Gamma-aminobutyric acid contents and glutamate decarboxylase activity in local Thai rice and transgenic rice overexpressing oscam1-1 gene

Abstract

In rice grain, many considerable nutrients are shown to have potential functional food benefits. GABA is a metabolic product that is primarily produced by the decarboxylation of L-glutamic acid (L-Glu), catalyzed by glutamate decarboxylase (GAD), which is a cytosolic enzyme regulated by Ca2+-calmodulin (CaM) complex. The accumulation of GABA is related to the activity of GAD and substrate concentration of L-Glu. In this study, analysis of GABA contents in local Thai rice (Oryza sativa L.) during 0-72 h of germination revealed that GABA concentrations rapidly increased and the pattern of increment varied among different rice varieties. Genome-wide association mapping using 2,009,083 SNPs from a population of 100 varieties based on the GABA concentrations 48 hours after germination was carried out. As a result, 23 SNPs associated to five genes were identified. LOC Os02g40320, LOC Os02g40330, and LOC Os11g35580 were three genes having significant SNPs in their coding regions. The additional genes with upstream significant SNPs were LOC_Os01g18390 and LOC_Os04g13480. The protein encoded by LOC_Os02g40320 was related to numerous other proteins by STRING analysis, including nitrate reductase 1 (NIA1) and sucrose synthase 6. (SUS6). The response regulator receiver domain protein, which is encoded by LOC_Os4g13480 and functions in the His-to-Asp phosphorelay signal transduction pathway, was discovered to interact with a number of proteins in the two-component system responding to cytokinin, which has previously been shown to have a functional relationship with GABA.

To investigate the role of calmodulin in GABA accumulation, GABA contents in the three transgenic ‘Khoa Dawk Mali 105’ (KDML105) rice overexpressing OsCaM1-1 gene (L1, L2, and L7) were examined compared with the wild-type ‘KDML105’ (WT), which are differing in salt tolerance. Under normal condition, GABA contents in the transgenic rice overexpressing OsCam1-1 calmodulin gene increased in the first 24 hr of germination while that of WT did not. Under salt stress (100 mM NaCl), their levels slightly increased and were not different among the rice lines. After 4 days with visible sprouting under both normal and salt stress conditions, GABA contents in all rice lines rapidly increased. GABA contents under salt stress condition continued to increase and reached higher levels at day 6 and 8 in all transgenic overexpressing lines when compared with that of WT. After 10 and 12 days, GABA levels under normal condition then rapidly increased whereas those under salt stress when the seedlings were severely affected significantly decreased. In addition, GAD activity in all transgenic rice lines and WT appeared to increase in the first week of germination and those of the transgenic rice were higher than WT at day 12 after germination. Together these results indicated that higher GABA content in the transgenic rice overexpressing OsCam1-1 gene may result from the induced GAD activity, which in turn confers the salt tolerance ability to the transgenic rice.