JXB Advance Access originally published online on November 8, 2004
Journal of Experimental Botany 2005 56(409):73-80; doi:10.1093/jxb/eri020
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RESEARCH PAPER |
Co-ordinated gene expression of photosynthetic glyceraldehyde-3-phosphate dehydrogenase, phosphoribulokinase, and CP12 in Arabidopsis thaliana
Laboratory of Molecular Plant Physiology, Department of Biology, University of Bologna, Via Irnerio 42, I-40126 Bologna, Italy
* To whom correspondence should be addressed. Fax: +39 051 242576. E-mail: trost{at}alma.unibo.it
Received 22 June 2004; Accepted 25 August 2004
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Abstract |
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Photosynthetic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) interact in the chloroplast stroma through the action of the small peptide CP12. This supramolecular complex concurs with the light-dependent modulation in vivo of GAPDH and PRK activities. The expression patterns of several genes potentially involved in the formation of the complex have been studied. The genome of Arabidopsis thaliana includes seven genes for phosphorylating GAPDH isozymes, one PRK gene, and three genes for CP12. The expression of four GAPDH genes was analysed, i.e. GapA-1 and GapB for photosynthetic GAPDH of chloroplasts (NAD(P)-dependent), GapC-1 for cytosolic GAPDH, and GapCp-1 for plastid GAPDH (both NAD-dependent). A similar analysis was performed with PRK and two CP12 genes (CP12-1, CP12-2). The expression of GapA-1, GapB, PRK, and CP12-2 was found to be co-ordinately regulated with the same organ specificity, all four genes being mostly expressed in leaves and flower stalks, less expressed in flowers, and little or not expressed in roots and siliques. The expression of all these genes in leaves was terminated during prolonged darkness or following sucrose treatments, and their transcripts decayed with similar kinetics. At variance with CP12-2, gene CP12-1 appeared to be expressed more in flowers, it was totally insensitive to darkness, and less affected by sucrose. The expression of glycolytic GapC was strong and ubiquitous, insensitive to dark treatments, and unaffected by sucrose. GapCp transcripts were also found to be ubiquitous at lower levels, slowly decreasing in the dark and stable in sucrose-treated leaves. The co-ordinated expression of genes GapA-1, GapB, PRK, and CP12-2 is consistent with their specific involvement in the formation of the photosynthetic regulatory complex of chloroplasts.
Key words: Calvin cycle, light regulation, sugar sensing, supramolecular complexes, transcriptional control
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Calvin cycle enzymes exist at high concentration in the stroma and several of these enzymes have been suggested to be components of organized supramolecular complexes (Süss et al., 1993
; Gontero et al., 2002). The interaction between glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) is one of the best documented examples (Müller, 1972; Wara-Aswapati et al., 1980; Clasper et al., 1991; Lebreton and Gontero, 1999; Mouche et al., 2002). A specific explanation for this long-known multienzyme interaction came when the chloroplast peptide CP12 was discovered (Pohlmeyer et al., 1996) and its role as a linker between GAPDH and PRK demonstrated (Wedel and Soll, 1998; Scheibe et al., 2002; Graciet et al., 2003). CP12 is a small peptide present in chloroplasts of higher plants and green algae and in cyanobacteria. It contains two pairs of fully conserved cysteines which can form disulphide bridges. Following oxidation of both cysteine pairs, the in vivo supramolecular complex appears to include two PRK dimers linked to two GAPDH tetramers by means of a CP12 dimer (Wedel and Soll, 1998; Graciet et al., 2003, 2004).
Photosynthetic GAPDH consists of two subunits, GapA and GapB. The latter is 30 amino acids longer than GapA, but apart from this C-terminal extension (CTE) the two subunits are 80% identical (Brinkmann et al., 1989). The CTE is implicated in kinetic enzyme regulation (inhibition in the dark) and aggregation of GAPDH into high-molecular weight polymers such as A8B8 (Zapponi et al., 1993; Baalmann et al., 1996; Li and Anderson, 1997; Sparla et al., 2002). Since the C-terminal portion of CP12 is co-linear in sequence with the last 20 amino acids of the CTE, it has been proposed that GapB was evolutionary derived by fusion between GapA and part of the CP12 (Pohlmeyer et al., 1996) and both domains may interact with GAPDH with similar effects. Less is known of CP12 binding to PRK. There is evidence from Chlamydomonas that complex formation with PRK requires a previous interaction between CP12 and GAPDH (Graciet et al., 2003).
Both GAPDH and PRK are kinetically activated by light in vivo. The activity of both enzymes is regulated by thioredoxins (Wolosiuk and Buchanan, 1978; Brandes et al., 1996; Sparla et al., 2002) and GAPDH activity is also dependent on metabolites including NAD(P)(H) and BPGA (Pupillo and Giuliani Piccari, 1975; Trost et al., 1993; Baalmann et al., 1995). Moreover, both GAPDH and PRK are inhibited when embedded in the supramolecular complex involving CP12 (Scheibe et al., 2002). During dark-to-light transitions, GAPDH and PRK activation in vivo seems to depend on the accumulation of reduced thioredoxins and metabolites in the chloroplast stroma (Buchanan, 1980; Scagliarini et al., 1993; Baalmann et al., 1994). Besides activating freely soluble GAPDH and PRK, these effectors promote supramolecular complex dissociation (Wara-Aswapati et al., 1980; Clasper et al., 1991; Wedel and Soll, 1998; Scheibe et al., 2002).
GAPDH and PRK gene expression has been investigated (Shih and Goodman, 1988; Raines et al., 1989; Yang et al., 1993; Lemaire et al., 1999). The expression of GapA and GapB is co-ordinately regulated by light at the transcriptional level and several cis acting elements and cognate binding factors have been identified (Chan et al., 2002). Both cryptochrome and phytochrome are involved in the light signal (Conley and Shih, 1995). GapA and GapB were reported to be induced by anaerobiosis but unaffected by sucrose in arabidopsis leaves or tobacco callus (Shih and Goodman, 1988; Yang et al., 1993). PRK expression is also regulated by light under the control of the circadian clock and down-regulated during senescence (Raines et al., 1989; Lemaire et al., 1999). The existence of a supramolecular complex including GAPDH, PRK, and CP12 in chloroplasts also suggests that the expression of the relevant genes might be co-ordinately regulated. The expression pattern of five genes of A. thaliana potentially involved in the formation of the GAPDH/CP12/PRK complex (GapA, GapB, PRK, and two CP12 genes; no data are available for the latter), and two further genes coding for GAPDH isozymes unlikely to be involved in the complex (cytosolic GapC and plastid GapCp) are reported here. Results indicate that the expression of GapA-1, GapB, PRK, and one CP12-2 genes are co-ordinately regulated under manifold conditions, whereas different regulatory mechanisms may govern the expression of the other genes tested.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Plant material and treatments
Arabidopsis thaliana plants (ecotype Columbia) were grown on a sterile mix of humus/perlite (3:1, v:v) at 22 °C under 14/10 h light/dark cycle in the growth chamber.
For dark treatments, 8-week-old plants were placed in the dark for up to 5 d at 22 °C. For sugar treatments, leaves were harvested from 6-week-old plants after 16 h adaptation to continuous light or continuous dark. Detached leaves were put in Petri dishes containing either bidistilled water or solutions of sucrose, glucose or sorbitol (200–300 mM) and incubated under gentle shaking for up to 72 h at 22 °C, either in continuous light or continuous dark. To prevent contaminations, solutions were renewed every 24 h.
Gene cloning
A. thaliana cDNAs corresponding to plastidial and cytosolic GAPDH isoforms have been cloned by RT-PCR. Total RNA was extracted from A. thaliana plants (Nawrath and Mètraux, 1999) and DNA complementary strands were synthesized using M-MLV reverse transcriptase (Sigma) and Oligo dT(15) primers (Promega). Specific DNA primers (see below), derived from genomic sequences (At3g26650 for GapA-1, At1g42970 for GapB, At3g04120 for GapC-1, and At1g79530 for GapCp-1; Table 1), were used to clone GAPDH fragments into a p-Drive cloning vector (QIAGEN PCR cloning kit). Resulting recombinant plasmids were sequenced to confirm the nature of cloned fragments. Plasmids containing the cDNA sequence for CP12-1 (U17174
[GenBank]
), CP12-2 (U10360
[GenBank]
), and PRK (C63669
[GenBank]
) were kindly provided by Arabidopsis Biological Resource Centre, Ohio State University.
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RNA extraction and northern blot analysis
Total RNA was extracted from different plant organs according to Nawrath and Métraux (1999)
. Leaves, stalks, flowers, and siliques were harvested from 6-week-old plants and immediately frozen in liquid nitrogen. Total RNA from roots was obtained from 4-week-old plants grown on sterile MS agar pots at 22 °C with a 14/10 h light/dark cycle. RNA pellets were resuspended in H2O-diethylpyrocarbonate. Concentration and purity were spectrophotometrically determined. For northern blot analysis 10 µg of total RNA were heat denaturated, separated by electrophoresis on a 6% formaldehyde-1% agarose gel, and transferred by capillarity to a nylon membrane (Hybond-N+, Amersham Pharmacia Biotech). Electrophoresis buffer and the blotting buffer consisted of 20 mM MOPS, 5 mM Na-acetate, pH 6.0 and 1.5 M NaCl, 150 mM Na-citrate, pH 7.0, respectively. Blotted membranes were hybridized with about 50 ng of gene-specific probes obtained by PCR with different plasmids as template and specific primer sets were designed as follows: GapA-1: up 5'-AGGTGGCCATTAATGG-3' and down 5'-TATCATACCAAGCAATCACC-3'; GapB: up 5'-TTAGGTGTTGGCATGGT-3' and down 5'-CTCGTTATCATACCAAGC-3'; GapC-1: up 5'-ATGGCTGACAAGAAGATTAG-3' and down 5'-ACATGTGGACGATCAAGTC-3'; CP12-1: up 5'-ATGACAACCATAGCTGCAGC-3' and down 5'-TTAATTATCATAAGTACGACACTC-3'; CP12-2: up 5'-ATGGCAACTATAGCTACTGGTC-3' and down 5'-TCAGTTGTCGTAAGTACGGCAC-3'; GapCp-1: up 5'-ATCGAGGTTGTAGCAGTC-3' and down 5'-GTCATACCAGGAGACAAG-3'; PRK: up 5'-ATGGCTGTCTCAACTATCTAC-3' and down 5'-TTAGGCTTTAGCTTCTGCACG-3'. PCR programs included a hot start at 95 °C for 5 min followed by 30 cycles of 1 min at 95 °C plus 1 min at 48 °C (GapA-1), or 54 °C (GapB, GapC-1, GapCp-1, and PRK) or 58 °C (CP12-1 and CP12-2), and finally 72 °C for 1.5 min.
Probes were radiolabelled with the kit Ready-To-Go DNA labelling beads (Amersham Pharmacia Biotech) according to the manufacturer's instructions. Hybridizations were performed overnight at 55 °C in 1% w/v BSA, 1 mM EDTA, 0.5 M sodium phosphate buffer pH 7.2, and 7% w/v sodium dodecylsulphate.
Enzyme activity assays
Enzyme activities were assayed on fresh leaf homogenates, obtained as for western blotting experiments. Both GAPDH and PRK activities were assayed by following the oxidation of NAD(P)H at 340 nm (
NAD(P)H=6.23 mM–1) in an Uvikon 941 plus spectrophotometer (Kontron). NAD(P)H-GAPDH activity assay was performed at 25 °C in a reaction mixture containing 50 mM TRIS–HCl pH 7.5, 1 mM EDTA, 3 mM 3-phosphoglyceric acid, 5 mM MgCl2, 0.5 mM KCN, 2 mM ATP, 5 U ml–1 3-phosphoglycerate kinase, and 5 mM dithiothreitol. Reactions were started by the addition of 0.2 mM NAD(P)H. Activated GAPDH activity was measured after incubation of the sample for 5 min at room temperature in the reaction mixture (without NAD(P)H).
Fully activated PRK activity was assayed at 25 °C after incubation for 30 min at room temperature in the assay mixture including 50 mM TRIS-HCl pH 7.5, 1 mM EDTA, 40 mM KCl, 10 mM MgCl2, 0.5 mM KCN, and 5 mM DTT. Baseline was acquired after addition of 2 mM ATP, 2.5 mM phosphoenolpyruvate, 5 U ml–1 pyruvate kinase, 6 U ml–1 lactate dehydrogenase, 0.2 mM NADH, and the reaction was initiated by the addition of 0.5 mM ribulose-5-phosphate.
Protein content was determined according to Bradford (1976).
Activity data were analysed with the ANOVA procedure (CoStat, CoHort Software, Monterey, CA) for the existence of significantly different means (P <0.05).
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Results |
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In silico search and molecular cloning of members of GAPDH and CP12 gene families, and of PRK from Arabidopsis thaliana
The genome of Arabidopsis thaliana contains seven nuclear genes of the family of phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Table 1). Photosynthetic GAPDH isoforms catalysing the reductive step of the Calvin cycle and using either NADPH or NADH as coenzyme are encoded by three genes, all including an N-terminal transit peptide for plastid location. Two of these (GapA-1 and GapA-2) code for GapA subunits and are 93% identical in terms of amino acid sequence, the third one codes for a unique GapB subunit. Cytosolic NAD-specific GAPDH, involved in glycolysis and gluconeogenesis, is encoded by two closely related genes in A. thaliana (GapC-1, GapC-2; Table 1). Finally, the primary sequence of a third type of GAPDH closely resembling GapCp of Pinus sylvestris (Meyer-Gauen et al., 1998
) features an NAD-specific isozyme located in A. thaliana plastids. It is encoded by two similar genes (GapCp-1 and GapCp-2; Table 1) both including putative transit peptides. Based on the relative abundance of ESTs in the TAIR database (http://www.arabidopsis.org; Table 1) it was observed that GapCp-2 is much less expressed than GapCp-1. All NAD-specific GAPDH isozymes are less than 50% identical to photosynthetic counterparts. In this work, an RT-PCR approach has been followed to clone the entire coding sequences of GapA-1, GapB, GapC-1, and GapCp-1 of A. thaliana. Sequencing of the resulting cDNA clones confirmed the coding sequences predicted by in silico analysis of respective genes.
The PRK of A. thaliana is encoded by a single gene, while three genes (CP12-1, CP12-2, CP12-3; Table 1) have been identified for CP12. The three CP12 genes include putative N-terminal transit peptides and are predicted by TargetP software (Emanuelsson et al., 2000) to encode plastid proteins. CP12-1 and CP12-2 encode peptides which are 86% identical between them and 78–80% identical to CP12 of spinach (Pohlmeyer et al., 1996). Within the CP12 gene family, CP12-3 has the lowest degree of conservation with CP12 of spinach (47% identical in amino acids) and, as judged from the relative abundance of ESTs in TAIR, it seems to be very little expressed in A. thaliana (Table I). The cDNAs for PRK, CP12-1, and CP12-2 were cloned and sequenced and their in silico predicted sequences were confirmed.
Organ-specific expression
The similarity of expression levels of GapA-1, GapB, CP12-1, CP12-2, and PRK genes in different plant organs has been investigated by northern blot analysis (Fig. 1). High expression levels of all these genes have been detected in photosynthetic organs (leaves and stalks) while transcripts were hardly detectable in roots and siliques. In flowers, the expression of GapA-1, GapB, PRK, and CP12-2 was very low, but the expression of CP12-1 was higher and almost approached the expression in leaves. GapC-1 was abundantly expressed in all organs, with higher transcript levels in leaves, stalks, and siliques. The expression of GapCp-1 was generally low but ubiquitous.
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Light-dependent expression
The expression of photosynthetic genes is regulated by light (Terzaghi and Cashmore, 1995
). Accordingly, total RNA was extracted from leaves of 8-week-old plants either grown under a normal light–dark regime or after transfer to total darkness for 5 d (Fig. 2). Northern blot analysis showed the expression of Calvin cycle genes GapA-1, GapB, and PRK to be declining after 24 h in the dark, and to drop to very low levels during the following days. Interestingly, CP12-2 was also down-regulated in darkness with similar kinetics, whereas the amounts of CP12-1 transcripts failed to change until the end of dark treatment (5 d). GapC-1 expression was also independent of the light/dark conditions. The expression of GapCp-1 was inhibited in the dark, although a decrease of the transcripts was slow and GapCp-1 messengers were easily detectable after as much as 5 d of darkness.
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Effect of sugars
To check whether the genes under study were sugar responsive, mature leaves from 6-week-old plants were harvested and incubated either with sterile water or 300 mM sucrose solution. Experiments using a 300 mM sorbitol solution as a control failed to influence the expression of these genes, thereby excluding the possibility of mere osmotica-dependent effects (not shown). To avoid interference between sugar-dependent and light-dependent control of gene expression, all experiments were performed either under continuous light or darkness (16 h acclimation, 48 h treatment). During the experiments, protein content in the control or treated leaves did not change significantly on a fresh weight basis, whereas chlorophyll decreased dramatically in sucrose-treated leaves but not in control leaves. Purple pigmentation of cells surrounding major veins, due to enhanced synthesis of anthocyanins, was apparent in sucrose-treated leaves following prolonged incubation.
Expression of three Calvin cycle genes (GapA, GapB, PRK) and two CP12 genes in the light was strongly inhibited by sucrose (Fig. 3). The transcripts of GapA-1, GapB, PRK, and CP12-2 rapidly declined with similar time-courses, down to undetectable levels between 6 h and 24 h of treatment. The response of CP12-1 was slower, but inhibition was complete 48 h after the start of the treatment. By contrast, neither GapC-1 nor GapCp-1 expression were significantly influenced by sucrose. When similar experiments were performed in the dark, lowering of GapA and GapB transcript levels were again observed with slower kinetics than in the light (not shown), whereas GapC and GapCp remained constant. The expression of GAPDH genes was also investigated using glucose as the permeant sugar and the results confirmed the effect, but 300 mM glucose proved to be somewhat less effective than 300 mM sucrose (not shown).
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To test whether the dramatic decrease of GapA and GapB transcript abundance caused by sucrose had consequences on enzyme activities, protein extracts were obtained for enzyme assays. Photosynthetic GAPDH activity (NADPH-dependent) stayed costant in control leaves, but showed a slow decline in sucrose-incubated leaves. After 72 h treatments the activity of sucrose-incubated leaves (0.36±0.16 µmol min–1 mg–1) was half the activity of control leaves (0.71±0.11 µmol min–1 mg–1). The effect was statistically significant (P <0.05). A very similar inhibition was observed when photosynthetic GAPDH was assayed under activating conditions, indicating that the activation state of the enzyme was not affected. In the presence of NADH rather than NADPH, the overall GAPDH activity referred to all GAPDH isozymes (A4, AnBn, GapC, and GapCp) was not significantly affected by sucrose treatments. The activity of PRK steadily decreased during the experiments in both control and sucrose-treated leaves and no significant variations could be ascribed to the treatment (not shown).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The expression of genes involved in photosynthesis is influenced by several factors including organ and developmental specificity, nutrient status of the plant, environmental conditions, and stress (Terzaghi and Cashmore, 1995
; Koch, 1996). In some instances, for example, during greening of etiolated leaves, the regulation of expression was found to be strictly co-ordinated (Raines et al., 1991). Co-ordinated transcription of light-regulated genes may depend on common light-regulatory elements, although these elements are not always conserved among promoters (Arguello-Astorga and Herrera-Estrella, 1998).
The family of GAPDH genes in Arabidopsis thaliana includes seven members. Here the focus has been on four genes representing the four types of GAPDH isozymes: GapA-1 and GapB for the dominant photosynthetic GAPDH isozyme (NAD(P)-dependent), GapC-1 for cytosolic GAPDH involved in glycolysis and gluconeogenesis, and GapCp-1 for NAD-specific GAPDH of plastids (Meyer-Gauen et al., 1998). PRK is encoded by a single gene, while three genes constitute the CP12 family with CP12-1 and CP12-2 being more strongly expressed than CP12-3 (Table 1). Calvin cycle GapA, GapB, and PRK transcripts were especially abundant in Arabidopsis leaves and flower stalks, less prominent in flowers, and virtually undetectable in roots and siliques. The basic expression pattern of these photosynthetic genes was also found for CP12 genes, supporting a role of CP12 proteins in photosynthesis. Minor differences in CP12 expression patterns with respect to Calvin cycle genes include the relatively higher expression of CP12-1 in flowers and CP12-2 in stalks.
While the absence of PRK transcripts in roots and its low levels in flowers and siliques are consistent with an exclusive role of this enzyme in the Calvin cycle, the analogous patterns of GapA-1 and GapB gene expression are unexpected since GAPDH activity is required for the glycolytic pathway of non-photosynthetic plastids leading to lipids, isoprenoids, and other end-products. The NADPH-dependent GAPDH activity of non-photosynthetic plastids is generally low, but the NADH-dependent activity can be substantial (Neuhaus et al., 1993) and is apparently catalysed by GapCp product (Backhausen et al., 1998; Petersen et al., 2003). Accordingly, GapCp-1 is expressed in Arabidopsis roots and siliques where GapA-1 and GapB transcripts are undetectable. The amino acid sequence of GapCp-1 is 82.5% identical to GapCp of Pinus sylvestris, which has been shown to be chloroplast located and NADH-dependent (Meyer-Gauen et al., 1998). Arabidopsis GapCp has a putative transit peptide for plastid localization and the typical sequence signature of NADH-specific GAPDH (Koksharova et al., 1998; Falini et al., 2003).
In leaves of adult plants transferred from a normal light/dark cycle to continuous darkness, the transcripts of GapA-1, GapB, PRK, and CP12-2 slowly decreased with similar kinetics. On the other hand, CP12-1 transcript abundance remained unaffected, indicating a different type of transcriptional control reminiscent of GapC. Pohlmeyer et al. (1996) found that spinach CP12 was equally expressed in green and etiolated cotyledons, confirming the light-independent expression of certain CP12 genes.
Sucrose treatment of detached leaves in the light also led to a rapid loss of GapA-1, GapB, and PRK transcripts, again with parallel time-courses. Both CP12 genes behaved similarly, but CP12-2 closely matched the drop of Calvin cycle transcipts, while the response of CP12-1 was somewhat delayed. In this study, GapC was steadily expressed during incubations with sucrose. By contrast, Shih and colleagues reported that Arabidopsis GapA-1 and GapB genes were not influenced by sucrose and GapC expression was stimulated (Shih and Goodman, 1988; Yang et al., 1993). The discrepancy with the present results is obvious and unexplained. However, it is commonly observed that those plant genes whose expression is increased by sugars are involved in the synthesis of carbohydrate reserves or in sugar catabolism, whereas sugar-repressed genes are usually involved in photosynthesis or the mobilization of storage compounds (Koch, 1996). Among Calvin cycle genes only rbcs has been clearly shown to be repressed by sugars, and the present demonstration that GapA-1, GapB, and PRK also behave in this way corroborates the sugar-sensing concept outlined above.
The effect of sucrose on photosynthetic GAPDH of chloroplasts was also notable in that the corresponding enzyme activities appeared to be far less affected than transcript levels. Although NADPH-GAPDH activity slowly decreased during lengthy treatments, it was still high in leaf material which had been depleted of either GapA-1 or GapB messengers for 48 h at least. The delay between the decrease of transcript levels and significant changes in GAPDH activity is remarkable. However, Chan et al. (2002) detected normal levels of GapA and GapB subunits and normal NADPH-GAPDH activity in an Arabidopsis mutant impaired in light-stimulated GapA-1 expression, i.e. deficient of GapA-1 transcripts, suggesting that post-transcriptional control was involved in the up-regulation of the GapA protein in the mutant.
In conclusion, the expression of two CP12 genes in organs of A. thaliana has been examined under different conditions, in parallel with genes (GapA, GapB, and PRK) whose products are known to interact with CP12 proteins in vivo. The expression of cytosolic GapC and plastid GapCp which do not interact with CP12 was also investigated for comparison. The expression of CP12-2, but not CP12-1, was found to be co-ordinately regulated with that of GapA-1, GapB, and PRK under all conditions tested. On the the other hand, the expression patterns of GapC-1 and GapCp-1 did not correlate with photosynthetic genes, and the latter finding is especially worth noting. The co-ordinated regulation of GapA-1, GapB, CP12-2, and PRK at the gene expression level is fully consistent with the existence and likely physiological relevance of a supramolecular complex involving GAPDH, CP12, and PRK proteins in chloroplasts (Wedel and Soll, 1998; Scheibe et al., 2002; Graciet et al., 2004).
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Acknowledgements |
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This work supported by the Ministero dell'Istruzione, Università e Ricerca (PRIN).
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Footnotes |
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Abbreviations: EST, expressed sequence tag; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PRK, phosphoribulokinase.
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