Identification of the maize amyloplast stromal 112-kD protein as a plastidic starch phosphorylase

Amyloplast is the site of starch synthesis in the storage tissue of maize (Zea mays). The amyloplast stroma contains an enriched group of proteins when compared with the whole endosperm. Proteins with molecular masses of 76 and 85 kD have been identified as starch synthase I and starch branching enzyme IIb, respectively. A 112-kD protein was isolated from the stromal fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to tryptic digestion and amino acid sequence analysis. Three peptide sequences showed high identity to plastidic forms of starch phosphorylase (SP) from sweet potato, potato, and spinach. SP activity was identified in the amyloplast stromal fraction and was enriched 4-fold when compared with the activity in the whole endosperm fraction. Native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses showed that SP activity was associated with the amyloplast stromal 112-kD protein. In addition, antibodies raised against the potato plastidic SP recognized the amyloplast stromal 112-kD protein. The amyloplast stromal 112-kD SP was expressed in whole endosperm isolated from maize harvested 9 to 24 d after pollination. Results of affinity electrophoresis and enzyme kinetic analyses showed that the amyloplast stromal 112-kD SP preferred amylopectin over glycogen as a substrate in the synthetic reaction. The maize shrunken-4 mutant had reduced SP activity due to a decrease of the amyloplast stromal 112-kD enzyme.     

Protein phosphorylation regulates maize endosperm starch synthase IIa activity and protein−protein interactions

Starch synthesis is an elaborate process employing several isoforms of starch synthases (SSs), starch branching enzymes (SBEs) and debranching enzymes (DBEs). In cereals, some starch biosynthetic enzymes can form heteromeric complexes whose assembly is controlled by protein phosphorylation. Previous studies suggested that SSIIa forms a trimeric complex with SBEIIb, SSI, in which SBEIIb is phosphorylated. This study investigates the post-translational modification of SSIIa, and its interactions with SSI and SBEIIb in maize amyloplast stroma. SSIIa, immunopurified and shown to be free from other soluble starch synthases, was shown to be readily phosphorylated, affecting V_max but with minor effects on substrate K_d and K_m values, resulting in a 12-fold increase in activity compared with the dephosphorylated enzyme. This ATP-dependent stimulation of activity was associated with interaction with SBEIIb, suggesting that the availability of glucan branching limits SSIIa and is enhanced by physical interaction of the two enzymes. Immunoblotting of maize amyloplast extracts following non-denaturing polyacrylamide gel electrophoresis identified multiple bands of SSIIa, the electrophoretic mobilities of which were markedly altered by conditions that affected protein phosphorylation, including protein kinase inhibitors. Separation of heteromeric enzyme complexes by GPC, following alteration of protein phosphorylation states, indicated that such complexes are stable and may partition into larger and smaller complexes. The results suggest a dual role for protein phosphorylation in promoting association and dissociation of SSIIa-containing heteromeric enzyme complexes in the maize amyloplast stroma, providing new insights into the regulation of starch biosynthesis in plants.     

Measurement of Metabolites Associated with Nonaqueously Isolated Starch Granules from Immature Zea mays L. Endosperm

Starch granules with associated metabolites were isolated from immature Zea mays L. endosperm by a nonaqueous procedure using glycerol and 3-chloro-1,2-propanediol. The soluble extract of the granule preparation contained varying amounts of neutral sugars, inorganic phosphate, hexose and triose phosphates, organic acids, adenosine and uridine nucleotides, sugar nucleotides, and amino acids. Based on the metabolites present and on information about translocators in chloroplast membranes, which function in transferring metabolites from the chloroplast stroma into the cytoplasm, it is suggested that sucrose is degraded in the cytoplasm, via glycolysis, to triose phosphates which cross the amyloplast membrane by means of a phosphate translocator. It is further postulated that hexose phosphates and sugars are produced from the triose phosphates in the amyloplast stroma by gluconeogenesis with starch being formed from glucose 1-phosphate via pyrophosphorylase and starch synthase enzymes. The glucose 1-phosphate to inorganic phosphate ratio in the granule preparation was such that starch synthesis by phosphorylase is highly unlikely in maize endosperm.     

Surface Localization of Zein Storage Proteins in Starch Granules from Maize Endosperm : Proteolytic Removal by Thermolysin and in Vitro Cross-Linking of Granule-Associated Polypeptides

Starch granules from maize (Zea mays) contain a characteristic group of polypeptides that are tightly associated with the starch matrix (C. Mu-Forster, R. Huang, J.R. Powers, R.W. Harriman, M. Knight, G.W. Singletary, P.L. Keeling, B.P. Wasserman [1996] Plant Physiol 111: 821–829). Zeins comprise about 50% of the granule-associated proteins, and in this study their spatial distribution within the starch granule was determined. Proteolysis of starch granules at subgelatinization temperatures using the thermophilic protease thermolysin led to selective removal of the zeins, whereas granule-associated proteins of 32 kD or above, including the waxy protein, starch synthase I, and starch-branching enzyme IIb, remained refractory to proteolysis. Granule-associated proteins from maize are therefore composed of two distinct classes, the surface-localized zeins of 10 to 27 kD and the granule-intrinsic proteins of 32 kD or higher. The origin of surface-localized δ-zein was probed by comparing δ-zein levels of starch granules obtained from homogenized whole endosperm with granules isolated from amyloplasts. Starch granules from amyloplasts contained markedly lower levels of δ-zein relative to granules prepared from whole endosperm, thus indicating that δ-zein adheres to granule surfaces after disruption of the amyloplast envelope. Cross-linking experiments show that the zeins are deposited on the granule surface as aggregates. In contrast, the granule-intrinsic proteins are prone to covalent modification, but do not form intermolecular cross-links. We conclude that individual granule intrinsic proteins exist as monomers and are not deposited in the form of multimeric clusters within the starch matrix.It has long been known that starch granules contain bound polypeptides, with protein levels of isolated starch granules from maize (Zea mays) ranging from 0.3 to 1.0% based upon measurement of N₂ (May, 1987). A recent study by our laboratory demonstrates that isolated starch granules from maize contain several dozen strongly bound polypeptides (Mu-Forster et al., 1996). The granule-associated proteins include starch-biosynthetic enzymes such as the waxy protein, SSI, and SBEIIb. These polypeptides are not removed from intact starch granules by protease treatment or detergent washing; therefore, they are believed to bind to the starch and to become irreversibly entrapped within the starch matrix.Based upon staining intensities of polypeptides extracted from the starch granule (Mu-Forster et al., 1996), approximately one-half of the granule-associated proteins in maize consist of low-molecular-mass polypeptides ranging between 10 and 27 kD. These bands fall within the size range displayed by the zein storage proteins, however, the spatial distribution of these polypeptides within the starch granule is unknown. Zeins have been defined as alcohol-soluble proteins that occur principally in protein bodies of maize endosperm and that may or may not require reduction before extraction (Wilson, 1991). The association of zeins with starch granules during endosperm development would not be expected because zein genes do not contain transit peptides that would target these proteins through the amyloplast envelope into the amyloplast stroma.The objective of this study was to establish the topology of granule-associated zeins in starch granules from maize endosperm. To accomplish this, it was necessary to distinguish between surface-localized and internalized polypeptides. Our working hypothesis defines polypeptides localized at the starch granule surface as those that are susceptible to hydrolysis upon treatment of intact granules with exogenous proteases. Conversely, internal granule proteins are defined as those that (a) become susceptible to proteolysis only following thermal disruption of the starch matrix, and (b) resist extraction by 2% SDS at room temperatures (Denyer et al., 1993; Rahman et al., 1995; Mu-Forster et al., 1996).In this study we were able to distinguish between surface-localized and internalized granule-associated polypeptides in starch granules from maize endosperm by use of the thermophilic protease thermolysin. Thermolysin is well suited for this purpose because it is highly active at starch-gelatinization temperatures, and has also been shown to effectively hydrolyze hydrophobic proteins located at the surfaces of chloroplasts and other subcellular organelles (Cline et al., 1984; Xu and Chitnis, 1995). Upon extended incubation of intact starch granules with thermolysin at subgelatinization temperatures, we found that zeins were selectively removed from the starch granule surface. All other granule-associated polypeptides remained inaccessible to proteolytic attack or to extraction by 2% SDS, unless the starch matrix was first disrupted by gelatinization. Our results distinguish between the surface-localized and granule-intrinsic proteins of maize endosperm, and establish that zeins are localized at the starch-granule surface. In addition, cross-linking experiments were conducted to determine nearest-neighbor relationships among zein subunits localized at the granule surface and granule intrinsic polypeptides localized within the starch matrix.     

Allelic variants of the amylose extender mutation of maize demonstrate phenotypic variation in starch structure resulting from modified protein-protein interactions

Amylose extender (ae(-)) starches characteristically have modified starch granule morphology resulting from amylopectin with reduced branch frequency and longer glucan chains in clusters, caused by the loss of activity of the major starch branching enzyme (SBE), which in maize endosperm is SBEIIb. A recent study with ae(-) maize lacking the SBEIIb protein (termed ae1.1 herein) showed that novel protein-protein interactions between enzymes of starch biosynthesis in the amyloplast could explain the starch phenotype of the ae1.1 mutant. The present study examined an allelic variant of the ae(-) mutation, ae1.2, which expresses a catalytically inactive form of SBEIIb. The catalytically inactive SBEIIb in ae1.2 lacks a 28 amino acid peptide (Val272-Pro299) and is unable to bind to amylopectin. Analysis of starch from ae1.2 revealed altered granule morphology and physicochemical characteristics distinct from those of the ae1.1 mutant as well as the wild-type, including altered apparent amylose content and gelatinization properties. Starch from ae1.2 had fewer intermediate length glucan chains (degree of polymerization 16-20) than ae1.1. Biochemical analysis of ae1.2 showed that there were differences in the organization and assembly of protein complexes of starch biosynthetic enzymes in comparison with ae1.1 (and wild-type) amyloplasts, which were also reflected in the composition of starch granule-bound proteins. The formation of stromal protein complexes in the wild-type and ae1.2 was strongly enhanced by ATP, and broken by phosphatase treatment, indicating a role for protein phosphorylation in their assembly. Labelling experiments with [γ-(32)P]ATP showed that the inactive form of SBEIIb in ae1.2 was phosphorylated, both in the monomeric form and in association with starch synthase isoforms. Although the inactive SBEIIb was unable to bind starch directly, it was strongly associated with the starch granule, reinforcing the conclusion that its presence in the granules is a result of physical association with other enzymes of starch synthesis. In addition, an Mn(2+)-based affinity ligand, specific for phosphoproteins, was used to show that the granule-bound forms of SBEIIb in the wild-type and ae1.2 were phosphorylated, as was the granule-bound form of SBEI found in ae1.2 starch. The data strongly support the hypothesis that the complement of heteromeric complexes of proteins involved in amylopectin synthesis contributes to the fine structure and architecture of the starch granule.     

Brittle-1, an Adenylate Translocator, Facilitates Transfer of Extraplastidial Synthesized ADP-Glucose into Amyloplasts of Maize Endosperms

Amyloplasts of starchy tissues such as those of maize (Zea mays L.) function in the synthesis and accumulation of starch during kernel development. ADP-glucose pyrophosphorylase (AGPase) is known to be located in chloroplasts, and for many years it was generally accepted that AGPase was also localized in amyloplasts of starchy tissues. Recent aqueous fractionation of young maize endosperm led to the conclusion that 95% of the cellular AGPase was extraplastidial, but immunolocalization studies at the electron- and light-microscopic levels supported the conclusion that maize endosperm AGPase was localized in the amyloplasts. We report the results of two nonaqueous procedures that provide evidence that in maize endosperms in the linear phase of starch accumulation, 90% or more of the cellular AGPase is extraplastidial. We also provide evidence that the brittle-1 protein (BT1), an adenylate translocator with a KTGGL motif common to the ADP-glucose-binding site of starch synthases and bacterial glycogen synthases, functions in the transfer of ADP-glucose into the amyloplast stroma. The importance of the BT1 translocator in starch accumulation in maize endosperms is demonstrated by the severely reduced starch content in bt1 mutant kernels.     

Physical association of starch biosynthetic enzymes with starch granules of maize endosperm. Granule-associated forms of starch synthase I and starch branching enzyme II.

Antibodies were used to probe the degree of association of starch biosynthetic enzymes with starch granules isolated from maize (Zea mays) endosperm. Graded washings of the starch granule, followed by release of polypeptides by gelatinization in 2% sodium dodecyl sulfate, enables distinction between strongly and loosely adherent proteins. Mild aqueous washing of granules resulted in near-complete solubilization of ADP-glucose pyrophosphorylase, indicating that little, if any, ADP-glucose pyrophosphorylase is granule associated. In contrast, all of the waxy protein plus significant levels of starch synthase I and starch branching enzyme II (BEII) remained granule associated. Stringent washings using protease and detergent demonstrated that the waxy protein, more than 85% total endosperm starch synthase I protein, and more than 45% of BEII protein were strongly associated with starch granules. Rates of polypeptide accumulation within starch granules remained constant during endosperm development. Soluble and granule-derived forms of BEII yielded identical peptide maps and overlapping tryptic fragments closely aligned with deduced amino acid sequences from BEII cDNA clones. These observations provide direct evidence that BEII exits as both soluble and granule-associated entities. We conclude that each of the known starch biosynthetic enzymes in maize endosperm exhibits a differential propensity to associate with, or to become irreversibly entrapped within, the starch granule.     

Evidence for independent genetic control of the multiple forms of maize endosperm branching enzymes and starch synthases

Soluble starch synthase and starch-branching enzymes in extracts from kernels of four maize genotypes were compared. Extracts from normal (nonmutant) maize were found to contain two starch synthases and three branching enzyme fractions. The different fractions could be distinguished by chromatographic properties and kinetic properties under various assay conditions. Kernels homozygous for the recessive amylose-extender (ae) allele were missing branching enzyme IIb. In addition, the citrate-stimulated activity of starch synthase I was reduced. This activity could be regenerated by the addition of branching enzyme to this fraction. No other starch synthase fractions were different from normal enzymes. Extracts from kernels homozygous for the recessive dull (du) allele were found to contain lower branching enzyme IIa and starch synthase II activities. Other fractions were not different from the normal enzymes. Analysis of extracts from kernels of the double mutant ae du indicated that the two mutants act independently. Branching enzyme IIb was absent and the citrate-stimulated reaction of starch synthase I was reduced but could be regenerated by the addition of branching enzyme (ae properties) and both branching enzyme IIa and starch synthase II were greatly reduced (du properties). Starch from ae and du endosperms contains higher amylose (66 and 42%, respectively) than normal endosperm (26%). In addition, the amylopectin fraction of ae starch is less highly branched than amylopectin from normal or du starch. The above observations suggest that the alterations of the starch may be accounted for by changes in the soluble synthase and branching enzyme fractions.     

Identification of Multiple Phosphorylation Sites on Maize Endosperm Starch Branching Enzyme IIb,a Key Enzyme in Amylopectin Biosynthesis

Starch branching enzyme IIb (SBEIIb) plays a crucial role in amylopectin biosynthesis in maize endosperm by defining the structural and functional properties of storage starch and is regulated by protein phosphorylation. Native and recombinant maize SBEIIb were used as substrates for amyloplast protein kinases to identify phosphorylation sites on the protein. A multidisciplinary approach involving bioinformatics, site-directed mutagenesis, and mass spectrometry identified three phosphorylation sites at Ser residues: Ser⁶⁴⁹, Ser²⁸⁶, and Ser²⁹⁷. Two Ca²⁺-dependent protein kinase activities were partially purified from amyloplasts, termed K1, responsible for Ser⁶⁴⁹ and Ser²⁸⁶ phosphorylation, and K2, responsible for Ser⁶⁴⁹ and Ser²⁹⁷ phosphorylation. The Ser²⁸⁶ and Ser²⁹⁷ phosphorylation sites are conserved in all plant branching enzymes and are located at opposite openings of the 8-stranded parallel β-barrel of the active site, which is involved with substrate binding and catalysis. Molecular dynamics simulation analysis indicates that phospho-Ser²⁹⁷ forms a stable salt bridge with Arg⁶⁶⁵, part of a conserved Cys-containing domain in plant branching enzymes. Ser⁶⁴⁹ conservation appears confined to the enzyme in cereals and is not universal, and is presumably associated with functions specific to seed storage. The implications of SBEIIb phosphorylation are considered in terms of the role of the enzyme and the importance of starch biosynthesis for yield and biotechnological application.     

Analysis of protein complexes in wheat amyloplasts reveals functional interactions among starch biosynthetic enzymes

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 gamma-(32)P-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 branching enzyme IIb in wheat is expressed at low levels in the endosperm compared to other cereals and encoded at a non-syntenic locus

Studies of maize starch branching enzyme mutants suggest that the amylose extender high amylose starch phenotype is a consequence of the lack of expression of the predominant starch branching enzyme II isoform expressed in the endosperm, SBEIIb. However, in wheat, the ratio of SBEIIb and SBEIIa expression are inversely related to the expression levels observed in maize and rice. Analysis of RNA at 15 days post anthesis suggests that there are about 4-fold more RNA for SBE IIa than for SBE IIb. The genes for SBE IIa and SBE IIb from wheat are distinguished in the size of the first three exons, allowing isoform-specific antibodies to be produced. These antibodies were used to demonstrate that in the soluble fraction, the amount of SBE IIa protein is two to three fold higher than SBIIb, whereas in the starch granule, there is two to three fold more SBE IIb protein amount than SBE IIa. In a further difference to maize and rice, the genes for SBE IIa and SBE IIb are both located on the long arm of chromosome 2 in wheat, in a position not expected from rice–maize–wheat synteny.     

Enzyme activities associated with maize kernel amyloplasts

Activities of the enzymes of gluconeogenesis and of starch metabolism were measured in extracts of amyloplasts isolated from protoplasts derived from 14-day-old maize (Zea mays L., cv Pioneer 3780) endosperm. The enzymes triosephosphate isomerase, fructose-1,6-bisphosphate aldolase, fructose-1,6-bisphosphatase, phosphohexose isomerase, phosphoglucomutase, ADPG pyrophosphorylase, UDPG pyrophosphorylase, soluble and bound starch synthases, and branching enzyme were found to be present in the amyloplasts. Of the above enzymes, ADPG pyrophosphorylase had the lowest activity per amyloplast. Invertase, sucrose synthase and hexokinase were not detected in similar amyloplast preparations. Only a trace of the cytoplasmic marker enzyme alcohol dehydrogenase could be detected in purified amyloplast fractions. In separate experiments, purified amyloplasts were lysed and then supplied with radioactively labeled glucose-6-phosphate, glucose-1-phosphate, fructose-1,6-bisphosphate, dihydroxyacetone phosphate, glucose, fructose, sucrose, and 3-0-methylglucose in the presence of adenosine triphosphate or uridine triphosphate. Of the above, only the phosphorylated substrates were incorporated into starch. Incorporation into starch was higher with added uridine triphosphate than with adenosine triphosphate. Dihydroxyacetone phosphate was the preferred substrate for uptake by intact amyloplasts and incorporation into starch. In preliminary experiments, it appeared that glucose-6-P and fructose-1,6-bisphosphate may also be taken up by intact amyloplasts. However, the rate of uptake and incorporation into starch was relatively low and variable. Additional study is needed to determine conclusively whether hexose phosphates will cross intact amyloplast membranes. From these data, we conclude that: (a) Triose phosphate is the preferred substrate for uptake by intact amyloplasts. (b) Amyloplasts contain all enzymes necessary to convert triose phosphates into starch. (c) Sucrose breakdown must occur in the cytosol prior to carbohydrate transfer into the amyloplasts. (d) Under the conditions of assay, amyloplasts are unable to convert glucose or fructose to starch. (e) Uridine triphosphate may be the preferred nucleotide for conversion of hexose phosphates to starch at this stage of kernel development.     

Maize starch-branching enzyme isoforms and amylopectin structure. In the absence of starch-branching enzyme IIb, the further absence of starch-branching enzyme Ia leads to increased branching

Previous studies indicated that the deficiency of starch-branching enzyme (SBE) Ia in the single mutant sbe1a::Mu (sbe1a) has no impact on endosperm starch structure, whereas the deficiency of SBEIIb in the ae mutant is well known to reduce the branching of starch. We hypothesized that in maize (Zea mays) endosperm, the function of SBEIIb is predominant to that of SBEIa, and SBEIa would have an observable effect only on amylopectin structure in the absence of SBEIIb. To test this hypothesis, the mutant sbe1a was introgressed into lines containing either wx (lacking the granule-bound starch synthase GBSSI) or ae wx (lacking both SBEIIb and GBSSI) in the W64A background. Both western blotting and zymogram analysis confirmed the SBEIa deficiency in sbe1a wx and sbe1a ae wx, and the SBEIIb deficiency in ae wx and sbe1a ae wx. Using zymogram analysis, no pleiotropic effects of sbe1a genes on SBEIIa, starch synthase, or starch-debranching enzyme isoforms were observed. High-performance size exclusion chromatography analysis shows that the chain-length profiles of amylopectin as well as beta-limit dextrin were indistinguishable between wx and sbe1a wx, whereas significant differences for both were observed between ae wx and sbe1a ae wx, suggesting an effect of SBEIa on amylopectin biosynthesis that is observable only in the absence of SBEIIb. The amylopectin branch density and the average number of branches per cluster were both higher in endosperm starch from sbe1a ae wx than from ae wx. These results indicate possible functional interactions between SBE isoforms that may involve enzymatic inhibition. Both the cluster repeat distance and the distance between branch points on the short intracluster chains were similar for all genotypes however, suggesting a similar pattern of individual SBE isoforms in cluster initiation and the determination of branch point location.     

Enzymic properties of amyloplasts form suspension cultures of soybean

The aim of this work was to determine which enzymes of carbohydrate metabolism are present in amyloplasts. Protoplasts from 4- to 5-day-old suspension cultures of soybean, Glycine max, were lysed and fractionated on a sucrose gradient. This gave an amyloplast fraction that contained stromal enzymes and was not seriously contaminated by cytosol or by organelles likely to be involved in carbohydrate metabolism. Studies of this fraction provide evidence that, in soybean cells, starch synthase and ADPglucose pyrophosphorylase are confined to amyloplasts; invertase, sucrose synthetase and UDPglucose pyrophosphorylase are absent from the amyloplast and probably confined to the cytosol; the following enzymes, though predominantly cytosolic, are present in the amyloplasts in activities high enough to mediate the rate of starch synthesis observed in vivo: glyceraldehyde-phosphate dehydrogenase (NAD), triosephosphate isomerase, fructose-1, 6-bisphosphate aldolase, fructose-bisphosphatase, glucosephosphate isomerase and phosphoglucomutase. The pathway from sucrose to starch in non-photosynthetic cells is discussed; particularly the possibility that sucrose is converted to triose phosphate for entry into the amyloplast.     

Protein phosphorylation in amyloplasts regulates starch branching enzyme activity and protein-protein interactions

Protein phosphorylation in amyloplasts and chloroplasts of Triticum aestivum (wheat) was investigated after the incubation of intact plastids with gamma-(32)P-ATP. Among the soluble phosphoproteins detected in plastids, three forms of starch branching enzyme (SBE) were phosphorylated in amyloplasts (SBEI, SBEIIa, and SBEIIb), and both forms of SBE in chloroplasts (SBEI and SBEIIa) were shown to be phosphorylated after sequencing of the immunoprecipitated (32)P-labeled phosphoproteins using quadrupole-orthogonal acceleration time of flight mass spectrometry. Phosphoamino acid analysis of the phosphorylated SBE forms indicated that the proteins are all phosphorylated on Ser residues. Analysis of starch granule-associated phosphoproteins after incubation of intact amyloplasts with gamma-(32)P-ATP indicated that the granule-associated forms of SBEII and two granule-associated forms of starch synthase (SS) are phosphorylated, including SSIIa. Measurement of SBE activity in amyloplasts and chloroplasts showed that phosphorylation activated SBEIIa (and SBEIIb in amyloplasts), whereas dephosphorylation using alkaline phosphatase reduced the catalytic activity of both enzymes. Phosphorylation and dephosphorylation had no effect on the measurable activity of SBEI in amyloplasts and chloroplasts, and the activities of both granule-bound forms of SBEII in amyloplasts were unaffected by dephosphorylation. Immunoprecipitation experiments using peptide-specific anti-SBE antibodies showed that SBEIIb and starch phosphorylase each coimmunoprecipitated with SBEI in a phosphorylation-dependent manner, suggesting that these enzymes may form protein complexes within the amyloplast in vivo. Conversely, dephosphorylation of immunoprecipitated protein complex led to its disassembly. This article reports direct evidence that enzymes of starch metabolism (amylopectin synthesis) are regulated by protein phosphorylation and indicate a wider role for protein phosphorylation and protein-protein interactions in the control of starch anabolism and catabolism.     

Distinct isoforms of ADPglucose pyrophosphorylase occur inside and outside the amyloplasts in barley endosperm

This paper addresses the controversial idea that ADPglucose pyrophosphorylase may be located in the cytosol in some non-photosynthetic plant organs. The intracellular location of the enzyme in developing barley endosperm has been investigated by isolation of intact amyloplasts. Amyloplast preparations contained 13–17% of the total endosperm activity of two plastidial marker enzymes, and less than 0.5% of the total endosperm activity of two cytosolic marker enzymes. Amyloplast preparations contained about 2.5% of the ADPglucose pyrophosphorylase activity, indicating that approximately 15% of the ADPglucose pyrophosphorylase activity in young endosperms is plastidial. Immunoblotting of gels of endosperm and amyloplast extracts also indicated that the enzyme is both inside and outside the amyloplast. Antibodies to the small subunits of the enzyme from barley and maize revealed two bands of protein of different sizes, one of which was located inside and the other outside the amyloplast. The plastidial protein was of the same size as a protein in the chloroplasts of barley leaves which was also recognized by these antibodies. It is suggested that the barley plant contains two distinct isoforms of ADPglucose pyrophosphorylase: one located in plastids (chloroplasts and amyloplasts) and the other in the cytosol of the endosperm. The role of the cytosolic ADPglucose pyrophosphorylase is unknown. Although it may contribute ADPglucose to starch synthesis, the total activity of ADPglucose pyrophosphorylase in the endosperm is far in excess of the rate of starch synthesis and the plastidial isoform is probably capable of catalysing the entire flux of carbon to starch.     

Association of Ferredoxin-NADP Oxidoreductase with the Chloroplastic Pyridine Nucleotide Dehydrogenase Complex in Barley Leaves

Barley (Hordeum vulgare L.) leaves were used to isolate and characterize the chloroplast NAD(P)H dehydrogenase complex. The stroma fraction and the thylakoid fraction solubilized with sodium deoxycholate were analyzed by native polyacrylamide gel electrophoresis, and the enzymes detected with NADH and nitroblue tetrazolium were electroeluted. The enzymes electroeluted from band S from the stroma fraction and from bands T1 (ET1) and T2 from the thylakoid fraction solubilized with sodium deoxycholate had ferredoxin-NADP oxidoreductase (FNR; EC 1.18.1.2) and NAD(P)H-FeCN oxidoreductase (NAD[P]H-FeCNR) activities. Their NADPH-FeCNR activities were inhibited by 2′-monophosphoadenosine-5′-diphosphoribose and by enzyme incubation with p-chloromercuriphenylsulfonic acid (p-CMPS), NADPH, and p-CMPS plus NADPH. They presented Michaelis constant NADPH values that were similar to those of FNRs from several sources. Their NADH-FeCNR activities, however, were not inhibited by 2′-monophosphoadenosine-5′-diphosphoribose but were weakly inhibited by enzyme incubation with NADH, p-CMPS, and p-CMPS plus NADH. We found that only ET1 contained two polypeptides of 29 and 35 kD, which reacted with the antibodies raised against the mitochondrial complex I TYKY subunit and the chloroplast ndhA gene product, respectively. However, all three enzymes contained two polypeptides of 35 and 53 kD, which reacted with the antibodies raised against barley FNR and the NADH-binding 51-kD polypeptide of the mitochondrial complex I, respectively. The results suggest that ET1 is the FNR-containing thylakoidal NAD(P)H dehydrogenase complex.     

The localization and expression of the class II starch synthases of wheat.

The starch granules of hexaploid wheat (Triticum aestivum) contain a group of three proteins known as SGP-1 (starch granule protein-1) proteins, which have apparent molecular masses of 100, 108, and 115 kD. The nature and role of these proteins has not been defined previously. We demonstrate that these polypeptides are starch synthases that are present in both the starch granule and the soluble fraction at the early stages of wheat endosperm development, but that are exclusively granule bound at mid and late endosperm development. A partial cDNA clone encoding a fragment of the 100-kD protein was obtained by screening a wheat endosperm cDNA expression library using monoclonal antibodies. Three classes of cDNA were subsequently isolated from a wheat endosperm cDNA library by nucleic acid hybridization and were shown to encode the 100-, 108-, and 115-kD proteins. The cDNA sequences are highly homologous to class II starch synthases and have the highest homology with the maize SSIIa (starch synthase IIa) gene. mRNA for the SGP-1 proteins was detected in the leaf, pre-anthesis florets, and endosperm of wheat and is highly expressed in the leaf and in the grain during the early to mid stages of development. We discuss the roles of the SGP-1 proteins in starch biosynthesis in wheat.