Journal of Experimental Botany, Vol. 54, No. 383, pp. 715-725,
February 1, 2003
© 2003 Oxford University Press
Subcellular localization of ADPglucose pyrophosphorylase in developing wheat endosperm and analysis of the properties of a plastidial isoform
Received 1 May 2002; Accepted 17 October 2002
1 Department of Botany, College of Biological Science, University of Guelph, Guelph, Ontario N1G 2W1, Canada
2 School of Biological Sciences, University of Manchester, 3.614 Stopford Building, Oxford Road, Manchester M13 9PT, UK
3 Advanced Technologies (Cambridge) Ltd., 210, Cambridge Science Park, Cambridge CB4 4WA, UK
4 To whom correspondence should be addressed. Fax: +1 519 767 1991. E-mail: itetlow{at}uoguelph.ca
5 Present address: Ciphergen Biosystems Ltd., Prior Road, Camberley, Surrey, GU15 1DA, UK.
Abbreviations: AGPase, adenosine 5' diphosphate glucose pyrophosphorylase; AGP-L, large subunit AGPase; AGP-S, small subunit AGPase; APPase, alkaline inorganic pyrophosphatase; DAP, days after pollination; DTT, dithiothreitol; 3-PGA, 3-phosphoglyceric acid; Pi, inorganic orthophosphate; PPi, inorganic pyrophosphate; UGPase, uridine 5' diphosphate glucose pyrophosphorylase.
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The intracellular location of ADPglucose pyrophosphorylase (AGPase) in wheat during endosperm development was investigated by analysis of the recovery of marker enzymes from amyloplast preparations. Amyloplast preparations contained 20–28% of the total endosperm activity of two plastidial marker enzymes and less than 0.8% of the total endosperm activity of two cytosolic marker enzymes. Amylo plasts prepared at various stages of development, from 8–30 d post anthesis, contained between 2% and 10% of the total AGPase activity; this implies that between 7% and 40% of the AGPase in wheat endosperm is plastidial during this period of development. Two proteins were recognized by antibodies to both the large and small subunits of wheat AGPase. The larger of the two AGPases was the major form of the enzyme in whole cell extracts, and the smaller, less abundant, form of AGPase was enriched in plastid preparations. The results are consistent with data from other graminaceous endosperms, suggesting that there are distinct plastidial and cytosolic isoforms of AGPase composed of different subunits. The plastidial isoform of AGPase from wheat endosperm is relatively insensitive to the allosteric regulators 3-phosphoglycerate and inorganic orthophos phate compared with plastidial AGPase from other species. Amyloplast AGPase showed no sensitivity to physiological concentrations of inorganic orthophosphate. 15 mM 3-phosphoglycerate caused no stimulation of the pyrophosphorolytic reaction, and only 2-fold stimulation of the ADPglucose synthesizing reaction.
Key words: ADPglucose pyrophosphorylase, endosperm, localization, regulation, wheat.
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Introduction |
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Adenosine 5' diphosphate glucose (ADPglucose) pyrophosphorylase (AGPase, EC 2.7.7.27) carries out the first committed step in the synthesis of both transient starch in chloroplasts and storage starch in amyloplasts. AGPase from higher plants is heterotetrameric, consisting of two large subunits and two small subunits encoded by at least two different genes (Preiss and Sivak, 1996). Plants possess multiple genes encoding either the large or small subunits, or both, and these are differentially expressed in different plant organs. This means that the AGPase subunit composition may vary in different parts of the same plant in tissues such as potato (La Cognata et al., 1995), rice (Nakamura and Kawaguchi, 1992) and barley (Villand et al., 1992). Biochemical evidence indicates the presence of at least two distinct AGPase enzymes in maize (Denyer et al., 1996), barley (Thorbjørnsen et al., 1996) and rice (Sikka et al., 2001) which have been shown to correspond to plastidial and cytosolic isoforms of AGPase. There is, however, no evidence that a cytosolic isoform of AGPase exists in the storage tissues of non-graminaceous species (Beckles et al., 2001).
AGPase provides the immediate soluble precursor for starch synthesis, ADPglucose, and it is thought that in vivo, the reaction is shifted out of equilibrium in favour of ADPglucose synthesis by the action of alkaline inorganic pyrophosphatase (APPase) within the plastid (Gross and ap Rees, 1986), and by inorganic pyrophosphate (PPi)-consuming reactions such as UDPglucose pyrophosphorylase (UGPase) in the cytosol (Kleczkowski, 1994). The AGPase reaction is characterized by its activation by micromolar amounts of 3-phosphoglyceric acid (3-PGA) and inhibition by inorganic phosphate (Pi), respectively. The ratio of these two allosteric effectors is believed to play a key role in the control of starch synthesis in photosynthetic tissues (Preiss, 1991). Some AGPases from certain storage tissues appear to have a lower sensitivity to both 3-PGA and Pi regulation; for example, the AGPase activities from developing pea embryos, bean cotyledons, and a proteolytically modified AGPase from maize endosperm all showed weak activation by 3-PGA and relative insensitivity to Pi inhibition (Hylton and Smith, 1992; Weber et al., 1995; Plaxton and Preiss, 1987). The AGPase activity from a low starch mutant of Chlamydomonas also showed reduced sensitivity to 3-PGA and Pi (Ball et al., 1991). For both the Chlamydomonas and maize endosperm AGPase, the decrease in sensitivity to 3-PGA activation was accompanied by a several fold decrease in specific activity. The AGPase extracted from barley endosperm, either crude or partially purified, was markedly insensitive to regulation by 3-PGA and Pi (Kleczkowski et al., 1993a), unlike the leaf enzyme which showed pronounced response to these effectors (Kleczkowski et al., 1993b). The barley endosperm AGPase was shown to undergo proteolytic degradation of the large subunit (Kleczkowski et al., 1993a) in contrast to the maize endosperm enzyme whose small subunit was apparently more susceptible to proteolysis (Plaxton and Preiss, 1987). However, when full length cDNAs encoding both subunit proteins of barley endosperm cytosolic AGPase were heterologously expressed, the enzyme showed the same insensitivity to 3-PGA and Pi regulation as AGPase from endosperm extracts, indicating that insensitivity to effectors may represent an intrinsic property of AGPases from some non-photosynthetic tissues (Villand and Kleczkowski, 1994; Doan et al., 1999). By contrast, both the potato tuber and the rice endosperm AGPases exhibit similar sensitivities to both 3-PGA and Pi as the corresponding chloroplast AGPase (Sikka et al., 2001; Ballicora et al., 1995). Recent studies of AGPase purified from wheat endosperm, presumed to be a cytosolic isoform, showed that the enzyme was insensitive to activation by 3-PGA. However, inhibition of the purified AGPase by Pi, ADP and fructose-1, 6-bisphosphate could be reversed by 3-PGA (Gómez-Casati and Iglesias, 2002). In preceding studies of cereal endosperm AGPase, there has been no separation of the cytosolic and plastidial isoforms, and so it is not possible to determine whether the observed insensitivity of such AGPases to 3-PGA and Pi is a feature of one or both isoforms. In the developing endosperms of maize and barley the cytosolic isoform of AGPase appears to be the most abundant, representing 95% and 85% of the total endosperm activity, respectively (Denyer et al., 1996; Thorbjørnsen et al., 1996), although the contribution that each isoform makes towards starch synthesis during endosperm development in these, and other tissues, is not known. It is likely that the lack of sensitivity to effectors is a function of the cytosolic isoform, but information on the kinetic properties of AGPase in endosperm amyloplasts is lacking.
The present study investigates the kinetic properties of the plastidial isoform of AGPase from developing wheat endosperm, and the relative abundance of the plastidial and cytosolic isoforms during grain development.
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Plant material and amyloplast isolation
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 was obtained from developing grains taken from the mid-ear region of the head at various stages of endosperm development between 6 d and 45 d after pollination (DAP) during November 2000. The different stages of endosperm development were determined by measuring the fresh weight of harvested seed from the tagged wheat plants. Amyloplasts were prepared from endosperm tissue between 8 DAP and 30 DAP using seeds which were individually weighed following removal from the inflorescence in order to determine the exact stage of development. Between 300 and 800 individually weighed grains (approximately 8–25 g fresh weight), depending on the stage of seed development, were used to prepare amyloplasts mechanically. Amyloplasts were prepared according to the method described by Tetlow et al. (1993), except that bovine serum albumin was omitted from the extraction medium and amyloplasts were centrifuged once through a layer of 3% (w/v) N-2,3-dihydroxypropylacetamido-2,4,6-triiodo-N,N1-bis-(2,3-dihydroxypropyl) isophthalamide (Nycodenz) (100 g for 25 min at 4 °C). Amyloplasts prepared in this way were 50–65% intact as judged by the latency of the plastid marker enzyme, alkaline inorganic pyrophosphatase (APPase) (Tetlow et al., 1993). Amyloplast pellets were lysed in rupturing buffer (100 mM N-tris(hydroxymethyl)methyl glycine (Tricine)-KOH (pH 7.8), 1 mM Na2-EDTA, 1 mM dithiothreitol (DTT), 5 mM MgCl2, 10 µg cm–3 chymostatin and 100 µM each of antipain dihydrochloride, bestatin, leupeptin, pepstatin, E-64, and aprotinin; 10 µM phosphoramidon and 1,10-phenanthroline, and 500 µM 3,4 dichloroisocoumarin). All fractions from the different stages of the various amyloplast preparations were frozen once in liquid nitrogen and then stored at –80 °C before enzyme assays were subsequently performed. The maximum mitochondrial contamination of the amyloplast preparations at each of the various stages of development was less than 0.2%, based on recoveries of the marker enzymes citrate synthase (EC 4.1.3.7) and cytochrome c oxidase (EC 1.9.3.1) (data not shown).
For analysis of the properties of plastidial AGPase, large-scale amyloplast preparations were made from wheat plants at 15–20 DAP (40–55 mg individual fresh weight of seed). Amyloplasts were prepared at this stage of endosperm development using between 80–110 g fresh weight of seed and the methods described above. AGPase assays were performed on amyloplast pellets lysed in rupturing buffer, after storage at –80 °C. After thawing on ice, starch was removed from the lysates by centrifugation at 13 500 g for 2 min at 4 °C in a pre-cooled microcentrifuge. The resulting supernatant was centrifuged at 100 000 g for 20 min at 4 °C in a Beckman TLX 100 ultracentrifuge to remove organelle membranes. The supernatant, termed plastid stroma, was used immediately for AGPase assays. The maximum cytosolic contamination of the large-scale amyloplast preparations was 0.4%, based on the recovery of uridine 5' diphosphate glucose (UDPglucose) pyrophosphorylase (UGPase, EC 2.7.7.9).
Chloroplasts were prepared from the primary leaves of 10-d-old wheat using the methods described by Bruce et al. (1994). Isolated chloroplasts were treated in the same way as amyloplast pellets (above).
Protein extraction
Whole cell extracts of AGPase, used for enzyme assays, were prepared by rapidly homogenizing approximately 0.5 g endosperm (various seed weights corresponding to different stages of development) in 1 cm3 of ice-cold rupturing buffer, followed by centrifugation at 13 500 g for 2 min at 4 °C. The resulting supernatant was desalted at 4 °C on a NAP-10 column (Pharmacia) pre-equilibrated in rupturing buffer. The desalted crude extract was immediately added to assays for AGPase activity (within 10 min of extraction).
Protein extracts of whole wheat endosperm and amyloplast preparations taken at various stages of development were also prepared for immunoblotting. Approximately 0.5 g of endosperm was ground in liquid nitrogen. 1 cm3 of extraction buffer (62 mM orthophosphoric acid, 120 mM ß-mercaptoethanol, 1 mM sodium monoiodoacetic acid, 1 mM phenylmethylsulphonylfluoride, 5% (v/v) glycerol) was added, mixed to a fine slurry, and left to thaw. Lithium dodecyl sulphate was then added to a final concentration of 2% (w/v). Amyloplast preparations were prepared for immunoblotting by vigorous mixing with 0.5–1 cm3 of extraction buffer and lithium dodecyl sulphate. The homogenate or plastid extract was boiled for 45 s and particulates removed by centrifugation for 15 min at 13 000 g, prior to loading onto polyacrylamide gels.
Enzyme assays
The following enzymes were assayed at 25 °C as described previously: APPase (EC 3.6.1.1), UGPase, alcohol dehydrogenase (EC 1.1.1.1), citrate synthase (EC 4.1.3.7) (Tetlow et al., 1993), cytochrome c oxidase (EC 1.9.3.1) (MacDonald and ap Rees, 1983), 1,4-
-D-glucan-6--(1,4--glucan)-4-transferase (starch branching enzyme, EC 2.4.1.18) and ADPglucose: 1,4--D-glucan-4-glucosyl transferase (soluble starch synthase, EC 2.4.1.21) (Smith, 1990).
Two assays were employed for the measurement of AGPase activity. Both assays were optimized with respect to pH and substrates/effectors, and activities were linear with respect to time and amount of extract added. Unless otherwise indicated, the assay concentration of 3-PGA was 15 mM. The pyrophosphorolytic activity of AGPase was assayed in a spectrophotometer monitoring the formation of NADH at 340 nm and 25 °C. The standard assay mixture contained in 1 cm3; 100 mM glycylglycine-NaOH (pH 7.5), 10 mM MgCl2, 2 mM DTT, 5 mM NaF, 0.5 mM NAD+, 1 mM ADPglucose, 100 µM glucose 1,6-bisphosphate, 1 mM tetra-sodium pyrophosphate (PPi), and 2 units each of phosphoglucomutase (from rabbit muscle) and glucose 6-phosphate dehydrogenase (from Leuconostoc mesenteroides). After correcting for non-specific reduction of NAD+ by samples, the reaction was initiated by the addition of PPi.
AGPase activity was also measured in the direction of ADPglucose synthesis at 37 °C. Assays (0.2 cm3) contained 100 mM N-(2-hydroxyethyl) piperazine-N1-ethanesulphonic acid (HEPES)-NaOH pH 7.8, 10 mM MgCl2, 0.05% (w/v) bovine serum albumin, 0.5 units inorganic pyrophosphatase (from baker’s yeast), and, unless otherwise indicated, 0.5 mM (U-14C) glucose 1-phosphate (specific radioactivity, 900 cpm nmol–1) and 1 mM ATP. After 2–10 min, the reactions were stopped by boiling for 30–50 s. Enzyme activity was determined by measuring the 14C-ADPglucose adsorbed onto DE81 paper (Whatman) following treatment with alkaline phosphatase according to methods described in Ghosh and Preiss (1966). Under these optimized assay conditions, the activity was linear with respect to time and amount of extract added.
Kinetic studies
The Km values for the substrates of AGPase were determined from double-reciprocal plots using near-saturating concentrations of the non-varied substrate. Ki values for Pi were determined by varying the Pi concentration at several fixed concentrations of ADPglucose (pyrophosphorolytic reaction). In these experiments, the concentrations of PPi were kept constant at 1 mM. The Ki value of Pi for the pyrophosphorolytic reaction was estimated from Dixon plots (Segel, 1975).
Preparation of antibody
GST fusion proteins of the AGPase small subunit (AGP-S, Ainsworth et al., 1993) and the AGPase large subunit (AGP-L, Ainsworth et al., 1995) were produced in pGEX (Pharmacia) according to the manufacturer’s protocol. Antibodies were raised to the purified protein in rabbits as described by Kruger et al. (1998).
SDS-PAGE and immunoblotting
Protein samples were diluted with a 20% volume of five-times strength gel sample buffer and heated to 100 °C for 3 min prior to loading on 10% gels following the method of Laemmli (1970). Proteins were transferred to nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany) by electroblotting, blocked with 3% (w/v) bovine serum albumin, and exposed to antibodies raised against AGP-S or AGP-L (diluted 1:2000). Bound antibodies were detected with horseradish peroxidase-conjugated donkey anti-rabbit antibody using the Amersham ECL detection kit (Amersham Life Sciences, Little Chalfont, Bucks, UK).
Protein determination
The protein content of wheat endosperm whole cell extracts and plastid preparations was determined using the Biorad protein assay (Biorad, Maylands Ave, Hemel Hempstead, Herts, UK) according to the manufacturer’s instructions and using thyroglobulin as a standard.
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Subcellular localization of AGPase in wheat endosperm
A rapid, mechanical technique was used to prepare amyloplasts from wheat endosperm at a number of developmental stages during the major grain-filling period (8–30 DAP). It was impractical to harvest sufficient tissue to prepare amyloplasts from younger endosperm (0–7 DAP), and the yield of amyloplasts from endosperms older than 30 DAP was extremely low, probably due to the density of the tissue. The distribution of AGPase activity (measured in the pyrophosphorolytic direction) between the pellet and supernatant fractions was compared with the distributions of marker enzymes assumed to be confined either to the amyloplast stroma (plastidial marker enzymes, APPase and starch branching enzyme) or to the cytosol (cytosolic marker enzymes, alcohol dehydrogenase and UGPase). In order to determine the extent of loss of enzyme activity during the various amyloplast preparations, the activity of each marker enzyme in the pellet and supernatant fractions was compared with its activity in the initial homogenate (Table 1). The recoveries of AGPase and the plastidial and cytosolic marker enzymes indicated no significant loss in activity (or activation) during the preparation of amyloplasts from wheat endosperm at these various stages of development.
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The distributions of the plastidial and cytosolic marker enzymes were significantly different (all preparations made from tissue 8 DAP to 30 DAP; Student’s t-test, P <0.001). Recoveries of plastidial marker enzymes in the pellets of amyloplast preparations ranged between 20% and 31%, compared with less than 0.8% for the cytosolic marker enzymes. Amyloplasts isolated at all stages of endosperm development were, therefore, essentially free from contamination by cytosol. In amyloplast preparations made throughout all of the stages of endosperm development, the proportion of AGPase activity in the pellet was significantly higher than that of the cytosolic marker enzymes and considerably lower than that of the plastidial marker enzymes (Table 1). These differences were statistically significant (Student’s t-test; P <0.01 for 8 DAP, and P <0.001 for 11–30 DAP), indicating that most of the AGPase activity in wheat endosperm is extra-plastidial, at least from 8 DAP and up to 30 DAP, but that a significant proportion is plastidial. Based on the recovery of AGPase and the percentages of the cytosolic and plastidial marker enzymes found in the pellets in the various amyloplast preparations, it was estimated that between 7% (at 8 DAP) and 43% (at 20 DAP) of the total AGPase activity in the endosperm is associated with amyloplasts (Table 1). The estimate is based on the assumption that the AGPase recovered in the pellets comprises both plastidial and extra-plastidial (cytosolic) activities, and that these sediment to the same extent as the relevant plastidial and cytosolic marker enzymes (Beckles et al., 2001; Denyer et al., 1996). Accordingly, the values presented above have been corrected for cytosolic contamination.
In order to check that the activity of AGPase in the pellet fraction is inside amyloplasts, the latency of AGPase activity was determined and compared with that of a cytosolic and a plastidial enzyme with amyloplast preparations made from endosperm 14–20 DAP. The activities of the enzymes were assayed on samples of intact amyloplasts and on samples in which amyloplasts had been osmotically lysed prior to assay to determine percentage latency, which is a measure of intactness (Trimming and Emes, 1993). The latency for AGPase (measured in the ADPglucose synthesis direction) was 54–78% (n=3), and for the plastidial enzyme APPase was 58–82% (n=3). These values were significantly higher (P <0.001) than the range of latency values for the cytosolic marker enzyme UGPase which was 0–3% (n=3). These results suggest that the AGPase recovered in the amyloplast pellet is located inside the organelles.
Different AGPase proteins are present in plastidial and cytosolic fractions of wheat endosperm
All studies with graminaceous plants to date have shown that, in the endosperm, the plastidial and extra-plastidial forms of the small subunit of AGPase are distinct proteins of different molecular masses (Denyer et al., 1996; Thorbjørnsen et al., 1996; Beckles et al., 2001). The distribution of the large and small subunits of AGPases in developing wheat endosperm was analysed by western blotting. Samples of whole cell extracts (containing plastidial and extra-plastidial AGPases) and of amyloplasts were extracted using a procedure designed to minimize proteolytic degradation. Blots were developed with antisera against the major forms of the small subunit (AGP-S) and large subunit (AGP-L) of AGPase from wheat endosperm (Fig. 1A, B, respectively). Both antisera recognized two polypeptides in crude extracts of wheat endosperm; these were 50–51 kDa and 55 kDa as detected with the AGP-S antiserum (Fig. 1A) and 50–51 kDa and 58 kDa detected with the AGP-L antiserum (Fig. 1B). However, with amyloplast preparations the antisera only recognized the smaller of the polypeptides seen in the crude extract; the 50–51 kDa polypeptide with the AGP-S antiserum and the 50–51 kDa polypeptide with the AGP-L antiserum. When the amyloplast sample was combined with the sample made from crude extract, the 55 kDa polypeptide was still clearly separated and recognized in addition to the 50–51 kDa polypeptide by the AGP-S antiserum (Fig. 1A). This indicates that the 55 kDa and 58 kDa AGPase subunits are not plastidial.
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Properties of plastidial AGPase in developing wheat endosperm
The maximum catalytic activity of wheat endosperm AGPase in tissue extracts (measured in the pyrophosphorolytic direction) was determined from 5 DAP (the youngest stage at which endosperms could be dissected) until 50 DAP (close to grain maturity), and compared with AGPase activity from amyloplasts (8–30 DAP). The latter was calculated from estimates of plastidial AGPase based on the recovery of organelle-specific marker enzymes as given in Table 1. Enzyme activity increased from before 10 DAP and was maximal around 12 DAP for whole cell extracts and plastidial AGPase (Fig. 2). Whole cell AGPase activity declined steadily after 11 DAP and continued throughout the later stages of development. Plastidial AGPase activity was maintained from 11 DAP until around 20 DAP, and then declined by approximately 50% at later stages of development (Fig. 2).
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Properties of AGPase in plastidial and whole cell extracts
The activity of AGPase from desalted whole cell extracts of wheat endosperm (15–20 DAP) was compared with desalted stromal extracts from amyloplasts isolated at an equivalent stage of endosperm development (Table 2). Extracts of wheat endosperm (containing 60–70% cytsolic AGPase and approximately 30–40% plastidial AGPase) contained high synthetic and pyrophosphorolytic activities of AGPase when assayed in the absence of 3-PGA compared with samples assayed in the presence of 15 mM 3-PGA. AGPase activity in whole cell extracts was insensitive to the addition of 15 mM 3-PGA when assayed in either direction. Pi (20 mM) inhibited both the synthetic and pyrophosphorolytic reactions by approximately 30% and 40%, respectively (Table 2). Addition of 3-PGA had no effect on the degree of Pi inhibition of AGPase from whole cell extracts when measured in the pyrophosphorolytic direction. However, 3-PGA did appear to prevent Pi inhibition in whole cell extracts when measured in the direction of ADPglucose synthesis. By contrast, plastidial AGPase activity was stimulated more than 2-fold by 15 mM 3-PGA when measured in the direction of ADPglucose synthesis. The activation of amyloplast AGPase by 3-PGA was much less than that observed with chloroplasts; a 25-fold activation of wheat chloroplast AGPase was observed with 1 mM 3-PGA (data not shown), which is in broad agreement with previous work by others (Sanwal et al., 1968; Dickinson and Preiss, 1969). When amyloplast AGPase was measured in the pyrophosphorolytic direction, however, no activation by 3-PGA was observed. Amyloplast AGPase was more sensitive to Pi inhibition than AGPase in whole cell extracts when measured in either direction; 20 mM Pi causing 88% and 64% inhibition of amyloplast AGPase when assayed in the forward and reverse directions, respectively. Addition of 15 mM 3-PGA had no effect on the degree of inhibition of amyloplast AGPase by Pi in either the forward or reverse directions (Table 2). Of particular note is the observation that, in whole cell extracts, the ratio of the pyrophosphorolytic reaction to the synthetic reaction is 3.8 to 1 (in the absence of any effector), whereas in the case of amyloplast activity the situation is reversed and a ratio of 0.45 to 1 is observed (Table 2). The ratio of pyrophosphorolysis to synthesis in whole cell extracts was unaffected by 3-PGA or Pi, unlike the amyloplast AGPase in which the ratio was decreased by 3-PGA and increased more than 3-fold by Pi (Table 2).
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The effects of 3-PGA and Pi on the forward and reverse reactions of the amyloplast isoform of AGPase were investigated in more detail (Fig. 3A, B, respectively). 3-PGA had no effect on the pyrophosphorolytic activity of plastidial AGPase at any concentration used (Fig. 3A). Maximal activation by 3-PGA in the synthetic direction was only achieved at 3-PGA concentrations greater than 10 mM, suggesting that the wheat amyloplast AGPase is far less sensitive to 3-PGA activation than the enzyme found in chloroplasts. When assayed in the synthetic direction, the amyloplast AGPase was relatively insensitive to inhibition by Pi at concentrations up to 10 mM; addition of 15 mM 3-PGA caused no significant modification of the effects of Pi. Only at 20 mM Pi was there any substantial inhibition of ADPglucose synthesis (Fig. 3B). The pyrophosphorolytic AGPase reaction showed more sensitivity to Pi inhibition than the forward reaction at lower Pi concentrations (40% inhibition with less than 5 mM Pi), but the effects of Pi were unaltered by the presence of 15 mM 3-PGA (Fig. 3B)
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The kinetic constants of the forward and reverse reactions of plastidial AGPase are summarized in Table 3. With the exception of the kinetics with respect to ATP, the Km values of the other substrates of AGPase were not affected by 3-PGA. 3-PGA reduced the Km for ATP by approximately 35%. Saturation curves with these substrates were hyperbolic in the presence or absence of 15 mM 3-PGA (data not shown). The Ki value with Pi for the pyrophosphorolytic reaction was 1.67±0.20 mM (n=3) and at least an order of magnitude greater than that of the barley leaf AGPase (Kleczkowski et al., 1993b).
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The analysis of the location of AGPase and organelle marker enzymes in wheat amyloplast preparations leads to the conclusion that wheat endosperm, in common with maize, barley and rice (Denyer et al., 1996; Thorbjørnsen et al., 1996; Sikka et al., 2001), possesses plastidial and extra-plastidial (probably cytosolic) isoforms of AGPase. The percentage of AGPase activity recovered in the amyloplast pellet was considerably higher than that of the cytosolic marker enzymes, and measurements of enzyme latency showed that this activity was inside intact plastids. Calculation of the recoveries of each of the marker enzymes (Table 1) showed that there was little or no loss of enzyme activity in any of the experiments which could alter the interpretation of the data. These experiments have shown that the cytosolic isoform of AGPase is present in the endosperm from at least 8 DAP until 30 DAP (the period when amyloplasts can be reliably and consistently isolated, and which includes the time of most active grain-filling). It was found that the proportion of plastidial AGPase activity was higher in wheat endosperm than with other cereals; at 11 DAP approximately 30% of AGPase activity in wheat was plastidial compared with around 15% in barley (Beckles et al., 2001) and only 6% in maize (Denyer et al., 1996) at equivalent stages of endosperm development. From 11 DAP until 30 DAP there was a similar proportion of plastidial AGPase activity in the developing wheat endosperm. The reason for the large difference between the proportions of plastidial and cytosolic AGPases in these cereals is unclear. It has been suggested that the function of a cytosolic AGPase is to allow more efficient partitioning of large amounts of carbon from sucrose into starch when there is a plentiful supply of sucrose in the endosperm (Beckles et al., 2001). If this is the case, then the relative proportions of cytosolic and plastidial AGPase within a tissue may be subject to any environmental factors which affect the sucrose content of the endosperm. Differences have been observed in the proportions of plastidial and extra-plastidial forms of AGPase from the endosperms of spring wheat (cv. Axona) grown at the same time of year in different locations (K Denyer, personal communications). Varied local growth conditions may, therefore, be responsible for the observed differences in the amount of plastidial AGPase in the endosperms of the three cereals studied to date, as well as species differences. Previous studies which have determined the proportions of AGPase in different subcellular compartments of cereal endosperm (Denyer et al., 1996; Thorbjørnsen et al., 1996; Beckles et al., 2001) have exploited the pyrophosphorolytic assay. However, the data in Table 2 suggest that for the amyloplast enzyme this assay is less sensitive than the biosynthetic assay, whereas the converse is true in whole cell extracts. The measurement of AGPase distribution based on the pyrophosphorolytic reaction is, therefore, likely to underestimate the proportion of plastidial activity with respect to the in vivo synthetic capacity.
The sizes of the large and small subunits for the cytosolic and plastidial forms of AGPase are different. Amyloplast AGPase is a smaller protein, with both subunits having smaller mass than their extra-plastidial (cytosolic) counterpart. Earlier studies with maize and Hordeum species also showed that the major cytosolic form of AGPase has a small subunit which is larger than that of the plastidial isoform (Denyer et al., 1996; Thorbjørnsen et al., 1996; Beckles et al., 2001).
The kinetic properties of the wheat amyloplast AGPase have not previously been determined. In common with studies of AGPases from some storage tissues, the wheat amyloplast AGPase was found to be largely insensitive to the allosteric effectors 3-PGA and Pi (Hylton and Smith, 1992; Kleczkowski et al., 1993a; Weber et al., 1995) and in marked contrast to chloroplast AGPases (Kleczkowski et al., 1993b). The activity of the plastidic AGPase from wheat endosperm determined in the direction of ADPglucose synthesis required 15 mM 3-PGA to achieve a 2-fold stimulation in rate, and was only inhibited approximately 40% by a high concentrations of Pi (20 mM). The major (cytosolic) form of AGPase from wheat endosperm has recently been purified and shown to be insensitive to 3-PGA, but sensitive to inhibition by Pi, ADP and fructose 1, 6-bisphosphate (Gómez-Casati and Iglesias, 2002). Inhibition by these metabolites could, however, be reversed by 3-PGA. The subcellular location of the AGPase purified by Gómez-Casati and Iglesias (2002) was not established, and the specific activity of the purified enzyme in the direction of ADPglucose synthesis was relatively low, about 820 nmol min–1 mg protein–1 (see Fig. 2 of that paper); only 2-fold higher than that of the plastid AGPase reported here. Further, the reported molecular size of the AGPase subunits purified by Gómez-Casati and Iglesias was 52–53 kDa, but the separate large and small subunits were not identified. This value is larger than either of the plastidial subunits reported here, but smaller than the cytosolic subunits (Fig. 1). Consequently, it seems more likely that the kinetic properties reported for the putative cytosolic AGPase (Gómez-Casati and Iglesias, 2002) were performed on a truncated version of the protein. It is well known that the kinetic properties of AGPase can be modified by proteolysis (Plaxton and Preiss, 1987) and, consequently, care must be taken in interpretation of kinetic data. In the present study, the calculated sizes of the non-plastidic large and small subunits (58 and 55 kDa, respectively) are commensurate with the known sequences (Ainsworth et al., 1993, 1995) which align to the cytosolic sequences reported for other species, for example, barley endosperm subunits (Villand et al., 1992; Eimert et al., 1997), reinforcing the above point. The calculated sizes of the plastidial large and small subunits determined in this paper (50–51 kDa) are typical of those reported for other plastidial isozymes (Denyer et al., 1996), and there is little sign of degradation, although a minor band which cross-reacted with antibody to the AGPase large subunit was detected in amyloplast preparations. Whether this small amount of contaminant represents a partial degradation product or is due to a non-specific cross-reaction has not been determined, but the catalytic small subunit appears intact and it is this which is sensitive to 3-PGA and Pi (Doan et al., 1999; Ballicora et al., 1995).
The plastidial and extra-plastidial (cytosolic) forms of AGPase appear to differ not only in their subunit sizes (see above), but also in their kinetic properties. Consideration of the ratio of pyrophosphorolytic to synthetic activity indicates a bias toward pyrophosphorolysis in whole cell AGPase (which is predominantly extra-plastidial) and toward synthesis in plastidial AGPase. Further, Pi inhibition of the synthetic reaction in whole cell extracts could be relieved by 3-PGA, whereas the synthetic reaction in amyloplasts was more sensitive to Pi, and this inhibition was not relieved by up to 15 mM 3-PGA. The basis for these differences is unclear, but may be a reflection of differences in the subunit isomers found in the two subcellular compartments (Fig. 1) and/or may be due to post-translational modification. Given the significant Pi inhibition (88%) of the plastidial AGPase in the synthetic direction, in vivo there may be less than maximal enzyme capacity within the amyloplast, since presumably Pi will always be present in this compartment.
Assuming that the plastidial enzyme accounts for approximately 35% of the total AGPase activity present in whole cell extracts, arithmetic calculation of the effect of Pi on the synthetic reaction indicates that the relatively small effect of Pi on the AGPase biosynthetic reaction in whole cell extracts is accounted for entirely by the plastidial enzyme. The implication of this is that the cytosolic enzyme is insensitive to Pi, and contrasts with the results obtained on a putative wheat endosperm cytosolic AGPase (Gómez-Casati and Iglesias, 2002) which was Pi sensitive. Surprisingly, however, the activation of plastidial AGPase by 3-PGA, when assayed in the biosynthetic reaction, is not reflected by a change in activity in whole cell extracts. The reason for this is not clear.
The amyloplast AGPase has substrate Km values that are similar to those previously reported for the major (cytosolic) form of AGPase in other cereal endosperms (Plaxton and Preiss, 1987; Kleczkowski et al., 1993a; Doan et al., 1999). The relative contribution of each form of AGPase to starch synthesis (ADPglucose synthesis) may, therefore, be dependent on local concentrations of ADPglucose, and Pi concentrations within the amyloplast. The contribution of extra-plastidial AGPase to starch biosynthesis (by the provision of ADPglucose for subsequent import across the amyloplast envelope) may also be dependent on PPi-consuming reactions in the cytosol. Inside the plastid, PPi is effectively removed by an active APPase, whereas in the cytosol, where PPi levels are greater (ap Rees and Morrell, 1990), a number of reactions could contribute to the removal of PPi, and thus influence extra-plastidial ADPglucose synthesis. These include a PPi-dependent phosphofructokinase and UGPase. It therefore appears that both the plastidic and cytosolic forms of AGPase in cereal endosperm are less sensitive to allosteric regulation than the chloroplast enzymes studied to date. Potato tuber AGPase has been shown to be activated by the reversible reduction of an intermolecular disulphide bridge in the presence of substrates and the absence of 3-PGA (Fu et al., 1998; Tiessen et al., 2002). However, reductive activation of AGPases from the storage tissues of monocots has yet to be demonstrated. In the present study, the effects of reducing agents on wheat endosperm AGPase were not determined, as all the buffers used throughout the experiments contained 1 mM DTT to preserve the integrity of the amyloplast envelope. The relatively high proportion of AGPase activity found in wheat amyloplasts leaves open the possibility that ADPglucose may be synthesized in both compartments in vivo. Future investigations will be concerned with the factors which regulate the relative proportions of cytosolic and amyloplast AGPase activity.
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The authors gratefully acknowledge the financial support of the BBSRC, the Royal Society and Biogemma (UK) plc. We are also grateful to Mr TW Heaton for growing the wheat at the Botany Experimental Grounds, University of Manchester.
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