1. *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

2. Explosive increase of population In developing countries Environmental degradation and climatic change around the world Progress in genetic engineering Development of crop plants tolerant to environmental stresses such as drought, salt loading and freezing

3. Drought environmental stress Plant response to environmental stresses Temperature stress Plant Cell High salinity Signal Perception Signal Transduction Gene Expression Stress Response and Tolerance

4. Strategies to engineer environmental stress tolerance in plants Promoters control gene expression under environmental stress conditions Transgenic techniques for various crops to introduce useful genes PromoterGene PromoterGene Genes function in tolerance to environmental stresses

5. Arabidopsis thaliana as a model plant Good for genetic analysis (2n). Small plants easily grown. Short life cycle (6-8 weeks). Smallest genome in plant kingdom (1.2 X 10 8 bp, 5 chromosomes). Gene number is about 26,000. Genome sequence was determined in 2000. Arabidopsis resource centers supply mutants and DNA libraries. Efficient transformation for functional analysis of genes.

6. Functions of water-stress-inducible gene products Water channel protein Detxification enzymes Protection factors of Macromolecules (LEA protein) Detoxication enzyme ABA biosynthetic enzyme Transcription factors (MYB, MYC, DREB, AREB) Protein kinases (CDPK, MAPK) Phospholipid metabolism (PLC, PIP5K) Water stress Key enzymes for Osmolyte biosynthesis (proline, sugar)

7. Environmental stress tolerance Environmental Stress Tolerance gene expression Overexpression of DREB1A DREB1A gene regulates a lot of target genes to enhance tolerance to environmental stresses Expression of more than 40 genes Tolerance gene expression Tolerance gene expression Enhanced stress tolerance Drought and High salinity Signal Perception DREB1A Tolerance gene expression Enhanced expression of more than 40 genes Tolerance gene expression Tolerance gene expression DREB1A Signal Perception

8. Combinations of promoters and transcription factors ① Constitutive promoter (35S) ＋ DREB1A DREB1A gene Constitutive promoter The constitutive promoter leads to generate DREB1A products all the time ② Stress-inducible promoter (RD29A) ＋ DREB1A DREB1A gene Stress-inducible promoter The stress-inducible promoter leads to generate DREB1A products only under stress conditions

9. droughthigh salinity 76.7%65.0% 16.7%42.8% 0.0% 13.8% Transgenic Arabidopsis plants overexpressing DREB1 plants display enhanced tolerance to dorught and salt stresses No treatment Stress-inducible promoter (RD29A) ＋ DREB1 Constitutive promoter (35S) ＋ DREB1 Wild type

10. Stress-inducible promoter is useful to generate DREB1A products without affecting plant growth Negative effect on plat growth Non-negative effect on plant growth stress condition Normal growth condition ON OFF ON Normal growth condition stress condition DREB1A gene Constitutive promoter DREB1A gene Stress-inducible promoter

11. Engineering environmental stress tolerance Model plants Arabidopsis Developing technologies Wheat Rice Agriculturally important crops Engineering for stress tolerance in crops MaizeSugarcane Legumes

12. The DREB1A transgenic rice plants display enhanced tolerance to drought stress Wild type DREB1A transgenic rice plants Drought stress test: Watering was withheld for 9 days, then rewatering for 13days, before the photograph was taken.

13. Wild Type Transgenic plants The DREB1A transgenic rice plants display enhanced tolerance to salt stress Salt stress test: Treated with 250mM NaCl for 3 days

15. CGIAR rice Arabidopsis rice Indica ricemaizewheat Collaboration for development of stress tolerant crops JIRCAS Biotechnology Laboratory Public Research Institute NIAS Lotus Japonicus (a model legume ) NIVTS cruciferous cropper and vegetables groundnutpigeonpea cassava Central Res. Lab. Hitachi, Ltd. petunia Oji Paper Co., Ltd. Forestry Res. Inst. eucalypt Kaisui Chemical Industry Co., Ltd. turf Company Institute The University Tokyo

16. RD29A:DREB1A Drought stress tolerance of the transgenic petunia dehydrated for １４ days wt Yoshiba et al. unpublished

17. Drought stress tolerance of the transgenic wheat RD29A:DREB1A wt RD29A:DREB1A wt Pellegrineschi et al. genome 2004

18. Laboratory test Improving stress tolerance by gene transfer Exploring appropriate stress-tolerance gene Exploring appropriate promoters Select best combination for sugarcane Stress-tolerance gene promoter Making transgenic sugarcane Drought and salt stress Improving stress tolerance by gene transfer Field test to evaluate stress tolerance and yield

19. Useful transcription factors to improve stress tolerance Useful transcription factors to improve stress tolerance Wounding Pathogen infection Drought, Salinity High temperatureLow temperature Stress tolerance Expression of stress-responsive genes Transcription factors cis- elements