*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

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

Droughtenvironmental

stress

Plant response to environmental stresses

Temperaturestress Plant Cell

High salinity

Signal Perception

Signal Transduction

Gene Expression

Stress Responseand Tolerance

Strategies to engineer environmental stress tolerance

in plants

Promoterscontrol gene expression underenvironmental stress conditions

Transgenic techniquesfor various crops to introduce useful genes

Promoter Gene Promoter Gene

Genesfunction in tolerance to environmental stresses

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 centerssupply mutants and DNA libraries.

Efficient transformation for functional analysis of genes.

Functions of water-stress-inducible gene products

Water channelprotein

Detxification enzymes

Protection factors ofMacromolecules

(LEA protein)

Detoxicationenzyme

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)

Environmental stress tolerance

Environmental Stress

Tolerancegene

expression

Overexpression of DREB1A

DREB1A gene regulates a lot of target genes to enhance tolerance to environmental stresses

Expression of more than 40 genes

Tolerancegene

expression

Tolerancegene

expression

Enhanced stress tolerance

Drought and High salinity

Signal Perception

DREB1A

Tolerancegene

expression

Enhanced expression of more than 40 genes

Tolerancegene

expression

Tolerancegene

expression

DREB1A

Signal Perception

Combinations of promoters and transcription factors

① 　 Constitutive promoter (35S) ＋ DREB1A

DREB1A geneConstitutive promoter

The constitutive promoter leads　　　　　　　　　　 to generate DREB1A products all the time

② 　 Stress-inducible promoter (RD29A) ＋ DREB1A

DREB1A geneStress-inducible promoter

The stress-inducible promoter leads to generate DREB1A products only under stress conditions

drought high salinity

76.7%65.0%

16.7%42.8%

0.0% 13.8%

Transgenic Arabidopsis plants overexpressing DREB1 plantsdisplay enhanced tolerance to dorught and salt stresses

No treatment

Stress-induciblepromoter (RD29A)

＋DREB1

Constitutivepromoter (35S)

＋DREB1

Wild type

Stress-inducible promoter is useful to generate DREB1A products without affecting plant growth

Negative effect on plat growth

Non-negative effecton plant growth

stress condition

Normal growth condition

ON

OFF

ON

ON

Normal growth condition

stress condition

DREB1A geneConstitutive promoter

DREB1A geneStress-inducible promoter

Engineering environmental stress tolerance

Model plants

Arabidopsis

Developing technologiesWheat

Rice

Agriculturally important crops

Engineering for stress tolerance in crops

Maize Sugarcane

Legumes

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.

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

CIMMYT IRRI ICRISAT CIATCGIAR

rice

Arabidopsis rice

Indica ricemaizewheat

Collaboration for development of stress

tolerant crops

JIRCAS Biotechnology Laboratory

Public ResearchInstitute

NIAS

Lotus Japonicus(a model legume)

NIVTS

cruciferous cropperand vegetables

groundnut pigeonpea cassava

Central Res. Lab.

Hitachi, Ltd.

petunia

Oji Paper Co., Ltd.Forestry Res. Inst.

eucalypt

Kaisui ChemicalIndustry Co., Ltd.

turf

Company Institute

The University Tokyo

RD29A:DREB1A

Drought stress tolerance of the transgenic petunia

dehydratedfor １４ days

wt

Yoshiba et al. unpublished

Drought stress tolerance of the transgenic wheat

RD29A:DREB1A wt RD29A:DREB1A wt

Pellegrineschi et al. genome 2004

Laboratory test

Improving stress tolerance by gene transferImproving stress tolerance by gene transfer

Exploring appropriate stress-tolerance gene

Exploring appropriate promoters

Select best combination for

sugarcane Stress-tolerance genepromoter

Making transgenic sugarcane

Drought and salt stress

Improving stress tolerance by gene transfer

Field test to evaluate stress tolerance and yield

Useful transcription factors to improve stress tolerance

Useful transcription factors to improve stress tolerance

WoundingPathogen infection

Drought, Salinity High temperature Low temperature

Stress tolerance

Expression of stress-responsive genes

Transcriptionfactors

cis-elements