Improvement of Plant Yield

Abstract

The invention relates to a method for improving the yield of a plant, comprising overexpressing in said plant a glutamine synthetase, said glutamine synthetase being both constitutively overexpressed in said plant and specifically overexpressed in the bundle sheath cells of said plant. The invention also relates to means for obtaining plants with improved yield.

Claims

1. A method of improving the yield of a plant, wherein said method comprises overexpressing in said plant a glutamine synthetase having at least 90% identity with the polypeptide of SEQ ID NO: 1 and, said glutamine synthetase being both constitutively overexpressed in said plant and specifically overexpressed in the bundle sheath cells of said plant, wherein said glutamine synthetase constitutively overexpressed and said glutamine synthetase specifically overexpressed have the same amino acid sequence.

2. The method according to claim 1, wherein said glutamine synthetase is from a plant selected from the group consisting of Zea mays, Setaria italica, Saccharum officinarum, Oryza sativa, Oryza brachyantha, Oryza glaberrima, Brachypodium distachyon, Hordeum vulgare, Triticum aestivum, Secale cereale, Secale cerealeTriticum turgidum, Lolium perenne and Aegilops tauschii.

3. The method according to claim 1, wherein said glutamine synthetase is selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 9 to SEQ ID NO: 26.

4. The method according to claim 1, wherein said plant is a C4 plant.

5. The method according to claim 1, wherein said plant is maize and said glutamine synthetase has the amino acid sequence SEQ ID NO: 1 (ZmGS1-b).

6. An isolated polynucleotide comprising a recombinant expression cassette comprising a polynucleotide encoding a glutamine synthetase as defined in claim 1 under the control of an heterologous constitutive promoter functional in a plant cell and a recombinant expression cassette comprising a polynucleotide encoding a glutamine synthetase as defined in claim 1 under the control of a bundle sheath cell specific promoter, provided that both glutamine synthetase enzymes have the same amino acid sequence.

7. The isolated polynucleotide comprising a recombinant expression cassette according to claim 6 wherein the heterologous constitutive promoter functional in a plant cell is SEQ ID NO: 3 (pCsVMV) and the bundle sheath cell specific promoter is SEQ ID NO: 8 (proZmRbcS).

8. A recombinant vector comprising a polynucleotide of claim 6.

9. A host cell comprising: a polynucleotide of claim 6.

10. The host cell according to claim 9 which is a plant cell.

11. The host cell according to claim 10 which is a maize cell.

12. A method of producing a transgenic plant having an improved seed yield, wherein said method comprises: providing the plant cell of claim 10; regenerating from said plant cell a transgenic plant overexpressing a glutamine synthetase having at least 90% identity with the polypeptide of SEQ ID NO: 1.

13. The method according to claim 12, wherein the transgenic plant is a C4 plant.

14. A transgenic plant, or an isolated organ or tissue thereof comprising, stably integrated in its genome, a recombinant expression cassette comprising a polynucleotide encoding a glutamine synthetase as defined in claim 1 under the control of an heterologous constitutive promoter functional in a plant cell and a recombinant expression cassette comprising a polynucleotide encoding a glutamine synthetase as defined in claim 1 under the control of a bundle sheath cell specific promoter, provided that both glutamine synthetase enzymes have the same amino acid sequence.

15. The transgenic plant according to claim 14, wherein said plant is a C4 plant.

16. Seed or kernel containing a recombinant expression cassette comprising a polynucleotide encoding a glutamine synthetase having at least 90% identity with the polypeptide of SEQ ID NO: 1 under the control of an heterologous constitutive promoter functional in a plant cell and a recombinant expression cassette comprising a polynucleotide encoding a glutamine synthetase having at least 90% identity with the polypeptide of SEQ ID NO: 1 under the control of a bundle sheath cell specific promoter, wherein both glutamine synthetase enzymes have the same amino acid sequence, obtained from a transgenic plant of claim 14.

17. A host cell comprising a recombinant expression cassette comprising a polynucleotide encoding a glutamine synthetase as defined in claim 1 under the control of an heterologous constitutive promoter functional in a plant cell and a recombinant expression cassette comprising a polynucleotide encoding a glutamine synthetase as defined in claim 1 under the control of a bundle sheath cell specific promoter, provided that both glutamine synthetase enzymes have the same amino acid sequence.

18. A host cell comprising the recombinant vector of claim 8.

19. The host cell according to claim 10, wherein the plant cell is a C4 plant cell.

Description

[0052] Foregoing and other objects and advantages of the invention will become more apparent from the following detailed description and accompanying drawings. It is to be understood however that this foregoing detailed description is exemplary only and is not restrictive of the invention.

[0053] FIG. 1: Schematic representation of the T-DNA fragment introduced into maize plants. RB and LB represent the right and left borders of the T-DNA. Ds (3Ac) and Ds (5Ac) represent the Ds transposable elements used to further remove the selection marker conferring resistance to kanamycin. NptII is the neomycin phosphotransferase gene with the Actin promoter and the Actin intron and nopaline synthetase terminator (3Nos) conferring kanamycin resistance. An Actin intron was placed between the maize ZmGln1-b cDNA and the CsVMV promoter.

[0054] FIG. 2: Western-blot (A) using tobacco antibodies recognizing GS1 and GS2, or enzymatic activity (B) analyses on leaf or root samples. The upper band seen on the western-blot corresponds to the plastidic GS (GS2) subunit, and the lower band corresponds to the cytosolic GS (GS1) subunit. (A) Western-blot analysis of GS1-b transformed maize lines (LXX) or from corresponding hybrid lines (HYY), leaf samples. (B) Enzymatic assays with samples of GS1-b transformed maize lines. A188: untransformed control line HA188: corresponding hybrid from untransformed A188 line.

[0055] FIG. 3: Yield data and grain humidity of GS1-b over-expressing lines. The yield data is expressed as percentage of the control lines and has been normalized to grain moisture at 15%. The larger symbols correspond to the average value obtained with all transformed lines (large triangle) or with the control lines (large square). For each data point calculated, the corresponding p-value is also indicated by a cross. The p-value scale is on the left side of the figure.

[0056] FIG. 4: Kernel number per ear and yield data of GS1-b over-expressing lines. The kernel number per ear is expressed as percentage of the control lines. The yield data, normalized to grain moisture at 15%, is expressed as quintal per hectare.

EXAMPLE 1: OVEREXPRESSION OF GS1-B (SEQ ID NO: 1) IN LEAF MESOPHYLL AND BUNDLE SHEATH CELLS IN MAIZE

[0057] The strategy, described below, is to transform maize with a T-DNA expressing a copy of the Gln1-b cDNA under control of the pCsVMV promoter, together with another copy of the Gln1-b cDNA under control of the maize bundle sheath cell specific promoter of the Rubisco (rbcS) gene (Katayama et al., 2000), in order to obtain transgenic maize plants expressing GS1-b both in the mesophyll and in the bundle sheath cells.

[0058] Plant Transformation, Regeneration and Characterization

[0059] Maize transformation of the inbred line A188 with Agrobacterium tumefaciens strain LBA4404 harbouring a super-binary plasmid was performed essentially as described by Ishida et al., 1996. In particular, the composition of all media cited hereafter is detailed in this reference. The protocol was slightly modified concerning the selective marker, which was the NPTII gene instead of the bar gene.

[0060] Super-Binary Plasmid pRec 1459

[0061] The super-binary plasmid used for transformation was the result of a recombination between plasmid pBIOS 1459 and the plasmid pSB1 (harbouring the virB and virG genes isolated from the super-virulent strain A281) within the Agrobacterium strain LBA4404 (pSB1) (Komari et al., 1996) forming the plasmid pRec 1459. pBIOS 1459 is a derivative of pSB11 (Komari et al., 1996) harbouring between the T-DNA borders, a neomycin resistance cassette (NPTII gene) (Bevan et al., 1992; Berg and Berg, 1983) flanked by an actin promoter (McElroy et al., 1990) and 3Nos terminator (Depicker et al., 1982;), and the ZmGln1-b cDNA (Sakakibara et al., 1992, SEQ ID NO: 5) flanked by the Cassava vein mosaic virus promoter (pCsVMV) (Verdaguer et al., 1996; SEQ ID NO: 3) linked to an actin intron (McElroy et al., 1990; SEQ ID NO: 4), the ZmGln1-b terminator (SEQ ID NO: 6) and 3Nos terminator (SEQ ID NO: 7), and another copy of the ZmGln1-b cDNA (SEQ ID NO: 5) flanked by the rbcS promoter (SEQ ID NO: 8), the ZmGln1-b terminator (SEQ ID NO: 6) and 3Nos terminator (SEQ ID NO: 7). The resulting nucleic sequence proCsVMV-OsActin_intron-ZmGln1-b-terminator ZmGln1-b-terminator NOS-proZmRbcS-ZmGln1-b -terminator ZmGln1-b-terminator NOS is referred to as SEQ ID NO: 2. The resulting agrobacterial strain used for transformation was LB4404 (pRec 1459). pRec 1459 is schematized in FIG. 1.

[0062] Transgenic Plants

[0063] Plant transformation was conducted using immature maize embryos isolated at 10 days after pollination. Immature embryos were incubated for 5 min with A. tumefaciens and cultured for 3 days on a LSAs medium without antibiotic selection in the dark at 25 C. Upon transfer to the LSD5 medium, A. tumefaciens was counter-selected by the presence of 250 mg L-1 cefotaxime, and the transformed calli were selected by the presence of 50 mg mL-1 kanamycin. After 2 weeks of culture, developing calli were transferred to LSD10 medium containing 50 mg mL-1 kanamycin and grown for 3 weeks. Type I calli were excised and cultured for another 3 weeks on kanamycin. For regeneration, well-developed type I calli were cultured on LSZ medium at 22 C. under continuous selective pressure and on kanamycin. After 2 weeks, calli bearing shoots were transferred to RMG2 medium and cultured another 2 weeks to allow the development of roots before the transfer of the plantlets to soil and gradual acclimatization to ambient humidity. Plants were then cultivated in a glasshouse (18 C.-24 C.) and selfed or pollinated with line A188 to produce seeds.

[0064] A number of transgenic lines were selected and tested by Quantitative PCR for the number of inserted T-DNA copies and by conventional PCR for the integrity of the inserted T-DNA. Only plants with up to 2 full length T-DNA copies were kept for further analysis.

[0065] Biochemical Characterization of Transgenic Plants Over-Expressing GS1-b

[0066] The presence of the introduced GS1-b protein was determined by western-blot (FIG. 2A) using tobacco antibodies recognizing GS1 and GS2, or enzymatic activity (FIG. 2B) analyses on leaf or root samples of greenhouse grown plants.

[0067] It can be seen that leaves of GS1-b transformed lines contain a higher level of GS1 proteins, as determined by western-blot analysis, and that GS1 enzymatic activity levels are higher (more than a 2 fold increase) than those of control lines. The same results hold true for the derived hybrids.

[0068] Field Trials

[0069] Hybrids with a tester line were obtained from T3 plants issued from the selected GS1-b over-expressing transgenic maize lines.

[0070] The transformant (T0) plants were first crossed with the A188 line thereby producing T1 plants. T1 plants were then self-pollinated twice, producing T3 plants which are homozygous lines containing the transgene. These T3 plants were then crossed with the tester line thereby leading to a hybrid. This hybrid is at a T4 level with regards to the transformation step and is hemizygous for the transgene. These hybrid plants are used in field experiments.

[0071] Control Hybrids are Obtained as Follows:

[0072] Control Equiv corresponds to a cross between A188 line (the line used for transformation) and the tester line.

[0073] Control HNS corresponds to a cross between a null segregant (isolated after the second self-pollination of the T1 plants) and the tester line. Said null segregant is a homozygous line which does not bear the transgene. Although the null segregant theoretically presents the same genome as A188, it has undergone in vitro culture (via the steps of callus differentiation and regeneration) and may thus present mutations (either genetic or epigenetic) with regards to an A188 line that has not undergone in vitro culture.

[0074] Yield was calculated as follows:

[0075] During harvest, grain weight and grain moisture are measured using an on-board equipment on the combine harvester. Grain weight is then normalized to moisture at 15%, using the following formula:

Normalized grain weight=measured grain weight(100measured moisture (as a percentage))/85 (which is 100normalized moisture at 15%).

[0076] As an example, if the measured grain moisture is 25%, the normalized grain weight will be: normalized grain weight=measured grain weight75/85.

[0077] Yield is then expressed in a conventional unit (such as quintal per hectare).

[0078] Experimental Design:

[0079] The experimental block comprised 5 replicates. Each replicate comprised about 58.5 plants per plot at a density of 69600 plants/ha.

[0080] Two controls were used in this experiment as described above (a null segregant (HNS) and a control equivalent (A188 crossed with the tester line).

[0081] Grain moisture, thousand kernel weight yield and ear kernel number data are represented in the Table 1 below.

TABLE-US-00001 TABLE 1 Grain moisture, TKW yield and ear kernel number measured for different transformation events with the ZmGln1-b cDNA. Data is given as per se or as a percentage compared to the control sample. Kernel Grain P-value P-value P-value number P-value moisture compared Thousand TKW compared Yield compared per ear compared Grain as % of to the Kernel as % of to the as % of to the Number of as % of to the moisture control control Weight control control Yield control control Kernels control control Sample (%) average average (TKW) (g) average average (Qx/Ha) average average per ear average average Control Equiv 19.3 267.8 64.8 480 T01617_027 19.1 99 0.7366 284.4 103 0.401 71.9 111 0.012 519 106 0.0679 T01617_022 19.3 100 0.8984 267.6 97 0.313 70.2 108 0.055 500 102 0.4971 T01617_032 18.8 97 0.2621 272.0 98 0.598 69.8 108 0.075 544 111 0.0008 T01617_020 18.8 97 0.2431 279.0 101 0.806 69.7 108 0.079 507 104 0.2527 T01617_023 18.8 97 0.2621 282.8 102 0.507 68.4 106 0.194 525 108 0.0294 T01617_014 18.6 96 0.1044 283.4 102 0.465 67.5 104 0.338 495 101 0.7142 T01617_017 18.9 98 0.3727 284.2 103 0.413 66.7 103 0.495 501 103 0.4316 T01617_031 19.0 99 0.5700 270.6 98 0.496 66.3 102 0.596 487 100 0.9554 T01617_018 19.1 99 0.7366 274.8 99 0.827 66.2 102 0.607 504 103 0.3380 T01617_012 19.3 100 0.8984 279.6 101 0.755 65.0 100 0.935 501 103 0.4328 Control HNS 19.3 285.8 64.8 497 Control 19.3 100 276.8 64.8 100 488 100 average T01617 19.0 98 277.8 68.2 105 0.041 508 104 0.0417 average

[0082] FIG. 3 represents the yield expressed as percentage of the control samples and in function of grain moisture.

[0083] Improved yield (between 5 and 10% improvement compared to the controls) was observed for the hybrid plants of most GS1-b over-expressing lines. Grains from GS1-b over-expressor plants were also less humid than grains from control plants (in average 19% vs 19.3%). The transgenic lines can be grouped by those leading to a yield increase (large circle) and those given similar values to the control samples (small circle).

[0084] FIG. 4 represents the ear kernel number expressed as percentage of the control samples and in function of yield.

[0085] Improved yield may thus be explained by the increased number of kernels per ear of the transgenic plants (see FIG. 4), since a correlation between yield and ear kernel number can be seen.

EXAMPLE 2: PORTABILITY OF THE TRAIT IMPROVED YIELD IN SEVERAL HYBRID BACKGROUNDS

[0086] The portability of the trait improved yield was tested in several hybrid backgrounds.

[0087] Hybrids were obtained from T3 plants issued from the selected GS1-b over-expressing transgenic maize lines obtained in Example 1 above crossed with either the tester line 1 (Stiff Stalk heterotic group, same tester line used in the previous example), or the tester line 2 (OH43 heterotic group) or the tester line 3 (Iodent heterotic group).

[0088] The transformed (T0) plants were first crossed with the A188 line thereby producing T1 plants. T1 plants were then self-pollinated twice, producing T3 plants which are homozygous lines containing the transgene. These T3 plants were then crossed with the tester line 1 or the tester line 2 or the tester line 3 thereby leading to a hybrid. These hybrids are at the T4 level with regards to the transformation step and are hemizygous for the transgene. These hybrid plants are used in field experiments.

[0089] Control hybrids are obtained as follows:

[0090] Control Equivalent corresponds to a cross between A188 line (the line used for transformation) and the tester line 1 or the tester line 2 or the tester line 3.

[0091] The yield was calculated as described in Example 1.

[0092] Experimental Design:

[0093] The experimental block comprised 5 replicates. Each replicate comprised about 58.5 plants per plot at a density of 69600 plants/ha.

[0094] One control was used in this experiment as described above (a control equivalent: A188 crossed with the tester line 1 or 2 or 3).

[0095] Yield measurements are represented in the Table 2 below.

TABLE-US-00002 TABLE 2 Yield comparison between transgenic plants and controls. Mean square (Qx/ha) Sample T 01617 Control Equiv Percentage % Prob. > F Across tester 69.16 64.41 107.4 0.0106 Tester 2 72.29 68.04 106.2 0.1840 Tester 3 73.40 68.40 107.3 0.1136 Tester 1 61.80 56.78 108.8 0.1124

[0096] Yield improvement (increase) of more than 7% across testers was observed showing a good portability of the trait in several genetic backgrounds.

EXAMPLE 3: COMPARISON OF THE YIELD IMPROVEMENT BETWEEN A MAIZE TRANSGENIC PLANT OVEREXPRESSING GS1-B (SEQ ID NO: 1) IN LEAF MESOPHYLL AND BUNDLE SHEATH CELLS VERSUS A MAIZE TRANSGENIC PLANT OVEREXPRESSING GS1-B IN LEAF MESOPHYLL AND A MAIZE TRANSGENIC PLANT OVEREXPRESSING GS1-B IN BUNDLE SHEATH CELLS

[0097] Maize transgenic plants overexpressing GS1-b of SEQ ID NO: 1 in leaf mesophyll and bundle sheath cells are obtained according to the method described in Example 1.

[0098] Maize transgenic plants overexpressing GS1-b of SEQ ID NO: 1 in leaf mesophyll are obtained by transforming a maize plant with a T-DNA expressing a copy of the Gln1-b cDNA under control of the pCsVMV promoter. Maize transgenic plants overexpressing GS1-b of SEQ ID NO: 1 in bundle sheath cells are obtained by transforming a maize plant with a T-DNA expressing a copy of the Gln1-b cDNA under control of the maize bundle sheath cell specific promoter of the Rubisco (rbcS) gene (Katayama et al., 2000). The plant transformation, regeneration and biochemical characterization are carried out according to the method of Example 1. The plasmid used for the transformation of a maize plant with a T-DNA expressing a copy of the Gln1-b cDNA under control of the pCsVMV promoter comprises the ZmGln1-b cDNA (Sakakibara et al., 1992, SEQ ID NO: 5) flanked by the Cassava vein mosaic virus promoter (pCsVMV) (Verdaguer et al., 1996; SEQ ID NO: 3) linked to an actin intron (McElroy et al., 1990; SEQ ID NO: 4), the ZmGln1-b terminator (SEQ ID NO: 6) and 3Nos terminator (SEQ ID NO: 7). The plasmid used for the transformation of a maize plant with a T-DNA expressing a copy of the Gln1-b cDNA under control of the maize bundle sheath cell specific promoter of the Rubisco (rbcS) gene comprises the ZmGln1-b cDNA (SEQ ID NO: 5) flanked by the rbcS promoter (SEQ ID NO: 8), the ZmGln1-b terminator (SEQ ID NO: 6) and 3Nos terminator (SEQ ID NO: 7).

[0099] The field trials are carried out according to the method of Example 1. Hybrids are obtained from T3 plants issued from the selected GS1-b over-expressing transgenic maize lines crossed with a tester line, according to the method of Example 1. The control hybrids and the experimental design are also carried out according to the method of Example 1.

[0100] The yield is calculated as described in Example 1 and compared between the different transgenic hybrid maize plants.

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