JXB Advance Access originally published online on June 11, 2007
Journal of Experimental Botany 2007 58(10):2699-2707; doi:10.1093/jxb/erm120
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RESEARCH PAPER |
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
1Department of Biological Science, Shimane University, Nishikawatsu 1060, Matsue, Shimane 690-8504, Japan
2Transgenic Crop Research and Development Center, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan
* To whom correspondence should be addressed. E-mail: akama{at}life.shimane-u.ac.jp
Received 17 April 2007; Accepted 1 May 2007
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Abstract |
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Glutamate decarboxylase (GAD) converts L-glutamate to
-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. 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 Ca2+/CaM. However, a truncated version of GAD2 (OsGAD2
C) 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 OsGAD2C had about 6-fold and 100-fold the GABA content of wild-type calli, respectively. Regenerated OsGAD2C 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.
Key words:
Agrobacterium, amino acid, calmodulin, GABA, GAD, -aminobutyric acid, glutamate decarboxylase, overexpression, rice
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Glutamate decarboxylase (GAD) is an enzyme that catalyses the conversion of L-glutamate to
-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. An alteration in the GABA content of animal cells can result in severe diseases (Kriegstein, 2005). By contrast, although the role of GABA in plants remains controversial (Bouché and Fromm, 2004; Bown et al., 2006), various environmental stresses, such as oxygen, water, temperature, and mechanical stresses, prompt the accumulation of GABA in plant cells (Shelp et al., 1999). Because these stresses result in a concurrent increase in intracellular Ca2+ concentration (Bush, 1995), it has been hypothesized that the Ca2+/calmodulin pathway is involved in cross-talk with the GAD activation pathway. Baum et al. (1993) isolated a nuclear gene coding for GAD from petunia, in which the CaM-binding domain (CaMBD) was present at the C-terminal proximal region. Interestingly, in dicotyledonous plants, without any exception, the genes encoding putative GADs reported so far carry at their C-termini the Ca2+/CaMBD, which is lacking in bacterial and animal GADs (Turano and Fang, 1998; Yun and Oh, 1998; Zik et al., 1998; Yevtushenko et al., 2003; Liu et al., 2004; Oh et al., 2005). In in vitro studies in these reports it has also been found that increases in plant GAD activity are dependent upon Ca2+/CaM. Therefore, Ca2+ signalling and enhancement of GAD enzymatic activity would connect environmental stress and GABA production. Furthermore, Chen et al. (1994) reported that GABA synthesis is developmentally regulated in petunia. Recent studies indicate that the C-terminal extension of GAD plays an important role in normal growth and development of plants (Baum et al., 1996). Furthermore, biochemical analysis has shown that Ca2+/CaM induces dimerization of the C-terminal domain of petunia GAD (Yuan and Vogel, 1998; Yap et al., 2003). It has also recently been shown that the C-terminal extension of petunia GAD is required for oligomerization of the GAD complex and activation via Ca2+/CaM (Zik et al., 2006). Therefore, functional expression of plant GAD is closely regulated via the C-terminal extension, which interacts with Ca2+/CaM.
Two nuclear GAD genes have previously been identified in the rice genome from cDNA and genomic DNA libraries, each encoding a distinct GAD isoform, designated OsGAD1 and OsGAD2 (Akama et al., 2001). The coding regions of these two genes have significant homology to other known dicotyledonous GAD genes, suggesting that both genes are functional in rice. Furthermore, an in vitro CaM-binding assay using recombinant proteins that contain the C-terminal peptides of OsGAD1 and OsGAD2, respectively, overexpressed in E. coli, and that were purified with an affinity chromatography, revealed that the former protein binds CaM, whereas the latter protein does not. Because this is the only known example of a plant GAD that does not have a CaM-binding site and it is suggested that the GAD–CaMBD from petunia functions as autoinhibition in the absence of Ca2+/CaM binding (Snedden et al., 1996), it was necessary to determine whether GAD2 is actually Ca2+/CaM-independent with respect to enzyme activation and whether the C-terminal extension acts as an autoinhibitory domain. In order to address these questions, two recombinant enzymes, GAD2 and a GAD with a truncated version of the C-terminal extension (GAD2C) were analysed comparatively, with respect to their enzymatic activity. Moreover, transgenic lines of rice and tobacco were produced that overexpressed these rice GADs.
It was found that the truncated version of GAD2 had higher enzyme activity in vitro than its wild-type counterpart in the presence or the absence of Ca2+/CaM, and an extremely high level of GABA accumulated in GAD2C transgenic plant cells, showing that the C-terminal extension of GAD2 functions as a negative regulatory domain for enzymatic activity both in vitro and in vivo. Overexpression of GAD2C in plants caused an aberrant phenotype: transgenic plants had pale green, curled leaves, and were sterile.
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Plant materials and growth conditions
Oryza sativa L. cv. Kitaake was used in this study. For in vitro tissue culture, rice seeds were immersed in 70% (v/v) ethanol for 30 s, rinsed three times with distilled water, surface-sterilized in 50% (v/v) bleach (Kaoh, Japan) for 30 min, then rinsed five times with distilled water. The seeds were transferred onto 2N6 medium to germinate at 28 °C under light conditions of 35 µmol m–2 s–1 illuminated by white fluorescent tubes and 16/8 h light/dark for 2 weeks (Hiei et al., 1994). When calli appeared they were transferred to fresh 2N6 media until infection with Agrobacterium.
Plasmid construction
OsGAD2, a cDNA clone coding for GAD in rice (accession no. AB056061; Akama et al., 2001) was used in this study. First, two different recombinant plasmids for overexpressing GAD in E. coli were constructed using pET32a (Novagen) as follows. cDNA coding for wild-type OsGAD2 was used as a template for PCR-amplification of the N-terminal coding regions using a specific primer set (5'-CGTGCGTAGCCATGGTTCTG-3' and 5'-GATGTTCACGCACCGGTTCT-3', NcoI site underlined). The resulting fragment was cleaved using NcoI/XhoI, and the DNA fragment obtained (235 bp in length) was subcloned into the NcoI/XhoI site of pET32a to produce pET32a::N-terminal. The C-terminal coding region of GAD2 cDNA was digested using XhoI. The fragment was inserted into the XhoI site of the pET32a::N-terminal, yielding pET32a::GAD2. Finally, clones with sequences in the correct orientation were selected by using restriction enzyme analysis. A GAD construct with a truncated C-terminal (GAD2C, lacking 31 amino acid residues at the C terminal) was produced as follows. The C-terminal coding region of GAD2 (amino acid numbers 301–469) was PCR-amplified using two primer sets specific for GAD2C (5'-AGCTCATCTTCCACATCAAC-3' and 5'-AAAGCATGCCTAGGCGGTCCGGGCGGGG-CC-3'). The PCR fragment was cleaved with ApaI and cloned into the ApaI/EcoRV site of pBluescript to yield pBluescript containing the C-terminal coding region of GAD2. After the plasmid was confirmed to contain the truncated C-terminal region by sequencing, a KpnI/ApaI fragment of pET32a::GAD2, harbouring the N-terminal coding region of GAD2 and the pET32a vector-coding sequence, was cloned into the KpnI/ApaI site of the C-terminal coding region of GAD2 in pBluescript, producing pBluescript containing GAD2C. This plasmid was then cleaved with NcoI and EcoRI. The open reading frame of GAD2C was cloned into the NcoI/EcoRI site of pET32a to produce pET32a::GAD2C. For rice transformation, two different Ti plasmids were constructed. Ti plasmid pCAMBIA1302 (CAMBIA, Australia) was first cleaved with NcoI/PlmI. The two E. coli expression vectors produced were cleaved with XhoI, followed by filling-in, and then the vectors were cleaved with NcoI. These two OsGAD fragments were purified using agarose gel electrophoresis and were each cloned into the pCAMBIA vector prepared as described above.
Overexpression of GAD fusion proteins in E. coli
Two pET plasmids were each introduced into E. coli strain BL21 (DE3) (Novagen) for production of recombinant GAD proteins in bacteria. Induction and purification of fusion proteins were carried out in accordance with the method of Akama et al. (2001). Because recombinant proteins GAD2 and GAD2C have proteolytic traits, these proteins underwent a further purification step using anion-exchange chromatography (HiTrap Q FF, Amersham) in accordance with the manufacturer's protocol. The purity of the two recombinant proteins was confirmed by SDS-PAGE, and their concentration was determined as described by Bradford (1976).
Agrobacterium-mediated plant transformation
The constructed Ti plasmids were introduced into Agrobacterium tumefaciens strain GV3101 (Deblaere et al., 1985) via electroporation. Approximately 2-week-old calli cultured in 2N6 medium were used for infection with Agrobacterium, essentially using the procedure described by Hiei et al. (1994). The resulting calli were selected in medium containing 50 mg l–1 hygromycin B for 4 weeks. Clonal calli were subjected to quantitative analysis of amino acids. Other clonal calli were transferred to N6S3-CH medium (Hiei et al., 1994) to regenerate plants. In parallel, tobacco leaf discs (Nicotiana tabacum cv. Xanthi) were infected with the Agrobacterium strains. The transformation procedure was essentially as described by De Block et al. (1987).
In vitro GAD assay
A rice GAD enzymatic assay was performed by directly measuring GABA production after the GAD reaction as follows: 2 µg of recombinant protein was incubated in 150 µl of reaction mixture containing 100 mM of an adequate buffer (bis-tris–HCl for pH 7.0; pyridine-HCl for pH 5.0, pH 6.0), 1 mM DTT, 5 mM glutamate, 0.5 mM pyridoxal 5'-phosphate, 0.5 mM CaCl2, 0.1 µM bovine calmodulin (Sigma), 10% (v/v) glycerol at 30 °C. An aliquot of the reaction mixture was mixed with a 1/10 volume of 1 N HCl to stop the reaction. The mixture was directly applied to an automated amino acid analyser (JLC-300, JEOL, Japan), which was based on a HPLC separation and quantification with a lithium buffer system after derivatization with ninhydrin of amino acid samples.
RNA extraction and RT-PCR
Total RNA was isolated from rice calli by using Sepasol RNAI Super (Nacalai Tesque, Japan). Single-stranded cDNAs coding for GAD2C were synthesized from total RNA as the template using reverse transcriptase (SuperScript II, Gibco BRL) and the reverse primer 5'-TCCATCTCGTCCAGGGCCAT-3'. Double-stranded cDNAs were PCR-amplified with the reverse primer described above and the forward primer 5'-AGCTCATCTTCCACATCAAC-3'. The PCR products (439 bp) were analysed by 2% agarose gel electrophoresis. As a control, rice actin mRNA (accession no. AU312086) underwent the same RT-PCR procedure described above with the primer set 5'-TCCATCTTGGCATCTCTCAG-3' and 5'-GTACCCGCATCAGGCATCTG-3'.
Extraction of free amino acid and quantification of their content
Rice calli maintaining in hygromycin-containing 2N6 medium and transgenic rice and tobacco plants (To generation) cultured in tubes containing Murashige and Skoog basal salts (Sigma) for 1 month after regeneration were subjected to determination of GABA contents. Free amino acids were isolated from these materials by using trichloroacetic acid (TCA) as follows. Tissues were homogenized with 8% (v/v) TCA. After centrifugation, the supernatant was mixed with an equal volume of diethyl ether, and the procedure was repeated twice. The samples were dried by using a centrifuge concentrator (Tomy, Japan). The resulting samples were suspended in 200 µl of 0.1 N HCl to apply for an amino acid analyser as described above.
Protein isolation and western blot analysis
Total proteins were isolated from calli by using an extraction buffer (50 mM NaPO4, pH 7.0, 0.1 mM β-mercaptoethanol, 10 mM EDTA, 0.1% sodium N-laurosyl sarcosinate, 0.1% Triton X-100). A polyclonal antibody against recombinant OsGAD2 was prepared in Medical and Biological Laboratories (MBL, Japan). SDS–PAGE, electroblotting and immunodetection were essentially carried out as described by Akama et al. (2001).
Microscopic analysis
Longitudinal sections of tobacco stems were prepared by using a plant microtome (model MTH-1, MBL, Japan). Samples were stained with 0.1% toluidine blue O for observation by bright field microscopy (Nikon Biophot, Japan).
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Comparison of the GAD C-terminal extension in rice and petunia
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). The overall similarity among the C-terminal extensions of these plant GADs is not high. However, except for GAD2, which lacks CaM-binding ability (Akama et al., 2001), there exists a high proportion of identity between the rice GADs and the petunia GAD: several amino acids indicated in the figure are conserved among several GADs. In particular, the Trp (W) residue and the Lys (K) cluster, which contribute to hydrophobic and electrostatic interactions, respectively, are both critical for efficient binding of CaM to the petunia GAD-CaMBD (Arazi et al., 1995). Yap et al. (2003) reported that pseudosubstrate Glu residues (E476 and E480) are present in the N-terminal region of the PhGAD-CaMBD, and that these residues play a role in autoinhibition in the absence of Ca2+/CaM binding, and are frequently observed in other plant GADs. The Glu residues were conserved at corresponding positions in the C-terminal extension of rice GAD1, 3, 4, and 5, implying an autoinhibitory role for the C-terminal regions of these four GADs. Taken together, these results show that the typical features found in PhGAD are conserved in all rice GAD isoforms except GAD2. Although GAD genes have been reported from various plant species, OsGAD2 is the only one found so far that is unable to bind CaM.
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Effect of truncating the C-terminus of OsGA2 on in vitro enzymatic activity
In order to explore the role of the 31-amino acid C-terminal extension in rice GAD2, two expression vectors were constructed harbouring rice GAD cDNAs: one corresponded to wild-type OsGAD2 and the other was based on wild-type OsGAD2, but lacked the coding region for the C-terminal extension, OsGAD2
C. These plasmids were introduced into E. coli BL21 (DE3) for overexpression of the recombinant proteins. Each bacterial strain was incubated to induce recombinant protein expression with IPTG. Figure 2 shows the recombinant proteins after two rounds of purification by nickel-affinity chromatography and anion-exchange chromatography. The band sizes of these two recombinant proteins, calculated with a molecular size marker, were 78 kDa and 74 kDa for OsGAD2 and OsGAD2C, respectively, that corresponded well to the protein molecular mass elucidated from the sequence information and a tag sequence encoding the expression vector, i.e. 76.6 kDa for the former and 73.4 kDa for the latter. The resulting recombinant proteins were assayed for enzymatic activity in vitro (Table 1). Recombinant GAD2 had relatively low activity at pH 7.0, but activity increased as the pH became lower. As expected, Ca2+/CaM did not stimulate this enzyme. Because recombinant OsGAD1 which possesses an authentic CaMBD resulted in a 5-fold increase in its enzyme activity in the presence of Ca2+/CaM (data not shown), the possibility could be ruled out of the reaction system used in this study that is essentially Ca2+/CaM independent. The tendencies observed in OsGAD2 were also done in OsGAD2C. Comparing the activity between these two enzymes, C-terminal truncated OsGAD2 had higher activity than the wild-type enzyme at any pH tested and it was over 40-fold higher than the wild-type at physiological pH.
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Accumulation of GABA in calli overexpressing rice GADs
In order to determine whether the rice GAD2 enzyme behaved the same way in plant cells as in vitro, wild-type and truncated OsGAD2 cDNAs were inserted into the T-DNA region of the Ti plasmid, under the control of the cauliflower mosaic virus (CaMV) 35S promoter and the nopaline synthase gene terminator (Fig. 3). These plasmids were then introduced into a strain of Agrobacterium. Scutellum-derived rice calli were infected with these strains of Agrobacterium (with hygromycin selection) to establish independent transformed cell lines. Free amino acids were then extracted from the callus cells to determine the GABA content. As shown in Fig. 4, 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 OsGAD2
C 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). In order to confirm that the GABA accumulation was due to overexpression of OsGAD2C, RT-PCR and western blot analysis were performed. RT-PCR revealed that seven clones containing OsGAD2C had a higher level of OsGAD2C mRNA expression than non-transformed calli, and that OsGAD2C mRNA was more abundant in calli with an extremely high level of GABA (clone nos 1 and 12; Fig. 5A). Protein analysis using an anti-GAD2 antibody showed that the accumulated GABA (as shown in Fig. 4) is directly linked to overexpression of the OsGAD2C protein (Fig. 5B). Therefore, taken together with the results of the comparative in vitro enzyme assay of these two proteins shown in Table 1, it is speculated that overexpression of C-terminal truncated GAD2 generates a much higher level of GAD activity than overexpression of the wild-type enzyme, leading to accumulation of very high levels of GABA in cells. Crude proteins were extracted from callus cells to measure GAD activity. In the absence of Ca2+/CaM, calli that accumulated GABA had activity that was five times as high as wild-type calli (wild-type, GAD2C-1 and GAD2C-12 produced 655, 3483, and 3884 pmol of GABA min–1 mg–1 protein, respectively). Furthermore, as shown in Table 2, a comparison of the content of the free amino acids of wild-type and truncated GAD2 callus cells revealed that not only the GAD substrate Glu, but also many other amino acids are present at lower levels in the cells in which GABA is accumulated to a very high level.
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Characteristics of transgenic plants overexpressing OsGADs
From calli overexpressing the rice GAD2
C gene, transgenic shoots were regenerated. Amino acid content was determined for samples from three different tissues (leaves, roots, stems) of regenerated rice plants. Compared with samples from wild-type plants, GABA levels were increased in every tissue for transgenic rice and in stems and roots for tobacco (Table 3). Interestingly, plants overexpressing OsGAD2C had a dwarf phenotype, with pale, curled leaves, and were infertile, whereas plants overexpressing OsGAD2 had a normal phenotype (Fig. 6A). The same Agrobacterium strains were used for tobacco transformation. The same tendencies (higher GABA content, dwarfism, infertility) were observed in the OsGAD2C-overexpressing tobacco plants (Fig. 6B). Histological analysis of tobacco plant tissues showed that cells in the stem cortex were distinct in their size among wild-type and transformed plants: wild-type parenchyma cells were large and elongated, but they were smaller and shorter in GAD2C plants (Fig. 6C, D).
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Discussion |
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Functional analysis of a glutamate decarboxylase (GAD2) from rice, a monocotyledon, was carried out in this study. Many kinds of calmodulin-binding proteins (CaMBPs) have been found in plants (Reddy et al., 2002; Bouché et al., 2005), indicating that a major regulatory pathway for plant enzymes involves Ca2+/CaM (Yang and Poovaiah, 2003; McCormack et al., 2005). It is possible that plants have more varied CaMBPs than other eukaryotes, and GAD is one of the most abundant CaMBPs in plants (Ling et al., 1994). Arabidopsis and rice are commonly used as model plants, and the genomes of both have been sequenced, with the result that both plants have been found to possess five GAD genes each. Arabidopsis GADs all contain the typical CaMBD; however, the GAD C-terminal extension (e.g. the basic amphiphilic
-helix and conserved Trp and Lys-cluster), which is ubiquitously found in dicotyledonous plants, is not found in rice GAD2 (Fig. 1). Extensive database searches against all plant DNA sequences so far reported did not identify any putative GAD2 orthologue. Therefore, it is tempting to speculate that OsGAD2 is involved in a unique regulation pathway, which most probably occurs via a novel Ca2+/CaM-independent route.
In order to determine the role of the C-terminal extension of OsGAD2, a truncated version was constructed to allow a comparison of its activity with that of the non-truncated version. It was found that the recombinant OsGAD2 enzyme was not activated in the presence of Ca2+/CaM (Table 1). This is the first evidence of a Ca2+/CaM-independent GAD on its activation in plants. This phenomenon might occur because the C-proximal region of OsGAD2 functions as a negative regulatory domain, and because the C-terminus of this enzyme is not a target for Ca2+/CaM, no conformational change of this domain would take place in the presence of Ca2+/CaM to stimulate enzyme activity. Under the different pH conditions tested, as pH became lower, enzyme activity tended to increase, indicating that GAD activity is high under acidic conditions, and that GABA production is stimulated by reducing cytosolic pH (Crawford et al., 1994; Snedden et al., 1995). Truncation of the C-terminal extension of OsGAD2 conferred a 7–40-fold increase in enzyme activity relative to wild-type in the conditions at pH 5 to pH 7 and in the presence or absence of Ca2+/CaM (Table 1). As far as is known, this is the first instance in plants where removal of a limited C-terminal region has been found to contribute directly to drastic enhancement of enzyme activity. The CaMBD in CaMBP is known to overlap with an autoinhibitory domain (Hoeflich and Ikura, 2002). The extremely high levels of accumulated GABA observed in the present study led to the hypothesis that the C-terminal extension of OsGAD2 functions as a strong autoinhibitory domain, thus artificially truncating it would result in abolition of this autoinhibition. Alternative approaches to activating enzyme(s) via a CaMBD, such as monoclonal antibodies directed against the CaMBD and controlled proteolysis have been described (Rasi-Caldogno et al., 1993; Snedden et al., 1996). Zik et al. (2006) used petunia GADC9, in which nine C-terminal amino acid residues were removed (a similar construct to the one used in the present study) and found that in vitro enzymatic activity was lower than that of wild-type GAD, and that Ca2+/CaM-induced activation did not occur. GADC9 bound to Ca2+/CaM, but could not form a higher structure (
680 kDa), unlike wild-type GAD, thus causing lower activity and no induction of activity.
In the present study, the two constructs used for the in vitro assay were both introduced in the Ti plasmid vector via Agrobacterium (Fig. 3). After infection with Agrobacterium, calli were incubated on media containing hygromycin to establish clonal cell lines. GABA content was determined for eight or four clones per construct (Fig. 4). Calli overexpressing GAD2 had a 6-fold greater GABA concentration compared with wild-type calli. Some of the GAD2C-overexpressing clones produced had an extremely high concentration of GABA (a 100-fold increase relative to wild-type). To date, other than ourselves, three different research groups have produced transgenic plants (tobacco and rice) and bacteria into which a plant GAD gene lacking a portion of the CaMBD has been introduced (Baum et al., 1996; McLean et al., 2003; Park et al., 2005). In all cases, transgenes were expressed at high levels, and transgenic organisms contained about 10–50 times higher concentrations of GABA than controls. These values are higher than that found for OsGAD2 in the present study. When OsGAD2C was overexpressed in rice cells in the present study, a GABA concentration of 17–22 nmol mg–1 FW calli was measured (Fig. 4), corresponding to 34–46% of total free amino acids, almost equivalent to the values reported by Baum et al. (1996). In transgenic plants the content of the free amino acids was reduced, meaning that the increase in GABA concentration influenced the proportion of the other amino acids examined. It is noted that the substrate of GABA, Glu, was present at lower levels than in wild-type plants (Table 2). Due to the close connections between the metabolic pathways of Glu and most of the other amino acids, a decrease in Glu directly might influence the concentrations of many of the other amino acids.
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 GAD2C protein concentration and GABA accumulation (Fig. 5B). Fourth, crude protein extracts isolated from wild-type and GAD2C calli showed that, in the absence of Ca2+/CaM, GAD in GAD2C cells had a higher level of activity than in wild-type cells. Taken together, these data show without question that overexpression of GAD2C increases GAD enzyme activity and leads to extremely high levels of GABA in plant cells. By contrast, transgenic tobacco plants overexpressing truncated petunia GAD (PhGADC), which accumulated extremely high levels of GABA, had a GAD enzyme activity in crude protein extract that was Ca2+/CaM-independent, but at a lower level than that in wild-type plants (Baum et al., 1996), unlike the GAD2C discussed in the present study. Baum et al. concluded that abnormal regulation of GAD activity in the PhGADC-overexpressing plant was the main reason for the extremely high GABA level.
In a recent structural study it was found that E. coli GAD that does not contain the CaMBD forms a 330 kDa homohexamer in acidic conditions, which is important for enzyme activation (Capitani et al., 2003). Inatomi and Slaughter (1975) reported tissue-specific GAD complexes occurring in barley. Because the structure that the truncated GAD2 forms in the present study was not investigated, the possibility cannot be excluded that GAD2C forms a complex with endogenous GAD subunits, which would contribute to the activity of the complex in cells.
Overexpression of OsGAD2C conferred aberrant phenotypes in both rice (line 12) and tobacco plants (Fig. 6). Rice plants had small, curled, etiolated leaves, indicating some interference with the normal development of plastids. Transgenic tobacco plants with OsGAD2C had a typical dwarf phenotype, with a reduction in the size of parenchyma cells in the stem cortex. In both cases, GABA content in the plant tissues was higher than that of controls, indicating an imbalance of amino acids in cells, i.e. an increase in GABA and a decrease in many other amino acids would be involved in these phenotypes in rice as well as tobacco. However, in leaves of tobacco there was reproducibly little difference of GABA content among wild-type and transgenic lines, although there is no explanation for this. Interestingly, a very similar phenotype was observed by Baum et al. (1996), irrespective of the kind of GAD tested, suggesting that an extremely high level of GABA and a decrease in Glu at least has similar effects on development and morphogenesis in plants. Recently, functional genomic studies in Arabidopsis have revealed the importance of close regulation of the GABA shunt in plant development and stress responses (Palanivelu et al., 2003; Bouché et al., 2003, 2004).
Bown et al. (2006) proposed that plants produce GABA for defence against invertebrate pests. The transgenic rice plants generated in this study that show constitutive expression of GABA may thus acquire enhanced resistance against various natural pests. Because it was possible to establish transgenic rice plants that accumulated extremely high levels of intracellular GABA, by controlling this expression using tissue- or stage-specific promoters or stress-inducible promoters, many different kinds of dominant-negative rice mutants could potentially be produced, which would contribute to exploring GABA function. Also, because GABA plays a role in reduction of blood pressure (Takahashi et al., 1955), a molecular breeding approach involving the accumulation of GABA in the rice grain might lead to development of a ‘functional food’ strain of rice in the near future.
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Acknowledgements |
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This research was supported by grants for functional analysis of genes relevant to agriculturally important traits in the rice genome from the Ministry of Agriculture, Forestry and Fisheries of Japan (Green Technology Project IP-2004).
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References |
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