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WO2006063963A1 - Methode pour accroitre la biomasse des vegetaux dans des conditions de stress - Google Patents

Methode pour accroitre la biomasse des vegetaux dans des conditions de stress Download PDF

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WO2006063963A1
WO2006063963A1 PCT/EP2005/056629 EP2005056629W WO2006063963A1 WO 2006063963 A1 WO2006063963 A1 WO 2006063963A1 EP 2005056629 W EP2005056629 W EP 2005056629W WO 2006063963 A1 WO2006063963 A1 WO 2006063963A1
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wee1
gene
stress
plants
dna
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PCT/EP2005/056629
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English (en)
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Lieven De Veylder
Dirk Gustaaf INZÉ
Kristof De Schutter
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Vib Vzw
Universiteit Gent
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Publication of WO2006063963A1 publication Critical patent/WO2006063963A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance

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  • the present invention relates to the use of WEE1 to increase crop yield in stress conditions. More specifically, it relates to a downregulation of Wee 1 activity in conditions of non-lethal stress.
  • a preferred embodiment is the use of a WEE1 knock out to increase seed weight, whereby the stress applied is drought stress.
  • DNA damage by exogenous or endogenous agents is a constant treat to the integrity of the genome cells have developed a series of surveillance mechanisms that monitor the status and structure of the DNA during cell cycle progression.
  • signal transduction cascades are activated, which halt cell cycle progression until completion of DNA repair.
  • DNA damage is signaled by the ATM (ataxia telangiectasia mutated) and ATR (Rad3-related) phosphatidyl inositol 3-kinase-like protein kinases (Abraham, 2001; Bartek and Lucas, 2001; Kurz and Lees-Millar, 2004; Zhou and Elledge, 2000).
  • ATM responds specifically to double stranded breaks, whereas ATR is primary activated by a block of the replication fork.
  • ATM/ATR transduce the signal to CHK1/CHK2 kinases, which in turn modulate the activity of the effectors that directly participate in the G1/S transition, S-phase progression, or the G2/M transition (Chen and Sanchez, 2004; Sancar et al., 2004).
  • Progression through the cell cycle is controlled by the ordered action of protein complexes that are at least composed of a catalytic subunit and a positive regulatory element, named Cyclin-Dependent Kinase (CDK) and cyclin, respectively.
  • CDK Cyclin-Dependent Kinase
  • the association of the CDK with its cyclin partner determines the activity of the complex, its stability, localization and substrate specificity (Murray et al., 2004; Pines and Rieder, 2001).
  • the sequential activation of different cyclin/CDK complexes drives the cell cycle through the phosphorylation of many target substrates such as, nuclear envelop proteins, histons, cytoskeletal elements, components of the proteolysis machinery, and transcriptionally regulatory proteins such as the retinoblastoma- related protein.
  • Cyclin/CDK activity is highly regulated on several levels. Control mechanisms include the regulated destruction of the cyclin subunit (Murray, 2004; Peters, 1998), the association of CDKs with regulatory proteins being different from cyclins (Lees, 1995), and the subcellular targeting of the cyclin/CDK complexes (Ohi and Gould, 1999).
  • the most elaborated mechanism of regulation of CDK activation involves protein phosphorylation, that is largely responsible for explaining why the onset of mitosis occurs abruptly, whereas the accumulation of mitotic cyclins during S and G2 phase is a gradual process.
  • CDK activity is positively regulated by the phosphorylation of a conserved threonine residue within the T-loop (Thr161 or equivalent residue) by the CDK activating kinases, and negatively through its phosphorylation of a single Tyrosine15 (in yeasts), or both Tyrosine15 and Threonine14 (in higher eukaryotes), by WEE1- family kinases (Berry and Gould, 1996). Phosphorylation of Tyr15 and Thr14 of the CDK subunit inhibits ATP fixation and blocks substrate binding.
  • Prompt activation of cyclin/CDK activity at the G2/M boundary in yeast and mammals is achieved by a dual-specificity phosphatase CDC25, encoded by one gene in yeast, three isoforms in mammals.
  • WEE1 preferentially phosphorylates the central cell cycle regulatory protein p34Cdc2. Therefore, WO0037645 suggested that down-regulation of WEE1, especially maize WEE1 , could be used to increase crop yield
  • WEE1 takes part in the mechanism that ensures the onset of mitosis is coupled to the completion of DNA repair in cells that have suffered DNA damage, resulting in a slower growth and a decreased crop yield when the WEE1 gene is down-regulated under normal (stressed) field conditions.
  • WEE1 gene is stress induced, down-regulation of WEE1 would not affect crop yield at all under hypothetical stress free conditions.
  • down-regulation of the WEE1 gene has no negative effect under conditions of non-lethal stress. Down-regulation of WEE1 under conditions of sublethal stress could avoid an early stop of the cell cycle, and may therefore result in an increased crop yield under those conditions.
  • a first aspect of the invention is the use of the WEE1 gene and/ or protein to increase crop yield in plants under conditions of sublethal stress.
  • the Wee1 protein is a protein with the sequence set forth in genbank accession number Gl:22761819 , or with a sequence that is at least 70% identical to said sequence, preferentially 80% identical, even more preferentially 90% identical, and whereby said protein functions as a protein kinase.
  • the Wee1 gene is a nucleic acid sequence encoding a WEE1 protein as described above. Gene as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule.
  • the term includes double- and single- stranded DNA and RNA. It also includes known types of modifications, for example methylation, "caps" substitution of one or more of the naturally occurring nucleotides with an analogue. It includes, but is not limited to, the coding sequence, and may include non-translated intron sequences. However, as used here, the promoter sequence is not included; this sequence will be referred as "endogenous promoter" when it indicates the promoter naturally occurring upstream of the gene.
  • Sublethal stress are those growth conditions in which a weel knock out is not negatively affected in growth, when compared to the WEE1 parental strain. Stress may be sublethal either because the level of stress is low as such and not affecting the plant growth, or it may be sublethal because it is local and only affecting some non-vital plant parts; And example of the latter non-lethal stress is the drought stress during seed ripening, which only affects the seeds, but not the total plant.
  • said use of WEE1 is a down-regulation.
  • said sublethal stress is abiotic stress.
  • said abiotic stress is drought stress.
  • a preferred embodiment of the invention is the use of WEE1 , according to the invention to increase see yield, preferably by a down-regulation of WEE1 during the phase of drought stress during seed ripening.
  • Another aspect of the invention is a method to increase crop yield in plants comprising the down-regulation of WEE1 in function of the stress applied.
  • said stress applied is abiotic stress.
  • WEE1 stops the cell cycle at a stress level that as such is not harmful for the plant, resulting in a loss of crop yield. Delaying the WEE1 response to higher stress levels will result in a later block of the cell cycle and an increase of crop yield.
  • the down-regulation of WEE1 should be carried out as an inverse function of the stress applied, being maximal under stress-free conditions, and being switched off under high stress conditions.
  • This can be realized by operably linking the gene encoding WEE1 to a promoter that is down regulated in stress conditions. Operably linking refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a promoter sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the promoter sequence.
  • Suitable stress down regulated promoters are known to the person skilled in the art and include, but are not limited too the promoters of the stress repressed genes genbank accession number AT1G74670, AT2G40610, AT1G69530 and AT5G51720. Delaying the WEE1 response can also be realized at protein level, eg by the production of an antibody that neutralizes the WEE1 kinase activity.
  • the gene, encoding said antibody is operably linked to a promoter, preferably a weak, constitutive promoter, the WEE1 kinase activity will be neutralized, unless the production of WEE1 protein is high enough to overcome the inactivation by the antibody.
  • FIG. 1 CDK phosphorylation in response to checkpoint activation.
  • Arabidopsis cell cultures were treated with 10 ⁇ l/ml aphidicolin (A) or with 3 ⁇ M propyzamide (P) and mock-treated in controls (C).
  • CDKs were purified using p10 CKS1At -Sepharose matrix, resolved by SDS-PAGE and immunoblotted using indicated antisera.
  • FIG. 2 Transcriptional response of the WEE1 gene after activation of the DNA replication checkpoint.
  • a and B Transcript levels of WEE1 and CDKA;1 in Arabidopsis cells treated with 40 rtiM hydroxyurea (A) or 10 ⁇ l/ml aphidicoline (B). Samples were harvested at the indicated time- points after adding the drugs. Gene expression was analysed by semi quantitative RT-PCR. The actine ACT2 gene was used as loading control.
  • C and D Transgenic Arabidopsis roots harboring the WEE1 promoter fused to the GUS gene are growth in the absence (C) or presence (D) of hydroxyurea. Plants were stained for GUS activity 24 hrs after applying the drug.
  • FIG. 3 Spatial expression pattern of WEE1. Promoter activity was visualized through histochemical GUS staining.
  • A Young seedling showing strong GUS staining in the SAM
  • B Root tissue showing GUS staining of vascular cells.
  • C Young developing flower bud.
  • D Mature flowers.
  • Figure 4 Transcriptional activation of WEE1 in response to DNA damage checkpoint. Seedling harboring the WEE1 or PARP-2 promoter fused to the GUS reporter gene (WEE1:GUS and PARP-2:GUS) were treated with increasing concentrations of zeocin for 24 hrs. Activation of promoter activity was visualized through histochemical GUS staining.
  • Figure 5 ATM-dependent induction of WEE1 in response to ⁇ -radiation.
  • Figure 6 Molecular and phenotypic analysis of wee1-1 Arabidopsis plants.
  • A Intron-exon organization of the WEE1 gene. Black and grey boxes represent exons, and lines indicate introns. Exons are numbered. The coding regions corresponding with the kinase domain are indicated in grey. The triangle corresponds to the T-DNA insertion in the GABI-Kat 270E05 line (wee1-1 mutant allele).
  • B Two-step RT-PCR analysis performed with equal amounts of total RNA prepared from 8-day-old wild-type (CoI-O) and wee1-1 seedlings with primers that specifically amplify the WEE1 -coding sequence flanking the T-DNA insertion site.
  • the actine 2 (ACT2) gene was used as loading control.
  • C-H Wild type (C, E and G) and wee1-1 (D, F, and H) seeds germinated on MS plates, grown for 5 days and then transferred to control medium (C and D) or plates containing 1 rtiM HU (E and F) or 12 ⁇ g/ml of aphidicoline (G and H) for 5 days.
  • J and K Wild type (J) and wee1-1 (K) seeds germinated on 12 ⁇ g/ml of aphidicoline.
  • FIG. 7 Schematic diagram depicting the CRE-inducible transcriptional activation of the WEE1 transgene.
  • A Under non-inducing conditions, the CRE recombinase, cloned under the control of the heat-shock inducible PHSP18.2 promoter, is not expressed and the Arath;CDK/l;7 promoter (PCDKA) drives the expression of the EGFP gene.
  • the EGFP gene is followed by a stopcodon and polyadenylation signal, preventing the synthesis of WEE1.
  • Figure 8 Molecular and phenotypic analysis of Arabidopsis plants after induction of WEE1 expression.
  • A WEE1 expression levels as determined by semiquantitative RT-PCR analysis in two independent transgenic lines harboring the inducible WEE1 gene expression cassette. RNA was isolated from non-induced (-) and induced (+) plants. CDKA;1 and ACT2 expression levels were measured as control.
  • B Phenotype (i, iii) and EGFP fluorescence (ii, iv) of transgenic roottips before (i, ii) and 3 days after (iii, iv) induction of WEE1 expression.
  • Arabidopsis thaliana plants were grown under long-day conditions (16 h of light, 8 h of darkness) at 22°C on germination medium (Valvekens et al., 1988).
  • the wee1-1 allele was obtained from the GABI-Kat T-DNA mutant collection (http://www.mpiz-koeln.mpq.de/GABI-
  • Fragments corresponding to the promoter of the Arabidopsis HSP18.2 gene, the CRE recombinase-coding sequence, and the octopine synthase terminator (OCS3 1 ) sequence were assembled into the pZErOTM-2 vector and subsequently inserted into the pCAMBIA2200 vector, resulting into the pJCRE vector.
  • the T-DNA region of the pJCRE vector contains the kanamycin resistance element which consists of the coding sequence of the neomycin phosphotransferase Il gene (nptll), flanked by the CaMV 35S promoter (P35S) and terminator (35S3 1 ) sequences.
  • the Arath;CDK/l;1 promoter, the enhanced GFP gene (£GFP)-coding sequence flanked at a 5' end by the proximal loxP site, the OCS3 1 sequences (flanked at a 3' end by a distal loxP site), and the GATEWAYTM recombination site were assembled into the pZErOTM-2 vector.
  • the cassette containing all the elements was subsequently cloned into the pCAMBIA1200 vector, resulting into the pJLOX vector.
  • the T-DNA region of the pJLOX vector contains the hygromycine resistance element, which consists in the coding sequence of the hygromycin phosphotransferase Il gene (hptll), flanked by the CaMV 35S promoter (P35S) and terminator (35S3 1 ) sequences. Cloning details are available upon request.
  • the WEE1 cDNA was introduced into the pJLOX vector by the GATEWAYTM recombinational cloning technology.
  • the obtained Agrobacterium strains were used to generate stably transformed Arabidopsis using the floral dip transformation method (Clough and Bent, 1998). Transgenic plants were selected on kanamycine- and/or hygromycine-containing media and later transferred to soil for seed stock amplification.
  • Genomic DNA was extracted from Arabidopsis leaves using the DNeasy Plant kit (Qiagen).
  • RNA was extracted from Arabidopsis tissues and cultured cells using the Trizol reagent (Invitrogen).
  • First-strand cDNA was prepared from 500 ng of total RNA with the Superscript RT Il kit (Invitrogen) and oligo(dT) 18 according to the manufacturer's instructions.
  • a 0.2 ⁇ L aliquot of the total RT reaction volume (20 ⁇ L) was used as a template in a semi-quantitative RT-mediated PCR amplification ensuring that the amount of amplified product remained in linear proportion to the initial template present in the reaction.
  • Protein extracts were prepared by grinding material in homogenization buffer (HB) (De Veylder et al., 1997). Protein concentrations were determined with the Protein Assay kit (Bio-Rad, Hercules, CA). CDKs were purified by p10 CKS1At -affinity chromatography using p10 CKS1At - Sepharose beads as described (Hemerly et al., 1995).
  • SDS-PAGE and protein gel blots were performed according to standard procedures with primary anti-CDKA;1 and anti-CDKB1;1 (Porceddu et al., 2001) antibodies diluted 1/5000 and 1/2500, respectively, and a secondary horseradish peroxidase (HRP)-conjugated sheep anti-rabbit antibody (Amersham Pharmacia Biotech) diluted 1/5000, or with the mouse monoclonal HRP-conjugated anti-phosphotyrosine p- Tyr (PY99; Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibody diluted 1/50000. Protein detection was done by chemiluminescent procedure (NEN Life Science Products Inc., Boston, MA, USA).
  • Leaves were harvested 21 days after sowing, cleared overnight in ethanol, stored in lactic acid for microscopy, and observed under a microscope fitted with differential interference contrast optics (Leica, Wetzlar, Germany).
  • the total (blade) area was determined from pictures digitized directly with a digital camera (Axiocam; Zeiss, Jena, Germany) mounted on a binocular (Stemi SV11 ; Zeiss).
  • DAPI 4,6-diamidino-2-phenylindole
  • Example 1 Arabidopsis CDKA; 1 is tyrosine phosphorylated upon activation of the DNA replication checkpoint.
  • CDK Tyr phosphorylation has been shown to occur upon G2/M arrest after cytokinin deprivation in tobacco and DNA replication checkpoint in Fucus zygotes (Zhang et al., 1996; Corellou et al., 2000).
  • the replication inhibitor aphidicolin being an inhibitor of DNA polymerases ⁇ and ⁇ .
  • Propyzamide was used to block cell cycle progression in mitosis by depolymerising the mitotic spindle (Planchais et al., 2000).
  • CDKA;1 belongs to the archetype group of CDKs, characterized by the presence of a PSTAIRE amino-acid sequence motif in the cyclin binding protein domain.
  • CDKB1;1 belongs to the group of plant-specific CDKs (Boudolf et al., 2004). As compared to control cells, neither drug treatment had effect on the abundance of CDKA;1 protein ( Figure 1).
  • CDKB1;1 levels were slightly increased in the propyzamide-treated cells, which can be explained by the preferential expression of the CDKB1;1 gene during M phase (Porceddu et al., 2001; Boudolf et al., 2004).
  • protein blots were probed with an anti-phosphotyrosine antibody. Whereas no antibody binding was detected in protein samples from control or propyzamide-treated cells ( Figure 1), a polypeptide band of the same electrophoretic mobility as CDKA; 1 was cross-reacting with the antibody in extracts prepared from the aphidicolin-treated cells. This analysis strongly indicates that CDKA; 1 is the target for Tyr phosphorylation upon activation of the DNA replication checkpoint.
  • Example 2 The WEE1 gene is expressed in response to activation of the DNA replication checkpoint in cultured Arabidopsis cells and seedlings.
  • transgenic Arabidopsis lines that contained as a transgene the 591-bp-long WEE1 promoter region fused to the ⁇ - glucuronidase (GUS) reporter gene.
  • GUS ⁇ - glucuronidase
  • Our data were also coherent with and the pattern of expression that can be deduced from a repository of 1434 publicly available microarray datasets using the GENEVESTIGATOR microarray analysis tool (Zimmermann et al., 2004).
  • Example 3 ATM kinase is required for the activation of the WEE1 gene in response to DNA damage
  • Figure 4 illustrates a typical reporter gene response in one-week-old seedlings treated with zeocin at concentration range from 1 ⁇ g/ml to 100 ⁇ g/ml.
  • Zeocin induced a strong dose-dependent induction of GUS activity in PARP-2.GUS reporter lines, demonstrating the efficiency of zeocin as a genotoxic drug (Figure 4).
  • WEE1 promoter activity was induced by the zeocin treatment; both in the root ( Figure 4) and the shoot tissues.
  • Example 4 Plants with a loss-of-function mutation in WEE1 are hypersensitive to perturbations of DNA metabolism
  • Seedlings of both genotypes had mature cotyledons and the first fully- expanded leaves of similar size and contained the same number of abaxial epidermal pavement cells (Table 1 ). Also root growth rates were similar for wild type and mutant plants ( Figure 6C, 6D and 6I). As it has been proposed that in maize the WEE1 gene can control the endocycle, the distribution profiles of ploidy levels in leaf cells of wild type and mutant plants were compared, but no differences were found (Table 2). These data illustrate that in Arabidopsis leaves and roots the WEE1 activity is not required for cell division or endoreduplication under normal growth conditions. Next, we characterized the growth of wee1-1 mutant plants in the presence of drugs that interfere with DNA replication.
  • Wild type and wee1-1 plants were germinated and grown on control medium for 5 days, and then transferred on control medium, or medium supplemented with either HU or aphidicolin at concentrations that had mild but perceptible effects on growth of the wild type root (Culligan et al., 2004).
  • the length of wild type roots was reduced by 32% and 72% respectively, as compared to non-treated plants (Fig 6C, 6E, 6G and 6I).
  • wee1-1 root growth was reduced by more than 70% in the presence of HU (Fig 6F and 6I), and mutant root growth was totally arrested in the presence of aphidicolin (Fig 6H and 61).
  • Example 5 Lack of WEE1 activity results in cell death in the presence of genotoxic stress
  • wee1-1 mutant is hypersensitive to DNA replication stress.
  • control and wee1- ⁇ plants were transferred 5 days after germination to control medium or medium containing aphidicolin.
  • Four days after transfer root tips of these plants were harvested and a real time PCR was performed.
  • WEE1 expression was three fold upregulated in control root tips under stress, no WEE1 expression was observed in the wee1-1 mutant.
  • CYCB1;1 expression was found to be three fold upregulated in the control root tip, suggesting a G2 arrest (Culligan et al., 2004), but was 7.5 fold upregulated in the wee1-1 mutant.
  • This accumulation of CYCB1;1 transcripts in stressed wee1-1 root tips could be due to an arrest of the cell cycle in the G2- or M-phase. It is expected that the wee1-1 cells, because of a lack of WEE1, cannot arrest their cell cycle to allow DNA repair on activation of the replication checkpoint and will enter the mitosis with incompletely replicated genomes. This supposedly results in an arrest of mitosis leading to meristematic failure and cell death. To analyze the amount of cells dying in the root tip, an Evans blue staining was performed. This cell-death staining showed more dying cells in the wee1-1 mutant than in control plants when growing under genotoxic conditions.
  • ATM and ART operate as signal transducing kinases in regulatory cascades that elicit the DNA damage response, which include cell cycle arrest.
  • Previous results showed that ATM-dependent activation of the WEE1 gene is a part of the DNA damage response in Arabidopsis.
  • WEE1 kinase activity was a part of the DNA damage response in Arabidopsis.
  • induced WEE1 transcript levels result in higher WEE1 protein production, which ultimately lead to the arrest of the plant cell cycle.
  • Seeds of the WEE1 knock out were analysed on size and weight.
  • the WEE1 knock out seeds are clearly bigger than the control seeds ( Figure 9).
  • For analysing the weight a certain weight of seeds was taken from the seeds stock, and the number of seeds was counted. Average seed weight was calculated by dividing the total weight by the number of seeds counted. The results are summarized in Table 3.
  • a S/M DNA replication checkpoint prevents nuclear and cytoplasmic events of cell division including centrosomal axis alignment and inhibits activation of cyclin-dependent kinase-like proteins in fucoid zygotes. Development 127, 1651-1660. Christensen, P.U., Bentley, N.J., Martinho, R.G., Nielsen, O., and Carr, A.M. (2000). Mik1 levels accumulate in S phase and may mediate an intrinsic link between S phase and mitosis.
  • Arabidopsis CksiAt protein binds to the cyclin-dependent kinases Cdc2aAt and Cdc2bAt.
  • Ionising radiation induces the expression of PARP-1 and PARP-2 genes in Arabidopsis.
  • AtATM is essential for meiosis and the somatic response to DNA damage in plants. Plant
  • SimpleSearch a flanking sequence tag (FST) database for the identification of T-DNA insertion mutants in Arabidopsis thaliana. Bioinformatics 19, 1441-1442.
  • Chem. 276, 36354-36360 Rhind, N., and Russell, P. (2001). Roles of the mitotic inhibitors Wee1 and Mik1 in the G 2 DNA damage and replication checkpoints. MoI. Cell. Biol. 21 , 1499-1508. Sancar, A., Lindsey-Boltz, L.A., ⁇ nsal-Kagmaz, K., and Linn, S. (2004). Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu. Rev. Biochem. 73,
  • a WEE1 homologue from Arabidopsis thaliana Planta 215, 518-522.
  • Valvekens D., Van Montagu, M. and Van Lijsebettens, M. (1988) Agrobacterium tumefaciens- mediated transformation ofArabidopsis thaliana root explants by using kanamycin selection. Proc. Natl. Acad. Sci USA 85, 5536-5540. Vandepoele, K., Raes, J., De Veylder, L., Rouze, P., Rombauts, S., and Inze, D. (2002).

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Abstract

La présente invention porte sur l'utilisation de WEE1 pour accroître le rendement des récoltes dans des conditions de stress. C'est invention porte plus spécifiquement sur la sensibilisation de la cellule au facteur de l'activité de Wee1 dans des conditions de stress non létales. Un mode de réalisation préféré est l'utilisation d'un knock out WEE1 pour augmenter le poids des graines, le stress induit étant un stress dû à la sécheresse.
PCT/EP2005/056629 2004-12-14 2005-12-09 Methode pour accroitre la biomasse des vegetaux dans des conditions de stress WO2006063963A1 (fr)

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EP2304036A4 (fr) * 2008-06-13 2012-01-25 Performance Plants Inc Procédés et moyens d augmentation de l efficacité d utilisation d eau par des plantes

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