γ-aminobutyric acid (GABA) is a four-carbon non-protein amino acid that is produced from glutamate by glutamate decarboxylase (GAD) and is ubiquitously present in organisms ranging from bacteria to plants and vertebrates (Bouché and Fromm 2004). GABA is known to function as an inhibitory neurotransmitter in animals, but its role in plants still remains unclear. Interestingly, only plant GADs have a C-terminal extension that acts as a calmodulin binding domain (CaMBD) (Baum et al. 1993). Therefore, it is thought that transient accumulation of intracellular calcium in response to various stresses may activate GAD via interaction of the CaMBD with a Ca2+/CaM complex. We recently identified a cDNA encoding a novel GAD isoform in rice (OsGAD2) (Akama et al. 2001). Like the GADs from dicotyledonous plants, OsGAD2 has a C-terminal extension, but it has no ability to bind to Ca2+/CaM. In transgenic rice cells that overexpressed OsGAD2 lacking the coding region for the C-terminal 30 amino acids (OsGAD2ΔC), extremely high levels of GABA were accumulated (300 pmol/mg FW and 30,000 pmol/mg FW in wild-type and OsGAD2ΔC calli, respectively), whereas the remaining free amino acids analyzed were present at almost the same levels as in wild-type cells, except for a several-fold increase in Gln and a several-fold decrease in Glu, Asp and Asn. Given that GABA production is induced by stress (Bouché and Fromm 2004), and that GABA functions to guide the pollen tube in Arabidopsis (Palanivelu et al. 2003), it is probable that GABA plays a key role in physiological regulation, for example by acting as a phytohormone, in plant cells. In order to investigate this possibility, we compared the transcription patterns of wild-type and GABA-accumulating calli using oligo microarray analysis.
Plant binary vector pCAMBIA1302 (Cambia) or pCAMBIA1302 carrying CaMV35S::OsGAD2ΔC were introduced into fresh rice calli (Oryza sativa L. cv. Kitaake) that had been maintained in 2N6 solid media. The cells were then transferred to 100 ml flasks containing 20 ml of liquid 2N6 media to initiate a suspension culture. The flasks were placed on a shaker 120 rpm at 28 °C in the dark. To maintain uniformity of cell size in the rice suspension cultures, cells were passed through mesh (pore size: 1 mm) and were transferred to fresh medium every 7 days to repeat for one month. Then, two days after transfer of the cells to the same fresh medium, total RNAs were extracted from cultured cells from these two different lines. For expression profiling by microarray analysis, we used 800 ng of total RNA per microarray slide (Rice Oligo Microarray Kit, 22K; Agilent). To eliminate false-positive results, we performed a dye-swap--experiment using two arrays. The procedure used for the microarray analysis was essentially as described by Yazaki et al. (2000). After hybridization and washing, the arrays were scanned using an Agilent Microarray Scanner. For spots that showed more than 5.55-fold higher signal intensity (see below) after normalization in samples corresponding to the GABA-accumulating cells, a BLAST search was performed. cDNAs from a total of 60 spots were selected and Table 1 lists these putative genes with elevated expression according to function. First, OsGAD2 expression was up-regulated 5.55-fold. Endogenous and introduced GAD2 genes were both monitored in the present system, and the observed up-regulation was considered to be due to overexpression of the truncated form of OsGAD2. The observed up-regulation of ACC oxidase (AK058296), a key enzyme in the synthesis of ethylene, is to be expected, given that GABA induces accumulation of ACC oxidase mRNA (Kathiresan et al. 1998). These results suggest that expression patterns of the GABA-inducible genes could be faithfully monitored in this experiment. Of the functional classes of proteins shown in Table 1, the lipid and cell wall-related proteins are the most numerous, with ten different genes found to be up-regulated. Because previous reports have indicated that GABA controls the elongation of stems and pollen tubes (Kathiresan et al. 1998, Palanivelu et al. 2003), it is tempting to conclude that the GABA-triggered expression of genes related to cell wall structure is involved in this dynamic growth. The transcription factor, transporter and stress-response-related classes of proteins each comprised three or four genes that were up-regulated. In particular, expression of YABBY ortholog (AK070205), which is known to play an important role in leaf and flower formation in plants (Bowman et al. 2002), was up-regulated about 20-fold in the GABA-accumulating cells. To date, little is known about the relationship between GABA and regulation of gene expression, so in future studies it would be worth investigating the possibility that GABA functions as a signal molecule that induces expression of transcription factors such as YABBY.
Acknowledgment
We thank Dr. Y. Nagamura (Rice Genome Research Center, National Institute of Agrobiological Sciences, Tsukuba, Japan) for his help with carrying out the oligo microarray experiment.
References
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