*yasunari fujita (jircas, japan) kazuko yamaguchi-shinozaki (jircas/univ. tokyo, japan) kazuo...

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*Yasunari Fujita (JIRCAS, JAPAN) Kazuko Yamaguchi-Shinozaki (JIRCAS/Univ. Tokyo, JAPAN) Kazuo Shinozaki (RIKEN, JAPAN) 4th Biomass-Asia Workshop Nov. 21, 2007 Improving drought and salt stress tolerance in plants by gene transfer

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  • *Yasunari Fujita (JIRCAS, JAPAN)Kazuko Yamaguchi-Shinozaki (JIRCAS/Univ. Tokyo, JAPAN)Kazuo Shinozaki (RIKEN, JAPAN)4th Biomass-Asia Workshop Nov. 21, 2007Improving drought and salt stress tolerance in plants by gene transfer

  • Explosive increase of population In developing countriesEnvironmental degradation and climatic change around the worldProgress in genetic engineeringDevelopment of crop plants tolerant to environmentalstresses such as drought, salt loading and freezing

  • DroughtenvironmentalstressPlant response to environmental stressesTemperaturestressPlant CellHigh salinitySignal PerceptionSignal TransductionGene ExpressionStress Responseand Tolerance

  • Strategies to engineer environmental stress tolerance in plantsPromoterscontrol gene expression underenvironmental stress conditionsTransgenic techniquesfor various crops to introduce useful genesPromoterGenePromoterGeneGenesfunction in tolerance to environmental stresses

  • Arabidopsis thaliana as a model plantGood for genetic analysis (2n).Small plants easily grown.Short life cycle (6-8 weeks).

    Smallest genome in plant kingdom(1.2 X 108 bp, 5 chromosomes).Gene number is about 26,000.Genome sequence was determined in 2000.

    Arabidopsis resource centerssupply mutants and DNA libraries.

    Efficient transformation for functional analysis of genes.

  • Environmental stress toleranceEnvironmental StressTolerancegeneexpressionOverexpression of DREB1ADREB1A gene regulates a lot of target genes to enhance tolerance to environmental stressesExpression of more than 40 genesTolerancegeneexpressionTolerancegeneexpression

    Enhanced stress toleranceDrought and High salinitySignal PerceptionDREB1ATolerancegene expressionEnhanced expression of more than 40 genesTolerancegeneexpressionTolerancegeneexpressionDREB1ASignal Perception

  • Combinations of promoters and transcription factorsConstitutive promoter (35S) DREB1ADREB1A geneConstitutive promoterThe constitutive promoter leads to generate DREB1A products all the timeStress-inducible promoter (RD29A) DREB1ADREB1A geneStress-inducible promoterThe stress-inducible promoter leads to generate DREB1A products only under stress conditions

  • droughthigh salinity76.7%65.0%16.7%42.8%0.0%13.8%Transgenic Arabidopsis plants overexpressing DREB1 plantsdisplay enhanced tolerance to dorught and salt stressesNo treatmentStress-induciblepromoter (RD29A)DREB1Constitutivepromoter (35S)DREB1Wild type

  • Stress-inducible promoter is useful to generate DREB1A products without affecting plant growthNegative effect on plat growthNon-negative effecton plant growthstress conditionNormal growth conditionONOFFONONNormal growth conditionstress conditionDREB1A geneConstitutive promoterDREB1A geneStress-inducible promoter

  • Engineering environmental stress tolerance Model plantsArabidopsisWheatRiceAgriculturally important cropsEngineering for stress tolerance in cropsMaizeSugarcaneLegumes

  • The DREB1A transgenic rice plants display enhanced tolerance to drought stressWild typeDREB1A transgenic rice plantsDrought stress test: Watering was withheld for 9 days, then rewatering for 13days, before the photograph was taken.

  • Wild TypeTransgenic plantsThe DREB1A transgenic rice plants display enhanced tolerance to salt stressSalt stress test: Treated with 250mM NaCl for 3 days

  • CGIARriceArabidopsisriceIndica ricemaizewheatCollaboration for development of stress tolerant cropsJIRCASBiotechnology LaboratoryPublic ResearchInstituteNIASLotus Japonicus(a model legume)NIVTS

    cruciferous cropperand vegetablesgroundnutpigeonpeacassavaCentral Res. Lab.Hitachi, Ltd.petuniaOji Paper Co., Ltd.Forestry Res. Inst.eucalyptKaisui ChemicalIndustry Co., Ltd.turfCompany InstituteThe University Tokyo

  • RD29A:DREB1A Drought stress tolerance of the transgenic petuniadehydratedfor dayswtYoshiba et al. unpublished

  • Drought stress tolerance of the transgenic wheatRD29A:DREB1AwtRD29A:DREB1AwtPellegrineschi et al. genome 2004

  • Improving stress tolerance by gene transferExploring appropriate stress-tolerance geneExploring appropriate promotersSelect best combination for sugarcaneStress-tolerance genepromoterMaking transgenic sugarcaneDrought and salt stressImproving stress tolerance by gene transferField test to evaluate stress tolerance and yield

  • Useful transcription factors to improve stress toleranceWoundingPathogen infectionDrought, SalinityHigh temperatureLow temperatureStress toleranceExpression of stress-responsive genesTranscriptionfactorscis-elements

    Let me first thank the organizers, for giving me this opportunity to present our work and also thank Dr. Shirai for your introduction. Im very happy to have the opportunity to give a talk today. In this talk, Id like to talk aboutFirstly, I am going to talk about the outline of the research in our group.The purpose of our research is to elucidate the mechanisms underlying environmental-stress tolerance in plants by mainly using techniques in molecular biology and to contribute to create useful environmental-stress tolerant crops. The main target is drought-, high-salinity-, and temperature stresses. Plants respond to environmental stresses such asIn the plant cell level, plant cells perceive the signal of environmental stresses. And signal transduction. And gene expression leads to stress response and tolerance.Especially, our group focus on the promoters and genes involved in the tolerance to environmental stresses. PromoterGenesIn our group, over the last decade we have looked for and found the useful promoters and genes, in addition, appropriate combinations of the promoters and the genes for various crops.In our group, at first, by using a model plant, Arabidopsis, we have revealed key promoters and genes implicated in the tolerance to environmental stresses. Compared to the other plants, this model plant Arabidopsis has better characters to analyze key genes and promoters. diploid,..By using Arabidopsis plants, we have also revealed functions of water-stress-inducible gene products. These are actual functional genes for the tolerance to water stresses such as drought and high salinity. These are regulatory genes for them. We have focused on the transcription factors involved in regulating gene expression of these functional genes.For example, DREB1A is a key transcription factor involved in the environmental stress tolerance. DREB1A is one of the most famous fruit in our laboratory. Our group has revealed that DREB1A gene regulates a lot of such stress-inducible genes that are involved in the stress tolerance. As depicted in this cartoon, overexpression of DREB1A leads to enhance the expression of stress-inducible genes

    Overexpression strategy is important, in addition, the promoters regulate the gene expression under environmental stress conditions and are also important factor. We made two types of gene constructs. One is a constitutive pro was fused to the key TF DREB1A gene. In the case of this, The constitutiveanother is a stress inducible pro was fused to DREB1A gene. In this case, the stress-inducible..These are wt, non-transgenic plants. Survival rates under drought and salt stress conditions are very low. In contrast, transgenic Arabidopsis plants display enhanced stress tolerance. But, in the case with the constitutive promoter, plant growth was greatly inhibited. By contrast, in the case with stress-inducible promoter, plant growth was not damaged and the transgenic plants showed highest survival rates.In the case of constitutive pro, all the time the plants generate DREB1A gene products. This is good for stress tolerance. But, under non-stress normal conditions, the transgenic plants show negative effect on plant growth. In contrast, in the case of stress-inducible pro, only under stress conditions, the plants generate DREB1A gene product, so that the transgenic plants show non-negative effect on plant growth. In summary, stress-In our lab., based on the fruit of the study by using a model plant Arabidopsis, we are now trying to create transgenic agriculturally important crops such as..In the case of sugarcane, now this project is just beginning.

    The DREB1A transgenic rice also display enhanced tolerance to drought stress. WateringThe DREB1A transgenic rice also display enhanced tolerance to drought stress. These plants were treated with 250mM NaCl for 3 days.Now we are mainly collaborating with CGIAR(Consultative Group on International Agricultural Research) centers to create useful transgenic crops. Based on our research in Arabidopsis and rice, we provides useful vectors containing appropriate combinations of promoters and genes. In these inst., collaborators perform the field tests to evaluate the ability of stress tolerance and yields in order to create transgenic crops for actual use in each field.

    In addition to the CGIAR centers, we have also collaborated with several National and company institutes.For examples, in the case of Petunia in collaboration with HITACHI company, the DREB1A transgenic petunia plants display enhanced tolerance to drought stress.In the case of wheat in collaboration with CIMIT, the DREB1A transgenic wheat plants also display enhanced tolerance to drought stress.In summary, for example, in the case of sugarcane, at first, we need to explore appropriate promoters and stres-tolerance genes. After that we need to select appropriate combination of promoters and gense for sugarecane. Then we are going to make transgenic sugarcane. After that we will conduct stress tolerance, safety and yield tests in the laboratory, green house, and the field levels. For example, in the laboratory test, temerature and humidity and the other conditions are all controlled. But, in the case of actual field, various situations always occur. So, it takes not a short time to develop transgenic plants.In our group including JIRCAS, RIKEN and the University of Tokyo, we have identified a lot of useful transcription factors to improve stress tolerance. Further, we are going to explore useful regulatory genes to improve stress tolerance in plants. Thank you for your attention.