The sugary-2 mutation in maize (Zea mays L.) is a result of the loss of catalytic activity of the endosperm-specific SS (starch synthase) IIa isoform causing major alterations to amylopectin architecture. The present study reports a biochemical and molecular analysis of an allelic variant of the sugary-2 mutation expressing a catalytically inactive form of SSIIa and sheds new light on its central role in protein–protein interactions and determination of the starch granule proteome. The mutant SSIIa revealed two amino acid substitutions, one being a highly conserved residue (Gly522→Arg) responsible for the loss of catalytic activity and the inability of the mutant SSIIa to bind to starch. Analysis of protein–protein interactions in sugary-2 amyloplasts revealed the same trimeric assembly of soluble SSI, SSIIa and SBE (starch-branching enzyme) IIb found in wild-type amyloplasts, but with greatly reduced activities of SSI and SBEIIb. Chemical cross-linking studies demonstrated that SSIIa is at the core of the complex, interacting with SSI and SBEIIb, which do not interact directly with each other. The sugary-2 mutant starch granules were devoid of amylopectin-synthesizing enzymes, despite the fact that the respective affinities of SSI and SBEIIb from sugary-2 for amylopectin were the same as observed in wild-type. The data support a model whereby granule-bound proteins involved in amylopectin synthesis are partitioned into the starch granule as a result of their association within protein complexes, and that SSIIa plays a crucial role in trafficking SSI and SBEIIb into the granule matrix.

INTRODUCTION

Storage starch produced in cereal endosperms accounts for a significant proportion of our daily caloric intake, and also represents an important raw material for various industrial purposes. ADP-Glc (ADP-glucose) is the substrate for SSs (starch synthases; EC 2.4.1.21), glucanotransferases, which catalyse the transfer of the glucosyl moiety to the non-reducing end of a pre-existing α-(1→4)-O-linked glucan primer to synthesize glucan polymers. GBSSI (granule-bound starch synthase I) is responsible for the synthesis of the unbranched amylose component of starch [1,2] as well as very long amylopectin chains [3,4]. The principal component of the starch granule is amylopectin, and this complex polymer is synthesized by the co-ordinated actions of multiple isoforms of soluble SS, SBEs (starch-branching enzymes; EC 2.4.1.18) and DBEs (debranching enzymes; EC 3.2.1.41 and EC 3.2.1.68). In addition, SSI, SSIIa, and either SBEIIa or SBEIIb (SBEIIa and SBEIIb of cereals/monocots are highly homologous and appear to have essentially interchangeable functions) are also found in the starch granule [5,6].

The biochemical function of the individual enzymes of the amylopectin biosynthetic pathway has been elucidated through a combination of mutation analysis in selected species, and detailed characterization of purified enzymes [7]. The complex hierarchical structure of amylopectin [8] probably arises from the co-ordinated interplay of this large array of enzyme activities that coexist in the amyloplast.

Although mutation analysis has proved valuable in assigning putative functions for the various amylopectin-synthesizing enzymes, interpretation of the data is complicated by increasing evidence of protein–protein interactions shown to occur within the pathway in cereal endosperms [912]. In particular, the phenotype arising from mutations in a single enzyme is likely to be a product of not only its specificity of function, but also its interactions (both physical and/or functional) with other enzymes of the pathway.

One of the most striking known mutants, in terms of starch phenotype, is caused by the loss of SSIIa activity in the cereal endosperm, and is termed sugary-2 (su2) in maize [13,14]. SSII mutants have been identified and characterized in several other species, including wheat [15,16], barley (termed the sex6 mutant) [17], japonica-type rice [18], pea [19], potato [20], sweet potato [21] and Arabidopsis thaliana [22], and the similarities of the starch phenotypes in these mutants imply a common function for this SS isoform in amylopectin biosynthesis. In cereals, there are two SSII isoforms: SSIIa, found in amyloplasts of developing endosperm, and SSIIb, found in vegetative tissues, whereas dicotyledonous plants have a single form of SSII. SSIIa isoforms are thought to be responsible for the formation of intermediate-length glucan chains [DP (degree of polymerization) 12–24] [17,19,22] utilizing the shorter-chain glucan products that are formed by SSI [23]. The products of SSII are thought to be the preferred substrates of the SBEII class [24]. Therefore SSIIa synthesizes a critical structural intermediate for amylopectin biosynthesis. Similar to SSI and SBEII, SSIIa is also partitioned between the stroma and the nascent starch granule. In developing wheat endosperm, it is found predominantly as a granule-bound protein from midgrain filling onwards [25,26] and in other tissues, such as pea [27] and Arabidopsis (Q. Zhao, personal communication), the majority of SSII is granule-bound. Studies with wheat and maize have shown that a fraction of the SSI, SSIIa and SBEII in the endosperm form a trimeric protein complex [10,11]. Given the putative biochemical functions of SSI, SSIIa and SBEII, their physical interactions with each other, and their partitioning between the soluble stroma and the insoluble granule, it is proposed that the trimeric assembly plays some, as yet undefined, role in amylopectin cluster synthesis.

In storage tissues from a number of species, loss of SSII is characterized by major alterations in starch structure, granule morphology and rheology manifested by an elevated amylose/amylopectin ratio and altered CLD (chain-length distribution) [2831]. Notably, loss of SSII activity in cereal endosperm causes alterations in amylopectin crystallinity [17] that are not observed with other SS mutations, e.g. SSI and SSIII mutants that retain the A-type crystallinity characteristic of cereal storage starches [32,33].

In the present study we test the hypothesis that SSIIa is the critical component of a trimeric protein complex, which includes SSI and SBEIIb, and is essential for the organization of amylopectin structure. We show that an allelic variant of the su2 mutation, caused by one critical amino acid substitution, leads to the expression of a catalytically inactive SSIIa and that the starch phenotype is typical of su2 mutations in cereals. Significantly, the mutant SSIIa is still able to form a trimeric protein complex with SSI and SBEIIb, but possesses no amylopectin-binding capacity. SSI and SBEIIb are lost from the granule, from which we deduce that the binding of SSIIa to starch/α-glucan determines whether the other proteins in the complex become granule-bound.

EXPERIMENTAL

Plant material

The su2 alleles were obtained from the Maize Genetics Cooperation Stock Center (Urbana/Champaign, IL, U.S.A.) and were introgressed into CG119 and CGR04 by back-crossing for four generations followed by two generations of self-pollinations. The resulting BC3S4 lines are homozygous for the su2 mutant alleles and on average possess 93.75% of the CG119 genome. Wild-type CG119 and su2 mutant maize plants were grown at 25–27°C in the glasshouse at the University of Guelph, under conditions described previously for growing wheat [10]. Self-pollinated kernels, obtained through controlled pollinations, were collected at 9–12 DAP (days after pollination), 20–25 DAP and 29–35 DAP, and used to prepare endosperm amyloplasts, starch and whole-cell soluble extracts. Plant materials were flash-frozen in liquid nitrogen and stored in −80°C until future use.

Amyloplast isolation

Maize endosperm amyloplasts were isolated using a modification of the methods described by Tetlow et al. [10]. Plastids were osmotically lysed in a buffer containing 100 mM Tricine/KOH, pH 7.8, 1 mM disodium EDTA, 1 mM DTT (dithiothreitol), 5 mM MgCl2 and a protease inhibitor cocktail (ProteCEASE™, G-Biosciences, used at 10 μl per cm3).

Isolation and analysis of starch granule-bound proteins

Isolation of starch granule-bound proteins (i.e. proteins trapped inside the granule matrix as opposed to proteins attached to the granule surface) was performed as described previously [9,34] using starch granules separated from plastid lysates (see above).

In addition to the acetone/SDS-washing methods described above, we also prepared granule-bound proteins using protease treatment using a modification of the method described in [5]. Starch granules (15 mg) isolated from plastid lysates were washed in buffer as described above and then incubated with 12.5 units of trypsin (Promega) in 470 μl of a buffer containing 50 mM Tris/HCl, pH 7.6, and 1 mM CaCl2 for 30 min at 37°C on a rotator. After incubation the starch granules were washed in buffer, acetone and SDS as described previously [9].

Expression and purification of recombinant enzymes in Escherichia coli

Expression and purification of recombinant maize SSIIa

The SSIIa cDNA sequences from CG119 were ligated to pET29a vectors (Novagen) after removing sequences encoding transit peptides. The recombinant plasmids were transformed into ArcticExpress competent E. coli cells (Stratagene) and protein expression was induced using 1 mM IPTG (isopropyl β-D-thiogalactopyranoside) at 10°C, under constant agitation for 24 h. E. coli (ArcticExpress) cells were collected by centrifugation and lysed using BugBuster Protein Extraction Reagent (Novagen). Recombinant SSIIa was purified from soluble fractions and inclusion bodies. A protein refolding kit (Novagen) was employed for the purification of inclusion bodies and refolding of recombinant proteins following the manufacturer's instructions. Soluble recombinant SSIIa was precipitated by 40% (w/v) ammonium sulfate followed by dialysis for 4 h at 4°C. The dialysates were centrifuged for 30 min at 27000 g at 4°C, and the supernatant was applied to a 1 ml ResourceQ™ column (connected to an ÄKTA Explorer FPLC, GE Healthcare) at 4°C, pre-equilibrated with 50 mM Tris/acetate buffer, pH 7.5, containing 0.05% Triton X-100. The columns were initially washed with five column volumes of the equilibration buffer, followed by elution with 0.6 M KC1 in the equilibration buffer. The flow rates were 1 ml/min, and 0.5 ml/fraction was collected. Eluates were tested by SDS/PAGE and Coomassie Blue staining for recombinant SSIIa. Partially purified SSIIa was concentrated on a Centricon YM50 filter (Millipore) and then applied to a Superdex 200 10/300GL gel permeation column connected to an ÄKTA Explorer FPLC (GE Healthcare) at 4°C. Purified recombinant protein was collected from fractions corresponding to a molecular mass of approximately 85–90 kDa (identified by immunoblotting). The biochemical functions of SSIIa were measured using 14C-labelled substrate assays and native gel zymogram assays (see below). Recombinant proteins were stored at −20°C in 40% (v/v) glycerol and catalytic activities were checked every 2–3 months (see Enzyme assays section below).

Expression and purification of recombinant maize SSI and SBEIIb

Recombinant maize SSI was produced in E. coli and purified following the protocol described in [34] and recombinant maize SBEIIb following the protocol described in [35].

Expression and purification of recombinant E. coli AGPase (adenosine 5′diphosphate glucose pyrophosphorylase)

The E. coli-derived AGPase gene (glgc) was built in the pETM-11 vector with a His6 tag fused at its N-terminus (kindly provided by Dr John Lunn, Max Planck Institute of Molecular Plant Physiology, Golm, Germany). The recombinant plasmid was transformed into E. coli strain ArcticExpress cells. A single clone picked up from an overnight plate was inoculated into LB (Luria–Bertani) medium and grown at 37°C overnight. Overnight media were subcultured in 2 litres of LB medium in the morning, and grown at 37°C for approximately 5 h, until the cell density was 0.3–0.4 at D600. Expression of recombinant protein was induced by adding IPTG to a final concentration of 1 mM and the cultures were grown for 24 h at 10°C. E. coli cells were collected by centrifugation and lysed using BugBuster Protein Extraction Reagent. Recombinant AGPase was purified using the Invitrogen Ni-NTA (Ni2+-nitrilotriacetate) Purification System (following the manufacturer's protocol), and further purification by anion-exchange chromatography (ResourceQ column, GE Healthcare). The column was equilibrated and run in a buffer containing 50 mM Tris/HCl, pH 8.0, and 5 mM MgCl2, and separation was achieved by applying a linear salt gradient of 0–0.5 M NaCl at a flow rate of 1 ml/min. AGPase was recovered as a single peak at 0.25–0.3 M NaCl concentration.

Enzyme assays

Starch-branching enzyme

SBE was assayed indirectly by stimulation of incorporation of 14C from [U-14C]Glc1P (α-D-glucose 1-phosphate) into glucan by phosphorylase a according to methods previously described in [10].

Starch phosphorylase

SP (starch phosphorylase) activity could be detected on SBE zymograms, producing a dark blue-coloured band of glucan product; the position of this activity band aligned with an immunoreactive band with anti-SP antibodies on corresponding native gels. A quantitative assay for SP was also employed measuring glucan-synthesizing activity as described in [35].

Starch synthase

SS activity was measured using ADP-[U-14C]Glc as described previously [10] with minor modifications. The activity of soluble SS was assayed by following the incorporation of 14C from ADP-[U-14C]Glc into glucan using ADP-[U-14C]Glc (5 kBq per assay) synthesized from [U-14C]Glc1P (see below).

Immunopurification and assay of SSIIa

SSIIa from wt (wild-type) and the su2 mutant amyloplasts was immunopurified using peptide-specific anti-(maize SSIIa) antibodies recognizing amino acids 7–27 at the N-terminus. Purified anti-SSIIa antibodies (35 μg) were incubated with both wt and su2 mutant amyloplast lysates (1 ml, equivalent to 1–1.5 mg protein) at room temperature (22°C) on a rotator for 50 min and immunoprecipitation of the antibodies was performed by adding 100 μl of Protein A–Sepharose (Sigma–Aldrich) made up as a 50% (w/v) slurry with PBS (137 mM NaCl, 10 mM Na2HPO4, 2.7 mM KCl and 1.8 mM KH2PO4, pH 7.4) at room temperature for 40 min. The Protein A–Sepharose–antibody–protein complex was centrifuged at 2000 g for 2 min at 4°C in a refrigerated microcentrifuge, and the supernatant was discarded. The pellet was washed five times (1.3 ml each) with PBS containing 1 M NaCl followed by three washes with a buffer containing 10 mM Hepes/NaOH, pH 7.5. Protein bound to the Protein A–Sepharose was analysed by SDS/PAGE and Western blots. The amount of protein on Protein A–Sepharose was quantified by titration calibrated with purified recombinant SSIIa protein. A 20 μl volume of Protein A–Sepharose–antibody–SSIIa matrix were used in each 200 μl SS assay system (described above). Reactions were initiated with 2 mM ADP-[U-14C]Glc. The mixtures were incubated at 25°C on an orbital rotator for 20 min. Reactions were terminated by heating the mixture at 95°C for 5 min, and the 14C-labelled glucan was collected by methanol/KCl (75%, v/v, methanol and 1%, w/v, KCl) precipitation.

Synthesis of ADP-[U-14C]Glc

ADP-[U-14C]Glc was synthesized from [U-14C]Glc1P (PerkinElmer) using purified E. coli AGPase following a protocol modified from [36]. The reaction mixture contained in a total volume of 200 μl; 10 mM Tris/HCl, pH 7.5, 1 mM MgCl2, 10 units of inorganic pyrophosphatase (from E. coli, MCLAB IPE-200), 80 μg of AGPase (equivalent to 96 units of recombinant E. coli AGPase), 0.02 μmol of [U-14C]Glc1P (~4 μCi) and 0.025 μmol of ATP, and was incubated at 30°C for 5 h. The reaction was stopped by heating at 95°C for 5 min, followed by centrifugation at 14000 g for 10 min. The supernatant was collected and tested by TLC for conversion efficiency. Protein was removed from the stopped reaction by precipitation with ice-cold acetone, and the supernatant was dried down. The dried product was resuspended in 200 mM Bicine/KOH, pH 8.5, and stored at −20°C before use in SS assays.

Zymograms and native PAGE

Zymograms for measuring SS and SBE activity were run according to the methods modified previously [9,10].

Affinity electrophoresis and calculation of Kd

Affinity electrophoresis was used as a means of measuring protein–glucan interactions, and dissociation constants were calculated from the retardation of the electrophoretic mobility of enzyme/protein by the substrate contained in the supporting medium following methods described in [10].

Size-exclusion chromatography of amyloplast proteins

Recombinant proteins were separated by size-exclusion chromatography in order to separate catalytically active monomers from inactive aggregates before experiments. Amylopast lysates from the two genotypes were fractionated by size-exclusion chromatography to separate high-molecular-mass forms of specific amylopectin-synthesizing enzymes (in protein complexes) from their respective monomeric forms using methods described previously [35].

Preparation and analysis of polyclonal maize antibodies

Polyclonal rabbit antisera targeted to maize AGPase small subunit (Bt2), SSI, SSIIa, SBEI, SBEIIa, SBEIIb, SP, SSIV, pullulanase (ZPU1) and Iso (isoamylase)-1 were raised against synthetic peptides prepared commercially (by Anaspec; http://www.anaspec.com/services/antibody.asp). The specific peptide sequence used for anti-(maize Bt2) antibody was NADNVQEAARETDGYF, corresponding to amino acid residues 482–497 of the full-length sequence (GenBank® accession number AAZ82467.1). The specific peptide sequences used for antibodies against maize SSI, SSIIa, GBSSI, SBEI, SBEIIa, SBEIIb, SP, Iso-1 and Iso-2 were as described in [34], and for SSIV and ZPU1 as described in [35].

Crude antisera were purified further using peptide-affinity columns according to the methods described in [34]. Pre-immune sera for each of the antibodies used above were employed as negative controls, and showed no cross-reaction with proteins from plastid lysates and co-immunoprecipitation experiments (results not shown).

Cross-linking protein complexes with sulfo-SBED

Sulfo-SBED {sulfo-N-hydroxysuccinimidyl-2-(6-[biotinamido]-2-(p-azido benzamido)-hexanoamido) ethyl-1,3′-dithioproprionate} contains a biotin backbone and two chemically reactive groups, a sulfo-NHS (sulfo-N-hydroxysuccinimide) arm for ligand coupling with a cleavable disulfide bridge, and a photosensitive phenyl azide that is activated by long-wave UV light. Since the sulfo-NHS and phenyl azide arms are only 22.8 Å (1 Å=0.1 nm) apart, bait proteins can be cross-linked to a prey proteins via the phenyl azide group. Purified recombinant maize SSI, SSIIa and SBEIIb were labelled with sulfo-SBED (Thermo Scientific, 0.1 mg·ml−1 dissolved in dimethylformamide) using the manufacturer's protocol and all steps were performed at 25°C in the dark. Then, 600–200 μg of bait proteins individually were mixed with 5-fold molar excess of sulfo-SBED and incubated at 25°C for 30 min. The mixture was dialysed against 500 ml of dialysis buffer containing 50 mM Hepes, pH 7.5, and 150 mM NaCl at 4°C overnight to remove unreacted sulfo-SBED. The labelled proteins (SSI-SBED, SSIIa-SBED and SBEIIb-SBED) were stored in multiple aliquots (50 μl) at −80°C, protected from light.

Sulfo-SBED-labelled recombinant SSI, SSIIa and SBEIIb were incubated with wt amyloplast lysates individually at 25°C for 60 min without light. Following incubation, samples were cross-linked by exposure to long-wavelength UV light (302 nm) for 5 min at a distance of 5 cm using a UV Model UV light (Entela). The cross-linked recombinant proteins were then bound to S-protein–agarose and washed with 250 ml of wash buffer containing 20 mM Tris/HCl, pH 7.5, 150 mM NaCl and 0.2% Triton X-100. Proteins cross-linked to the sulfo-SBED-labelled recombinant bait proteins were identified using MS and immunoblotting with anti-biotin antibodies (Pierce Scientific).

Immunoprecipitation

Immunoprecipitation and co-immunoprecipitation experiments were conducted using the methods described in [35].

SDS/PAGE and immunoblotting

Preparation of protein samples for SDS/PAGE and immunoblotting were as described in [35].

Molecular characterization of SSIIa sequences

Total mRNA was extracted with RNeasy Plant Mini Kit (Qiagen) from endosperms of CG119, su2/CG119, CGR04 and su2/CGR04 at 22–25 DAP grown in a glasshouse. The first-strand cDNA was synthesized by reverse transcription of total RNA using oligo-d(T) and the Qiagen kit. The SSIIa sequences were amplified using primers covering the full length of coding regions plus the parts of 5′-UTR (untranslated region) and 3′-UTR (5′-CCCATTGGACGAGCTTCCGCC-3′ and 5′-GCGGCGCGTCACTGCATATG-3′). PCR products were sequenced by the Advanced Analysis Centre at the University of Guelph using the above primers and two more primers (5′-TCCTACAGTTGAGCCATTAGTA-3′ and 5′-CATTGATCTTCCAGTCGTTAGA-3′). SSIIa cDNA sequences from different genotypes were analysed using GeneRunner 3.05 and polypeptide sequences were multi-aligned online (http://mafft.cbrc.jp/alignment/server/).

Measurement of endosperm starch content

The amount of starch present in the endosperms from the maize kernels from the different genotypes was determined as described previously for wheat endosperm [37].

Estimation of apparent amylose content by iodometry

Apparent amylose content was estimated following the colorimetric method of Morrison and Laignelet [38] with slight modifications as described in [39].

CLD analysis by capillary electrophoresis

CLD of starch was analysed following the method of O’Shea et al. [40] using a P/ACE 5510 capillary electrophoresis system (Beckman) with argon-laser-induced fluorescence detection.

Isoamylase-debranched reducing end assay

The method for the Iso-debranched reducing end assay used was a modified assay as described in [35].

DSC (differential scanning calorimetry)

Thermal properties of starches were analysed using a differential scanning calorimeter (2920 Modulated DSC; TA Instruments) equipped with an RCS (refrigerated cooling system) using the methods described in [35].

Estimation of protein content

Protein was determined using the Bio-Rad protein assay (Bio-Rad Laboratories) according to the manufacturer's instructions and using thyroglobulin as a standard.

Mass spectrometry

In-gel digestion with trypsin and preparation of peptides for MS were as described previously [10]. UPLC (ultra-performance liquid chromatography)–MS/MS (tandem MS) analyses were performed using a nanoAcquity™ UPLC System (Waters) in combination with a Q–TOF (quadrupole–time-of-flight)–micro mass spectrometer (Waters Micromass). The column used was a 75 μm×150 mm Atlantis™ dC18 column packed with 3 μm particles with an initial Symmetry™ C18 trapping column of 180 μm×20 mm with 5 μm particles. The LC (liquid chromatograph) was coupled with the mass spectrometer using a Universal Nanoflow Sprayer (Waters) operated with a PicoTip Emitter (New Objective) with an inner diameter of 10 μm. UPLC–MS/MS analyses were carried out at a flow rate of 400 nl/min and a column temperature of 35°C. Samples were loaded on to the trapping column and washed for 3 min with 2% solution B (acetonitrile with 0.1% formic acid). Peptides were separated using a linear gradient from 90% solution A (water with 0.1% formic acid), 2% solution B to 60% solution A and 40% solution B in 40 min. The mass spectrometer was operated in positive-ion mode with a capillary voltage of 3600 V and a cone voltage of 35 V. Data were acquired in data-dependent acquisition mode, one survey scan of 2 s was carried out followed by up to three MS/MS scans (of 2 s) of each of the three most intense precursor ions. MS/MS spectra were processed using Peaks Studio 4.5 (Bioinformatics Solutions) and searched against the NCBI protein database [41]. These searches were performed using trypsin specificity with the possibility of one missed cleavage at an MS tolerance of 100 p.p.m. and a MS/MS tolerance of 0.1 Da, and included the following variable modifications: carbamidomethyl-cysteine, and oxidized methionine and biotinylation.

RESULTS

Physicochemical analysis of starch from a su2 mutant

A comparison of starch from wt endosperm with starch extracted from the su2 mutant indicated that the starch properties of the particular allelic variant of su2 were typical of other SSII mutants.

Table 1 summarizes some of the physicochemical characteristics of the two starches. The total starch content of the su2 mutant was approximately 10% lower than the wt. However, the amylose content of the su2 genotype was significantly (P<0.0001) higher, nearly double that of the wt. Further analysis of the amylose and amylopectin from Iso-debranched starch separated by gel-permeation chromatography (Supplementary Figure S1 at http://www.BiochemJ.org/bj/448/bj4480373add.htm) suggested that the increase in apparent amylose (as measured by iodine binding) in su2 was due to an increase in longer DP glucan chains in amylopectin, as the specific amylose peak was similar between the two genotypes. However, the methods employed could not distinguish between shorter amylose chains, or a mixture of longer amylopectin chains and shorter amylose, within the fractions classed as longer DP glucan chains in amylopectin (fractions 70–100, Supplementary Figure S1). It was noteworthy that the su2 genotype showed a significant reduction in amylopectin content compared with wt (Supplementary Figure S1).

Table 1
Comparison of the physicochemical properties of wt and su2 starches isolated from developing maize endosperm at 20–25 DAP

Results are means±S.E.M. of three to five biological replicates.

    Gelatinization temperature (°C) 
Genotype Granule size (μm) Starch content (%) Amylose content (%) Onset Peak Completion ΔH (J/g) 
wt 8.2±1.4 70.3±0.8 22.9±2.5 64.1±0.4 69.9±0.1 86.2±0.1 15.8±0.9 
su2 8.2±0.9 61.1±1.2 42.8±2.7 48.1±0.7 54.4±0.1 67.1±0.1  4.2±0.2 
    Gelatinization temperature (°C) 
Genotype Granule size (μm) Starch content (%) Amylose content (%) Onset Peak Completion ΔH (J/g) 
wt 8.2±1.4 70.3±0.8 22.9±2.5 64.1±0.4 69.9±0.1 86.2±0.1 15.8±0.9 
su2 8.2±0.9 61.1±1.2 42.8±2.7 48.1±0.7 54.4±0.1 67.1±0.1  4.2±0.2 

The glucan CLD of starch following debranching with Iso was determined using FACE (fluorophore-assisted carbohydrate electrophoresis). The results summarized in Figure 1 show the normalized glucan chain distribution of wt starch as a reference to su2 starch where wt values are subtracted from the su2 values. The results show an increase in the proportion of chains of DP 7–10, and a significant reduction in the proportion of glucan chains of DP 12–22 in the su2 mutant. The chain-length profile, in particular the doublet peak in the DP 12–22 region, is typical of that found in other cereal SSIIa mutants [17,42].

Glucan CLD profiles of debranched wt and su2 starches expressed as mol% difference of su2 compared with wt

Figure 1
Glucan CLD profiles of debranched wt and su2 starches expressed as mol% difference of su2 compared with wt

Data presented are representative of three replicate analyses.

Figure 1
Glucan CLD profiles of debranched wt and su2 starches expressed as mol% difference of su2 compared with wt

Data presented are representative of three replicate analyses.

The GT (gelatinization temperature) of starch is a measure of granule order, and represents the temperature at which the granule loses order following heating in water. The GTs of purified wt and su2 starches were analysed by DSC, and the data, summarized in Table 1, show that su2 starch has a significantly (P<0.0001) lower GT than wt starch.

Starch granule morphology was investigated using SEM (scanning electron microscopy) (Supplementary Figure S2 at http://www.BiochemJ.org/bj/448/bj4480373add.htm). Although there was no significant difference in granule diameter between the two genotypes (Table 1), su2 starches showed altered granule morphology, having a higher proportion of irregularly shaped granules.

X-ray diffraction patterns from both wt and su2 mutant starch granules are shown in Supplementary Figure S3 (http://www.BiochemJ.org/bj/448/bj4480373add.htm). The wt diffraction is characterized by typical A- and V-type crystallinity. A main A-type diffraction doublet is observed at 17 and 18° with further peaks at 15, 20 and 23°. Characteristic V-type reflections are also observed at 7, 13 and 20°. The wt starch exhibited 37.5% crystallinity including 1.28% corresponding to V-type reflections. It should also be noted that the proportion of V-type crystallinity was calculated based on the area under the V-type reflections and may overestimate the actual proportion of single helical amylose crystals due to potential overlap corresponding to A-type crystal structures. In contrast, the su2 mutant starch exhibits less extensive crystallinity as is evident from the poorly resolved 17 and 18° doublet. Total crystallinity accounts for 12.2% including 6.1% of V-type crystallinity. In addition, there is no evidence for B-type crystallinity; this is most obvious from a review of the low-angle region where the characteristic interhelix repeat from B-type crystallinity would be present at 5.5°. Although there are significantly reduced levels of crystallinity, and thus severe disruption in chain packing, the mutant starch largely retains the crystal form of the wt (i.e. A- plus V-type). This is in contrast with the sex6 mutant in barley [17], but consistent with previous X-ray diffraction data on maize su2 starch [30].

Biochemical characterization of the su2 mutant

Amyloplast stromal proteins

Amyloplasts were isolated from developing maize endosperms at 20–25 DAP, during maximal rates of starch deposition, and used to prepare lysates for immunoblot analysis of enzymes of starch synthesis. Figure 2 shows immunoblot analysis of amyloplast stromal enzymes and indicates that all of the major enzymes of amylopectin synthesis, including SSIIa, are present in the wt and the su2 mutant of the same cultivar in comparable amounts as judged by their immunoreactivity with their respective antibodies. GBSSI, which is involved in amylose biosynthesis and is normally exclusively localized to starch granules as a granule-bound protein, was detected in the stroma of su2 (but not wt) amyloplasts. Immunoblot analysis of SSIII in wt and su2 amyloplast lysates showed extensive truncation of the protein had occurred before electrophoresis. However, analysis of the truncation products on Western blots indicated equivalent levels of SSIII on both wt and su2 amyloplasts (results not shown).

Immunological characterization of amyloplast lysates from wt and su2 maize
Figure 2
Immunological characterization of amyloplast lysates from wt and su2 maize

Amyloplast lysates (approximately 0.8 mg/ml) were prepared from developing wt and su2 endosperms at 20–25 DAP (individual kernel fresh weight of approximately 300 mg). Approximately 12–15 μg of soluble (stromal) proteins was loaded on to each gel lane and separated on 12% acrylamide gels, electroblotted on to nitrocellulose membranes, and developed with various peptide-specific anti-maize antibodies as shown. Arrows indicate cross-reactions of each of the antibodies with its corresponding target protein; AGPase (Bt2) (approximately 54 kDa), GBSSI (granule-bound starch synthase) (approximately 56 kDa), Iso-1 (approximately 80 kDa, but with a predicted mass of 90 kDa), SBEIIb (approximately 85 kDa), SSI (approximately 74 kDa), SSIIa (approximately 76 kDa, but with a predicted mass of 85 kDa), SSIV (approximately 95 kDa), SBEI (approximately 80 kDa), SBEIIa (approximately 90 kDa), SP (approximately 112 kDa) and ZPU1 (approximately 105 kDa). MW, molecular-mass markers (in kDa).

Figure 2
Immunological characterization of amyloplast lysates from wt and su2 maize

Amyloplast lysates (approximately 0.8 mg/ml) were prepared from developing wt and su2 endosperms at 20–25 DAP (individual kernel fresh weight of approximately 300 mg). Approximately 12–15 μg of soluble (stromal) proteins was loaded on to each gel lane and separated on 12% acrylamide gels, electroblotted on to nitrocellulose membranes, and developed with various peptide-specific anti-maize antibodies as shown. Arrows indicate cross-reactions of each of the antibodies with its corresponding target protein; AGPase (Bt2) (approximately 54 kDa), GBSSI (granule-bound starch synthase) (approximately 56 kDa), Iso-1 (approximately 80 kDa, but with a predicted mass of 90 kDa), SBEIIb (approximately 85 kDa), SSI (approximately 74 kDa), SSIIa (approximately 76 kDa, but with a predicted mass of 85 kDa), SSIV (approximately 95 kDa), SBEI (approximately 80 kDa), SBEIIa (approximately 90 kDa), SP (approximately 112 kDa) and ZPU1 (approximately 105 kDa). MW, molecular-mass markers (in kDa).

Granule-bound proteins

A specific group of proteins consistently remain bound within the starch granule matrix in plastids following extensive washing with buffers, acetone and SDS; these are termed granule-bound proteins [6,34] and are distinct from proteins that adhere to the surface of the granules [5]. We observed no difference between the granule-bound protein profiles of starches washed using the buffer/acetone/SDS method and the protease treatment method (results not shown). All data presented for granule-bound proteins were obtained from starch samples washed with buffer, acetone and 2% (w/v) SDS. The granule-bound proteins of wt and su2 endosperms were analysed by SDS/PAGE and immunoblotting (Figure 3). The major granule-bound proteins were identified by Q–TOF–MS analysis of silver-stained bands subjected to in-gel trypsin digestion following silver-staining (Figure 3A, and MS results not shown). Four proteins previously identified as being granule-bound were observed in wt starch granules: GBSSI, SSI, SSIIa and SBEIIb. However, in su2 starch granules, only GBSSI was detected in amounts comparable with wt starch (Figure 3A), whereas SSI, SSIIa and SBEIIb were not detectable using Western blotting or MS analysis. Immunoblot experiments confirmed the presence and identity of the proteins observed in silver-stained gels, and also indicated that AGPase (small subunit), Iso-1, ZPU1, SSIV and SP were not present as granule-bound proteins in wt or su2 amyloplasts (results not shown).

Analysis of starch granule-bound proteins from wt and su2 amyloplasts
Figure 3
Analysis of starch granule-bound proteins from wt and su2 amyloplasts

Starch granules were isolated from amyloplasts at 20–25 DAP and washed extensively to remove proteins that were loosely bound to the granule surface. A total of 40 mg of purified starch from each genotype was boiled in 600 μl of SDS-loading buffer, and 35 μl of the supernatant from the boiled sample was loaded on to gels. (A) Granule-bound proteins from wt and su2 plastids were separated by SDS/PAGE using 4–12% acrylamide gradient gels and visualized by silver staining. Horizontal arrows indicate the major polypeptides which were excised from wt and su2 starch granules and identified by Q–TOF–MS analysis; a, GBSSI (found in both wt and su2); b, SSI (absent from su2); c, SSIIa (absent from su2); and d, SBEIIb (absent from su2). (B) Starch granule proteins from wt and su2 starch were separated by SDS/PAGE and subjected to immunoblot analysis using various peptide-specific anti-maize antibodies. MW, molecular-mass markers (in kDa).

Figure 3
Analysis of starch granule-bound proteins from wt and su2 amyloplasts

Starch granules were isolated from amyloplasts at 20–25 DAP and washed extensively to remove proteins that were loosely bound to the granule surface. A total of 40 mg of purified starch from each genotype was boiled in 600 μl of SDS-loading buffer, and 35 μl of the supernatant from the boiled sample was loaded on to gels. (A) Granule-bound proteins from wt and su2 plastids were separated by SDS/PAGE using 4–12% acrylamide gradient gels and visualized by silver staining. Horizontal arrows indicate the major polypeptides which were excised from wt and su2 starch granules and identified by Q–TOF–MS analysis; a, GBSSI (found in both wt and su2); b, SSI (absent from su2); c, SSIIa (absent from su2); and d, SBEIIb (absent from su2). (B) Starch granule proteins from wt and su2 starch were separated by SDS/PAGE and subjected to immunoblot analysis using various peptide-specific anti-maize antibodies. MW, molecular-mass markers (in kDa).

su2 mutant expresses a catalytically inactive SSIIa

The maximum catalytic activities of SS, SBE and SP (all enzymatic activities that have been shown previously to be associated with protein complexes in amyloplasts) were measured in amyloplasts from su2, and compared with amyloplast lysates from wt maize endosperm at equivalent stages of development (20–25 DAP). Total branching enzyme activity in the su2 mutant was lower than that of wt maize (wt 54±0.8 nmol/min per mg of protein, and su2, 45±0.1 nmol/min per mg of protein; P<0.0001), whereas SP activity (measured in the glucan-synthesizing direction, as the incorporation of [U-14C]Glc-1P into glycogen) in the su2 mutant was unchanged compared with wt activity (wt, 47±7 nmol/min per mg of protein, and su2, 49+6 nmol/min per mg of protein). However, measurable soluble SS activity in su2 was significantly higher than in wt with either rabbit liver glycogen or gelatinized corn amylopectin as the source of glucan primer (Figure 4A). SSIIa activity was measured directly by exploiting the fact that the epitope for the anti-SSIIa antibody was on the extreme N-terminus of the mature protein (Figure 5) enabling catalytically active native SSIIa to be immunopurified. Equivalent amounts of wt and su2 SSIIa were immunoprecipitated from amyloplast lysates and washed in PBS containing 1 M NaCl to remove other proteins, including other SS isoforms (see immunoblots in Figure 4B) before assaying SSIIa activity. Figure 4(B) shows that only SSIIa activity is being measured, as no other SS isoforms are present. Figure 4(B) also shows that the measurable SSIIa activity using glycogen as a primer was more than 2-fold greater than with gelatinized corn amylopectin. SSIIa assays using 2 mM ADP-[U-14C]Glc (Figure 4B) clearly show that the su2 SSIIa is catalytically inactive, irrespective of the source of α-glucan used as primer.

Changes in catalytic activities of SS isoforms in su2 endosperm

Figure 4
Changes in catalytic activities of SS isoforms in su2 endosperm

(A) Total SS activity was measured in amyloplast lysates from wt and su2 amyloplasts isolated at 20–25 DAP. Approximately 20 μg of stromal proteins were used in each assay to measure total soluble SS activity (see the Experimental section). Results are the means±S.E.M. of three to five independent amyloplast preparations. (B) Immunopurification of SSIIa from wt and su2 amyloplast lysates using peptide-specific anti-maize SSIIa antibodies raised against the N-terminus of SSIIa (amino acids 7–27). The total catalytic activity of SS isoforms (A) and of immunopurified SSIIa (B) was measured using ADP-[U-14C]Glc and glucan primers as indicated (see the Experimental section for details).

Figure 4
Changes in catalytic activities of SS isoforms in su2 endosperm

(A) Total SS activity was measured in amyloplast lysates from wt and su2 amyloplasts isolated at 20–25 DAP. Approximately 20 μg of stromal proteins were used in each assay to measure total soluble SS activity (see the Experimental section). Results are the means±S.E.M. of three to five independent amyloplast preparations. (B) Immunopurification of SSIIa from wt and su2 amyloplast lysates using peptide-specific anti-maize SSIIa antibodies raised against the N-terminus of SSIIa (amino acids 7–27). The total catalytic activity of SS isoforms (A) and of immunopurified SSIIa (B) was measured using ADP-[U-14C]Glc and glucan primers as indicated (see the Experimental section for details).

Structural analysis of SSIIa from su2 endosperm

Figure 5
Structural analysis of SSIIa from su2 endosperm

(A) Catalytically inactive SSIIa from su2 maize endosperm showing the two amino acids substituted in the mutant protein, the epitope recognized by the anti-maize SSIIa antibodies, putative ADP-Glc-binding domain and catalytic glycosyltransferase domains determined from previous structural studies. (B) Amino acid sequence alignments of SSIIa from different plant species in the regions of the two amino acid substitutions found in su2. Comparison of the aspartic acid (D) to valine (V) and glycine (G) to arginine (R) substitutions (boxed) in su2 maize show that Gly522 is a conserved residue of the glycosyltransferase domain of SSIIa.

Figure 5
Structural analysis of SSIIa from su2 endosperm

(A) Catalytically inactive SSIIa from su2 maize endosperm showing the two amino acids substituted in the mutant protein, the epitope recognized by the anti-maize SSIIa antibodies, putative ADP-Glc-binding domain and catalytic glycosyltransferase domains determined from previous structural studies. (B) Amino acid sequence alignments of SSIIa from different plant species in the regions of the two amino acid substitutions found in su2. Comparison of the aspartic acid (D) to valine (V) and glycine (G) to arginine (R) substitutions (boxed) in su2 maize show that Gly522 is a conserved residue of the glycosyltransferase domain of SSIIa.

Amino acid sequence of su2 SSIIa

The amino acid sequence of the catalytically inactive SSIIa in su2 was determined following reverse transcription–PCR of total RNA. Structural analysis of SSIIa from the su2 mutant indicates that the catalytically inactive enzyme has two amino acid substitutions, Asp146→Val and Gly522→Arg, the latter being at a highly conserved site (Figure 5). No difference in mobility between the mutant and wt SSIIa was observed when the respective native or recombinant proteins were separated by SDS/PAGE or native PAGE (results not shown).

The inactive SSIIa in su2 is unable to bind glucan

The absence of granule-bound amylopectin-synthesizing enzymes in the su2 mutant led us to examine the glucan-binding capacities of SSI, SSIIa and SBEIIb (the major amylopectin-synthesizing granule-bound proteins in wt starch granules) in the two maize genotypes. Affinity gel electrophoresis was employed to quantify the dissociation constant (Kd) of SSI, SSIIa and SBEIIb in wt and su2 plastid lysates by measuring their Rm (relative migration) in the presence of different concentrations of α-(1→4)-linked glucans (gelatinized starch and amylopectin) (Figure 6). Given the high sensitivity of the various peptide-specific antibodies, we used Western blotting to visualize the migration of individual enzymes on α-glucan-containing gels. 1/Rm values of SSI and SBEIIb forms from wt and su2 plastid lysates, and wt SSIIa in gelatinized amylopectin-containing gels, were linearly related to the concentration of glucan substrate in the gels (Figures 6A–6C and Table 2). The mobilities of both SSI and SBEIIb in amylopectin- and starch-containing gels in wt amyloplasts were identical with those of su2 (Table 2). Three forms of SBEIIb, each showing different mobilities in α-glucan-containing gels, were resolved in both genotypes. The glucan affinity determined for each SBEIIb form in wt extracts was identical with the corresponding form in the su2 mutant (Table 2).

Affinity of SSI, SSIIa and SBEIIb for amylopectin in wt and su2 amyloplasts by affinity gel electrophoresis

Figure 6
Affinity of SSI, SSIIa and SBEIIb for amylopectin in wt and su2 amyloplasts by affinity gel electrophoresis

Amyloplast extracts (20–25 DAP) were separated on 10% (w/v) native gels containing various concentrations of corn amylopectin. The mobility of SSI, SSIIa and SBEIIb forms from wt and su2 amyloplasts in the amylopectin-containing native gels was determined by immunoblotting and probing with respective peptide-specific antibodies. (AC) Mobility of SSIIa (A), SBEIIb (B) and SSI (C) in native gels with 0 and 0.5% amylopectin. (D) Plots of the 1/Rm of SSIIa from wt (■) and su2 (●) amyloplast lysates against the concentration of maize endosperm amylopectin in the gels at room temperature. Immunoblots of SSIIa mobility in the full range of amylopectin concentrations used to construct the plot are shown in Supplementary Figure S4 at http://www.BiochemJ.org/bj/448/bj4480373add.htm). See Table 2 for calculated dissociation constants.

Figure 6
Affinity of SSI, SSIIa and SBEIIb for amylopectin in wt and su2 amyloplasts by affinity gel electrophoresis

Amyloplast extracts (20–25 DAP) were separated on 10% (w/v) native gels containing various concentrations of corn amylopectin. The mobility of SSI, SSIIa and SBEIIb forms from wt and su2 amyloplasts in the amylopectin-containing native gels was determined by immunoblotting and probing with respective peptide-specific antibodies. (AC) Mobility of SSIIa (A), SBEIIb (B) and SSI (C) in native gels with 0 and 0.5% amylopectin. (D) Plots of the 1/Rm of SSIIa from wt (■) and su2 (●) amyloplast lysates against the concentration of maize endosperm amylopectin in the gels at room temperature. Immunoblots of SSIIa mobility in the full range of amylopectin concentrations used to construct the plot are shown in Supplementary Figure S4 at http://www.BiochemJ.org/bj/448/bj4480373add.htm). See Table 2 for calculated dissociation constants.

Table 2
Comparison of Kd values and affinity constants of wt and su2 maize SSI, SSIIa and SBEIIb

Summary of kinetic data derived from affinity chromatography experiments with amyloplast lysates from developing maize endosperm (20–25 DAP) in gels containing varied concentrations of gelatinized corn amylopectin (see Figures 6A and 6D for SSIIa mobility data). Results are means±S.E.M. for three independent experiments. The results for SBEIIb are from three distinct migratory forms of SBEIIb detected by anti-(maize SBEIIb) antibodies on native gels that are present in both wt and su2 amyloplasts. The results represent the mobility of each of the three forms in gelatinized amylopectin-containing gels, comparing the wt and su2 for each corresponding form. N/A, not applicable.

 SSI SSIIa SBEIIb 
 wt su2 wt su2 wt su2 
Kd 0.25±0.13 0.24±0.02 0.31±0.02 0.18±0.02 0.17±0.01 
     0.23±0.01 0.23±0.01 
     0.33±0.03 0.31±0.02 
Affinity (1/Kd) 4.12±0.35 4.10±0.29 3.20±0.22 N/A 6.15±0.31 6.19±0.29 
     4.43±0.14 4.41±0.11 
     3.25±0.23 3.30±0.25 
 SSI SSIIa SBEIIb 
 wt su2 wt su2 wt su2 
Kd 0.25±0.13 0.24±0.02 0.31±0.02 0.18±0.02 0.17±0.01 
     0.23±0.01 0.23±0.01 
     0.33±0.03 0.31±0.02 
Affinity (1/Kd) 4.12±0.35 4.10±0.29 3.20±0.22 N/A 6.15±0.31 6.19±0.29 
     4.43±0.14 4.41±0.11 
     3.25±0.23 3.30±0.25 

Unlike the wt protein, su2 SSIIa showed no alteration in migration, as a function of the amount of amylopectin added to the gel (Figures 6A and 6D). In contrast with the behaviour of the SSI, SBEIIb and wt SSIIa forms, control proteins (BSA and molecular mass standards) also showed no change in migration, irrespective of the glucan concentration in the gel, as would be expected (results not shown). The behaviour of the control proteins indicates that the reduced mobility observed with the different SS and SBE forms in the affinity gels is a result of their specific affinity for the glucan provided, rather than being caused by a dense polyacrylamide/glucan matrix in the gel. Furthermore, the lack of mobility of the su2 SSIIa indicates that the mutation has caused a loss of affinity for glucan in addition to a loss of catalytic capacity. The dissociation constants determined for SBEIIb and SSI with amylopectin as substrate in both wt and su2 are similar to those previously determined for other branching enzymes [44] and recombinant maize SSI [23].

Protein–protein interactions in amyloplasts of su2 maize

Co-immunoprecipitation of enzymes of amylopectin synthesis in wt and su2 amyloplasts

Peptide-specific antibodies were used in reciprocal co-immunoprecipitation experiments to analyse protein–protein interactions among starch-synthesizing enzymes in wt and su2 maize amyloplasts at 20–25 DAP. All of the antibodies used in co-immunoprecipitation experiments (anti-SSI, anti-SSIIa and anti-SBEIIb) were able to recognize, and precipitate, their respective target protein [34]. Figure 7 shows that antibodies against SSI, SSIIa or SBEIIb co-precipitated each of the target proteins in both wt and su2 amyloplast lysates. In both wt and su2 amyloplast extracts, no interactions were observed between SSs (SSI and SSIIa), SBEIIb, and SBEI or SP. Pre-incubation of wt and su2 plastid lysates with glucan-degrading enzymes (amyloglucosidase and α-amylase) did not prevent co-immunoprecipitation of SSI, SSIIa and SBEIIb isoforms, indicating that their association in each of the genotypes tested is due to specific protein–protein interactions, and not a result of these enzymes binding to a common glucan chain.

Co-immunoprecipitation of stromal proteins from wt and su2 amyloplasts to determine protein–protein interactions
Figure 7
Co-immunoprecipitation of stromal proteins from wt and su2 amyloplasts to determine protein–protein interactions

Volumes of 0.5–1 ml of amyloplast lysates (0.8–1 mg of protein per ml) prepared from wt and su2 endosperm 20–25 DAP were incubated with peptide-specific anti-SS and anti-SBEIIb antibodies at 25°C for 50 min, and then immunoprecipitated with Protein A–Sepharose. The washed Protein A–Sepharose–antibody–antigen complexes were boiled in 200 μl of SDS-loading buffer and 30 μl was loaded on to 4–12% pre-cast polyacrylamide gradient gels, electroblotted on to nitrocellulose, and developed with various maize antisera as shown. (A) Co-immunoprecipitation using anti-SSI antibodies, (B) co-immunoprecipitation using anti-SSIIa antibodies, (C) co-immunoprecipitation using anti-SBEIIb antibodies. Cross-reactions with the various antisera used were as follows: SSI at 74 kDa, SSIIa at 76 kDa and SBEIIb at 85 kDa. When present, SBEI and SP are visualized at 80 kDa and 112 kDa respectively. The large band observed at approximately 50 kDa in all lanes is due to auto-recognition of the IgG heavy chain. MW, molecular-mass markers (in kDa).

Figure 7
Co-immunoprecipitation of stromal proteins from wt and su2 amyloplasts to determine protein–protein interactions

Volumes of 0.5–1 ml of amyloplast lysates (0.8–1 mg of protein per ml) prepared from wt and su2 endosperm 20–25 DAP were incubated with peptide-specific anti-SS and anti-SBEIIb antibodies at 25°C for 50 min, and then immunoprecipitated with Protein A–Sepharose. The washed Protein A–Sepharose–antibody–antigen complexes were boiled in 200 μl of SDS-loading buffer and 30 μl was loaded on to 4–12% pre-cast polyacrylamide gradient gels, electroblotted on to nitrocellulose, and developed with various maize antisera as shown. (A) Co-immunoprecipitation using anti-SSI antibodies, (B) co-immunoprecipitation using anti-SSIIa antibodies, (C) co-immunoprecipitation using anti-SBEIIb antibodies. Cross-reactions with the various antisera used were as follows: SSI at 74 kDa, SSIIa at 76 kDa and SBEIIb at 85 kDa. When present, SBEI and SP are visualized at 80 kDa and 112 kDa respectively. The large band observed at approximately 50 kDa in all lanes is due to auto-recognition of the IgG heavy chain. MW, molecular-mass markers (in kDa).

The trimeric assembly observed in su2 amyloplasts containing SSI, SSIIa and SBEIIb was separated by gel-permeation chromatography and showed the same elution pattern as the wt complex, which has been observed previously ([34] and results not shown).

Topological analysis of the SSI–SSIIa–SBEIIb protein complex using sulfo-SBED

We employed a cross-linking approach to determine the spatial relationship between the components of the trimeric protein complex assembly (SSI, SSIIa and SBEIIb) in amyloplasts. Functional recombinant maize proteins were purified from E. coli lysates and cross-linked with the heterobifunctional reagent sulfo-SBED to be used as bait proteins in protein–protein interaction experiments with plastid lysates. Proteins in close proximity (up to 14.3 Å) to the bait protein can be cross-linked, and subsequently labelled with the thiol-cleavable biotin tag and then identified using anti-biotin antibodies. The results of cross-linking experiments are shown in Figure 8. In each case, using a specific recombinant protein as bait, Western blot analyses indicated the interacting protein(s) cross-linked to the bait protein and were tagged with biotin (non-cross-linked proteins are washed away). The interacting target proteins were also identified by MS, which was also employed to identify specific biotinylated peptides on each of the target proteins. Biotinylation of each target protein was determined from MS data as an increase in mass of 226.08, representing biotinylation of a lysine residue on the peptide. Figure 8 shows that when either recombinant SSI or recombinant SBEIIb was used as bait, only SSIIa was biotinylated in each case. However, when recombinant SSIIa was the bait protein, both SSI and SBEIIb were cross-linked and biotinylated, suggesting that SSIIa lies in a central position within the trimeric protein complex.

Cross-linking the components of the trimeric protein complex in amyloplasts using the heterobifunctional reagent sulfo-SBED
Figure 8
Cross-linking the components of the trimeric protein complex in amyloplasts using the heterobifunctional reagent sulfo-SBED

Purified functional recombinant proteins were cross-linked with sulfo-SBED and used as bait in interaction experiments with amyloplast stromal proteins. The reconstituted protein complex containing the recombinant bait protein was UV-cross-linked and washed to remove all interacting proteins that had not been cross-linked by the biotin-containing tag on the bait protein. The identities of the cross-linked proteins are indicated. Silver-stained biotin-tagged polypeptides were excised from polyacrylamide gels and digested with trypsin, and the recovered polypeptides sequenced using Q–TOF–MS to identify proteins and biotinylated polypeptides. Only biotinylated peptides are shown, acquired from a single representative in-gel digest from each experiment involving a cross-linked recombinant bait protein. MW, molecular-mass markers (in kDa).

Figure 8
Cross-linking the components of the trimeric protein complex in amyloplasts using the heterobifunctional reagent sulfo-SBED

Purified functional recombinant proteins were cross-linked with sulfo-SBED and used as bait in interaction experiments with amyloplast stromal proteins. The reconstituted protein complex containing the recombinant bait protein was UV-cross-linked and washed to remove all interacting proteins that had not been cross-linked by the biotin-containing tag on the bait protein. The identities of the cross-linked proteins are indicated. Silver-stained biotin-tagged polypeptides were excised from polyacrylamide gels and digested with trypsin, and the recovered polypeptides sequenced using Q–TOF–MS to identify proteins and biotinylated polypeptides. Only biotinylated peptides are shown, acquired from a single representative in-gel digest from each experiment involving a cross-linked recombinant bait protein. MW, molecular-mass markers (in kDa).

Measurement of the catalytic activity of SSIIa-containing protein complexes in wt and su2 amyloplasts

The trimeric protein complexes containing SSI, SSIIa and SBEIIb, which have been observed previously in wt cereal amyloplasts and also shown to be present in su2 amyloplasts (see above), were isolated using the SSIIa immunopurification method (see the Experimental section) to measure the catalytic capacity of the protein complexes. In order to study the protein complex assemblies, the immunopurified complexes from wt and su2 amyloplasts were washed gently in aqueous buffers, and immunoblotting was used to determine the presence of the protein complex components. Figure 9 shows that the isolated protein complex from wt amyloplasts is catalytically active with respect to SS and SBE activities. The SS activity associated with the immunoprecipitated wt ‘complex’ (5.6 nmol/h per μg of protein, Figure 9A), represents a combination of SSI and SSIIa activities, and is comparable with the activity associated with SSIIa alone (4.3 nmol/h per μg of protein, see Figure 4B) with equal amounts of immunopurified SSIIa. By contrast, SS and SBE activities in the immunopurified protein complex from su2 amyloplasts were significantly lower than their respective wt activities (Figure 9). The SS activity in the su2 protein complex (0.4 nmol/h per μg of protein, Figure 9A) is likely to represent only SSI activity since the SSIIa is catalytically inactive, and immunoblots indicated that other SS isoforms were not present. SBEIIb activity in the su2 complex was reduced to more than 15-fold in comparison with the corresponding wt activity, despite the fact that equal amounts of SBEIIb (as judged by immunoblot analysis) were present in the protein complexes in the two genotypes. Previous studies have shown that protein phosphorylation stimulates the formation of such trimeric protein complexes [1012]. When amyloplasts were pre-treated with 1 mM ATP before immunopurification of the protein complex, the amount of SSI and SBEIIb associated with SSIIa was markedly enhanced as judged by immunoblotting (Figures 9A and 9B). Following ATP treatment, the catalytic activities of SS and SBE were increased in the immunoprecipitated wt complexes. In the su2 complex, only SS activity increased following ATP treatment, whereas there was no significant change in SBE activity.

Measurement of the catalytic activity associated with SSIIa-containing protein complexes in wt and su2 amyloplasts
Figure 9
Measurement of the catalytic activity associated with SSIIa-containing protein complexes in wt and su2 amyloplasts

The trimeric protein complex present in the two genotypes containing SSI, SSIIa and SBEIIb was isolated using the immunopurification technique used for isolation of SSIIa (see the Experimental section) except that the immunoprecipitated protein complex was washed five times in PBS followed by a single wash with 10 mM Hepes/KOH, pH 7.5. All assays were performed on material immunoprecipitated from amyloplast lysates with anti-SSIIa antibodies. Proteins associated with SSIIa on the Protein A–Sepharose beads were identified by immunoblot analysis and quantified by titration with the respective purified recombinant protein. SS activity (A) was determined using 2 mM ADP-Glc and 5 mg·ml−1 gelatinized amylopectin, and SBE activity (B) was determined using the phosphorylase a-stimulation assay. Controls were Protein A–Sepharose beads incubated with amyloplast lysates. (A) and (B) also show the respective SS and SBE activities of the immunopurified complex following treatment of amyloplasts with 1 mM ATP (30 min pre-incubation). Results are means±S.E.M. for three independent experiments.

Figure 9
Measurement of the catalytic activity associated with SSIIa-containing protein complexes in wt and su2 amyloplasts

The trimeric protein complex present in the two genotypes containing SSI, SSIIa and SBEIIb was isolated using the immunopurification technique used for isolation of SSIIa (see the Experimental section) except that the immunoprecipitated protein complex was washed five times in PBS followed by a single wash with 10 mM Hepes/KOH, pH 7.5. All assays were performed on material immunoprecipitated from amyloplast lysates with anti-SSIIa antibodies. Proteins associated with SSIIa on the Protein A–Sepharose beads were identified by immunoblot analysis and quantified by titration with the respective purified recombinant protein. SS activity (A) was determined using 2 mM ADP-Glc and 5 mg·ml−1 gelatinized amylopectin, and SBE activity (B) was determined using the phosphorylase a-stimulation assay. Controls were Protein A–Sepharose beads incubated with amyloplast lysates. (A) and (B) also show the respective SS and SBE activities of the immunopurified complex following treatment of amyloplasts with 1 mM ATP (30 min pre-incubation). Results are means±S.E.M. for three independent experiments.

DISCUSSION

The present study is a biochemical analysis of an allelic variant of the su2 mutant of maize which has been used to examine the role of SSIIa in directing the association of other components of heteromeric protein complexes to the starch granule.

Comparative analysis of the physicochemical characteristics of the su2 starch with wt indicates properties typical of other SSII mutants (e.g. reduced GT and increased amylose/amylopectin ratio and modified glucan CLD). Significantly, starch in amyloplasts of the su2 mutant is devoid of the subset of granule-bound proteins associated with amylopectin synthesis (see Figure 3A), including SSI and SBEIIb, which also appears to be a characteristic feature of the SSII mutation in the endosperms of other cereals such as barley (sex6), wheat and rice [15,17,45]. The su2 mutation described in the present paper expresses a catalytically inactive form of SSIIa with two single amino acid substitutions, Asp146→Val and Gly522→Arg. Given that the substitution at position 146 is actually found in the wt of other cereal forms of SSIIa, and is not conserved across species in general (Figure 5B), this strongly suggests that it is the mutation at Gly522 which is significant for glucan binding as it is found in the glycosyltransferase domain of all forms of SSII. Thus a single amino acid mutation in one protein leads to the loss of two others from the granule, and, as is discussed below, affects their respective measurable activities differentially.

Western blot analyses of native PAGE and SDS/PAGE gels of soluble proteins from amyloplast lysates show that the su2 mutant used in the present study expressed SSIIa protein, other SS and SBE isoforms, DBEs, plastidial SP and AGPase at levels comparable with the wt. However, analysis of the catalytic activities of SS and SBE isoforms in isolated amyloplasts revealed unpredicted changes associated with the su2 mutation. In particular, total immunoprecipitable SS activity was significantly increased with the loss of SSIIa activity. The measurable SS activity in the su2 amyloplasts must therefore be a result of other soluble SS isoforms since immunopurification of endogenous SSIIa indicated that the su2 SSIIa was catalytically inactive (Figure 4). Analysis of SS mutants has previously shown increases in total SS activity. For example, loss of SSIII in maize endosperm led to an increase in total SS activity [46]. Recent studies with single and double mutants of SSII and SSIII in Arabidopsis and barley have also noted stimulation of SSI activity in this genetic background, leading to speculation that SSII may be a negative regulator of SSI [47,48].

In contrast with the effect on SS activity, su2 amyloplast lysates showed a significant reduction in total SBE activity. Importantly, the present study showed that the SBEIIb present in a protein complex with SSI and SSIIa (as opposed to free monomeric SBEII) displayed very low catalytic activity in the su2 mutant (Figure 9B) and provides the most likely explanation for the decrease in total measurable SBE activity in amyloplast lysates. Analysis of the trimeric protein complex in su2 amyloplasts indicated the same complement of proteins as found in wt amyloplasts, and no indication (on the basis of immunoprecipitation and gel-permeation chromatography) that other proteins were associated with the protein complex in su2.

Analysis of the glucan affinities of each of the components of the wt protein complex show that all enzymes possess affinity for α-glucan substrates, measured by the retardation of the respective proteins in native gels containing various concentrations of gelatinized amylopectin or starch (Figure 6 and Table 2, and Supplementary Figure S4 at http://www.BiochemJ.org/bj/448/bj4480373add.htm), and all are found as granule-bound proteins. The exact mechanism by which proteins involved in starch synthesis become granule-bound is not known. The glucan-binding kinetics for SSI and SBEIIb (Table 2) observed in maize amyloplasts are similar to those observed for recombinant maize SSI [23] and SBEII isoforms from wheat endosperm amyloplasts [10]. The glucan-binding properties of the monomeric forms of SSI and SBEIIb from amyloplast extracts were unaffected in the su2 mutant compared with the corresponding wt enzyme, whereas the catalytically inactive SSIIa from su2 showed no affinity for amylopectin. This leads to the suggestion that the ability of proteins to become granule-bound may not be a function of the glucan-binding properties of individual enzymes, but rather their association within a multimeric protein complex that binds all of the proteinaceous components through the binding domain(s) of one of the proteins. Support for this also comes from the recent analysis of an ae (amylose extender) mutant of maize expressing a catalytically inactive SBEIIb that was unable to bind to α-glucan substrates, but was found as a granule-bound protein in the starch granules [35]. Since the mutant SBEIIb in the ae mutant was part of a functional protein complex in the amyloplasts, it was argued that glucan binding, and eventual granule-association of proteins, which are components of active protein complexes, was mediated by a protein other than SBEIIb within the trimeric assembly [35].

The fact that ATP-stimulated association of SSI, SSIIa and SBEIIb in both genotypes is consistent with previous data reinforces the view that protein complex formation is a phosphorylation-dependent process [9,10,34]. The presence of a catalytically inactive, possibly misfolded, SSIIa may be the cause of reduced SS and SBE activities in the su2 protein complex. However, it is also interesting to note that SS activity in both the wt and su2 protein complexes increased in response to ATP (Figure 9A). This suggests that the additional SSI recruited to the mutant complex as a result of protein phosphorylation is still functional, whereas the SBEIIb in the complex lacks measureable catalytic activity, possibly caused by the lack of provision of substrate from SSIIa. Cross-linking experiments (Figure 8) indicate that SSIIa is in close proximity to the other two components of the trimeric assembly, possibly playing a central role in the functioning of the complex. The central position of SSIIa in the complex could have a physical influence on the folding of both SSI and SBEIIb. Examination of the data in Figure 9 adds support to this idea. For example, the assay used to measure the activity of SBE in the complex (phosphorylase a-stimulation) is independent of the effects of the activities of other enzymes in the complex (SS) since SBE branches glucan produced by phosphorylase a. Suppression of SBEIIb activity in the su2 complex could therefore be a result of its inability to access the substrate, from SSIIa in vivo, or phosphorylase a in the in vitro assay, resulting from its close proximity to the inactive SSIIa. We postulate that the functioning of SBEIIb in the su2 complex in vivo is inhibited by reduced substrate supply, being dependent on elongated glucan chains supplied by, in turn, SSI and SSIIa (the glucan products produced by SSII have been shown to be of minimal DP for the catalytic action of the SBEII class [49]). Despite the inactivity of SSIIa and the low catalytic activity of the trimeric complex in the mutant, su2 endosperm nevertheless contain significant amounts of starch, which implies that the non-granule-bound forms of SSI and SBEIIb are able to contribute to the synthesis of starch granules. The altered granule morphology and crystallinity observed in su2 starch granules may therefore provide a clue as to the function of protein complexes in the structural organization of amylopectin.

The results of the present study support a model whereby granule-association of SSI, SSIIa and SBEIIb is mediated through the glucan-binding ability of SSIIa, which forms the core component of a protein complex that is formed in the amyloplast stroma (Figure 10). Loss of glucan-binding ability of SSIIa leads to the inability of the other components of the protein complex to become granule-bound, reduction in functional branching enzyme activity within the complex, and altered physicochemical properties of starch granules.

Proposed model for granule protein deposition in amyloplasts

Figure 10
Proposed model for granule protein deposition in amyloplasts

Granule-bound proteins involved in amylopectin biosynthesis are the components of a trimeric protein complex assembly consisting of SSI, SSIIa and SBEIIb, which is implicated in amylopectin cluster formation. SSIIa is the central core of this trimeric assembly, and directs itself, and the other proteins interacting with it, into the granule matrix via a glucan-binding domain at as yet unspecified α-glucan chains or regions of the granule. The partially active trimeric protein complex in su2 amyloplasts contains a catalytically inactive SSIIa whose glucan-binding domain has no affinity for amylopectin and is unable to bind α-glucans. Consequently, all of the proteins in the su2 trimeric complex remain in the plastid stroma and the starch granules are devoid of granule-bound proteins involved in amylopectin biosynthesis.

Figure 10
Proposed model for granule protein deposition in amyloplasts

Granule-bound proteins involved in amylopectin biosynthesis are the components of a trimeric protein complex assembly consisting of SSI, SSIIa and SBEIIb, which is implicated in amylopectin cluster formation. SSIIa is the central core of this trimeric assembly, and directs itself, and the other proteins interacting with it, into the granule matrix via a glucan-binding domain at as yet unspecified α-glucan chains or regions of the granule. The partially active trimeric protein complex in su2 amyloplasts contains a catalytically inactive SSIIa whose glucan-binding domain has no affinity for amylopectin and is unable to bind α-glucans. Consequently, all of the proteins in the su2 trimeric complex remain in the plastid stroma and the starch granules are devoid of granule-bound proteins involved in amylopectin biosynthesis.

Abbreviations

     
  • ADP-Glc

    ADP-glucose

  •  
  • AGPase

    adenosine 5′-diphosphate glucose pyrophosphorylase

  •  
  • CLD

    chain-length distribution

  •  
  • DAP

    days after pollination

  •  
  • DBE

    debranching enzyme

  •  
  • DP

    degree of polymerization

  •  
  • DSC

    differential scanning calorimetry

  •  
  • GBSSI

    granule-bound starch synthase I

  •  
  • Glc1P

    α-D-glucose 1-phosphate

  •  
  • GT

    gelatinization temperature

  •  
  • IPTG

    isopropyl β-D-thiogalactopyranoside

  •  
  • Iso

    isoamylase

  •  
  • LB

    Luria–Bertani

  •  
  • MS/MS

    tandem MS

  •  
  • Q–TOF

    quadrupole–time-of-flight

  •  
  • SBE

    starch-branching enzyme

  •  
  • SP

    starch phosphorylase

  •  
  • SS

    starch synthase

  •  
  • SSI

    soluble starch synthase I

  •  
  • sulfo-SBED

    sulfo-N-hydroxysuccinimidyl-2-(6-[biotinamido]-2-(p-azido benzamido)-hexanoamido) ethyl-1,3′-dithioproprionate

  •  
  • sulfo-NHS

    sulfo-N-hydroxysuccinimide

  •  
  • UPLC

    ultra-performance liquid chromatography

  •  
  • UTR

    untranslated region

  •  
  • wt

    wild-type

AUTHOR CONTRIBUTION

Fushan Liu contributed to the discussion and was responsible for the electron microscopy and all of the biochemical experiments except for the sulfo-SBED cross-linking, which was performed by Nadya Romanova. Elizabeth Lee performed back-crossing of the su2 allele into maize lines and genotyping. Regina Ahmed was responsible for the separation of amylose and amylopectin, the CLD analysis of starches and analysis and interpretation of these experiments. Martin Evans was responsible for the X-ray diffraction experiments and, with Elliot Gilbert, was responsible for the analysis and interpretation of these results. Matthew Morell, Michael Emes and Ian Tetlow researched the data, contributed to the discussion, wrote the paper and reviewed/edited the paper before submission.

We are grateful to Dr John Lunn (Max Planck Institute of Molecular Plant Physiology, Golm, Germany) for the cDNA for E. coli AGPase. We gratefully acknowledge Dr Alan Myers and Dr Martha James (Iowa State University, Ames, IA, U.S.A.) for providing cDNAs for the mature sequences of maize SSI, SBEI, SBEIIa and SBEIIb. We thank Dr Dyanne Brewer (University of Guelph Advanced Analysis Center) for Q–TOF–MS analyses and Mr Zaheer Ahmed (University of Guelph) for starch granule size and gel-permeation chromatography analyses.

FUNDING

This work was supported by the Ontario Ministry of Agriculture, Food and Rural Affairs Bio-Products Research Grants [grant numbers 026262 (to M.J.E. and I.J.T.) and 200172 (to I.J.T.)], Natural Sciences and Engineering Research Council (NSERC) Discovery Grants [grant numbers 262209 (to M.J.E.) and 341722 (to I.J.T.)], an NSERC Strategic Grant [grant number 048237 (to M.J.E. and I.J.T.)] and a Commonwealth Scientific and Industrial Research Organization (CSIRO) Sir Frederick McMaster Fellowship awarded to I.J.T.

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Author notes

The nucleotide sequence data reported for Zea mays glucose-1-phosphate adenyltransferase have been deposited in GenBank®, EMBL, DDBJ and GSDB Nucleotide Sequence Databases under the accession number AAZ82467.1.

Supplementary data