JXB Advance Access published online on February 21, 2008
Journal of Experimental Botany, doi:10.1093/jxb/erm340
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© 2008 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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RESEARCH PAPER |
NAD(H)-dependent glutamate dehydrogenase is essential for the survival of Arabidopsis thaliana during dark-induced carbon starvation
Department of Biological Sciences, University of Alberta, Edmonton, AB T6G 2E9, Canada
* To whom correspondence should be addressed. E-mail: yo{at}ualberta.ca
Received 5 November 2007; Revised 3 December 2007 Accepted 4 December 2007
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Abstract |
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Interconversion between glutamate and 2-oxoglutarate, which can be catalysed by glutamate dehydrogenase (GDH), is a key reaction in plant carbon (C) and nitrogen (N) metabolism. However, the physiological role of plant GDH has been a controversial issue for several decades. To elucidate the function of GDH, the expression of GDH in various tissues of Arabidopsis thaliana was studied. Results suggested that the expression of two Arabidopsis GDH genes was differently regulated depending on the organ/tissue types and cellular C availability. Moreover, Arabidopsis mutants defective in GDH genes were identified and characterized. The two isolated mutants, gdh1-2 and gdh2-1, were crossed to make a double knockout mutant, gdh1-2/gdh2-1, which contained negligible levels of NAD(H)-dependent GDH activity. Phenotypic analysis on these mutants revealed an increased susceptibility of gdh1-2/gdh2-1 plants to C-deficient conditions. This conditional phenotype of the double knockout mutant supports the catabolic role of GDH and its role in fuelling the TCA cycle during C starvation. The reduced rate of glutamate catabolism in the gdh2-1 and gdh1-2/gdh2-1 plants was also evident by the growth retardation of these mutants when glutamate was supplied as the alternative N source. Furthermore, amino acid profiles during prolonged dark conditions were significantly different between WT and the gdh mutant plants. For instance, glutamate levels increased in WT plants but decreased in gdh1-2/gdh2-1 plants, and aberrant accumulation of several amino acids was detected in the gdh1-2/gdh2-1 plants. These results suggest that GDH plays a central role in amino acid breakdown under C-deficient conditions.
Key words: Amino acid metabolism, carbon starvation, glutamate, glutamate dehydrogenase
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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Carbon (C) and nitrogen (N) metabolism is fundamental to plant growth and development. The complex co-ordination of C and N metabolism is achieved by sensing the cellular C/N balance and regulating the transcription of many genes involved in photosynthesis, respiration, and N assimilation (Stitt and Krapp, 1999; Coruzzi and Zhou, 2001; Palenchar et al., 2004; Plaxton and Podestá, 2006). Metabolically, this intricate linkage involves the interconversion between keto-acids and amino acids (Lea and Ireland, 1999). Glutamate dehydrogenase (GDH; EC 1.4.1.2) catalyses in vitro the reductive amination of 2-oxoglutarate to glutamate as well as the oxidative deamination of glutamate to 2-oxoglutarate. Since the conversion of these metabolites constitutes the major link between C and N metabolism in plants, GDH appears to play a pivotal role in plant metabolism.
As to the genes encoding GDH in plants, there are at least two GDH genes in all plant species studied to date, each encoding distinct polypeptides, 
or β subunits of GDH (reviewed by Purnell et al., 2005). The random assembling of the and β subunits in the hexameric holoenzyme yields seven isoforms that can be visualized on a non-denaturing polyacrylamide gel (Loulakakis and Roubelakis-Angelakis, 1991, 1996). In Arabidopsis, two GDH genes have been cloned and characterized (Turano et al., 1997). Studies conducted on these two Arabidipsis GDH genes suggest that they are responsible for most of the GDH activity in Arabidopsis although two more genes were subsequently identified and annotated as putative GDH genes (Purnell et al., 2005; Fontaine et al., 2006). Also, the expression of the two Arabidopsis GDH genes has been shown to be dependent on the organ type, developmental stage, and nutritional conditions (Turano et al., 1997). Moreover, the mitochondrial localization of GDH has been demonstrated by subcellular fractionation (Turano et al., 1996, 1997). Immunohistochemical studies showed the specific localization of GDH in the mitochondria of phloem companion cells and on occasion in the cytoplasm of these cells (Dubois et al., 2003; Tercé-Laforgue et al., 2004a; Fontaine et al., 2006).
GDH had been originally viewed as the major ammonium assimilatory enzyme; however, Lea and Miflin (1974) demonstrated that the glutamine synthetase (GS)/glutamate synthase (GOGAT) cycle was the major route for ammonium assimilation in plants. Since then, the in vivo direction of the GDH reaction and hence the role of the enzyme has remained ambiguous despite much research targeted at addressing this question. Currently, it seems accepted that GDH is not essential for the primary N assimilation; however, the potential of GDH playing a role in ammonium assimilation under certain physiological conditions cannot be discarded (Dubois et al., 2003). For instance, the up-regulation of GDH in response to elevated ammonium levels suggests that GDH is important in the detoxification of ammonium by assimilating some of the excess ammonium ions (Tercé-Laforgue et al., 2004a, b). A recent study also reported that ammonium assimilation could be attributed to GDH under salt-stress conditions (Skopelitis et al., 2006). On the other hand, the catabolic role of GDH has been evident in several labelling experiments using carrot cell suspensions (Robinson et al., 1991), isolated potato mitochondria (Aubert et al., 2001) and the transgenic tobacco plants possessing higher GDH activity (Purnell and Botella, 2007). These results suggest that GDH functions to funnel the C skeletons of glutamate into the TCA cycle for energy production under C-limiting conditions. The strict regulation of GDH genes by the cellular C availability further supports this notion (Robinson et al., 1991; Melo-Oliveira et al., 1996; Masclaux-Daubresse et al., 2002; Restivo, 2004).
The analysis of mutant plants deficient for a specific enzyme is often critical in elucidating metabolic pathways and the in vivo function of the enzyme. GDH-deficient mutants have been isolated in maize (Magalhaes et al., 1990; Pryor, 1990) and in Arabidopsis (Melo-Oliveira et al., 1996; Fontaine et al., 2006). The maize GDH-deficient mutant, which had 10–15-fold decreases in root GDH activity, did not allow the determination of the anabolic role of GDH. Although a significant decrease in the rate of [15N]NH
assimilation was observed in the mutant roots, the inhibition of ammonium assimilation by a potent GS inhibitor, methionine sulphoximine (MSX), argued against the N assimilatory role of GDH (Magalhaes et al., 1990). In another study, feeding [15N]glutamate to the root system resulted in reduced [15N]glutamate catabolism in the maize gdh mutant, indicating the catabolic role of GDH (Stewart et al., 1995). However, these studies lacked an isogenic control line, making the results difficult to interpret. Conversely, the growth retardation of the Arabidopsis GDH1-deficient mutant (gdh1-1) grown on high inorganic N media suggested a non-redundant role of GDH for ammonium assimilation under the conditions of excess inorganic N (Melo-Oliveira et al., 1996). Nonetheless, considering the exceptionally high concentration of inorganic N used in their study, the suggested ammonium-assimilating role of GDH seems questionable under physiologically relevant conditions.
It should be noted that all the gdh mutants identified to date were not true GDH-null mutants because plants contain multiple GDH genes, each encoding either or β of the GDH subunits. Consequently, the elucidation of the role of GDH has awaited the isolation of a mutant lacking both of the subunits. Here, the isolation and characterization of the gdh1 mutant, the gdh2 mutant, and the gdh double knockout mutant in Arabidopsis are reported. The susceptibility of the double knockout mutant to dark-induced C-limiting conditions suggests the potential role of GDH in amino acid breakdown to supply the carbon skeletons of amino acids to the respiratory pathway.
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Materials and methods |
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Plant materials and plant growth conditions
The Columbia ecotype of Arabidopsis thaliana was used in all experiments. The T-DNA mutant lines, Salk_042736 (gdh1-2), SAIL_191_F12 (gdh1-3), SAIL_560_C10, Salk_102709, Salk_102710, Salk_102711 (gdh2-1), and SAIL_192_B08, were obtained from ABRC. The mutant plants homozygous for the T-DNA insertions were isolated from the obtained seed stocks using the PCR screening protocol described on the Salk Institute web page (http://signal.salk.edu.). Typically, plants were grown on potting mixture, MetroMix 220 (Scotts Co, Marysville, OH), at 22 °C and at approximately 150 µmol m–2 s–1 light (with white fluorescent light; measured at the soil surface or Petri dish surface levels) on a 16/8 h light/dark cycles unless otherwise indicated. For some experiments, plants were also grown on vertical plates containing sucrose-free 0.5x MS media in the similar light conditions as described above. The root tissues used in this study were harvested from 17-d-old plants grown on 0.5x MS media with 0.4% agar for 10 d and subsequently grown on the liquid 0.5x MS media for 7 d as described by Miyashita et al. (2007).
Dark treatment
To test the survival of plants under prolonged dark conditions, plants were initially grown under the normal light/dark cycle as described above and then transferred to darkness for the specified days. For this test, plants grown on soil for 25 d and the plants grown on vertical plates containing sucrose-free 0.5x MS media for 14 d were used. To assess the survival of the plants, the viability of the shoot apex was checked after a 5 d recovery period.
To study the response of GDH to the prolonged dark conditions, plants grown in the dark for up to 4 d were also used for RNA and protein extractions. For this purpose, 32-d-old soil-grown plants and 18-d-old plate-grown plants were used for the dark treatments. In addition, 18-d-old plate-grown plants of WT as well as gdh mutant lines were used for the dark treatment, and the above-ground tissues were harvested and used for metabolite extractions.
Growth experiments on controlled N media
To characterize the growth phenotype of the gdh mutants, plants were grown on controlled 0.5x MS media containing 1% sucrose and 0.6% agar with varying concentrations of inorganic N (either KNO3:NH4NO3=1:1 or NH4Cl only). To assess the ability of the plants to utilize glutamate as a N source, 5 mM glutamate was added to the controlled 0.5x MS media containing the varying amount of inorganic N (KNO3:NH4NO3=1:1). In all experiments, triplicates were used, and the fresh weight and the dry weight of 12 plants were measured after a 2-week-growth period.
RT-PCR and quantitative real-time RT-PCR analysis
RNA extraction, DNase treatment, and first-strand cDNA synthesis were performed as described by Miyashita et al. (2007). For the RT-PCR to demonstrate the absence of the corresponding transcripts in the gdh mutant lines, amplifications were performed with the synthesized cDNA as templates and with the following gene-specific primers; GDH1, 5'-ACCGAAGCTTTGCTTAACGA-3' and 5'-CTTTAAGCTCCCCAGCCTCT-3', GDH2, 5'-GGGAAATCGATTCAGGGTTT-3' and 5'-GCGACTCGGTTAACTCCAAG-3' and β-Tubulin (Kang and Singh, 2000).
For quantitative real-time RT-PCR, PCR reaction and the SYBR Green detection were performed on a light cycler ABI Prism 7000 (Applied Biosystems, Foster City, CA). Each reaction was performed in triplicate, and the specific amplification of the target was checked with a heat dissociation protocol (60–95 °C) after the completion of the PCR reaction. The gene-specific primers for real-time RT-PCR were designed to amplify 125–150 bp sequences found in the coding regions near the 3'-end of each gene; GDH1, 5'-CAAGGCTTTATGTGGGAGGA-3' and 5'-TGAGCCACACGATTAACACC-3', GDH2, 5'-GGATTCATGTGGGAAGAGGA-3' and 5'-GCGACTCGGTTAACTCCAAG-3', ACT2, 5'-TATCGCTGACCGTATGAGCA-3' and 5'-TTACCTGCTGGAATGTGCTG-3'. The amplified fragments were cloned into pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA). To quantify the transcripts of each gene, the copy numbers of each target were determined by the standard curve constructed using the diluted plasmid samples. The determined copy numbers of GDH1 and GDH2 were then normalized against the copy numbers of ACT2 transcripts.
Enzyme assays
Protein extractions and enzyme assays were performed as described by Turano et al. (1996, 1997), however, the assay was slightly modified to measure the activity using a 96-well ELISA plate reader, SpectraMax+ (Molecular Devices, Sunnyvale, CA). In this study, the reductive amination was routinely measured as the activity of GDH. The protein concentrations in each protein extract were determined with protein assay kit (Bio-Rad, Philadelphia, PA). For staining non-denaturing gels for GDH activity, proteins were separated by non-denaturing polyacrylamide gel electrophoresis using a Mini-Protein II system (Bio-Rad, Philadelphia, PA) as described by Turano et al. (1997). The polyacrylamide gels were then stained for the oxidative deamination activity of GDH as described by Turano et al. (1996).
Metabolites assays
For amino acid extractions, samples were ground to a fine powder in liquid N2 using a mortar and a pestle. Amino acids were extracted with a methanol:chloroform:water (12:5:3 by vol.) (MCW) mixture at 4 °C for 1 h. 150 µl water and 100 µl chloroform were then added to 500 µl of the MCW extract. After the phase-separation, the upper aqueous phase was transferred to a new tube and vacuum dried. The dried samples were redissolved in water and used for amino acids analysis. Amino acids were analysed by reverse-phase HPLC of their o-phthaldialdehyde derivatives as described by Sedgwick et al. (1991).
For soluble carbohydrate measurements, the metabolites were extracted with 0.6 M perchloric acids as described by Miyashita et al. (2007). The Glucose (HK) Assay Kit, the Fructose Assay Kit, and the Sucrose Assay kit (Sigma, St Louis, MO) were used to measure the levels of each soluble sugar in the extracts.
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Results |
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Arabidopsis GDH genes and their expression
Two GDH genes, GDH1 (At5g18170) and GDH2 (At5g07440), have been previously cloned in Arabidopsis (Turano et al., 1997). Phylogenetic analysis indicated that GDH1 and GDH2 encode the β and
subunits of the NAD(H)-dependent GDH, respectively (Purnell et al., 2005). A database search for additional GDH genes has identified a putative GDH (At3g03910) encoding a β subunit-like protein of NAD(H)-dependent GDH and another distinctive gene encoding NADP(H)-dependent GDH (At1g51720) (Purnell et al., 2005). Therefore, Arabidopsis has a total of four genes annotated as GDH, including two previously characterized GDH genes (GDH1 and GDH2), and the other two putative GDH genes. In the current study, only the two previously known GDH genes, GDH1 and GDH2, were investigated in depth since there had been no clear evidence supporting the contribution of the other GDH genes (At3g03910 and At1g51720) to the GDH activity in Arabidopsis. Also, in this paper, NAD(H)-dependent GDH is abbreviated simply as ‘GDH’ unless otherwise indicated, although plants appear to contain both NAD(H) - and NADP(H)-dependent GDH. The organ-dependent regulation of GDH genes has previously been shown in Arabidopsis (Turano et al., 1997). In this study, the expression of GDH1 and GDH2 was analysed in various organs using quantitative real-time RT-PCR. The results indicted that the two GDH genes were differently regulated depending on the organ types. Briefly, the transcripts detected in roots of the plants grown semi-hydroponically were mostly GDH2, whereas those detected in rosette leaves of soil-grown mature plants were mostly GDH1 (Fig. 1A). In other organs, both GDH1 and GDH2 transcripts were detected at relatively similar levels (Fig. 1A). In terms of GDH enzyme activity levels, the activity in the roots was exceptionally strong, whereas rosette leaves had the lowest activity levels among the organs tested (Fig. 1B). The banding pattern of the seven isoenzymes visualized on a non-denaturing gel also coincided well with the relative abundance of GDH1 and GDH2 transcripts; for example, the bands for isoenzyme 5–7 (the hexameric form of GDH is rich in the GDH2 protein) and those for isoenzyme 1–3 (the haxameric form of GDH is rich in GDH1 protein) were evident in roots and rosette leaves, respectively (Fig. 1C).
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Isolation and characterization of Arabidopsis gdh mutants
The T-DNA insertion lines (Alonso et al., 2003) obtained from the Arabidopsis Biological Resource Center (ABRC, Ohio State University, Columbus, OH) were screened in search of GDH-deficient mutants. Two mutant lines (gdh1-2 and gdh1-3), each containing a T-DNA insertion in GDH1, were isolated. One mutant line (gdh2-1), containing a T-DNA insertion in GDH2, was also recovered. The exact locations of the T-DNA insertions were confirmed by sequencing the PCR products flanking the insertion junctions (Fig. 2A, B). Subsequently, gdh1-2 and gdh2-1 were crossed, creating a double knockout mutant (gdh1-2/gdh2-1) defective in both GDH genes. RT-PCR analysis confirmed the absence of the corresponding gene transcripts in each mutant line (Fig. 2C). In gdh2-1 and gdh1-2/gdh2-1, the reduction in GDH enzyme activity was evident in both shoots and roots of the 17-d-old plants grown semi-hydroponically on liquid 0.5x Murashige and Skoog (MS) media (Fig. 2D). Typically, GDH activity was negligible in the double knockout mutant both in shoots and roots of the 17-d-old plants (Fig. 2D) as well as in the stems and leaves of soil-grown mature plants (data not shown). In addition, in-gel GDH staining detected only the single band of isoenzyme 1 (homohexamer of GDH1 protein) and that of isoenzyme 7 (homohexamer of GDH2 protein) in gdh2-1 and in the two gdh1 mutants, respectively (Fig. 2E). In gdh1-2/gdh2-1, no band was visible on the non-denaturing gel in shoots and roots (Fig. 2E), indicating that NAD(H)-dependent GDH activity in this line was not detectable. These results clearly demonstrate that the T-DNA insertions in each GDH gene eliminated the functional gene products in the corresponding mutant lines.
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An attempt to measure NADP(H)-dependent activity in the gdh mutant plants was also made. Since NADP(H) can be used as a cofactor by NAD(H)-GDH (Bhadula and Shargool, 1991; Turano et al., 1996), a similar reduction in NADP(H)-GDH activity was observed in the gdh mutants (data not shown). Nonetheless, a small but finite NADP(H)-dependent activity was detected in both shoots and roots of gdh1-2/gdh2-1 plants (data not shown) although the NAD(H)-dependent GDH activity was negligible in this line. Therefore, this small activity remaining in gdh1-2/gdh2-1 suggests the presence of the NADP(H)-dependent GDH in Arabidopsis. In contrast to the mitochondrial NAD(H)-GDH, plant NADP(H)-dependent GDH has been proposed to be localized in plastids, but little is known about plant NADP(H)-dependent GDH as only limited evidence has suggested the presence of this enzyme in higher plants (Lea and Thurman, 1972; Bhadula and Shargool, 1991; Turano et al., 1996, 1997).
Induction of GDH by prolonged dark conditions
The regulation of GDH in response to cellular C status has been demonstrated in several plant species, characterized by the induction of GDH under dark conditions (Melo-Oliveira et al., 1996; Turano et al., 1997; Mascaux-Daubresse et al., 2002; Restivo, 2004). In this study, the dark-induction of GDH was investigated at the transcript and protein levels in the above-ground tissues of young seedlings grown on sucrose-free 0.5x MS media and in the rosette leaves of soil-grown plants. For the enzyme activity assays and the non-denaturing gel staining, the gdh mutant plants were used along with WT plants to distinguish the induction patterns of the two GDH subunits.
In the above-ground tissues of the plate-grown seedlings, the transcriptional induction was observed for GDH1 after 1 d of the dark treatment (increased 1.75-fold), while a gradual repression was seen for GDH2 during the dark treatment (Fig. 3A). An increase in the enzyme activity was noticeable in WT plants during the first 2 d of the treatment (Fig. 3B). Similarly, gdh1-2 plants showed an increase in the enzyme activity after 1 d of the treatment, but was followed by a small decline at subsequent time points (Fig. 3B). In gdh2-1 plants, the enzyme activity was very low compared with that in WT plants, and the activity change during the dark treatment appeared minor at the all time points (Fig. 3B). In gdh1-2/gdh2-1 plants, GDH activity was nearly undetectable throughout this time-course experiment (Fig. 3B). The band pattern seen on the non-denaturing gel supported the changes in GDH activity in each line during the dark treatments (Fig. 3C).
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In the rosette leaves of soil-grown plants, a dramatic increase in the GDH2 transcripts was detected in response to the dark treatments (increased 6.1-fold after 1 d of the treatment and 24.3-fold after 4 d of the treatment; see Fig. 3D). On the other hand, GDH1 expression did not change significantly during the treatments (Fig. 3D). At the enzyme activity levels, an increase was observed for all lines until day 2 of the treatment, except for the double knockout mutant. However, these increases were followed by a subsequent enzyme activity reduction in the WT and gdh2-1 plants (Fig. 3E). The decrease in the activity in these lines may be attributed to the inactivation or degradation of the GDH1 protein under prolonged dark conditions. In gdh1-2 plants, the enzyme activity in rosette leaves was very low compared with that of WT, but the induction in the enzyme activity during the dark treatment continued throughout the time-course of the experiment (Fig. 3E). The apparent transcriptional up-regulation of GDH2 supports the large increase in the enzyme activity found in gdh1-2 plants. The non-denaturing gel staining further substantiated the increase of GDH2 protein during the dark treatments because the bands of isoenzyme 5–6 (rich in GDH2 protein) appeared in the dark-treated WT plants and the intensity of the band of isoenzyme 7 increased after the dark treatments in gdh1-2 plants (Fig. 3F). In gdh1-2/gdh2-1 plants, GDH activity was negligible at all time points (Fig. 3E) and no band was detected by non-denaturing gel staining (data not shown).
Conditional phenotypes of the gdh mutants
Under optimal growth conditions, all gdh mutant lines grew to maturity and did not display noticeable growth phenotypes at any developmental stages. To determine if there is any conditional phenotype of the gdh mutant plants, growth experiments under several different environmental conditions were performed.
To assess the catabolic role of GDH during C starvation, survival of the plants subjected to prolonged dark conditions was monitored. In this experiment, plants were initially grown under long-day conditions and subsequently transferred to darkness for various periods of time. The survival rate of the plants was scored after a 5-d recovery period (Figs 4A, 5A). When soil-grown plants were tested for survival under prolonged dark conditions, an increased susceptibility of gdh1-2/gdh2-1 plants to the dark treatment was observed (Fig. 4B). Typically, necrosis was observed at the tip of the rosette leaves in gdh1-2/gdh2-1 plants after 3 d of the dark treatment, and gdh1-2/gdh2-1 plants rarely survived the 6-d dark treatment (Fig. 4B). The single gdh mutants, (gdh1-2 and gdh2-1) behaved much like WT plants and the death of these plants was observed after 12 d of the treatment. The similar conditional phenotype of gdh1-2/gdh2-1 was also observed when the plants were grown on sucrose-free 0.5x MS media (Fig. 5B). Approximately 30% of gdh1-2/gdh2-1 plants survived the 4-d dark treatment and the survival rate decreased to 0% after 6 d of the treatment (Fig. 6A). To demonstrate that dark-induced C starvation was the cause of the conditional phenotype of gdh1-2/gdh2-1 plants, subsets of plants were transferred to 0.5x MS media containing 1% sucrose before the onset of the dark treatment. The sucrose supplementation rescued the conditional phenotype of gdh1-2/gdh2-1, although gdh1-2/gdh2-1 plants were still slightly more susceptible to dark treatments than WT plants (Figs 5C, 6B). This suggested that the death of the dark-treated gdh1-2/gdh2-1 plants were caused by C starvation. Therefore, this conditional phenotype of gdh1-2/gdh2-1 supports the hypothesis that GDH plays a catabolic role in supplying C source to the respiratory pathway for energy production under the conditions of C deficit. Moreover, the absence of this conditional phenotype in both of the single gdh mutants indicates that both of the homohexameric GDH isoenzymes (isoenzyme 1 and 7) catalyse glutamate breakdown under C-deficient conditions. Since it is likely that heterohexameric isoenzymes (isoenzymes 2–6) also catalyse the glutamate catabolism, both GDH1 and GDH2 proteins play a redundant role under C starvation.
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In order to give insights into the physiological role of GDH in other conditions, several growth experiments using the gdh mutants were performed. In one experiment, the concentrations of inorganic N (used at the ratio, NO
: NH= 2:1) were varied from 0 mM to 30 mM. This dose–response experiment did not result in any growth phenotypes in the gdh mutants (Fig. 7; Table 1). However, the supplementation of 5 mM glutamate as the alternative N source to these media revealed the growth retardation of gdh2-1 and gdh1-2/gdh2-1 plants (Fig. 7; Table 1). Because glutamate N has to be utilized to support plant growth under N-limiting conditions, the reduced growth of gdh2-1 and gdh1-2/gdh2-1 plants can be attributed to the reduced mobilization of glutamate N caused by the diminished GDH activity in these lines. In another experiment, plants were grown on media containing various levels of ammonium as the sole nitrogen source, but the growth of the plants on these media was very poor and no difference was observed between WT and the gdh mutant lines (data not shown). Also, the addition of GS inhibitor, MSX (as low as 10 µM), to the growth media resulted in seedling death at the very early stage of development in all lines (data not shown). In a separate experiment, it was confirmed that MSX does not inhibit in vitro GDH activity (data not shown). Thus, the results suggest that the involvement of GDH in primary N assimilation is unlikely. Overall, the results obtained through these various experiments suggest that GDH plays a negligible role in ammonium assimilation but support its role in glutamate catabolism.
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Metabolic changes under the conditions of dark-induced C starvation
To monitor the changes in the levels of metabolites under dark conditions, 18-d-old plants grown on sucrose-free 0.5x MS media were dark-treated for various days. The above-ground tissues of the dark-treated plants were then harvested and used for the metabolite analysis. Table 2 shows the major metabolic differences found between WT and gdh1-2/gdh2-1 plants (see Supplementary Table 1 at JXB online for the complete data set). In the untreated plants, the amino acid profiles of WT and gdh1-2/gdh2-1 plants were similar except for a few amino acids (e.g. ornithine). However, the levels of many amino acids differed substantially between WT and gdh1-2/gdh2-1 plants under dark conditions. For example, the levels of glutamate increased in WT but decreased in gdh1-2/gdh2-1 plants during the dark treatment (Table 2). Also, alanine and aspartate showed a constant decline in WT, but they accumulated in gdh1-2/gdh2-1 plants in the dark (Table 2). Several other amino acids (i.e. histidine, tyrosine, tryptophan, valine, phenylalanine, isoleucine, leucine, and lysine) showed increases under dark conditions, however, the magnitude of the increases were often different between WT and gdh1-2/gdh2-1 plants; in fact, the levels of tyrosine, valine, isoleucine, leucine, and lysine were significantly higher in gdh1-2/gdh2-1 than in WT plants (Table 2). In gdh1-2 plants, the amino acid profiles were relatively similar to WT plants, whereas the amino acid levels in gdh2-1 plants were more comparable to gdh2-1/gdh1-2 plants (see Supplementary Table 1 at JXB online). Combined with the GDH activity found in the above-ground tissues of the young seedlings (a large reduction in gdh2-1 but not in gdh1-2; see Figs 2D, 3B), GDH activity appears to be an important factor affecting the levels of many amino acids under prolonged dark conditions. In addition, dark-induced asparagine accumulation occurred in WT and all gdh mutant plants. However, the observed asparagine increase in this study was relatively smaller than those reported previously (Lam et al., 2003; Lin and Wu, 2004; Lea et al., 2007), possibly due to the young age of the samples used in this study (18-d-old seedlings grown on sucrose-free 0.5x MS media). For the levels of soluble carbohydrates (i.e. glucose, fructose, and sucrose), rapid decreases during the dark treatment were observed in both WT and gdh1-2/gdh2-1 plants (Table 2). This confirmed that the prolonged dark treatment mimicked the C-limiting conditions.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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In an attempt to elucidate the long-disputed role of plant GDH, the regulation of GDH in Arabidopsis was examined and the Arabidopsis gdh mutants was characterized, focusing mainly on the role of GDH under dark-induced C deficiency. Although GDH has been extensively studied using biochemical, genetic, and physiological approaches, previous attempts did not unequivocally define the in vivo role of GDH in plants. The difficulty in elucidating the function of GDH has been in part due to the lack of a true GDH-null mutant. In the current study, Arabidopsis mutants defective in GDH1 and GDH2 were isolated. The cross between the two mutants allowed us to recover the double knockout mutant whose GDH activity was reduced to negligible levels. In the strict sense, this double knockout mutant was not a true GDH-null mutant due to the potential expression of another putative GDH gene (At3g03910) and the presence of NADP(H)-dependent GDH activity. It is possible that this NADP(H)-dependent GDH activity allows the double knockout mutant to grow in the absence of NAD(H)-GDH activity. However, the use of this mutant proved useful to unravel the physiological function of plant GDH.
In order to understand the complex nature of GDH regulation in plants, the levels of GDH transcripts, enzyme activity, and isoenzyme patterns in several organs of Arabidopsis were examined. The results indicated that the expression of the two GDH genes was reciprocal in roots and rosette leaves (Fig. 1A). This finding supports the previous results that showed high levels of GDH1 mRNA in leaves but not in roots (Melo-Oliveira et al., 1996). On non-denaturing gels stained for GDH activity, root samples were rich in anodal isoforms (isoenzymes 5–7), whereas cathodal isoforms (isoenzymes 1–3) predominated in leaf samples (Fig. 1C; Turano et al., 1997; Fontaine et al., 2006). This band pattern difference between roots and leaves can be well explained by the organ-dependent expression of GDH1 and GDH2. The large reduction in GDH activity in the roots of gdh2-1 plants (Fig. 2B) and that in the rosette leaves of gdh1-2 plants (Fig. 3E) also supports the contrasting regulation of the two GDH genes in these tissues. It is now generally accepted that C availability is a key factor controlling the levels of GDH transcripts and enzyme activity (Sahulka and Lisá, 1980; Robinson et al., 1991; Melo-Oliveira et al., 1996; Athwal et al., 1997; Masclaux-Daubresse et al., 2002; Restivo, 2004). The C starvation triggered by prolonged dark conditions is thus the probable cause of the dark-induction of GDH because the up-regulation can be reversed by an externally supplied C source (Melo-Oliveira et al., 1996; Masclaux-Daubresse et al., 2002; Restivo, 2004). In this study, the dark-induction was apparent for GDH2 in rosette leaves of mature plants (Fig. 3D). Similarly, about a 20-fold increase in GDH2 transcripts and a significantly smaller increase in GDH1 transcripts have been reported previously (Turano et al., 1997). On the other hand, the dark-induction of GDH genes was distinctly different in the above-ground tissues of young Arabidopsis seedlings (Fig. 3A). This suggests that the developmental stage and/or the tissue type affects the inducibility of GDH genes during C starvation. Furthermore, the induction of Arabidopsis GDH2 has also been found during the senescence process (see Genevestigator Response Viewer; Zimmermann et al., 2004). This is consistent with the increase of GDH transcripts and activity in senescing leaves (Masclaux et al., 2000; Tercé-Laforgue et al., 2004b).
With regard to the physiological role of GDH, two conditional phenotypes of the gdh double knockout mutant, both of which are consistent with the catabolic role of GDH, were observed. It has been hypothesized that the glutamate catabolized by GDH enters the TCA cycle as an alternative C source during C starvation (Robinson et al., 1991, 1992; Melo-Oliveira et al., 1996; Aubert et al., 2001; Miflin and Habash, 2002; Dubois et al., 2003; Purnell and Botella, 2007). The increased susceptibility of gdh1-2/gdh2-1 plants to prolonged dark conditions and the phenotype rescue by externally supplied sucrose support this hypothesis. It should be noted that the GDH reaction itself might play a role in energy production as it produces extra NADH, which can be used to produce ATP through oxidative phosphorylation. Under the conditions of C deficit, protein degradation is a key process for recycling the cellular components. In fact, decreases in the soluble protein content were observed during the dark treatment in the crude enzyme extracts (data not shown), and there have been several reports of proteolysis induction during C starvation (Moriyasu and Ohsumi, 1996; Brouquisse et al., 1998; Thompson and Vierstra, 2005). The amino acids catabolized as an alternative energy source during C starvation are likely to derive from the protein turnover through an autophagic process, which is a non-selective bulk degradation of cyctoplasmic components (Klionsky and Emr, 2000; Thompson and Vierstra, 2005). Interestingly, Arabidopsis mutants deficient in autophagy exhibit increased susceptibility to C starvation, similar to gdh1-2/gdh2-1 plants (Hanaoka et al., 2002; Thompson et al., 2005). The phenotype of these mutants substantiates the importance of proteolysis and the subsequent amino acid degradation for the production of alternate energy source under C-limiting conditions. The second conditional phenotype, the reduced utilization of glutamate N observed in gdh2-1 and gdh1-2/gdh2-1 plants, supports the role of GDH in catalysing the oxidative deamination in vivo. The growth of the plants on the media containing glutamate as the sole N source also suggests that the ammonium ions released by the GDH reaction can be readily reassimilated within the plant. Supporting the breakdown of glutamate by GDH, the transgenic tobacco plants overexpressing the β subunit of GDH exhibited increased [15N]NH accumulation after [15N]glutamate feeding (Purnell and Botella, 2007).
It has been hypothesized that GDH assimilates ammonium to alleviate the toxic effects when the levels of ammonium exceed a certain threshold level (Tercé-Laforgue et al., 2004a, b; Skopelitis et al., 2006). In the current study, the gdh mutant and WT plants were shown to be equally susceptible to ammonium when the plants were grown on the media containing various levels of ammonium as the sole nitrogen source (data not shown). Thus, our results did not demonstrate the proposed role of GDH in ammonium detoxification. In ammonium-sensitive species, plant roots take up ammonium, but most of the ammonium ions remain unassimilated and are subsequently excreted from root cells (Britto et al., 2001). This futile transmembrane ammonium cycling can consume substantial amount of cellular energy, lowering the adenylate energy state; therefore, this has been suggested as the cause of ammonium toxicity (Britto et al., 2001). This model may explain the GDH induction by ammonium because GDH appears to be regulated by cellular carbohydrate levels and ultimately by cellular ATP levels (Athwal et al., 1997). However, the role of GDH under toxic levels of ammonium remains unclear. The response of the gdh mutant lines to ammonium stress should be studied further to uncover the role of GDH in coping with toxic levels of ammonium.
The observed metabolic changes under the dark-induced C deficiency were somewhat puzzling because both the increase in glutamate levels in WT and the fall of glutamate in gdh mutants during the dark treatment can easily be explained by the reductive amination of 2-oxoglutate by GDH (Table 2; see Supplementary Table 1 at JXB online). However, this result is not definitive proof that glutamate is produced by GDH since glutamate can be metabolized by diverse pathways in plants (Lea and Ireland, 1999; Forde and Lea, 2007). Considering the results of previous studies and the current study, an explanation for the observed metabolic changes by the catabolic role of GDH is contemplated. The two unexpected results that need to be addressed are the fall in glutamate levels and the significant accumulation of several amino acids in the dark-treated gdh1-2/gdh2-1 mutants (Table 2). The observed increase in the levels of several amino acids in the dark conditions may be due to the result of increased protein degradation under C-deficient conditions, and GDH may play a role in the breakdown of amino acids to provide alternate energy sources (Fig. 8). It should be noted that the conversion of glutamate to 2-oxoglutarate by GDH does not necessarily result in the decline in glutamate levels if 2-oxoglutarate is immediately used as the substrate for transaminations (Fig. 8). The exaggerated amino acid accumulation (i.e. alanine, aspartate, tyrosine, valine, leucine, isoleucine, and ornithine) in the gdh mutants during the dark treatment may have occurred due to the reduced breakdown of these amino acids in the mutants. For example, the initial step of the catabolism of branched-chain amino acids (valine, leucine, and isoluecine) occurs in mitochondria and is catalysed by aminotransferases using 2-oxoglutarate as an amino acceptor (Taylor et al., 2004). Assuming that GDH catabolize glutamate to 2-oxoglutarate, the reduced supply of 2-oxoglutarate in the gdh mutants may have inhibited the aminotransferase reaction, causing the branched-chain amino acids to accumulate in the gdh mutants. Taylor et al. (2004) also suggested that the catabolism of these branched-chain amino acids is important in providing the alternate oxidative phosphorylation energy source during C starvation. Similarly, the accumulation of alanine, aspartate, tyrosine, and ornithine in the gdh mutants could have been caused by the reduced conversion of these amino acids to the corresponding keto-acids since the aminotransferase reactions require a supply of 2-oxoglutarate. Supporting this idea, the other amino acids that did not over-accumulate in the gdh mutants appears degradable in pathways not requiring 2-oxoglutarate, with one exception for lysine, whose first step of degradation is catalysed by lysine-ketoglutarate reductase which requires 2-oxoglutarate (Tang et al., 1997; Zhu et al., 2002). On the other hand, the fall in glutamate in the dark-treated gdh mutants may have been caused by the malfunctioning of the GS/GOGAT cycle in the gdh mutants under carbon deficiency since this pathway requires energy input (ATP and NADH) and a constant supply of 2-oxoglutarate. Indeed, the GS/GOGAT cycle seems more severely affected in the gdh1-2/gdh2-1 than in WT plants, evidenced by the markedly low levels of glutamine found in gdh1-2/gdh2-1 plants after 4 d of the dark treatment (Table 2). Because the GS/GOGAT cycle is the major pathway of glutamate synthesis under normal conditions (Lea and Ireland, 1999; Miflin and Habash, 2002), the decline in glutamate levels in the gdh mutants may have been caused by the inhibition of the GS/GOGAT cycle during the dark treatment. Interestingly, the pattern of these metabolic changes observed under dark conditions was quite similar to the metabolic changes observed when GS was inhibited by MSX; for example, glutamine, glutamate, asparagine, aspartate, alanine, and serine levels declined, whereas valine, leucine, isoleucine, proline, and threonine levels accumulated most likely due to the enhanced protein degradation (Rhodes et al., 1986). Considering these possibilities, the overall metabolic phenotype of the gdh mutants seems better explained by the catabolic role of GDH. Overall, the obtained data suggest that GDH is a key enzyme in the breakdown of not only glutamate, but also several amino acids under the conditions of C deficiency (Fig. 8), and this proposed role of GDH is consistent with the increased susceptibility of gdh1-2/gdh2-1 to prolonged dark conditions.
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The proposed role of GDH in amino acid breakdown appears important for survival of essentially all cells/tissues under C-limiting conditions. However, plants often sacrifice some cells/tissues (in some cases, whole organs) to allow more important metabolically active cells/tissues to survive severe stress conditions. Considering this, the specific localization of GDH in the phloem companion cells (Dubois et al., 2003; Tercé-Laforgue et al., 2004a; Fontaine et al., 2006) could be a strategy of plants to allow phloem cells to survive and remain active under C-deficient conditions because heterotrophic meristems are dependent on the transport of respiration substrates through the phloem. Supporting this idea, more severe damage often occurred to rosette leaves after the dark treatments compared to stems or shoot apical meristems. The mitochondrial localization of GDH also correlates well with the proposed role of the enzyme in funnelling the carbon skeletons of amino acids into the TCA cycle during C deficiency. Interestingly, Takenaka et al. (2007) reported that the ability of GDH proteins to bind to RNA inhibits in vitro RNA editing. They reported that this inhibition was relieved by the addition of NADH, NADPH, and ATP, demonstrating the ability of GDH to bind to these molecules (Takenaka et al., 2007). It would be interesting to see if the binding of GDH to NADH and/or ATP regulates in vivo GDH activity in mitochondria, reflecting the co-ordination of GDH activity in accordance with the cellular energy state.
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Supplementary data |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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The complete data for the changes in amino acid contents in WT, gdh1-2, gdh2-1, and gdh1-2/gdh2-1 plants during the dark treatment is available at JXB online.
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Acknowledgements |
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The authors would like to thank Gary Sedgwick for the help with the HPLC analysis. This work was supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery grant to AGG.
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