|Budget Amount *help
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2003: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2002: ¥2,200,000 (Direct Cost: ¥2,200,000)
1 Occurrence of two forms of radish γ-glutamyltransferases (GGT; EC.220.127.116.11), soluble and cell wall bound ones, were demonstrated in many plants including radish (Raphanus sativus).
2 We cloned three cDNAs (rsggt1, AB098475; rsggt2, AB102676;rsggt3, unregistered) predicted to encode radish soluble GGTs, sequenced them and analyzed their properties. These cDNA seems to encode a heterodimeric type of GGTs homologous to mammalians GGTs.
3 These three genes were transcribed by organ and tissue specific manner: rsggt1 was expressed characteristically in immature seeds and cotyledons; rsggt2 in cotyledons, leaves and roots; and rsggt 3 in leaves, roots and immature seeds.
4 We transformed tobacco plants with rsggt1 and rsggt2. Transformed tobacco plants grew normally. They developed higher level of GGT activity, although wild type tobacco plants exhibited a very low GGT activity. Expressed GGT activity was found in the bound fraction, although cDNA used encoded soluble radish GGT. To understand this discrepancy remains to be solved in future.
5 We purified bound GGTs from radish cotyledons. The bound GGT was a monomeric enzyme unlike soluble GGTs. cDNA was presumed to be highly homologous to Arabidopsis cDNA (AK118068) encoding putative β-1,3-glucanase. Tobacco plants transformed with putative β-1,3-glucanase cDNA exhibited elevated level of GGT activity, but not β-1,3-glucanase.
6 We measured thiol compounds in apoplastic space of leaves of transgenic and wild type tobacco plants. Decreased level of glutathione (GSH) and elevated level of cysteinylglycine, a product of GGT reaction, were found in transgenic plants. These findings suggest that cell wall bound GGTs participate in GSH catabolism in apoplastic space.