*Yasunari Fujita (JIRCAS, JAPAN) Kazuko Yamaguchi-Shinozaki (JIRCAS/Univ. Tokyo, JAPAN) Kazuo Shinozaki (RIKEN, JAPAN)

1. 4th Biomass-Asia Workshop Nov. 21, 2007 Improving drought and salt stress tolerance in plants by gene transfer *Yasunari Fujita (JIRCAS, JAPAN) Kazuko Yamaguchi-Shinozaki (JIRCAS/Univ. Tokyo, JAPAN) Kazuo Shinozaki (RIKEN, JAPAN)

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

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

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

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 108 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 Transcription factors (MYB, MYC, DREB, AREB) Detxification enzymes Water stress Protein kinases (CDPK, MAPK) Protection factors of Macromolecules (LEA protein) Phospholipid metabolism (PLC, PIP5K) Key enzymes for Osmolyte biosynthesis (proline, sugar) ABA biosynthetic enzyme Detoxication enzyme

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

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

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

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

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

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

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

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

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

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

18. Laboratory test Improving stress tolerance by gene transfer Exploring appropriate promoters Exploring appropriate stress-tolerance gene Select best combination for sugarcane promoter Stress-tolerance gene 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 High temperature Low temperature Drought, Salinity Wounding Pathogen infection Transcription factors cis- elements Expression of stress-responsive genes Stress tolerance