PLANTS WITH INCREASED YIELD AND METHOD FOR PRODUCING SAID PLANTS

Abstract

The invention relates to a method for increasing the yield and biomass of a plant, by means of an increase in the expression of the L-aspartate oxidase in the plant. The method according to the invention allows an increase in the photosynthetic capacities of the plants as a result of an increase in the quantities of NAO and the derivatives thereof in said plants. The invention relates to the plants produced by such a method.

Claims

1. A method for improving at least one phenotypic trait of a plant, said method comprising transforming the plant with a nucleic acid sequence, wherein the nucleic acid sequence comprises a sequence encoding L-aspartate oxidase and comprises an exogenous promoter functionally linked to the sequence encoding L-aspartate oxidase, wherein the at least one phenotypic trait is one or more selected from the group consisting of seed yield, abiotic stress resistance, biotic stress resistance, germination rate, and growth rate, wherein the abiotic stress resistance comprises at least one of intense sunlight, lack of light, excess water, cold, or excessive heat, wherein the nucleic acid sequence is present in a genome, wherein the resulting plant overexpresses L-aspartate oxidase, wherein the overexpression of L-aspartate oxidase leads to increased synthesis of NAD and derivatives thereof, and wherein said sequence comprises SEQ ID NO:1 or a sequence with at least 80% identity to SEQ ID NO:1, with the proviso that the sequence having at least 80% identity is not SEQ ID NO:15.

2. The method according to claim 1, further comprising cultivation of the plant to maturity, wherein the method improves the seed yield of the plant.

3. The method according to claim 1, further comprising cultivation of the plant to maturity, wherein the method improves the germination rate of the plant.

4. The method according to claim 1, further comprising cultivation of the plant to maturity, wherein the method improves the abiotic stress resistance and/or the biotic stress resistance of the plant.

5. The method according to claim 1, wherein the plant is selected from the group consisting of wheat, barley, rice, maize, sorghum, sunflower, rapeseed, soybean, cotton, pea, common bean, cassava, mango, banana, potato, tomato, pepper, melon, zucchini, watermelon, lettuce, cabbage, eggplant, and poplar.

6. The method according to claim 1, wherein the plant is rice (Oryza sativa), wheat, barley or maize.

7. The method according to claim 1, wherein the sequence encoding L-aspartate oxidase comprises a sequence having at least 90% homology with SEQ ID NO: 1.

8. The method according to claim 1, wherein the sequence encoding L-aspartate oxidase comprises a sequence having at least 95% homology with SEQ ID NO: 1.

9. The method according to claim 1, wherein the sequence encoding L-aspartate oxidase comprises a sequence having at least 98% homology with SEQ ID NO: 1.

10. A method for improving at least one phenotypic trait of a plant, said method comprising transformation of a nucleic acid sequence into the plant, wherein the nucleic acid sequence comprises a sequence encoding L-aspartate oxidase and comprises an exogenous promoter functionally linked to the sequence encoding L-aspartate oxidase, wherein the at least one phenotypic trait is one or more selected from the group consisting of seed yield, abiotic stress resistance, biotic stress resistance, germination rate, and growth rate, wherein the abiotic stress resistance is at least one of intense sunlight, lack of light, excess water, cold, or excessive heat, wherein the nucleic acid sequence is present in a genome, wherein the resulting plant overexpresses L-aspartate oxidase, and wherein the overexpression of L-aspartate oxidase leads to increased synthesis of NAD and derivatives thereof, wherein the sequence encoding L-aspartate oxidase comprises SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 16 or a sequence having at least 80% identity with SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 16, with the proviso that the sequence having at least 80% identity is not SEQ ID NO:15.

11. The method according to claim 10, further comprising cultivation of the plant to maturity, wherein the method improves the seed yield of the plant.

12. The method according to claim 10, further comprising cultivation of the plant to maturity, wherein the method improves the germination rate of the plant.

13. The method according to claim 10, further comprising cultivation of the plant to maturity, wherein the method improves the abiotic stress resistance and/or the biotic stress resistance of the plant.

14. The method according to claim 10, wherein the plant is selected from the group consisting of wheat, barley, rice, maize, sorghum, sunflower, rapeseed, soybean, cotton, pea, common bean, cassava, mango, banana, potato, tomato, pepper, melon, zucchini, watermelon, lettuce, cabbage, eggplant, and poplar.

15. The method according to claim 10, wherein the plant is rice (Oryza sativa), wheat, barley or maize.

16. The method according to claim 10, wherein the sequence encoding L-aspartate oxidase comprises a sequence having at least 90% homology with SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 16.

17. The method according to claim 10, wherein the sequence encoding L-aspartate oxidase comprises a sequence having at least 95% homology with SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 16.

18. The method according to claim 10, wherein the sequence encoding L-aspartate oxidase comprises a sequence having at least 98% homology with SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 16.

Description

FIGURES

[0241] FIG. 1 is a description of the NAD biosynthesis pathway in plants, and the use thereof for energy metabolism and stress-related signaling (L-aspartate oxidase (AO) activity is shown in the figure).

[0242] FIG. 2 is a schematic representation of the construction of a plant transformation vector, pCW162, comprising the L-aspartate oxidase (AO) overexpression cassette.

[0243] FIG. 3 is a graph representing the level of expression (A) and the activity (B) of L-aspartate oxidase in the leaves of control plants (ctrl), of plants overexpressing L-aspartate oxidase (35S::AO1 and 35S::AO2) and of plants mutant-negative for L-aspartate oxidase (mAO), 6 weeks of age.

[0244] FIG. 4 is a graph representing energy-related metabolites (ATP (B) and pyridine nucleotides (A)) in the leaves of control plants (ctrl), of plants overexpressing L-aspartate oxidase (35S::AO1 and 35S::AO2) and of plants mutant-negative for L-aspartate oxidase (mAO), 6 weeks of age.

[0245] FIG. 5 represents the photosynthetic capacities of leaves of control plants () and of plants overexpressing (.box-tangle-solidup.) L-aspartate-oxidase, 6 weeks of age.

[0246] FIG. 6 is a photograph of control Arabidopsis thaliana plants and of plants overexpressing L-aspartate oxidase (35S::AO1 and 35S::AO2), 6 weeks of age.

[0247] FIG. 7 is a graph representing the biomass (B) and the size (A) of control plants (ctrl), of plants overexpressing L-aspartate oxidase (35S::AO1 and 35S::AO2) and of plants mutant-negative for L-aspartate oxidase (mAO).

[0248] FIG. 8 is a graph representing the seed biomass collected from control plants (ctrl), from plants overexpressing L-aspartate oxidase (35S::AO1 and 35S::AO2) and from plants mutant-negative for L-aspartate oxidase (mAO).

[0249] FIG. 9 is a graph representing the correlation between L-aspartate oxidase activity (A), NAD levels (B) and biomass expressed as rosette diameter.

[0250] FIG. 10 is a photograph of control plants (ctrl) and of plants overexpressing L-aspartate oxidase (35S::AO) under abiotic stress conditions corresponding to intense heat combined with intense light.

[0251] FIG. 11 is a germination curve for control plants (.diamond-solid.) and for plants overexpressing L-aspartate oxidase (35S::AO1 ( and X) and 35S::AO2 (.box-tangle-solidup.)) under nitrogen (nitrate)-rich medium conditions (A) and nitrogen (nitrate)-poor medium conditions (B).

[0252] FIG. 12 is a graph representing biotic stress conditions corresponding to proliferation of Myzus persicae aphids on control plants (ctrl) and on plants overexpressing L-aspartate oxidase (35S::AO1 and 35S::AO2).

EXAMPLES

[0253] Materials and Methods

[0254] Generation of Transgenic Plants

[0255] Arabidopsis thaliana cDNA (coding sequence, CDS) encoding L-aspartate-oxidase (L-AO) (SEQ ID NO: 1) was amplified by PCR with primers having the following sequences:

TABLE-US-00001 sense primer (SEQ ID NO: 17) (GAG AGA CCC GGG ATG GCG GCT CAT GTT TCT AC); antisense primer (SEQ ID NO: 18) (GAG AGA CAG CTG AAT CGT TAG TTA TTC ACT CGA C);

[0256] The amplification product was then subcloned between the Smal1 and Sal1 sites of the binary vector pCW162, under the control of the CaMV35S promoter in order to generate transgenic plants overexpressing L-aspartate oxidase. The nptII cassette of pCW162 was used for the selection of transgenic plants on medium containing kanamycin. The resulting plasmid was then used for the stable transformation of Arabidopsis thaliana plants in order to overexpress the L-AO gene, using Agrobacterium tumefaciens strain GV3101. Primary transformants were selected on Murashige and Skoog medium containing 50 mg/I kanamycin monosulfate. After about 10 days of culture in vitro (23° C., under light intensity of 100 μmol of photons/m.sup.2/s), resistant seedlings were transferred to containers of potting soil in a long-day (LD: 16-hour day, 8-hour night) culture chamber in order to produce seeds that have undergone a new selection scheme. The number of putative transgenic plants was noted in order to select lines that inserted a single copy of the transgene (ratio of ¾ non-resistant, ¼ resistant). Progeny were selected until stable lines homozygous for the T-DNA insertion were obtained.

[0257] Measurement of Levels of L-Aspartate Oxidase Transcripts

After a total RNA extraction using the NucleoSpin RNA II kit (Macherey-Nagel) according to the supplier's instructions, 1 μg of total RNA was used as template for synthesis of first-strand cDNA and reverse transcription with the first-strand synthesis system SuperScript III (Invitrogen). Overexpression of the L-aspartate oxidase gene was examined by RT-PCR with primers having the following sequences:

TABLE-US-00002 sense primer (SEQ ID NO: 19) (GAT CGT TCA CCG TGC TGA TA) and antisense primer (SEQ ID NO: 20) (TGT GTT CAA GCC ATC CTG AG);

[0258] The control (ctrl) line, or plant, was produced from ecotype Columbia (Col 0) transformed with the empty vector pCW1628.

[0259] Plant Culture

[0260] The transgenic lines of Arabidopsis thaliana used in this study were produced from Arabidopsis thaliana ecotype Columbia plants (Col 0). After 48 hours of stratification at 4° C. in the dark, the seeds were sown and cultivated under short-day (SD: 8-hour day, 16-hour night) conditions in a culture chamber under illumination of 100 μmol photons/m.sup.2/s at the leaf, at 18-20° C. and 65% humidity (except for the determination of silique number and the measurement of seed amount, long-day (LD: 16-hour day, 8-hour night) conditions were used). Nutrient solution was supplied twice per week.

[0261] Sampling for Metabolic Analyses

[0262] Leaf samples were taken in the middle of the photoperiod, rapidly frozen in liquid nitrogen and stored at −80° C. until subsequent analysis. For metabolomic and transcriptomic analyses, the plants were analyzed and sampled at 6 weeks of age (SD), and at 8 weeks of age (SD) for gas-exchange analysis.

[0263] Assay of L-Aspartate Oxidase Activity

[0264] A method for assaying L-aspartate oxidase activity was developed using a spectrophotometer: 0.5 g of a sample of frozen leaves was ground in liquid nitrogen and taken up in 2 ml of extraction buffer (Tris-HCl, pH 8). After centrifugation, the crude extract was desalted by size-exclusion chromatography on a PD10 column. For 0.7 ml of desalted extract, 100 μl of 10 mM L-aspartate, 100 μl of 10 mM fumarate and 100 μl of 200 μM FAD were added to start the reaction, which was followed at 30° C. for 30 minutes. The reaction was stopped by heating at 100° C. for 2 minutes in order to precipitate the proteins, followed by centrifugation. To 1 ml of reaction supernatant were successively added: [0265] 0.5 ml of 0.33 M sodium phenolate, pH 13; [0266] 0.5 ml of 0.1% sodium nitroprusside; [0267] 0.5 ml of 0.2% NaClO.

[0268] L-Aspartate oxidase activity was measured by spectrophotometry at OD 635 nm by assay, against a standard range of 0 to 100 nmol of (NH.sub.4)SO.sub.4, NH.sub.4.sup.+ coming from the near instantaneous degradation at pH 8 of the iminoaspartate formed during the reaction.

[0269] Metabolomic Measurements

[0270] Assays of metabolites with antioxidant properties, such as the pyridine nucleotides NAD.sup.+ and NADH, were carried out by spectrophotometry via enzymatic coupling on a microplate reader. These metabolites were quantified by a recycling reaction by following the reduction of DCPIP (2,6-dichlorophenol-indophenol) at 600 nm in the presence of alcohol dehydrogenase and ethanol. NAD.sup.+ is assayed after acid extraction: About 100 mg of leaves was ground with a mortar in liquid nitrogen, to which 1 ml of 0.2 N HCl is added. After the ground material was thawed, it was transferred to a 2 ml Eppendorf tube. The extract is then clarified by centrifugation for 10 minutes at 14,000 g, at 4° C. 200 μl of supernatant was heated for 1 minute at 100° C., then neutralized by adding 20 μl of NaH.sub.2PO.sub.4 (200 mM, pH 5.6) and a sufficient volume of 0.2 M NaOH (about 200 μl) to reach pH 7. For the assay of NADH, alkaline extraction was necessary. In the same manner as for the acid extraction, 100 mg of leaves was ground with a mortar in liquid nitrogen then 1 ml of 0.2 M NaOH was added. The mixture was then centrifuged for 10 minutes at 14,000 g at 4° C. 200 μl of supernatant was heated for 1 minute at 100° C., then neutralized by adding 20 μl of NaH.sub.2PO.sub.4 (200 mM, pH 5.6) and a sufficient volume of 0.2 N HCl (about 150 μl) to reach pH 7.

[0271] Spectrophotometric measurement of the extracted metabolites is carried out as follows: In each measurement well were successively added 100 μl of 100 mM HEPES/2 mM EDTA buffer (pH 7.5), 20 μl of 1.2 mM DCPIP, 10 μl of 10 mM PMS (phenazine methosulfate) and 10 μl of ADH (25 U) in a final volume of 200 μl. For the test samples, 20 μl of extract and 25 μl of double-distilled water are added. After shaking the plate, the reaction is initiated by adding 15 μl of absolute EtOH. NAD measurements are carried out at 600 nm by a microplate reader in reference to a standard range of NAD.sup.+ or of NADH.

[0272] The ATP assay was carried out using the ENLITEN ATP Assay System Bioluminescence kit (Promega) following the procedure recommended by the supplier.

[0273] Measurements of Gas Exchange

[0274] Measurements of gas exchange and of chlorophyll fluorescence were carried out using the LI-6400XT system (LI-COR, Lincoln, Nebr., USA) and the parameters were calculated with the software provided by the manufacturer. The conditions were: photon flux density ¼ 1,000 mmol m.sup.2/s, chamber temperature 22° C., flow rate 100 mmol/s, relative humidity 60%. The net carbon assimilation (An) responses and the molar fraction of internal CO.sub.2 (An/Ci curves) carried out under ambient oxygen content conditions (21%) were measured on attached leaves with an infrared gas analysis system equipped with a fluorimeter chamber (LI-COR 6400-40; LI-COR Inc., Lincoln, Nebr., United States).

[0275] Germination Test

[0276] In order to ensure that the differences in germination rates observed are not due to seed quality, wild plants and mutant plants were cultivated side by side under identical conditions in a culture chamber in order to produce fresh seeds under long-day conditions. Fully mature and sterilized seeds were sown on plates of ¼ Hoagland's medium, nitrate-free (0.2 mM) or nitrate-rich (2.25 mM). After stratification for 2 days at 4° C. in the dark, the seeds were placed in a culture chamber at 23° C. Radicle protrusion was used as the criterion for evaluating germination differences between wild-type and mutant seeds.

[0277] Test for Resistance to Abiotic Stress Conditions

[0278] Seven-week-old plants cultivated under short-day (SD) conditions were transferred for one week under conditions of continuous light of 350 μmol photons/m.sup.2/s at 37° C. and 65% humidity.

[0279] Test for Resistance to Biotic Stress

[0280] Myzus persicae aphids from the same colony maintained in the laboratory on wild Arabidopsis thaliana plants of the same ecotype as that of the wild and mutant plants tested were collected and transferred to fresh 5-week-old plants. In 2 days, they produced larvae. The adult aphids were removed and only the larvae were kept. This made it possible to produce aphids of the same age ±1 day. Seven days later, 3 aphids were transferred to each 18-day-old plant of each genotype. After 5 days, the number of aphids was counted with a magnifying glass for each plant. By statistical analysis (ANOVA), it was confirmed that rosette diameter did not influence aphid proliferation.

[0281] Statistical Analyses

[0282] Unless otherwise specified, the data are the means and standard deviations of three to five independent samples of different plants; significant differences are expressed using Student's t-test with p<0.05. All the experiments were repeated at least three times and gave similar results. The Student's t-test and the two-way analysis of variance (ANOVA) were implemented using the Excel software (Microsoft).

[0283] Results

[0284] The results indicate that the Arabidopsis thaliana nucleotide sequence used for transformation of the plants, and which is homologous to that of the bacterial L-aspartate oxidase characterized in the literature, indeed corresponds to L-aspartate oxidase activity and that the transformed lines exhibit L-aspartate oxidase activity that is increased by a factor of 2 to 4 (FIG. 3).

[0285] The results show that the constitutive overexpression of L-aspartate oxidase cDNA indeed leads to an increase in levels of NAD and of related energy metabolites (FIG. 4).

[0286] The results show that lines overexpressing L-aspartate oxidase have higher rates of photosynthetic CO.sub.2 assimilation (FIG. 5).

[0287] The growth and the yield of plants transformed with the L-aspartate oxidase expression cassette were evaluated. Overexpression of L-aspartate oxidase leads to an increase in root growth and in leaf surface area. The size of transgenic plants overexpressing L-aspartate oxidase is larger than the size of control plants of the same age (FIGS. 6 and 7). The opposite is observed in mutant plants with greatly reduced levels of aspartate oxidase (FIG. 7). The fresh weight of the plantlets is also significantly increased (FIG. 7) and the ratio of fresh weight to dry weight is unchanged in relation to control plants. In addition to the plant's increased development, an increase in the mean size of epidermal cells can be detected. No ploidy variation was observed between plants overexpressing L-aspartate oxidase and plants not overexpressing L-aspartate oxidase, which confirms that the increased size of lines overexpressing L-aspartate oxidase is not linked to increased ploidy. A strong correlation between the diameter of the rosette and the abundance of transcripts, from the activity and level of L-aspartate oxidase and NAD, was observed (FIG. 9), showing that the increase in plant growth depends directly on the level of L-aspartate oxidase expression. Overexpression of L-aspartate oxidase thus causes an increase in cell growth and an increase in the growth and development of the entire plant.

[0288] The seed yield of transgenic plants overexpressing L-aspartate oxidase proves to be higher than that of control plants. The 45% increase in seed yield observed (FIG. 8) correlates with the number of siliques per plant. This increase in seed production is not accompanied by any silique-filling problem in plants overexpressing L-aspartate oxidase in relation to control plants. Furthermore, silique size is identical between all the plants. Overexpression of L-aspartate oxidase thus results in increased seed yield. Conversely, mutant plants with low L-aspartate-oxidase activity have a seed yield 42% lower than control lines.

[0289] The increase in seed yield is concomitant with increased germination quality. Indeed, seeds produced by plants overexpressing L-aspartate oxidase germinate faster than control seeds (FIG. 11). The germination capacity of plants overexpressing L-aspartate oxidase is not modified in a nitrogen-poor environment as is observed for control seeds (FIG. 11). Overexpression of L-aspartate oxidase thus stimulates germination, and seeds can germinate better under nitrogen-deficient conditions.

[0290] Plants overexpressing L-aspartate oxidase continuously exposed to 350 μmol photons/m.sup.2/s and 37° C. survived whereas control plants dried out and died under the same extreme conditions (FIG. 10). Overexpression of L-aspartate oxidase thus strengthens the resistance of plants to severe abiotic stress conditions.

[0291] Plants overexpressing L-aspartate oxidase cultivated in the presence of Myzus persicae aphids limited aphid development in relation to control plants infested under the same conditions (FIG. 12). Overexpression of L-aspartate oxidase thus strengthens the resistance of plants to biotic stress conditions such as aphid attack.