Fructose sensing and signaling in plants

Abstract

The present invention relates to fructose sensing and signaling mechanisms in plants to improve plant properties.In particular, the present invention relates to vectors or mobile genetic elements comprising nucleic acid molecules encoding tonoplast proton-fructose symporters.Furthermore, the present invention relates to host cells, plants, seeds, methods for producing plants, methods for increasing the yield of plant storage compounds, methods for increasing drought tolerance in plants, the use of tonoplast proton-fructose symporters in plants to increase the concentration of plant storage compounds, or methods for selecting plants with increased fructose levels.

Description

[Background technology]

[0001] The present invention relates to providing and generating plants with beneficial properties. In particular, the present invention relates to providing and generating crop plants that produce increased amounts of biomass and / or increased amounts of desired plant storage compound(s), e.g., sugars such as sucrose, starches, lipids and / or proteins, and / or have increased resistance to adverse environmental conditions, such as drought.

[0002] The inventors have surprisingly found that increasing the concentration of fructose in the cytosol of a plant alters sugar signaling in the plant, for example in the leaves of the plant, which results in an alteration of the long-distance transport of sugars, such as sucrose in the phloem of the plant. In particular, the long-distance transport in the plant is altered so that more sugars are transported from the plant site of carbohydrate synthesis (the source) to the plant site of storage of plant storage compounds (the sink). In plant organs that act as sinks for plant storage compounds, such compounds can be converted to other plant storage compounds. For example, sugars can be converted to starch or cellulose in the plant organ. Plant storage compounds can include, but are not limited to, sugars such as sucrose, starches, proteins and / or lipids. Furthermore, the inventors have found that increasing the concentration of fructose in the cytosol of a plant (particularly by overexpressing a tonoplast proton-fructose symporter, such as EDRL4) increases the biomass of the plant.

[0003] The inventors' observations strongly suggest that an increase in cytosolic fructose levels can be sensed by plants in different plant compartments, thereby resulting in changes in long-distance transport of sugars, which has a positive effect on biomass production, drought resistance, and especially on the accumulation of plant storage compounds (e.g., sucrose) in plant organs (such as fruit, seeds, roots, or storage organs). Increasing the amount of sucrose in the sucrose storage compartment of a plant is particularly desirable in the case of crop plants such as sugar beet. Therefore, as the skilled artisan will appreciate, any means of increasing the fructose concentration in the cytosol of a plant can be used to achieve the beneficial effects described herein. Summary of the Invention

[0004] In one embodiment, the present invention relates to a vector or mobile genetic element comprising a nucleic acid molecule, wherein the nucleic acid molecule encodes a tonoplast proton-fructose symporter. In particular, the nucleic acid molecule is selected from the group consisting of a) a nucleic acid molecule having at least 50% nucleic acid sequence identity to a nucleic acid sequence selected from the group consisting of any one of the sequences according to SEQ ID NOs: 40, 39, 1, and 129-152, b) a nucleic acid molecule having a nucleic acid sequence that is complementary to or hybridizes under stringent conditions to a nucleic acid molecule according to a), and c) a nucleic acid molecule having a nucleic acid sequence that encodes a protein having at least 50% amino acid sequence identity to an amino acid sequence selected from the group consisting of any one of the sequences according to SEQ ID NOs: 58, 57, 22, and 77-128.

[0005] In a further aspect, the invention includes a host cell comprising the above-described vector or mobile genetic element.

[0006] The present invention also includes a plant or a part of a plant comprising at least one host cell as described herein. The plant of the present invention may be a genetically modified plant. The plant may be a transgenic plant.

[0007] In one embodiment, a genetically modified plant is provided that contains increased levels of fructose in cytosol compared to the wild-type plant that it originates from.Compared to the wild-type plant that it originates from, the plant of the present invention can overexpress vacuolar membrane proton-fructose symporter.

[0008] In some embodiments, the plants of the present invention are characterized as follows. i) the plant comprises a modified promoter of an endogenous gene encoding a tonoplast proton-fructose symporter to be overexpressed, such that the tonoplast proton-fructose symporter is homologously overexpressed; ii) the plant comprises at least one additional copy of a nucleic acid encoding a tonoplast proton-fructose symporter that is overexpressed such that the tonoplast proton-fructose symporter is homologously overexpressed; or iii) the plant comprises a heterologous nucleic acid encoding a heterologous tonoplast proton-fructose symporter such that the tonoplast proton-fructose symporter is heterologously overexpressed.

[0009] In some embodiments of the invention, the plant comprises a vector or mobile genetic element of the invention, which can be introduced into the plant as described herein, as described in the cited literature, and by any other means known to one of skill in the art.

[0010] The present invention also includes the seed of the plant according to the present invention.The seed can be planted to provide the plant according to the present invention.The plant can be cultivated to solve the technical problems described herein, for example, to increase the yield of plant storage compounds, to increase biomass, to increase the concentration of plant storage compounds in at least one plant organ, or to increase the drought resistance of the plant.

[0011] In a further embodiment, the present invention includes a method of producing a plant, comprising: a) genetically modifying at least one plant cell to produce at least one plant cell that contains an increased level of fructose in the cytosol compared to the wild-type plant cell from which it is derived; and b) regenerating a plant from the genetically modified plant cell of step a), wherein the plant contains an increased level of fructose in the cytosol compared to the wild-type plant from which it is derived.

[0012] In some embodiments, the step of genetically modifying at least one plant cell (e.g., in step a)) comprises: i) modifying the promoter of an endogenous gene encoding a tonoplast proton-fructose symporter such that the tonoplast proton-fructose symporter is overexpressed; ii) introducing into the plant cell at least one additional copy of a nucleic acid encoding a tonoplast proton-fructose symporter such that the tonoplast proton-fructose symporter is homologously overexpressed; or iii) introducing into the plant cell a heterologous nucleic acid encoding a heterologous tonoplast proton-fructose symporter such that the tonoplast proton-fructose symporter is heterologously overexpressed; Includes.

[0013] In some embodiments, a vector or mobile genetic element of the invention is introduced into at least one plant cell.

[0014] The present invention also includes a method for increasing the yield of plant storage compounds, comprising increasing the concentration of fructose in the cytosol of the plant.In some embodiments, the plant that produces the plant storage compounds is produced as described herein.In some embodiments, the plant that produces the plant storage compounds is a plant as described herein.The concentration of fructose in the cytosol of the plant can be increased as described herein.

[0015] The plant storage compound can be selected from sugar, preferably sucrose, starch, protein or lipid.In particular, the sugar can be the sugar present in sugar beet, soybean or tomato fruit, the lipid can be the lipid present in sunflower seed or rapeseed, and the protein can be the protein present in soybean.Preferably, the plant storage compound is sucrose.

[0016] A method for increasing the yield of plant storage compounds can include: i) the biomass of at least one plant organ containing the plant storage compound is increased, preferably the plant organ is a fruit, a seed, a root or a storage organ, or ii) The concentration of a plant storage compound in at least one plant organ is increased, preferably the plant organ is a fruit, a seed, a root or a storage organ.

[0017] In some embodiments, the yield of a plant storage compound is increased by increasing the biomass of at least one plant organ, such as a fruit, a seed, a root, or a storage organ. In some embodiments, the yield of a plant storage compound is increased by increasing the concentration of the plant storage compound in at least one plant organ, such as a fruit, a seed, a root, or a storage organ.

[0018] In a further aspect, the present invention provides a method for increasing drought tolerance in a plant, comprising increasing the concentration of fructose in the cytosol of the plant. i) said plant having increased drought tolerance is produced according to the method of the invention, and / or ii) the plant having increased drought tolerance is a plant of the present invention; may include.

[0019] The plant may be a crop plant. In some embodiments, the plant is selected from the group consisting of sugar beet (Beta vulgaris), cabbage (Brassica rapa), rapeseed (Brassica napus), sunflower (Helianthus annuus), potato (Solanum tuberosum), corn (Zea mays), rice (Oryza sp., e.g. Oryza sativa), barley (Hordeum vulgare), rye (Secale cereale), or sorghum (Sorghum sp., e.g. Sorghum bicolor), preferably the plant is sugar beet.

[0020] In a preferred embodiment, the plant is a sugar beet, the plant organ is a sugar beet taproot, and the plant storage compound is sucrose.

[0021] The present invention also provides the use of a tonoplast proton-fructose symporter in a plant for increasing the concentration of a plant storage compound in a plant organ, optionally wherein the plant storage compound is selected from sugar, preferably sucrose, starch, protein or lipid, and the plant is selected from the group consisting of sugar beet (Beta vulgaris), cabbage (Brassica rapa), rapeseed (Brassica napus), sunflower (Helianthus annuus), potato (Solanum tuberosum), maize (Zea mays), rice (Oryza sp., e.g. Oryza sativa), barley (Hordeum vulgare), rye (Secale cereale), or sorghum (Sorghum sp., e.g. Sorghum bicolor), preferably the plant is sugar beet and the plant organ is the sugar beet taproot.

[0022] The present invention also provides a method for selecting a plant having an increased level of fructose in the cytosol of the plant, the method comprising the steps of: i) Cultivating plants; ii) measuring the fructose level in at least one plant cell of said plant; iii) comparing the measured fructose level to at least one fructose reference level; and iv) selecting plants having increased levels of fructose compared to said at least one reference level.

[0023] The reference level may be the level of fructose in the cytosol of the reference plant. The reference plant may be the plant from which the plant originates. The reference plant may be the same species of plant. For example, if the plant is genetically modified, the reference plant may be the wild-type plant from which the plant originates. In some embodiments, the plant is the plant of the present invention, for example comprising a vector or genetic element as described herein. The plant may also overexpress a tonoplast proton-fructose symporter as described herein. In some embodiments, the reference fructose level is determined in the same plant organ and / or the same plant cell type. [Brief description of the drawings]

[0024] [Figure 1]Phylogenetic tree of the MST transporter family (Figure 1A). Figure adapted from Pommerrenig et al. (2018). Phylogenetic tree of 53 Arabidopsis MST family proteins based on nucleotide sequences. Numbers at nodes indicate node support values. Only values ​​less than 100 are shown. MST subfamilies are abbreviated as follows: PMT, POLYOL MONOSACCHARIDE TRANSPORTER; VGT, VACUOLAR GLUCOSE TRANSPORTER; TST, TONOPLAST SUGAR TRANSPORTER; ERDL, EARLY-RESPONSE TO DEHYDRATION SIX-LIKE protein; STP, SUGAR TRANSPORTER; pHT, PLASTIDIC HEXOSE TRANSPORTER; INT, INOSITOL TRANSPORTER. The names of the individual transport proteins are shown along with their Arabidopsis designations (in parentheses in the figure). Letters outside the circle indicate the subcellular localization of the protein: PM, plasma membrane; T, tonoplast; C, chloroplast; G, Golgi. * indicates predicted chloroplast localization by identification of a chloroplast transit peptide sequence (ChloroP 1.1; Emanuelsson et al. 1999). Letters in parentheses indicate predicted localization (e.g., when localization has been shown in heterologous systems). Question marks indicate unknown subcellular localization. For each subfamily, tested and predicted transport substrates are listed. Larger font size indicates higher affinity of the subfamily member for the substrate. MST nucleotide sequences were obtained from the aramemnon database (Schwacke et al. 2003). Multiple sequence alignments for all MST sequences were constructed using Clustal Omega (Sievers et al. 2011).Bayesian phylogenetic analysis was performed with MrBayes version 3.2.6 (Ronquist and Huelsenbeck 2003) using the HKY+I+G model. MrBayes was run by conducting two parallel Metropolis-joint Monte Carlo Markov chain analyses with four chains for 2 million generations. The SD of split frequencies was <0.01. Tree files were visualized using FigTree v.1.4.3. Figure 1B shows a close-up of the region containing the AtERDL4 gene. [Diagram 2] Vacuolar membrane localization of an N-terminal fusion of ERDL4 with GFP. A-F) Confocal images of tobacco mesophyll protoplasts transformed with the PromoterUBQ10-ERDL4-GFP construct. Scale bar 10 μm. A-F) Images of tobacco protoplasts expressing an ERDL4-GFP fusion. Different confocal channels of the same protoplast are shown. A) Brightfield image. B) Chlorophyll-derived autofluorescence showing the location of the chloroplasts (spheres). C) GFP-derived fluorescence showing the location of the ERDL4-GFP fusion protein. D) Overlay of chlorophyll and GFP fluorescence. E and F) Images of lysed protoplasts expressing ERDL4-GFP fluorescence. E) GFP-derived fluorescence at the tonoplast. F) Overlay of GFP fluorescence and brightfield image. The white triangle in C-F points to the tonoplast. G) Schematic of the 2D structure of the ERDL4 protein fused to GFP at its C-terminus. Different numbers within the protein structure indicate different transmembrane helices of the ERDL4 protein. Black triangles indicate serine or threonine residues that are predicted phosphorylation sites. [Diagram 3]Schematic diagram of ERDL4 localization and proposed function. TST2- and VGT1 / 2-dependent transport of sugars, namely glucose (glc), fructose (frc), and sucrose (suc), is stimulated by cytosolic fructose. Vacuolar frc, derived from TST-mediated import or vacuolar suc hydrolysis, is transported to the cytosol via ERDL4 and activates TST2 and VGT1 / 2 activity (indicated by asterisks on the TST2 and VGT1 / 2 transporters in the schematic). The high specificity of ERDL4 for frc results in the accumulation of high concentrations of suc and glc, but not frc, in the vacuole overall. [Figure 4] Phenotypes and fresh weights of different erdl4 mutants and ERDL4 overexpressing plants. A) Phenotypes of 4-week-old WT, 35S-ERDL4, and erdl4 knockout plants grown in soil substrate under short-day conditions. B) Quantification of shoot biomass based on fresh weights of plants shown in (A). Bar graphs show mean ± SE from n=12 independent plants. Asterisks indicate significant difference from WT by Student's t-test (*p<0.05). [Diagram 5] Characterization of root parameters of various erdl4 mutants and ERDL4 overexpressing plants. A) Representative photographs of rosettes and roots of 5-week-old WT, 35S-ERDL4, and erdl4 knockout plants grown in hydroponics. ERDL4 overexpressing plants have longer roots and greater biomass. B) Root dry biomass from plants shown in (A). Bar graphs show mean ± SE from n=6 roots. Asterisks indicate significant difference from WT by Student's t test. C) Primary root length of WT and 35S-ERDL4 plants measured over 6 days. [Figure 6] 1,000 seed weight is increased in ERDL4 overexpressing plants. 1000 seed weights of dry mature seeds from WT, erdl4 knockout mutants and ERDL4 overexpressing plants. Bar graphs show the average 1000 seed weights of n=6 plants per line. Asterisks indicate significant differences by Student's t-test (**=p<0.01; ***=p<0.001). [Figure 7] Seed lipids contents of dry mature seeds from erdl4 knockout mutants and ERDL4 overexpressing plants. Bars show average values ​​from n=6 plants per line. Asterisks indicate significant differences by Student's t-test (*=p<0.05; **=p<0.01; ***=p<0.001). [Figure 8] Increased phloem sugar flux from detached leaves of ERDL4 overexpressing plants. Sugars were measured as hexose equivalents in phloem exudates harvested from detached leaves of 5-week-old WT, 35S-ERDL4, and erdl4 knockout plants. Bars are means ± SE from at least n = 12 leaves. Asterisks indicate significant differences from WT by Student's t test. [Figure 9] Total and intracellular sugar accumulation in WT, ERDL4 overexpressing, and mutant plants. A) Glucose (glc), fructose (frc), and sucrose (suc) contents in shoots. B-D) Intracellular contents of glucose (B), fructose (C), and sucrose (D) in vacuole, chloroplast, and cytosol fractions of shoots from WT, ERDL4 overexpressing, and mutant plants (obtained by non-aqueous fractionation). Bars show mean ± SE of n=5 plants. Different letters above the bars indicate significant differences (p<0.05) based on one-way ANOVA with post-hoc Tukey's test. nd=no quantifiable amount detected. [Figure 10]CIPK6 expression is increased by fructose and also in ERDL4 overexpressing lines. A) Relative expression of CIPK6 (At4g30960) and TST2 (At4g35300) in response to different sugars or mannitol. Leaf discs excised from Arabidopsis thaliana source leaves were treated with sugars by incubating in half-strength MS medium without (control) or with 2% of the respective sugar or mannitol. Expression was quantified by RT-qPCR and normalized to AtACT2 expression. B) Relative expression of CIPK6 (At4g30960) in WT and two 35S-ERDL4 lines. A-B) Bar graphs show mean ± SE of n=4 replicates. Asterisks indicate significant differences by Student's t-test (*=p<0.05; **=p<0.005; ***=p<0.001). [Figure 11] Dynamics of cold acclimation and deacclimation of sugar content in Arabidopsis thaliana leaves. Time course changes were recorded for Col-0, tst1-2 double knockout, and two BvIMP2 (=BvERDL4) overexpressing lines under cold stress (4°C) and deacclimation (20°C). A) Glucose content B) Fructose content C) Sucrose content. Each time point represents the average value from at least three plants per line. Error bars indicate SE. Asterisks indicate significant changes in BvIMP2 overexpressing lines compared to WT at a given time point. Asterisks of different colors indicate the relationship with the corresponding line (grey=BvIMP2 OX1, black=BvIMP2 OX2). [Figure 12]Fresh weight of plants grown under control or drought conditions. Drought stress was applied based on the field capacity (FC) of the soil substrate. (A) 100% FC=control, well-watered condition; (B) 60% FC=mild drought stress; (C) 40% FC= severe drought stress for Arabidopsis thaliana Col-0 (Wt, control), two ERDL4 overexpression lines (Oex1, Oex2), and an erdl4 knockout line (KO1, KO2). Plants were grown under 100% FC for 7 days and then stressed and grown under their respective FC for 3.5 weeks. Bars show mean ± SE of at least n=6 plants. Asterisks indicate significant differences compared to WT by Student's t-test (*=p<0.05; **=p<0.005; ***=p<0.001). [Figure 13]Venn diagram and correlation analysis between differentially expressed genes (DEGs) from ERDL4 overexpressing and WT plants and leaf discs incubated with fructose (Frc) or glucose (Glc) and mannitol (Man = control). A) and C) show Venn diagrams of DEGs. Numbers in circles represent the number of DEGs (significant at p<0.001 by two-tailed t-test with three replicates). There were 484 35S-ERDL4-dependent DEGs, 4340 Frc-dependent DEGs, and 5148 Glc-dependent DEGs. A) Diagram showing overlap of DEGs from 35S-ERDL4 and Frc treatments. There were 130 DEGs regulated by both ERDL4 overexpression and Frc. B) Correlation between the relative expression (based on log2 fold change) of 130 DEGs between 35S-ERDL4 (relative to WT) and Frc (relative to Man). r = Pearson coefficient calculated from ERDL4 / WT and Frc / Man matrices. C) Diagram showing overlap of DEGs from 35S-ERDL4 and Glc treatment. There were 155 DEGs regulated by both ERDL4 overexpression and Glc. D) Correlation between relative expression (based on log2 fold change) of 155 DEGs between 35S-ERDL4 (relative to WT) and Frc (relative to Man). r = Pearson coefficient calculated from ERDL4 / WT and Glc / Man matrices. [Figure 14] Figure 14 shows the genomic sequence of the Beta vulgaris gene for ERDL4 (BvERDL4;2 (BvIMP2; Bv6_128840_qhip.t1), SEQ ID NO: 76). The promoter region is shown in italics, the 5'-UTR and 3'-UTR regions are shown in bold, and the CDS region is underlined. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] All publications, including but not limited to patents, patent applications, and scientific publications, cited in this specification are herein incorporated by reference for all purposes to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art.

[0027] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0028] As used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, or the absence of a combination when interpreted in the alternative ("or").

[0029] The use of the term "comprising" and other grammatical forms such as "comprises" and "comprised" is not limiting. The terms "comprising", "comprises" and "comprised" should be understood as representing an open-ended description of an embodiment of the invention that may, but does not necessarily, include additional technical features in addition to the technical features explicitly described. In a similar sense, the use of the term "including" and other corresponding grammatical forms such as "involves" and "involved" is not limiting. The same applies to the term "including" and other grammatical forms such as "includes" and "included".

[0030] Section headings throughout the specification are for organizational purposes only, and in particular they are not intended to limit the various embodiments described therein, and it should be understood that embodiments (and features therein) described under one subheading may be freely combined with embodiments (and features therein) described under another subheading.

[0031] Furthermore, the terms "comprising," "involving," and "including" and all grammatical forms thereof should not be construed to refer only to embodiments that include additional features to those expressly recited. These terms likewise refer to embodiments consisting only of those features expressly recited.

[0032] As used herein, the term "nucleic acid" refers to an oligomer or polymer of naturally occurring or modified nucleotides. A nucleic acid can also contain nucleotide substitutions relative to a reference sequence. For example, a nucleic acid can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions. A nucleic acid can have 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55% or 50% sequence identity to a reference sequence. A reference sequence can be a nucleic acid having a sequence according to SEQ ID NO: 1 to SEQ ID NO: 152. A nucleic acid can be ribonucleic acid (RNA), or deoxyribonucleic acid (DNA), or a hybrid of RNA and DNA. A nucleic acid can be single-stranded or double-stranded. A double-stranded nucleic acid can be RNA-RNA, RNA-DNA (hybrid), or DNA-DNA. Duplex formation occurs via hydrogen bonding, for example, by Watson-Crick base pairing. The nucleic acid may have one or two 3' overhangs. The nucleic acid may have one or two 5' overhangs. The nucleic acid may have one or two blunt ends. Combinations of the above overhangs or blunt ends are also included.

[0033] A nucleic acid can be defined by the sequence of nucleotides, designated by the commonly accepted letter code for the nucleotide bases, i.e., A (adenine), C (cytosine), G (guanine), T (thymine) and U (uracil). As used herein, "sequence" refers to a sequence of nucleotides or a sequence of amino acids.

[0034] The sequence of amino acids may have 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55% or 50% sequence identity to a reference sequence and / or may contain amino acid substitutions relative to the reference sequence.

[0035] As used herein, the term "hybridizes under stringent conditions" means capable of hybridizing under stringent conditions, e.g., conditions corresponding to a Tm (melting temperature) of about 50°C, 51°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C or 70°C or higher.

[0036] By "increased sucrose concentration" or "increased sucrose concentration" or "higher sucrose concentration in sucrose storage organs of a plant" is meant an increase in the average sucrose concentration based on the fresh weight of the sucrose storage organs of at least 0.2%, 0.4%, 0.6%, 0.8% or 1%, preferably at least 1.2%, 1.4%, 1.6%, 1.8% or 2%, particularly preferably at least 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 7%, 8% or 10%, most preferably at least 15%, when compared to a reference plant cultivated under identical conditions.

[0037] In one embodiment, the increase in yield of plant storage compounds is an increase in sucrose concentration.

[0038] "Increased yield of plant storage compounds" means that the yield is increased relative to a reference plant. The reference plant may be a reference plant as defined herein, for example, a wild-type plant from which the plant with increased yield is derived. The plant with increased yield may be genetically modified, for example, the plant may contain a vector or a mobile genetic element of the present invention. The yield of the plant storage compounds is determined in a manner common in the art. For example, the yield may be the mass of the plant storage compounds per cultivated area.

[0039] A storage organ is a plant organ that stores plant storage compounds. A storage organ may be a sugar storage organ. As used herein, a "sugar storage organ" is a part of a plant where the plant stores sugars, such as sucrose, and can be harvested. In particular, a sugar storage organ may be a fruit, such as a beet or a corn kernel.

[0040] A "plant" may be a crop plant, in particular a protein plant such as sugar beet (Beta vulgaris), cabbage (Brassica rapa), rapeseed (Brassica napus), sunflower (Helianthus annuus), potato (Solanum tuberosum), corn (Zea mays), rice (Oryza sp., e.g. Oryza sativa), barley (Hordeum vulgare), rye (Secale cereale), sorghum (Sorghum sp., e.g. Sorghum bicolor), Brassica oleracea capitata, soybean (Glycine max) and pea (Pisum sativum), other cereals including wheat (Triticum aestivum), silage crops such as alfalfa (Medicago satvia), intercrops such as white mustard (Sinapis alba), radish (Raphanus sativum), and the like. The plant may be a flowering plant such as (Citrullus sativus), phacelia, buckwheat (Fagopyrum esculentum), wild turnip (Brassica rapa) or other vegetables including: spinach (Spinacia oleracea), red beet (Beta vulgaris ssp. vulgaris), Swiss chard (Beta vulgaris ssp. vulgaris), tomato (Lycopersicum esculentum), green beans (Phaseolus vigna spp.), peppers (Capsicum annuum), cucumber (Cucumis sativus), watermelon (Citrullus lanatus) and melon (Cucumis melo). In some embodiments, the plant is a transgenic plant. In some embodiments, the plant is modified, particularly genetically modified. In some embodiments, modification of the plant results in an increased level of fructose in the cytosol of the plant, particularly compared to a reference plant, such as a wild-type plant from which the modified plant is derived.Increased fructose levels can be achieved by overexpressing a tonoplast proton-fructose symporter, such as any one of the tonoplast proton-fructose symporters taught herein.

[0041] The term " tonoplast proton-fructose symporter is overexpressed " means that the amount of tonoplast proton-fructose symporter in plant, plant cell or tonoplast is greater than that of reference plant, plant cell or tonoplast that is not modified to overexpress.For example, when tonoplast proton-fructose symporter is overexpressed compared to the wild-type plant from which the plant originates, the plant contains a greater amount of tonoplast proton-fructose symporter in plant, plant cell or tonoplast than that of wild-type plant, plant cell or tonoplast.

[0042] The "vacuol membrane proton-fructose symporter" is a co-transporter protein that is located in the vacuole membrane and transports protons and fructose from the vacuole to the cytosol.

[0043] The present invention includes a vector or mobile genetic element comprising a nucleic acid molecule, said nucleic acid molecule encoding a vacuolar membrane proton-fructose symporter, said nucleic acid molecule being selected from the group consisting of: a) a nucleic acid molecule having at least 50% nucleic acid sequence identity to a nucleic acid sequence selected from the group consisting of any one of the sequences set forth in SEQ ID NOs: 40, 39, 1, and 129-152; b) a nucleic acid molecule having a nucleic acid sequence which is complementary to or hybridizes under stringent conditions to the nucleic acid molecule according to a); c) A nucleic acid molecule having a nucleic acid sequence encoding a protein having at least 50% amino acid sequence identity to an amino acid sequence selected from the group consisting of any one of the sequences set forth in SEQ ID NOs: 58, 57, 22, and 77-128.

[0044] In one embodiment, the nucleic acid molecule contained in the vector or mobile genetic element has at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a nucleic acid sequence selected from the group consisting of any one of the sequences set forth in SEQ ID NOs: 40, 39, 1, and 129-152.

[0045] In one embodiment, the protein encoded by the nucleic acid molecule has at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to an amino acid sequence selected from the group consisting of any one of the sequences set forth in SEQ ID NOs: 58, 57, 22, and 77-128.

[0046] The vector or mobile genetic element may further comprise any one or more of the nucleic acid sequences set forth in SEQ ID NOs: 1-152, or a nucleic acid molecule having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to a nucleic acid sequence selected from the group consisting of any one of the nucleic acid sequences set forth in SEQ ID NOs: 1-152.

[0047] The vector or mobile genetic element may further comprise one or more nucleic acid sequences encoding any one of the amino acid sequences set forth in SEQ ID NOs: 1-152, or one or more nucleic acid sequences encoding an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to an amino acid sequence selected from the group consisting of any one of the amino acid sequences set forth in SEQ ID NOs: 1-152.

[0048] A vector or a mobile genetic element can be used in the methods and uses taught herein, for example, to achieve overexpression of a tonoplast proton-fructose symporter. For example, a vector or a mobile genetic element can be used to produce a host cell as described herein, such that the host cell, e.g., a plant cell, contains the vector or the mobile genetic element. A vector or a mobile genetic element can be used to produce a plant as described herein. In particular, a plant as described herein can contain a vector or a mobile genetic element. A vector or a mobile genetic element can be used to increase the yield of a plant storage compound. A vector or a mobile genetic element can be used to increase the biomass of a plant, in particular the biomass of at least one organ of a plant. A vector or a mobile genetic element can be used to increase the concentration of at least one plant storage compound in at least one organ of a plant. In some embodiments, the yield of a plant storage compound can be increased by increasing the biomass of at least one organ of a plant, or by increasing the concentration of a plant storage compound in at least one plant organ, or by a combination of both increasing the biomass and the concentration. Furthermore, the vector or mobile genetic element can be used to increase the sucrose concentration in a sucrose storage organ of a plant, as described herein, in particular by using the vector or mobile genetic element in a method for increasing the sucrose concentration in a sucrose storage organ of a plant, as described herein. The vector or mobile genetic element can also be applied to the described use of the tonoplast proton-fructose symporter in a plant, for example, to increase the sucrose concentration in a sucrose storage organ of a plant by introducing the vector or mobile genetic element into a cell of the plant. The vector or mobile genetic element can also be used in the described method for increasing the biomass of a plant.In particular, overexpression of the tonoplast proton-fructose symporter described herein encoded by a nucleic acid molecule contained in a vector or mobile genetic element results in increased production of biomass in a plant containing the vector or mobile genetic element, which also results in increased sucrose concentration when contained in a plant as described herein.

[0049] A vector or mobile genetic element can be introduced into a cell, such as a eukaryotic or prokaryotic cell, by transformation, transfection, or other means known in the art. For example, a vector or mobile genetic element can be introduced into a plant cell by infecting the plant cell with Agrobacterium tumefaciens containing the vector or mobile genetic element. A vector or mobile genetic element can also be introduced into a plant cell by biolistic transfer or protoplast transformation. Preferably, the vector or mobile genetic element is stably maintained in the cell by integrating the vector or mobile genetic element into the genome of the cell. By stably integrating the vector or mobile genetic element into the genome of a cell, such as a plant cell, a plant can be regenerated from the cell stably carrying the vector or mobile genetic element, thereby producing a transgenic plant.

[0050] Genome editing can be used to introduce vector or mobile genetic element into plant genome.Genome editing can be used to modify the promoter of the endogenous gene that codes for tonoplast proton-fructose symporter, so that tonoplast proton-fructose symporter is overexpressed.Genome editing includes, for example, CRISPR gene editing using CRISPR-nuclease system.

[0051] In a further aspect, the present invention relates to a host cell comprising the vector or the mobile genetic element. The host cell may be a cell as described herein, in particular a plant cell of a plant as described herein.

[0052] In a further aspect, the present invention relates to a plant or part of a plant comprising at least one host cell, wherein the host cell preferably comprises a vector or a mobile genetic element as described herein.

[0053] The plants described herein are particularly characterized by being genetically modified to contain elevated levels of fructose in the cytosol compared to the wild-type plant from which they are derived.The level of fructose in the cytosol of the plant can be at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, or at least 500% higher than the level of fructose in the cytosol of the wild-type plant.

[0054] The fructose level in the cytosol of a plant, for example a transgenic plant, can be increased in different ways.For example, the fructose level in the cytosol of a plant can be increased by any method that leads to the overexpression of the tonoplast proton-fructose symporter, particularly the tonoplast proton-fructose symporter disclosed herein.

[0055] The overexpression of the tonoplast proton-fructose symporter can be achieved, for example, by activating the promoter of endogenous tonoplast proton-fructose symporter gene.The TATA box of the promoter can be modified to achieve overexpression.To achieve overexpression, cis-regulatory elements can be introduced, for example, via genome editing as described herein and known to those skilled in the art.Promoter activation in plants can be achieved, for example, as described in EP 3 546 582.

[0056] In some embodiments, overexpression of the tonoplast proton-fructose symporter is achieved by introducing at least one additional copy of the tonoplast proton-fructose symporter into the plant cell. The additional copy of the tonoplast proton-fructose symporter can be an additional copy of an endogenous tonoplast proton-fructose symporter or an additional copy of a heterologous tonoplast proton-fructose symporter.

[0057] In some embodiments, the tonoplast proton-fructose symporter can be any one of the tonoplast proton-fructose symporters disclosed herein, for example, ERDL4 (SEQ ID NO: 1 and SEQ ID NO: 22) of Arabidopsis thaliana (At), BvIMP (SEQ ID NO: 39 and SEQ ID NO: 57) of Beta vulgaris (Bv), BvIMP2 (SEQ ID NO: 40 and SEQ ID NO: 58) of Beta vulgaris, or any one of the sequences set forth in SEQ ID NO: 77-128. Preferably, the tonoplast proton-fructose symporter can be any one of the candidates identified in the examples herein. More preferably, the tonoplast proton-fructose symporter can be ERDL4 of Arabidopsis thaliana, BvIMP of Beta vulgaris, or BvIMP2 of Beta vulgaris.

[0058] In a particularly preferred embodiment, the plant is Beta vulgaris that overexpresses a tonoplast proton-fructose symporter, such as BvIMP or BvIMP2.

[0059] In some embodiments, the vacuolar membrane proton-fructose symporter comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to any of the vacuolar membrane proton-fructose symporter amino acid sequences taught herein, particularly any of SEQ ID NOs: 58, 57, 22, and 77-128.

[0060] In addition to overexpressing the tonoplast proton-fructose symporter, other means may be applied to increase the level of fructose in the cytosol in order to achieve the beneficial effects of the present invention.

[0061] For example, downregulation of the expression of SWEET (Sugars Will Eventually be Exported Transporter 17) can increase the amount of fructose in the cytosol. SWEET17 can transport fructose through the tonoplast. In an embodiment, overexpression of tonoplast proton-fructose symporter can be combined with downregulation of the expression of SWEET17. Downregulation of the expression of SWEET17 can be achieved by various means, for example, by genetically knocking out the SWEET17 gene or using RNAi. In an embodiment, SWEET17 is Arabidopsis thaliana SWEET17 (SEQ ID NO: 5, 28) or Beta vulgaris SWEET17 (SEQ ID NO: 45, 64). Downregulation of SWEET17 can be applied to any plant, especially crop plants that have orthologs of SWEET17.

[0062] The level of fructose in the cytosol can also be increased by reducing the activity of fructose kinase and / or hexokinase, which phosphorylates fructose and reduces the amount of free fructose in the cytosol.Therefore, it is contemplated to increase the level of fructose in the cytosol by reducing the expression level of fructokinase and / or hexokinase (e.g., by downregulation or knockout).In some embodiments, the fructokinase and / or hexokinase is selected from the group of fructokinase and / or hexokinase disclosed herein.Preferably, the reduction in the expression level of fructokinase and / or hexokinase can be combined with the overexpression of vacuolar membrane proton-fructose symporter and / or the downregulation of the expression of SWEET17.

[0063] In some embodiments, TILLING (Targeting Induced Local Lesions in Genomes) using chemical mutagens such as ethyl methanesulfonate (EMS) can be applied to identify mutant plants with increased cytosolic levels of fructose. For example, in some embodiments, TILLING can be used to identify plants that overexpress tonoplast proton-fructose symporters such as ERDL4, BvIMP, or BvIMP2.

[0064] Increasing the yield of plant storage compounds refers to increasing the yield of plant storage compounds with respect to a corresponding plant that is not a plant taught herein, particularly a corresponding plant that has a lower fructose level in the cytosol, and / or a plant that is not modified according to the present technical teachings. The corresponding plant is a plant of the same species.

[0065] The methods and uses of the present invention, such as methods of producing plants, methods of increasing the yield of plant storage compounds, and methods of increasing the drought resistance of plants, may comprise a step of growing a plant. EXAMPLES

[0066] Example 1: Overexpression of ERDL4 increases cytosolic fructose A tonoplast-localized transporter called ERDL4 (Early Response to Dehydration-Like 4) belongs to a large subgroup of transporters of the MST family (Figure 1; Buettner, 2007) and is a proton symporter that exports fructose from the vacuole to the cytosol (Figures 2 and 3). Its overexpression leads to an increase in fructose in the cytosol. Whether other sugars are also transported remains to be determined.

[0067] In order for fructose to be sensed and thereby contribute to nuclear signaling, it must be present in the cytosol. Otherwise, transcriptional regulation cannot occur, as the vacuole is considered "outside" the cell and the nucleus cannot see the outside of the cell. In non-aqueous fractionation (NAF) experiments, we could show that fructose content is specifically increased in the cytosol in Arabidopsis thaliana plants overexpressing ERDL4 (Figure 9).

[0068] Generation of ERDL4 Oex (overexpression) lines. The full-length coding region of ERDL4 was amplified with primers ERDL4_Fwd: (5′-ggggacaagtttgtacaaaaaagcaggcttaATGAGTTTTAGGGATGATAATACG-3′) and ERDL4_Rev: (5′-ggggaccactttgtacaagaaagctgggtaTCATCTGAACAAAGCTTGGATCTC-3′), cloned into pDON / Zeo, and subcloned into pK2GW7 GatewayTM vector (Karimi et al., 2002) to generate overexpression lines in Col-0 background. Agrobacterium tumefaciens strain GV3101 was used for transformation. Arabidopsis Col-0 strains were transformed by floral dip method (Clough and Bent, 1998). Positive lines were screened by kanamycin antibiotic resistance, and the strongest lines were further selected using RT-PCR.

[0069] Metabolite quantification Extraction of soluble sugars was performed by adding 1 ml of 80% ethanol to 100 mg of frozen, finely ground plant material at 80°C for 30 min. The extract was collected and evaporated in a vacuum concentrator (Eppendorf, Hamburg, Germany). The resulting pellet was dissolved in ddH2O and quantified using the NADP-linked enzyme test (Stitt et al., 1989) via a 96-well plate reader (Tecan Infinite 200; Tecan Group).

[0070] Example 2: Increasing cytosolic fructose is beneficial for plant biomass production. Arabidopsis thaliana ERDL4 overexpressing plants had larger leaf rosettes (Figure 4) and higher root biomass (Figure 5), higher 1000-seed weights (Figure 6) and higher seed lipid content (Figure 7) than control plants, and also showed a higher sugar flux exported from the leaves via the phloem than control plants (Figure 8).

[0071] Increased rosette size, root biomass and seed weight in ERDL4 overexpressing plants: Rosette size was determined by measuring the rosette fresh weight from 4-week-old plants grown on soil substrate ED73 (Patzer Einheitserde, Sinntal-Altengronau, Germany) under short-day (10 h light / 14 h dark) conditions at 21°C. Rosettes were cut above the hypocotyl and soil particles were removed.

[0072] Root biomass was measured from 4-week-old plantlets grown in a hydroponic system. Plants were removed from the hydroponic system and the roots of individual plants were cut below the hypocotyl. Excess water was removed by rolling the cut root stumps back and forth on a paper towel. Fresh weight of the roots was measured using a precision balance (Sartorius, Goettingen, Germany). After measuring the fresh weight, the roots were dried overnight in an oven at 55 °C and reweighed.

[0073] For seed weight measurements, plants from different lines were grown under short days (10 h light / 14 h dark) at 21°C for five weeks and then transferred to long days (18 h light / 6 h dark) at 21°C for seed maturation. Approximately 1000 mature seeds from at least four plants from WT, two ERDL4 overexpressing lines, and two erdl4 mutant lines were counted and weighed. The 1000 seed weight was calculated by dividing the weight of harvested seeds by the number of seeds and multiplying the value by 1000.

[0074] Determination of lipid content: Lipid quantification was performed according to a previously optimized protocol (Reiser et al., 2004). 100 mg of seeds were homogenized in a mortar with liquid nitrogen. 1.5 ml of isopropanol was added to the homogenate and the seeds were further homogenized. It was transferred to a 1.5 ml Eppendorf tube and incubated at 4°C for 12 h at 100 rpm. The sample was centrifuged at 13,000 g for 10 min. The supernatant was then transferred to a pre-weighed 1.5 ml Eppendorf tube and evaporated at 60°C for 8 h. The remaining lipid pellet was quantified gravimetrically.

[0075] Phloem exudate measurement: Phloem exudates were collected according to the protocol of Xu et al.

[2019] with some modifications. Source leaves of the same developmental stage from 6-week-old well-watered plants were cut at the base of the petiole with a sharp razor blade. The petiole was immersed in 20 mM K2-EDTA solution. Leaves from the same line were stacked and the petiole was re-cut while immersed in EDTA solution. The leaves were then transferred to 1.5 ml Eppendorf tubes with 500 ul of EDTA solution. Exudates were collected for 6 h in a dark, humid chamber. Leaves were weighed and the exudates were dried in a vacuum concentrator. The pellets were solubilized in 100 ul of ddH2O for sugar quantification.

[0076] Example 3: Increased cytosolic fructose is sensed in the nucleus and triggers transcriptional regulation Overexpression of ERDL4 increases cytosolic fructose, which is sensed in the nucleus. The transcription of several genes is deregulated by the presence of increased fructose in the cytosol. One example of such a gene is CIPK6 (Calcineurin B-like protein (CBL)-interacting protein kinase 6; Figure 10), which activates TST2 (Tonoplast Sugar Transporter 2) through phosphorylation, which then imports additional monosaccharides into the vacuole (Deng et al., 2020). Whether this activation of TST2 is part of the explanation for the increased biomass through fructose sensing remains to be elucidated. Overexpression of CIPK6 is one possibility to test this. Meanwhile, knockout of CIPK6 in ERDL4 overexpressors will likely lead to elucidation of the transcriptional and post-translational activation of TST2, which may give hints as to whether and which, if any, of these activations are important for the ERDL4 overexpression phenotype. However, overexpression of TST in Arabidopsis thaliana has been shown to result in a similar phenotype with increased biomass (Wormit et al., 2006; Wingenter et al., 2010). Other genes are also deregulated in ERDL4 overexpressors and / or in high cytosolic fructose plants, which may also explain some of the phenotypes seen.

[0077] For RNA-Seq analysis, RNA was isolated using the RNEasy KIT (Qiagen, Hilden, Germany) from source leaves of 6-week-old plants grown under short days (10 h light, 14 h dark at 21 °C) on soil substrate ED73 (Einheitserde Patzer, Sinntal-Altengronau, Germany). Briefly, approximately 100 mg of frozen and pulverized tissue was extracted with 1.5 ml of Qiazol lysis reagent in a 2 ml Eppendorf cup and centrifuged at 12.000 g at 4 °C. The supernatant was purified using RNEasy spin columns. RNA concentration was calculated using Nanodrop 2000 / 2000c (Thermo Fisher, Germany). Approximately 5 micrograms of total RNA was transported to NOVOGENE (Cambridge, UK) for RNA library construction and bioinformatics RNaseq analysis (https: / / en.novogene.com).

[0078] Example 4: Increasing cytosolic fructose by decreasing fructokinase and hexokinase activity to increase plant biomass production Furthermore, there are several enzymes that phosphorylate fructose to F6P to return it to carbon metabolism - thus, reduced activity of certain fructokinases (FrcKs) and hexokinases (HxKs), brought about by knocking out or downregulating their expression, could also result in higher fructose concentrations, thereby increasing fructose sensing and plant biomass production.

[0079] Example 5: Overexpression of ERDL4 and downregulation of SWEET17 synergize to increase cytosolic fructose levels and enhance plant biomass production Moreover, Arabidopsis SWEET17 (Sugars Will Eventually be Exported Transporter 17) has the ability to transport fructose across the tonoplast. However, because SWEET17 is a facilitator rather than an activated transporter, fructose does not accumulate in the cytosol (Chardon et al., 2013; Guo et al., 2014; Klemens et al., 2014; Valifard et al., 2021). The fructose gradient (fructose concentration in the vacuole versus the cytosol) potentially present across the tonoplast can only be equalized. Therefore, simultaneous overexpression of ERDL4 and downregulation of SWEET17 is expected to result in even higher cytosolic fructose levels. For example, in sugar beet, BvERDL4;2 (sequences are given in the Appendix) and BvSWEET17 (the sequence of the closest orthologue of SWEET17 from Arabidopsis thaliana is given in the Appendix) could be modified to increase cytosolic fructose.

[0080] Example 6: Overexpression of ERDL4 homologues in crop plants for increased plant biomass production Overexpression using transgenes As an example of the crop to be improved, the experiments contemplated for sugar beet are described. However, the improvement of other crops is also contemplated. Overexpression of the sugar beet orthologue of Arabidopsis thaliana ERDL4 is predicted to lead to increased biomass and yield. BvERDL4;2 (BvIMP2) is likely to be the corresponding orthologue with the appropriate function, since its overexpression leads to fructose export from the vacuole (Experiment 7; Figure 11). Expression of BvIMP2 under the control of a constitutive promoter (e.g. the 35S-CaMV promoter or the ubiquitin promoter) or a leaf or green tissue specific promoter (e.g. the chlorophyll a / b binding protein promoter or the StLS1 promoter, respectively) is expected to mimic the Arabidopsis thaliana ERDL4 overexpression phenotype.

[0081] Overexpression of endogenous genes by activating their promoters An alternative to the above GM approach is to activate the promoter of BvIMP2 by modifying core promoter elements such as the TATA box or by introducing cis-regulatory elements by genome editing. Such modified promoters lead to higher expression of BvIMP2 and thus to the above-mentioned phenotypes.

[0082] TILLING mutants leading to overexpression of ERDL4 Furthermore, promoter activation can be achieved by mutagenesis, e.g., by single point mutations improving the core promoter elements. Furthermore, mutagenesis of the coding sequence or other adjacent regulatory sequences (yet to be determined) may lead to improved activity in enhancing fructose export from the vacuole.

[0083] Gain of fructose transport function by mutagenesis of BvERDL4;1 Because the sequence of BvERDL4;1 (BvIMP) is similar to BvIMP2, it seems reasonable that its function could be redirected to fructose transport by mutagenesis. Such an extra fructose transporter could lead to an increase in cytosolic fructose and result in the same phenotypes as above.

[0084] Overexpression of ERDL4 coupled with downregulation / knockout of SWEET17 Similar to what was mentioned above, overexpression of BvIMP2 (either by mutagenesis or genome editing) in a sweet17 mutant background could result in even higher cytosolic fructose content (see Experiment 4). Alternatively, overexpression of BvIMP2 could be combined with RNAi against BvSWEET17 to achieve this goal.

[0085] Screening of plant populations for natural overexpressors of ERDL4 and subsequent enrichment of the allele in breeding pools Populations of elite or exotic breeding material of sugar beet (and other crops) are screened for genotypes with increased BvIMP2 expression. Such screening is performed via RNaseq in leaves. Detection of natural overexpressors will be used for crossing to elite material, increasing its corresponding allele in the breeding pool. This should ultimately lead to increased yield.

[0086] Example 7: Overexpression of a potential sugar beet ERDL4 homolog in Arabidopsis indicates fructose export from the vacuole Analysis of the evolution of sugar and starch contents in Arabidopsis thaliana (Col-0, attst1-2, BvIMP2 OX1 and OX2) under cold stress (4°C) and reacclimation (20°C; Fig. 11) allowed us to assign BvIMP2 (Bv6_128840_qhip.t1), the sugar beet orthologue of AtERDL4, to the vacuolar fructose export function. Glucose (a), fructose (b), sucrose (c) and starch (d) contents over a 14-day period are shown. The fructose content in BvIMP2 OX1 and OX2 did not reach the level of fructose in the control plants and decreased faster than in the control plants. This indicates that BvIMP2 (joining endogenous AtERDL4) exports fructose from the vacuole. Cytosolic fructose can then be sent to metabolism and utilized for assimilation reactions. Here, the extra fructose is thought to be a precursor for the accumulation of starch.

[0087] To analyze sugar and starch content in Arabidopsis thaliana (Col-0, attst1-2, BvIMP2 OX1 and OX2) under cold stress (4°C) and reacclimation (20°C), plants were grown in standard soil (ED-73; DIN1154080T) consisting of 50 l soil mixed with 15 l sand. Prior to the experiment, plants were grown at 20°C (10 h light / 14 h dark) 110 μmol m-2 s-1 for 21 days. After 3 weeks, plants were transferred to 4°C under the same light and dark conditions. Plant material was harvested before the 4°C treatment and 1, 2, 4 and 7 days after transfer to 4°C. After the entire 7-day 4°C treatment, the remaining plants were returned to 20°C and plant material was harvested 4 and 7 days after return. Four biological replicates, each consisting of a pool of four plant rosettes, were harvested at 10 am, immediately after light onset. Harvested plant material was transferred to liquid nitrogen, ground, and stored at −80°C until use.

[0088] Soluble sugars were extracted from the frozen material. For this purpose, approximately 50 mg of powdered plant material was weighed into 1 ml of 80% EtOH solution and extracted twice with 1 ml of 80% EtOH solution at 80 °C for 1 h with shaking at 350 rpm. After each extraction step, the insoluble components were centrifuged at 14,000 rpm for 10 min and the supernatants were combined in 2 ml reaction tubes. The combined metabolite extracts were then evaporated in a concentrator-vacuum centrifuge (Eppendorf, Hamburg, Germany). The evaporated pellet was dissolved in 1 ml ddH2O and stored at -20 °C until measurement. The pellet remaining after extraction was washed with 500 μl 80% EtOH and 1 ml ddH2O and used for starch extraction.

[0089] For starch extraction, the washed pellet remaining after extraction of soluble metabolites was mixed with 200 μl ddH2O and autoclaved at 121 °C for 40 min. The starch chains present in the pellet were then hydrolytically digested. For hydrolytic cleavage of starch, 200 μl of enzyme mixture (5 U α-amylase, Sigma-Aldrich, Munich, Germany; 5 U amyloglucosidase, Roche, Mannheim, Germany; 200 mM sodium acetate; pH 4.8) was added to the autoclaved pellet and incubated at 37 °C for at least 4 h. To stop the enzymatic reaction, the sample was subsequently heated at 95 °C for 10 min. After a final centrifugation at 14,000 rpm for 10 min, the supernatant was used to quantify starch.

[0090] The concentrations of sugars (glucose, fructose, sucrose) and hydrolyzed starch were measured by NAD+-coupled enzyme assay as described by Stitt et al., 1989. For this purpose, 190 μl of premix was added to 10–20 μl of sample. Glucose, fructose and sucrose were measured photometrically at a wavelength of 340 nm using an Infinite® M Nano microplate reader (Tecan, Maennedorf, Switzerland) after addition of the enzymes hexokinase (Roche, Mannheim, Germany), phosphoglucoisomerase (Roche, Mannheim, Germany) and invertase (Sigma-Aldrich, Munich, Germany). The sugar content was then calculated according to the Lambert-Beer law.

[0091] The premix for sugar determination contained: 100 mM HEPES (pH 7.5), 10 mM MgCl2, 2 mM ATP, 1 mM NADP, and 0.5 U of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides.

[0092] Table 1. Strains analyzed [Table 1]

[0093] Example 8: Overexpression of ERDL4 to enhance drought tolerance ERDL4 overexpressing Arabidopsis plants already have a higher biomass under well-watered control conditions (100% field capacity; compare Fig. 4). When plants are exposed to mild (60% field capacity) or more severe (40% field capacity) drought stress, the fresh weight biomass is reduced. However, in overexpressing plants the biomass reduction is less pronounced than in the control (Fig. 12). Calculation of the fresh weight ratio of overexpressing (Oex) to control shows that: 100% field capacity – Oex1 / Wt = 1.38; Oex2 / Wt = 1.29 60% field capacity – Oex1 / Wt = 1.72; Oex2 / Wt = 2.01 40% Field Capacity – Oex1 / Wt = 1.66; Oex2 / Wt = 1.49

[0094] The beneficial effect of ERDL4 overexpression for drought tolerance can be explained by the improved root growth of overexpressor plants (compare Figure 5), which can utilize their elongated root system to obtain water better than control plants and even erdl4 mutants (KO1, KO2). As a result, mutants with smaller root systems are especially sensitive to drought.

[0095] Application of drought stress to plants growing in soil substrate: Plants were exposed to drought conditions based on soil field capacity (FC) (Bouzid et al., 2019). Plants were maintained at defined soil moisture contents with 48-h irrigation intervals until harvest (control = 100%, 60%, 40% FC). Seeds of wild-type and ERDL4-overexpressing lines were germinated in standard soil (ED-73; Einheitserde Patzer; Sinntal-Altengronau, Germany) and plants were grown for one week before being transplanted into soil with 60% moisture / field capacity (FC) compared to the control soil (100%). To prevent osmotic shock, seedlings were not transplanted directly from 100% FC to 40% FC. Instead, two-week-old seedlings grown for one week in 100% FC and one week in 60% FC were not watered until soil moisture reached 40%. Afterwards, the soil moisture level was kept constant by monitoring the pot weight (Xt) at 48-h intervals (precision balance with an accuracy of 0.01 g). To calculate the field water capacity of the soil, three pots were filled with the corresponding soil, then dried in an oven at 60 °C for 4 days, and the dry soil weight of each pot (X0) was measured. Consequently, three more pots were filled with the same amount of soil, dried, and then watered until the soil was saturated (Xf). The pots were then transferred to a growth chamber. After 48 h, the pots were weighed again (Xt). The percentage of the soil's field water capacity was calculated using the following formula: [(Xt-X0) / (Xf-X0)] *100 (Bouzid et al., 2019).

[0096] Drought stress was applied based on the field water capacity of the soil (100%, 60%, 40%). Plants were subjected to stress 7 days after germination. Stress was applied for 3.5 weeks. Fresh weight was determined by the fresh weight of rosettes immediately after harvest.

[0097] Example 9: Many genes deregulated in Arabidopsis thaliana ERDL4 overexpressors are also deregulated in fructose- but not glucose-floated Arabidopsis thaliana leaf discsBecause ERDL4 overexpression leads to elevated fructose and glucose levels in the cytosol, it is reasonable to check the effects on global gene expression caused by changes in intracellular sugar distribution by RNASeq analysis. To test whether the transcriptome changes in ERDL4 overexpressors are caused by intracellular glucose or fructose accumulation, we performed control experiments in which excised discs from mature Arabidopsis leaves were incubated overnight in a 0.5 MS buffer solution at pH 5.7 supplemented with 1% glucose, 1% fructose, or 1% mannitol (as an osmolarity control). Under these conditions, supplemented sugars diffuse through the apoplasm into intact mesophyll cells, where they are imported into the cytosol. Such flooding with pure sugar species has been shown to trigger sugar species-specific responses at the gene expression level (Wormit et al., 2006; Luo et al., 2012). Genes that show significant differential expression between different plant lines or between different treatments are called “differentially expressed genes” (DEGs). DEGs between ERDL4 overexpressors and wild type (WT) were identified by testing for significant differences in the expression of individual genes using two-tailed t-tests. DEGs between fructose and mannitol incubations or between glucose and mannitol incubations were identified by testing for significant differences in the expression of individual genes using two-tailed t-tests. Conditional analysis identified DEGs overlapping between ERDL4 overexpressors and fructose treatment or between ERDL4 overexpressors and glucose treatment (Figure 13). In total, 130 common DEGs were identified between ERDL4 overexpressors and fructose treatment, and 155 common DEGs were identified between ERDL4 overexpressors and glucose treatment (Figure 13).Correlation analysis based on the individual amplitude of gene expression changes for 130 common DEGs from ERDL4 overexpressors and fructose treatment, and 155 common DEGs from ERDL4 overexpressors and glucose treatment, showed a Pearson coefficient of r = 0.48 for ERDL4 overexpressors and fructose treatment, and ERDL4 overexpressors and glucose treatment, respectively (r 2 =0.23) and r=0.15 (r 2 = 0.0225). This analysis revealed a ten-fold higher correlation between ERDL4 overexpression-dependent and fructose-dependent gene expression changes compared with ERDL4 overexpression-dependent and glucose-dependent gene expression, indicating that the transcriptional changes recorded in ERDL4 overexpressors were driven by changes in fructose rather than alterations in glucose signaling.

[0098] Example 10: Identification of orthologous sequences of AtERDL4 in several dicotyledonous and monocotyledonous crop species To identify crop genes orthologous to the Arabidopsis thaliana ERDL4 gene, a series of sequence comparisons were performed with the corresponding protein (amino acid) sequences of these genes. Genes / proteins from sugar beet (Beta vulgaris), cabbage (Brassica rapa), oilseed rape (Brassica napus), sunflower (Helianthus annuus), potato (Solanum tuberosum), maize (Zea mays), rice (Oryza sativa), barley (Hordeum vulgare), rye (Secale cereale) and sorghum (Sorghum bicolor) were analyzed. To show that Arabidopsis thaliana proteins can be distinguished from each other using a BLAST search (Altschul et al., 1997; standard conditions: Expect: 10; Matrix: pairwise alignments generated with BLOSUM62), a comparison was first made between AtERDL4, 6 and 7 (Table 2, compare also Fig. 1). The higher the score, the more similar the sequences are. ERDL6 and 7 were included in the analysis because ERDL6 is the most similar paralog of ERDL4 in Arabidopsis thaliana, and ERDL7 is the second most similar but is already quite divergent from ERDL4 and 6 (compare also Fig. 1).

[0099] Table 2. Comparison of Arabidopsis thaliana ERDL4, 6, and 7. [Table 2]

[0100] AtERDL4, 6 and 7 are distinguishable from each other.

[0101] To define putative orthologues of AtERDL4 in Brassica rapa, three BLAST searches were performed against protein sequences of the Brassica rapa genome using (i) AtERDL4, (ii) AtERDL6, and (iii) AtERDL7 as query sequences, respectively. The obtained BLAST scores are shown in Table 3.

[0102] Table 3. Comparison of AtERDL4, AtERDL6 and AtERDL7 with Brassica rapa proteins, respectively. [Table 3]

[0103] BrA and BrB (see full sequences in the Appendix) are candidates for AtERDL4 orthologues in cabbage, while BrC, BrD, and BrE are candidates for AtERDL6. BrF is a candidate orthologue for AtERDL7. Other sequences from Arabidopsis thaliana can also be included in the analysis (Figure 1). However, the purpose of the sequence comparison here is only to define the most likely candidates for the orthologues of AtERDL4 (not all AtERDL proteins).

[0104] To define putative orthologues of AtERDL4 in Brassica napus, three BLAST searches were performed against protein sequences of the Brassica napus genome using (i) AtERDL4, (ii) AtERDL6, and (iii) AtERDL7 as query sequences, respectively. The resulting BLAST scores are shown in Table 4.

[0105] Table 4. Comparison of AtERDL4, AtERDL6 and AtERDL7 with Brassica napus proteins, respectively. [Table 4]

[0106] BnA, BnB, BnC and BnD are candidates for the AtERDL4 orthologues in Brassica napus. BnE, BnF, BnG and BrH are more likely to be orthologues of AtERDL6. BnI and BnJ are unlikely to be candidates for the AtERDL4 orthologues.

[0107] To define putative orthologues of AtERDL4 in Beta vulgaris, three BLAST searches were performed against protein sequences of the Beta vulgaris genome using (i) AtERDL4, (ii) AtERDL6, and (iii) AtERDL7 as query sequences, respectively. The obtained BLAST scores are shown in Table 5.

[0108] Table 5. Comparison of AtERDL4, AtERDL6 and AtERDL7 with Beta vulgaris proteins, respectively. [Table 5]

[0109] BvIMP and BvIMP2 are candidates for the AtERDL4 (and AtERDL6) orthologues in sugar beet. BvC does not appear to be a candidate for the orthologue of AtERDL4 or 6. These data are supported by the functional analysis of BvIMP2, which shows that BvIMP2 is a functional orthologue of AtERDL4 (see Figure 11).

[0110] To define putative orthologues of AtERDL4 in Helianthus annuus, three BLAST searches were performed against protein sequences of the Helianthus annuus genome using (i) AtERDL4, (ii) AtERDL6, and (iii) AtERDL7 as query sequences, respectively. The resulting BLAST scores are shown in Table 6.

[0111] Table 6. Comparison of AtERDL4, AtERDL6, and AtERDL7 with Helianthus annuus proteins, respectively. [Table 6]

[0112] HaA and HaB are candidates for the AtERDL4 (and AtERDL6) orthologues in sunflower. HaD does not appear to be a candidate for the orthologue of AtERDL4 or 6.

[0113] To define putative orthologues of AtERDL4 in Solanum tuberosum, three BLAST searches were performed against protein sequences of the Solanum tuberosum genome using (i) AtERDL4, (ii) AtERDL6 and (iii) AtERDL7 as query sequences, respectively. The obtained BLAST scores are shown in Table 7.

[0114] Table 7. Comparison of AtERDL4, AtERDL6 and AtERDL7 with Solanum tuberosum proteins, respectively. [Table 7]

[0115] StA and StB are candidates for the AtERDL4 (and AtERDL6) orthologues in potato. StF does not appear to be a candidate for the orthologue of AtERDL4 or 6.

[0116] To define putative orthologues of AtERDL4 in several monocotyledonous plant species, three BLAST searches were performed against protein sequences of several monocotyledonous plant species genomes using (i) AtERDL4, (ii) AtERDL6 and (iii) AtERDL7 as query sequences, respectively. The obtained BLAST scores are shown in the table below. To define putative orthologues of AtERDL4 in Zea mays, three BLAST searches were performed against protein sequences of Zea mays genome using (i) AtERDL4, (ii) AtERDL6 and (iii) AtERDL7 as query sequences, respectively. The obtained BLAST scores are shown in Table 8 below.

[0117] Table 8. Comparison of AtERDL4, AtERDL6 and AtERDL7 with Zea mays proteins, respectively. [Table 8]

[0118] ZmA and ZmB are the best candidates for the AtERDL4 (and AtERDL6) orthologues in maize. ZmF is not a candidate for the orthologue of AtERDL4 or 6.

[0119] To define putative orthologues of AtERDL4 in Oryza sativa, three BLAST searches were performed against protein sequences of the Oryza sativa genome using (i) AtERDL4, (ii) AtERDL6, and (iii) AtERDL7 as query sequences, respectively. The obtained BLAST scores are shown in Table 9.

[0120] Table 9. Comparison of AtERDL4, AtERDL6 and AtERDL7 with Oryza sativa proteins, respectively. [Table 9]

[0121] OsA, OsB and OsC are candidates for the orthologues of AtERDL4 (and AtERDL6) in rice. OsD, OsE and OsF are unlikely candidates for the orthologues of AtERDL4 (and AtERDL6).

[0122] To define putative orthologues of AtERDL4 in Hordeum vulgare, three BLAST searches were performed against protein sequences of the Hordeum vulgare genome using (i) AtERDL4, (ii) AtERDL6 and (iii) AtERDL7 as query sequences, respectively. The obtained BLAST scores are shown in Table 10.

[0123] Table 10. Comparison of AtERDL4, AtERDL6 and AtERDL7 with Hordeum vulgare proteins, respectively. [Table 10]

[0124] HvA, HvB and HvC are candidates for the orthologues of AtERDL4 (and AtERDL6) in barley. HvD is not a candidate for the orthologues of AtERDL4 (and AtERDL6).

[0125] To define putative orthologues of AtERDL4 in Secale cereale, three BLAST searches were performed against protein sequences of the Secale cereale genome using (i) AtERDL4, (ii) AtERDL6 and (iii) AtERDL7 as query sequences, respectively. The resulting BLAST scores are shown in Table 11.

[0126] Table 11. Comparison of AtERDL4, AtERDL6 and AtERDL7 with Secale cereale proteins, respectively. [Table 11]

[0127] ScA, ScB, or ScC are candidates for the orthologues of AtERDL4 (and AtERDL6) in rye. ScD and especially ScE are unlikely candidates for the orthologues of AtERDL4 (and AtERDL6).

[0128] To define putative orthologues of AtERDL4 in Sorghum bicolor, three BLAST searches were performed against protein sequences of the Sorghum bicolor genome using (i) AtERDL4, (ii) AtERDL6, and (iii) AtERDL7 as query sequences, respectively. The obtained BLAST scores are shown in Table 12.

[0129] Table 12. Comparison of AtERDL4, AtERDL6 and AtERDL7 with Sorghum bicolor proteins, respectively. [Table 12]

[0130] SbA, SbB, or SbC are candidates for the orthologues of AtERDL4 (and AtERDL6) in sorghum. SbD and especially SbE are unlikely candidates for the orthologues of AtERDL4 (and AtERDL6).

[0131] The foregoing examples are illustrative of the invention and should not be construed as limiting the invention to the embodiments disclosed therein. Although the invention has been described in detail with reference to preferred embodiments, variations and modifications exist within the scope and spirit of the invention as described and defined in the following claims.

[0132] References 1) In concert: Orchestrated changes in carbohydrate homeostasis are critical for plant abiotic stress tolerance; Pommerrenig B et al. (2018), DOI: 10.1093 / pcp / pcy037 2) ChloroP, a neural network based method for predicting chloroplast transit peptides and their cleavage sites; Emanuelsson O et al., (2008), DOI: 10.1110 / ps.8.5.978 3) ARAMEMNON, a novel database for Arabidopsis integral membrane proteins; Schwacke R et al. (2003), DOI: 10.1104 / pp.011577 4) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega; Sievers F et al. (2011), DOI: 10.1038 / msb.2011.75 5) MrBayes: Bayesian phylogenetic inference under mixed models; Ronquist F & Huelsenbeck JP (2003), DOI: 10.1093 / bioinformatics / btg180 6) The monosaccharide transporter(-like) gene family in Arabidopsis; Buettner M (2007), DOI: 10.1016 / j.febslet.2007.03.016 7) GATEWAY TMvectors for Agrobacterium-mediated plant transformation; Karimi M et al. (2002), DOI: 10.1016 / S1360-1385(02)02251-3 8) "Floral dip: a simplified method for Agrobacterium‐mediated transformation of Arabidopsis thaliana; Clough S & Bent AF (1998), DOI: 10.1046 / j.1365-313x.1998.00343.x 9) Metabolite levels in specific cells and subcellular compartments of plant leaves; Stitt M (1989), DOI: 10.1016 / 0076-6879(89)74035-0 10) Molecular physiological analysis of the two plastidic ATP / ADP transporters from Arabidopsis; Reiser J et al (2004), DOI: 10.1104 / pp.104.049502 11) Regulation of sucrose transporters and phloem loading in response to environmental cues; Xu Q et al (2018), DOI: 10.1104 / pp.17.01088 12) The Calcium Sensor CBL2 and Its Interacting Kinase CIPK6 Are Involved in Plant Sugar Homeostasis via Interacting with Tonoplast Sugar Transporter TST2(1); Deng JW et al. (2020), DOI: 10.1104 / pp.19.01368 13) Molecular identification and physiological characterization of a novel monosaccharide transporter from Arabidopsis involved in vacuolar sugar transport; Wormit A et al. (2006), DOI: 10.1105 / tpc.106.047290 14) Increased Activity of the Vacuolar Monosaccharide Transporter TMT1 Alters Cellular Sugar Partitioning, Sugar Signaling, and Seed Yield in Arabidopsis; Wingenter K et al (2010), DOI: 10.1104 / pp.110.162040 15) Leaf Fructose Content Is Controlled by the Vacuolar Transporter SWEET17 in Arabidopsis; Chardon F et al (2013), DOI: 10.1016 / j.cub.2013.03.021 16) SWEET17, a Facilitative Transporter, Mediates Fructose Transport across the Tonoplast of Arabidopsis Roots and Leaves; Guo, WJ et al. (2014), DOI: 10.1104 / pp.113.232751 17) Overexpression of a proton‐coupled vacuolar glucose exporter impairs freezing tolerance and seed germination; Klemens, PAW et al. (2014), DOI: 10.1111 / nph.12642 18) Vacuolar fructose transporter SWEET17 is critical for root development and drought tolerance; Valifard M et al (2021), DOI: 10.1093 / plphys / kiab436 19) Arabidopsis species deploy distinct strategies to cope with drought stress; Bouzid M et al. (2019), DOI: 10.1093 / aob / mcy237 20) An autoregulatory feedback loop involving PAP1 and TAS4 in response to sugars in Arabidopsis; Luo, QJ et al. (2012), DOI: 10.1007 / s11103-011-9778-9 21) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs; Altschul, SF et al. (1997), DOI: 10.1093 / nar / 25.17.3389

Claims

1. A vector or mobile genetic element comprising a nucleic acid molecule, the nucleic acid molecule encoding a vacuolar membrane proton-fructose symporter, the nucleic acid molecule comprising: a) a nucleic acid molecule having at least 50% nucleic acid sequence identity to a nucleic acid sequence selected from the group consisting of any one of the sequences set forth in SEQ ID NOs: 40, 39, 1, and 129-152; b) a nucleic acid molecule having a nucleic acid sequence that is complementary to or hybridizes under stringent conditions to the nucleic acid molecule described in a); and c) a nucleic acid molecule having a nucleic acid sequence encoding a protein having at least 50% amino acid sequence identity to an amino acid sequence selected from the group consisting of any one of the sequences set forth in SEQ ID NOs: 58, 57, 22, and 77-128. A vector or mobile genetic element selected from the group consisting of:

2. A host cell comprising the vector or mobile genetic element of claim 1.

3. A plant or part of a plant comprising at least one host cell according to claim 2.

4. A plant comprising the vector or mobile genetic element of claim 1, which comprises elevated levels of fructose in the cytosol compared to the wild-type plant from which it was derived.

5. A plant according to claim 4, which overexpresses a vacuolar membrane proton-fructose symporter compared to the wild-type plant from which it is derived.

6. i) the plant comprises a modified promoter of an endogenous gene encoding the tonoplast proton-fructose symporter to be overexpressed, thereby resulting in homologous overexpression of the tonoplast proton-fructose symporter; or ii) the plant comprises at least one additional copy of a nucleic acid encoding the tonoplast proton-fructose symporter to be overexpressed, thereby resulting in homologous overexpression of the tonoplast proton-fructose symporter, or iii) the plant comprises a heterologous nucleic acid encoding a heterologous tonoplast proton-fructose symporter, whereby the tonoplast proton-fructose symporter is heterologously overexpressed; The plant according to claim 5.

7. A seed of the plant according to claim 4.

8. a) modifying at least one plant cell so as to produce at least one plant cell that contains an elevated level of fructose in the cytosol relative to the wild-type plant cell from which it was derived; and b) regenerating a plant from the modified plant cell of step a), wherein the plant contains elevated levels of fructose in the cytosol compared to the wild-type plant from which it was derived. A method for producing a plant, comprising:

9. modifying the at least one plant cell in step a) i) modifying the promoter of an endogenous gene encoding a vacuolar membrane proton-fructose symporter, thereby overexpressing said vacuolar membrane proton-fructose symporter; ii) introducing into said plant cell at least one additional copy of a nucleic acid encoding a tonoplast proton-fructose symporter, thereby homologously overexpressing said tonoplast proton-fructose symporter; or iii) introducing a heterologous nucleic acid encoding a heterologous tonoplast proton-fructose symporter into said plant cell, thereby resulting in homologous overexpression of said tonoplast proton-fructose symporter. The method of claim 8, comprising:

10. 9. The method of claim 8, wherein the vector or mobile genetic element of claim 1 is introduced into the at least one plant cell.

11. A method for increasing the yield of plant storage compounds, comprising increasing the concentration of fructose in the cytosol of a plant, wherein the plant producing the plant storage compounds is produced by the method of claim 8.

12. A method for increasing the yield of a plant storage compound, comprising increasing the concentration of fructose in the cytosol of a plant, wherein the plant producing the plant storage compound is a plant described in claim 4.

13. 13. The method according to claim 12, wherein the plant storage compound is selected from sugars, preferably sucrose, starches, proteins or lipids.

14. i) increasing the biomass of at least one plant organ containing said plant storage compounds, preferably said plant organ being a fruit, a seed, a root or a storage organ; or ii) increasing the concentration of said plant storage compound in at least one plant organ, preferably said plant organ being a fruit, a seed, a root or a storage organ; The method of claim 11.

15. 10. A method for increasing drought tolerance in a plant, comprising increasing the concentration of fructose in the cytosol of the plant, wherein the plant having increased drought tolerance is produced by the method of claim 8.

16. A method for increasing drought tolerance in a plant, comprising increasing the concentration of fructose in the cytosol of the plant, wherein the plant having increased drought tolerance is a plant described in claim 4.

17. 5. The plant according to claim 4, wherein the plant is selected from the group consisting of sugar beet (Beta vulgaris), cabbage (Brassica rapa), rapeseed (Brassica napus), sunflower (Helianthus annuus), potato (Solanum tuberosum), maize (Zea mays), rice (Oryza sp., e.g. Oryza sativa), barley (Hordeum vulgare), rye (Secale cereale), or sorghum (Sorghum sp.), preferably sugar beet.

18. 12. The method of claim 11, wherein the plant is a sugar beet, the plant organ is a sugar beet taproot, and the plant storage compound is sucrose.

19. 1. A method for selecting plants having increased fructose levels in the cytosol of the plant, comprising: i) Growing plants; ii) measuring the fructose level in at least one plant cell of said plant; iii) comparing the measured fructose level with at least one fructose reference level; and iv) Selecting plants with increased fructose levels relative to at least one reference level. A method comprising:

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