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Journal of Experimental Botany, Vol. 54, No. 386, pp. 1351±1360, May 2003 DOI: 10.1093/jxb/erg151 RESEARCH PAPER Molecular and biochemical characterization of cytosolic phosphoglucomutase in wheat endosperm (Triticum aestivum L. cv. Axona) Emma J. Davies1, Ian J. Tetlow2, Caroline G. Bowsher1 and Michael J. Emes2,3 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 Received 26 August 2002; Accepted 12 February 2003 Abstract Introduction 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 puri®ed from wheat endosperm (Triticum aestivum L. cv. Axona). Both isoforms required glucose 1,6-bisphosphate for their activity with Ka values of 4.5 mM and 3.8 mM 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 signi®cant 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. Phosphoglucomutase (PGM, EC 2.7.5.1) 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 (MuÈhlbach 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 ®rst 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. Key words: Phosphoglucomutase, starch synthesis, Triticum aestivum. 3 To whom correspondence should be addressed. Fax: +1 519 767 1991. E-mail: [email protected] 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 ¯uoride; UGPase, uridine 5¢ diphosphate glucose pyrophosphorylase. Journal of Experimental Botany, Vol. 54, No. 386, ã Society for Experimental Biology 2003; all rights reserved 1352 Davies et al. 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 ®rst 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 (Thorbjùrnsen 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 puri®cation 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. Materials and methods 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 ®rst 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 puri®ed by centrifuging twice through a layer of 3% (w/v) 5-(N-2,3dihydroxypropylacetamido)-2, 4, 6-triiodo-N, N¢-bis-(2,3-dihydroxypropyl) isophthalamide (Nycodenz, Robbins Scienti®c, 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 ¯uoride (PMSF), 5 mM MgCl2, 1 mM bestatin, 1 mM E64, 1 mM pepstatin, and 1 mM leupeptin). The extract was clari®ed 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) 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 mM 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 af®nities for each partially puri®ed 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) (Gross and ap Rees, 1986), UGPase (EC 2.7.7.9) (MuÈller-RoÈber et al., 1992), AGPase (EC 2.7.7.27) (Journet and Douce, 1985, except that G16BP was omitted), alcohol dehydrogenase (ADH, EC 1.1.1.1) (MacDonald and ap Rees, 1983) and cytochrome c oxidase (cyt c oxidase, EC 1.9.3.1) (Darley-Usmar et al., 1987). Partial puri®cation of PGM isoforms Unless otherwise stated, all enzyme puri®cation 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, Characterization of cytosolic phosphoglucomutase in wheat endosperm 1353 3 was dissolved in 1 cm buffer containing 50 mM 2-amino-2hydroxymethylpropane-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 ¯ow 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 ¯ow 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 ¯ow rate of 1 cm3 min±1. The column was washed with 2 vols of buffer A and then 1 cm3 fractions collected at a ¯ow 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 lZap 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 [a-32P]-dCTP using a random primer (Stratagene). Filters were washed under low stringency conditions for three 30 min washes using 23 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 mg 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 [a-32P]-dCTP using a random priming kit (Stratagene), prehybridized, hybridized and exposed to ®lm as described previously (Fonseca et al., 1997). Autoradiograms were scanned and relative amounts of mRNAs determined as described previously (Fonseca et al., 1997). Results Enzyme puri®cation Prior to puri®cation 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, Table 1. Distribution of enzymes between cytosol and amyloplasts in wheat endosperm Activities of marker enzymes in preparations of amyloplasts puri®ed from cell-free extracts of developing wheat endosperm at approximately 14 dpa. Values in bold parentheses are estimates of the percentage of the total AGPase and PGM activity that is plastidial, and are calculated from the following equation of Beckles et al. (2001) which is used to estimate the percentage of extra-plastidial AGPase activity (using APPase as the plastidial marker enzyme and UGPase as the cytosolic marker enzyme): % Extra-plastidial activity=(% Plastid marker activity in pellet±% Total AGPase activity in pellet)/(% Plastid marker activity in pellet±% Cytosol marker activity in pellet) All values are means 6SEM of three amyloplast preparations with each sample assayed in triplicate. Enzyme Activity recovered in supernatant and pellet as % of whole cell extracts % Recovery in amyloplasts from whole cell extracts APPase UGPase ADH Cyt c oxidase AGPase PGM 98.963.7 111.566.2 102.364.1 89.361.3 97.863.2 105.267.1 25.3964.61 0.2460.07 0.1560.06 0.2060.08 8.2062.12 [32%] 6.7360.99 [26%] 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). 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 ®rst 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 puri®ed 120-fold and 50-fold respectively (Table 2). 1354 Davies et al. Table 2. Puri®cation of PGM isoforms from wheat endosperm whole cell extracts and amyloplast stroma Wheat endosperm whole cell extracts (WSE) were prepared from 200 ears of developing wheat taken at approximately 12±20 dpa. Puri®cation of the two PGM isoforms was carried out as described in the Materials and methods section. Amyloplasts (AP) from wheat endosperm (isolated at approximately 12±20 dpa) were isolated and puri®cation of the plastidic PGM isoform was carried out as described in the Materials and methods section. Data shown is from a single representative puri®cation experiment. Puri®cation stage Homogenate Ammonium sulphate precipitation (40± 60% saturation) Phenyl sepharose Hi Trap Q PGM peak 1 PGM peak 2 Volume (ml) Total protein (mg) Speci®c activity (mmol min±1 mg±1 protein) WSE AP WSE AP 430 14 10 4 229.6 31.0 22.64 3.39 0.24 0.78 0.13 1.85 1 3.22 1 14.57 2 2 3.5 0.78 2.93 6.84 12.12 53.86 2 0.23 0.36 0.33 ± 26.92 12.04 13.59 ± 111.20 49.96 106.61 ± 2 2 ± Biochemical characterization of the partially puri®ed PGM isoforms The two partially puri®ed isoforms of PGM were characterized with respect to pH, substrate af®nities 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 99.761 mM (n=3) and 12269 mM (n=3), respectively. The two isoforms had an absolute requirement for G16BP for their activity and the Ka values were 4.4760.31 mM (n=3) and 3.7860.36 mM (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 signi®cant 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). The full-length cDNA clone of cytosolic PGM from the WSE Puri®cation -fold AP WSE AP 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 con®rmation 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 identi®ed any sequence area that would suggest the isolated clone could be targeted to the plastid. Comparison of the deduced amino acid sequences of the cytosolic PGM from wheat endosperm to that of PGM sequences of cytosolic Bromus inermis (AF197925), plastidic Solanum tuberosum (AJ240053), Homo sapiens (M83088), and Saccharomyces cerevisiae (P33401), 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), Pisum sativum (AJ250769), Arabidopsis thaliana (AJ242650), Populus tremula (AF097938), Mesembryanthemum crystallinum (U84888), Zea mays 1 (U89341), Zea mays 2 (U89342), and Bromus inermis (AF197925). 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). However, it had considerably lower homology (approximately 25%) to PGM sequences of E. coli (U08369) and spinach chloroplasts (X75898). 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 Characterization of cytosolic phosphoglucomutase in wheat endosperm 1355 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 identi®ed 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. Fig. 1. Elution pro®les of PGM activity. (A) Elution pro®le of PGM activity from wheat endosperm whole cell extract following chromatography on a 1 cm3 phenyl sepharose column. Approximately 2 mg of protein was loaded onto the column. Data are means of three replicates 6SEM and 1 unit is equivalent to 1 mmol min±1. In (A) to (C), the ®lled squares show PGM activity and the open squares show protein content. (B) Elution of PGM activity from wheat endosperm whole cell extracts following anion exchange chromatography. Approximately 1 mg of protein containing PGM activity eluted from the phenyl sepharose column was loaded onto a 1 cm3 HiTrap Q column. (C) Elution of PGM activity from wheat endosperm amyloplast stroma following anion exchange chromatography. Approximately 1 mg of amyloplast stromal protein containing PGM activity was loaded onto a 1 cm3 HiTrap Q column following elution from the phenyl sepharose column. the parsimony method (Clustal W). The relationship between PGM sequences from different species is shown in Fig. 3. 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 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 ®gures 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 pro®le observed for PGM activity in whole cell extracts follows the same pro®le 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). 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 3.9660.90 (n=4) mg. per endosperm (5 dpa) to 16.7260.62 (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. 1356 Davies et al. Discussion 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 puri®ed 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 af®nities. The Km values for G1P for the partially puri®ed isoforms from wheat endosperm are approximately 2-fold higher than those reported for the PGM isoforms of spinach leaves (MuÈhlbach and Schnarrenberger, 1978), which have Km values of 40 mM and 60 mM for plastidic and cytosolic isoforms respectively, and a PGM isoform from isolated amyloplasts of wheat endosperm (40 mM, Esposito et al., 1999). Two isoforms of PGM have been identi®ed 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 ®rst 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 con®rm 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 mM and 3.8 mM for cytosolic and plastidic isoforms, respectively) are lower than those reported for spinach leaf PGM (12.5 mM for the cytosolic isoform and 10 mM for the plastidic isoform) (MuÈhlbach and Schnarrenberger, 1978), but substantially higher than that previously reported for the plastid PGM in wheat endosperm (0.6 mM, 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 Fig. 2. Amino acid sequence alignment of PGMs from different species. Key to ®gure: 1, wheat endosperm cytosolic PGM (AJ313311); 2, Bromus inermis cytosolic PGM (AF197925); 3, Solanum tuberosum plastidic PGM (AJ240053); 4, Homo sapiens PGM (M83088), and 5, Saccharomyces cerevisiae PGM (P33401). The star (*) represents amino acids that are identical and the dot (.) indicates where the amino acids are similar or conserved. The catalytic centre and the metal ion-binding site, the two conserved regions of PGM, are indicated by the grey boxes. The sequence alignment was performed using the program Clustal W (Thompson et al., 1994). Characterization of cytosolic phosphoglucomutase in wheat endosperm 1357 Fig. 3. Unrooted phylogenetic tree of PGMs from different species. An optimal amino acid alignment was created with the program Clustal W and a phylogenetic tree was constructed. The tree was displayed with the Treeview program. The plant cytosolic PGM sequences used in the alignment were wheat (AJ313311), M. crystallinum (U84888), Bromus grass (AF197925), maize 1 (U89341), maize 2 (U89342), potato (AJ240054), Populus (AF097938), pea (AJ250769), and Arabidopsis (AJ242650); and the plastidic sequences used were pea (AJ250770), potato (AJ240053), oilseed rape (AJ250771), Arabidopsis (AJ242601), and spinach (X75898). The other PGM sequences used in the alignment were human (M83088), yeast (P33401), E. coli (U08369), and A. tumefaciens (P39671). differences in enzyme preparation; the PGM isoforms in the present study had each been puri®ed several-fold from whole cell extracts, whereas the plastid PGM analysed by Esposito et al. (1999) was prepared by lysing puri®ed amyloplasts. The intracellular concentrations of the activator are not known and it cannot be concluded that these differences are signi®cant. However, estimates of the cytosolic G16BP concentration in developing wheat endosperm cells (approximately 8 mM) 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 signi®cantly 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 modi®cation, 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 Fig. 4. RNA expression of the cytosolic isoform of PGM during wheat endosperm development between 5 and 30 dpa. (A) Northern analysis of PGM expression, (B) hybridization of rRNA speci®c cDNA probe to the same blot in (A), and (C) normalized densitometry readings of probe hybridization shown in (A) and (B). Approximately 10 mg of RNA was loaded in each lane. included the catalytic centre (T/S-A-S-H-N motif), as identi®ed 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 1358 Davies et al. Fig. 5. Changes in PGM activity during the development of wheat endosperm. (A) PGM activity measured in whole cell extracts of wheat endosperm between 8 and 30 dpa; (B) plastidial PGM activity measured in amyloplast stroma between 8 and 30 dpa and (C) changes in the relative levels of plastidic (®lled squares) and cytosolic (®lled diamonds) PGM compared with the starch content (®lled circles) measured in the endosperm between 5 and 30 dpa. PGM assays were performed as described in the Materials and methods section under optimized conditions with saturating concentrations of G1P and G16BP. Each value is the mean 6 SEM of four independent experiments. kingdoms diverged from each other early in evolution and then evolved independently. A PGM transcript of approximately 2 kb was identi®ed (Fig. 4) in the wheat endosperm and was found to be developmentally regulated con®rming 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 (Thorbjùrnsen 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 ®lling, 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. 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