JXB Advance Access originally published online on March 12, 2007
Journal of Experimental Botany 2007 58(7):1761-1770; doi:10.1093/jxb/erm034
© 2007 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.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER |
The glycine decarboxylase complex multienzyme family in Populus
Biotechnology Research Center, School of Forest Resources and Environmental Science, Michigan Technological University, Houghton, MI 49931, USA
* To whom correspondence should be addressed. E-mail: chtsai{at}mtu.edu
Received 11 December 2006; Revised 1 February 2007 Accepted 5 February 2007
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Abstract |
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In plants, the glycine decarboxylase complex (GDC) cooperates with serine hydroxymethyltransferase (SHMT) to mediate photorespiratory glycine–serine interconversion. GDC is also postulated to be an integral component of one-carbon (C1) metabolism in heterotrophic tissues, although molecular evidence in plants is scarce. An initial report of a xylem-specific isoform of GDC component H-protein, PtgdcH1, in aspen (Populus tremuloides Michx.) provided molecular evidence consistent with an important role for GDC in plant C1 metabolism. PtgdcH1 is phylogenetically distinct from the leaf-abundant photorespiratory PtgdcH3, but both isoforms restored GDC activity in a yeast H-protein knockout mutant, suggesting their functional equivalence. The Populus genome contains eight transcriptionally active GDC genes, encoding four H-proteins, two T-proteins, and single P- and L-proteins. The two Populus T-protein isoforms, PtgdcT1 and PtgdcT2, exhibited differential expression in leaves and xylem, similar to PtgdcH3 and PtgdcH1. In silico identification of AC elements in the promoters of xylem-abundant PtgdcH1 and PtgdcT2, as well as many lignin biosynthetic genes of Populus is consistent with a prominent role for GDC in methyl-intensive lignification during wood formation. The AC element is absent from Arabidopsis GDC promoters, and GDC expression has not been linked to secondary growth in this herbaceous annual. Taken together, the results suggest that the association of distinct H-protein and T-protein isoforms with photorespiration and C1 metabolism is a distinguishing feature of Populus, and may signify molecular adaptation of GDC to cope with the C1 demands of lignification in woody perennials.
Key words: One-carbon metabolism, lignin, photorespiration, Populus genome
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Introduction |
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The glycine decarboxylase complex (GDC) is a mitochondrial multienzyme system involved in the generation of one-carbon (C1) units for biosynthetic reactions in all organisms. It catalyses the oxidation of glycine in the presence of cofactor tetrahydrofolate (THF) to CO2, NH3, and 5,10-methylene-THF (CH2-THF), with a concomitant reduction of NAD+ to NADH (Kikuchi, 1973; Walker and Oliver, 1986). The CH2-THF is then transferred to a second molecule of glycine by mitochondrial serine hydroxymethyltransferase (SHMT) to yield serine (Kikuchi, 1973; Rao and Rao, 1982; Bourguignon et al., 1988). In photosynthetic tissues, GDC and SHMT comprise about half of the soluble mitochondrial matrix protein (Oliver et al., 1990), and cooperate to salvage photorespiratory glycine for regeneration of C3 units that can re-enter the Calvin cycle (Kisaki et al., 1971; Oliver, 1994). Glycine and serine catabolism also supplies C1 units for the biosynthesis of primary (e.g. nucleic acids and proteins) and secondary metabolites (Cossins and Chen, 1997). It has been estimated that the C1 demands of secondary metabolism can exceed those of primary metabolism by >20-fold in tissues rich in methylated secondary products, such as lignin (Hanson and Roje, 2001).
GDC is composed of four subunits, designated P-, H-, T-, and L-proteins, encoded by nuclear genes with N-terminal mitochondrial targeting pre-sequences (Oliver, 1994). The lipoamide-containing H-protein (gdcH) is a non-catalytic carrier protein that forms the core of the GDC and serves as a mobile co-substrate for the reactions catalysed by the other subunits (reviewed in Douce et al., 2001). The lipoate cofactor is covalently bound to a lysine residue of the H-protein (Merand et al., 1993; Pares et al., 1994), and the resultant lipoamide arm undergoes a catalytic cycle of reductive methylamination, catalysed by the P-protein (gdcP); methylamine transfer, catalysed by the T-protein (gdcT); and oxidative electron transfer, catalysed by the lipoamide dehydrogenase (LPD or L-protein), during the course of glycine oxidation (Douce et al., 2001). Genes encoding GDC subunits have been characterized in pea (Kim and Oliver, 1990; Kim et al., 1991; Bourguignon et al., 1992, 1993; Macherel et al., 1992; Turner et al., 1992a, b), Arabidopsis (Srinivasan and Oliver, 1992, 1995), and Flaveria (Bauwe et al., 1995; Kopriva and Bauwe, 1995; Kopriva et al., 1995). With the exception of LPD which also functions in other multienzyme complexes (Turner et al., 1992a; Vauclare et al., 1996), GDC components are reportedly much more abundant in green leaves than in heterotrophic tissues. The reported low abundance of GDC in non-photosynthetic tissues has spurred speculation that SHMT may function without GDC in non-photorespiratory C1 metabolism (Ireland and Hiltz, 1995). However, demand for C1 units in roots and leaves of etiolated seedlings (Walton and Woolhouse, 1986; Rogers et al., 1991) may be abnormally low in these model systems. In contrast, recent work in aspen (Populus tremuloides Michx.) underscores the importance of GDC in heterotrophic tissues where demand for C1 units is high, in large part, to support active secondary metabolism (Wang et al., 2004). PtgdcH1 encodes a protein that is phylogenetically distinct from photorespiration-associated H-proteins, including aspen homologue PtgdcH3, and is expressed at high levels in developing xylem and root tips, but weakly in green tissues (Wang et al., 2004). Photorespiratory and C1-specific H-protein isoforms are present in all three sequenced plant genomes (Arabidopsis, Oryza, and Populus), with poor sequence homology in all cases (i.e. 70–75% sequence similarity at the amino acid level). The low degree of sequence similarity may help explain why C1-specific H-protein isoforms have escaped detection by photorespiratory gdcH gene probes or antibodies in heterotrophic tissues.
The discovery of a C1-specific H-protein isoform in Populus (Wang et al., 2004) provides molecular evidence to support previous findings, based on 13C-NMR spectroscopy, that GDC and SHMT activities are coupled for the interconversion of glycine and serine in heterotrophic tissues (Mouillon et al., 1999; Hartung and Ratcliffe, 2002). In this study, PtgdcH1 and PtgdcH3 isoform function is demonstrated, and it is shown that spatiotemporally distinct isoforms occur for some but not all of the GDC subunits in Populus. Finally, phylogenetic analysis and gene expression are coupled with in silico promoter analysis to evaluate the relationship between GDC subunit co-evolution and GDC function in photosynthetic and wood-forming tissues of Populus.
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Materials and methods |
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Plant materials
Shoot apices, fully expanded mature leaves, young stems (internodes 10–14 from the top). and root tips (terminal 2–3 mm) were obtained from greenhouse-grown aspen. Developing secondary xylem and phloem were harvested from field-grown aspen on the campus of Michigan Technological University. Tissues were snap-frozen in liquid nitrogen and stored at –80 °C until use.
PCR-based cloning and DNA sequencing
Total RNA was extracted from various tissues according to Chang et al. (1993) and treated with TURBOTM DNase (Ambion, Austin, TX, USA). First-strand cDNA was synthesized using SuperScript II reverse transcriptase with oligo(dT) primers, according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). Gene-specific primers were designed for RT-PCR amplification of PtgdcH2, H4, T1, T2, P1, and LPD1 open reading frames (ORFs), based on the Populus genome sequence (v1.1) hosted at the DOE Joint Genome Institute (JGI), and GenBank Populus expressed sequence tags (ESTs). Additional gene-specific primers were designed to obtain 5'- and 3'-untranslated regions (UTRs) using the SMART RACE cDNA amplification kit (Clontech, Palo Alto, CA, USA). PCR products were purified using the UltraClean PCR Clean-up Kit (MoBio, Carlsbad, CA, USA) and cloned into the pCRII vector using the TA cloning kit (Invitrogen). Positive clones were sequenced fully from both directions using the CEQ dye terminator cycle sequencing Quick Start kit and the CEQ8000 Genetic Analysis System (Beckman Coulter, Fullerton, CA, USA). The sequences were deposited in GenBank under accession numbers EF015601, AY369261, and EF150632–EF150636.
Sequence analysis
cDNA sequences were translated and polypeptide parameters computed using the ExPASy Translate and Protparam tools (http://us.expasy.org/). N-terminal targeting peptides were predicted using TargetP v1.1, ChloroP v1.1 (Emanuelsson et al., 2000) and Predotar v1.03 (Small et al., 2004), and cleavage sites predicted by the TargetP program. Phylogenetic analysis was performed using the Molecular Evolutionary Genetics Analysis (MEGA) package v3.1 (Kumar et al., 2004) and unrooted trees were constructed using the neighbour-joining method. For in silico promoter analysis, a 1500 bp sequence upstream of the start codon of each gene was retrieved from the Populus genome sequence. For genes that contain introns in the 5'-UTR (PtgdcT2, PtHCT6, and PtCCR2) or a long (>1300 bp) 5'-UTR (e.g. PtCCoAOMT2), only sequences upstream of the predicted 5'-UTR were used. Putative regulatory elements were identified using the plant cis-acting regulatory DNA elements (PLACE) database (Higo et al., 1999), or the RSA (Regulator Sequence Analysis, http://rsat.scmbb.ulb.ac.be/rsat/) tools (van Helden et al., 2000).
Functional complementation in yeast
A yeast (Saccharomyces cerevisiae) knockout strain (YSC1021-546384) for the H-protein (Scgcv3) and its parental strain BY4739 (MAT
-leu2
0 lys20 ura30) were obtained from Open Biosystems (Huntsville, AL, USA). Deletion of Scgcv3 and its replacement by a kanamycin-resistant gene in the knockout strain (Wach et al., 1994) were confirmed by PCR. The ORFs of PtgdcH1, PtgdcH3, and Scgcv3 were PCR amplified, cloned into pCRII for sequence confirmation, and subcloned into the yeast expression vector pYES2-NTC (Invitrogen) under control of the GAL1 promoter. Plasmids were transformed to the knockout strain using the lithium acetate method (Gietz and Woods, 2002). Transformants were selected on uracil-deficient medium and screened by colony PCR using a combination of vector- and gene-specific primers. Positive colonies were grown alongside wild-type and knockout strains on glycine minimal medium, containing yeast nitrogen base, 2% (w/v) galactose to induce the GAL1 promoter, 1% (w/v) raffinose as carbon source, and 200 mM glycine as the sole nitrogen source. Auxotrophic requirements of the yeast strains were fulfilled by supplementing the medium with leucine, lysine, and uracil.
Quantitative real-time RT-PCR analysis
The relative transcript abundance of all GDC isoforms in various aspen tissues was analysed by real-time RT-PCR using the ABsolute QPCR SYBR Green Mix (Abgene, Rochester, NY, USA) and the Mx3000P Real-Time PCR system (Stratagene, La Jolla, CA, USA). Gene-specific primers (Table 1) flanking 240–260 bp amplicons in the 3'-UTRs were designed based on both cloned and JGI-predicted cDNA sequences and, whenever possible, GenBank Populus EST sequences. Three housekeeping genes (cyclophilin, elongation factor 1ß, and ubiquitin) were included. Each reaction was performed in duplicate with two biological replicates, using cDNA synthesized from 10 ng of total RNA. Since the housekeeping genes were expressed relatively strongly in all tissues, their PCR amplification was performed using 100-fold diluted cDNA in order to improve relative transcript abundance estimates of weakly expressed genes. The specificity of amplification was assessed by dissociation curve analysis at the end of each run using the MxPro software (Stratagene), and confirmed by cloning and sequencing of the PCR products. Relative transcript abundance of GDC genes was determined according to Tsai et al. (2006) using the geometric mean of the values for the housekeeping genes.
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Results |
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PtgdcH1 and PtgdcH3 complement a yeast null mutant
The mechanism of glycine cleavage by GDC is conserved among animals, bacteria, and plants (reviewed in Oliver, 1994; Douce et al., 2001). Functional equivalence of GDC subunits across species has also been demonstrated using reconstituted pea leaf H-, P-, and T-proteins with exogenous yeast LPD that exhibited active glycine decarboxylation activity (Walker and Oliver, 1986). We therefore sought to substantiate involvement of PtgdcH1 and PtgdcH3 in glycine catabolism by yeast complementation analysis using a knockout strain (YSC1021-546384) that is deficient in Scgcv3, the yeast H-protein homologue. The knockout strain is not viable if glycine is supplied as the sole nitrogen source (Fig. 1). Transformants expressing either PtgdcH1 or PtgdcH3 constructs grew as well as the gcv3-complemented cells and the wild-type strain on minimal glycine medium (Fig. 1). No complementation was observed with the vector alone (not shown). The results provide functional evidence that the divergent PtgdcH1 and PtgdcH3 isoforms are both involved in glycine catabolism.
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The Populus GDC family contains four gdcH, two gdcT, one gdcP, and one LPD gene
The complementation data point to spatiotemporal regulation, rather than functional divergence of PtgdcH1 and PtgdcH3 isoforms, as the mechanism for plant-wide coordination of C1 versus photorespiratory GDC activities. To determine whether spatiotemporally distinct isoforms are a common feature of GDC component proteins, we searched the Populus genome sequence (Tuskan et al., 2006) to identify all GDC family members. Four, two, two, and four genes encoding gdcH, gdcT, gdcP, and LPD, respectively, are annotated in the Populus genome (Table 2). PCR-based strategies (see Materials and methods) were used to clone two additional gdcH, designated PtgdcH2 and PtgdcH4, two gdcT (PtgdcT1 and PtgdcT2), one gdcP (PtgdcP1), and one LPD (PtLPD1) cDNAs from aspen. The annotated PtgdcP2 and PtLPD2 genes carry premature termination codons and find no specific hits in the GenBank Populus EST database of >369 000 entries (dbEST release 042806). In addition, RT-PCR of PtgdcP2 and PtLPD2 with multiple sets of primers and in different tissues yielded no amplification, suggesting that they represent pseudogenes (hence the ps suffix in PtgdcP2ps and PtLPD2ps). A truncated gdcH pseudogene (PtgdcH5ps) is also annotated in the poplar genome, in addition to the four gdcHs cloned (Table 2). Subcellular localization of the Populus GDC family was not determined, but all GDC isoforms, except for PtLPD3 and PtLPD4, contain the conserved mitochondrial targeting peptide cleavage signature R-X-X-A/S-S/T (Glaser et al., 1998), and are predicted to be localized in the mitochodria by the TargetP and Predotar programs. PtLPD3 and PtLPD4 are predicted to be localized in the plastids by the TargetP and ChloroP programs, and are most similar to plastidic LPD isoforms of Arabidopsis that are components of the chloroplastic pyruvate dehydrogenase complex (Lutziger and Oliver, 2000). Since GDC is localized exclusively in mitochondria, PtLPD3 and PtLPD4 were not analysed further, leaving only PtLPD1 with its predicted mitochondrial targeting sequence as the candidate GDC subunit. The Populus GDC family therefore comprises four gdcH, two gdcT, one gdcP, and one LPD gene.
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Of particular interest are the two new gdcH members, PtgdcH2 and PtgdcH4, that are paralogues of PtgdcH1 and PtgdcH3, respectively, derived from genome-wide gene duplications after the divergence of Arabidopsis and Populus (Tuskan et al., 2006). As such, the paralogous isoforms are highly similar to each other (97% amino acid similarity) but share only moderate homology (74% amino acid similarity) between groups. As described previously (Wang et al., 2004), the two groups are phylogenetically distinct, with PtgdcH3/4 clustering with other well-characterized photorespiratory (class I) isoforms, and PtgdcH1/2 forming a separate branch (class II) with isoforms from Arabidopsis (At2g35120), rice (Os2g07410, Os6g45670), and alfalfa (ABC75361 [GenBank] , Fig. 2). Analysis of GenBank loblolly pine (Pinus taeda) ESTs (>329 000 entries, dbEST release 042806) also uncovered two distinct gdcH contigs, represented by CX712701 [GenBank] and DT62519 (Fig. 2), suggesting evolutionary conservation of the two classes of H-proteins in gymnosperms as well. The two Populus gdcT isoforms have not been associated with genome-wide duplications (Tuskan et al., 2006), but are highly similar to each other, with 98% similarity at the amino acid level. They are most probably derived from an independent, segmental gene duplication event. PtgdcT1 and PtgdcT2 cluster together in the phylogenetic tree (Fig. 3), and are most closely related to the potato isoform.
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Real-time RT-PCR analysis
Expression of the entire GDC family in green tissues (mature leaves, shoot apices, and green stems) and non-photosynthetic tissues (xylem, phloem, and root tips) was assessed by quantitative real-time RT-PCR (Fig. 4). Consistent with previous findings, PtgdcH1 transcripts were most abundant in xylem and root tips, and low in the other tissues. Expression of its paralogue PtgdcH2 was two orders of magnitude lower. PtgdcH3 was expressed specifically in photosynthetic tissues as reported by Wang et al. (2004), with higher levels in leaves and shoot apices than stems. The transcript levels of the PtgdcH3 paralogue, PtgdcH4, were high in leaves, lower in shoot apices, and very low in the other tissues. Interestingly, the two paralogous T-proteins exhibited distinct expression. PtgdcT1 was primarily expressed in leaves while PtgdcT2 was most abundant in xylem, reminiscent of the differential expression of PtgdcH1 and PtgdcH3/4, respectively. PtgdcP1 expression was highest in leaves and xylem, lower in root tips, shoot apices and stems, and least in phloem. Compared with the other GDC genes, PtLPD1 expression was low overall.
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In silico analysis of GDC promoters
To identify putative cis regulatory elements coordinating GDC isoform expression, the promoter sequence 1500 bp upstream of the start codon of each GDC gene was retrieved from the JGI Populus genome sequence. The core (CTCACCAAC) of the AC element, originally identified in a parsley 4-coumarate:CoA ligase (4CL) promoter (Hauffe et al., 1991), was found in the promoters of the xylem-abundant PtgdcH1, PtgdcT2, and PtgdcP1, as well as the PtgdcH1 paralogue PtgdcH2 (Fig. 5). PtgdcT1, exhibiting lower levels of xylem/root expression, also contains a putative AC element, with one mismatch, in its promoter. In Arabidopsis, the AC element is present in the promoter of most of the lignin biosynthetic genes (Raes et al., 2003), and this was confirmed to be the case for Populus as well (Fig. 5). Interestingly, the AC element is not found in any of the Arabidopsis GDC promoters (not shown). The AC element also resembles the experimentally verified Myb-binding motif (MACCWAMC, where M=A/C and W=A/T) that is conserved in the promoters of phenylalanine ammonia-lyase (PAL), 4CL, and several flavonoid biosynthetic genes, including chalcone synthase, chalcone isomerase, and dihydroflavonol reductase (Sablowski et al., 1994). Taken together, these results support the idea that component proteins of the GDC, except photorespiration-associated PtgdcH3/4 and multifunctional LPD1, are coordinately regulated with phenylpropanoid genes in heterotrophic tissues to support methylation-intensive biosynthesis of lignin and flavonoids.
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Several cis elements implicated in light, phytochrome A (phyA), or circadian regulation were found in the aspen GDC gene promoters (Table 3), consistent with the reported light- (Srinivasan and Oliver, 1995; Vauclare et al., 1996) and phytochrome-mediated (Morohashi, 1987) regulation of GDC. The most prominent motifs were ‘sequences over-represented in light-induced promoters’ (SORLIPs), that are present in many phyA-induced Arabidopsis gene promoters (Hudson and Quail, 2003). The SORLIP1 (GCCAC) motif was found in the promoter of all GDC genes, except PtgdcH4, while the SORLIP2 (GGGCC) motif was identified in the promoters of PtgdcH1, PtgdcH3, and PtgdcH4. The GT-1 box (GGTTAA), originally identified in ribulose 1,5-bisphosphate carboxylase/oxygenase small subunit (rbcS) gene promoters and subsequently found in other circadian- and light-regulated promoters (Giuliano et al., 1988), was recognized in PtgdcH2, PtgdcH3, PtgdcH4, PtgdcT1, and PtgdcT2 promoters. The G-box core (ACGTG), also found in the promoters of rbcS and various other light-induced genes (Gilmartin et al., 1990; Hudson and Quail, 2003), was identified in the promoters of PtgdcH1, PtgdcT2, and PtLPD1. Interestingly, the evening element (AAAATATCT), conserved and over-represented in many circadian-regulated phenylpropanoid gene promoters (Harmer et al., 2000; Hudson and Quail, 2003), was found only in the PtgdcH1 promoter, consistent with its coordinated expression with lignin pathway genes. These results suggest that both photorespiration- and C1-associated GDC genes are regulated by circadian rhythms.
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Discussion |
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Genes encoding subunits of the photorespiratory GDC have been well characterized in pea and Arabidopsis (reviewed in Oliver, 1994; Douce et al., 2001). However, active expression of GDC genes in heterotrophic tissues had not been reported until recently, when a divergent H-protein (PtgdcH1) was isolated from lignifying xylem of aspen (Wang et al., 2004). It is shown in this study that aspen H-proteins differentially expressed in photosynthetic (PtgdcH3) and non-photosynthetic (PtgdcH1) tissues are functionally equivalent to the yeast isoform. The data suggest that whole-plant coordination of photorespiratory and heterotrophic GDC activities is under strict transcriptional control. Indeed, expression analysis of the aspen GDC family revealed another subunit, T-protein, that is also encoded by spatiotemporally distinct isoforms. In contrast, single genes for P- and L-proteins were expressed in all aspen tissues examined. It thus appears that expression, specifically of H-protein and T-protein isoforms, dictates the timing and magnitude of tissue-specific GDC activity in Populus.
Analysis of the Populus genome suggests that eight of the 10 annotated GDC genes were impacted by the Salicoid genome-wide duplication event (Tuskan et al., 2006). The PtgdcH1/2, PtgdcP1/2, and PtLPD1/2 pairs are located on homeologous chromosomes (Tuskan et al., 2006), and the PtgdcH3/4 pair is similar in age to the Salicoid event (GA Tuskan, personal communication). This genome-wide duplication pre-dates the divergence of Populus and Salix 60–65 million years ago (mya) according to the fossil record, or 8–13 mya according to molecular clock analysis, an estimation discrepancy probably resulting from the clonal growth habit of Populus (Tuskan et al., 2006). The only homologous pair, PtgdcT1 and PtgdcT2, not associated with the Salicoid event probably arose from an independent, segmental duplication 3–6 mya by molecular clock analysis (not shown), much more recently than the polyploidization. H-protein represents the most diverse and largest gene family of GDC. The photorespiratory and C1-specific H-protein isoforms are evolutionarily conserved in monocot, dicot, and gymnosperm taxa (Fig. 2), pointing to their divergence prior to the angiosperm and gymnosperm split >300 mya (Bowe et al., 2000). It appears that the ancestral GDC gene family structure (two H-, one P-, one T-, and one L-protein) is conserved among Populus, Arabidopsis, and rice, but lineage-specific genome-wide duplication events (Goff et al., 2002; Bowers et al., 2003; Paterson et al., 2004; Tuskan et al., 2006) along with subsequent chromosomal rearrangements have differentially shaped present-day family structures (Table 4). For instance, Arabidopsis contains duplicate genes for the photorespiratory H-protein but a single gene for the C1-associated isoform. The reverse is true for rice, while Populus retains duplicate genes for each. The remaining GDC gene families are conserved between Arabidopsis and rice, each with duplicate gdcP and LPD genes and a single gdcT gene. Populus, however, retains only one functional gene each for the P- and L-proteins.
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Evolutionarily, the GDC component genes exhibiting tissue-specific expression in aspen are the oldest (PtgdcH3/4 versus PtgdcH1/2) and youngest (PtgdcT1 versus PtgdcT2) homologous pairs in this woody dicot. The strong expression of PtgdcH3/4 in leaves is consistent with what is observed in pea and Arabidopsis; however, the abundance of PtgdcH1 transcripts in xylem (Fig. 4; Wang et al., 2004) and its further up-regulation during wood formation (Hertzberg et al., 2001) is not observed in Arabidopsis (see Supplementary data of Ko and Han, 2004; Ehlting et al., 2005). PtgdcH1 might have acquired xylem-specific expression, following its divergence from PtgdcH2, as an adaptive feature to cope with extensive stem lignification. PtgdcH2, with its weak expression, resembles more closely the Arabidopsis orthologue At2g35120. An emerging scenario is that the phylogenetically more ancient PtgdcH1 might have facilitated a functional recruitment of PtgdcT2 that is advantageous for lignification in wood-forming tissues. Associated with the gene duplication events is the evolution of AC element(s) in the promoters of xylem-expressing GDC genes, including the single-copy PtgdcP1. The AC element, implicated in transcriptional regulation of many lignin and flavonoid biosynthetic genes (Sablowski et al., 1994; Raes et al., 2003), is absent in the promoters of the photorespiratory PtgdcH3/4 and the multifunctional PtLPD1, and is not found in any of the Arabidopsis GDC promoters. Coordinated regulation of xylem-specific PtgdcH1 and PtgdcT2 with many phenylpropanoid genes, therefore, appears to have evolved as a tree-specific adaptation for methyl-intensive lignification in wood-forming tissues.
Light-dependent regulation of GDC is well established (reviewed in Cossins and Chen, 1997; Douce et al., 2001), and, as expected, several light- or phyA-responsive elements were found in the promoters of Populus GDC genes. For instance, all promoters but one (PtgdcH4) contain multiple copies of the SORLIP1 motif over-represented in light- and phyA-induced promoters (Hudson and Quail, 2003). Interestingly, the G-box core sequence and the evening element associated with light and circadian regulation were identified in the promoters of C1-specific GDC genes. Both elements were also found in the promoters of several lignin biosynthetic genes, including PtPAL4, Pt4CL1, PtHCT6, PtC3H3, PtCCoAOMT2, and PtCCR2 (data not shown). Using microarray analysis, many of the Arabidopsis lignin and flavonoid biosynthetic genes, and at least one Myb transcription factor (PAP1), were shown to be coordinately regulated in a circadian cycle, peaking before dawn (Harmer et al., 2000). Promoter analysis thus reveals at least two suites of regulatory machinery that differentially modulate GDC expression in response to circadian rhythm for photorespiration or phenylpropanoid metabolism.
The two mitochondrially localized LPD genes of Arabidopsis exhibit distinct expression in leaves (AtLPD1) and roots (AtLPD2), but are similarly expressed in all other tissues (Lutziger and Oliver, 2001). This has prompted the authors to suggest their differential, albeit interchangeable, association with the GDC and the -ketoacid dehydrogenase complexes, respectively. The single PtLPD1 gene, on the other hand, was similarly expressed in aspen leaves and roots, a pattern that was also reported for the single-copy LPD gene in pea (Bourguignon et al., 1992; Turner et al., 1992a, 1993; Vauclare et al., 1996). The disproportionately high level of PtLPD1 transcripts in roots in relation to other GDC genes is consistent with its presumed involvement in both glycine and -ketoacid metabolism, with the latter being particularly active in roots (Titus et al., 1968; Thelen et al., 1999; Lutziger and Oliver, 2001).
The serine–glycine cycle proposed by Mouillon et al. (1999) involves the concerted action of mitochondrial GDC and SHMT to produce serine that enters the cytosol as the primary C1 donor, via cytosolic SHMT, for methylation reactions. In both mammalian and non-photosynthetic plant cells, the cytosolic SHMT activity was shown to be dependent on folate cofactor availability, driven by cellular C1 demand, and intimately connected to the mitochondrial SHMT and GDC (Cossins and Chen, 1997; Mouillon et al., 1999). Consistent with this, one mitochondrial (PtSHMT3) and two cytosolic (PtSHMT1 and PtSHMT6) SHMT isoforms were also abundantly expressed in aspen xylem (M Rajinikanth and C-J Tsai, unpublished results). Lignification is one of the most C1-intensive processes in higher plants (Hanson and Roje, 2001), and the identification of xylem-preferential PtgdcH1, PtgdcT2, and PtSHMT1/3/6 isoforms now provides molecular support for glycine–serine interconversion as a major C1 source in non-photosynthetic tissues (Mouillon et al., 1999). The regulation of glycine levels in plants serves many important functions, associated with the biosynthesis of purines (Zrenner et al., 2006), glutathione (Kopriva and Rennenberg, 2004), glycine-betaine (Sakamoto and Murata, 2002), and other methyl-rich secondary metabolites. In mammals, glycine acts as an important neurotransmitter in the central nervous system for transducing sensory information (López-Corcuera et al., 2001). Recently, an important signalling role for glycine in the regulation of intracellular calcium gating has been demonstrated in Arabidopsis (Dubos et al., 2003). Considering that mitochondrial GDC-SHMT is proposed to be the only significant route of glycine catabolism in plants (Mouillon et al., 1999), regulation of tissue-adapted GDC warrants further investigation in light of its significance to both metabolism and signalling in photosynthetic and methyl-rich vascular tissues.
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Acknowledgements |
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This work was supported by the USDA McIntire-Stennis Cooperative Forestry Research Program.
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References |
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