JXB Advance Access originally published online on March 31, 2003
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Journal of Experimental Botany, Vol. 54, No. 386, pp. 1351-1360,
May 1, 2003
© 2003 Oxford University Press
Molecular and biochemical characterization of cytosolic phosphoglucomutase in wheat endosperm (Triticum aestivum L. cv. Axona)
Received 26 August 2002; Accepted 12 February 2003
1 School of Biological Sciences, 3.614 Stopford Building, Oxford Road, University of Manchester, Manchester M13 9PT, UK
2 Department of Botany, College of Biological Sciences, University of Guelph, Guelph, Ontario, N1G 4C4, Canada
Abbreviations: ADH, alcohol dehydrogenase; ADPG, ADPglucose; AGPase, ADPglucose pyrophosphorylase; APPase, alkaline inorganic pyrophosphatase; cyt c oxidase, cytochrome c oxidase; dpa, days post-anthesis; Na2-EDTA, disodium ethylenediaminetetra-acetic acid; G1P, glucose 1-phosphate; G6P, glucose 6-phosphate; G16BP, glucose 1,6-bisphosphate; PGM, phosphoglucomutase; PMSF, phenyl methyl sulphonyl fluoride; UGPase, uridine 5' diphosphate glucose pyrophosphorylase.
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Abstract |
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Evidence from a number of plant tissues suggests that phosphoglucomutase (PGM) is present in both the cytosol and the plastid. The cytosolic and plastidic isoforms of PGM have been partially purified from wheat endosperm (Triticum aestivum L. cv. Axona). Both isoforms required glucose 1,6-bisphosphate for their activity with Ka values of 4.5 µM and 3.8 µM for cytosolic and plastidic isoforms, respectively, and followed normal Michaelis–Menten kinetics with glucose 1-phosphate as the substrate with Km values of 0.1 mM and 0.12 mM for the cytosolic and plastidic isoforms, respectively. A cDNA clone was isolated from wheat endosperm that encodes the cytosolic isoform of PGM. The deduced amino acid sequence shows significant homology to PGMs from eukaryotic and prokaryotic sources. PGM activity was measured in whole cell extracts and in amyloplasts isolated during the development of wheat endosperm. Results indicate an approximate 80% reduction in measurable activity of plastidial and cytosolic PGM between 8 d and 30 d post-anthesis. Northern analysis showed a reduction in cytosolic PGM mRNA accumulation during the same period of development. The implications of the changes in PGM activity during the synthesis of starch in developing endosperm are discussed.
Key words: Phosphoglucomutase, starch synthesis, Triticum aestivum.
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Introduction |
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Phosphoglucomutase (PGM, EC 2.7.5.1 [EC] ) catalyses the interconversion of glucose 1-phosphate (G1P) and glucose 6-phosphate (G6P), with glucose 1,6-bisphosphate (G16BP) being a cofactor in this reaction (Ray et al., 1983). In plant tissues, PGM is present in the cytosol and the plastid (Mühlbach and Schnarrenberger, 1978; Sangwan and Singh, 1987; Popova et al., 1998). The PGM reaction is thought to be important in the partitioning of carbon between the pathways of starch synthesis and carbohydrate oxidation (Tetlow et al., 1998; Esposito et al., 1999); alterations in the levels of plastidic and cytosolic PGM in dicotyledenous species and mutations in plastidic PGM have been shown to have a deleterious effect on starch synthesis (Harrison et al., 2000; Tauberger et al., 2000; Fernie et al., 2001, 2002).
The plastidic PGM isoform provides the substrate (G1P) for the ADP-glucose pyrophosphorylase (AGPase) reaction, which is the first committed step of the starch synthetic pathway. In plants that rely upon the import of G6P into amyloplasts, there is an absolute requirement for the plastidial isoform of PGM. The absence or reduction of plastidial PGM in mutants of Arabidopsis thaliana (Caspar et al., 1985), Nicotiana sylvestris (Hanson and McHale, 1988), pea (Harrison et al., 1998, 2000), and in transgenic potato plants (Tauberger et al., 2000; Fernie et al., 2001) have resulted in starchless or near-starchless phenotypes, indicating that in these dicotyledonous tissues the plastidic isoform plays an essential role in starch synthesis.
The antisense repression of the cytosolic PGM isoform in potato tubers results in dramatic alterations in both plant morphology and in carbon partitioning between sink and source organs. As G6P is viewed as the first glycolytic intermediate it is suggested that the cytosolic PGM isoform may play an important role in the partitioning of carbon between the starch synthetic (G1P-utilizing) and glycolytic pathways (G6P-utilizing) in these tissues (Fernie et al., 2002).
There is now evidence that in the developing endosperm of maize (Denyer et al., 1996), barley (Thorbjørnsen et al., 1996) and wheat (Beckles et al., 2001; Tetlow et al., 2003) ADP-glucose (ADPG) can be synthesized in the cytosol and taken up directly by the amyloplast as well as being synthesized within the organelle. The possession of a cytosolic AGPase could bypass the need for the G1P produced by the plastidial PGM reaction, since ADPG synthesis could be directly linked to the uridine 5' diphosphate glucose pyrophosphorylase (UGPase) reaction. Wheat endosperm can import G1P, G6P and ADPG into amyloplasts, but whilst G6P can be transported by these organelles it is not able to support starch synthesis in vitro (Tetlow et al., 1994, 1998). PGM catalyses a reaction that at equilibrium results in a 20-fold excess of G6P over G1P (King, 1970) and, therefore, in this tissue, PGM may be a potential drain on the G1P pool either in the cytosol or the amyloplast with respect to starch synthesis. Consequently, the relationship between PGM and starch synthesis in developing endosperm is likely to be different from that observed in other species and organs such as the potato tuber.
This study aims to determine how the plastidic and cytosolic isoforms are regulated in relation to starch synthesis in the developing wheat grain. The purification and biochemical characterization of plastidic and cytosolic PGM isoforms from wheat endosperm amyloplasts, are described here. The isolation of a full-length cDNA encoding cytosolic PGM of wheat endosperm is also described. RNA analysis was performed to examine the developmental regulation of cytosolic PGM mRNA accumulation. The biochemical activity of PGM was measured in isolated amyloplasts and whole cell extracts to examine the developmental changes occurring during grain development.
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Materials and methods |
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Materials
All reagents, substrates and enzymes were purchased from Sigma–Aldrich Co. Ltd. (Poole, Dorest, UK) and BDH–Merck Ltd. (Lutterworth, Leicestershire, UK), unless otherwise stated. All coupling enzymes used in enzymes assays had (NH4)2SO4 removed by centrifugation at 13 000 g for 5 min; the supernatant was discarded and the pellet resuspended in the appropriate assay buffer. All other reagents were of analytical grade and aqueous solutions were made up with distilled water.
Protease inhibitors (bestatin, E63, pepstatin, and leupeptin) were purchased from Roche Diagnostics Ltd. (Lewes, East Sussex, UK). Agarose, RNA ladder (high range) and restriction enzymes (EcoRI and XhoI) were purchased from Helena Biosciences (Sunderland, Tyne and Wear, UK). Phenol was purchased from BioGene Ltd. (Kimbolton, UK).
Plant material, amyloplast isolation, and whole cell extract preparation
Spring wheat (Triticum aestivum L. cv. Axona) was grown under conditions previously described (Tetlow et al., 1993) and the developing ears tagged at the onset of anthesis (the first appearance of anthers). Endosperm tissue for amyloplast preparations was obtained from developing grains taken from the mid-ear region of the head between 5 d and 30 d post-anthesis (dpa). Amyloplasts were purified by centrifuging twice through a layer of 3% (w/v) 5-(N-2,3-dihydroxypropylacetamido)-2, 4, 6-triiodo-N, N'-bis-(2,3-dihydroxy propyl) isophthalamide (Nycodenz, Robbins Scientific, Knowle, Sohihul, UK) as previously described except that the 0.1% (w/v) bovine serum albumin was omitted (Tetlow et al., 1993). Amyloplast stroma was prepared from lysates using the methods described by Tetlow et al. (2003). To determine total enzyme activity from wheat endosperm whole cell extracts, 10–20 grains were isolated, the endosperm dissected out and homogenized in 1 cm3 of ice-cold rupturing buffer (100 mM N-[2-hydroxy-1,1-bis (hydroxymethyl) ethyl] glycine (Tricine)–NaOH (pH 7.8), 1 mM disodium-ethylenediaminetetra-acetic acid (Na2-EDTA), 1 mM dithiothreitol, 1 mM phenyl methyl sulphonyl fluoride (PMSF), 5 mM MgCl2, 1 µM bestatin, 1 µM E64, 1 µM pepstatin, and 1 µM leupeptin). The extract was clarified by centrifugation at 13 000 g for 30 min at 4 °C, and the supernatant removed and assayed immediately for PGM and subcellular marker enzymes (see below).
Enzyme assays
PGM (EC. 2.7.5.1
[EC]
) was assayed at 25 °C in a 1 cm3 reaction mixture containing 30 mM 1,3-diaza 2,4-cyclopentadiene (imidazole)–HCl (pH 7.4), 3.3 mM magnesium chloride (MgCl2), 0.85 mM nicotinamide adenine dinucleotide, oxidized (NAD), 0.9 mM Na2-EDTA, 15 µM G16BP, and 0.7 U glucose 6-phosphate dehydrogenase (from Leuconostoc mesenteroides). Assays were initiated with 1.5 mM G1P and increased absorbance was measured at 340 nm.
The substrate affinities for each partially purified isoform were measured by varying G1P and G16BP independently. In all assays the G1P used was a preparation essentially free from G16BP (Sigma, grade IV).
For each preparation of isolated amyloplasts the following enzyme assays were carried out at 25 °C according to methods described previously: alkaline inorganic pyrophosphatase (APPase, EC 3.6.1.1 [EC] ) (Gross and ap Rees, 1986), UGPase (EC 2.7.7.9 [EC] ) (Müller-Röber et al., 1992), AGPase (EC 2.7.7.27 [EC] ) (Journet and Douce, 1985, except that G16BP was omitted), alcohol dehydrogenase (ADH, EC 1.1.1.1 [EC] ) (MacDonald and ap Rees, 1983) and cytochrome c oxidase (cyt c oxidase, EC 1.9.3.1 [EC] ) (Darley-Usmar et al., 1987).
Partial purification of PGM isoforms
Unless otherwise stated, all enzyme purification steps were carried out at 4 °C.
Solid ammonium sulphate was added to wheat endosperm whole cell extracts and isolated amyloplast stroma to 40% saturation and allowed to precipitate for 30 min. The precipitate was removed by centrifugation at 10 000 g for 20 min and the supernatant brought to 60% saturation by adding further amounts of ammonium sulphate. The precipitate, collected by centrifugation at 10 000 g for 20 min, was dissolved in 1 cm3 buffer containing 50 mM 2-amino-2-hydroxymethylpropane-1,3-diol (Tris)–HCl (pH 8.0), 1 mM Na2-EDTA and 1 mM PMSF (buffer A).
The above enzyme preparations (2 cm3) from both whole cell extracts and amyloplasts were applied to a 1 cm3 phenyl sepharose column (Amersham Pharmacia, Little Chalfont, Buckinghamshire, UK) previously equilibrated with buffer A containing 1 M ammonium sulphate at a flow rate of 1 cm3 min–1. The column was washed with 2 vols of buffer A with 1 M ammonium sulphate and then 1 cm3 fractions collected at a flow rate of 1 cm3 min–1 using a stepwise gradient of ammonium sulphate from 1.0 to 0 M. Fractions containing PGM activity were pooled and dialysed for 2 h in 2.0 l of buffer A.
The pooled fractions (2 cm3) were applied to a 1 cm3 HiTrap Q column (Amersham Pharmacia, Little Chalfont, Buckinghamshire, UK) pre-equilibrated with buffer A at a flow rate of 1 cm3 min–1. The column was washed with 2 vols of buffer A and then 1 cm3 fractions collected at a flow rate of 1 cm3 min–1 using a stepwise gradient of KCl from 0 to 0.2 M.
Starch and protein determination
Starch was extracted and measured according to the methods described in Tetlow et al. (1994). Protein was measured using a Bio-Rad protein assay dye reagent (Bio-Rad, Hemel Hempstead, Hertfordshire, UK) based on the method of Bradford (1976) using thyroglobulin as the standard.
Library screening
A cDNA library was initially prepared with mRNA isolated from wheat (Triticum aestivum cv. Axona) endosperm 10–14 dpa using a Stratagene 
Zap library kit and according to the manufacturer’s instructions. The library was hybridized at 50 °C overnight with a full-length 2.1 kbp oilseed rape plastidic PGM cDNA clone (Harrison et al., 2000) radioactively labelled with [
-32P]-dCTP using a random primer (Stratagene). Filters were washed under low stringency conditions for three 30 min washes using 2x SSC and 0.1% SDS at 50 °C.
RNA isolation and hybridization
Total RNA was isolated from different developmental stages (5–30 dpa) of wheat endosperm according to the method described by Lahners et al. (1988). For RNA blot hybridization 10 µg of the isolated total RNA from each developmental stage was denatured with formaldehyde and subjected to electrophoresis and blotted onto a Hybond N+ membrane as described previously (Fonseca et al., 1997). The probe used for the hybridization was either the isolated wheat cPGM or a wheat rRNA cDNA clone. In all cases, DNA probes were radiolabelled with [-32P]-dCTP using a random priming kit (Stratagene), prehybridized, hybridized and exposed to film as described previously (Fonseca et al., 1997). Autoradiograms were scanned and relative amounts of mRNAs determined as described previously (Fonseca et al., 1997).
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Results |
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Enzyme purification
Prior to purification of the plastidial PGM isoform from isolated amyloplasts, marker enzyme assays were carried out to check that the amyloplasts were essentially free from contamination by other cell compartments (Table 1). Less than 1% of the total activities of the cytosolic marker enzymes, ADH and UGPase, and the mitochondrial marker enzyme, cyt c oxidase, were recovered in the pellet, indicating that the level of contamination of plastids by cytosol and mitochondria was low. The sum of the enzyme activities in all the supernatant and pellet fractions obtained during an amyloplast preparation was approximately 100% of those in the original homogenate, indicating that there was no serious loss of enzyme activity during the preparation of amyloplasts. Allowing for organelle breakage and cytosolic contamination, approximately 26% of total cellular PGM is calculated as being present in amyloplasts (calculated from Table 1).
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PGM activity was precipitated between 40% and 60% ammonium sulphate saturation using either wheat endosperm tissue extracts or isolated amyloplasts. The total PGM activity of the amyloplast preparations more than doubled following ammonium sulphate fractionation, but this was not the case in the whole cell extracts (Table 2). PGM activity eluted in a single peak from the phenyl sepharose column between 1.0 M and 0.8 M ammonium sulphate (Fig. 1A). The enzyme preparation from whole cell extracts was resolved into two peaks of PGM activity following anion exchange chromatography (Fig. 1B). PGM peak 1 was eluted at approximately 0.1 M KCl and PGM peak 2 at approximately 0.15 M KCl. The enzyme preparation from isolated amyloplasts when loaded onto a Hi Trap Q column was resolved in a single peak of PGM activity eluting at 0.1 M KCl (Fig. 1C), coincident with the first peak obtained from whole cell extracts. This suggests that PGM peak 1 is the plastidic isoform of PGM and PGM peak 2 is the cytosolic isoform of PGM. The two isoforms, cytosolic and plastidic were purified 120-fold and 50-fold respectively (Table 2).
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Biochemical characterization of the partially purified PGM isoforms
The two partially purified isoforms of PGM were characterized with respect to pH, substrate affinities and molecular weight. The two isoforms of PGM had the same pH optimum, but in different buffers. The optimum assay conditions for the plastidic isoform was found to be pH 8.0 in glycylglycine buffer whereas the cytosolic isoform had optimal activity at pH 8.0 in Tris buffer. Both isoforms of PGM, at their respective pH optima, followed normal Michaelis–Menten kinetics with respect to G1P as the substrate. Km values for the cytosolic and plastidic isoforms as determined by Lineweaver–Burk plots were 99.7±1 µM (n=3) and 122±9 µM (n=3), respectively. The two isoforms had an absolute requirement for G16BP for their activity and the Ka values were 4.47±0.31 µM (n=3) and 3.78±0.36 µM (n=3), respectively, for the cytosolic and plastidic isoforms. The two isoforms had identical molecular weights of 61 kDa as determined by gel permeation chromatography and SDS electrophoresis (data not shown). A number of metabolites (ATP, AMP, ADP, 3-phosphoglyceric acid, inorganic pyrophosphate, ribulose 1,5-bisphosphate, and inorganic orthophosphate) were added to the PGM assay to determine whether they caused an activation or inhibition of the PGM isoforms. However, it was found that none of these metabolites had any significant effect on PGM activity (data not shown).
Sequence analysis of wheat endosperm cytosolic PGM
Screening of a wheat endosperm cDNA library with a plastidic oil seed rape cDNA clone that had sequence similarity to known PGM sequences resulted in the isolation of a cDNA clone that encoded a cytosolic isoform of PGM (EMBL accession number AJ313311
[GenBank]
). The full-length cDNA clone of cytosolic PGM from the wheat endosperm library was 2358 bp in length. This comprised a 339 bp 5' untranslated region, a 1743 bp open reading frame and a 276 bp 3' untranslated region. The open reading frame encoded a predicted protein of 581 amino acids (Fig. 2), with a predicted molecular mass of 63 kDa. Further confirmation that the PGM cDNA clone isolated was cytosolic came from analysis using PSORT and Chloro V1.1 computer programs (Emanuelsson et al., 1999). Neither of these transit peptide site prediction programs identified any sequence area that would suggest the isolated clone could be targeted to the plastid.
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Comparison of the deduced amino acid sequences of the cytosolic PGM from wheat endosperm to that of PGM sequences of cytosolic Bromus inermis (AF197925 [GenBank] ), plastidic Solanum tuberosum (AJ240053 [GenBank] ), Homo sapiens (M83088 [GenBank] ), and Saccharomyces cerevisiae (P33401 [GenBank] ), revealed long stretches of identical residues as shown in Fig. 2. The wheat endosperm cytosolic PGM had the highest amino acid sequence identity (80–90%) with the deduced cytosolic sequences of PGMs of Solanum tuberosum (AJ240054 [GenBank] ), Pisum sativum (AJ250769 [GenBank] ), Arabidopsis thaliana (AJ242650 [GenBank] ), Populus tremula (AF097938 [GenBank] ), Mesembryanthemum crystallinum (U84888 [GenBank] ), Zea mays 1 (U89341 [GenBank] ), Zea mays 2 (U89342 [GenBank] ), and Bromus inermis (AF197925 [GenBank] ). The cytosolic cDNA clone showed approximately 60% homology to plastidic sequences and approximately 50% homology to sequences from Homo sapiens, Saccharomyces cerevisiae and Agrobacterium tumefaciens (P39671 [GenBank] ). However, it had considerably lower homology (approximately 25%) to PGM sequences of E. coli (U08369 [GenBank] ) and spinach chloroplasts (X75898 [GenBank] ).
Phylogenetic analysis of PGM proteins
An alignment of protein sequences with homology to wheat endosperm cytosolic PGM was analysed to determine the probable phylogeny of the proteins by the parsimony method (Clustal W). The relationship between PGM sequences from different species is shown in Fig. 3.
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Analysis of an unrooted phylogenetic tree generated from an alignment of PGM protein sequences revealed three distinct classes corresponding to plant, bacterial and mammalian origin. The plant class of PGM is further divided into two sections corresponding to cytosolic and plastidic isoforms of the enzymes, indicating how the different PGMs have diverged during evolution.
Expression of cytosolic PGM mRNA transcripts during wheat endosperm development
A PGM transcript of approximately 2 kb was identified in the wheat endosperm and was found to be developmentally regulated. Figure 4 shows the normalized densitometry readings and indicates that the transcript levels peak at 8 dpa and decline thereafter throughout the period of endosperm development studied.
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Activity of PGM isoforms during wheat endosperm development
Whole cell extracts and amyloplasts were prepared from wheat endosperm at different stages of development. Marker enzyme assays were performed on isolated amyloplasts and whole cell extracts from each developmental stage, which showed the same level of contamination from cytosol and mitochondria in the plastids and the same enzyme recovery in the supernatant and pellet to those figures shown in Table 1.
The total activity of PGM, in whole cell extracts, peaks at 11 dpa and then declines by approximately 80% over the period of development studied (Fig. 5A). Plastidial PGM activity declines between 8 and 30 dpa by approximately 90% (Fig. 5B). The activity profile observed for PGM activity in whole cell extracts follows the same profile obtained for RNA expression of the cytosolic isoform of PGM (Fig. 4), and is consistent with the observation that the cytosolic enzyme constitutes the greater part of the total activity (Table 1).
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The amount of PGM activity in amyloplasts varied from 13% to 32% during development based on marker enzyme recoveries (data not shown). At 14 dpa approximately 26% of the total PGM activity is plastidial, which is consistent with previous determinations made in developing wheat endosperm (Esposito et al., 1999). Taking account of this and the measured total PGM activity per endosperm the calculated changes in the amount of cytosolic and plastidic PGM isoforms were determined (Fig. 5C). Between 5 and 30 dpa the starch content of the developing endosperm increased steadily from 3.96±0.90 (n=4) mg. per endosperm (5 dpa) to 16.72±0.62 (n=4) mg per endosperm at 30 dpa (Fig. 5C).
The relative changes of both isoforms of PGM, when activity is expressed per endosperm, peak at approximately 11 dpa and then steadily decline throughout the period of development studied (Fig. 5C). The relative changes of plastidial PGM are slightly different to those obtained when expressed per mg protein (Fig. 5B). This may be due to the fact that the protein content of the tissue also changes during development.
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Discussion |
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PGM activity in wheat endosperm was found to be present in two cell compartments (cytosol and plastid) and the properties of the two isoforms are very similar. Many of these properties are shared with PGMs from other plant species, mammalians and micro-organisms. The two partially purified PGM isoforms were characterized biochemically and it was shown that they have many basic properties in common with each other and with PGMs from other species such as potato tubers (Pressey, 1967) and developing seeds of Cassia corymbosa (Small and Matheson, 1979): with respect to pH optima, molecular size and substrate and activation affinities.
The Km values for G1P for the partially purified isoforms from wheat endosperm are approximately 2-fold higher than those reported for the PGM isoforms of spinach leaves (Mühlbach and Schnarrenberger, 1978), which have Km values of 40 µM and 60 µM for plastidic and cytosolic isoforms respectively, and a PGM isoform from isolated amyloplasts of wheat endosperm (40 µM, Esposito et al., 1999). Two isoforms of PGM have been identified in wheat endosperm previously (Sangwan and Singh, 1987). In that report, the peaks of PGM activity were resolved after anion exchange chromatography of tissue extracts, but the authors assigned the first peak (corresponding to peak 1 of Fig. 1B) as being cytosolic in origin and the second peak as plastidic. No sequence analysis of the isoforms, or separation of organelles was performed to confirm this and the conclusions drawn were based on small differences in kinetic properties of the two isozymes. It has been clearly shown in this report that this annotation was incorrect and that it is the PGM isozyme which elutes, from anion exchange chromatography, at the lower salt concentration which is plastidic in origin. Ka values for G16BP reported here (4.5 µM and 3.8 µM for cytosolic and plastidic isoforms, respectively) are lower than those reported for spinach leaf PGM (12.5 µM for the cytosolic isoform and 10 µM for the plastidic isoform) (Mühlbach and Schnarrenberger, 1978), but substantially higher than that previously reported for the plastid PGM in wheat endosperm (0.6 µM, Esposito et al., 1999). Although these Ka values for G16BP in wheat amyloplasts are in the same order of magnitude, they may be a result of differences in enzyme preparation; the PGM isoforms in the present study had each been purified several-fold from whole cell extracts, whereas the plastid PGM analysed by Esposito et al. (1999) was prepared by lysing purified amyloplasts. The intracellular concentrations of the activator are not known and it cannot be concluded that these differences are significant. However, estimates of the cytosolic G16BP concentration in developing wheat endosperm cells (approximately 8 µM) would suggest that both wheat PGM isoforms will be fully activated in vivo (Esposito et al., 1999). Although it has previously been observed that the mass action ratio for PGM in wheat endosperm amyloplasts can be displaced significantly from the equilibrium constant (Tetlow et al., 1998), no simple explanation for this phenomenon was found based on kinetic properties and response to other metabolites and it is concluded that other mechanisms, possibly including post-translational modification, must be involved in the regulation of this enzyme.
The sequence of the cytosolic wheat endosperm cDNA cloned here contains many regions that are conserved in human, yeast, cytosolic Bromus inermis and plastidic potato PGM sequences (Fig. 2). Regions of high homology included the catalytic centre (T/S-A-S-H-N motif), as identified in rat PGM by Milstein and Sanger (1961), and the metal ion-binding loop (D-G-D-G/A-D motif), as described in the crystal structure of muscle PGM by Dai et al. (1992). The serine (Ser) located at residue 116 in human PGM serves as the phosphate acceptor/donor in the catalytic process (Ray and Peck, 1972). The catalytic site was found at positions 121 to 125 in cytosolic wheat endosperm PGM, with Ser-123 being the acceptor/donor and the metal ion-binding loop was located at residues 298 to 302, similar to the enzyme from maize (Manjunath et al., 1998).
Phylogenetic analysis (Fig. 3) indicates the separation of the PGMs into classes corresponding to plant, bacterial and mammalian origin. The plant enzyme is further divided into two classes corresponding to plastidic and cytosolic, as there is a distinct separation between the two isoforms. The plastidic sequences have approximately equal distances between plant and prokaryote branches and is consistent with the hypothesis that plastids are derived from ancient prokaryote symbionts. Branches from cytosolic and plastidic plant sequences, yeast, bacteria, and mammalian have approximate equal distances indicating the general homology between all PGM sequences, and suggesting that the PGM sequences from the different kingdoms diverged from each other early in evolution and then evolved independently.
A PGM transcript of approximately 2 kb was identified (Fig. 4) in the wheat endosperm and was found to be developmentally regulated confirming that regulation is transcriptional. Interestingly, the major period of starch synthesis in wheat endosperm begins at approximately 10 dpa (Briarty et al., 1979) when the PGM transcript levels start to decline. The reason for the failure to isolate a wheat plastidic cDNA clone using the 2.1 kbp oilseed rape plastidic cDNA clone is unclear. It may be that, at the developmental stage the endosperm cDNA library was prepared (10–14 dpa), mRNA transcript levels for this protein are too low for its isolation.
Recent work carried out by Tetlow et al. (2003) measured the activity of AGPase throughout wheat endosperm development. This work together with the data presented here suggests that there is an inverse relationship between the two enzymes. When PGM activity is high, the AGPase activity is low giving rise to a high ratio between PGM and AGPase. The ratio between PGM and AGPase changes as AGPase activity increases and PGM activity decreases (as the starch content of the endosperm is increasing). The high activity of the PGM isoforms early in development may be due to the fact that, at this stage of development, there is a high demand for substrates for energy, cell division, respiration, and glycolysis.
In tissues containing both a cytosolic and a plastidic AGPase, for example, maize (Denyer et al., 1996), barley (Thorbjørnsen et al., 1996), rice (Sikka et al., 2001), and wheat (Tetlow et al., 2003), it may be that neither the plastidic nor cytosolic PGM isoforms contribute greatly to starch synthesis to the same extent as in other tissues, for example, potato, where AGPase is exclusively plastidial (Fernie et al., 2001, 2002). The decline observed in PGM expression and activity in the cytosol and amyloplasts of developing wheat endosperm during the major period of grain filling, is consistent with the notion that its role is not as important as in tissues containing only plastidic AGPase. Interestingly, Pan et al. (1990) tested a number of maize inbred lines and found that the majority had only one form of PGM activity and that this activity was due to the plastidic isozyme. The lack of the cytosolic isozyme in these maize inbred lines had no discernible phenotypic effects and caused no reduction in starch content (Pan et al., 1990) reinforcing the view that its role in starch synthesis is not critical in cereal endosperms. The import of ADPG from the cytosol in such tissues would bypass the need for the import of hexose phosphates, therefore decreasing the need for the interconversion of hexose phosphates by the PGM isoforms in the cytosol and the amyloplast.
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Acknowledgements |
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This work was financially supported by the Biotechnology and Biological Sciences Research Council (BBSRC) through a RASP studentship. We thank Dr T Wang (John Innes Centre, Norwich) for the gift of the plastidic oil seed rape clone and Mr TW Heaton for growing the wheat at the Botany Experimental Grounds (Manchester).
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References |
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