Planta (1990, 182:599-604
P l a n t a 9 Springer-Verlag1990
Evidence that the "waxy" protein of pea (Pisum sativum L.) is not the major starch-granule-bound starch synthase Alison M. Smith John Innes Institute and AFRC Institute of Plant Science Research, ColneyLane, Norwich NR4 7UH, UK Received 1 March; accepted 2 June 1990
Abstract. The aim of this work was to identify the starchgranule-bound starch synthase of developing pea embryos. When starch-granule-bound proteins were solubilised by digestion of granules with a-amylase and fractionated on a Mono Q anion-exchange column, activity of starch synthase eluted as three peaks. The distribution of activity in fractions from the column coincided with that of a 77-kDa protein. An antibody to this protein inhibited starch-synthase activity both in solubilised, starch-granule-bound protein and on intact starch granules. Recoveries of activity through extraction, solubilisation and chromatography indicate that this protein is the major, if not the only, form of starch synthase on the starch granule. The major, 59-kDa protein of the pea starch granule is antigenically related to the product of the waxy locus of potato, which has previously been identified as the starch-granule-bound starch synthase of the tuber. However, the distribution of the 59-kDa protein did not coincide with that of starchsynthase activity in fractions from the Mono Q column. An antibody to the 59-kDa protein did not inhibit starch-synthase activity. The results raise questions about the relationship between " w a x y " proteins and starch-granule-bound starch synthases generally. Key words: Embryo (starch synthase) Pisum (starch synthase) - Starch granule - Starch synthase (purification)
Introduction It is widely accepted that the starch-granule-bound starch-synthase activity in plants is a function of a protein of about 60 kilodaltons (kDa) (Preiss 1988). Evidence for this comes from three sources. First, " w a x y " mutations of the starch-storing organs of a wide range of species result in the loss of both a major protein of Abbreviations: kDa = kilodalton; SDS = sodium dodecylsulphate
this size and most of the starch-synthase activity from the starch granule (Tsai 1974; Echt and Schwartz 1981 ; Sano 1984; Konishi et al. 1985; Hovenkamp-Hermelink et al. 1987). For some species - for example maize it has been shown that the 60-kDa protein is the product of the waxy locus (Shure etal. 1983; K16sgen etal. 1986). The products of waxy loci - which will be referred to as " w a x y " proteins - of different species are closely related to each other both antigenically and at the level of their gene sequences (Vos-Scheperkeuter et al. 1986; Rohde et al. 1988; Visser et al. 1989). Second, an antibody raised to the " w a x y " protein of potato tubers has been shown to inhibit the starch-synthase activity of Amaranthus starch granules (Vos-Scheperkeuter et al. 1986). However, in these experiments 75% of the total starch-synthase activity had been lost from the granules during treatments prior to incubation with the antibody, and an inhibition of only 40% of the remaining activity was demonstrated. Third, two forms of starch synthase have been solubilised and separated from the starch granules of developing maize endosperm (MacDonald and Preiss 1983, 1985). The major form had a molecular weight of 60 kDa - the same as the product of the waxy gene - and the minor form a molecular weight of 92 kDa (measured by sucrose-density-gradient centrifugation). Both forms were eliminated from the starch granule by a mutation at the waxy locus, and it was not established whether either was the product of this locus. There is no comparable information about forms of starch-granule bound starch synthase for any other species. Thus, although a large body of evidence is consistent with the view that " w a x y " proteins are the starchgranule-bound starch synthases, direct and unequivocal evidence is lacking. To study this problem in pea, the starch-granulebound proteins from the starch of developing embryos were solubilised and fractionated by an adaptation of the methods used for maize (MacDonald and Preiss 1983), and antibodies raised to specific starch-granulebound proteins were used to determine which proteins have starch-synthase activity.
600
Material and methods Material. Freshly harvested, developing embryos (450-600 mg FW) of a round-seeded line of Pisum sativum L. (BC1/9 RR), derived from JI 430 (John Innes germplasm collection) by Hedley et al. (1986), were used for all experiments. Plants were grown in a greenhouse at a minimum temperature of 12~ C. Acarbose and BAY e 4609 were the kind gift of Drs. W.R. Wuttke and E. M611er, Bayer AG, Wuppertal, FRG, and antibodies to legumin and vicilin were the kind gift of Dr. R. Casey, John Innes Institute.
Preparation of starch. Embryos (50 g) were crushed in a mortar with 50 ml 100 mM Tris-acetate (pH 7.0), 0.5 M NaC1, 1 mM dithiothreitol (DTT), 1 mM ethylenediaminetetraacetic acid (EDTA) at 2~ C. The homogenate was squeezed through four layers of cheesecloth and the residue re-extracted with a further 50 ml extraction medium and filtered again. The combined filtrate was stirred at 4 ~ C for 30 min then spun at 10000.g for 10 min at 4 ~ C. The supernatant and green and yellowish material from the surface of the pellet were discarded and the pellet resuspended in 10(~ 200 ml extraction medium. This centrifugation and resuspension step was repeated once more with extraction medium, then three times with 50 m M Tris-acetate (pH 8.0), 1 mM DTT, 1 mM EDTA. The final suspension was allowed to settle under gravity, the supernatant discarded and the pellet resuspended in 50 ml acetone at - 2 0 ~ C. The suspension was allowed to settle for about 60 s, then the supernatant - containing the finer fraction of particulate material - was discarded. The remaining material was washed twice more with acetone, dried and stored at - 2 0 ~ for up to two months before use. Solubilisation and fractionation of starch-granule-bound proteins. Pea embryo starch (1-3 g) was ground in 0.5-g aliquots in a mortar at 2 ~ for 2.5 min, suspended at 0.125 g.m1-1 in 100mM Trisacetate (pH 7.7), 100 mM KC1, 1 mM DTT, t mM EDTA, 5% (v/v) glycerol that contained 1 unit. ml-1 c~-amylase (porcine pancreas: BCL, Lewes, Sussex, UK), stirred for 90 min at 25 ~ C, then spun at 40000.g, 4 ~ C for 30 min. The supernatant is referred to as the solubilised, starch-granule-bound protein. The presence of a-amylase in this preparation did not interfere with the assay of starch synthase. Addition of the inhibitors acarbose and BAY e 4609 to the assay and removal of unreacted ADP glucose with Dowex 1 according to Macdonald and Preiss (1983) did not affect the measured activity. The inhibitors abolished activity of a-amylase in the preparation. For anion-exchange chromatography, the supernatant was dialysed for 2 h at 4 ~ C against two changes, each of 1 1, of 50 mM Tris-acetate (pH 8.0), ! mM DTT, 1 mM EDTA, 5% (v/v) glycerol, then applied to a Mono Q column (HR 5 x 5, Pharmacia FPLC system; Pharmacia, Milton Keynes, UK) equilibrated with this medium. The column was washed with 6 ml of this medium, then eluted at 1 ml.min 1 with a linear gradient of 30 ml from 0 to 0.5 M KC1 in this medium. Fractions of 1 ml were collected.
A.M. Smith: Starch-granule-bound starch synthase from pea
Polyacrylamide-gel electrophoresis. Samples were analysed on 7.5% sodium dodecyl sulphate (SDS)-polyacrylamide gels (100-50 mm 2, 1 mm thick) according to Laemmli (1970), and visualised by staining with Brilliant Blue R. Samples of high protein concentration were diluted appropriately then mixed 1:1 with double-strength gel sample buffer (Laemmli 1970). Dilute samples were dialysed against water, freeze-dried, and dissolved in gel sample buffer. Starch granules were heated to 100~ for 3 min at 50 mg.m1-1 in gel sample buffer (Laemmli 1970) and spun at 10000.g for 10 min. The supernatant was applied to the gel. This procedure did not result in complete extraction of protein from the starch granules but ratios of the proteins in the first extraction were the same as those obtained after repeated and exhaustive further extraction of the pellet. Extracts of embryos were made with a pestle and mortar in 100 mM 3-(N-morpholino)propanesulphonic acid (Mops, pH 7.4), 1 mM DTT at 4 ~ C and spun at 10000-g for I0 min. The supernatant is referred to as the soluble fraction. The pellet was washed four times by resuspension in extraction medium and centrifugation. This reduced contamination with soluble proteins to about 1% (measured as alcohol-dehydrogenase activity, data not shown). The pellet was suspended in gel sample buffer, heated to 100 ~ C for 3 min and spun at 10000.g for I0 min. The supernatant was applied to the gel.
Preparation of antibody and Western blotting. Antigen was prepared by excision of stained bands from preparative SDS-polyacrylamide gels followed by electroelution of the protein. Rabbits were immunised and serum collected precisely according to Bhattacharyya et al. (1990). Western blots were prepared and developed according to Bhattacharyya et al. (1990).
Immunoprecipitation experiments. Samples of 0.1 ml of solubilised, starch-granule-bound protein were incubated with 50 lal Protein A-Sepharose at 60 mg.m1-1 in 25 mM Tris (pH 7.5) and 21~100 ~tl serum for 2 h at room temperature, then spun at 10000-g for 10 min. The supernatant was assayed for starch-synthase activity. Controls contained bovine serum albumin at 20 mg. m l - 1 in phosphate-buffered saline in place of serum. Samples of intact starch granules (0.125 g.ml-1 in the medium described above for incubation of granules with a-amylase) were treated in the same way except that Protein A-Sepharose was omitted and incubations were not spun prior to assay.
Experiments with potato tubers. Immature tubers of Solanum tuberosum L. cv. King Edward were obtained from a local greengrocer. Starch was prepared according to Vos-Scheperkeuter et al. (1986). In mixing experiments with pea embryo, polyvinylpolypyrrolidone (200 mg-g-1 potato) was added with the extraction medium to samples containing potato. Approximately 1 g of tuber and 0.4 g of embryo were used in mixed extractions. Polyvinylpolypyrrolidone had no effect on the extractable activity of starch synthase from pea embryos (data not shown).
Results
Assay of starch synthase. The assay contained 100 mM N,N-bis(2-
Purification o f starch. In o r d e r to m i n i m i s e c o n t a m i n a -
hydroxyethyl)glycine (Bicine), 0.5 M Na-citrate (pH 8.6), 2.6 mM ADP[U-14C]glucose (Amersham plc, Amersham, Bucks., UK) at 2 GBq . m o l - 1, 1.5 mg amylopectin (from potato), and 20 ~tl extract or starch-granule suspension (0.125 g.m1-1 in the medium described above for incubation of granules with a-amylase) in a final vol of 115 ~tl. Controls contained either boiled extract or, where activity on starch grains was being assayed, starch that had been incubated in 1 M perchloric acid for 30 min at room temperature, washed four times with water and resuspended in the appropriate medium. Assays were incubated for 10 min at 25 ~ C, then heated to 100 ~ C for 2 rain. Assays containing granules were shaken vigorously throughout the incubation. Starch was precipitated, washed, and radioactivity in it determined as described previously (Smith et al. (1989).
tion of starch by protein bodies, the insoluble material f r o m p e a e m b r y o s w a s w a s h e d i n i t i a l l y in 0.5 M NaC1, a n d at a l a t e r s t a g e in p u r i f i c a t i o n t h e l e a s t - d e n s e fraction of the insoluble material - containing the remaining p r o t e i n b o d i e s - w a s d i s c a r d e d . P u r i f i e d s t a r c h f r o m developing pea embryos contained very few major proteins. O n S D S - p o l y a c r y l a m i d e gels, t h e t h r e e m o s t p r o m i n e n t b a n d s h a d a p p a r e n t m o l e c u l a r w e i g h t s o f 59, 60 a n d 77 k D a , t h e 5 9 - k D a p r o t e i n c o n s t i t u t i n g 7 0 % o r m o r e o f the p r o t e i n o n the g r a n u l e (Fig. 1). N o n e o f these proteins cross-reacted on Western blots with antib o d i e s to l e g u m i n a n d vicilin ( d a t a n o t s h o w n ) .
A.M. Smith: Starch-granule-bound starch synthase from pea P r e p a r a t i o n o f pea starch granules resulted in little or no loss o f activity o f starch synthase. First, a mixing experiment with p o t a t o tuber indicated that there was no general loss o f starch-synthase activity u p o n initial extraction o f embryos. W h e n samples o f pea e m b r y o and p o t a t o tuber were co-extracted, activity in the crude h o m o g e n a t e was 9 7 % o f that expected f r o m activities in crude h o m o g e n a t e s o f samples o f e m b r y o and tuber extracted separately. Second, after five washings o f the insoluble material f r o m a crude h o m o g e n a t e o f pea embryos, activity in the c o m b i n e d washings plus this material a c c o u n t e d for 89% o f that originally present in the crude h o m o g e n a t e . A d d i t i o n o f the protease inhibitors p h e n y l m e t h y l s u l p h o n y l fluoride ( P M S F , 1 m M ) , leupeptin (0.1 m M ) , or c h y m o s t a t i n (5 g g . m l - 1), or o f 10 m M E D T A , to the extraction and washing media h a d no effect o n the recovery o f starch-synthase activity (data n o t shown).
Fig. 1. Starch-granule-bound proteins of developing pea embryo and potato tuber. Tracks a, b,f, g, are 7.5% SDS-polyacrylamide gels of: a, protein from 1 mg pea starch; b, g, molecular-weight standards (sizes indicated in kDa); f, protein from 1 mg potato starch. Tracks c, d, e, are Western blots of proteins from pea starch as in track a. Tracks h, i, j, are Western blots of potato starch as in track f, except that protein was from about 7 mg starch. Tracks c, h, are developed with serum (1/5000 dilution) containing antibody to the 59-kDa protein of pea starch. Tracks d, i, are developed with serum (1/15000 dilution) containing antibody to the 77-kDa protein of pea starch. Tracks e, j, are developed with pre-immune serum (1/5000 dilution) from the rabbit subsequently immunised with the 59-kDa protein. Blots developed with preimmune serum from the rabbit subsequently immunised with the 77-kDa protein were identical to e andj (not shown)
601 Acetone-washed, purified starch stored at - 2 0 ~ lost up to 30% o f starch-synthase activity over two months. However, results o b t a i n e d with this starch were always qualitatively the same as those obtained with freshly purified starch. o f s t a r c h - g r a n u l e - b o u n d protein. A b o u t 50% o f the protein on purified starch granules was released by grinding followed by i n c u b a t i o n with a - a m y lase at 2 5 ~ for 1.5 h. On S D S - p o l y a c r y l a m i d e gels, the proteins released a p p e a r e d identical to those o n the Solubilisation
Fig. 2. A Release and fractionation of proteins from starch granules of pea by e-amylase and Mono Q chromatography. 7.5% SDSpolyacrylamide gels of: track a, molecular-weight standards (sizes indicated in kDa); track b, protein solubilised from 2.5 mg pea starch by digestion with e-amylase; track c, protein remaining on 2 mg pea starch after digestion with e-amylase; track d, protein from 1 mg undigested pea starch; track e, a-amylase used in digestion; track f, fraction of solubilised protein that failed to absorb to the Mono Q column; track g, fraction of solubilised protein that eluted from the Mono Q column with the major peak of starch-synthase activity. B Occurrence of the 59- and 77-kDa proteins of pea starch granules in the soluble fraction of pea embryos. Western blots of the total insoluble (tracks a, c) and soluble (tracks, b, d) fractions of pea embryos of 450 mg FW, prepared as described in Material and methods. Each track contains 0.1% of the material from a single embryo. Tracks a, b, developed with serum (1/1 500 dilution) containing antibody to the 59-kDa protein of pea starch granules. Tracks, c, d, developed with serum (1/10000 dilution) containing antibody to the 77-kDa protein of pea starch granules. Tracks e, f, developed with pre-immune serum (1/1 500 dilution) from rabbit subsequently immunised with the 59-kDa protein. Blots developed with pre-immune serum from the rabbit subsequently immunised with the 77-kDa protein were identical to e andf(not shown)
602 intact granule and those remaining on the granule after the incubation (Fig. 2A). Disruption of the granules by grinding caused an increase of four- to sevenfold in their starch-synthase activity. For example, in one, typical experiment activity increased f r o m 0.2 n m o l - m i n - ~ - m g - 1 starch on intact granules to 1.3 nmol. m i n - ~ - r a g - ~ starch on disrupted granules. Incubation with e-amylase solubilised about 50% of this activity. The solubilised activity plus that remaining in the insoluble fraction accounted for at least 80%, and often m o r e than 90%, o f the activity on the disrupted granules prior to incubation. Addition of the protease inhibitors P M S F , leupeptin or chymostatin to the incubation medium at the concentrations given above had no effect on the recovery of starch-synthase activity. The ratio of citrate-stimulated to unstimulated activity in the presence of primer was altered during the solubilisation o f starch synthase. This ratio was 1.2 + 0.1 for disrupted granules, 2.5_+0.2 for solubilised activity, and 1.0_+ 0.2 for activity that remained insoluble after incubation (means-t-SE from three experiments, each on a separate batch o f starch). Activity with UDP-glucose was less than 10% of that with ADP-glucose at both low (2.6 m M ) and high (26 m M ) concentrations. Activity with glycogen as a primer was only 16% or less of that with amylopectin at the same concentration (10 mgml-~ in the assay).
Identification and separation of solubilised proteins. Because of the prominence o f the 59-kDa protein of pea starch granules, and its similarity in size to the " w a x y " proteins of other species, it seemed likely that it might be a starch-granule-bound starch synthase. On Western blots an antibody raised to the 59-kDa protein specifically recognised this protein and the 60-kDa protein of pea starch granules, and the major, 57-kDa protein of potato starch granules (Fig. 1). It did not recognise proteins in the soluble fraction of pea embryos (Fig. 2 B). However, the antibody did not precipitate starch-synthase activity f r o m preparations of solubilised, starchgranule-bound protein (Fig. 3). This was not because the antibody failed to recognise or precipitate the 59k D a protein under the experimental conditions. The antibody strongly recognised the native protein (not shown), and antibody-antigen complexes were precipitated from the incubation with Protein A-Sepharose. When solubilised, starch-granule-bound proteins were fractionated on a M o n o Q anion-exchange column, activity of starch synthase was consistently recovered in three peaks which eluted between 0.15 and 0.35 M KC1 and accounted for at least 80% of the activity loaded. Less than 10% of the recovered activity failed to adsorb to the column. This pattern was seen with both freshly prepared and acetone-washed starch. A typical fraction is shown in Fig. 4. The ratios o f citrate-stimulated to unstimulated activity, and primed to unprimed activity, of the three peaks varied considerably from one preparation to another. Variation between experiments was greater than variation between the peaks in any one experiment. The
A.M. Smith: Starch-granule-bound starch synthase from pea
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Fig. 3A-D. Precipitation experiments with antibodies to the 59and 77-kDa proteins of the pea starch granule. Solubilised, starchgranule-bound proteins or intact starch granules were incubated with serum for 2 h, then assayed for starch-synthase activity. Incubations with solubilised, starch-granule-bound proteins also contained Protein A-Sepharose which was removed by centrifugation prior to assay. For each experiment, the ratio of serum containing antibody to pre-immune serum was varied so that all incubations contained the same concentration of serum. In all experiments, control incubations without serum had the same activities (+ 15%) of starch synthase as incubations with pre-immune sera. A Solubilised starch-granule-bound protein. B Intact starch granules. Activities of starch synthase in A and B are expressed as percentages of activity in incubations containing pre-immune sera alone, e - - e , Incubation with serum containing antibody to the 77-kDa protein; o - - o , incubation with serum containing antibody to the 59-kDa protein. C, D Western blots of 7.5% SDS-polyacrylamide gels of incubations with antibody to the 77-kDa protein shown in A. Tracks 1~4 are equal loadings of the supernatants after centrifugation of incubations with, in the order given, 0, 5, 10 and 15 gl of serum. C is developed with serum (1/15000 dilution) containing antibody to the 77-kDa protein, D with serum (1/5000 dilution) containing antibody to the 59-kDa protein. Note that the amount of 77-kDa protein declines with increasing antibody concentration (C) whereas the amount of 59/60-kDa protein does not (D). The strong band common to both blots, and also to blots developed with pre-immune sera (not shown), is rabbit IgG
relative activities of the peaks also varied from one experiment to another, but in six out of seven experiments the first peak contained considerably more activity than either of the other two. The distribution of activity eluting from the M o n o Q column did not match that of the 59- and 60-kDa proteins. Approximately 80% of these proteins failed to adsorb to the column (Fig. 2A, estimate made from scanning gels of column fractions). The remainder eluted as a broad, single peak at approx. 0.25 M KC1 (Fig. 4). The distribution of activity closely matched that of the 77-kDa protein. In particular, it was often the only pro-
A.M. Smith: Starch-granule-bound starch synthase from pea
603
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starch synthase from preparations o f solubilised, starchgranule-bound protein from pea starch granules (Fig. 3). In these experiments, little or none of the 59- and 60-kDa proteins was precipitated (Fig. 3). The antibody also inhibited activity of starch synthase on intact pea starch granules (Fig. 3).
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Fig. 4. Analysis of fractions of solubilised, starch-granule-bound protein eluted from a Mono Q anion-exchange column. Solubilised, starch-granule-bound protein from freshly prepared starch which had not been treated with acetone was applied to a Mono Q column in Tris-acetate buffer (pH 8.0) and eluted with a gradient of KC1 ( . . . . . ). One-ml fractions were collected and monitored for protein content ( ) and activity of starch synthase @ - - - -). W, wash fraction. Western blots of 7.5% SDS-polyacrylamide gels of individual fractions (wash and 8-18) were developed with: A, serum (l/15 000 dilution) containing antibody to the 77-kDa protein of pea starch granules, and B, serum (1/5000 dilution) containing antibody to the 59-kDa protein of pea starch granules. Each track contains approx. 1/20 of the material from the fraction, except for the wash fraction where the track contains approx. 1/175 of the material. Sizes of bands are indicated in kDa. Note the presence of large amounts of the 59-kDa protein, and the absence of the 77-kDa protein and starch-synthase activity, in the wash fraction. Fractions containing the first two peaks of starch-synthase activity also contain the largest amounts of the 77-kDa protein
tein visible on gels of the first, major peak of activity (Fig. 2A), most of it adsorbed to the column, and it eluted as two peaks and a tail that coincided with the three peaks of starch-synthase activity (Fig. 4). On Western blots, an antibody raised to the 77-kDa protein recognised this protein and - much more weakly - the 59- and 60-kDa proteins and a minor, 45-kDa protein of peak starch granules (Fig. 1). It recognised a very minor 77-kDa protein in the soluble fraction o f pea embryos (Fig. 2 B). It also recognised strongly a minor 92-kDa protein and much more weakly - the major, 57-kDa protein, of potato starch granules (Fig. 1). The antibody precipitated almost all of the activity of
It is highly likely that the activity eluted from the M o n o Q column represents by far the major, if not the only, form of starch-granule-bound starch synthase in developing pea embryos. Direct measurement of the recovery of activity from the crude homogenate in fractions from the column is not possible because of the large increase in assayable activity brought about by disruption of the granules. However, estimates o f recoveries and mixing experiments indicate strongly that only minimal losses of activity occurred at all other stages. Activity of starchgranule-bound starch synthase in crude homogenates o f embryos of this cultivar o f peas is about three times greater than is required to account for the rate of starch synthesis (Smith et al. 1989). Most, if not all, of the starch-granule-bound starch synthase o f the pea embryo is attributable to a 77-kDa protein 9 This was the only protein which consistently co-purified with the major peak of starch-synthase activity solibilised from the starch granule, and its distribution in fractions from the column generally matched that of starch-synthase activity. An antibody to this protein precipitated starch-synthase activity from preparations of solubilised, starch-granule-bound protein, and inhibited activity on the intact granule. The 77-kDa protein is predominantly bound to the starch granule in the developing pea embryo, although a closely related or identical protein is a very minor component o f the soluble protein of the embryo. The reason why activity apparently attributable to a single protein eluted as three peaks from the Mono Q column is unclear. Solubilisation of starch-granulebound proteins led to reproducible changes in the ratio of citrate-stimulated to unstimulated starch-synthase activity in the presence of primer, similar to those reported for the enzyme from maize endosperm (MacDonald and Preiss 1983). However, in starch synthase eluted from the M o n o Q column, this ratio and the ratio o f primed to unprimed activity varied widely from one experiment to another, as did the ratios of activity in the three peaks. It is possible that both the effects of citrate and primer on the activity, and the point at which it elutes from the column, are functions of the amount and size of endogenous glucan primer bound to it, and that this varies from one preparation to the next. My results provide no evidence that the major, antigenically similar, 59- and 60-kDa proteins o f pea starch granules have starch-synthase activity. Their distribution in fractions from the M o n o Q column did not match that o f the activity, and an antibody which strongly recognised both of these proteins failed to precipitate starch-synthase activity from preparations of solubilised,
604 s t a r c h - g r a n u l e - b o u n d protein. Conversely, starch-synthase activity could be precipitated with a n t i b o d y to the 7 7 - k D a starch synthase u n d e r conditions in which the 59- and 6 0 - k D a proteins remained in solution. However, the possibility that a starch-synthase activity o f these proteins was lost on extraction, or n o t revealed by the assays I used, c a n n o t be ruled out. The very weak crossreaction between the a n t i b o d y to the 7 7 - k D a starch synthase and the 59- and 6 0 - k D a proteins on Western blots indicates that they are related in some way to starch synthases, but their role in the synthesis o f the starch granule remains to be elucidated. The strong cross-reaction between the a n t i b o d y to the 5 9 - k D a protein o f the pea starch granule and the m a j o r protein o f the p o t a t o starch granule indicates that the 59- and 6 0 - k D a proteins o f pea belong to the " w a x y " class o f proteins f o u n d on the starch granules o f all species so far examined. The m a j o r protein o f the p o t a t o starch granule is eliminated by a " w a x y " m u t a t i o n , and is antigenically, related to the w a x y gene p r o d u c t o f maize (Vos-Scheperkeuter et al. 1986; H o v e n k a m p - H e r m e l i n k et al. (1987). It has been generally accepted that " w a x y " proteins are the m a j o r or only f o r m o f s t a r c h - g r a n u l e - b o u n d starch synthase in plants (Preiss 1988) M y results indicate that this is n o t universally the case. First, I can find no evidence that significant starchsynthase activity is associated with the " w a x y " proteins o f pea. Second, I can attribute essentially all o f the s t a r c h - g r a n u l e - b o u n d starch-synthase activity o f pea to a protein which is a different size from, and only very weakly antigenically related to, the " w a x y " class o f proteins. Third, the 9 5 - k D a protein o f p o t a t o starch granules is m u c h m o r e closely related to the starch synthase o f pea starch granules than is the " w a x y " protein o f p o t a t o . It seems likely that this 9 5 - k D a protein is an i m p o r t a n t s t a r c h - g r a n u l e - b o u n d starch synthase. I suggest that a re-examination, species by species, o f the identity o f the s t a r c h - g r a n u l e - b o u n d starch synthase and the role o f the " w a x y " protein m a y be required. I am grateful to my colleagues Kay Denyer, Ian Dry (CSIRO, Adelaide, Australia), Rob Ireland (Mount Allison University, New Brunswick, Canada), Cathie Martin and Steve Rawsthorne for useful discussions during the course of this work, Cliff Hedley for the gift of pea seeds, and Ian Bedford for preparing pea starch and gels of starch-granule-bound proteins. This work was supported by the Agriculture and Food Research Council via a grantin-aid to the John Innes Institute.
References Bhattacharyya, M.K., Smith, A.M., Ellis, T.H.N., Hedley, C., Martin, C. (1990) The wrinkled-seed character of pea described
A.M. Smith: Starch-granule-bound starch synthase from pea by Mendel is caused by a transposon-like insertion in a gene encoding starch-branching enzyme. Cell 60, 115 121 Echt, S., Schwartz, D. (1981) Evidence for the inclusion of controlling elements within the structural gene at the waxy locus in maize. Genetics 99, 275-284 Hedley, C.L., Smith, C.M., Ambrose, M.J., Cook, S., Wang, T.L. (1986) An analysis of seed development in Pisum sativum. II. The effect of the r locus on the growth and development of the seed. Ann. Bot. 58, 371-379 Hovenkamp-Hermelink, J.H.M., Jacobsen, E., Ponstein, A.S., Visser, R.G.F., Vos-Scheperkeuter, G.H., Bijmolt, E.W., de Vries, J.N., Witholt, B. Feenstra, W.J. (1987) Isolation of an amylose-free mutant of the potato (Solanum tuberosum L.). Theor. Appl. Genet. 75, 217 221 Hseih, J.-S. (1988) Genetic studies on the Wx gene of sorghum [Sorghum bicolor (L.) Moench.] I. Examination of the protein product of the waxy locus. Bot. Bull. Academia Sinica 29, 293299 K16sgen, R.B., Gierl, A., Schwartz-Sommer, Z., Saedler, H. (1986) Molecular analysis of the waxy locus of maize. Mol. Gen. Genet. 203, 232244 Konishi, Y., Nojima, H., Okuno, K., Asaoka, M., Fuwa, H. (1985) Characterisation of starch granules from waxy, nonwaxy and hybrid seeds of Amaranthus hypochrondriacus L. Agric. Biol. Chem. 49, 1965-1971 Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685 MacDonald, F.D., Preiss, J. (1983) Solubilization of the starchgranule-bound starch synthase of normal maize kernels. Plant Physiol. 73, 175-178 MacDonald, F.D., Preiss, J. (1985) Partial purification and characterization of granule-bound starch synthases from normal and waxy maize. Plant Physiol. 78, 849-852 Preiss, J. (1988) Biosynthesis of starch and its regulation. In: The biochemistry of plants, vol. 14, pp. 181-254, Stumpf, P.K., Conn, E.E., eds. Academic Press, New York Rohde, W., Becker, D., Salamini, F. (1988) Structural analysis of the waxy locus from Hordeum vulgate. Nucleic Acids Res. 16, 7185-7186 Sano, Y. (1984) Differential regulation of waxy gene expression in rice endosperm. Theor. Appl. Genet. 68, 462473 Shure, M., Wessler, S., Fedoroff, N. (1983) Molecular identification and isolation of the waxy locus in maize. Cell 35, 225-233 Smith, A.M. (1988) Major differences in isoforms of starch-branching enzyme between developing embryos of round- and wrinkled-seeded peas (Pisum sativum L.). Planta 175, 270-279 Smith, A.M., Bettey, M., Bedford, I.D. (1989) Evidence that the rb locus alters the starch content of developing pea embryos through an effect on ADP glucose pyrophosphorylase. Plant Physiol. 89, 1279-1284 Tsai, C.-Y. (1974) The function of the waxy locus in starch synthesis in maize endosperm. Biochem. Genet. 11, 83-96 Visser, R.G.F., Hergersberg, M., Van der Leij, F.R., Jacobsen, E., Witholt, B., Feenstra, W.J. (1989) Molecular cloning and partial analysis of the gene for granule-bound starch synthase from a wildtype and an amylose-free potato (Solanum tuberosum L.). Plant Sci. 64, 185 192 Vos-Scheperkeuter, G.H., Boer, W. de, Visser, R.F.G., Feenstra, W.J., Witholt, B. (1986) Identification of granule-bound starch synthase in potato tubers. Plant Physiol. 82, 411-416
P l a n t a 9 Springer-Verlag1990
Evidence that the "waxy" protein of pea (Pisum sativum L.) is not the major starch-granule-bound starch synthase Alison M. Smith John Innes Institute and AFRC Institute of Plant Science Research, ColneyLane, Norwich NR4 7UH, UK Received 1 March; accepted 2 June 1990
Abstract. The aim of this work was to identify the starchgranule-bound starch synthase of developing pea embryos. When starch-granule-bound proteins were solubilised by digestion of granules with a-amylase and fractionated on a Mono Q anion-exchange column, activity of starch synthase eluted as three peaks. The distribution of activity in fractions from the column coincided with that of a 77-kDa protein. An antibody to this protein inhibited starch-synthase activity both in solubilised, starch-granule-bound protein and on intact starch granules. Recoveries of activity through extraction, solubilisation and chromatography indicate that this protein is the major, if not the only, form of starch synthase on the starch granule. The major, 59-kDa protein of the pea starch granule is antigenically related to the product of the waxy locus of potato, which has previously been identified as the starch-granule-bound starch synthase of the tuber. However, the distribution of the 59-kDa protein did not coincide with that of starchsynthase activity in fractions from the Mono Q column. An antibody to the 59-kDa protein did not inhibit starch-synthase activity. The results raise questions about the relationship between " w a x y " proteins and starch-granule-bound starch synthases generally. Key words: Embryo (starch synthase) Pisum (starch synthase) - Starch granule - Starch synthase (purification)
Introduction It is widely accepted that the starch-granule-bound starch-synthase activity in plants is a function of a protein of about 60 kilodaltons (kDa) (Preiss 1988). Evidence for this comes from three sources. First, " w a x y " mutations of the starch-storing organs of a wide range of species result in the loss of both a major protein of Abbreviations: kDa = kilodalton; SDS = sodium dodecylsulphate
this size and most of the starch-synthase activity from the starch granule (Tsai 1974; Echt and Schwartz 1981 ; Sano 1984; Konishi et al. 1985; Hovenkamp-Hermelink et al. 1987). For some species - for example maize it has been shown that the 60-kDa protein is the product of the waxy locus (Shure etal. 1983; K16sgen etal. 1986). The products of waxy loci - which will be referred to as " w a x y " proteins - of different species are closely related to each other both antigenically and at the level of their gene sequences (Vos-Scheperkeuter et al. 1986; Rohde et al. 1988; Visser et al. 1989). Second, an antibody raised to the " w a x y " protein of potato tubers has been shown to inhibit the starch-synthase activity of Amaranthus starch granules (Vos-Scheperkeuter et al. 1986). However, in these experiments 75% of the total starch-synthase activity had been lost from the granules during treatments prior to incubation with the antibody, and an inhibition of only 40% of the remaining activity was demonstrated. Third, two forms of starch synthase have been solubilised and separated from the starch granules of developing maize endosperm (MacDonald and Preiss 1983, 1985). The major form had a molecular weight of 60 kDa - the same as the product of the waxy gene - and the minor form a molecular weight of 92 kDa (measured by sucrose-density-gradient centrifugation). Both forms were eliminated from the starch granule by a mutation at the waxy locus, and it was not established whether either was the product of this locus. There is no comparable information about forms of starch-granule bound starch synthase for any other species. Thus, although a large body of evidence is consistent with the view that " w a x y " proteins are the starchgranule-bound starch synthases, direct and unequivocal evidence is lacking. To study this problem in pea, the starch-granulebound proteins from the starch of developing embryos were solubilised and fractionated by an adaptation of the methods used for maize (MacDonald and Preiss 1983), and antibodies raised to specific starch-granulebound proteins were used to determine which proteins have starch-synthase activity.
600
Material and methods Material. Freshly harvested, developing embryos (450-600 mg FW) of a round-seeded line of Pisum sativum L. (BC1/9 RR), derived from JI 430 (John Innes germplasm collection) by Hedley et al. (1986), were used for all experiments. Plants were grown in a greenhouse at a minimum temperature of 12~ C. Acarbose and BAY e 4609 were the kind gift of Drs. W.R. Wuttke and E. M611er, Bayer AG, Wuppertal, FRG, and antibodies to legumin and vicilin were the kind gift of Dr. R. Casey, John Innes Institute.
Preparation of starch. Embryos (50 g) were crushed in a mortar with 50 ml 100 mM Tris-acetate (pH 7.0), 0.5 M NaC1, 1 mM dithiothreitol (DTT), 1 mM ethylenediaminetetraacetic acid (EDTA) at 2~ C. The homogenate was squeezed through four layers of cheesecloth and the residue re-extracted with a further 50 ml extraction medium and filtered again. The combined filtrate was stirred at 4 ~ C for 30 min then spun at 10000.g for 10 min at 4 ~ C. The supernatant and green and yellowish material from the surface of the pellet were discarded and the pellet resuspended in 10(~ 200 ml extraction medium. This centrifugation and resuspension step was repeated once more with extraction medium, then three times with 50 m M Tris-acetate (pH 8.0), 1 mM DTT, 1 mM EDTA. The final suspension was allowed to settle under gravity, the supernatant discarded and the pellet resuspended in 50 ml acetone at - 2 0 ~ C. The suspension was allowed to settle for about 60 s, then the supernatant - containing the finer fraction of particulate material - was discarded. The remaining material was washed twice more with acetone, dried and stored at - 2 0 ~ for up to two months before use. Solubilisation and fractionation of starch-granule-bound proteins. Pea embryo starch (1-3 g) was ground in 0.5-g aliquots in a mortar at 2 ~ for 2.5 min, suspended at 0.125 g.m1-1 in 100mM Trisacetate (pH 7.7), 100 mM KC1, 1 mM DTT, t mM EDTA, 5% (v/v) glycerol that contained 1 unit. ml-1 c~-amylase (porcine pancreas: BCL, Lewes, Sussex, UK), stirred for 90 min at 25 ~ C, then spun at 40000.g, 4 ~ C for 30 min. The supernatant is referred to as the solubilised, starch-granule-bound protein. The presence of a-amylase in this preparation did not interfere with the assay of starch synthase. Addition of the inhibitors acarbose and BAY e 4609 to the assay and removal of unreacted ADP glucose with Dowex 1 according to Macdonald and Preiss (1983) did not affect the measured activity. The inhibitors abolished activity of a-amylase in the preparation. For anion-exchange chromatography, the supernatant was dialysed for 2 h at 4 ~ C against two changes, each of 1 1, of 50 mM Tris-acetate (pH 8.0), ! mM DTT, 1 mM EDTA, 5% (v/v) glycerol, then applied to a Mono Q column (HR 5 x 5, Pharmacia FPLC system; Pharmacia, Milton Keynes, UK) equilibrated with this medium. The column was washed with 6 ml of this medium, then eluted at 1 ml.min 1 with a linear gradient of 30 ml from 0 to 0.5 M KC1 in this medium. Fractions of 1 ml were collected.
A.M. Smith: Starch-granule-bound starch synthase from pea
Polyacrylamide-gel electrophoresis. Samples were analysed on 7.5% sodium dodecyl sulphate (SDS)-polyacrylamide gels (100-50 mm 2, 1 mm thick) according to Laemmli (1970), and visualised by staining with Brilliant Blue R. Samples of high protein concentration were diluted appropriately then mixed 1:1 with double-strength gel sample buffer (Laemmli 1970). Dilute samples were dialysed against water, freeze-dried, and dissolved in gel sample buffer. Starch granules were heated to 100~ for 3 min at 50 mg.m1-1 in gel sample buffer (Laemmli 1970) and spun at 10000.g for 10 min. The supernatant was applied to the gel. This procedure did not result in complete extraction of protein from the starch granules but ratios of the proteins in the first extraction were the same as those obtained after repeated and exhaustive further extraction of the pellet. Extracts of embryos were made with a pestle and mortar in 100 mM 3-(N-morpholino)propanesulphonic acid (Mops, pH 7.4), 1 mM DTT at 4 ~ C and spun at 10000-g for I0 min. The supernatant is referred to as the soluble fraction. The pellet was washed four times by resuspension in extraction medium and centrifugation. This reduced contamination with soluble proteins to about 1% (measured as alcohol-dehydrogenase activity, data not shown). The pellet was suspended in gel sample buffer, heated to 100 ~ C for 3 min and spun at 10000.g for I0 min. The supernatant was applied to the gel.
Preparation of antibody and Western blotting. Antigen was prepared by excision of stained bands from preparative SDS-polyacrylamide gels followed by electroelution of the protein. Rabbits were immunised and serum collected precisely according to Bhattacharyya et al. (1990). Western blots were prepared and developed according to Bhattacharyya et al. (1990).
Immunoprecipitation experiments. Samples of 0.1 ml of solubilised, starch-granule-bound protein were incubated with 50 lal Protein A-Sepharose at 60 mg.m1-1 in 25 mM Tris (pH 7.5) and 21~100 ~tl serum for 2 h at room temperature, then spun at 10000-g for 10 min. The supernatant was assayed for starch-synthase activity. Controls contained bovine serum albumin at 20 mg. m l - 1 in phosphate-buffered saline in place of serum. Samples of intact starch granules (0.125 g.ml-1 in the medium described above for incubation of granules with a-amylase) were treated in the same way except that Protein A-Sepharose was omitted and incubations were not spun prior to assay.
Experiments with potato tubers. Immature tubers of Solanum tuberosum L. cv. King Edward were obtained from a local greengrocer. Starch was prepared according to Vos-Scheperkeuter et al. (1986). In mixing experiments with pea embryo, polyvinylpolypyrrolidone (200 mg-g-1 potato) was added with the extraction medium to samples containing potato. Approximately 1 g of tuber and 0.4 g of embryo were used in mixed extractions. Polyvinylpolypyrrolidone had no effect on the extractable activity of starch synthase from pea embryos (data not shown).
Results
Assay of starch synthase. The assay contained 100 mM N,N-bis(2-
Purification o f starch. In o r d e r to m i n i m i s e c o n t a m i n a -
hydroxyethyl)glycine (Bicine), 0.5 M Na-citrate (pH 8.6), 2.6 mM ADP[U-14C]glucose (Amersham plc, Amersham, Bucks., UK) at 2 GBq . m o l - 1, 1.5 mg amylopectin (from potato), and 20 ~tl extract or starch-granule suspension (0.125 g.m1-1 in the medium described above for incubation of granules with a-amylase) in a final vol of 115 ~tl. Controls contained either boiled extract or, where activity on starch grains was being assayed, starch that had been incubated in 1 M perchloric acid for 30 min at room temperature, washed four times with water and resuspended in the appropriate medium. Assays were incubated for 10 min at 25 ~ C, then heated to 100 ~ C for 2 rain. Assays containing granules were shaken vigorously throughout the incubation. Starch was precipitated, washed, and radioactivity in it determined as described previously (Smith et al. (1989).
tion of starch by protein bodies, the insoluble material f r o m p e a e m b r y o s w a s w a s h e d i n i t i a l l y in 0.5 M NaC1, a n d at a l a t e r s t a g e in p u r i f i c a t i o n t h e l e a s t - d e n s e fraction of the insoluble material - containing the remaining p r o t e i n b o d i e s - w a s d i s c a r d e d . P u r i f i e d s t a r c h f r o m developing pea embryos contained very few major proteins. O n S D S - p o l y a c r y l a m i d e gels, t h e t h r e e m o s t p r o m i n e n t b a n d s h a d a p p a r e n t m o l e c u l a r w e i g h t s o f 59, 60 a n d 77 k D a , t h e 5 9 - k D a p r o t e i n c o n s t i t u t i n g 7 0 % o r m o r e o f the p r o t e i n o n the g r a n u l e (Fig. 1). N o n e o f these proteins cross-reacted on Western blots with antib o d i e s to l e g u m i n a n d vicilin ( d a t a n o t s h o w n ) .
A.M. Smith: Starch-granule-bound starch synthase from pea P r e p a r a t i o n o f pea starch granules resulted in little or no loss o f activity o f starch synthase. First, a mixing experiment with p o t a t o tuber indicated that there was no general loss o f starch-synthase activity u p o n initial extraction o f embryos. W h e n samples o f pea e m b r y o and p o t a t o tuber were co-extracted, activity in the crude h o m o g e n a t e was 9 7 % o f that expected f r o m activities in crude h o m o g e n a t e s o f samples o f e m b r y o and tuber extracted separately. Second, after five washings o f the insoluble material f r o m a crude h o m o g e n a t e o f pea embryos, activity in the c o m b i n e d washings plus this material a c c o u n t e d for 89% o f that originally present in the crude h o m o g e n a t e . A d d i t i o n o f the protease inhibitors p h e n y l m e t h y l s u l p h o n y l fluoride ( P M S F , 1 m M ) , leupeptin (0.1 m M ) , or c h y m o s t a t i n (5 g g . m l - 1), or o f 10 m M E D T A , to the extraction and washing media h a d no effect o n the recovery o f starch-synthase activity (data n o t shown).
Fig. 1. Starch-granule-bound proteins of developing pea embryo and potato tuber. Tracks a, b,f, g, are 7.5% SDS-polyacrylamide gels of: a, protein from 1 mg pea starch; b, g, molecular-weight standards (sizes indicated in kDa); f, protein from 1 mg potato starch. Tracks c, d, e, are Western blots of proteins from pea starch as in track a. Tracks h, i, j, are Western blots of potato starch as in track f, except that protein was from about 7 mg starch. Tracks c, h, are developed with serum (1/5000 dilution) containing antibody to the 59-kDa protein of pea starch. Tracks d, i, are developed with serum (1/15000 dilution) containing antibody to the 77-kDa protein of pea starch. Tracks e, j, are developed with pre-immune serum (1/5000 dilution) from the rabbit subsequently immunised with the 59-kDa protein. Blots developed with preimmune serum from the rabbit subsequently immunised with the 77-kDa protein were identical to e andj (not shown)
601 Acetone-washed, purified starch stored at - 2 0 ~ lost up to 30% o f starch-synthase activity over two months. However, results o b t a i n e d with this starch were always qualitatively the same as those obtained with freshly purified starch. o f s t a r c h - g r a n u l e - b o u n d protein. A b o u t 50% o f the protein on purified starch granules was released by grinding followed by i n c u b a t i o n with a - a m y lase at 2 5 ~ for 1.5 h. On S D S - p o l y a c r y l a m i d e gels, the proteins released a p p e a r e d identical to those o n the Solubilisation
Fig. 2. A Release and fractionation of proteins from starch granules of pea by e-amylase and Mono Q chromatography. 7.5% SDSpolyacrylamide gels of: track a, molecular-weight standards (sizes indicated in kDa); track b, protein solubilised from 2.5 mg pea starch by digestion with e-amylase; track c, protein remaining on 2 mg pea starch after digestion with e-amylase; track d, protein from 1 mg undigested pea starch; track e, a-amylase used in digestion; track f, fraction of solubilised protein that failed to absorb to the Mono Q column; track g, fraction of solubilised protein that eluted from the Mono Q column with the major peak of starch-synthase activity. B Occurrence of the 59- and 77-kDa proteins of pea starch granules in the soluble fraction of pea embryos. Western blots of the total insoluble (tracks a, c) and soluble (tracks, b, d) fractions of pea embryos of 450 mg FW, prepared as described in Material and methods. Each track contains 0.1% of the material from a single embryo. Tracks a, b, developed with serum (1/1 500 dilution) containing antibody to the 59-kDa protein of pea starch granules. Tracks, c, d, developed with serum (1/10000 dilution) containing antibody to the 77-kDa protein of pea starch granules. Tracks e, f, developed with pre-immune serum (1/1 500 dilution) from rabbit subsequently immunised with the 59-kDa protein. Blots developed with pre-immune serum from the rabbit subsequently immunised with the 77-kDa protein were identical to e andf(not shown)
602 intact granule and those remaining on the granule after the incubation (Fig. 2A). Disruption of the granules by grinding caused an increase of four- to sevenfold in their starch-synthase activity. For example, in one, typical experiment activity increased f r o m 0.2 n m o l - m i n - ~ - m g - 1 starch on intact granules to 1.3 nmol. m i n - ~ - r a g - ~ starch on disrupted granules. Incubation with e-amylase solubilised about 50% of this activity. The solubilised activity plus that remaining in the insoluble fraction accounted for at least 80%, and often m o r e than 90%, o f the activity on the disrupted granules prior to incubation. Addition of the protease inhibitors P M S F , leupeptin or chymostatin to the incubation medium at the concentrations given above had no effect on the recovery of starch-synthase activity. The ratio of citrate-stimulated to unstimulated activity in the presence of primer was altered during the solubilisation o f starch synthase. This ratio was 1.2 + 0.1 for disrupted granules, 2.5_+0.2 for solubilised activity, and 1.0_+ 0.2 for activity that remained insoluble after incubation (means-t-SE from three experiments, each on a separate batch o f starch). Activity with UDP-glucose was less than 10% of that with ADP-glucose at both low (2.6 m M ) and high (26 m M ) concentrations. Activity with glycogen as a primer was only 16% or less of that with amylopectin at the same concentration (10 mgml-~ in the assay).
Identification and separation of solubilised proteins. Because of the prominence o f the 59-kDa protein of pea starch granules, and its similarity in size to the " w a x y " proteins of other species, it seemed likely that it might be a starch-granule-bound starch synthase. On Western blots an antibody raised to the 59-kDa protein specifically recognised this protein and the 60-kDa protein of pea starch granules, and the major, 57-kDa protein of potato starch granules (Fig. 1). It did not recognise proteins in the soluble fraction of pea embryos (Fig. 2 B). However, the antibody did not precipitate starch-synthase activity f r o m preparations of solubilised, starchgranule-bound protein (Fig. 3). This was not because the antibody failed to recognise or precipitate the 59k D a protein under the experimental conditions. The antibody strongly recognised the native protein (not shown), and antibody-antigen complexes were precipitated from the incubation with Protein A-Sepharose. When solubilised, starch-granule-bound proteins were fractionated on a M o n o Q anion-exchange column, activity of starch synthase was consistently recovered in three peaks which eluted between 0.15 and 0.35 M KC1 and accounted for at least 80% of the activity loaded. Less than 10% of the recovered activity failed to adsorb to the column. This pattern was seen with both freshly prepared and acetone-washed starch. A typical fraction is shown in Fig. 4. The ratios o f citrate-stimulated to unstimulated activity, and primed to unprimed activity, of the three peaks varied considerably from one preparation to another. Variation between experiments was greater than variation between the peaks in any one experiment. The
A.M. Smith: Starch-granule-bound starch synthase from pea
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Fig. 3A-D. Precipitation experiments with antibodies to the 59and 77-kDa proteins of the pea starch granule. Solubilised, starchgranule-bound proteins or intact starch granules were incubated with serum for 2 h, then assayed for starch-synthase activity. Incubations with solubilised, starch-granule-bound proteins also contained Protein A-Sepharose which was removed by centrifugation prior to assay. For each experiment, the ratio of serum containing antibody to pre-immune serum was varied so that all incubations contained the same concentration of serum. In all experiments, control incubations without serum had the same activities (+ 15%) of starch synthase as incubations with pre-immune sera. A Solubilised starch-granule-bound protein. B Intact starch granules. Activities of starch synthase in A and B are expressed as percentages of activity in incubations containing pre-immune sera alone, e - - e , Incubation with serum containing antibody to the 77-kDa protein; o - - o , incubation with serum containing antibody to the 59-kDa protein. C, D Western blots of 7.5% SDS-polyacrylamide gels of incubations with antibody to the 77-kDa protein shown in A. Tracks 1~4 are equal loadings of the supernatants after centrifugation of incubations with, in the order given, 0, 5, 10 and 15 gl of serum. C is developed with serum (1/15000 dilution) containing antibody to the 77-kDa protein, D with serum (1/5000 dilution) containing antibody to the 59-kDa protein. Note that the amount of 77-kDa protein declines with increasing antibody concentration (C) whereas the amount of 59/60-kDa protein does not (D). The strong band common to both blots, and also to blots developed with pre-immune sera (not shown), is rabbit IgG
relative activities of the peaks also varied from one experiment to another, but in six out of seven experiments the first peak contained considerably more activity than either of the other two. The distribution of activity eluting from the M o n o Q column did not match that of the 59- and 60-kDa proteins. Approximately 80% of these proteins failed to adsorb to the column (Fig. 2A, estimate made from scanning gels of column fractions). The remainder eluted as a broad, single peak at approx. 0.25 M KC1 (Fig. 4). The distribution of activity closely matched that of the 77-kDa protein. In particular, it was often the only pro-
A.M. Smith: Starch-granule-bound starch synthase from pea
603
0.5
starch synthase from preparations o f solubilised, starchgranule-bound protein from pea starch granules (Fig. 3). In these experiments, little or none of the 59- and 60-kDa proteins was precipitated (Fig. 3). The antibody also inhibited activity of starch synthase on intact pea starch granules (Fig. 3).
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15
Fig. 4. Analysis of fractions of solubilised, starch-granule-bound protein eluted from a Mono Q anion-exchange column. Solubilised, starch-granule-bound protein from freshly prepared starch which had not been treated with acetone was applied to a Mono Q column in Tris-acetate buffer (pH 8.0) and eluted with a gradient of KC1 ( . . . . . ). One-ml fractions were collected and monitored for protein content ( ) and activity of starch synthase @ - - - -). W, wash fraction. Western blots of 7.5% SDS-polyacrylamide gels of individual fractions (wash and 8-18) were developed with: A, serum (l/15 000 dilution) containing antibody to the 77-kDa protein of pea starch granules, and B, serum (1/5000 dilution) containing antibody to the 59-kDa protein of pea starch granules. Each track contains approx. 1/20 of the material from the fraction, except for the wash fraction where the track contains approx. 1/175 of the material. Sizes of bands are indicated in kDa. Note the presence of large amounts of the 59-kDa protein, and the absence of the 77-kDa protein and starch-synthase activity, in the wash fraction. Fractions containing the first two peaks of starch-synthase activity also contain the largest amounts of the 77-kDa protein
tein visible on gels of the first, major peak of activity (Fig. 2A), most of it adsorbed to the column, and it eluted as two peaks and a tail that coincided with the three peaks of starch-synthase activity (Fig. 4). On Western blots, an antibody raised to the 77-kDa protein recognised this protein and - much more weakly - the 59- and 60-kDa proteins and a minor, 45-kDa protein of peak starch granules (Fig. 1). It recognised a very minor 77-kDa protein in the soluble fraction o f pea embryos (Fig. 2 B). It also recognised strongly a minor 92-kDa protein and much more weakly - the major, 57-kDa protein, of potato starch granules (Fig. 1). The antibody precipitated almost all of the activity of
It is highly likely that the activity eluted from the M o n o Q column represents by far the major, if not the only, form of starch-granule-bound starch synthase in developing pea embryos. Direct measurement of the recovery of activity from the crude homogenate in fractions from the column is not possible because of the large increase in assayable activity brought about by disruption of the granules. However, estimates o f recoveries and mixing experiments indicate strongly that only minimal losses of activity occurred at all other stages. Activity of starchgranule-bound starch synthase in crude homogenates o f embryos of this cultivar o f peas is about three times greater than is required to account for the rate of starch synthesis (Smith et al. 1989). Most, if not all, of the starch-granule-bound starch synthase o f the pea embryo is attributable to a 77-kDa protein 9 This was the only protein which consistently co-purified with the major peak of starch-synthase activity solibilised from the starch granule, and its distribution in fractions from the column generally matched that of starch-synthase activity. An antibody to this protein precipitated starch-synthase activity from preparations of solubilised, starch-granule-bound protein, and inhibited activity on the intact granule. The 77-kDa protein is predominantly bound to the starch granule in the developing pea embryo, although a closely related or identical protein is a very minor component o f the soluble protein of the embryo. The reason why activity apparently attributable to a single protein eluted as three peaks from the Mono Q column is unclear. Solubilisation of starch-granulebound proteins led to reproducible changes in the ratio of citrate-stimulated to unstimulated starch-synthase activity in the presence of primer, similar to those reported for the enzyme from maize endosperm (MacDonald and Preiss 1983). However, in starch synthase eluted from the M o n o Q column, this ratio and the ratio o f primed to unprimed activity varied widely from one experiment to another, as did the ratios of activity in the three peaks. It is possible that both the effects of citrate and primer on the activity, and the point at which it elutes from the column, are functions of the amount and size of endogenous glucan primer bound to it, and that this varies from one preparation to the next. My results provide no evidence that the major, antigenically similar, 59- and 60-kDa proteins o f pea starch granules have starch-synthase activity. Their distribution in fractions from the M o n o Q column did not match that o f the activity, and an antibody which strongly recognised both of these proteins failed to precipitate starch-synthase activity from preparations of solubilised,
604 s t a r c h - g r a n u l e - b o u n d protein. Conversely, starch-synthase activity could be precipitated with a n t i b o d y to the 7 7 - k D a starch synthase u n d e r conditions in which the 59- and 6 0 - k D a proteins remained in solution. However, the possibility that a starch-synthase activity o f these proteins was lost on extraction, or n o t revealed by the assays I used, c a n n o t be ruled out. The very weak crossreaction between the a n t i b o d y to the 7 7 - k D a starch synthase and the 59- and 6 0 - k D a proteins on Western blots indicates that they are related in some way to starch synthases, but their role in the synthesis o f the starch granule remains to be elucidated. The strong cross-reaction between the a n t i b o d y to the 5 9 - k D a protein o f the pea starch granule and the m a j o r protein o f the p o t a t o starch granule indicates that the 59- and 6 0 - k D a proteins o f pea belong to the " w a x y " class o f proteins f o u n d on the starch granules o f all species so far examined. The m a j o r protein o f the p o t a t o starch granule is eliminated by a " w a x y " m u t a t i o n , and is antigenically, related to the w a x y gene p r o d u c t o f maize (Vos-Scheperkeuter et al. 1986; H o v e n k a m p - H e r m e l i n k et al. (1987). It has been generally accepted that " w a x y " proteins are the m a j o r or only f o r m o f s t a r c h - g r a n u l e - b o u n d starch synthase in plants (Preiss 1988) M y results indicate that this is n o t universally the case. First, I can find no evidence that significant starchsynthase activity is associated with the " w a x y " proteins o f pea. Second, I can attribute essentially all o f the s t a r c h - g r a n u l e - b o u n d starch-synthase activity o f pea to a protein which is a different size from, and only very weakly antigenically related to, the " w a x y " class o f proteins. Third, the 9 5 - k D a protein o f p o t a t o starch granules is m u c h m o r e closely related to the starch synthase o f pea starch granules than is the " w a x y " protein o f p o t a t o . It seems likely that this 9 5 - k D a protein is an i m p o r t a n t s t a r c h - g r a n u l e - b o u n d starch synthase. I suggest that a re-examination, species by species, o f the identity o f the s t a r c h - g r a n u l e - b o u n d starch synthase and the role o f the " w a x y " protein m a y be required. I am grateful to my colleagues Kay Denyer, Ian Dry (CSIRO, Adelaide, Australia), Rob Ireland (Mount Allison University, New Brunswick, Canada), Cathie Martin and Steve Rawsthorne for useful discussions during the course of this work, Cliff Hedley for the gift of pea seeds, and Ian Bedford for preparing pea starch and gels of starch-granule-bound proteins. This work was supported by the Agriculture and Food Research Council via a grantin-aid to the John Innes Institute.
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