1. Engineering Tomato that hyper accumulate GABA Takashi AKIHIRO

2. Permission from Ministry of Health, Labour and Welfare Food for Specified Health Use （ FOSHU ） Introduction

3. € 6.7 €13.4 € 20.3 €27.0 € 33.7 € 40.4 € 47.1 billion year 2005 6300 Ttransition of FOSHU market in Japan

4. GABA Gamma-aminobutyric acid In vertebrates, GABA is an inhibitory neurotransmitter in the brain. GABA is able to induce relaxation, analgesia, and sleep. GABA are known to stimulate GABA receptors. Several neurological disorders, such as epilepsy, sleep disorders, and Parkinson's disease are affected by this neurotransmitter. In 2005, some “Dietary Supplement” companies started producing and selling various formulas containing GABA itself.

5. GABA Gamma-aminobutyric acid In plant, GABA accumulation has been associated with a variety of environmental stress conditions, including hypoxia, temperature shock, low pH and mechanical manipulation.

6. Philippe Raymond et al (2000) Austrian J of plant phys Why huge amount of GABA accumulated in the tomato fruit is not cleared.

8. 140mg/50g Chocolate Supplement 250mg/ 60 capsules 150mg/kg Rice Tomato 40-60 mg/100gFW 70mg/2L Chinese Tea

9. GABA 40 ～ 60mg/100gFW 100 ～ 120 mg/100gFW 200 ～ 250 mg/100gFW Normal Tomato Salt treated Tomato (Dr Saito et al.,) Targeted value GABA- Hyper accumulator

12. Working hypothesis

13. Workflow Mining databases (Tiger and Mibase) for putative GADs,GABATs,SSADHs and SSRs EST ↓ Isolation of these genes by RT-PCR ↓ Expression analysis of these genes using (Semi-)quantitative RT-PCR ↓ Measurement of these genes activity using E coli expression system (or in vitro translation system) ↓ Mutant screening using TILLING. Create transgenic tomato that GAD,GABAT,SSADH,and SSR Over express or Knockout (RNAi)

14. Fig. 1. Sequence alignment of GADs from various plants. Identical and similar amino acid residues are indicated by black and gray boxes, respectively. Gaps are shown by hyphens. the C-terminal peptide extension that is unique to plant GADs is shown by a dashed line. GAD

15. Fig. 2. Alignment of the amino acid sequences of the C-terminus of plant GADs.

16. LeGADA LeGADB LeGADF Petunia OsGAD1OsGAD2 Fig. 3. Structural features of C-terminal portions of GADs from tomato, rice, lotus Indian mustard and petunia.

17. Fig. 4. Sequence alignment of GABATs from tomato and Arabidopsis. Identical and similar amino acid residues are indicated by black and gray boxes, respectively. Gaps are shown by hyphens. GABAT

18. Table 2. Prediction of the subcellular location of GABATs using TargetP, MitoProII,PSORT and PSORTII program.

19. Fig. 5. Sequence alignment of SSADHs from tomato and Arabidopsis. Identical and similar amino acid residues are indicated by black and gray boxes, respectively. Gaps are shown by hyphens. SSADH

20. Fig. 6. Sequence alignment of SSRs from tomato and Arabidopsis. Identical and similar amino acid residues are indicated by black and gray boxes, respectively. Gaps are shown by hyphens. SSR

21. Fig. 7. Isolation of total RNAs from tomato Control DAF3DAF6 DAF9-1DAF9-2 DAF12DAF15DAF18DAF21DAF24DAF27DAF30DAF33DAF36 DAF39DAF42DAF45 ROOT SHOOT LEAF FLOWER FRUIT

22. Summary GAD GABAT SSADH SSR X3 X2 Isolated from MicroTom

23. Acknowledgement Dr Watanabe Tani Tominaga