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Engineering Tomato that hyper accumulate GABA Takashi AKIHIRO Introduction Food for Specified Health Use （FOSHU） Permission from Ministry of Health, Labour and Welfare billion € 47.1 6300 € 40.4 € 33.7 €27.0 € 20.3 €13.4 € 6.7 2005 year Ttransition of FOSHU market in Japan In vertebrates, GABA is an inhibitory neurotransmitter in the brain. GABA is able to induce relaxation, analgesia, and sleep. GABA Gamma-aminobutyric acid 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. 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. Why huge amount of GABA accumulated in the tomato fruit is not cleared. Philippe Raymond et al (2000) Austrian J of plant phys Supplement Chinese Tea Tomato 250mg/ 60 capsules Chocolate 70mg/2L Rice 40-60 mg/100gFW 140mg/50g 150mg/kg Normal Tomato 40～60mg/100gFW Salt treated Tomato (Dr Saito et al.,) 100～120 mg/100gFW GABA Targeted value 200～250 mg/100gFW GABA- Hyper accumulator Working hypothesis 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) GAD 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. Fig. 2. Alignment of the amino acid sequences of the C-terminus of plant GADs. OsGAD1 LeGADA Petunia LeGADB OsGAD2 LeGADF Fig. 3. Structural features of C-terminal portions of GADs from tomato, rice, lotus Indian mustard and petunia. GABAT 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. Table 2. Prediction of the subcellular location of GABATs using TargetP, MitoProII,PSORT and PSORTII program. SSADH 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. SSR 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. DAF45 DAF42 DAF39 DAF36 DAF33 DAF30 DAF27 DAF24 DAF21 DAF18 DAF15 DAF12 DAF9-2 DAF9-1 DAF6 DAF3 Control FRUIT FLOWER LEAF SHOOT ROOT Fig. 7. Isolation of total RNAs from tomato Summary GAD GABAT SSADH SSR X3 X3 X2 X2 Isolated from MicroTom Acknowledgement Dr Watanabe Tani Tominaga