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First published online February 8, 2008; 10.1104/pp.108.116244 Plant Physiology 146:1878-1891 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
Analysis of Protein Complexes in Wheat Amyloplasts Reveals Functional Interactions among Starch Biosynthetic Enzymes1,[C],[W],[OA]Department of Molecular and Cellular Biology, College of Biological Sciences, University of Guelph, Guelph, Ontario, N1G 2W1, Canada (I.J.T., K.G.B., S.C., A.M., F.L., N.S.B., M.J.E.); Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College, London W6 8LH, United Kingdom (R.W.); and Division of Plant Industry, Commonwealth Scientific and Industrial Research Organization, Canberra, Australian Capital Territory 2601, Australia (M.K.M.)
Protein-protein interactions among enzymes of amylopectin biosynthesis were investigated in developing wheat (Triticum aestivum) endosperm. Physical interactions between starch branching enzymes (SBEs) and starch synthases (SSs) were identified from endosperm amyloplasts during the active phase of starch deposition in the developing grain using immunoprecipitation and cross-linking strategies. Coimmunoprecipitation experiments using peptide-specific antibodies indicate that at least two distinct complexes exist containing SSI, SSIIa, and either of SBEIIa or SBEIIb. Chemical cross linking was used to identify protein complexes containing SBEs and SSs from amyloplast extracts. Separation of extracts by gel filtration chromatography demonstrated the presence of SBE and SS forms in protein complexes of around 260 kD and that SBEII forms may also exist as homodimers. Analysis of cross-linked 260-kD aggregation products from amyloplast lysates by mass spectrometry confirmed SSI, SSIIa, and SBEII forms as components of one or more protein complexes in amyloplasts. In vitro phosphorylation experiments with  -32P-ATP indicated that SSII and both forms of SBEII are phosphorylated. Treatment of the partially purified 260-kD SS-SBE complexes with alkaline phosphatase caused dissociation of the assembly into the respective monomeric proteins, indicating that formation of SS-SBE complexes is phosphorylation dependent. The 260-kD SS-SBEII protein complexes are formed around 10 to 15 d after pollination and were shown to be catalytically active with respect to both SS and SBE activities. Prior to this developmental stage, SSI, SSII, and SBEII forms were detectable only in monomeric form. High molecular weight forms of SBEII demonstrated a higher affinity for in vitro glucan substrates than monomers. These results provide direct evidence for the existence of protein complexes involved in amylopectin biosynthesis.
Starch is produced by the majority of higher plant species inside plastids and represents a major storage product of many of the seeds and storage organs produced agriculturally and used for human consumption as well as many important industrial applications. The starch granule is a complex polymeric structure with a hierarchical order, allowing efficient packing of large amounts of Glc into a water-insoluble form, and is composed of two distinct types of Glc polymer: amylose and amylopectin.
Amylose comprises largely unbranched
The basic enzymatic steps required for the formation of amylopectin (the core pathway) are known and have been determined using a combination of biochemical techniques and forward and reverse genetics approaches. Amylopectin biosynthesis is initiated by the formation of
To date, information on how coordination between amylopectin-synthesizing enzymes is achieved is sparse. Recent work in wheat (Triticum aestivum) endosperm amyloplasts suggests protein phosphorylation is involved in modulating the catalytic activity of some key enzymes (the SBEII class) and their ability to form physical interactions with other starch-metabolizing enzymes (Tetlow et al., 2004b The objective of the research described in this communication was to investigate interactions between SSs and SBEs in starch-synthesizing plastids. This article describes the isolation and characterization of protein complexes comprising SSI, SSII, and SBEII from amyloplast extracts of developing wheat endosperm. The data indicate that formation of the SS-SBE protein complexes and SBEII homodimers in the developing endosperm occurs from around 10 to 15 d after pollination (DAP), the major grain-filling period, and is phosphorylation dependent. This article presents the first direct evidence of catalytically active protein complexes involved in amylopectin biosynthesis and indicates that the kinetic properties of branching enzymes are altered in higher ordered states, most likely dimers.
Fractionation and Measurement of SS and SBE Activities in Developing Wheat Endosperm Soluble proteins extracted from developing wheat endosperm at early stages of grain filling were separated by gel filtration chromatography to examine possible changes in aggregation state of specific enzyme activities involved in amylopectin biosynthesis. Measurements of the extractable catalytic activities of SS and SBE at early stages of endosperm development (6–9 DAP) following separation of protein extracts by gel filtration chromatography showed single, broad peaks of SS and SBE activities (Fig. 1, A and B ). Both activities eluted from the gel filtration column in fractions with apparent molecular mass within the size range expected of monomeric forms of SBEII (approximately 88 kD) and of the SS isoforms SSI and SSII, which are approximately 75 and 85 kD, respectively. At later stages of endosperm development (10–15 DAP and later), there was a notable shift in the elution patterns of both SS and SBE activities, indicating an apparent increase in molecular mass or aggregation state of the enzymes contributing to these measured activities. At later stages of endosperm development, the total eluted SS and SBE activity was divided almost equally between an apparently high Mr peak of around 200 to 300 kD (termed HMW), as well as the peak originally observed at the earlier stages of endosperm development (6–9 DAP) with an apparently lower molecular mass, corresponding to the size of the monomeric proteins (termed LMW). Analysis of SS and SBE activities by gel filtration chromatography at later stages of endosperm development (beyond 15 DAP) showed essentially the same separation of peaks of SS and SBE activities as observed in Figure 1 at 10 to 15 DAP (data not shown).
The results described above were obtained with whole cell homogenates. Similar results were also obtained when amyloplast lysates prepared from endosperm were separated by gel filtration chromatography and fractions assayed for SBE and SS activities (data not shown).
Analysis of the fractions (following gel filtration chromatography of whole cell extracts) by immunoblotting with anti-SBE antibodies (Fig. 1C) showed that both forms of SBEII (SBEIIa and SBEIIb) were responsible for the measured SBE activity in both LMW and HMW fractions and that SBEI was not expressed at the stages of endosperm development used in these experiments, consistent with previous findings (Morell et al., 1997
Previous work has shown that many stromal proteins rapidly become phosphorylated when amyloplasts and chloroplasts of wheat are incubated with [
Amyloplast lysates were used as a source of material for coimmunoprecipitation experiments to examine protein-protein interactions and the possibility that phosphorylation has a role in facilitating such interactions (Fig. 2 ). Plastid lysates were employed to reduce the cross-reactivity of antibodies with nonspecific proteins associated with the use of whole cell extracts.
Figure 2 shows the results of experiments using anti-SBEII antibodies as the immunoprecipitation agent. The data in Figure 2A show that each of the peptide-specific anti-SBEII antibodies (and the anti-SS antibodies; Supplemental Fig. S1) precipitates only the respective form of SBE and SS from plastid lysates and that phosphorylation (preincubation with ATP) or dephosphorylation (preincubation with APase) does not affect the ability of any of the antibodies to bind to the respective forms of SBE and SS.
Experiments with stroma prepared from amyloplasts isolated from endosperm at 6 to 9 DAP showed no coimmunoprecipitation of other starch-synthesizing enzymes as judged by immunoblotting, regardless of the pretreatment conditions (Fig. 2A). However, at 10 to 15 DAP, when higher molecular mass/aggregation states of SSs and SBEs were observed in the gel filtration experiments (Fig. 1), both SBEII antibodies were able to coimmunoprecipitate SSI and SSII, indicating potential protein-protein interactions between each of the SBEII forms and SSI and SSII. Preincubation of plastid lysates with glucan-degrading enzymes (amyloglucosidase and
Significantly, the SS-SBE interactions observed at 10 to 15 DAP of endosperm development in Figure 2B were not observed when plastid lysates were preincubated with APase (which causes nonspecific dephosphorylation), suggesting that the SS-SBE interactions observed in wheat endosperm are developmentally regulated and also phosphorylation dependent. Lysates of late-stage endosperm amyloplasts that were not incubated with ATP showed the same coimmunoprecipitation phenomena as those incubated with ATP (data not shown), suggesting that the phosphorylation state of the enzymes is not markedly altered by exogenous ATP (given at 1 mM). Reciprocal experiments demonstrate that both forms of SBEII are co-precipitated by antibodies to SSI and SSII. Figure 2C illustrates the coimmunoprecipitation of SBEIIb in a phosphorylation-dependent manner. Immunoblots of proteins immunoprecipitated with anti-SBEII antisera were also developed with other available wheat-specific antibodies: anti-plastidial starch phosphorylase, anti-plastidial 1,4-
Stromal proteins from amyloplasts isolated from endosperm at 10 to 15 DAP were separated by gel filtration chromatography. Eluted column fractions were then immediately incubated with the homobifunctional cross-linking reagent bis (sulfosuccinimidyl) suberate (BS3), and the proteins were separated by SDS-PAGE, electroblotted and developed with various anti-SBE and anti-SS antisera. Figure 3A shows the electroblotted, cross-linked proteins from column fractions containing the two major peaks of SS and SBE activities, corresponding to the HMW fractions (containing putative protein complexes) and the LMW fractions (containing monomeric forms of SS and SBE; Fig. 1). Cross linking of proteins in the HMW fraction facilitated detection of a product of approximately 260 kD that displays cross-reactivity with anti-SBEII, anti-SSI, and anti-SSII antisera (Fig. 3A). Another cross-linked product of approximately 180 kD was observed in the HMW fraction, which showed cross-reactivity only with anti-SBEIIa or anti-SBEIIb antisera (Fig. 3A). Cross-linking experiments with the LMW fractions demonstrated the presence of only monomeric forms of SBEII (88 kD), SSI (75 kD), and SSII (85 kD), as expected. Similar HMW, cross-linked products were obtained when amyloplast lysates were incubated with BS3 prior to separation of proteins by gel filtration chromatography (data not shown). Figure 3A shows that when proteins separated by gel filtration chromatography are incubated with APase prior to cross linking with BS3 (eluted column fractions were pretreated with APase prior to the addition of BS3), the protein complexes in the HMW fraction dissociate into monomers, and no aggregated SS or SBE products could be detected.
The cross-linked polypeptides that cross reacted with the various antisera described above and shown in Figure 3A were in-gel digested with trypsin (from corresponding silver-stained SDS-gels), and some of the resulting peptides were sequenced using quadrupole-orthogonal-acceleration-time of flight mass spectrometry (Q-TOF-MS). The MS survey acquisition data obtained from single representative analyses are shown in Figure 3B. The sequence data in Figure 3B shows that the amyloplast proteins present in the 260-kD cross-linked complex(es) were SSI, SSIIa, and forms of SBEII (the close sequence homology between SBEIIa and SBEIIb means that the two forms cannot be distinguished on the basis of the peptide sequences acquired by the mass spectrometer), whereas only peptides from SBEII forms could be detected in the cross-linked complexes of approximately 180 kD (Fig. 3B). Figure 3C demonstrates that SSI protein could be detected in association with SSII following immunoprecipitation with a monospecific SSII antibody (Supplemental Fig. S1), confirming that SSI and SSII can coexist in the same complex. The proteins from the LMW fraction that cross-reacted with the various antisera shown in Figure 3A were also in-gel digested with trypsin, and the peptides were analyzed by Q-TOF-MS; these analyses confirmed that each of the antibodies recognized the respective monomeric protein (data not shown). Immunoblotting of column fractions also revealed other cross-linked aggregation products containing SBEII, SSI, and SSII, with molecular masses greater than 260 kD, but these were of low abundance, and no measurable MS spectra could be obtained from them.
Washed starch granules were also incubated with BS3 to determine whether any of the granule-associated proteins formed aggregates/complexes. BS3 is a low-Mr, water-soluble, cross-linking reagent (mass of 368.4) that would be predicted to penetrate the starch granule structure. Nevertheless, we found no evidence to suggest that any of the granule-associated proteins were present as aggregation products (determined by immunoblotting and silver-stained SDS-gels), even when starch granules were partially digested by
In parallel to the previously described analysis of protein pellets after immunoprecipitation, the supernatants were also assayed for residual SS activity. The data presented in Figure 4A show that soluble SS activity is immunoprecipitated by both of the SBEII antisera only in the HMW fractions. In total, both of the anti-SBEII antibodies precipitated approximately 90% of the measurable SS activity in the HMW fraction, each accounting for approximately equal amounts of the soluble SS activity. Analysis of the pellets following immunoprecipitation of the HMW protein fractions by anti-SBEII antisera indicated the presence of SSI and SSIIa (Fig. 4B). However, addition of anti-SBEIIa or anti-SBEIIb antisera to LMW fractions caused no reduction in soluble SS activity in the supernatant, and only the respective forms of SBEII could be detected in the pellets following immunoblot analysis, indicating no interaction with SS forms (Fig. 4B). Similar results were obtained in an experiment in which both SBEII antibodies were added to the HMW and LMW fractions; approximately 90% of the measurable SS activity in the HMW fraction was precipitated (data not shown).
Analysis of the Glucan-Binding Properties of LMW and HMW SBE Activities
Affinity gel electrophoresis was employed to quantify the dissociation constant (Kd) of branching enzyme from the HMW fractions compared with the corresponding monomeric proteins by measuring relative migration (Rm) in the presence of different concentrations of
This article presents evidence for the existence of functional interactions between the SBEII class and the SSI and SSII classes of amylopectin-synthesizing enzymes in amyloplasts from developing wheat endosperm. Changes in elution profile following gel permeation chromatography, demonstration of coimmunoprecipitation of SSs with branching enzymes, chemical cross linking, and the loss of protein complexes in the presence of alkaline phosphatase provide direct evidence for their physical interaction. The observation that these complexes are seen at specific stages of development adds weight to the argument that they represent functional biochemical activities in vivo. In the endosperms of Festucoideae, such as wheat and barley (Hordeum vulgare), large A-type starch granules are formed first, with the synthesis of small B-type granules occurring later at around 15 DAP, depending on environmental conditions. While it is tempting to speculate that the formation of protein complexes is associated with B-granule synthesis, this seems unlikely, because of the presence of complexes in maize (Zea mays) endosperm (Hennen-Bierwagen et al., 2008  [accompanying article]).
Coimmunoprecipitation experiments in wheat and maize, as well as yeast (Saccharomyces cerevisiae) two-hybrid experiments (Hennen-Bierwagen et al., 2008
Other forms of SS and SBE are known to be expressed in the endosperm: SSIII, SSIV, and SBEI. These isoforms were not detected when the 260-kD cross-linked aggregates were analyzed by Q-TOF-MS. However, we cannot rule out that SSIII and SSIV may also be complexed, because we do not have specific probes for these proteins in wheat. Evidence suggests that SSIII also interacts with other starch-synthesizing enzymes in maize (Hennen-Bierwagen et al., 2008 ), although in gel permeation chromatography experiments, similar to those described here, SSIII was associated only with fractions of much higher molecular size (approximately 600 kD). In the case of SSIV, it is probable that the protein is not expressed at the stage of endosperm used in these experiments, since work with rice indicates that SSIV, like SBEI, is not expressed until late in endosperm development (Dian et al., 2005). SBEI could not be detected within the period of endosperm development chosen for this study (up to 15 DAP; Fig. 1), which is in accordance with previous findings (Morell et al., 1997) and explains why the previously observed complex involving SBEI, SBEIIb, and starch phosphorylase was not observed here (Tetlow et al., 2004b).
The results show that as much as 40% to 50% of the measurable SS catalytic activity in the endosperm is in a high-Mr form at around 10 to 15 DAP (Fig. 1), although it should be noted that activities of SSs (Mu et al., 1994 In addition to demonstrating their presence in heterocomplexes, evidence has been provided from chemical cross-linking studies that SBEII isoforms also form homodimers. To our knowledge, these experiments in wheat and maize are the first published evidence for homodimer formation among SBEII forms. SBEIIa and SBEIIb show a high degree of sequence identity, so it must be assumed that the less homologous regions at the amino- and carboxy-terminal ends of the proteins drive dimerization. Analysis of the mobility of HMW and LMW forms of SBEII in gels containing presumptive glucan substrates (Fig. 5) suggests that the former may produce a catalytically functional unit with increased affinity for glucan substrates compared with the monomeric SBEII forms. This difference in affinity could represent the activity due to formation of homodimers of either SBEII isoform, or it could be the result of physical interaction with SSs in heteromeric complexes. The HMW and LMW forms of SBEII showed identical mobilities in native gels containing no glucan substrate (Fig. 5), which is more likely to be consistent with the activity of dimers. This is reinforced by the observation that immunodetection of SSI on the same blots did not show the same relative mobility of either form of SBEII (data not shown). The possibility that the differing affinity for starch may have arisen from the formation of a heteromeric complex cannot be ruled out completely, because it is possible that the complexes containing SBEII and SS might disassemble during electrophoresis. What is clear is that SBE isoforms in the HMW fraction show markedly different kinetic properties to their monomeric counterparts. Further work is needed to define the biochemical characteristics of SSs and branching enzymes when part of larger Mr complexes.
Some of the components of the SS-SBE complexes have been shown to be phosphorylated (SBEIIa, SBEIIb, and SSII, but not SSI; Tetlow et al., 2004b
Previous genetic and biochemical data are consistent with the existence of protein-protein interactions among amylopectin biosynthetic enzymes, in particular, between SBE and SS forms. For example, the du1– maize mutant that conditions a loss of SSIII function (Gao et al., 1998
Because amylopectin is made by the ordered elongation and branching of glucan chains, the SS and SBE classes of enzymes would be logical partners in any amylopectin-synthesizing protein complex. In addition to the genetic evidence for interactions between the SSs and SBEs (see above), additional in vitro evidence exists for functional interactions between these enzyme classes. In maize kernel extracts, the activity of SSI was greatly stimulated by the addition of purified SBEI or SBEII (Boyer and Preiss, 1979
Previous models have attempted to explain how individual enzymes contribute to the distinct unit structure of the cluster in amylopectin, often making use of well-characterized mutants or using antisense or Mutator transposon approaches. However, recent genetic and biochemical evidence (Colleoni et al., 2003
Plants and Growth Conditions
Spring wheat (Triticum aestivum) ‘Taho’ was grown under glasshouse conditions in a soil medium containing local topsoil, Turface MVP (Profile Products), peat moss, lime, and Nutricote (14-14-14; Morton's Horticultural Products) in a ratio of 3:1:1:0.01:0.01. All other plant growth conditions are as previously described (Tetlow et al., 1993
Amyloplasts were isolated from the endosperm obtained from developing grains taken from the mid-ear region of the head at various DAP (see above) using the methods described by Tetlow et al. (2003)
Whole cell extracts were prepared by rapidly homogenizing approximately 0.5 to 0.8 g endosperm (of various individual seed weights corresponding to different stages of development) in 1 cm3 of ice-cold rupturing buffer followed by centrifugation at 13,500g for 2 min at 4°C. The resulting supernatant was subjected to ultracentrifugation as described above and immediately loaded onto the size exclusion column (below).
Amyloplast lysates and whole cell extracts were separated by size exclusion chromatography using a Superdex 200 10/300GL column using an ÄKTA FPLC (Amersham Biosciences) at 4°C. The column was pre-equilibrated and run in a buffer containing 10 mM HEPES-NaOH, pH 7.5, and 100 mM NaCl at a flow rate of 0.25 cm3/min. Protein extracts (between 1.2 and 2.2 mg/cm3 protein) were loaded onto the column in a final volume of 0.5 cm3 and fractions of 0.5 cm3 were collected using a Frac 950 fraction collector (Amersham Biosciences). The column was routinely calibrated using commercially available standards; those used for calibration were: thyroglobulin (669 kD), ferritin (440 kD), catalase (232 kD), aldolase (158 kD), albumin (67 kD), ovalbumin (43 kD), chymotrypsinogen (25 kD), and ribonuclease (13.7 kD) purchased from Amersham Biosciences.
Subcellular marker enzyme assays were performed as previously described (Tetlow et al., 2003
Soluble SS activity was assayed using a modification of the method described by Jenner et al. (1994) Amylolytic activity was estimated by measuring the release of 14C-labeled products from a 14C-labeled glycogen substrate. The 14C-labeled glycogen substrate was prepared by incubating 1.5 units of phosphorylase a (product no. P-1261, Sigma-Aldrich) in a buffer containing 100 mM sodium citrate, pH 8, 1 mM Na2-EDTA, 1 mM DTT, 2.5 mM AMP, 4.8 mg cm–3 glycogen (from rabbit liver, type III; Sigma-Aldrich), and 10 mM [U-14C]Glc 1-P (3.7–7.4 kBq per preparation) for 2 h at 25°C. The reaction was terminated by heating at 95°C for 5 min and the radio-labeled glucan washed in methanol-KCl as for SBE assays above. The assay for amylolytic activity involved incubating 0.1 cm3 of washed 14C-labeled glucan with 0.1 cm3 protein fractions for 10 min at 25°C. The reaction was terminated by heating to 95°C for 5 min and the remaining 14C-labeled glucan was precipitated using methanol-KCl; the 14C-labeled products released into the supernatant were taken as a measure of amylolytic activity and counted using a liquid scintillation counter.
Phosphorylation reactions in which intact amyloplasts were incubated with
Polyclonal antibodies were raised in rabbits against the synthetic peptides derived from the N-terminal sequences of wheat SBEI (VSAPRDYTMATAEDGV) and wheat SBEIIa (AASPGKVLVPDGESDDLASY; Rahman et al., 2001
Immunoprecipitation experiments were performed with samples of amyloplast stroma and size exclusion column fractions, using methods previously described (Tetlow et al., 2004b
Prior to electrophoresis, proteins were mixed with SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% [w/v] SDS, 10% [w/v] glycerol, 5% [v/v] β-mercaptoethanol, 0.001% [w/v] bromophenol blue) and boiled for 2 min. Protein samples were separated on gels using precast NUPAGE Novex 4% to 12% BisTris gradient gels (Invitrogen Canada) with MOPS running buffer and following the manufacturer's instructions for sample preparation and electrophoresis. SDS-solubilized cross-linked proteins were either separated using four to 12 BisTris gradient gels (see above) or with precast NUPAGE Novex 3% to 8% Tris-acetate gels following the manufacturer's instructions for sample preparation and electrophoresis. Gels were either stained with a colloidal Coomassie G-250 kit (Simply Blue Safestain, Invitrogen) or silver-stained according to methods described by Shevchenko et al. (1996)
Samples for immunoblot analysis were transblotted onto nitrocellulose membranes (Pall Life Sciences), blocked with 1.5% bovine serum albumen, and exposed to antibodies using the methods described by Harlow and Lane (1999)
For native zymogram analysis, protein samples were mixed with native gel sample buffer (50% [v/v] glycerol, 0.2% [w/v] bromophenol blue) in a ratio of 20:1 and separated on native 5% (w/v) polyacrylamide gels in 1.5 M Tris-HCl, pH 8.8. For analysis of SBE activities, gels contained 0.2% (w/v) maltotetrose, 1.4 units phosphorylase a (from rabbit muscle; Sigma-Aldrich, catalog no. P–1261) and 10 mg of the For affinity electrophoresis, various concentrations of glucan substrates (corn starch or amylopectin) were added to the native gel polymerization mixture. Protein samples (0.5–2 mg) from gel filtration experiments corresponding to either complexes or monomeric forms of SS and SBE were loaded on these gels, and electrophoresis was carried out at 100V constant at 25°C in running buffer (25 mM Tris, 192 mM Gly) containing 1 mM DTT. The migration distances of the proteins were measured after immunoblotting and probing for specific enzymes as described above.
Affinity electrophoresis was used as a means of measuring protein-glucan interactions, and Kd are calculated from the retardation of the electrophoretic mobility of enzyme/protein by the substrate contained in the supporting medium. We followed the methods described by Commuri and Keeling (2001)
Amyloplasts were prepared for cross-linking experiments using buffers free of EDTA and DTT and were lysed in gel filtration chromatography running buffer containing a protease inhibitor cocktail (see section above on plastid isolation). Fractions of amyloplast lysates separated by size exclusion chromatography were immediately incubated with 1 mM of the homobifunctional cross-linking reagent BS3 (Pierce/BioLynx) on a rotating table at 25°C for 30 min. The cross-linking reaction was quenched by the addition of 10 µL of a 1-M solution of Tris, pH 8, to the 110-µL reaction mix, and the sample volume reduced to 40 to 50 µL using MicroCon concentrators (Millipore). The concentrated cross-linked samples were mixed with SDS sample buffer prior to electrophoresis (see above).
In-gel digestion with trypsin and preparation of peptides for MS were as described previously (Tetlow et al., 2004b
The capillary voltage was set to 3,500 V, and data-dependent tandem mass spectra acquisitions were performed on precursors with charge states of 2, 3, or 4 over a survey mass range of 400 to 1,300. Known trypsin autolysis products and keratin-derived precursor ions were automatically excluded. The collision voltage was varied between 18 and 45 V depending on the charge and mass of the precursor. Product ion spectra were charge-state de-encrypted and de-isotoped with a maximum entropy algorithm (MaxEnt 3, Micromass). Proteins were identified by correlation of uninterpreted tandem mass spectra to entries in SwissProt/TREMBL, using ProteinLynx Global Server (Version 1, Micromass). One missed cleavage per peptide was allowed, and an initial mass tolerance of 50 ppm was used in all searches. Cysteines were assumed to be carbamidomethylated, but other potential modifications were not considered in the first pass search. When this approach failed, amino acid sequences were deduced manually from the charge state de-encrypted spectra (Wait et al., 2002
The protein content of wheat endosperm whole cell extracts and plastid preparations was determined using the Bio-Rad protein assay (Bio-Rad Laboratories Canada) according to the manufacturer's instructions and using thyroglobulin as a standard.
The following materials are available in the online version of this article.
We thank K. Gale, B. Kosar-Hashemi, and J. Higgins (Commonwealth Scientific and Industrial Research Organization, Canberra, Australia) for the preparation of the anti-wheat-SBEIIb and anti-barley starch phosphorylase antisera, respectively. The authors gratefully acknowledge Dr. Alan Myers for constructive comments on the manuscript, Zhenxiao Lu for technical assistance, and Mark Burrell for help with figure preparation. Received January 14, 2008; accepted February 7, 2008; published February 8, 2008.
1 This work was supported by the Natural Sciences and Engineering Research Council Grant (no. 262209 to M.J.E.), the Ontario Ministry of Agriculture and Food Bio-Products Research Grant (project no. 026262 to I.J.T. and M.J.E.), and the New South Wales Agricultural Genomics Centre (to M.K.M.). 
2 Present address: Technische Universität Kaiserslauten, Abteilung Pflanzenphysiologie, Erwin-Schrödinger-Strasse 22, D–67633, Kaiserslautern, Germany.
3 Present address: Centre for Research in Neuroscience, University of Montreal, Sherbrooke Street West, Montreal, Quebec, H3A 2T5, Canada. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Michael J. Emes (memes{at}uoguelph.ca).
[C] Some figures in this article are displayed in color online but in black and white in the print edition.
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.108.116244 * Corresponding author; e-mail memes{at}uoguelph.ca.
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ABSTRACT
RESULTS
-(1
4)-linked glucan chains and does not appear to participate in the formation of the ordered part of the matrix. Amylopectin is a branched glucan polymer typically comprising between 65% and 85% of the starch granule mass, produced by the formation of 
