Os04g0447800

Glutamate decarboxylase (GAD) converts L-glutamate to g-aminobutyric acid (GABA), which is a non-protein amino acid present in all organisms. Plant GADs carry a C-terminal extension that binds to Ca2+/calmodulin(CaM) to modulate enzyme activity.

Annotated Information

Function

• Plant GADs carry a C-terminal extension that binds to Ca2+/calmodulin(CaM) to modulate enzyme activity.Glutamate decarboxylase (GAD) is an enzyme that catalyses the conversion of L-glutamate to c-aminobutyric acid (GABA), which is a non-protein amino acid that is commonly present in both prokaryotes and eukaryotes. In animals, GABA is known to be a major inhibitory neurotransmitter.

Expression

• Recent sequencing of the rice genome revealed the presence of five GAD isoforms, including OsGAD1 and OsGAD2 (International Rice Genome Sequencing Project,2005). All five isoforms are at least transcriptionally active, given the presence of corresponding EST clones.Figure 1 shows the amino acid sequences of the C-terminal regions of the five rice GADs and the petunia GAD (PhGAD), which is currently the most intensively analysed GAD (Baum et al., 1993).

'Fig. 1. Sequence of the C-terminal portions of GADs from plants.Comparison of the C-terminal amino acid sequences of rice GADs(OsGAD1 to OsGAD5) and petunia GAD (PhGAD). Identical and similar amino acids are indicated by black and grey boxes, respectively.Trp (W) and the cluster of Lys (K) present in the C-proximal region of petunia GAD, which are important for in vitro binding to CaM (Arazi et al., 1995), are indicated by asterisks and a thick line, respectively.The positions of two pseudosubstrate residues (E476 and 480) in PhGAD predicted by Yap et al. (2003) are indicated by hashes.Accession numbers: OsGAD1 (AB56060), OsGAD2 (AB56061),OsGAD3 (AK071556), OsGAD4 (AF377946.3), OsGAD5 (AK070858), PhGAD (L16797).'

• The concentration of GABA present in non-transformed rice calli was reproducibly within the range of 200–300 pmol mg�1 FW. When calli overexpressing GAD2 were analysed, the GABA concentration varied from 300 pmol to 2000 pmol mg�1 FW, an up to 6-fold increase in GABA concentration. Some of the cells overexpressing OsGAD2DC also had an extremely high concentration of GABA: clone nos 1 and 12 had about a 100-fold increase in GABA content relative to non-transformed cells(Fig. 4).

'Fig. 4. GABA levels in calli of a wild-type (wt) rice line and transgenic lines overexpressing either OsGAD2 or OsGAD2DC. Total free amino acids were extracted from callus cells by using the TCA method. The GABA content of each sample was determined with an automated amino acid analyser.'

• Data presented here show that the mutant GAD2 gene contributes to the extremely high level of GABA accumulated in cells. First, the mutant GAD2 enzyme had a much higher level of Ca2+/CaM-independent activity than the wild-type enzyme in vitro (Table. 1). Second,RT-PCR analysis indicated overexpression of the transgene at the RNA level (Fig. 5A). Third, western blot analysis using an anti-GAD2 antibody indicated a positive correlation between GAD2DC protein concentration and GABA accumulation (Fig. 5B).

'Fig. 5. Analyses of rice calli overexpressing OsGAD2DC. (A) RT-PCR analysis. Total RNAs were isolated by using Sepasol RNAI Super(Nacalai Tesque). Rice actin mRNA was used as a control. wt, wildtype calli. Seven independent rice cell lines (nos 1 to 12) overexpressed OsGAD2DC. GAD2 cDNA, OsGAD2 cDNA cloned into the p Bluescript vector with different concentrations as a PCR standard. (B)Western blot of the crude proteins extracted from callus cells. Rabbit anti-GAD2 antibody and a goat anti-rabbit IgG antibody alkaline phosphatase conjugate were used as primary and secondary antibodies,respectively. Samples no. 1 to 12 correspond to those in (A).2704 Akama and Takaiwa Down'

Evolution

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Labs working on this gene

• Department of Biological Science, Shimane University, Nishikawatsu 1060, Matsue, Shimane 690-8504, Japan
• Transgenic Crop Research and Development Center, National Institute of Agrobiological Sciences,Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan

References

• Akama K, Akihiro T, Kitagawa M, Takaiwa F. 2001. Rice(Oryza sativa) contains a novel isoform of glutamate decarboxylase that lacks an authentic calmodulin-binding domain at the C-terminus. Biochimica et Biophysica Acta 1522, 143–150.