JXB Advance Access originally published online on February 28, 2005
Journal of Experimental Botany 2005 56(414):1129-1142; doi:10.1093/jxb/eri105
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RESEARCH PAPER |
Characterization and functional investigation of an Arabidopsis cDNA encoding a homologue to the d-PGMase superfamily
1Laboratoire de Physiologie et de Génétique Moléculaire des Plantes, Université Libre de Bruxelles, CP 242, Bd du Triomphe, B-1050 Bruxelles, Belgium
2Vakgroep Moleculaire Genetica and Department of Plant Systems Biology, Vlaams Interuniversitair Instituut voor Biotechnologie (VIB), Universiteit Gent, Technologiepark 927, B-9052 Gent, Belgium
3Institute for Plant Biotechnology, University of Stellenbosch, Private Bag, 7602 Matieland, South Africa
4National Centre of Genetic Resources and Biotechnology, Cenargen/Embrapa, S.A.I.N. Parque Rural, Final W3, Asa Norte, 70770-900 Brasilia, Brazil
To whom correspondence should be addressed. Fax: +32 2 650 5421. E-mail: nverbru{at}ulb.ac.be
Received 17 August 2004; Accepted 16 December 2004
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Abstract |
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An Arabidopsis thaliana cDNA (At-74) has been isolated that encoded an uncharacterized protein showing homology with members of the d-PGMase superfamily: cofactor-dependent phosphoglycerate mutases (d-PGM-ases) and the phosphatase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (6PF2Kase/F2, 6Pase). Preliminary phylogenetic studies indicated that At-74 cDNA and its close homologue in Arabidopsis, At-74H, belong, however, to an equally distinct group. At-74 was ubiquitously expressed in vegetative organs and induced by glucose. The At-74 cDNA was overexpressed in A. thaliana to investigate its function, but this overexpression did not result in a clear phenotype. Enzymatic assays performed on At-74-overproducing transgenic plants or E. coli cells showed no increase in either the activities of cofactor-dependent and -independent phosphoglycerate mutases (i-PGMases) and F2,6Pase or that of acid phosphatases. The possible role of At-74 in plant metabolism was further investigated by carbon partitioning experiments with [U-14C] glucose and measurements of soluble sugars in both young leaves and roots. Two overexpressing At-74 lines showed a clear increase in glucose uptake. This paper introduces the At-74 homologue of the d-PGMase superfamily members and supports a possible role of At-74 in carbohydrate metabolism.
Key words: d-PGMase family, transgenic plants, [U-14C]glucose feeding
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionSummaryReferences |
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Glycolysis is a central metabolic pathway, common to the vast majority of organisms (Plaxton, 1996
; Fothergill-Gillmore and Michels, 1993). In plants, it is only part of a complex network of pathways of carbohydrate metabolism, distributed over several compartments of the cell.
Phosphoglycerate mutase (PGMase) activity is central to glycolysis, being responsible for the interconversion of two monophosphoglycerates: 3-phosphoglycerate (3PGA) and 2-phosphoglycerate (2PGA) (Fothergill-Gilmore and Watson, 1989). In nature, two unrelated families of enzyme possess PGMase activity, the cofactor-dependent PGMases and the cofactor-independent PGMases (Jedrzejas, 2000). The former class is dependent on the 2,3-bisphosphoglycerate (2,3BPGA) cofactor for the priming (phosphorylation) of the enzyme necessary for activity. The latter is independent of the cofactor, but absolutely dependent on the presence of divalent Mn2+ (Singh and Setlow, 1979) or Co2+ (Collet et al., 2001) metal ions for activity. In both cases, PGMase activity involves the exchange of a phospho group between a retained intermediate that undergoes enzyme-bound reorientation and protein. In the case of d-PGMases, the enzyme is phosphorylated on a His residue (Bond et al., 2001), while in i-PGMases a serine is transiently phosphorylated (Murphy et al., 1997). Both groups of PGMases are evolutionarily related to classes of phosphatases, acid phosphatases, and alkaline phosphatases for d-PGMases and i-PGMases, respectively (Jedrzejas, 2000). These relationships are sometimes apparent from sequence analysis (Bazan et al., 1989) but, in other cases, only apparent after structural determinations. d-PGMases are related to narrowly specific phosphatases with which they form the ‘d-PGMase superfamily’. Among these phosphatases are fructose-2,6-bisphosphatase (Okar et al., 2001), 
-ribitol-5'-phosphate phosphatase (O'Toole et al., 1994), and mannitol-1-phosphatase (Liberator et al., 1998). They are also related to broad specificity phosphatases (Rigden et al., 2001) and protein phosphate phosphatases (Rigden, 2003). All members of this superfamily have a phosphohistidine signature in common. In the plant kingdom i-PGMases have been well characterized (Carreras et al., 1982; Graña et al., 1995) in contrast to d-PGMAses for which up to now very little information has been available (Carreras et al., 1982; Mazarei et al., 2003). i-PGMases exist as monomers of 60 kDa encoded by single genes and are highly conserved, as are glycolytic enzymes in general (Fothergill-Gillmore and Michels, 1993). Multiple i-PGMases isozymes are present in plants. Some are reported to be localized exclusively in the cytosol (Huang et al., 1993; Westram et al., 2002) while others were found in both the cytosol and plastids (Botha and Dennis, 1986a). A third isozyme was observed in the nucleus (Wang et al., 1996). This distribution suggests that i-PGMases might have several roles in the plant cell. In this way it has been shown that i-PGMases impairs photosynthesis and growth (Westram et al., 2002).
After d-PGMase itself, the best-studied member of the d-PGMase superfamily is fructose-2,6-bisphosphatase (F2,6Pase), typically found with a 6-phosphofructo-2-kinase (6PF2Kase) domain in a bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase enzyme (6PF2Kase/F2,6Pase; EC 2.7.1.105
[EC]
and EC 3.1.3.46
[EC]
, respectively) 6PF2Kase/F2,6Pase catalyses the synthesis and degradation of fructose-2,6-bisphosphate (F2,6P2), a non-metabolite sugar phosphate only present in eukaryotic organisms (Stitt, 1990a). The function of F2,6P2 in the photosynthetic metabolism of plants, like spinach, which predominantly accumulate starch has been extensively studied. In these plants F2,6P2 is involved in the regulation of sucrose synthesis with photosynthetic activity and starch synthesis. Experiments on transgenic Arabidopsis and tobacco plants confirmed that a decrease in the level of F2,6P2 increases the flux towards sucrose, whereas an increase of this metabolite increases the flux towards starch (Draborg et al., 2001; Scott et al., 1995, 2000). In wheat, which predominantly accumulates sucrose, F2,6P2 is also involved in the co-ordination of sucrose synthesis and photosynthesis in the source tissues. By contrast, in starch-accumulating plants F2,6P2 does not regulate the partitioning of fixed carbon between sucrose and starch synthesis during the photoperiod (Trevanion, 2002). The role of F2,6P2 in the plant sink tissue has not been very well studied and remains to be clarified. Concerning the structure of 6PF2Kases/F2,6Pases in plants, it is clearly different from that found in other eukaryotes. For a long time only biochemical data and kinetics analyses were available and relied on the characterization of relatively crude enzyme preparations (Pilkis et al., 1995). In spinach, two forms of F2,6Pase were identified, a bifunctional 6PF2Kase/F2,6Pase enzyme of about 90 kDa (Larondelle et al., 1986) and a monofunctional F2,6Pase which is a dimer of 33 kDa subunits (Macdonald et al., 1987, 1989). However, more recently, plant cDNAs encoding bifunctional 6PF2Kases/F2,6Pases have been isolated from several plants, including potato, maize, spinach, and Arabidopsis (Draborg et al., 1999; Villadsen et al., 2000; Markham and Kruger, 2002). These plant 6PF2Kase/F2,6Pase isoforms are homotetramers of about 95 kDa subunits whereas 6PF2Kases/F2,6Pases from other organisms form a dimer of a single subunit of about 50–100 kDa. However, like the bifunctional animal enzyme (Pilkis et al., 1995), plant 6PF2Kases/F2,6Pases present both kinase and phosphatase activities. These activities were measured in bacteria (Draborg et al., 1999; Villadsen et al., 2000) and thus supported the presence of a bifunctional F2KPase in plants. Surprisingly, an antisense approach in Arabidopsis to reduce F2,6Pase activity showed that there is apparently no monofunctional F2,6Pase activity (Draborg et al., 2001). This conclusion is strengthened by the availability of the complete Arabidopsis thaliana genome sequence (Arabidopsis Genome Initiative, 2000) in which one bifunctional 6PF2Kase/F2,6Pase sequence is present and no sequence corresponding to an isolated F2,6Pase domain.
The PFAM protein domain database (Bateman et al., 2002) predicts the presence in the A. thaliana genome of about 15 possible genes, not all of which are known to be expressed, coding for proteins containing domains homologous to d-PGMases and F2,6Pases. However, as only one of these is a bifunctional 6PF2Kase/F2,6Pase, the question arises as to the function(s) of the remaining (putative) proteins? In recent years studies indicated that the spectrum of catalytic activities present in the d-PGMase superfamily is broader than initially expected (Rigden et al., 2002, 2003) and that novel activities probably remain to be discovered. In this paper, the identification of the At-74 cDNA from A. thaliana is described, which was initially annotated as coding for an unknown protein showing homology mainly with monofunctional d-PGMase and F2,6Pase. During this study, Arabidopsis genome automatic annotation revealed five more d-PGMase-like genes. However, none of these newly-annotated proteins have yet been confirmed at the enzymatic level. In silico analyses clearly indicate that At-74 and its closest homologues form a new family linked to the d-PGMase superfamily, but distinct from the annotated d-PGMase-like genes. The At-74 gene has been characterized at the transcript and protein level. Moreover, in order to assess a possible role for this gene, transgenic Arabidopsis lines overexpressing the At-74 cDNA were generated. Analysis of the plants is presented, as well as a first functional investigation of the role of At-74 in plant metabolism.
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Materials and methods |
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Computational analysis
Searches in sequence databases were carried out using BLAST (Altschul et al., 1990
) and sequence alignments with T-COFFEE (Notredame et al., 2000). Manual manipulation was with JALVIEW (http://www.ebi.ac.uk/
michele/jalview). A consensus, Neighbor–Joining phylogenetic tree was constructed and drawn with the PHYLIP package (Felsenstein, 1989).
Plant material and growth conditions
Arabidopsis thaliana (L.) Heynh. ecotype Columbia (Col) was used for both in vivo and in vitro experiments. To select the different transgenic populations, seeds were surface-sterilized and germinated in vitro in a growth chamber on MS agar medium containing (Murashige and Skoog, 1962): 4.3 g l–1 Murashige and Skoog salts 0.5 g l–1 MES (pH 5.7), 1% sucrose, 0.68 g l–1 agar or 2 g l–1 gelrite and 75 µg l–1 kanamycin (Hua et al., 1997) at 22 °C with a 16/8 h photoperiod (60 µE m–2 s–1 light intensity). Three-week-old seedlings were then transferred to soil and grown to maturity in a greenhouse at 22 °C with a 16/8 h photoperiod (100 µE m–2 s–1 light intensity) and 60% relative humidity.
For experiments with 6% glucose, 13-d-old wild-type Col seedlings (50) germinated on nylon discs (3x2 cm) and grown on MS agar medium without kanamycin were transferred from in vitro to MS medium containing 4.3 g l–1 Murashige and Skoog salts, 0.5 g l–1 MES (pH 5.7) and 6% glucose. Seedlings were harvesting every 4 h for 24 h (60 µE m–2 s–1 light intensity). Arabidopsis seedlings used as control were transferred to MS medium without sugars.
For western analyses in normal conditions, young seedlings (transgenic and wild-type plants) germinated on MS agar medium (4.3 g l–1 Murashige and Skoog salts, 0.5 g l–1 MES pH 5.7, and 1% sucrose) were used. For western analyses performed with different glucose concentrations, three week-old wild-type Columbia seedlings were grown on MS agar medium supplemented with different glucose concentrations (4.3 g l–1 Murashige and Skoog salts, 0.5 g l–1 MES pH 5.7 and 0, 2, 4, 5, and 6% glucose).
Plasmid construction and Arabidopsis transformation
The At-74 cDNA was isolated in a screen for Arabidopsis cDNAs that could increase the osmotolerance in a yeast strain by overexpression (Sun et al., 2001). However, the enhanced osmotolerance could not be reproduced in yeast. The At-74 cDNA was inserted into the Agrobacterium pGiBin19 binary vector (Bevan, 1984) under the control of a cauliflower mosaic virus (CaMV) 35S RNA promoter and a 3' nopaline synthase terminator (nos). The gus fragment in the pGiBin19-(35S)-Gus recombinant vector was removed by digestion with BamHI and SacI (blunt-ended by T4 DNA polymerase). The At-74 cDNA cloned in the pYES2 (Gibco-BRL) yeast vector was digested with BamHI and SphI (blunt-ended with T4 DNA polymerase) and subcloned into the pGiBin19-(35) binary vector between the BamHI and SacI sites. Root explant transformation of Arabidopsis wild-type Col ecotype was performed as described by Sun et al. (2001). Analyses were carried out in the T2 and T3 generations.
Northern blot analysis
Total RNA was isolated as described by Verwoerd et al. (1989) or using TRizol reagent (Gibco-BRL). Equal amounts of RNA were separated by electrophoresis with 1% agarose/formaldehyde gel electrophoresis and transferred onto a Hybond N+ membrane (Amersham-Pharmacia). DNA probes for hybridization were labelled with [-32P]-dCTP using Rediprime system II (Amersham Biosciences). Membranes were hybridized with Na2HPO4 buffer at 65 °C overnight and washed twice at 65 °C with 2x SSC, 0.1% SDS for 20 min and then once at 65 °C with 0.2x SSC, 0.1% SDS for 10–15 min.
Southern analysis
Genomic DNA was extracted according to Doyle and Doyle (1990). The digested DNA was resolved by electrophoresis on an agarose gel (0.8%) and blotted onto a Hybond N+ membrane. Probe labelling and hybridizations were carried out as for northern blot analysis.
Antibody production
Antibodies against At-74 were synthesized by immunizing rabbits with the At-74 recombinant protein (At-74-His) overexpressed in Escherichia coli. The full sequence of the At-74 cDNA was amplified by PCR with primers 5'-AGTCCGGACAATAAACTGCTTCCG-3' and 5'-CGCGGATCCGAGCAGCATTGTCCATTGTATAAC-3'. The PCR product was digested with BamHI and cloned into the expression vector pET21d(+) (Novagen) in the BamHI and NcoI (blunt-ended by T4 DNA polymerase) restriction sites in the frame of a 6 histidine tag at the C terminus. At-74-His was overexpressed in E. coli BL21-codonPlus-(DE3)-RP (Stratagene). The fusion protein was partially purified on a Ni2+ column according to Novagen instructions and a modified protocol (Rogl et al., 1998). Purified proteins were separated by 10% SDS-PAGE and the main band of polyacrylamide containing At-74-His was excised and used for immunization of the rabbits. Preimmuned sera were tested and no immunoreactive signal was detected.
SDS-PAGE and western blot analysis
Protein extraction and western blots were carried out as described by Sun et al. (2001). After incubation with primary antibody (dilution at 1/2500), protein blots were probed with either goat antirabbit IgG horse peroxidase (Sigma) or antirabbit horse alkaline phosphatase (dilution at 1/25 000). Immune complexes were detected with BCIP and NBT (Sigma) or by the enhanced chemiluminescence system (NEN).
RT-PCR analysis
RT-PCR analyses were performed on 1–3 µg of total plant RNA according to the manufacturer's instructions (Gibco-BRL). For experiments with Arabidopsis organs, total RNA was treated with DNase I before reverse transcription. Reverse transcription was performed in a final volume of 20 µl with Oligo(dT)12–18 for analysis using the actin gene as the control or random hexamers for analysis using the 18S rRNA gene as control. Two µl of single-stranded cDNA was used for each PCR (94 °C for 2 min: 1 cycle of 94 °C for 30 s, 55 °C for 1 min, 72 °C for 1 min: 15 cycles to analyse the expression of the actin gene, 18 cycles to analyse the expression of the 18S rRNA gene and 20 cycles to analyse the expression of At-74 ; 72 °C for 10 min:1 cycle). Amplifications were carried out with At-74, At-74H, Actin2, and 18S rRNA primers. Actin2 and 18S rRNA were used as loading controls. 18S rRNA was used because Actin2 is not constitutive in seeds (Sun et al., 2001). At-74 primers: 5'-CCAATCATCCATGAGTCCGGACAA-3' and 5'-CAGCAGCATTGTCCATTGTATAACTC-3'. At-74H primers: 5'-GAGAGCCCTCATATCAACCCAAAG-3' and 5'-ACACGACACCCATCAACACGATC-3'. Actin2 primers: 5'-GTGCCAATCTACGAGGGTTTCT-3' and 5'-CAATGGGACTAAAACGCAAAAC-3'. 18S RNA primers: 5'-CTGTCGGCAAGGTGTGAACTC-3' and 5'-GTATGGTCGCAAGGCTGAAAC-3'. PCR products (20 µl) were loaded on 1% agarose gels and blotted onto Hybond N+ membranes (Amersham-Pharmacia). Hybridizations were carried out as described in RNA blot analyses. Signals quantification was performed using a phosphoimager (Storm, Molecular Dynamics) and the program Image Quant (Molecular Dynamics).
Protein extraction
Three week-old transgenic and wild-type seedlings grown in vitro were used for protein extractions. Seedlings were frozen in liquid nitrogen and stored at –80 °C until extraction. The frozen tissue was weighed and extracted in a 2:1 ratio with ice-cold extraction buffer A (100 mM HEPES-KOH pH 7.5, 10% glycerol, 5 mM DTT, and 0.5 mM Perfabloc inhibitors cocktail). After vortexing, the homogenate was centrifuged at 8 000 rpm (Sorvall RC Plus, SLA-600TC rotor) for 25 min at 4 °C. The supernatant was removed and centrifuged at 13 000 rpm for 15 min at 4 °C. The clear supernatant was rapidly frozen in liquid nitrogen and stored at –80 °C. Proteins were desalted on a Sephadex G-25 column (PD-10, Amersham-Pharmacia) equilibrated with buffer B (100 mM HEPES-KOH pH 7.5, 10% glycerol, and 5 mM DTT) and eluted with the same buffer. Aliquots of eluates containing proteins were rapidly frozen in liquid nitrogen and stored at –80 °C for protein determination and enzyme activities.
Proteins were extracted from BL21-codonPlus-(DE3)-RP bacteria (Stratagene) transformed with the pET21(d) vector or the recombinant pET21(d) expressing At-74 and BL21-(DE3) cells (Stratagene) containing either pGEX plasmid (Amersham Biosciences), or the recombinant pGEX vector expressing a rat liver F2,6P2ase (gift from Dr Nicholas J Kruger, Oxford University, UK). Bacteria cultures were induced at OD O.6 with 1 mM IPTG (final concentration) at 37 °C for 3 h and centrifuged at 4000 g for 10 min at 4 °C. The pellet was washed in buffer C (100 mM HEPES-KOH pH 7.5, 10% glycerol, and 1 mM DTT) and centrifuged as before. The pellet was frozen in liquid nitrogen and stored at –80 °C. For protein extraction, frozen cells were washed in buffer A and centrifuged at 4000 g for 10 min at 4 °C. The pellet was resuspended in the same buffer with 1 mg ml–1 of lysozyme. The homogenate was kept on ice for 30 min and then submitted to three freeze–thaw cycles with dry-ice and ethanol. The extract was treated for 1 h with DNase I (0.1 mg ml–1) before centrifugation at 13 000 g for 15 min at 4 °C. Soluble proteins were desalted as previously. Aliquots were frozen in liquid nitrogen and stored at –80 °C for protein determination and F2,6Pase activity measurement. Protein concentration was determined according to Bradford (1976) with serum bovine albumin used as a standard.
Enzymatic assays
PGMase assays were performed at room temperature (Botha and Dennis, 1986b). i-PGMase activities were measured with 100 µl of desalted protein in a 1 ml final volume (100 mM TRIS/HCl pH 7.5, 10 mM MgCl2, 0.250 mM NADH, 2,5 mM ADP, 5 U ml–1 pyruvate kinase, 6 U ml–1 lactate dehydrogenase, and 1 U ml–1 enolase). The reaction was initiated by the addition of 60 µl of 80 mM 3PGA. Absorbance at 340 nm was measured over time with a spectrophotometer (PerkinElmer). d-PGMase assays were performed in the same conditions but in the presence of cofactor 2,3-bisphosphoglycerate (2,3BPGA 0.20 mM).
F2,6Pase activity was measured by the disappearance of F2,6P2 monitored by a PFPase bioassay (pyrophosphate-dependent phosphofructokinase) (Stitt, 1990b) Fifteen µl of desalted protein were assayed at room temperature in a final volume of 140 µl (50 mM TRIS/HCl pH 7.8, 5 mM MgCl2, 1 mM EGTA, and 1 mM EDTA) by adding 21 pmol F2,6P2. Immediately, and at certain time points thereafter, 20 µl aliquots were removed and alkalinized (to stop the reaction) in 30 µl KOH (0.25 N). The amount of F2,6P2 in these aliquots was quantified through the stimulation of PFPase in a final volume of 200 µl (150 mM TRIS/HCl pH 7.8, 3 mM MgCl2, 3 mM F6P acidified, 300 mM NADH, 0.4 mM PPi, 1 mU PFPase potato, 0.1 U aldolase, 0.8 U glycerol-3phosphate-dehydrogenase, and 2.3 U triose phosphate isomerase). The decrease of extinction was measured at 340 nm in a plate reader (PowervaveX, Biotek Instruments) over 30 min. The amount of F2,6P2 was estimated by reference to a standard curve based on the activation of PFPase activity against F2,6P2 concentration (prepared in reactions similar to the time zero aliquot but spiked with 0–2 pmol F2,6P2 assay–1).
Phosphatase assays were conducted (Basha, 1983) in a 1 ml vol. (100 mM sodium acetate pH 4.9, 50 mM p-nitrophenyl phosphate: PNPP). The reaction was initiated by the addition of 7 µl of desalted protein and terminated by the addition of 200 µl of 4 M NaOH. Release of p-nitrophenol was determined by measuring the absorption at 410 nm.
Incubation of leaves and roots with [U-14C]-glucose
Transgenic and wild-type Arabidopsis seeds were germinated and grown in vitro for 3 weeks in square plates laid vertically (Mani et al., 2002) and containing MS gelrite medium without kanamycin. Shoots/leaves and roots were separately rinsed twice in sterile water and incubated at room temperature in 20 ml of fresh buffer I (25 mM K-MES pH 5,7, 1 mM CaCl2). Before incubation, roots were blotted on paper to remove the gelrite and washed again in distilled water. After 10 min of incubation, leaves and roots were blotted on paper and divided into several samples which were weighed (100–140 mg per sample). Leaf and root samples were incubated separately in the dark at 28–30 °C with gentle shaking in sealed 250 ml Erlenmeyer flasks containing 1.5 ml of buffer II (25 mM K-MES pH 5.7, 250 mM mannitol, 5 mM glucose, 2 µCi ml–1 [U-14C]-glucose). The 14CO2 produced was collected in 0.5 ml of 12% (w/v) KOH contained in a central well inside each flask. After 4 h of incubation, samples were rinsed separately, once with ice-cold distilled water and twice in 20 ml of ice-cold 1 mM CaCl2 for 15–20 min. Leaves and roots were blotted on paper and incubated for 24 h at 65 °C in 1.5 ml tubes containing 1 ml of 70% ethanol (v/v) and 30 mM TRIS pH 7. After incubation samples were stored at 4 °C before fractionation.
Total 14C uptake occurring in young leaves and roots
Leaf and root samples were centrifuged at 10 000 g for 10 min at room temperature. Each supernatant was processed through a 1 ml cation exchange LC-WCX column (SUPELCO) coupled to a 1 ml anion exchange LC-SAX column (SUPELCO). Soluble sugars were eluted through these columns with 5 ml of 30% ethanol. Amino acids contained in the LC-WCX column were eluted with 5 ml of 30% ethanol, 4 M ammonia and organic acids contained in the LC-SAX column extracted with 5 ml of 30% ethanol, 2 M formic acid. Pellets corresponding to the insoluble matter were considered to be the starch fraction. They were washed twice with 10 ml of sterile water to eliminate unincorporated label, centrifuged at 3000 rpm at room temperature (Hettisch universal/K2S) and filtered separately through a nylon disc. To determine total 14C uptake occurring in each Arabidopsis leaf and root sample, an aliquot of eluted [U-14C]-sugars, -amino acids and -organic acids was diluted 1:4 with Ultima FloTM M scintillation cocktail and counted for 10 min in a Beckman LS 1801 scintillation counter. For starch samples, 1 ml of water was added to the scintillation cocktail.
HPLC separation of [14C]-sugars
HPLC was used for the determination of [14C]-labelled sugars contained in samples labelled with [U-14C]-glucose. Eluted [U-14C]-sugars were reduced completely in a vacuum centrifuge and resuspended in 500 µl of sterile water. Two hundred µl were centrifuged at 10 000 g and 20 µl separated on an Allosphere amino (NH2) column (Alltech) witth 80% (v/v) acetonitrile (HPLC Shimadzu SCL-10AVP system). 14C in glucose, fructose, and sucrose was determined by liquid scintillation spectroscopy (Radiomatic A-500 inline radio-chromatography detector). Peak elution times were obtained using standards of [U-14]-glucose, [U-14]-fructose, and [U-14]-sucrose. The integration of the peak areas for the fractionated sugars was performed with a Shimadzu Class-VP software package.
Statistical analysis
The distribution of the sugar and metabolite values measured after [U-14C]-glucose feeding between At-74OE lines and the control line was compared by analysis of variance (ANOVA). A two-factor ANOVA test was performed with the Statistica program (StatSoft Inc). (P <0.005 line effect significant).
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Results and discussion |
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At-74 and the d-PGMase superfamily
In silico analysis of At-74 highlighted a phosphohistidine motif:
At-74 (Genbank code: 15229917; At3g05170) was isolated in a screen of an Arabidopsis cDNA library in Saccharomyces cerevisiae to identify genes involved in salt tolerance. However, the enhanced salt-tolerant phenotype initially observed could not be reproduced in yeast (data not shown). The 1280 bp At-74 cDNA insert contains a 922 bp open reading frame coding for an unknown protein with a calculated molecular weight of 37 kDa and no predicted peptide signal sequence. Blast searches using the At-74 sequence against the Arabidopsis genome revealed a closely related ORF (Genbank code: 15223983; At1g08940) that was named At-74H and that shares 64% sequence identity at the protein level with At-74. Analysis of the genomic sequences displays a similar gene structure between At-74 and At-74H. Both have a single intron of about 500 bp located at the same position, suggestive of a probable duplication event (Blanc et al., 2003
). Further database searches revealed three other possibly homologous proteins, one in Streptomyces coelicolor (Genbank code: 21224153; 41% sequence identity between the deduced amino acid sequences), the second in Saccharomyces cerevisiae (Genbank code: 6320256; ORF YDR051c; 37% sequence identity between the deduced amino acid sequences) and the third in Oryza sativa (Genbank code: 6069661; 27% sequence identity between the deduced amino acid sequences). These three proteins were also annotated as unknown. Much lower in the list of the Blast search hits, but with significant scores, were several proteins belonging to bifunctional 6PF2K/F2,6Pase and d-PGMase families. The presence of the characteristic phosphohistidine motif [LIVM]-x-R-H-G-[EQ]-x(3)-N in At-74 and in the four presumed homologous proteins, provided some proof of a relationship between the At-74 protein and these two enzyme families, which belong to the d-PGMases superfamily.
Comparison of At-74 family sequences with members of the d-PGMase superfamily:
The At-74 and its closest homologous sequences (15229917, 15223983, 6069661, 21224153, 6320256), which form a group by themselves, were compared with representatives of the d-PGMase superfamily with known catalytic activities in an attempt to draw inferences about the likely catalytic activity of At-74 (Fig. 1). Three of these representatives, Escherichia coli d-PGMase, rat F2,6Pase, and Bacillus stearothermophilus PhoE (Rigden et al., 2003), are of known structure and a further two, E. coli -ribazole-5'-phosphate phosphatase (O'Toole et al., 1994) and Eimeria tenella mannitol-1-phosphatase (Liberator et al., 1998) are of unknown structure. The related families of phosphoprotein phosphatases (Rigden, 2003) were not included in the comparison since they contain a characteristic deletion that is not present in the At-74 family protein.
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Successive structural determinations combined with sequence analysis have defined a catalytic core, so far common to all catalytically active members of the d-PGMase superfamily, containing just four residues; His10, His183, Arg9, and Arg61 in the E. coli d-PGMase numbering (Fig. 1). A fifth residue, Glu88, is common to all except the phosphoprotein (Rigden, 2003
). These five core residues are conserved in the At74 family (Fig. 1). Outside this core, each family has its own set of conserved residues involved in substrate binding (Fig. 1) which are predictive of the specificity of each family. These are known from crystallographic studies for d-PGMase, F2,6Pase, and PhoE (Yuen et al., 1999; Bond et al., 2002; Rigden et al., 2002). For the families of undetermined structure, inference even from analysis of sequence conservation is difficult since each family contains few members. Comparison of the At-74 family sequences with these key sets of specificity-determining residues shows that they are most similar to those of PhoE, which has a strikingly hydrophobic binding site (Rigden et al., 2002). All but one the hydrophobic positions involved in PhoE substrate binding is occupied by hydrophobic residues in the At-74 family. Nevertheless the exception (PhoE Trp203, in E. coli d-PGMase numbering, is a conserved Glu in the At-74 family) combined with some large differences in residue size, suggest that At-74 and PhoE substrate specificities are significantly different. These analyses support the deduction that the At-74 gene family is likely to encode proteins having a novel catalytic activity. Currently, Arabidopsis annotation has revealed five novel proteins annotated as d-PGMase-like proteins (Mazarei et al., 2003). Alignments of At-74 and At-74H protein sequences with these five newly-annotated sequences (Fig. 2) show four well-conserved residues common to the catalytically-active members of the large d-PGMase superfamily (Arg9, His10, His183, Glu203), except for two sequences (At5g04120, At3g50520). Phylogenetic analyses (Fig. 3) indicate that three (15237708, 15217922, 15227803) out of five d-PGMase-like proteins form a group which is distinctly related to the well-known members of the d-PGMase superfamily and to At-74 family members. Interestingly, the last two protein sequences (15237633, 15229783) could tend to be more related to the broad-specificity family of phosphatases even if a clear phosphohistidine motif is lacking.
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Expression study of At-74 in Arabidopsis
Influence of glucose/sugar concentration on At-74 expression:
To obtain information regarding the role of At-74 in Arabidopsis, its expression was analysed in various organs. Since the levels of expression were low (northern blot analysis; data not shown) RT-PCR analyses were performed with specific primers. Expression of At-74 in all organs was constitutive (Fig. 4A, B) and much higher than that of At-74H (data not shown).
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Since d-PGMases and F2,6Pases, which are among the best-characterized At-74 related proteins, participate in or regulate glycolysis, a possible influence of high glucose concentration on the level of expression of At-74 was assessed. Thirteen-day-old Arabidopsis wild-type seedlings grown in vitro were incubated for different time points over 24 h in a liquid medium containing 6% (m/v) glucose. RT-PCR analyses carried out on full seedlings indicated that At-74 was rapidly induced by a high glucose concentration (Fig. 5A, B). The expression of At-74 was highest after 4 h treatment and slightly decreased over the next 20 h. Antibodies were raised against recombinant At-74 protein. Western analyses performed with these antibodies confirmed the low level of expression in control conditions and the enhancement of At-74 protein level in the presence of high glucose concentration (Fig. 5C) (confirmed at different developmental stages, data not shown). These first results suggest that At-74 protein may effectively be involved in plant carbohydrate metabolism.
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Overexpression of At-74 in A. thaliana:
The presence in the At-74 sequence of a phosphohistidine motif and the conserved catalytic core currently believed to be essential for the activity of d-PGMase superfamily members suggested that At-74 encodes a catalytically-active protein, rather than a non-enzyme homologue (Todd et al., 2002
). Among the closely-related proteins, a deletion mutant was only available for ORF YDRO51c of S. cerevisiae. However, this mutant had no particular phenotype, grew on classical yeast medium and its transformation with At-74 cDNA produced no difference in growth rate (data not shown). Thus, At-74 was overexpressed in Arabidopsis under the control of the CaMV 35S promoter in order to obtain further insight into its function. Twenty-one transgenic T2 lines were screened by northern blot analysis (Fig. 6A) and eight clearly overproduced At-74 (At-74OE). To maximize stability of expression in the next generations, homozygous lines showing one copy of the transgene were selected by segregation analysis on kanamycin (data not shown) and Southern analysis (Fig. 6B). Three independent At-74OE lines: nos 9, 12, and 17 were used in subsequent experiments. Western analyses with antibodies raised against recombinant At-74 confirmed the overproduction of the At-74 protein in tested At-74OE plants (Fig. 6C). The possible influence of At-74 overexpression in plant sugar metabolism was investigated by germinating At-74OE lines on different concentrations of glucose, fructose, and sucrose. At-74OE lines were affected neither in growth nor in phenotypic appearance, although a slight sugar-sensitive phenotype was sometimes observed compared with control plants (data not shown). This phenotype was only observed in the presence of high glucose concentrations (5–6%) and was never observed with fructose or sucrose. No particular phenotype appeared with sugar analogues and non-metabolizable osmotica (polyethylene glycol, sorbitol, mannitol) indicating that At-74 is probably not involved in sugar sensing and abiotic stress.
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Enzymatic activities:
PGMase and F2,6Pase enzyme activities were measured in At-74OE lines. Assays were performed in pH condition favourable to the measurement of d-PGMase activity, but no increase of cofactor-dependent PGMase was observed compared with the wild-type plants. The same result was observed after measuring i-PGMase activity (Table 1 A). This result is compatible with the unsuccessful complementation of the gpm1 PGMase-deficient (Rodicio and Heinisch, 1998; Fraser et al., 1999
) deletion mutant of S. cerevisiae with the At-74 cDNA (data not shown).
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F2,6Pase enzyme assays were also performed on the same transgenic lines and similar activity was also observed between At-74OE lines and the control plants (Table 1 B). To confirm that At-74 showed no F2,6Pase activity, enzymatic activities were also performed in E. coli, which is naturally devoid of F2,6Pase. At-74 and the cDNA of a functional rat F2,6Pase (gift from Dr Kruger, Oxford) were cloned in pET and pGEX bacterial expression vectors, respectively, and these recombinant vectors were then transformed into E.coli. Substantial activity was observed in extracts of induced bacteria containing the functional rat F2,6Pase, but none was detected in extracts overexpressing At-74 or the pET and pGEX control vectors (Table 1 B).
Acid phosphatase activity was also measured to determine whether At-74 was a phosphatase similar to PhoE. Assays were performed on plant extracts. p-nitrophenyl phosphate (PNPP) was used in the reaction as a broad substrate (Basha, 1983; Rigden et al., 2001). No difference in total phosphatase acid activity was observed between transgenic and control plants. Acid phosphatase assays in polyacrylamide gels of separated total crude protein extracts also gave similar results (data not shown). This confirms that At-74 is not a PhoE like phosphatase (Table 1 C).
Taken together, these results are in agreement with the in silico sequence analysis which groups At-74 with neither d-PGMases and F2,6Pases nor with phosphatase with a broad phosphatase specificity.
[U-14C]glucose uptake in leaves and roots
14C incorporation into detached leaves and roots supplied with [U-14C]glucose:
Since At-74 expression was induced by exogenous glucose, 14C-glucose labelling experiments were performed to determine whether At-74 overexpression could affect carbon partitioning in transgenic plants. The study was conducted on excised leaves and roots of a pool of young seedlings grown in vitro on normal MS agar medium. The experiment was carried out in the dark in order to observe the distribution of 14C without the influence of photosynthesis. After 4 h incubation with [U-14C]glucose, 14C incorporation into sugars, CO2, starch, organic acids, and amino acids was estimated separately in leaves and roots (Table 2). It is clear that the leaves of the two transgenic lines 9 and 12 have taken up much less 14C-glucose (difference >23%) than the WT and the transgenic line 17. Uptake of label by the roots was the same between the control and transgenic lines. In order to compare partitioning in the lines, despite the difference in uptake, the labelling data were compared on a percentage distribution basis. In the leaves most of the label (>60%) was present in the sugar pool in contrast to the roots in which much less label was present (<40%). To investigate whether this difference is primarily due to the inability of the photosynthetic tissue to mobilize glucose, once taken up, total sugars were fractionated to determine the label in the three main sugars: glucose, fructose, and sucrose. The partitioning indicated that the label portion of soluble sugars was significantly greater in leaves than in roots, as had already been observed in the label portion of total sugars. It also appeared that the ability to mobilize glucose was higher in the root tissue, where about 12–17% of the label remains in the glucose pool compared with leaves (Table 2). However, in leaves, both transgenic lines 9 and 12 were significantly more efficient in the mobilization of glucose than the WT (P <0.05). In root tissue the portion of labelled sucrose was higher (>25–30%) than in leaves except for lines 9 and 12 in which a larger portion of the label was found in the sucrose pool (>60%). The presence of labelled fructose in leaves and, to a lesser extent in roots, indicated that the sucrose was broken down in these tissues. In transgenic line 9 it is clear that the return of label from sucrose to fructose (P <0.05) was lowest compared with the wild type, probably accounting at least in part for the consistently larger partitioning to sucrose in this line.
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Concerning non-soluble sugar metabolites, overall mobilization of the radiolabel was between 80% and 88% in the leaf tissue and about 94–96% in the roots (Table 2). In leaves, between 21% and 24% of the mobilized label was respired to carbon dioxide. In the roots, complete oxidation was slightly higher, reaching 31–33%. Almost double the amount of label was partitioned to starch (25%) in the root tissue compared with the leaves (11–12%). No obvious differences were observed in partitioning to respiration, or the organic and amino acid fractions between the transgenic and control lines in leaves or in roots. As observed in other studies the percentage of 14C incorporation into amino acids was marginal (Schneider et al., 2002
). Taken together these data indicate that two At-740E lines on three tested present a significant increase of glucose uptake and mobilization in leaves compared with the wild type which indicates that At-74 could encode a functional protein in A. thaliana involved in carbohydrate metabolism.
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Summary |
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TopAbstractIntroductionMaterials and methodsResults and discussionSummaryReferences |
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In conclusion, sequence and phylogenetic analysis of the At-74 family suggest that it contains catalytically-active proteins possessing a novel substrate specificity in the d-PGMase superfamily. Experimental data do not allow the determination of the exact function of At-74. No activities were observed for d-PGMase, F2,6Pase, and acid phosphatase. However, if no direct interpretation about the role of At-74 can be drawn from these results, expression and feeding experiments with glucose support a role of At-74 in sugar metabolism in plants. The presence of close At-74 homologues in different kingdoms suggests that At-74 acts in a broadly-distributed pathway. At-74 is not expected to be a regulatory enzyme, as these are usually more abundantly expressed and overexpression would have affected carbon partitioning. Often the effect of overproduction of a single enzyme is constrained by regulatory mechanisms, which limit the effect of the expected genetic alteration. For this reason it would be interesting to generate Arabidopsis plants in which At-74 activity has been totally eliminated. To this end, At-74 antisense plants are currently being characterized. In order to investigate further the role of At-74 and its homologues in plant metabolism, enzyme assays performed on purified enzyme with various substrates will be required.
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Acknowledgements |
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We would like to thank Brigitte Van de Cotte and Sue Rose for help with protein purification and HPLC analyses, respectively. We are grateful to Richard Cooke for critical reading of the manuscript. This work was supported by grants from a European Union Training and Mobility fellowship (FMRX-CT96-0007) and the Flemish community for Bilateral Scientific & Technological Cooperation between Flanders and South Africa (BIL99/34). FB was indebted to the FNRS for a postdoctoral fellowship (contract 2.456502).
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Footnotes |
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* Present address: Institut de Recherche pour le Développement, 911 Avenue Agropolis, BP 64501; F-34394 Montpellier cedex 5, France.
Present address: South African Sugarcane Research Institute, Private Bag X02, Mount Edgecombe, 4300, South Africa.
Present address: Innogenetics, Technologiepark 7, B-9052 Gent, Belgium.
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References |
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|
TopAbstractIntroductionMaterials and methodsResults and discussionSummaryReferences |
|---|
Arabidopsis Genome Initiative. 2000. Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408, 796–815.[CrossRef][Medline]
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. Journal of Molecular Biology 215, 403–410.[CrossRef][Web of Science][Medline]
Basha SM. 1983. Purification and characterization of an acid phosphatase from peanut (Arachis hypogaea) seed. Canadian Journal of Botany 62, 385–391.[CrossRef][Web of Science]
Bateman A, Birney E, Cerruti L, Durbin R, Etwiller L, Eddy SR, Griffiths-Jones S, Howe KL, Marshall M, Sonnhammer EL. 2002. The Pfam protein families database. Nucleic Acids Research 30, 276–280.
Bazan JF, Fletterick RJ, Pilkis SJ. 1989. Evolution of a bifunctional enzyme: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Proceedings of the National Academy of Sciences, USA 86, 9642–9646.
Bevan M. 1984. Binary Agrobacterium vectors for plant transformation. Nucleic Acids Research 12, 8711–8721.
Blanc G, Hokamp K, Wolfe KH. 2003. A recent polyploidy superimposed an older large-scale duplications in the Arabidopsis genome. Genome Research 13, 137–144.
Bond CS, White MF, Hunter WN. 2001. High resolution structure of the phosphohistidine-activated form of Escherichia coli cofactor-dependent phosphoglycerate mutase. Journal of Biological Chemistry 276, 3247–3253.
Bond CS, White MF, Hunter WN. 2002. Mechanistic implications for Escherichia coli cofactor-dependent phosphoglycerate mutase based on the high-resolution crystal structure of a vanadate complex. Journal of Molecular Biology 316, 1071–1081.[CrossRef][Web of Science][Medline]
Botha FC, Dennis DT. 1986a. Isozymes of phosphoglyceromutase from the developing endosperm of Ricinus communis: isolation and kinetic properties. Archives of Biochemistry and Biophysics 245, 96–103.[CrossRef][Web of Science][Medline]
Botha FC, Dennis DT. 1986b. Phosphoglyceromutase activity and concentration in the endosperm of developing and germinating Ricinus communis seeds. Canadian Journal of Botany 65, 1908–1912.[Web of Science]
Bradford MM. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254.[CrossRef][Web of Science][Medline]
Carreras J, Mezquita J, Bosch J, Bartrons R, Pons G. 1982. Phylogeny and ontogeny of the phopshoglycerate mutases. IV. Distribution of glycerate-2,3-P2 dependent and independent phophoglycerate mutases in algae, fungi, plants, and animals. Comparative Biochemistry and Physiology 71B, 591–597.
Collet JF, Stroobant V, Van Schaftingen E. 2001. The 2,3-bisphosphoglycerate-independent phosphoglycerate mutase from Trypanosoma brucei: metal-ion dependency and phosphoenzyme formation. FEMS Microbiology Letters 204, 39–44.[CrossRef][Web of Science][Medline]
Doyle JJ, Doyle JL. 1990. Isolation of plant DNA from fresh tissues. Focus 12, 13–15.
Draborg H, Villadsen D, Nielsen TH. 1999. Cloning, characterization and expression of a bifunctional fructose-6-phosphate, 2-kinase/fructose-2,6-bisphosphatase from potato. Plant Molecular Biology 39, 709–720.[CrossRef][Web of Science][Medline]
Draborg H, Villadsen D, Nielsen TH. 2001. Transgenic Arabidopsis plants with decreased activity of fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase have altered carbon partitioning. Plant Physiology 126, 750–758.
Felsenstein J. 1989. PHYLIP-Phylogeny Inference Package (Version 3.2). Cladistics 5, 164–166.
Fothergill-Gilmore LA, Michels PAM. 1993. Evolution of glycolysis. Progress in Biophysics and Molecular Biology 59, 105–235.[CrossRef][Web of Science][Medline]
Fothergill-Gilmore LA, Watson HC. 1989. The phosphoglycerate mutases. Advances in Enzymology 62, 227–313.
Fraser HI, Kvaratskhelia M, White MF. 1999. The two analogous phosphoglycerate mutases of Escherichia coli. FEBS Letters 455, 344–348.[CrossRef][Web of Science][Medline]
Graña X, de la Ossa PP, Broceño C, Stöcker M, Garriga J, Puigdomènech P, Climent F. 1995. 2,3-bisphosphoglycerate-independent phosphoglycerate mutase is conserved among different phylogenic kingdoms. Comparative Biochemistry and Physiology 112B, 287–293.
Hua XJ, Vande Cotte B, Van Montagu M, Verbruggen N. 1997. Developmental regulation of pyrroline-5-carboxylate reductase gene expression in Arabidopsis. Plant Physiology 114, 1215–1224.[Abstract]
Huang Y, Blakeley SD, McAleese SM, Fothergill-Gillmore LA, Dennis DT. 1993. Higher-plant cofactor-independent phosphoglyceromutase: purification, molecular characterization and expression. Plant Molecular Biology 23, 1039–1053.[CrossRef][Web of Science][Medline]
Jedrzejas MJ. 2000. Structure, function, and evolution of phosphoglycerate mutases: comparison with fructose-2,6,-bisphosphatase, acid phosphatase, and alkaline phosphatase. Progress in Biophysics and Molecular Biology 73, 263–287.[CrossRef][Web of Science][Medline]
Larondelle Y, Mertens E, Van Schatingen E, Hers H-G. 1986. Purification and properties of spinach leaf phosphofructokinase 2/fructose 2,6-bisphosphatase. European Journal of Biochemestry 161, 351–357.[CrossRef]
Liberator P, Anderson J, Feiglin M, Sardana M, Griffin P, Schmatz D, Myers RW. 1998. Molecular cloning and functional expression of mannitol-1-phosphatase from the apicomplexan parasite Eimeria tenella. Journal of Biological Chemistry 273, 4237–4244.
MacDonald FD, Chou Q, Buchanan BB, Stitt M. 1989. Purification and characterization of fructose-2,6-bisphosphatase, a substrate-specific cytosolic enzyme from leaves. Journal of Biological Chemistry 264, 5540–5544.
MacDonald FD, Cséke C, Chou Q, Buchanan BB. 1987. Activities synthesizing and degrading fructose 2,6-bisphosphate in spinach leaves reside on different proteins. Proceedings of the National Academy of Sciences, USA 84, 2742–2746.
Mani S, Van de Cotte B, Van Montagu M, Verbruggen N. 2002. Altered levels of proline dehydrogenase cause hypersensitivity to proline and its analogs in Arabidopsis. Plant Physiology 128, 1–11.
Markham JE, Kruger NJ. 2002. Kinetic properties of bifunctional 6-phosphofructo-2 kinase/fructose-2,6-bisphosphatase from spinach leaves. European Journal of Biochemistry 269, 1267–1277.[Web of Science][Medline]
Mazarei M, Lennon KA, Puthoff DP, Rodermel SR, Baum TJ. 2003. Expression of an Arabidopsis phosphoglycerate mutase homologue is localized to apical meristems, regulated by hormones, and induced by sedentary plant–parasitic nematodes. Plant Molecular Biology 53, 513–530.[CrossRef][Web of Science][Medline]
Murashige T, Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15, 473–497.[CrossRef]
Murphy JE, Stec B, Ma L, Kantrowitz ER. 1997. Trapping and visualization of a covalent enzyme–phosphate intermediate. Nature Structural Biology 4, 618–622.[CrossRef][Web of Science][Medline]
Notredame C, Higgins DG, Heringa J. 2000. T-Coffee: a novel method for fast and accurate multiple sequence alignment. Journal of Molecular Biology 302, 205–217.[CrossRef][Web of Science][Medline]
Okar DA, Manzano A, Navarro-Sabate A, Riera L, Bartrons R, Lange AJ. 2001. PFK-2/FBPase-2: maker and breaker of the essential biofactor fructose-2,6-bisphosphate. Trends in Biochemical Sciences 26, 30–35.[CrossRef][Web of Science][Medline]
O'Toole GA, Trzebiatowski JR, Escalante-Semerena JC. 1994. The cobC gene of Salmonella typhimurium codes for a novel phosphatase involved in the assembly of the nucleotide loop of cobalamin. Journal of Biological Chemistry 269, 26503–26511.
Pilkis SJ, Claus TH, Kurland IJ, Lange AJ. 1995. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: a metabolic signaling enzyme. Annual Review of Biochemistry 64, 799–835.[CrossRef][Web of Science][Medline]
Plaxton WC. 1996. The organization and regulation of plant glycolysis. Annual Review of Plant Physiology and Plant and Molecular Biology 47, 185–214.
Rigden DJ. 2003. Unexpected catalytic site variation in phosphoprotein phosphatase homologues of cofactor-dependent phosphoglycerate mutase. FEBS Letters 536, 77–84.[CrossRef][Web of Science][Medline]
Rigden DJ, Littlejohn JE, Henderson K, Jedrzejas MJ. 2003. Structures of phosphate and trivanadate complexes of Bacillus stearothermophilus phosphatase PhoE: structural and functional analysis in the cofactor-dependent phosphoglycerate mutase superfamily. Journal of Molecular Biology 325, 411–420.[CrossRef][Web of Science][Medline]
Rigden DJ, Mello LV, Setlow P, Jedrzejas MJ. 2002. Structure and mechanism of action of a cofactor-dependent phosphoglycerate mutase homologue from Bacillus stearothermophilus with broad specificity phosphatase activity. Journal of Molecular Biology 315, 1129–1143.[CrossRef][Web of Science][Medline]
Rigden DJ, Bagyan I, Lamani E, Setlow P, Jedrzejas MJ. 2001. A cofactor-dependent phosphoglycerate mutase homologue from Bacillus stearothermophilus is actually a broad specificity phosphatase. Protein Science 10, 1835–1846.[CrossRef][Web of Science][Medline]
Rodicio R, Heinisch J. 1987. Isolation of the yeast phosphoglyceromutase gene and construction of deletion mutants. Molecular and General Genetics 206, 133–140.
Rogl H, Kosemund K, Kühlbrandt W, Collinson I. 1998. Refolding of Escherichia coli produced membrane protein inclusion bodies immobilized by nickel chelating chromatography. FEBS Letters 432, 21–26.[CrossRef][Web of Science][Medline]
Schneider A, Häusler RE, Kolukisaoglu U, Kunze R, van der Graaff E, Schwacke R, Catoni E, Desimone M, Flügge UI. 2002. An Arabidopsis thaliana knock-out mutant of the chloroplast triose phosphate/phosphate translocator is severely compromised only when starch synthesis, but not starch mobilization is abolished. The Plant Journal 32, 685–699.[CrossRef][Web of Science][Medline]
Singh RP, Setlow P. 1979. Purification and properties of phosphoglycerate phosphomutase from spores and cells of Bacillus megaterium. Journal of Bacteriology 137, 1024–1027.
Scott P, Lange AJ, Kruger NJ. 2000. Photosynthetic carbon metabolism in leaves of transgenic tobacco (Nicotiana tabacum L.) containing decreased amounts of fructose 2,6-bisphosphate. Planta 211, 864–873.[CrossRef][Web of Science][Medline]
Scott P, Lange AJ, Pilkis SJ, Kruger NJ. 1995. Carbon metabolism in leaves of transgenic tobacco (Nicotiana tabacum L.) containing elevated fructose 2,6-bisphosphate levels. The Plant Journal 7, 461–469.[CrossRef][Web of Science][Medline]
Stitt M. 1990a. Fructose 2,6-bisphosphate as a regulatory metabolite in plants. Annual Review of Plant Physiology and Plant Molecular Biology 41, 153–185.[CrossRef][Web of Science]
Stitt M. 1990b. Fructose 2,6-bisphosphate. In: Methods in plant biochemistry, Vol. 3. 87–92.
Sun W, Bernard C, Van de Cotte B, Van Montagu M, Verbruggen N. 2001. At-HSP17.6A encoding a small heat-shock protein in Arabidopsis can enhance osmotolerance upon overexpression. The Plant Journal 27, 407–415.[CrossRef][Web of Science][Medline]
Todd AE, Orengo CA, Thornton JM. 2002. Sequence and structural differences between enzyme and nonenzyme homologues. Structure 10, 1435–1451.[Medline]
Trevanion SJ. 2002. Regulation of sucrose and starch synthesis in wheat (Triticum aestinum L) leaves: role of fructose-2,6-bisphosphate. Planta 215, 653–665.[CrossRef][Web of Science][Medline]
Verwoerd TC, Dekker BMM, Hoekema A. 1989. A small scale procedure for the rapid isolation of plant RNAs. Nucleic Acids Research 17, 2362.
Villadsen D, Rung JH, Draborg H, Nielsen TH. 2000. Structure and heterologous expression of a gene encoding fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase from Arabidopsis thaliana. Biochimica et Biophysica Acta 1492, 406–413.[Medline]
Wang J-L, Walling LL, Jauh GY, GU Y-Q, Lord EM. 1996. Lily cofactor-independent phosphoglycerate mutase: purification, partial sequencing, and immunolocalization. Planta 200, 343–352.[Web of Science][Medline]
Westram A, Lloyd JR, Roessner U, Riesmeier JW, Kossmann J. 2002. Increases of 3-phosphoglyceric acid in potato plants through antisense reduction of cytoplasmic phosphoglycerate mutase impairs photosynthesis and growth, but does not increase starch contents. Plant, Cell and Environment 25, 1133–1143.[CrossRef]
Yuen MH, Mizuguchi H, Lee YH, Cook PF, Uyeda K, Hasemann CA. 1999. Crystal structure of the H256A mutant of rat testis fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase. Fructose 6-phosphate in the active site leads to mechanisms for both mutant and wild-type bisphosphatase activities. Journal of Biological Chemistry 274, 2176–2184.
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