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Plant Physiol. (1993) 103: 1355-1359 Biochemical Evidence for the Role of the Waxy Protein fron Pea (Pisum sativum 1.) as a Granule-Bound Starch Synthase Mirta Noemi Sivak*, Margaret Wagner, and Jack Preiss Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824 circumvented the problem by solubilizing starch from maize kemels with a-amylase and amyloglucosidase and found that the dominant of the two starch synthases so released had a molecular mass of 60 kD, as determined by ultracentrifugation on Suc density gradients (Macdonald and Preiss, 1985). Smith (1990), however, using essentially the same approach on pea (Pisum sativum L.) starch, obtained results that led her to propose that "the 'waxy' protein of pea. . . is not the major granule-bound starch synthase" and that re-examination, species by species, of the identity of the starchgranule-bound starch synthase" may be required. Because of the need to elucidate the mechanism of amylose synthesis and the relevance of this subject to the possible alteration of the amylose content of starch for industrial purposes, it was considered necessary to reinvestigate the GBSS from pea. Here we present new evidence for the role of the Wx protein of pea as a GBSS that contradicts the findings presented by Smith (1990). Proteins were solubilized from starch extracted from developing pea (Pisum sativum 1.) embryos and chromatography of these proteins on a Mono-Q column separated two peaks of starch synthase activity. The major activity peak comprised more than 80% of the total activity. This fraction contained only the Waxy protein, as shown by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate followed by staining for proteins or by immunoblot. A 77-kD polypeptide associated with the starch granules and presumed by others to be a starch synthase could not be detected in any of the active fractions. The native molecular weight of the solubilized starch synthase was 59,600 f 1700 as determined by sucrose density gradient. It is concluded that in pea seeds the Waxy protein and the starch synthase bound to the granule are the same protein. The final product of the Waxy locus is a protein of molecular mass 58 to 60 kD associated with the starch granule in a11 the plant species and tissues studied (for a review, see Preiss, 1991). A large body of genetic evidence indicates that the GBSS activity, which is responsible for the synthesis of amylose, is a function of the Wx protein. Indeed, in wx mutants there is virtually no amylose, GBSS activity is very low, and the Wx protein is missing (Nelson and Rines, 1962; Nelson et al., 1978; Echt and Schwartz, 1981; Shure et al., 1983). In an amylose-free potato mutant, activity of GBSS was very low and the Wx protein was absent (HovenkampHermelink et al., 1987). When potato plants were transformed to produce antisense RNA so that the expression of the Wx gene would be inhibited, the activity of GBSS was also inhibited and the tubers contained amylose-free starch (Visser et al., 1991). The similarity in the deduced amino acid sequences of the Wx proteins and the glycogen synthase from Escherichia coli, particularly at the ADP-Glc binding site (Preiss, 1991), strongly suggests that the Wx proteins are GBSS. However, no direct biochemical evidence of the identity between the GBSS and the Wx protein has been presented to date. The Wx protein can be extracted by heating the starch with SDS or by incubating at 37OC with 9 M urea (Frydman and Cardini, 1967; Shure et al., 1983), methods too drastic for the extraction of starch synthase activity. Macdonald and Preiss (1983) MATERIALS AND METHODS Materiais Pea (Pisum sativum L.) of the round-seeded var Alaska (Burpee, Warmister, PA) was grown in a greenhouse. The embryos were frozen on dry ice upon harvesting and stored at -8OOC until use. Mono-Q HR 5/5 and mo1 wt standards were from Pharmacia. Bicinchoninicacid protein reagent was from Pierce (Rockford, IL). [U-'4C]Glc-l-P (315.9 mCi/mmol) was from DuPont-New England Nuclear. ADP['4C]Glc was synthesized from ATP and [U-'4C]Glc-l-P (Preiss and Greenberg, 1972). Amylopectin was purchased from Pierce, aamylase (pig pancreas, PMSF treated, 1260 units/mg protein), prestained mo1 wt standards, pig heart malic dehydrogenase mitochondrion), and rabbit muscle lactic dehydrogenase came from Sigma; amyloglucosidase (Aspergillus niger, 14 units/mg protein) were from Boehringer Mannheim; Dowex 1-X8 resin (200-400 mesh, chloride form, analytical grade) was obtained from Bio-Rad. The amylase inhibitors acarbose and BAY e 4609 were gifts of Drs. E. Truscheit and D. Schmidt of Bayer AG (Wuppertal, Germany). Preparation of Starch Granules Starch was isolated from pea seeds as described previously for maize kemels (Macdonald and Preiss, 1983) with the following minor modifications. Embryos were crushed in a This work was supported by a grant to M.N.S. from the Research Excellence Funds of the state of Michigan and by the U.S. Department of Agriculture/Department of Energy/National Science Foundation Plant Science Program No. 88-37271-3964. * Corresponding author; fax 1-517-353-9334. Abbreviations: GBSS,granule-bound starch synthase; Wx, Waxy. 1355 Downloaded from on August 3, 2017 - Published by www.plantphysiol.org Copyright © 1993 American Society of Plant Biologists. All rights reserved. Sivak et al. 1356 blender. The starch-containing pellet obtained after the first centrifugation of the crude extract was resuspended in a medium containing 50 m~ Tris-acetate, pH 8.0, 2 m~ EDTA, 2.5 m~ DTT, and 30% (w/v) SUC,and filtered through four layers of cheesecloth. The filtrate was layered on top of a SUC cushion (2 volumes of suspended granules per volume of Suc cushion) containing 75% (w/v) SUCin the same medium as the 30% SUCsolution (Burr and Burr, 1976) and spun for 10 min at 12,OOOg. After remova1 of the supematant and the protein bodies that remained at the top of the cushion, the starch pellet was resuspended in the 30% SUCsoiution and the procedure was repeated. The starch was finally resuspended in the 30% SUCsolution, vacuum filtered through a glass fiber filter, and rapidly washed with 100 mL of acetone (-20°C) by resuspending the starch in the acetone and filtering under vacuum. The starch was then vacuum dried at room temperature and stored at -2OOC until use. Release and Fradionation of Granule-Bound Proteins Starch (3 g) was ground, suspended in buffer, and partially digested essentially as described by Macdonald and Preiss (1983), but a-amylase was used at 70 units mL-' and amyloglucosidase, when it was used, was at 0.56 unit mL-'. After treatment of the starch with a-amylase and amyloglucosidase, the starch suspension was centrifuged for 30 min at 25,OOOg. The precipitate was subjected to a second solubilization cycle and the two supematants were pooled and concentrated using an Amicon apparatus fitted with a PM30 membrane. The concentrate was diluted 10-fold with a medium containing 20 m~ triethanolamine/KOH, pH 7.3, 2.5 m~ DTT, 10% (v/v) glycerol, and 2 m~ EDTA (buffer A) and applied to a Mono-Q column (HR 5 X 5, Pharmacia fastprotein liquid chromatography system) equilibrated with buffer A. After washing the column with 5 mL of this medium, the enzyme was eluted with a gradient of KC1 in buffer A as indicated in the figure; the flow rate was 0.5 mL min-1. Fractions of 1 mL were collected, assayed for starch synthase activity, and subjected to SDS-PAGE and immunoblot as described below. Assay of Starch Synthase The transfer of Glc from ADP-Glc into primer was measured in an assay (essentially the same as assay B described by Macdonald and Preiss, 1983), which contained 140 nmol ADP['4C]Glc of specific activity 700 cpm nmol-', 20 pmol Bicine/KOH, pH 8.4, 0.5 M citrate, 2 pmol GSH, 1 pmol EDTA, 1 mg rabbit liver glycogen, and enzyme in a total volume of 0.2 mL. Reactions were incubated at 3OoC and the incorporation of radioactivity into methanol-insoluble polysaccharide was measured as described previously (Ghosh and Preiss, 1966). When the presence of amylases interfered with this assay, the reaction was performed in the presence of amylase inhibitors and the product was quantified by using Dowex 1-X8 to absorb the unreacted ADP['4C]Glc as described by Macdonald and Preiss (1983). Plant Physiol. Vol. 103, 1993 PACE and lmmunoblotting Samples were analyzed on 7% polyacrylamide gels in the presence of SDS according to Laemmli (1970). For the analysis of starch-bound proteins, these were extracted as described by Shure et al. (1983). After electrophoresis, proteins were stained using silver or Coomassie brilliant blue as indicated, or electroblotted onto nitrocellulose membrane according to Bumette (1981). After electroblotting, nitrocellulose membranes were treated with rabbit antiserum against the maize Wx protein (Shure et al., 1983) or with antiserum raised against the 77-kD protein (Smith, 1990). The antigen-antibody complex was visualized by treating the membrane with goat anti-rabbit immunoglobulin G linked to alkaline phosphatase, followed by incubation with a substrate mix containing 5-bromo-4-chloro-3-indoyl phosphate and nitroblue tetrazolium. Immunological specificity was tested by substituting rabbit preimmune serum. MOIWt Determination The mo1 wt of the partially purified enzymes was determined on 5 to 20% SUCdensity gradients according to Martin and Ames (1961). The 3-mL gradients were 5 to 20% SUCin 50 m~ Tris-acetate, pH 8.0, 2 m~ EDTA, 2.5 m~ DTT, and centrifugation was in a SW60 rotor, at 4OC, 125,OOOg for 18 h. 'The markers were pig heart malate dehydrogenase (mitochondrial, molecular mass 68 kD) assayed by the method of Ochoa (1955), and rabbit muscle lactate dehydrogenase (molecular mass 140 kD) assayed according to Komberg (1955). Other Methods Protein was measured by the method of Smith et al. (1985) using commercially obtained bicinchoninic acid reagent and BSA as standard. RESULTS AND DISCUSSION Starch granule-bound proteins from developing pea embryos were released from the starch using a-amylase. After two cycles of digestion-centrifugation,70 to 80%of the starch was recovered in the precipitate. The starch synthase activity of the starch is likely to be severely underestimated before digestion because of the difficulty of the ADP['4C]Glc in reaching the granule-bound enzyme. Therefore, the a-amylase digestion appeared to solubilize about 100% of the activity (rather than the 20-30% that could be expected from the amount of starch digested). The supematant containing the amylase and the proteins released from the starch were fractionated by chromatography on a Mono-Q column that resolved two peaks of starch synthase activity (Fig. l), the major one comprising more than 80% of the total. The activity recovered from the MonoQ column was higher than that applied to it, probably because in the presence of high amounts of amylases starch synthase activity is underestimated to some extent even when using the Dowex assay and amylase inhibitors. (Macdonald and Preiss, 1983). At the concentration of a-amylase used, amyloglucosidasedid not seem to be necessary for solubilization. Indeed, the recovery of starch synthase in the Downloaded from on August 3, 2017 - Published by www.plantphysiol.org Copyright © 1993 American Society of Plant Biologists. All rights reserved. Waxy Protein from Pea Is a Granule-Bound Starch Synthase KCIIM) 07 PS FT 10 II 12 13 14 15 MW 1357 13 14 15 MW 04 (-42.7 0.3 •O.Z O.I 03 10 Poss-Through Figure 1. Chromatography on Mono-Q of the proteins released from starch extracted from developing pea embryos by digestion with a-amylase. After injecting the sample and washing the column with buffer without KCI, the column was eluted with a gradient containing KCI as indicated in the figure and 1-mL fractions were collected and monitored for starch synthase activity (broken line) and A2so (continuous line). "Pass-Through" indicates the activity and AIM of the fraction that eluted from the column during injection. supernatant was similar and the starch synthase activity eluted at the same positions in the gradient (not illustrated). Another factor that did not affect recovery of activity or the activity profile obtained from the Mono-Q chromatography was whether the purified starch was used 'wet/ i.e. solubilized immediately after its purification, or whether it was first washed with acetone and dried as described above (see 'Materials and Methods'). The first activity peak (which eluted from the Mono-Q column at 0.085 M KCI) displayed a relatively high specific activity, i.e. 10 fanol [I4C]Glc incorporated min"1 mg"1 protein, when assayed in the presence of 0.5 M citrate. SDSPAGE of this fraction followed by staining for proteins showed the presence of a 60-kD polypeptide (Fig. 2), which in immunoblot (not illustrated) was recognized by the antiserum raised against the Wx protein of maize (Shure et al., 1983). Conversely, the 77-kD polypeptide present in pea starch (Fig. 2) could not be detected in any of the active fractions (peak I or II) by protein staining or by immunoblot using an antiserum raised against the latter protein (Smith, 1990). It is worth noting that the antiserum raised against the 77-kD polypeptide present in pea starch (Smith, 1990) also recognized other polypeptides, both in pea (Fig. 2) and in maize starch (not illustrated). For this reason, in experiments like the one illustrated in Figure 2, the antiserum against the 77-kD protein was used for immunoblots because it recognized both the 77-kD and the Wx protein and therefore simplified the characterization of the fractions. The anti-77kD antiserum clearly showed the presence of the 60-kD polypeptide in the active fraction. No Wx protein could be detected in the 'pass-through' fraction, which contained the «-amylase used to release the starch-bound proteins. Figure 3 shows an example of the cenrrifugation pattern on Sue density gradient of the GBSSI and of the internal standards, malic dehydrogenase from pig heart (mitochondrial) with a molecular mass of 67 kD (Noyes et al., 1974), Figure 2. SDS-PACE of proteins from a pea starch sample (1 mg) and of Mono-Q fractions (10 jiL) as shown in Figure 1. Left, Immunoblot using antiserum raised against the 77-kD protein associated with pea starch (Smith, 1990). Right, Protein (silver) staining. PS, Pea starch; PT, "pass through," i.e proteins that did not bind to the column. The numbers indicate the fraction number as in Figure 1. For the immunoblot the molecular mass standards were prestained with apparent molecular mass as follows: Fru-6-P kinase (rabbit muscle), 96.4 kD; pyruvate kinase (chicken muscle), 80.4 kD; ovalbumin (chicken egg), 55.7 kD; lactic dehydrogenase (rabbit muscle), 43.7 kD. For protein staining, the standards were: phosphorylase b, 97.4 kD; BSA, 66.2 kD; ovalbumin, 42.7 kD. A polypeptide of 60 kD, recognized by the antiserum, was present in fraction 12, which contained more than 80% of the total starch synthase activity recovered from the Mono-Q. Note that the antiserum recognized not only the 77-kD polypeptide present in starch but also two bands of 59 and 60 kD. 1 0.0 Fraction number Figure 3. Ultracentrifugation pattern on a continuous Sue density gradient (5-20% Sue) of the major starch synthase associated with pea starch (CBSSI) and of the internal standards, malic dehydrogenase (MDH) from pig heart (mitochondrial, molecular mass 67 kD) and lactic dehydrogenase (LDH) from rabbit muscle (molecular mass 140 kD). Enzyme activities are expressed for LDH and MDH as A/W min"1 ^L"1 of fraction, and for starch synthase as nmol [14C]Clc incorporated 15 min"' /iL"1 of fraction. Downloaded from on August 3, 2017 - Published by www.plantphysiol.org Copyright © 1993 American Society of Plant Biologists. All rights reserved. 1358 Sivak et al. and the lactic dehydrogenase from rabbit muscle with a molecular mass of 140 kD (Lovell and Winzor, 1974). From the migration of the GBSSI in the gradient it was calculated that its native molecular mass is 59.6 f 1.7 kD (average of three determinations). Although the first activity peak that eluted from the MonoQ comprised most of the starch synthase activity, the second peak was also studied. The properties of the two isoforms were very similar with respect to their stimulation by citrate and primer preference. Activity in the absence of added primer as a percentage of the activity with primer was higher for GBSSII, suggesting that the latter may contain more endogenous primer (a-glucans) associated with it (Sivak, 1992). The mo1 wt of the GBSSII was found to be similar to that of GBSSI, and SDS-PAGE showed that it also contained Wx protein plus other polypeptides, but not one of 77 kD (not illustrated). The fact that the antiserum raised against the 77-kD polypeptide from pea recognizes others, most significantly the Wx protein, would make neutralization experiments using the antiserum too ambiguous to interpret. This is especially true for the experiments of Smith (1990), in which very large volumes of undiluted serum (20-100 pL of serum for 100 pL of solubilized starch proteins, equivalent to about 0.008 starch synthase unit) were used in the neutralization experiments and high dilutions of it (1:15,000) were used for the immunoblots. Dry et al. (1992) compared the amino acid sequence of the mature pea 59-kD protein (named GBSSI in that paper) deduced from the corresponding cDNA sequence with those of the maize Wx and potato Wx proteins and the glycogen synthase of Escherichia coli (see also Preiss, 1991) and could find no obvious difference that could justify the lack of starch synthase activity of the pea 59-kD protein. The data presented here indicate that in developing pea embryos the Wx protein and the starch synthase bound to the granule are the same protein. The methodology used by Smith (1990) was somewhat different than the one used by Macdonald and Preiss (1983), i.e. she used only a-amylase (and not a combination of a-amylase and amyloglucosidase) for the solubilization of starch and only one step of purification, i.e. the chromatography on Mono-Q. These changes, however, cannot explain the different results obtained by Smith because when we used only a-amylase for starch solubilization and chromatographed on Mono-Q the proteins so released, the correlation between the presence of Wx protein and starch synthase activity was very clear and the 77-kD protein could not be detected in the active fractions. It is worth mentioning that in our experiments only aamylase and not Wx protein could be detected in the protein fraction that did not bind to the column. Conversely, in the results published by Smith, most of the Wx protein was in the “pass-through”fraction. The a-amylase does not bind to the Mono-Q in these conditions and would make the detection of starch synthase activity difficult. The cause for the failure of the Wx protein to bind to the Mono-Q column at pH 8.0 (Smith, 1990), when in our hands it did attach at pH 7.3, is unknown, but possible explanations are (a) the KCl present in the solubilization media was incompletely removed by inadequate dialysis, reducing the Plant Physiol. Vol. 103, 1993 capacity of the Mono-Q column and decreasing the capacity of the weaker-charged proteins to bind; (b) the use of Tris buffer by Smith negatively affected the chromatography, since Tris is not recommended for its use on Mono-Q because of the large pH variation experienced with temperature changes; and/or (c) the use by Smith (1990) of only a fraction of the a-amylase activity used by Macdonald and Preiss (1983) may have been insufficient. As discussed above, measurement of starch synthase activity present in the starch itself can be grossly underestimated, which could lead, in tum, to gross overestimation of the proportion of activity released by treatment with amylases. It is worth mentioning that the major GBSS described by Macdonald and Preiss, with a native molecular mass of 60 kD, is associated with the Wx protein according to the criteria described above for the pea GBSS (not illustrated). Thus, the biochemical reexamination of starch synthase present in starch from two species, i.e. maize and pea, strengthens the genetic evidence supporting the role of the Wx protein as a GBSS with a major role in the determination of amylose content of starch. ACKNOWLEDCMENTS We wish to thank Dr. Alison Smith for the gift of antiserum raised against the 77-kD protein of pea starch, and Dr. Susan Wessler for the antiserum raised against the Wx protein of maize. Received June 28, 1993; accepted August 31, 1993. Copyright Clearance Center: 0032-0889/93/l03/1355/05. LITERATURE ClTED Burnette WW (1981) Westem blotting. Electrophoretic transfer of proteins from SDS-polyacrylamidegels to nitrocelluloseand radiographic detection with antibody and radioiodinated protein A. Ana1 Biochem 112: 195-203 Burr B, Burr FA (1976) Zein synthesis in maize endosperm by polyribosomes attached to protein bodies. Proc Natl Acad Sci USA 7 3 515-519 Dry I, Smith A, Edwards A, Bhattacharyya M, Dunn P, Martin C (1992) Characterization of cDNAs encoding two isoforms of granule-bound starch synthase which show differential expression in developing storage organs of pea and potato. Plant J 2: 193-202 Echt CS, Schwarz D (1981) Evidence for the inclusion of controlling elements within the structural gene at the Waxy locus in maize. Genetics 9 9 275-284 Frydman RB, Cardini CE (1967) Studies on the biosynthesis of starch 11. J Biol Chem 242: 312-317 Ghosh HP, Preiss P (1966) Adenosine diphosphate glucose pyrophosphorylase. A regulatory enzyme in the biosynthesis of starch in spinach chloroplasts. J Biol Chem 241: 4491-4505 Hovenkampt-Hermelink JHM, Jacobsen E, Ponstein AS, Visser RGF, Vos-Sheperkeuter GH, Bijmolt EW, de Vries JN, Witholt B, Feenstra WJ (1987) Isolation of an amylose-free starch mutant of the potato (Solanum tuberosum L.). Theor Appl Genet 75: 217-221 Kornberg A (1955) Lactic dehydrogenase of muscle. Methods Enzymol 1:441-443 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685 Lovell SJ, Winzor DJ (1974) Effects of phosphate on the dissociation and enzymic stability of rabbit muscle lactate dehydrogenase. Biochemistry 13 3527-3531 Macdonald FD, Preiss J (1983) Solubilization of the starch-granulebound starch synthases of normal maize kemels. Plant Physiol73 175-178 Downloaded from on August 3, 2017 - Published by www.plantphysiol.org Copyright © 1993 American Society of Plant Biologists. All rights reserved. Waxy Protein from Pea 1s a Granule-Bound Starch Synthase Macdonald FD, Preiss J (1985) Partia1 purification and characterization of granule-bound starch synthases from normal and waxy maize. Plant Physiol78 849-852 Martin RG, Ames BN (1961) A method for determining the sedimentation behavior of enzymes: application to protein mixtures. J Biol Chem 2 3 6 1372-1379 Nelson OE,Chourey PS, Chang MT (1978) Nucleoside diphosphate sugar-starch glucosyl transferase activity of wx starch granules. Plant Physiol62: 383-386 Nelson OE, Rines HW (1962) The enzymatic deficiency in the waxy mutant of maize. Biochem Biophys Res C o m " 9 297-300 Noyes BE, Glatthaar BE, Caravelli JS, Bradshaw RA (1974) Structural and functional similarities between mitochondrial malate dehydrogenase and L-3-hydroxyacyl COA dehydrogenase. Proc Natl Acad Sci USA 71: 1334-1338 Ochoa S (1955) Malic dehydrogenase from pig heart. 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Ana1 Biochem 1 5 0 76-85 Visser RGF, Somhorst 1, Kuipers GJ, Ruys NJ,Feenstra WJ, Jacobsen E (1991) Inhibition of the gene for granule-bound starch synthase in potato by antisense constructs. Mo1 Gen Genet 2 2 5 289-296 Downloaded from on August 3, 2017 - Published by www.plantphysiol.org Copyright © 1993 American Society of Plant Biologists. All rights reserved.