (especially relevant for the plant's response to fungi, altered, especially reduced, production of ethylene and/or inhibition of its reception by the plant, ripening, flowering and the plant's general health), salt responsive protein 2 (especially relevant for the plant's resistance and/or tolerance to salt stress like soil sa- linity and/or plant's general health) and combinations thereof.
Superabsorbent polymers according to the present invention are well-known synthetic particulate organic polymers which are solid and hydrophilic, which are insoluble in water and which are capable of absorbing a multiple of their weight of water or aqueous solutions, thereby form- ing a water containing polymer gel, but which on drying again form particles. Superabsorbent polymers according to the present invention are generally capable of absorbing at least 100 parts by weight of water per one part by weight of superabsorbent polymer (deionised water at 25°C, pH 7.5, 1 bar). The amount of water or aqueous solution a superabsorbent polymer is capable of absorbing, is also termed as absorption capacity or maximal absorption. For purposes of the invention, superabsorbent polymers are preferred which have an absorption capacity for deionised water (pH 7.5, 25°C, 1 bar) of at least 150 g/g, e.g. 150 to 500 g/g, in particular 200 to 500 g/g, more preferably 300 to 500 g/g of superabsorbent polymers. For purposes of the invention, superabsorbent polymers are preferred which have an absorption capacity for a 0.1 % by weight aqueous solution of sodium chloride of at least 100 g/g, in particular 100 to 300 g/g of superabsorbent polymer (pH 7.5, 25°C, 1 bar). The maximal absorption or absorption capacity can be determined by routine methods known e.g. from F. L. Buchholz et a\. "Modern Super- absorbent Polymer Technology", Wiley-VCH 1998, p. 153 (absorbent capacity method) or EP 993 337, example 6. The superabsorbent polymer material is preferably in the form of granules. Preferred super- absorbent polymer granules are those which have a moderate swelling rate, i.e. superabsor- bents, wherein the time required to achieve 60% of the maximal absorption is at least 10 minutes, in particular from 10 to 100 minutes. These values can be determined according to standard methods as described in F. L. Buchholz et al., loc. cit, p. 154 (swelling kinetics meth- ods).
The superabsorbent polymers may be nonionic or ionic crosslinked polymers. For the purpose of the invention, the superabsorbent polymer is preferably selected from crosslinked anionic su- perabsorbent polymers, in particular from covalently crosslinked anionic superabsorbent polymers. A survey of suitable superabsorbent polymers is e.g. given in F. L. Buchholz et al., loc. cit., p. 11-14. Crosslinked anionic superabsorbent polymers are crosslinked polymers which comprise anionic functional groups or acidic groups, which can be neutralized in water, e.g. sulfonic acid groups (SO3H or SO3 ), phosphonate groups (PO3H2 or PO3 ) or carboxylate groups (CO2H or CO2 ). These polymers are in principle obtainable by a process which comprises co- polymerizing a monoethylenically unsaturated acidic monomer and a crosslinking monomer optionally in the presence of a grafting base and optionally in the presence of one or more further neutral monoethylenically unsaturated monomers. In preferred superabsorbent polymers the carboxylate groups make up at least 80 mol-%, in particular at least 95 mol-%, of the acidic groups.
Suitable acidic monomers include monoethylenically unsaturated mono- and dicarboxylic acids having preferably from 3 to 8 carbon atoms such as acrylic acid, methacrylic acid, ethacrylic acid, [alpha]-chloroacrylic acid, crotonic acid, maleic acid, maleic anhydride, itaconic acid, citra- conic acid, mesaconic acid, glutaconic acid, aconitic acid and fumaric acid; monoesters of monoethylenically unsaturated dicarboxylic acids having from 4 to 10 and preferably from 4 to 6 carbon atoms, for example monoesters of maleic acid such as monomethyl maleate; monoeth- ylenically unsaturated sulfonic acids and phosphonic acids, for example vinylsulfonic acid, al- lylsulfonic acid, sulfoethyl acrylate, sulfoethyl methacrylate, sulfopropyl acrylate, sulfopropyl methacrylate, 2-hydroxy-3-acryloyloxypropylsulfonic acid, 2-hydroxy-3-methacryloyloxypropyl- sulfonic acid, styrenesulfonic acid, 2-acrylamido-2-methylpropanesulfonic acid, vinylphosphonic acid and allylphosphonic acid and the salts, especially the sodium, potassium and ammonium salts, of these acids. The acidic monomers usually make up at least 15 %, by weight, preferably at least 20 % by weight, of the superabsorbent polymer, e.g. 15 to 99.9 % by weight, in particular from 20 to 99.8 % by weight, based on the acidic form of the anionic superabsorbent polymer. Preference is given to crosslinked anionic superabsorbent polymers, wherein the polymerized acidic monomers comprise at least one monoethylenically unsaturated carboxylic acid CA or a salt thereof. Preferably the monoethylenically unsaturated carboxylic acid CA or the salt thereof accounts for at least 80 mol-%, in particular at least 95 mol-% of the total amount of polymerized acidic monomeres.
Useful crosslinking monomers include compounds having at least two, for example 2, 3, 4 or 5, ethylenically unsaturated double bonds in the molecule. These compounds are also referred to as crosslinker monomers. Examples of crosslinker monomers are N,N'-methylenebisacrylamide, polyethylene glycol diacrylates and polyethylene glycol dimethacrylates, each derived from poly- ethylene glycols having a molecular weight from 106 to 8500 and preferably from 400 to 2000, trimethylolpropane triacrylate, trimethylol propane trimethacrylate, ethylene glycol diacrylate, ethylene glycol dimethacrylate, propylene glycol diacrylate, propylene glycol dimethacrylate, butanediol diacrylate, butanediol dimethacrylate, hexanediol diacrylate, hexanediol dimethacrylate, diethylene glycol diacrylate, diethylene glycol dimethacrylate, triethylene glycol diacrylate, trieth- ylene glycol dimethacrylate, dipropylene glycol diacrylate, dipropylene glycol dimethacrylate, tripropylene glycol diacrylate, tripropylene glycol dimethacrylate, allyl methacrylate, diacrylates and dimethacrylates of block copolymers of ethylene oxide and propylene oxide, di-, tri-, tetra- or pentaacrylated or -methacrylated polyhydric alcohols, such as glycerol, trimethylol propane, pentaerythritol or dipentaerythritol, esters of monoethylenicaily unsaturated carboxylic acids with ethylenically unsaturated alcohols such as allyl alcohol, cyclohexenol and dicyclopentenyl alcohol, e.g. allyl acrylate and allyl methacrylate, also triallylamine, dialkyldiallylammonium halides such as dimethyldiallylammonium chloride and diethyldiallylammonium chloride, tetraallyleth- ylenediamine, divinylbenzene, diallyl phthalate, polyethylene glycol divinyl ethers of polyethylene glycols having a molecular weight from 106 to 4000, trimethylolpropane diallyl ether, bu- tanediol divinyl ether, pentaerythritol triallyl ether, reaction products of 1 mol of ethylene glycol diglycidyl ether or polyethylene glycol diglycidyl ether with 2 mol of pentaerythritol triallyl ether or allyl alcohol, and divinylethyleneurea. The amount of crosslinker monomer is generally in the range from 0.05 to 20% by weight, preferably in the range from 0.1 to 10% by weight and especially in the range from 0.2 to 5% by weight, based on the weight of the superabsorbent polymer in the acidic form.
Suitable grafting bases can be of natural or synthetic origin. They include oligo- and polysac- charides such as starches, i.e. native starches from the group consisting of corn (maize) starch, potato starch, wheat starch, rice starch, tapioca starch, sorghum starch, manioca starch, pea starch or mixtures thereof, modified starches, starch degradation products, for example oxidative^, enzymatically or hydrolytically degraded starches, dextrins, for example roast dextrins, and also lower oligo- and polysaccharides, for example cyclodextrins having from 4 to 8 ring members. Useful oligo- and polysaccharides further include cellulose and also starch and cellulose derivatives. It is also possible to use polyvinyl alcohols, homo- and copolymers of N-vi- nylpyrrolidone, polyamines, polyamides, hydrophilic polyesters or polyalkylene oxides, especially polyethylene oxide and polypropylene oxide as a grafting base. The amount of grafting base may be up to 50 % by weight of the weight of the superabsorbent polymer in the acidic form, e.g. from 1 to 50 % by weight.
The monomers forming the superabsorbent polymer may also contain neutral monoethylenicaily unsaturated monomers which do not have a polymerizable group or an acidic group. Examples are monoethylenicaily unsaturated hydrophilic monomers, i.e. monomers having a water solubil- ity of at least 80 g/l at 25°C, 1 bar, including hydroxyalkyl esters of monoethylenicaily unsaturated monocarboxylic acids, e.g. the hydroxyalkyl acrylates and methacrylates, such as hydrox- yethyl acrylate and hydroxyethylmethacrylate, amides of monoethylenicaily unsaturated monocarboxylic acids such as acrylamide and methacrylamide, monomers having a polyether group, such as vinyl, allyl and methallyl ethers of polyethylene glycols and esters of monoethylenicaily unsaturated monocarboxylic acids and polyethers, such as polyethylenglykol acrylate and poly- ethyleneglycol methacrylate. In a preferred embodiment of the invention the neutral monomers make up from 10 to 84.9 % by weight, in particular from 20 to 79.9 % by weight of the super- absorbent polymer in the acidic form. Preferred anionic superabsorbent polymers have a moderate charge density, i.e. the amount of acidic groups in the superabsorbent polymer is preferably from 0.1 to 1.1 mol per 100 g of superabsorbent polymer, in particular form 0.2 to 1 mol per 100 g of superabsorbent polymer, based on the weight of the superabsorbent polymer in the acidic form.
In a very preferred embodiment of the invention, the water absorbent polymer is a crosslinked copolymer or graft copolymer of ethylenically unsaturated monomers M which comprise at least one monoethylenically unsaturated carboxylic acid CA or a salt thereof at least one amide of a monoethylenically unsaturated acid (monomer AM), and a crosslinking monomer in polymerized form.
Suitable monoethylenically unsaturated carboxylic acids CA comprise monoethylenically unsaturated mono-carboxylic acids having 3 to 8 carbon atoms, such as acrylic acid and meth- acrylic acid, and monoethylenically unsaturated dicarboxylic acids having from 4 to 8 carbon atoms, such as maleic acid, fumaric acid, itaconic acid and citraconic acid. Suitable salts of monoethylenically unsaturated carboxylic acids CA comprise the alkali metal salts and the ammonium salts, in particular the potassium or sodium salts. Preferred monoethylenically unsaturated carboxylic acids CA include mono-carboxylic acids having 3 to 8 carbon atoms, in particular acrylic acid and methacrylic acid and the salts thereof, in particular the alkalimetal salts thereof, and more preferably the alkali metal salts of acrylic acid, especially the sodium salt and the potassium salt of acrylic acid.
Suitable amides of monoethylenically unsaturated acids are the amides of monoethylenically unsaturated mono-carboxylic acids having 3 to 8 carbon atoms, in particular acrylamide and methacrylamide.
In this embodiment, the water absorbent polymer is preferably a covalently crosslinked copolymer, i.e. it contains a crosslinking monomer as defined above. Preferably, the carboxylic acid CA and the amide AM make up at least 80% by weight, e.g. from 80 to 99.95% by weight, and more preferably at least 90% by weight, e.g. from 90 to 99.9% by weight, of the ethylenically unsaturated monomers M forming the superabsorbent polymer. In this embodiment the crosslinking monomer will generally make up from 0.05 to 20% by weight, in particular from 0.1 to 10% by weight of the monomers M.
In a particular preferred embodiment, the monomers M comprise at least 90% by weight, e.g. from 90 to 99.9% by weight, based on the total weight of monomers M, of a mixture of acrylic acid or a salt thereof, in particular an alkali metal salt thereof, more preferably the potassium salt of acrylic acid, and acrylamide.
In particular, the superabsorbent polymer comprises in polymerized form:
15 to 89.9 %, in particular 20 to 79.8 % by weight of at least one carboxylic acid CA or a salt thereof, preferably acrylic acid or a salt thereof, in particular an alkalimetal salt thereof, more preferably the potassium salt of acrylic acid (calculated in the acidic form),
10 to 84.9 % in particular 20 to 79.8 % by weight of at least one amide AM, preferably an amide of a monoethylenically unsaturated mono-carboxylic acid having 3 to 8 carbon atoms, in particular acrylamide; and
0.1 to 10 %, in particular 0.2 to 5 % by weight of at least one crosslinker monomer, wherein the % by weight are based on the superabsorbent polymer in the acidic form, the amount of mono- mers AM and CA making up at least 90 %, e.g. 90 to 99.9 % of the monomers forming the superabsorbent polymer.
Suitable superabsorbent polymers of this type are known in the art, e.g. from US 4,417,992, US 3,669,103 and WO 01/25493. They are also commercially available, e.g. from SNF SA., France, under the trademark Aquasorb(R), e.g. Aquasorb(R) 3005 KL, 3005 KM, 3005 L and 3005 M.
In another very preferred embodiment of the invention, the water absorbent polymer is a cross- linked copolymer or graft copolymer of ethylenically unsaturated monomers M which comprise at least 80 % by weight, e.g. from 80 to 99.95% by weight, preferably at least 90 % by weight, e.g. from 90 to 99.9% by weight, based on the total amount of monomers M, of a mixture of at least one monoethylenically unsaturated carboxylic acid CA, preferably acrylic and at least one alkali metal salt of a monoethylenically unsaturated carboxylic acid CA, preferably a potassium salt or sodium salt thereof, more preferably the potassium salt or sodium salt of acrylic acid. In this embodiment, the water absorbent polymer is preferably a covalently crosslinked copolymer. In this embodiment the crosslinking monomer will generally make up from 0.05 to 20% by weight, in particular from 0.1 to 10% by weight of the monomers M.
In particular, the superabsorbent polymer of this embodiment comprises in polymerized form: 15 to 89.9 %, in particular 20 to 79.8 % by weight of at least one carboxylic acid CA, preferably acrylic acid;
10 to 84.9 % in particular 20 to 79.8 % by weight of at least one or a salt thereof, in particular an alkalimetal salt thereof, more preferably the potassium salt of acrylic acid (calculated in the acidic form);
0.1 to 10 %, in particular 0.2 to 5 % by weight of at least one crosslinker monomer, wherein the % by weight are based on the superabsorbent polymer in the acidic form, the amount of carboxylic acid CA and the salt of CA making up at least 90 %, e.g. 90 to 99.9 % of the monomers forming the superabsorbent polymer.
Very preferred superabsorbent polymers according to the present invention include polyacrylic acid and lignocellulose-modified polyacrylic acid, especially the alkali metal salts and the ammonium salts, in particular the potassium or sodium salts thereof. Such type of polymers are commercially available, e.g. from BASF AG under the trade names Luquasorb(R), e.g.
Luquasorb(R) 1280, Luquasorb(R) 1060, Luquasorb(R) 1 160, Luquasorb(R) 1061 and Hy- Sorb(R) and Phytogel and Vitola® of BASF, respectively.
Preferably, the average particle size of the superabsorbent polymer granules ranges from 0.1 to 5 mm, preferably from 0.2 to 5 mm, in particular from 0.5 to 4 mm. The average particle size is the weight average of the diameter which may be determined by microscopy or by sieving anal- ysis, preferably sieving analysis.
In a preferred embodiment of the invention the superabsorbent polymer granules are surface crosslinked (see F. L. Buchholz, loc. cit. pp. 97 to 103, and the literature cited therein). In the surface crosslinked polymer granules some of the functional groups in the surface region of the superabsorbent polymer granules have been crosslinked by reaction with polyfunctional compounds. Surface crosslinking can be a covalent or ionic crosslinking.
Apart from surface crosslinking, the surface of superabsorbent polymer granules, which are used for preparing the pesticide composition, may have been treated with additives to reduce their dustiness and/or to ease their flow, including treatment with anti-caking additives such as particulate silica, in particular fumed silica, optionally in combination with polyols, or quaternary surfactants.
The superabsorbent polymer material is dried using the established technology in the art includ- ing spray drying and the like. Drying will be carried out until a water content of not more than 20 % wt, preferably not more than 10 wt% and even more preferably until a water content of 0.5 to 20 wt%, most preferably 1 to 15 wt% is achieved.
The dried superabsorbent polymer material is preferably classified and milled using the estab- lished technology of this field to obtain the superabsorbent polymer in the form of granules.
The plant is treated with the superabsorbent polymer by applying superabsorbent polymer in an amount of about 1 to 1000 kg/ha culture soil, preferably 50 to 800 kg/ha culture soil and most preferably 100 to 500 kg/ha culture soil.
The usual agricultural methods can be used for applying the superabsorbent polymer to the culture soil either by simply spreading the superabsorbent over the soil or mixing it with the soil before applying a layer of superabsorbent polymer and soil to the ground or working the super- absorbent polymer into the soil using conventional agricultural techniques. The latter operations can be optionally combined with the application of fertilizer.
The superabsorbent polymer is applied to the soil either before the seed is brought to the soil, or before the seedling is planted into the ground or during the cultivation of the already growing plant.
Cultivation of the plant in the presence of the superabsorbent polymer includes leaving the su- perabsorbent polymer on the soil where the plant is grown for extended periods of time, prefera- bly for the whole growth cycle until harvesting the desired crop or fruit or plant product.
The term "plant" generally comprises all plants of economic importance and/or men- grown plants. They are preferably selected from agricultural, silvicultural and ornamental plants, more preferably agricultural plants and silvicultural plants, utmost preferably agricultural plants. The term "plant (or plants)" is a synonym of the term "crop" which is to be understood as a plant of economic importance and/or a men-grown plant. The term "plant" as used herein includes all parts of a plant such as germinating seeds, emerging seedlings, herbaceous vegetation as well as established woody plants including all belowground portions (such as the roots) and above- ground portions.
The plants to be treated according to the invention are selected from the group consisting of agricultural, silvicultural, ornamental and horticultural plants, each in its natural or genetically modified form, more preferably from agricultural plants. In one embodiment, the aforementioned methods for increasing the health of a plant and/or increasing the control of undesirable vegetation and/or increasing the control of phytopathogenic fungi comprises treating the plant propagules, preferably the seeds of an agricultural, horticultural, ornamental or silvicultural plant selected from the group consisting of transgenic or non- transgenic plants with a mixture according to the present invention.
In one embodiment, the plant to be treated according to the method of the invention is an agricultural plant. Agricultural plants are plants of which a part or all is harvested or cultivated on a commercial scale or which serve as an important source of feed, food, fibres (e.g. cotton, linen), combustibles (e.g. wood, bioethanol, biodiesel, biomass) or other chemical compounds. Agricul- tural plants also horticultural plants, i.e. plants grown in gardens (and not on fields), such as certain fruits and vegetables. Preferred agricultural plants are for example cereals, e.g. wheat, rye, barley, triticale, oats, sorghum or rice; beet, e.g. sugar beet or fodder beet; fruits, such as pomes, stone fruits or soft fruits, e.g. apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries, blackberries or gooseberries; leguminous plants, such as lentils, peas, al- falfa or soybeans; oil plants, such as rape, oil-seed rape, canola, juncea (Brassica juncea), linseed, mustard, olives, sunflowers, coconut, cocoa beans, castor oil plants, oil palms, ground nuts or soybeans; cucurbits, such as squashes, cucumber or melons; fiber plants, such as cotton, flax, hemp or jute; citrus fruit, such as oranges, lemons, grapefruits or mandarins; vegetables, such as spinach, lettuce, asparagus, cabbages, carrots, onions, tomatoes, potatoes, cu- curbits or paprika; lauraceous plants, such as avocados, cinnamon or camphor; energy and raw material plants, such as corn, soybean, rape, canola, sugar cane or oil palm; corn; tobacco; nuts; coffee; tea; bananas; vines (table grapes and grape juice grape vines); hop; turf; natural rubber plants or ornamental and forestry plants, such as flowers, shrubs, broad-leaved trees or evergreens, e.g. conifers; and on the plant propagation material, such as seeds, and the crop material of these plants.
More preferred agricultural plants are field crops, such as potatoes, sugar beets, cereals such as wheat, rye, barley, triticale, oats, sorghum, rice, corn, cotton, rape, sunflowers, oilseed rape, juncea and canola, legumes such as soybeans, peas and beans (fieldbeans), lentil, sugar cane, turf; ornamentals; or vegetables, such as cucumbers, tomatoes, or onions, leeks, lettuce, squashes, alfalfa, clover most preferred agricultural plants are potatoes, beans (fieldbeans), alfalfa, sugar cane, turf, sugar beets, cereals such as wheat, rye, triticale, barley, oats, sorghum, rice, corn, cotton, soybeans, oilseed rape, canola, juncea, sunflower, sugar cane, peas, lentils and alfalfa and utmost preferred plants are selected from soybean, wheat, sunflowers, canola, juncea, corn, cotton, sugar cane, peas, lentils and alfalfa and oilseed rape.
In another preferred embodiment of the present invention, the plants to be treated are selected from soybean, wheat, sunflower, canola, oilseed rape, corn, cotton, sugar cane, juncea, peas, lentils and alfalfa. The utmost preferred plant is soybean.
In an especially preferred embodiment of the present invention, the plants to be treated are selected from wheat, barley, corn, soybean, rice, canola and sunflower. In one embodiment, the plant to be treated according to the method of the invention is a horticultural plant. The term "horticultural plants" are to be understood as plants which are commonly used in horticulture - e. g. the cultivation of ornamentals, vegetables and/or fruits. Examples for ornamentals are turf, geranium, pelargonia, petunia, begonia and fuchsia. Examples for vegetables are potatoes, tomatoes, peppers, cucurbits, cucumbers, melons, watermelons, garlic, onions, carrots, cabbage, beans, peas and lettuce and more preferably from tomatoes, onions, peas and lettuce. Tomatoes are especially preferred.
Examples for fruits are apples, pears, cherries, strawberry, citrus, peaches, apricots and blue- berries.
In one embodiment, the plant to be treated according to the method of the invention is an ornamental plant. Ornamental plants" are plants which are commonly used in gardening, e.g. in parks, gardens and on balconies. Examples are turf, geranium, pelargonia, petunia, begonia and fuchsia.
In one embodiment, the plant to be treated according to the method of the invention is a silvicultural plants. The term "silvicultural plant" is to be understood as trees, more specifically trees used in reforestation or industrial plantations. Industrial plantations generally serve for the com- mercial production of forest products, such as wood, pulp, paper, rubber tree, Christmas trees, or young trees for gardening purposes. Examples for silvicultural plants are conifers, like pines, in particular Pinus spec, fir and spruce, eucalyptus, tropical trees like teak, rubber tree, oil palm, willow (Salix), in particular SaNx spec, poplar (cottonwood), in particular Populus spec, beech, in particular Fagus spec, birch, oil palm and oak.
The present invention also includes genetically modified plants. Genetically modified plants are plants, which genetic material has been so modified by the use of recombinant DNA techniques that under natural circumstances cannot readily be obtained by cross breeding, mutations or natural recombination. Typically, one or more genes have been integrated into the genetic material of a genetically modified plant in order to improve certain properties of the plant. Such genetic modifications also include but are not limited to targeted post-transtional modification of protein(s), oligo- or polypeptides e. g. by glycosylation or polymer additions such as prenylated, acetylated or farnesylated moieties or PEG moieties.
Plants that have been modified by breeding, mutagenesis or genetic engineering, e. g. have been rendered tolerant to applications of specific classes of herbicides, such as hydroxy- phenylpyruvate dioxygenase (HPPD) inhibitors; acetolactate synthase (ALS) inhibitors, such as sulfonyl ureas (see e. g. US 6,222,100, WO 01/82685, WO 00/26390, WO 97/41218,
WO 98/02526, WO 98/02527, WO 04/106529, WO 05/20673, WO 03/14357, WO 03/13225, WO 03/14356, WO 04/16073) or imidazolinones (see e. g. US 6,222,100, WO 01/82685, WO 00/026390, WO 97/41218, WO 98/002526, WO 98/02527, WO 04/106529, WO 05/20673, WO 03/014357, WO 03/13225, WO 03/14356, WO 04/16073); enolpyruvylshikimate-3-phos- phate synthase (EPSPS) inhibitors, such as glyphosate (see e. g. WO 92/00377); glutamine synthetase (GS) inhibitors, such as glufosinate (see e.g. EP-A 242 236, EP-A 242 246) or oxynil herbicides (see e. g. US 5,559,024) as a result of conventional methods of breeding or genetic engineering. Several cultivated plants have been rendered tolerant to herbicides by conventional methods of breeding (mutagenesis), e. g. Clearfield® summer rape (Canola, BASF SE, Germany) being tolerant to imidazolinones, e. g. imazamox. Genetic engineering methods have been used to render cultivated plants such as soybean, cotton, corn, beets and rape, tolerant to herbicides such as glyphosate and glufosinate, some of which are commercially available under the trade names Round up Ready® (glyphosate-tolerant, Monsanto, U.S.A.) and LibertyLink® (glufosinate-tolerant, Bayer CropScience, Germany).
Furthermore, plants are also covered that are by the use of recombinant DNA techniques capable to synthesize one or more insecticidal proteins, especially those known from the bacterial genus Bacillus, particularly from Bacillus thuringiensis, such as δ-endotoxins, e. g. CrylA(b), CrylA(c), CrylF, CrylF(a2), CryllA(b), CrylllA, CrylllB(bl) or Cry9c; vegetative insecticidal pro- teins (VIP), e. g. VIP1 , VIP2, VIP3 or VIP3A; insecticidal proteins of bacteria colonizing nematodes, e. g. Photorhabdus spp. or Xenorhabcfus spp.; toxins produced by animals, such as scorpion toxins, arachnid toxins, wasp toxins, or other insect-specific neurotoxins; toxins produced by fungi, such Streptomycetes toxins, plant lectins, such as pea or barley lectins; agglutinins; proteinase inhibitors, such as trypsin inhibitors, serine protease inhibitors, patatin, cystatin or papain inhibitors; ribosome-inactivating proteins (RIP), such as ricin, maize-RIP, abrin, luffin, saporin or bryodin; steroid metabolism enzymes, such as 3-hydroxysteroid oxidase, ecdyster- oid-IDP-glycosyl-transferase, cholesterol oxidases, ecdysone inhibitors or HMG-CoA-reductase; ion channel blockers, such as blockers of sodium or calcium channels; juvenile hormone esterase; diuretic hormone receptors (helicokinin receptors); stilben synthase, bibenzyl synthase, chitinases or glucanases. In the context of the present invention these insecticidal proteins or toxins are to be understood expressly also as pre-toxins, hybrid proteins, truncated or otherwise modified proteins. Hybrid proteins are characterized by a new combination of protein domains, (see, e. g. WO 02/015701 ). Further examples of such toxins or genetically modified plants capable of synthesizing such toxins are disclosed, e. g., in EP-A 374 753, WO 93/007278,
WO 95/34656, EP-A 427 529, EP-A 451 878, WO 03/18810 und WO 03/52073. The methods for producing such genetically modified plants are generally known to the person skilled in the art and are described, e. g. in the publications mentioned above. These insecticidal proteins contained in the genetically modified plants impart to the plants producing these proteins tolerance to harmful pests from all taxonomic groups of athropods, especially to beetles (Coelop- tera), two-winged insects (Diptera), and moths (Lepidoptera) and to nematodes (Nematoda). Genetically modified plants capable to synthesize one or more insecticidal proteins are, e. g., described in the publications mentioned above, and some of which are commercially available such as YieldGard® (corn cultivars producing the Cry1 Ab toxin), YieldGard® Plus (corn cultivars producing CrylAb and Cry3Bb1 toxins), Starlink® (corn cultivars producing the Cry9c toxin), Herculex® RW (corn cultivars producing Cry34Ab1 , Cry35Ab1 and the enzyme Phosphinothri- cin-N-Acetyltransferase [PAT]); NuCOTN® 33B (cotton cultivars producing the Cry1 Ac toxin), Bollgard® I (cotton cultivars producing the CrylAc toxin), Bollgard® II (cotton cultivars producing Cry1 Ac and Cry2Ab2 toxins); VIPCOT® (cotton cultivars producing a VIP-toxin); NewLeaf® (potato cultivars producing the Cry3A toxin); Bt-Xtra®, NatureGard®, KnockOut®, BiteGard®, Pro- tecta®, Bt1 1 (e. g. Agrisure® CB) and Bt176 from Syngenta Seeds SAS, France, (corn cultivars producing the CrylAb toxin and PAT enyzme), MIR604 from Syngenta Seeds SAS, France (corn cultivars producing a modified version of the Cry3A toxin, c.f. WO 03/018810), MON 863 from Monsanto Europe S.A., Belgium (corn cultivars producing the Cry3Bb1 toxin), IPC 531 from Monsanto Europe S.A., Belgium (cotton cultivars producing a modified version of the CrylAc toxin) and 1507 from Pioneer Overseas Corporation, Belgium (corn cultivars producing the Cry1 F toxin and PAT enzyme).
Furthermore, plants are also covered that are by the use of recombinant DNA techniques capable to synthesize one or more proteins to increase the resistance or tolerance of those plants to bacterial, viral or fungal pathogens. Examples of such proteins are the so-called "pathogenesis- related proteins" (PR proteins, see, e. g. EP-A 392 225), plant disease resistance genes (e. g. potato cultivars, which express resistance genes acting against Phytophthora infestans derived from the mexican wild potato Solanum bulbocastanum) or T4-lysozym (e. g. potato cultivars capable of synthesizing these proteins with increased resistance against bacteria such as Erwinia amylvora). The methods for producing such genetically modified plants are generally known to the person skilled in the art and are described, e. g. in the publications mentioned above.
Furthermore, plants are also covered that are by the use of recombinant DNA techniques capable to synthesize one or more proteins to increase the productivity (e. g. bio mass production, grain yield, starch content, oil content or protein content), tolerance to drought, salinity or other growth-limiting environmental factors or tolerance to pests and fungal, bacterial or viral pathogens of those plants.
Furthermore, plants are also covered that contain by the use of recombinant DNA techniques a modified amount of substances of content or new substances of content, specifically to improve human or animal nutrition, e. g. oil crops that produce health-promoting long-chain omega-3 fatty acids or unsaturated omega-9 fatty acids (e. g. Nexera® rape, DOW Agro Sciences, Canada). Furthermore, plants are also covered that contain by the use of recombinant DNA techniques a modified amount of substances of content or new substances of content, specifically to improve raw material production, e. g. potatoes that produce increased amounts of amylopectin (e. g. Amflora® potato, BASF SE, Germany). The up-regulation and down-regulation of the genes in the plant following treatment and cultivation of the plant with the superabsorbent polymer is measured by a differential gene expression approach.
In the differential gene expression approach the activity of a selected gene in the plant is meas- ured in a quantitative manner by determining the amount of RNA, preferably mRNA, present in a sample of the plant during or after treatment of the plant with the superabsorbent polymer. Similarly, in a control experiment, the amount of RNA, preferably mRNA, present in the same sample of the plant not treated with superabsorbent polymer but subjected otherwise to the same cultivation conditions is determined for comparative purposes. The relative increase or decrease in amount of RNA, preferably mRNA, is the parameter indicating the increase or decrease of gene activity.
The methods for measuring such differential gene expression are known in the art. A conventional differential (gene) display, particularly for mRNA, can be used to identify genes which are up-regulated or down-regulated in activity while quantitative real time PCR allows the quantitative measurement of up-regulation and down-regulation of genes. Quantitative real time PCR and related techniques can be carried out by the skilled person based on the established standard protocols. Preferably, DNA microarray technology, as known in the art and commercially available, is used for measuring the up-regulation and down-regulation of genes of interest. One preferred approach is based on the GeneChip® tomato genome array as available from Affymetrix. The methods for using such DNA microarray as well as the use of additional instrumentation and supporting kits can be found in the manufacturer's instructions, for instance in the GeneChip® Expression Analysis manual of Affimetrix, and in various textbook publications and review articles. In a further embodiment, the present invention is directed to a process of changing the gene expression in a plant comprising the steps of treating the plant with a superabsorbent polymer, cultivating the plant treated with the superabsorbent polymer, and up-regulating at least one gene of the plant by the addition of the superabsorbent polymer, and/or down-regulating at least one gene of the plant by the addition of the superabsorbent polymer.
In a further preferred embodiment of this process, the plant is cultivated in the presence of an external stress factor. In a further embodiment, the present invention is directed to a process of determining the change in gene expression in a plant upon treatment of the plant with a superabsorbent polymer, comprising the steps of treating the plant with a superabsorbent polymer, cultivating the plant treated with a superabsorbent polymer, optionally in the presence of an external stress factor, and measuring the up-regulation and/or down-regulation of at least one gene of the plant during and/or after cultivation of the plant.
In a further preferred embodiment of this process, measuring the up-regulation and/or down- regulation of at least one gene of the plant is carried out by use of a transcriptomic assay, preferably by use of a microarray.
Further embodiments according to the present invention include the following embodiments: Embodiment 1 :
Process for improving plant health by changing the gene expression in a plant characterized in that the plant is treated and/or cultivated with a superabsorbent polymer and at least one gene of the plant is up-regulated by the addition of the superabsorbent polymer, and/or at least one gene is down-regulated by the addition of the superabsorbent polymer, wherein improved plant health is determined by increased yield of plant product, improved plant vigor, enhanced quality of the plant and/or improved tolerance or resistance of the plant to abiotic and/or biotic stress factors.
Embodiment 2:
Process according to the embodiment 1 , wherein the increased yield of a plant product is determined by grains, fruits, vegetables, nuts, grains, seeds, wood and/or flowers and combinations thereof.
Embodiment 3:
Process according to embodiment 1 , wherein improved plant vigor is determined by improved vitality of the plant, improved plant growth, improved plant development, improved visual ap- pearance, improved plant stand, less plant verse/lodging, improved emergence, enhanced root growth and/or more developed root system, enhanced nodulation, in particular rhizobial nodula- tion, bigger leaf blade, bigger size, increased plant weight, increased plant height, increased tiller number, increased number of side shoots, increased number of flowers per plant, increased shoot growth, increased root growth like extensive root system, increased yield when grown on poor soils or unfavorable climate, enhanced photosynthetic activity, preferably based on increased stomatal conductance and/or increased CO2 assimilation rate, increased stomatal conductance, increased CO2 assimilation rate, enhanced pigment content like chlorophyll content, flowering, earlier fruiting, earlier and improved germination, earlier grain maturity, improved self-defense mechanisms, less non-productive tillers, less dead basal leaves, less input needed such as fertilizers or water, greener leaves, complete maturation under shortened vegetation periods, less fertilizers needed, less seeds needed, easier harvesting, ripening, longer shelf-life, longer panicles, delay of senescence, stronger and/or more productive tillers, better extractabil- ity of ingredients, improved quality of seeds for being seeded in the following seasons for seed production, reduced production of ethylene and/or the inhibition of its reception by the plant, growth repression, and combinations thereof. Embodiment 4:
Process according to embodiment 1 , wherein enhanced quality of the plant is determined by increased nutrient content, increased protein content, increased content of fatty acids, increased metabolite content, increased carotenoid content, increased sugar content, increased content of amino acids, including essential amino acids, improved nutrient composition, improved protein composition, improved composition of fatty acids, improved metabolite composition, improved carotenoid composition, improved sugar composition, improved amino acids composition, improved or optimal fruit color, improved leaf color, higher storage capacity, and/or higher pro- cessability of the harvested products and combinations thereof. Embodiment 5:
Process according to embodiment 1 , wherein improved tolerance or resistance of the plant to abiotic stress factors is determined by improved tolerance or resistance to heat stress including temperatures higher than 30°C, temperature conditions causing heat damage to a plant like heat damaged foliage or burnt leaves, cold stress like temperature conditions below 10°C, peri- ods of thawing and freezing, frost, variations in temperature like temperatures conditions that lead to the freezing of water either for extended periods of time or only temporary periods, temperature unusual for the season, drought stress, exposure to cold water, flood, water-logging, wind, sun light, particularly sun light causing signs of scorch, sun burn or similar signs of irradiation and heat stress to the plant, acid or alkaline pH conditions in the soil with pH values lower than pH 5 and/or pH values higher than 9, salt stress like soil salinity, soil erosion, inorganic pollution, soil contamination or soil pollution with chemicals, particularly with heavy metals, preferably chromium, lead, cadmium, arsenic, antimony, mercury, iron, thallium, barium, beryllium, polonium, uranium, toxic waste, nuclear waste, acid rain, air pollution, preferably radiation like high UV radiation due to the exposure to the decreasing ozone layer, increased ozone levels, nitro- gen oxides and/or sulfur oxides, oxidative stress, organic pollution, oil and/or fuel dumping or spilling, nuclear radiation, contact with sewage, over-fertilization, nutrient deficiencies, herbicide injuries, plant wounding, compaction, natural disasters, preferably tornadoes, hurricanes, wildfires, flooding and combinations thereof. Embodiment 6:
Process according to embodiment 1 , wherein improved tolerance or resistance of the plant to biotic stress factors is determined by the tolerance or resistance to the attack of living organ- isms including pests like insects, arachnides, nematodes, competing plants like weeds, microorganisms like fungi, bacteria and/or viruses and combinations thereof.
Embodiment 7:
Process according to embodiments 1 to 6, wherein the up-regulated plant gene is selected from pathogenesis related protein P4, xyloglucan-specific fungal endoglucanase inhibitor protein, sucrose synthase, beta-1 ,2-glucanase, chitinase, serine acetyltransferase and combinations thereof.
Embodiment 8:
Process according to embodiments 1 to 6, wherein the down-regulated plant gene is selected from gras 1 , lipoxygenase, verticillium wilt disease resistance protein Ve2, AVr9/CF-9 rapidly elicited protein 1 , ripening regulated protein DDTFR10/A, salt responsive protein 2 and combinations thereof. Embodiment 9:
Process according to embodiments 1 to 8, wherein the plant is an agricultural plant like wheat, rye, barley, triticale, oats, sorghum or rice, beet, sugar beet or fodder beet, fruits like pomes, apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries, blackberries or gooseberries, leguminous plants, such as lentils, peas, alfalfa or soybeans, oil plants, such as rape, oil-seed rape, canola, juncea, linseed, mustard, olives, sunflowers, coconut, cocoa beans, castor oil plants, oil palms, ground nuts or soybeans, cucurbits, such as squashes, cucumber or melons, fiber plants, such as cotton, flax, hemp or jute, citrus fruit, such as oranges, lemons, grapefruits or mandarins, vegetables, such as spinach, lettuce, asparagus, cabbages, carrots, onions, tomatoes, potatoes, cucurbits or paprika, lauraceous plants, such as avocados, cinna- mon or camphor, energy and raw material plants, such as corn, soybean, rape, canola, sugar cane or oil palm, corn, tobacco, nuts, coffee, tea, bananas, vines, hop, turf, natural rubber plants or ornamental and forestry plants, such as flowers, shrubs, broad-leaved trees or evergreens, like conifers. Embodiment 10:
Process according to embodiments 1 to 9, wherein the superabsorbent polymer is selected from polyacrylic acid and cellulose-modified polyacrylic acid or combinations thereof.
Embodiment 1 1 :
Process according to embodiments 1 to 10, wherein the superabsorbent polymer is in the form of granules having average particle size in the range of 0.1 to 5 mm, preferably from 0.2 to 4 mm. Embodiment 12:
Process for improving the plant health by changing the gene expression in a plant comprising the steps of treating and/or cultivating the plant with a superabsorbent polymer, and up-regulating at least one gene of the plant by the addition of the superabsorbent polymer, and/or down- regulating at least one gene of the plant by the addition of the superabsorbent polymer, wherein improved plant health is determined by increased yield of plant product, improved plant vigor, enhanced quality of the plant and/or improved tolerance or resistance of the plant to abiotic and/or biotic stress factors. Embodiment 13:
Process of determining the change in gene expression in a plant upon treatment of the plant with a superabsorbent polymer, comprising the steps of treating and/or cultivating the plant with a superabsorbent polymer and measuring the up-regulation and/or down-regulation of at least one gene of the plant during and/or after cultivation of the plant.
Embodiment 14:
Process of determining the change in gene expression in a plant according to claim 14, wherein measuring the up-regulation and/or down-regulation of at least one gene of the plant is carried out by use of a transcriptomic assay, preferably by use of a microarray.
The present invention is further illustrated, but not limited by the following examples: Examples Methods
Two preparations of tomato plants were evaluated, grown either in sterile or non-sterile soil. The plants were cultivated for different times and the roots of the tomato plants were first removed from the pots and then cleaned to remove all traces of soil and inert material (Figure 1 ). For microarray analysis of the collected samples were used after 10 days following the application of the superabsorbent polymers, together with control samples grown in sterile soil.
The plant material was collected and immediately frozen in liquid nitrogen. For RN A extraction, the commercially available kit NucleoSpin RNA plant (Macherey-Nagel) was used, following the manufacturer's instructions.
Three technical replicates for each sample were used for biological hybridization of the samples. Isolation of RNA from plants:
RNA was extracted from the leaves and the roots of tomato plants according to the following procedure. For RNA extraction, "TRIZOL® Reagent" (Invitrogen) was used following the supplier's instructions. The plant material was maintained at -80°C until use. 100 mg of leaves or root of the plant frozen in liquid nitrogen were homogenized in a mortar and placed in liquid nitrogen for storage of the frozen plant tissue.
To the frozen tissue, 1 ml of "TRIZOL® Reagent" was added per 100 mg of tissue and stirred in a Silamat S5 (Ivoclar Vivadent, Madrid ) for 5-10 sec. The samples were incubated for 5 min at room temperature and centrifuged at 12,000 g for 10 min at 4°C. The samples were then collected as the supernatant to which 200 μΙ chloroform was added. The samples were vortexed for 20 sec and incubated for 3 min at room temperature. Subsequently, they were centrifuged at 10,000 rpm for 15 min at 4°C, the aqueous layers were collected and 500 μΙ of isopropanol were added. It was stirred again and vortexed for 10 sec. The mixtures were allowed to incubate for 15 min at room temperature. The samples then were centrifuged at 10,000 rpm for 8 min at 4°C. Subsequently, 1 ml of cold 70% ethanol was added to the supernatant, vortexed and centri- fuged at 10,000 rpm for 5 min. Finally, the supernatants were removed, dried for 5 min and re- suspended in 20-50 μΙ DEPC-treated water to obtain the pure RNA material from the plant.
Quantification of the amount of RNA for each sample was carried out using a commercial NanoDrop spectrophotometer ND 1000.
Preparation of sample material for gene expression analysis:
From the isolated RNA of the corresponding plant samples, cDNA was prepared by reverse transcription and subsequently amplified by quantitative real time PCR of the selected marker genes of the plant samples collected at 10, 20, 30, 40, 50 and 60 days after application of the superabsorbent polymer.
- Standardization and filtering of sample data In a first filtering step, a robust multi array average (RMA) analysis was applied in order to remove background and weaker signals leading to decreased variability of the signals.
- Statistical analysis An ANOVA type analysis was carried out leading to a total of 10,209 transcripts of which 5,330 relate to relevant genes that pass the test of p < 0.01.
- Analysis of changes between groups A t-test and FDR (false discovery rate) to remove false positives allowing obtaining the results of differential expression relative to the control experiment.
The parameters were as follows: FDR < 0.1 ; p < 0.01 , fold change (FC) > 2 and < -2.
Experimental test: In this experiment, different genomic markers in tomato plants inoculated with different formulations of superabsorbent polymer at different times during cultivation, growth and development of the plant were identified and determined.
Untreated seeds of hybrid tomato, Juncal variety, lot no: 078-0122567549, size: 2.40-2.80, were used as plant species.
The seeds were sterilized with 70 % ethanol for 10 minutes, a commercial bleach (5 % sodium hypochlorite) was used for 10 minutes, followed by repeated washing of the seeds with sterile, distilled water. The seeds were planted same day. After 18 days the seedlings were transplanted into sockets of 0.1 I to have a good root development, 8 days later the seedlings were transferred into 16 liters pots (two seedlings for each pot). Two granular formulations of superabsorbent polymer (named H1 - a polyacrylic acid-type superabsorbent polymer - and H2 - a lignocellulose-modified polyacrylic acid -, 840 mg of each polymer product per pot) were homogenously applied to the tomato plants on the substrate, at the bottom of the planting furrow (4 cm) covering the substrate. In the control experiments, only water without superabsorbent polymer was applied.
A conventional NPK fertilizer (20-20-20) was used for cultivation of the plants. The NPK fertilizer was applied in doses of 1 grain / pot to each plant of the pots 50 days after application of the superabsorbent polymer. As the substrate, sterile peat and sterile or non-sterile soil brought from Utrera (Sevilla) was used as cultivation medium. Sterilization of the soil before use was carried out by two sterilization cycles for 1 h at 121 °C in autoclave. The same amount of water was added to each pot.
The plants were kept in the greenhouse CIALE throughout cultivation, under controlled conditions of 25-27°C during the day and 21 °C at night, with a photoperiod of 15 h and relative hu- midity of 40-60 %. The tomato plants were examined at 10, 20, 30, 40, 50 and 60 days after adding the different treatments.
After 10, 20, 30, 40, 50 and 60 days after application of the superabsorbent polymers, plant material was collected from the leaves and the roots of at least 3 plants of each treatment. Leaf samples collected after 10 days of the application of the superabsorbent polymer were used for microarray analysis, while the rest was used for verifying the data.
The comparative study for each condition relative to the control experiment led to the results as shown in the Volcano plots (Fig. 1 and Fig. 2).
Fig. 1 and Fig. 2 show the Volcano plots of p-values of the t-test of H 1 ("t-test-H 1 ") vs. the control experiment, the parameter on the horizontal x axis is "log2(fold change)", the parameter on the vertical y axis is "-logl O(p-value)", "FC" stands for "fold change", "P-val" stands for "p-value". The differential gene expression found in the present experiment for the cultivation of the plants in the presence of superabsorbent polymer can be summarized as follows:
Superabsorbent polymer H1 versus control:
230 differential genes (108 genes were up-regulated, 122 genes were down-regulated)
Superabsorbent polymer H2 versus control:
75 differential genes (20 genes up-regulated, 55 genes down-regulated) Additional preparation of a Venn diagram using the data from the above differential gene expression analysis allows the comparative analysis of the changes in gene expression caused by the presence of the two different superabsorbent polymers during cultivation of the corresponding plants (Fig. 3). Fig. 3 shows this Venn diagram, wherein the left oval refers to the t-test of H1 at FC2 ("t-test- H 1 -FC2"), the right oval refers to the t-test of H2 at FC2 ("t-test-H2-FC2").
The Venn diagram shows that 53 genes were differentially expressed during cultivation of the plants in the presence of both superabsorbent polymers H 1 and H2, while 46 genes were up- regulated and 7 genes were down-regulated in the presence of both superabsorbent polymers.
Most relevant up-regulated genes identified after PCR analysis:
Figure imgf000037_0001
Most relevant down-regulated genes identified after PCR analysis:
Gene Code Fold Related function
Probeset change
(expression
vs. control)
Grasl LesAffx.60966.2.S1_at -2,0338 Growth repressor.
Lipoxygenase Les.3632.1.S1_at -2,3182 Biosynthesis of fatty acids, plant health. Verticillium wilt diLes.3506.1 .S1_at -2,4313 Plant defense response sease resitance
protein Ve2
ACS6 Les.3769.1 .S1_at -2,6265 Biosynthesis of ethylene, plant health.
Cell-wall peroxiLesAffx.57363.1.S1_at -2,6388 Response to oxidative dase stress
Avr9/Cf-9 rapidly LesAff.51274.1.S1_at -2,8907 Ethylene response, plant elicited protein 1 health.
Ripening regulated Les.274.1.S1_at -3,13 Response to chitin, ripenprotein ing, plant health.
DDTFR10/A
Salt responsive Les.5038.1 .S1_at -3,6016 Salt tolerance, plant health. protein2

Claims

Use of a superabsorbent polymer for improving plant health by changing the gene expression in a plant, characterized in that the plant is treated with the superabsorbent polymer, the plant treated with the superabsorbent polymer is cultivated, and at least one gene of the plant is up-regulated by the addition of the superabsorbent polymer, and/or at least one gene is down-regulated by the addition of the superabsorbent polymer, wherein improved plant health is determined by increased yield of plant product, improved plant vigor, enhanced quality of the plant and/or improved tolerance or resistance of the plant to abiotic and/or biotic stress factors.
Use of a superabsorbent polymer according claim 1 , wherein increased yield of a plant product is determined by grains, fruits, vegetables, nuts, grains, seeds, wood and/or flowers and combinations thereof.
Use of a superabsorbent polymer according claim 1 , wherein improved plant vigor is determined by improved vitality of the plant, improved plant growth, improved plant development, improved visual appearance, improved plant stand, less plant verse/lodging, improved emergence, enhanced root growth and/or more developed root system, enhanced nodulation, in particular rhizobial nodulation, bigger leaf blade, bigger size, increased plant weight, increased plant height, increased tiller number, increased number of side shoots, increased number of flowers per plant, increased shoot growth, increased root growth like extensive root system, increased yield when grown on poor soils or unfavorable climate, enhanced photosynthetic activity, preferably based on increased sto- matal conductance and/or increased CO2 assimilation rate, increased stomatal conductance, increased CO2 assimilation rate, enhanced pigment content like chlorophyll content, flowering, earlier fruiting, earlier and improved germination, earlier grain maturity, improved self-defense response, less non-productive tillers, less dead basal leaves, less input needed such as fertilizers or water, greener leaves, complete maturation under shortened vegetation periods, less fertilizers needed, less seeds needed, easier harvesting, ripening, longer shelf-life, longer panicles, delay of senescence, stronger and/or more productive tillers, better extractability of ingredients, improved quality of seeds for being seeded in the following seasons for seed production, altered or reduced production of ethylene and/or the inhibition of its reception by the plant, growth repression, and combinations thereof.
Use of a superabsorbent polymer according claim 1 , wherein enhanced quality of the plant is determined by increased nutrient content, increased protein content, increased content of fatty acids, increased metabolite content, increased carotenoid content, increased sugar content, increased content of amino acids, including essential amino acids, improved nutrient composition, improved protein composition, improved composition of fatty acids, improved metabolite composition, improved carotenoid composition, improved sugar composition, improved amino acids composition, improved or optimal fruit color, improved leaf color, higher storage capacity, and/or higher processability of the harvested products and combinations thereof.
5. Use of a superabsorbent polymer according claim 1 , wherein improved tolerance or re- sistance of the plant to abiotic stress factors is determined by tolerance and/or resistance to heat stress including temperatures higher than 30°C, temperature conditions causing heat damage to a plant like heat damaged foliage or burnt leaves, cold stress like temperature conditions below 10°C, periods of thawing and freezing, frost, variations in temperature like temperatures conditions that lead to the freezing of water either for extended periods of time or only temporary periods, temperature unusual for the season, drought stress, exposure to cold water, flood, water-logging, wind, sun light, particularly sun light causing signs of scorch, sun burn or similar signs of irradiation and heat stress to the plant, acid or alkaline pH conditions in the soil with pH values lower than pH 5 and/or pH values higher than 9, salt stress like soil salinity, soil erosion, inorganic pollu- tion, soil contamination or soil pollution with chemicals, particularly with heavy metals, preferably chromium, lead, cadmium, arsenic, antimony, mercury, iron, thallium, barium, beryllium, polonium, uranium, toxic waste, nuclear waste, acid rain, air pollution, preferably radiation like high UV radiation due to the exposure to the decreasing ozone layer, increased ozone levels, nitrogen oxides and/or sulfur oxides, oxidative stress, organic pollution, oil and/or fuel dumping or spilling, nuclear radiation, contact with sewage, over- fertilization, nutrient deficiencies, herbicide injuries, plant wounding, compaction, natural disasters, preferably tornadoes, hurricanes, wildfires, flooding and combinations thereof.
6. Use of a superabsorbent polymer according claim 1 , wherein improved tolerance or re- sistance of the plant to biotic stress factors is determined by tolerance and/or resistance to the attack of living organisms including pests like insects, arachnides, nematodes, competing plants like weeds, microorganisms like fungi, bacteria and/or viruses and combinations thereof. 7. Use of a superabsorbent polymer according to one of the preceding claims, wherein the up-regulated plant gene is selected from pathogenesis related protein P4, xyloglucan- specific fungal endoglucanase inhibitor protein, sucrose synthase, beta-1 ,2-glucanase, chitinase, serine acetyltransferase and combinations thereof. 8. Use of a superabsorbent polymer according to one of the preceding claims, wherein the down-regulated plant gene is selected from gras 1 , lipoxygenase, verticillium wilt disease resistance protein Ve2, AVr9/CF-9 rapidly elicited protein 1 , ripening regulated protein DDTFR10/A, salt responsive protein 2 and combinations thereof. 9. Use according to one of the preceding claims, wherein the plant is an agricultural plant like wheat, rye, barley, triticale, oats, sorghum or rice, beet, sugar beet or fodder beet, fruits like pomes, apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries, blackberries or gooseberries, leguminous plants, such as lentils, peas, alfalfa or soybeans, oil plants, such as rape, oil-seed rape, canola, juncea, linseed, mustard, olives, sunflowers, coconut, cocoa beans, castor oil plants, oil palms, ground nuts or soybeans, cucurbits, such as squashes, cucumber or melons, fiber plants, such as cotton, flax, hemp or jute, citrus fruit, such as oranges, lemons, grapefruits or mandarins, vege- tables, such as spinach, lettuce, asparagus, cabbages, carrots, onions, tomatoes, potatoes, cucurbits or paprika, lauraceous plants, such as avocados, cinnamon or camphor, energy and raw material plants, such as corn, soybean, rape, canola, sugar cane or oil palm, corn, tobacco, nuts, coffee, tea, bananas, vines, hop, turf, natural rubber plants or ornamental and forestry plants, such as flowers, shrubs, broad-leaved trees or ever- greens, like conifers.
10. Use according to one of the preceding claims, wherein the superabsorbent polymer is selected from polyacrylic acid and cellulose-modified polyacrylic acid or combinations thereof.
11. Use according to one of the preceding claims, wherein the superabsorbent polymer is in the form of granules, having average particle size in the range of 0.1 to 5 mm, preferably from 0.2 to 4 mm. 12. Process for improving the plant health by changing the gene expression in a plant comprising the steps of treating the plant with a superabsorbent polymer, cultivating the plant treated with the superabsorbent polymer, and up-regulating at least one gene of the plant by the addition of the superabsorbent polymer, and/or down-regulating at least one gene of the plant by the addition of the superabsorbent polymer, wherein improved plant health is determined by increased yield of plant product, improved plant vigor, enhanced quality of the plant and/or improved tolerance or resistance of the plant to abiotic and/or biotic stress factors.
13. Process of determining the change in gene expression in a plant upon treatment of the plant with a superabsorbent polymer, comprising the steps of treating the plant with a superabsorbent polymer, cultivating the plant treated with a superabsorbent polymer, and measuring the up-regulation and/or down-regulation of at least one gene of the plant during and/or after cultivation of the plant.
Process of determining the change in gene expression in a plant according to claim 14, wherein measuring the up-regulation and/or down-regulation of at least one gene of the plant is carried out by use of a transcriptomic assay, preferably by use of a microarray.
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