C-terminal extension of rice glutamate decarboxylase (OsGAD2) functions as an autoinhibitory domain and overexpression of a truncated mutant results in the accumulation of extremely high levels of GABA in plant cells

Abstract

Glutamate decarboxylase (GAD) converts L-glutamate to gamma-aminobutyric acid (GABA), which is a non-protein amino acid present in all organisms. Plant GADs carry a C-terminal extension that binds to Ca(2+)/calmodulin (CaM) to modulate enzyme activity. However, rice possesses two distinct types of GAD, OsGAD1 and OsGAD2. Although they both have a C-terminal extension, the former peptide contains an authentic CaM-binding domain (CaMBD), which is common to dicotyledonous plants, while the latter does not. Therefore, the role of the C-terminal extension in functional expression of OsGAD2 was investigated. An in vitro enzyme assay using recombinant OsGAD2 proteins revealed low activity in the presence or absence of Ca(2+)/CaM. However, a truncated version of GAD2 (OsGAD2DeltaC) had over 40-fold higher activity than wild-type GAD at physiological pH. These two DNA constructs were introduced simultaneously into rice calli via Agrobacterium to establish transgenic cell lines. Free amino acids were isolated from several lines for each construct to determine GABA content. Calli overexpressing OsGAD2 and OsGAD2DeltaC had about 6-fold and 100-fold the GABA content of wild-type calli, respectively. Regenerated OsGAD2DeltaC rice plants had aberrant phenotypes such as dwarfism, etiolated leaves, and sterility. These data suggest that the C-terminal extension of OsGAD2 plays a role as a strong autoinhibitory domain, and that truncation of this domain causes the enzyme to act constitutively, with higher activity both in vitro and in vivo.

Characterization and developmental expression of a glutamate decarboxylase from maritime pine

“…S1). The typical sequence of a CaM-binding domain has been only reported in the GADs of angiosperms; it is absent in GAD sequences from other kingdoms, in two GAD sequences from O. sativa (Akama et al 2001;Akama and Takaiwa 2007), and in the full-length cDNA sequences from Bryophyta (Fig. 1).…”

“…6). The production of 2-oxoglutarate by cytosolic NADP + -IDH has been considered as a limiting factor in the biosynthesis of glutamate in plants (Hodges 2002), and the alteration of the glutamate/GABA ratio has been shown to aVect the elongation of stem internodes in tobacco and rice seedlings Akama and Takaiwa 2007). Therefore, regulation of the synthesis of glutamate and GABA can play a role in development and diVerentiation of the stem in plants.…”

“…The regulation of GAD by CaM strongly suggests that GABA production could be involved in signaling pathways. In Arabidopsis, it has been reported that GABA levels function as a signal that controls pollen tube growth (Palanivelu et al 2003), and in tobacco and rice, it was found that GABA levels aVect cellular elongation in the stem in vivo Akama and Takaiwa 2007). Although no molecular receptor for GABA has been characterized in plants so far, the regulation of a large set of 14-3-3 proteins by GABA was recently reported in Arabidopsis (Lancien and Roberts 2006), and the existence of binding sites for GABA in pollen protoplasts has been reported in tobacco (Yu et al 2006).…”

Contribution of Gamma amino butyric acid (GABA) to salt stress responses of Nicotiana sylvestris CMSII mutant and wild type plants

“…In that study, GAD activity decreased within the first 24 h of 150 mM NaCl treatment while it increased in 50, 100 and 150 mM NaCl treated groups at day 4. Moreover, rice plants over-expressing the GAD gene accumulate extremely high amounts of GABA in the free amino acid pool under salt stress treatment (Akama and Takaiwa, 2007).…”

Simultaneous Expression of Spinacia oleracea Chloroplast Choline Monooxygenase (CMO) and Betaine Aldehyde Dehydrogenase (BADH) Genes Contribute to Dwarfism in Transgenic Lolium perenne

“…Dwarfism is an aberrant plant phenotype that could be induced by over-expression of MYB transcription factors (Zhang et al 2009), single virulence factor (Hoshi et al 2009), brassinosteroid receptor gene (Morinaka et al 2006), extremely hydrophobic peptides (Dresselhaus et al 2005), glutamate decarboxylase (Akama and Takaiwa 2007), fungal pectin methylesterase (Hasunuma et al 2004), regulator of gene expression (Mussig et al 2000), phytochrome A (Stockhaus et al 1992), and the Agrobacterium tumefaciens nopaline transferred DNA iaaH gene (Sitbon et al 1992). Dwarfism was also observed in transgenic rice plant expressing an antisense cDNA for the alpha subunit of rice heterotrimeric G protein (Fujisawa et al 1999).…”