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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 Vmax but with minor effects on substrate Kd and Km 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.

Starch branching enzymes (SBEs) are one of the major classes of enzymes that catalyze starch biosynthesis in plants. Here, we utilized the clustered regularly interspaced short palindromic repeats–CRISPR associated protein 9 (CRISPR–Cas9)-mediated gene editing system to investigate the effects of SBE mutation on starch structure and turnover in the oilseed crop Brassica napus. Multiple single-guide RNA (sgRNA) expression cassettes were assembled into a binary vector and two rounds of transformation were employed to edit all six BnaSBE genes. All mutations were heterozygous monoallelic or biallelic, and no chimeric mutations were detected from a total of 216 editing events. Previously unannotated gene duplication events associated with two BnaSBE genes were characterized through analysis of DNA sequencing chromatograms, reflecting the complexity of genetic information in B. napus. Five Cas9-free homozygous mutant lines carrying two to six mutations of BnaSBE were obtained, allowing us to compare the effect of editing different BnaSBE isoforms. We also found that in the sextuple sbe mutant, although indels were introduced at the genomic DNA level, an alternate transcript of one BnaSBE2.1 gene bypassed the indel-induced frame shift and was translated to a modified full-length protein. Subsequent analyses showed that the sextuple mutant possesses much lower SBE enzyme activity and starch branching frequency, higher starch-bound phosphate content, and altered pattern of amylopectin chain length distribution relative to wild-type (WT) plants. In the sextuple mutant, irregular starch granules and a slower rate of starch degradation during darkness were observed in rosette leaves. At the pod-filling stage, the sextuple mutant was distinguishable from WT plants by its thick main stem. This work demonstrates the applicability of the CRISPR–Cas9 system for the study of multi-gene families and for investigation of gene-dosage effects in the oil crop B. napus. It also highlights the need for rigorous analysis of CRISPR–Cas9-mutated plants, particularly with higher levels of ploidy, to ensure detection of gene duplications.

Starch commands a central role in the carbon budget of the majority of plants on earth, and its biological role changes during development and in response to the environment. Throughout the life of a plant, starch plays a dual role in carbon allocation, acting as both a source, releasing carbon reserves in leaves for growth and development, and as a sink, either as a dedicated starch store in its own right (in seeds and tubers), or as a temporary reserve of carbon contributing to sink strength, in organs such as flowers, fruits, and developing non-starchy seeds. The presence of starch in tissues and organs thus has a profound impact on the physiology of the growing plant as its synthesis and degradation governs the availability of free sugars, which in turn control various growth and developmental processes. This review attempts to summarize the large body of information currently available on starch metabolism and its relationship to wider aspects of carbon metabolism and plant nutrition. It highlights gaps in our knowledge and points to research areas that show promise for bioengineering and manipulation of starch metabolism in order to achieve more desirable phenotypes such as increased yield or plant biomass.

The Sucrose Non-fermenting-1-Related Protein Kinase 1 (SnRK1) is a highly conserved heterotrimeric protein kinase in plants. It possesses a catalytic subunit (α) and two regulatory subunits (β and γ). The effects of altered expression of AKINβ1 on carbohydrate metabolism and gene expression in leaves were investigated in an Arabidopsis T-DNA insertion mutant. The contents of key intermediates in the tricarboxylic acid (TCA) cycle of the mutant leaves were markedly reduced throughout the diurnal cycle, coupled with a decrease in measurable respiration rate. Compared to wild-type, 2485 genes and 188 genes were expressed differentially in leaves of the akinβ1 mutant in response to light and darkness respectively. Among these, several genes exhibited very substantial decreases in expression. Notably, expression of particular isoforms of multigene families involved in malate and lipid metabolism, and nitrate uptake showed decreases of 40-240 fold during the light period, but not during darkness. The subcellular localization of AKINβ1 and the regulatory function of N-myristoylation on the subcellular localization of AKINβ1 were investigated, showing that AKINβ1 localizes to Golgi. A model is hypothesized to explain the effects of AKINβ1 on metabolism and gene expression in Arabidopsis.

Physical properties of resistant starch (RS) were examined in a range of barley genotypes to determine the contribution of starch and seed physical characteristics to the RS component. Thirty-three barley genotypes were studied, which varied significantly in their RS, amylose, and starch contents and grain yield. From 33 genotypes, 13 exhibiting high RS were selected for detailed physicochemical analysis of starch. In high-RS varieties, granule size and number were unimodal, compared with normal starches from a reference genotype, which showed a bimodal distribution. Principal component analysis (PCA) showed that a higher content of granules

Starch synthase 2 (SS2) is an important enzyme in leaf starch synthesis, elongating intermediate-length glucan chains. Loss of SS2 results in a distorted starch granule phenotype and altered physiochemical properties, highlighting its importance in starch biosynthesis, however, the post-translational regulation of SS2 is poorly understood. In this study, a combination of bioinformatic and in vitro analysis of recombinant SS2 was used to identify and characterize SS2 post-translational regulatory mechanisms. The SS2 N-terminal region, comprising the first 185 amino acids of the mature protein sequence, was shown to be highly variable between species, and was predicted to be intrinsically disordered. Intrinsic disorder in proteins is often correlated with protein phosphorylation and protein-protein interactions. Recombinant Arabidopsis thaliana SS2 formed homodimers that required the N-terminal region, but N-terminal peptides could not form stable homodimers alone. Recombinant SS2 was shown to be phosphorylated by chloroplast protein kinases and recombinant casein kinase II at two N-terminal serine residues (S63, S65), but mutation of these phosphorylation sites (Ser>Ala) revealed that they are not required for homo-dimerization. Heteromeric enzyme complex (HEC) formation between SS2 and SBE2.2 was shown to be ATP-dependent. However, SS2 homo-dimerization and protein phosphorylation are not required for its interaction with SBE2.2, as truncation of the SS2 N-terminus did not disrupt ATP-dependent HEC assembly. SS2 phosphorylation had no affect on its catalytic activity. Intriguingly, the removal of the N-terminal region of SS2 resulted in a 47-fold increase in its activity. As N-terminal truncation disrupted dimerization, this suggests that SS2 is more active when monomeric, and that transitions between oligomeric state may be a mechanism for SS2 regulation.

Starch synthesis requires several enzymatic activities including branching enzymes (BEs) responsible for the formation of α(1 → 6) linkages. Distribution and number of these linkages are further controlled by debranching enzymes that cleave some of them, rendering the polyglucan water-insoluble and semi-crystalline. Although the activity of BEs and debranching enzymes is mandatory to sustain normal starch synthesis, the relative importance of each in the establishment of the plant storage polyglucan (i.e. water insolubility, crystallinity and presence of amylose) is still debated. Here, we have substituted the activity of BEs in Arabidopsis with that of the Escherichia coli glycogen BE (GlgB). The latter is the BE counterpart in the metabolism of glycogen, a highly branched water-soluble and amorphous storage polyglucan. GlgB was expressed in the be2 be3 double mutant of Arabidopsis, which is devoid of BE activity and consequently free of starch. The synthesis of a water-insoluble, partly crystalline, amylose-containing starch-like polyglucan was restored in GlgB-expressing plants, suggesting that BEs' origin only has a limited impact on establishing essential characteristics of starch. Moreover, the balance between branching and debranching is crucial for the synthesis of starch, as an excess of branching activity results in the formation of highly branched, water-soluble, poorly crystalline polyglucan.

Protein–protein interactions between starch phosphorylase (SP) and other starch biosynthetic enzymes were investigated using isolated maize endosperm amyloplasts and a recombinant maize enzyme. Plastidial SP is a stromal enzyme existing as a multimeric protein in amyloplasts. Biochemical analysis of the recombinant maize SP indicated that the tetrameric form was catalytically active in both glucan-synthetic and phosphorolytic directions. Protein–protein interaction experiments employing the recombinant SP as an affinity ligand with amyloplast extracts showed that the multimeric state of SP determined interactions with other enzymes of the starch biosynthetic pathway. The monomeric form of SP interacts with starch branching enzyme I (SBEI) and SBEIIb, whereas only SBEI interacts with the tetrameric form of SP. In all cases, protein–protein interactions were broken when amyloplast lysates were dephosphorylated in vitro, and enhanced following pre-treatment with ATP, suggesting a mechanism of protein complex formation regulated by protein phosphorylation. In vitro protein phosphorylation experiments with [γ-32P]-ATP show that SP is phosphorylated by a plastidial protein kinase. Evidence is presented which suggests SBEIIb modulates the catalytic activity of SP through the formation of a heteromeric protein complex.

Starch-branching enzymes (SBEs) are one of the four major enzyme classes involved in starch biosynthesis in plants and algae, and their activities play a crucial role in determining the structure and physical properties of starch granules. SBEs generate α-1,6-branch linkages in α-glucans through cleavage of internal α-1,4 bonds and transfer of the released reducing ends to C-6 hydroxyls. Starch biosynthesis in plants and algae requires multiple isoforms of SBEs and is distinct from glycogen biosynthesis in both prokaryotes and eukaryotes which uses a single branching enzyme (BE) isoform. One of the unique characteristics of starch structure is the grouping of α-1,6-branch points in clusters within amylopectin. This is a feature of SBEs and their interplay with other starch biosynthetic enzymes, thus facilitating formation of the compact water-insoluble semicrystalline starch granule. In this respect, the activity of SBE isoforms is pivotal in starch granule assembly. SBEs are structurally related to the α-amylase superfamily of enzymes, sharing three domains of secondary structure with prokaryotic Bes: the central (β/α)8 -barrel catalytic domain, an NH2 -terminal domain involved in determining the size of α-glucan chain transferred, and the C-terminal domain responsible for catalytic capacity and substrate preference. In addition, SBEs have conserved plant-specific domains, including phosphorylation sites which are thought to be involved in regulating starch metabolism. SBEs form heteromeric protein complexes with other SBE isoforms as well as other enzymes involved in starch synthesis, and assembly of these protein complexes is regulated by protein phosphorylation. Phosphorylated SBEIIb is found in multienzyme complexes with isoforms of glucan-elongating starch synthases, and these protein complexes are implicated in amylopectin cluster formation. This review presents a comparative overview of plant SBEs and includes a review of their properties, structural and functional characteristics, and recent developments on their post-translational regulation.

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 γ-32P-ATP indicated that SSII and both forms of SBEII are phosphorylated. Treatment of the partially purified 260-kD SS-SBE complexes with alkaline phosphatase caused dissociation of the assembly into the respective monomeric proteins, indicating that formation of SS-SBE complexes is phosphorylation dependent. The 260-kD SS-SBEII protein complexes are formed around 10 to 15 d after pollination and were shown to be catalytically active with respect to both SS and SBE activities. Prior to this developmental stage, SSI, SSII, and SBEII forms were detectable only in monomeric form. High molecular weight forms of SBEII demonstrated a higher affinity for in vitro glucan substrates than monomers. These results provide direct evidence for the existence of protein complexes involved in amylopectin biosynthesis.

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.

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.

Multiple forms of starch synthases (EC 2.4.1.21) are critical for the synthesis of starch in higher plants. These enzymes catalyze the extension of linear glucan chains by the transfer of glucose from the nucleotide sugar ADPglucose. Recombinant maize starch synthase I (rSSI) was purified from Escherichia coli in order to study the regulation of this enzyme by protein phosphorylation. The rSSI was phosphorylated in vitro by incubation with [γ 32P]-ATP and maize amyloplast lysates, which were used as a source of protein kinase. Maximal phosphorylation of rSSI was achieved within 20 minutes, and there was no noticeable change in the amount of phosphorylation beyond this time. Phosphoamino acid analysis of rSSI indicated phosphorylation of one or more serine residues. In order to purify and identify the phosphopeptides, phosphorylated rSSI was digested with trypsin to yield smaller peptides, which were then concentrated using Immobilized Metal Affinity Chromatography (IMAC). Potential phosphopeptides eluting from the IMAC column were purified and putatively identified using reversed phase (C18 column) High Pressure Liquid Chromatography (HPLC). They were then analyzed by mass spectrometry using Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) which yielded the mass of potential phosphopeptides.

Most of the carbon used for starch biosynthesis in cereal endosperms is derived from ADP-glucose (ADP-Glc) synthesized by extra-plastidial AGPase activity, and imported directly across the amyloplast envelope. The properties of the wheat endosperm amyloplast ADP-Glc transporter were analysed with respect to substrate kinetics and specificities using reconstituted amyloplast envelope proteins in a proteoliposome-based assay system, as well as with isolated intact organelles. Experiments with liposomes showed that ADP-Glc transport was dependent on counter-exchange with other adenylates. Rates of ADP-Glc transport were highest with ADP and AMP as counter-exchange substrates, and kinetic analysis revealed that the transport system has a similar affinity for ADP and AMP. Measurement of ADP and AMP efflux from intact amyloplasts showed that, under conditions of ADP-Glc-dependent starch biosynthesis, ADP is exported from the plastid at a rate equal to that of ADP-Glc utilization by starch synthases. Photo-affinity labelling of amyloplast membranes with the substrate analogue 8-azido-[alpha-P-32]ADP-Glc showed that the polypeptide involved in substrate binding is an integral membrane protein of 38 kDa. This study shows that the ADP-Glc transporter in cereal endosperm amyloplasts imports ADP-Glc in exchange for ADP which is produced as a by-product of the starch synthase reaction inside the plastid.

In this study, the functional properties of A- and B-type wheat starch granules from two commercial wheat flours were investigated for digestibility in vitro, chemical composition (e.g., amylose, protein, and ash content), gelatinization, retrogradation, and pasting properties. The branch chain length and chain length distribution of these A- and B-type wheat starch granules were also determined using high-performance anion exchange chromatography (HPAEC). Wheat starches with different granular sizes not only had different degrees of enzymatic hydrolysis and thermal and pasting properties, but also different molecular characteristics. Different amylose content, protein content, and branch chain length of amylopectin in A- and B-type wheat starch granules could also be the major factors besides granular size for different digestibility and other functional properties of starch. The data indicate that different wheat cultivars with different proportion of A- and B-type granular starch could result ...

Starch synthesis requires several enzymatic activities including branching enzymes (BEs) responsible for the formation of α(1→6) linkages. Distribution and number of these linkages are further controlled by debranching enzymes (DBEs) that cleave some of them, rendering the polyglucan water-insoluble and semi-crystalline. Although the activity of BEs and DBEs is mandatory to sustain normal starch synthesis, the relative importance of each in the establishment of the plant storage polyglucan (i.e. water-insolubility, crystallinity, presence of amylose) is still debated. Here, we have substituted the activity of BEs inArabidopsiswith that of theEscherichia coliglycogen branching enzyme (GlgB). The latter is the BE counterpart in the metabolism of glycogen, a highly branched water-soluble and amorphous storage polyglucan. GlgB was expressed in thebe2 be3double mutant ofArabidopsisthat is devoid of BE activity and consequently free of starch. The synthesis of a water-insoluble, partly crystalline, amylose-containing starch-like polyglucan was restored in GlgB-expressing plants, suggesting that BEs origin only have a limited impact on establishing essential characteristics of starch. Moreover, the balance between branching and debranching is crucial for the synthesis of starch, as an excess of branching activity results in the formation of highly branched, water-soluble, poorly crystalline polyglucan.

Highlight Starch biosynthetic enzymes in rice endosperm are physically associated with each other and form enzymatically active multiple protein–protein complexes, several of which were common to cereals while others were unique., Amylopectin is a highly branched, organized cluster of glucose polymers, and the major component of rice starch. Synthesis of amylopectin requires fine co-ordination between elongation of glucose polymers by soluble starch synthases (SSs), generation of branches by branching enzymes (BEs), and removal of misplaced branches by debranching enzymes (DBEs). Among the various isozymes having a role in amylopectin biosynthesis, limited numbers of SS and BE isozymes have been demonstrated to interact via protein–protein interactions in maize and wheat amyloplasts. This study investigated whether protein–protein interactions are also found in rice endosperm, as well as exploring differences between species. Gel permeation chromatography of developing rice endosperm extracts revealed that all 10 starch biosynthetic enzymes analysed were present at larger molecular weights than their respective monomeric sizes. SSIIa, SSIIIa, SSIVb, BEI, BEIIb, and PUL co-eluted at mass sizes >700kDa, and SSI, SSIIa, BEIIb, ISA1, PUL, and Pho1 co-eluted at 200–400kDa. Zymogram analyses showed that SSI, SSIIIa, BEI, BEIIa, BEIIb, ISA1, PUL, and Pho1 eluted in high molecular weight fractions were active. Comprehensive co-immunoprecipitation analyses revealed associations of SSs–BEs, and, among BE isozymes, BEIIa–Pho1, and pullulanase-type DBE–BEI interactions. Blue-native-PAGE zymogram analyses confirmed the glucan-synthesizing activity of protein complexes. These results suggest that some rice starch biosynthetic isozymes are physically associated with each other and form active protein complexes. Detailed analyses of these complexes will shed light on the mechanisms controlling the unique branch and cluster structure of amylopectin, and the physicochemical properties of starch.

We have identified a novel means to achieve substantially increased vegetative biomass and oilseed production in the model plant Arabidopsis thaliana. Endogenous isoforms of starch branching enzyme (SBE) were substituted by either one of the endosperm-expressed maize (Zea mays L.) branching isozymes, ZmSBEI or ZmSBEIIb. Transformants were compared with the starch-free background and with the wild-type plants. Each of the maize-derived SBEs restored starch biosynthesis but both morphology and structure of starch particles were altered. Altered starch metabolism in the transformants is associated with enhanced biomass formation and more-than-trebled oilseed production while maintaining seed oil quality. Enhanced oilseed production is primarily due to an increased number of siliques per plant whereas oil content and seed number per silique are essentially unchanged or even modestly decreased. Introduction of cereal starch branching isozymes into oilseed plants represents a potentially useful strategy to increase biomass and oilseed production in related crops and manipulate the structure and properties of leaf starch.

Expression of one specific isoform of plastidic glucose 6-phosphate dehydrogenase (G6PDH) was manipulated in transgenic tobacco. Antisense and sense constructs of the endogenous P2 form of G6PDH were used to transform plants under the control of the cauliflower mosaic virus (CaMV) 35S promotor. Recombinant plants with altered expression were taken through to homozygosity by selective screening. Northern analyses revealed substantial changes in the expression of the P2 form of G6PDH, with no apparent impact on the activity of the cytosolic isoenzyme. Analysis of G6PDH activity in chloroplasts showed that despite the large changes in expression of P2-G6PDH, the range of enzyme activity varied only from approximately 50 to 200% of the wild type, reflecting the presence of a second G6PDH chloroplastic isoform (P1). Although none of the transgenic plants showed any visible phenotype, there were marked differences in metabolism of both sense and antisense lines when compared with wild-type/control lines. Sucrose, glucose and fructose contents of leaves were higher in antisense lines, whereas in overexpressing lines, the soluble sugar content was reduced below that of control plants. Even more striking was the observation that contents of glucose 6-phosphate (Glc6P) and 6-phosphogluconate (6PG) changed, such that the ratio of Glc6P:6PG was some 2.5-fold greater in the most severe antisense lines, compared with those with the highest levels of overexpression. Because of the distinctive biochemical properties of P2-G6PDH, we investigated the impact of altered expression on the contents of antioxidants and the response of plants to oxidative stress induced by methyl viologen (MV). Plants with decreased expression of P2-G6PDH showed increased content of reduced glutathione (GSH) compared to other lines. They also possessed elevated contents of ascorbate and exhibited a much higher ratio of reduced:oxidised ascorbate. When exposed to MV, leaf discs of wild-type and overexpressing lines demonstrated increased oxidative damage as measured by lipid peroxidation. Remarkably, leaf discs from plants with decreased P2-G6PDH did not show any change in lipid peroxidation in response to increasing concentrations of up to 15 micro m MV. The results are discussed from the perspective of the role of G6PDH in carbohydrate metabolism and oxidative stress. It is suggested that the activity of P2-G6PDH may be crucial in balancing the redox poise in chloroplasts.

Chromoplasts were extracted from the perianth of the wild Meadow Buttercup ( Ranunculus acris L.) and purified by sucrose density gradient centrifugation. Analysis of marker enzymes indicated that chromoplast preparations were essentially free of contamination by cytosol, mitochondria and microbodies. The distribution and recovery of glycolytic enzymes indicated an incomplete sequence of enzymes for this pathway in chromoplasts. Fructose 1, 6- bis phosphate aldolase (aldolase) was the only glycolytic enzyme for which unequivocal evidence of localisation within chromoplast preparations could not be obtained. Sub-cellular fractionation and immunological studies using antibodies raised against spinach cytosolic and chloroplastic aldolase, revealed that over 40% of its activity was associated with a washed particulate/membrane fraction in chromoplasts, but this could be removed with 1.2 M NaCl. It is concluded that Ranunculus chromoplasts do not possess a plastidial form of aldolase, and that the aldolase measured in the plastid fraction is a cytosolic form associating with plastid membranes. Isolated buttercup chromoplasts possess a functional oxidative pentose-phosphate pathway (OPPP) which could be monitored as 14 CO 2 release following incubation with [U- 14 C]-glucose 6-phosphate (Glc6P). 14 CO 2 was not evolved when [U- 14 C]-glucose 1-phosphate (Glc1P) was supplied to chromoplasts suggesting this compound is not transported into the intact organelle and that no interconversion of hexose-phosphate by phosphoglucomutase occurred outside the plastid. Transport experiments using native chromoplast vesicles preloaded with inorganic orthophosphate indicated that these organelles transported Glc6P and dihydroxyacetone phosphate (DHAP), but not Glc1P. The chromoplasts isolated from this wild plant have similar enzymic components and transport properties to other chromoplasts studied to date.

Second harmonic generation (SHG) microscopy is employed to study changes in crystalline organization due to altered gene expression and hydration in barley starch granules. SHG intensity and susceptibility ratio values (R'(SHG)) are obtained using reduced Stokes-Mueller polarimetric microscopy. The maximum R'(SHG) values occur at moderate moisture indicating the narrowest orientation distribution of nonlinear dipoles from the cylindrical axis of glucan helices. The maximum SHG intensity occurs at the highest moisture and amylopectin content. These results support the hypothesis that SHG is caused by ordered hydrogen and hydroxyl bond networks which increase with hydration of starch granules. (C) 2015 Optical Society of America

Starch biosynthesis requires the ordered assembly of glucose units into amylose and amylopectin from ADPglucose. Whilst amylose has a relatively simple linear structure, the synthesis of amylopectin requires the formation of regular, repeating clusters of glucan chains. These clusters have a 9 nm periodicity and comprise a crystalline, alpha helical region and an amorphous branched region, a unit which is repeated many times over to give rise to blocklets of amylopectin. Although the individual enzymes of starch biosynthesis have been studied extensively, our knowledge of the mechanisms which give rise to this highly ordered structure is limited. There is an increasing body of literature which has demonstrated that several reactions are regulated post-translationally, including via redox modulation, protein-protein interactions and protein phosphorylation. This chapter summarises our current knowledge of these mechanisms and offers a model of how they serve to coordinate the biosynthesis of amylopectin in particular.

The distribution of starch synthase I and starch branching enzyme IIb between the starch granule and amyloplast stroma plays an important role in determining endosperm amylose content of cereal grains. Starch synthase IIa (SSIIa) catalyses the polymerisation of intermediate length glucan chains of amylopectin in the endosperm of cereals. Mutations of SSIIa genes in barley and wheat and inactive SSIIa variant in rice induce similar effects on the starch structure and the amylose content, but the severity of the phenotypes is different. This study compared the levels of transcripts and partitioning of proteins of starch synthase I (SSI) and starch branching enzyme IIb (SBEIIb) inside and outside the starch granules in the developing endosperms of these ssIIa mutants and inactive SSIIa variant. Pleiotropic effects on starch granule-bound proteins suggested that the different effects of SSIIa mutations on endosperm amylose content of barley, wheat and rice are determined by the distribution of SSI and SBEIIb between the starch granule and amyloplast stroma in cereals. Regulation of starch synthesis in ssIIa mutants and inactive SSIIa variant may be at post-translational level or the altered amylopectin structure deprives the affinity of SSI and SBEIIb to amylopectin.

The present study investigated the role of protein phosphorylation, and protein complex formation between key enzymes of amylopectin synthesis, in barley genotypes exhibiting "high amylose" phenotypes. Starch branching enzyme (SBE) down-regulated lines (ΔSBEIIa and ΔSBEIIb), starch synthase (SS)IIa (ssiia(-), sex6) and SSIII (ssiii(-), amo1) mutants were compared to a reference genotype, OAC Baxter. Down-regulation of either SBEIIa or IIb caused pleiotropic effects on SSI and starch phosphorylase (SP) and resulted in formation of novel protein complexes in which the missing SBEII isoform was substituted by SBEI and SP. In the ΔSBEIIb down-regulated line, soluble SP activity was undetectable. Nonetheless, SP was incorporated into a heteromeric protein complex with SBEI and SBEIIa and was readily detected in starch granules. In amo1, unlike other mutants, the data suggest that both SBEIIa and SBEIIb are in a protein complex with SSI and SSIIa. In the sex6 mutant no protein complexes involving SBEIIa or SBEIIb were detected in amyloplasts. Studies with Pro-Q Diamond revealed that GBSS, SSI, SSIIa, SBEIIb and SP are phosphorylated in their granule bound state. Alteration in the granule proteome in ΔSBEIIa and ΔSBEIIb lines, suggests that different protein complexes are involved in the synthesis of A and B granules.

Little is known about the thermal properties and internal molecular structure of transitory starch. In this study, granule morphology, thermal properties, and the cluster structure of Arabidopsis leaf starch at beginning and end of the light period were explored. The structural properties of building blocks and clusters were evaluated by using diverse chromatographic techniques. On the granular level, starch from end of day had larger granule size, thinner crystalline lamellae thickness, lower free surface energy of crystals, and lower tendency to retrograde than that from end of night. On the molecular level, the starch had lower amylose content, larger cluster size, and higher number of blocks per cluster at the end of day than at end of night. It is concluded that the core of the granules contains a more permanent molecular and less-ordered physical structure different from the transitory layers laid down around the core at daytime.

elsewhere (Jones and Chapman, 1995a, b), but more to discuss the rationale of such approaches, including considArtificial membrane systems are being used increas- erations and controls. It is hoped that the references ingly to study the function and role of plant membrane provided will supply the reader with any more detailed proteins, particularly solute transporters which cata- information that is required. lyse counter-exchange of metabolites. Performing Model bilayer systems are often used for studying such studies requires (i) solubilization of the protein membrane transport as they negate many of the probwith a suitable surfactant, (ii) functional reconstitution lems associated with the use of membrane vesicles of the protein in a well characterized liposome system. ( Klingenberg, 1985; Hochstadt et al., 1975) and some Much of the technology has been derived from studies types of intact plant organelles ( Tetlow et al., 1996). The on non-plant systems and there are many pitfalls of model system approach involves removal of membrane which to be wary before applying it to a new protein. proteins, or specific protein(s), from the cell or purified This short review outlines the key parameters which organelle, followed by functional reconstitution into a should be considered when attempting the study of closed bilayer system produced specifically for that purplant membrane proteins in artificial lipid bilayers, pose. Transport measurements can then be made. The including types of surfactants, lipid composition advantage of such a system is that conditions on either of vesicles, membrane permeability, and protein side of the membrane can be manipulated in order to orientation. characterize the function of a single transporter or transport complex in isolation from other cellular or membrane

sketolase, ribose 5-phosphate isomerase, ribulose 5-phosphate 3-epimerase) appear to be restricted to between phosphorylated 3-, 4-, 5-, 6-, and 7-carbon sugars catalysed by the enzymes ribose 5-phosphate isomerase the plastid. In tobacco root and leaf, however, the non-oxidative enzymes were found in the cytosolic as (Rib5P isomerase), ribulose 5-phosphate 3-epimerase (Rul5P 3-epimerase), transaldolase ( TA), and transketolwell as the plastidic compartments. In the absence of ribose 5-phosphate isomerase and ribulose ase ( TK ). With the exception of the transaldolase, these reversible enzymes are amphibolic, and also play a part 5-phosphate 3-epimerase in the cytosol, the product of the oxidative limb of the pathway (ribulose in the Calvin cycle. Their function as part of the pentose phosphate pathway is to provide carbon skeletons for the 5-phosphate) must be transported into a compartment capable of utilizing it. Ribulose 5-phosphate was sup- shikimate pathway via erythrose 4-phosphate, and nucleotide synthesis utilizing ribose 5-phosphate, as well as plied to isolated intact pea root plastids and was shown to be capable of supporting nitrite reduction. recycling of sugar phosphate intermediates for use in the glycolytic pathway (ap Rees, 1985). The kinetics of ribulose 5-phosphate-driven nitrite reduction in isolated pea root plastids suggested that The enzymes of the oxidative portion of the pathway are commonly found in both the cytosolic and plastidic the metabolite was translocated across the plastid envelope in a carrier-mediated transport process, fractions of photosynthetic (Herbert et al., 1979) and non-photosynthetic cells (Ashihara and Komamine, 1976; indicating the presence of a translocator capable of transporting pentose phosphates.

Summary Phosphoglucomutase (PGM) activity was measured during the development of endosperm of spring wheat ( Triticum aestivum L. cv. Axona) in crude extracts and in isolated amyloplasts. Results show a 60 % reduction in activity of plastidial PGM activity between 5 and 15 days post anthesis. Measurements of the concentrations of the substrates and co-factors of the PGM reaction in the tissue during this period indicate that the enzyme should be saturated in vivo , suggesting that the reduction in activity of PGM during endosperm development is a function of the amount of PGM protein. The implications of this for starch synthesis are discussed.

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: Ser(649), Ser(286), and Ser(297). Two Ca(2+)-dependent protein kinase activities were partially purified from amyloplasts, termed K1, responsible for Ser(649) and Ser(286) phosphorylation, and K2, responsible for Ser(649) and Ser(297) phosphorylation. The Ser(286) and Ser(297) 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-Ser(297) forms a stable salt bridge with Arg(665), part of a conserved Cys-containing domain in plant branching enzymes. Ser(649) 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.

Starch synthesis and CO2 evolution were determined after incubating intact and lysed wheat (Triticum aestivum L. cv. Axona) endosperm amyloplasts with 14C-labelled hexose-phosphates. Amyloplasts converted [U-14C]glucose 1-phosphate (Glc1P) but not [U-14C]glucose 6-phosphate (Glc6P) into starch in the presence of ATP. When the oxidative pentose-phosphate pathway (OPPP) was stimulated, both [U-14C]Glc1P and [U-14C]Glc6P were metabolized to CO2, but Glc6P was the better precursor for the OPPP, and Glc1P-mediated starch synthesis was reduced by 75%. In order to understand the basis for the partitioning of carbon between the two potentially competing metabolic pathways, metabolite pools were measured in purified amyloplasts under conditions which promote both starch synthesis and carbohydrate oxidation via the OPPP. Amyloplasts incubated with Glc1P or Glc6P alone showed little or no interconversion of these hexose-phosphates inside the organelle. When amyloplasts were synthesizing starch, the stromal concentrations of Glc1P and ADP-glucose were high. By contrast, when flux through the OPPP was highest, Glc1P and ADP-glucose inside the organelle were undetectable, and there was an increase in metabolites involved in carbohydrate oxidation. Measurements of the plastidial hexose-monophosphate pool during starch synthesis and carbohydrate oxidation indicate that the phosphoglucose isomerase reaction is at equilibrium whereas the reaction catalysed by phosphoglucomutase is significantly displaced from equilibrium.

Plastids in non-photosynthetic tissue are the site of fatty acid, starch and amino acid synthesis. The intermediates to support these activities are imported from the cytoplasm or generated by carbohydrate oxidation within the organelle. This review considers the current understanding of the nature of the substrates which are transported into such plastids, the interaction between pathways within the organelle which have a supply and demand relationship, and pathways which potentially compete for substrates.

Resistant starch (RS) consumption can modulate postprandial metabolic responses, but its effects on carbohydrate (CHO) handling in type 2 diabetics (T2D) are unclear. It was hypothesized that a bagel high in RS would improve glucose and insulin homeostasis following the 1st meal, regardless of the amount of available CHO, and that in association with incretins, the effects would carry over to a 2nd meal. Using a randomized crossover design, 12 T2D ingested four different bagel treatments (their 1st meal) determined by available CHO and the weight or amount of bagel consumed: treatment A, without RS (50 g of available CHO); treatment B, with RS (same total CHO as in A); treatment C, with RS (same available CHO as in A); and treatment D, with the same RS as in B and available CHO as in A and C. A standard 2nd meal was ingested 3 h later. Following the first meal, B elicited a lower glucose incremental area under the curve (iAUC) than C (P < 0.05), D (P < 0.05), and A (trend; P = 0.07), lower insulin iAUC than A (P < 0.05) and C (P < 0.05), and lower glucose-dependent insulinotropic polypeptide (GIP) iAUC than A (P < 0.05). There was a positive correlation (P < 0.05) between GIP and insulin iAUCs after the 2nd meal, and C had a 3 times greater slope than the other treatments (r = 0.91, P < 0.001), yet lacked a significant concomitant improvement in glucose disposal. These results show that for the 1st meal, RS was effective when it replaced a portion of the available CHO, while ingesting more RS influenced the GIP–insulin axis following the 2nd meal.

The pyruvate dehydrogenase complex (PDC) and acetyl-CoA carboxylase (ACC, EC 6.4.1.2) have been characterized in pea root plastids. PDC activity was optimum in the presence of 1.0 mM pyruvate, 1.5 mM NAD + , 0.1 mM CoA, 0.1 mM TPP, 5 mM MgCI2, 3.0 mM cysteine-HCI, and 0.1 M Tricine (pH 8.0) and represents approximately 47% of the total cellular activity. ACC activity was greatest in the presence of 1.0 mM acetyl-CoA, 4 mM NaHC03, 3 mM ATP, 10 mM MgCI2, 2.5 mM dithiothreito l, and 100 mM Tricine (pH 8.0). Both enzymes were stimulated by reduced sutphydryl reagents and inhibited by sulphydryl inhibitors. ACC was also inhibited by malonyl-CoA while PDC was inhibited by both malonyl-CoA and NADH. Both enzymes were stimulated by DHAP and UDP-galactose while ACC was also stimulated by PEP and F1,6P. Palmitic acid and oleic acid both inhibited ACC, but had essentially no effect on PDC. Palmitoyl-CoA inhibited both enzymes while PA and Lyso-PA inhibited PDC, but stimulated ACC. The results presented support the hypothesis that PDC and ACC function in a co-ordinated fashion to promote glycolytic carbon flow to fatty acid biosynthesis in pea root plastids.

The rates of incorporation of various metabolites into starch by isolated amyloplasts from developing endosperm of spring wheat (Triticum aestivum L. cv. Axona) were examined. Of the metabolites tested that were likely to be present in the cytosol at concentrations sufficient to sustain starch synthesis, only glucose 1-phosphate (Glc1P) supported physiologically relevant rates of starch synthesis. Incorporation of Glc1P into starch was both dependent on the presence of ATP and intact organelles. The rate of incorporation of hexose into starch became saturated at a Glc1P concentration of less than 1 mol·m-3 in the presence of 1 mol·m-3 ATP. Starch synthesis from 5 mol · m-3 ADP-glucose supplied to the organelles occurred at rates 15-fold higher than from similar concentrations of Glc1P, but it is argued that this is probably of little physiological relevance. The net incorporation of hexose units into starch from GlclP was inhibited 50% by 100 mmol.m-3 carboxyatractyloside. Carbohydrate oxidation in the amyloplast was stimulated by the addition of 2-oxoglutarate and glutamine, and in such circumstances incorporation of14C-labelled metabolites into starch was reduced. Glucose 6-phosphate proved to be a better substrate for oxidative pathways than Glc1P. Our results suggest that Glc1P is the primary substrate for starch synthesis in developing wheat endosperm, and that ATP required for starch synthesis is imported via an adenylate translocator.

Summary The effect of tryptic digestion on hexosephosphate isomerase activity in pea root plastids and cytosol was investigated. Maintenance of organelle integrity conferred substantial protection from proteolysis when compared with broken organelles. Electrophoretic studies indicated the presence of two major isoenzymes of hexosephosphate isomerase in pea roots, one cytosolic the other plastidial. Further studies indicated that the loss of hexosephosphate isomerase activity from organelles during tryptic digestion is due to proteolysis of the enzyme within or released from the organelle and not loss of contaminating cytosolic activity which was not susceptible to trypsin proteolysis.

SUMMARY Uncoupled Photosystem I (PS I) activity driven by either reduced 2,6-dichlorophenolindophenol (DCPIPH2) or N,N,N',N'-tetramethyl-p-phenylene diamine (TMPDH2) showed a stimulation following pre-treatment at 40–50°C, followed by inhibition at higher temperatures. The stimulation was more marked in thylakoids isolated from warm-grown plants than in those isolated from cool-grown plants. Approximately maximal rates of PS I activity were achieved at physiological temperatures (25–35°C) using highly lipophilic-reduced diaminodurene (DADH2) as an electron donor with little stimulation occurring as a result of high temperature pre-treatment. Electron transport driven by all three electron donors was markedly inhibited by KCN/HgCl2 following all pre-treatment temperatures, but relatively insensitive to ethyldimethylaminopropyl-carbodiimide (EDAC). It is suggested that the stimulation of PS I activity involves a phase change in the thylakoid membrane leading to an increase in permeability which allows enhanced access of DCPIPH2 and TMPDH2 to a common electron acceptor site located in the region of cytochrome f. The greater stimulation of electron transport in thylakoids isolated from warm-grown plants may be due to a more pronounced phase change occurring as a consequence of altered membrane composition as modified by growth temperature.

Advances in the resolution of starch structure have played a critical role in our understanding of the physicochemical and functional properties associated with the wide variety of agriculturally produced starches. In addition, our knowledge of starch structure and ability to determine subtle structural differences between different starches has proved invaluable in ascribing a function to the many enzymes involved in its metabolism through the study of mutants, which is further improving our understanding of starch biosynthesis and degradation. However, we still need a greater understanding of the relationship between starch structure and starch functionality. An important gap in our understanding of structure–function relationships is our inability to directly determine interbranch distances in amylopectin (and therefore branching density). This is currently estimated by digestion of the polymer with β-amylase and β-limit dextrins, revealing the distribution of glucan chain lengths from the reducing ends of branch points to the outer branch point of the chain [137]. The long term applied goal of much research in this field is to be able to rationally manipulate amylopectin structure to produce functional starches for particular end-uses. This will require a combined effort including manipulation of postharvest starches by techniques such as cross-linking, but also by breeding and biotechnology. Underpinning the latter approach is a fundamental knowledge of the pathway of starch biosynthesis, and this is the subject of another article.

The amylose extender (ae(-)) mutant of maize lacks starch branching enzyme IIb (SBEIIb) activity, resulting in amylopectin with reduced branch point frequency, and longer glucan chains. Recent studies indicate isozymes of soluble starch synthases form high molecular weight complexes with SBEII isoforms. This study investigated the effect of the loss of SBEIIb activity on interactions between starch biosynthetic enzymes in maize endosperm amyloplasts. Results show distinct patterns of protein-protein interactions in amyloplasts of ae(-) mutants compared with the wild type, suggesting functional complementation for loss of SBEIIb by SBEI, SBEIIa, and SP. Coimmunoprecipitation experiments and affinity chromatography using recombinant proteins showed that, in amyloplasts from normal endosperm, protein-protein interactions involving starch synthase I (SSI), SSIIa, and SBEIIb could be detected. By contrast, in ae(-) amyloplasts, SSI and SSIIa interacted with SBEI, SBEIIa, and SP. All interactions in the wild-type were strongly enhanced by ATP, and broken by alkaline phosphatase, indicating a role for protein phosphorylation in their assembly. Whilst ATP and alkaline phosphatase had no effect on the stability of the protein complexes from ae(-) endosperm, radiolabelling experiments showed SP and SBEI were both phosphorylated within the mutant protein complex. It is proposed that, during amylopectin biosynthesis, SSI and SSIIa form the core of a phosphorylation-dependent glucan-synthesizing protein complex which, in normal endosperm, recruits SBEIIb, but when SBEIIb is absent (ae(-)), recruits SBEI, SBEIIa, and SP. Differences in stromal protein complexes are mirrored in the complement of the starch synthesizing enzymes detected in the starch granules of each genotype, reinforcing the hypothesis that the complexes play a functional role in starch biosynthesis.

The mechanisms involved in sensing oxidative signalling molecules, such as H2O2, in plant and animal cells are not completely understood. In the present study, we tested the postulate that oxidation of Met (methionine) to MetSO (Met sulfoxide) can couple oxidative signals to changes in protein phosphorylation. We demonstrate that when a Met residue functions as a hydrophobic recognition element within a phosphorylation motif, its oxidation can strongly inhibit peptide phosphorylation in vitro. This is shown to occur with recombinant soybean CDPKs (calcium-dependent protein kinases) and human AMPK (AMP-dependent protein kinase). To determine whether this effect may occur in vivo, we monitored the phosphorylation status of Arabidopsis leaf NR (nitrate reductase) on Ser534 using modification-specific antibodies. NR was a candidate protein for this mechanism because Met538, located at the P+4 position, serves as a hydrophobic recognition element for phosphorylation of Ser534 and its oxidation substantially inhibits phosphorylation of Ser534 in vitro. Two lines of evidence suggest that Met oxidation may inhibit phosphorylation of NR-Ser534 in vivo. First, phosphorylation of NR at the Ser534 site was sensitive to exogenous H2O2 and secondly, phosphorylation in normal darkened leaves was increased by overexpression of the cytosolic MetSO-repair enzyme PMSRA3 (peptide MetSO reductase A3). These results are consistent with the notion that oxidation of surface-exposed Met residues in kinase substrate proteins, such as NR, can inhibit the phosphorylation of nearby sites and thereby couple oxidative signals to changes in protein phosphorylation.

Mutations affecting specific starch biosynthetic enzymes commonly have pleiotropic effects on other enzymes in the same metabolic pathway. Such genetic evidence indicates functional relationships between components of the starch biosynthetic system, including starch synthases (SSs), starch branching enzymes (BEs), and starch debranching enzymes; however, the molecular explanation for these functional interactions is not known. One possibility is that specific SSs, BEs, and/or starch debranching enzymes associate physically with each other in multisubunit complexes. To test this hypothesis, this study sought to identify stable associations between three separate SS polypeptides (SSI, SSIIa, and SSIII) and three separate BE polypeptides (BEI, BEIIa, and BEIIb) from maize (Zea mays) amyloplasts. Detection methods included in vivo protein-protein interaction tests in yeast (Saccharomyces cerevisiae) nuclei, immunoprecipitation, and affinity purification using recombinant proteins as the solid phase ligand. Eight different instances were detected of specific pairs of proteins associating either directly or indirectly in the same multisubunit complex, and direct, pairwise interactions were indicated by the in vivo test in yeast. In addition, SSIIa, SSIII, BEIIa, and BEIIb all comigrated in gel permeation chromatography in a high molecular mass form of approximately 600 kD, and SSIIa, BEIIa, and BEIIb also migrated in a second high molecular form, lacking SSIII, of approximately 300 kD. Monomer forms of all four proteins were also detected by gel permeation chromatography. The 600- and 300-kD complexes were stable at high salt concentration, suggesting that hydrophobic effects are involved in the association between subunits.

The potential role of the plastidial oxidative pentose phosphate pathway (OPPP) in providing the NADPH for fatty acid synthesis in plastids from developing embryos of Brassica napus (L.) has been investigated. Measurements of distributions of enzyme activities in fractions obtained from homogenates of isolated embryos have revealed that the glucose 6-phosphate and 6-phosphogluconate dehydrogenases are present in both cytosol and plastid, as is ribose 5-phosphate isomerase. However, transketolase and transaldolase are most probably confined to the plastid, while ribulose 5-phosphate epimerase is essentially cytosolic, although a very small proportion of plastid-localized activity cannot be ruled out. The activity of the OPPP in intact plastids was measured by the release of 14 CO 2 from [1- 14 C]glucose 6-phosphate. Activity was detectable in the absence of electron sinks created by the addition of metabolites to the incubation media and was stimulated 1.3-, 3.2-, and 7.9-fold by the respective additions of glutamine plus 2-oxoglutarate, cofactors and substrates for fatty acid synthesis, or methyl viologen. An increase in OPPP activity in response to additions that are absolutely required for fatty acid synthesis in these isolated plastids provides direct evidence that these two processes are connected, most probably by NADP/NADPH metabolism. The OPPP activity with methyl viologen was more than twice that during fatty acid synthesis, suggesting that the latter is not limited by OPPP capacity. Light energy may also contribute to reductant provision and, consistent with the possibility of maintenance of a balance of NADPH from light and the OPPP, glucose 6-phosphate dehydrogenase activity in the isolated plastids was decreased by light or by DTT.

This article reviews current knowledge of starch metabolism in higher plants, and focuses on the control and regulation of the biosynthetic and degradative pathways. The major elements comprising the synthetic and degradative pathways in plastids are discussed, and show that, despite present knowledge of the core reactions within each pathway, understanding of how these individual reactions are co-ordinated within different plastid types and under different environmental conditions, is far from complete. In particular, recently discovered aspects of the fine control of starch metabolism are discussed, which indicate that a number of key reactions are controlled by post-translational modifications of enzymes, including redox modulation and protein phosphorylation. In some cases, enzymes of the pathway may form protein complexes with specific functional significance. It is suggested that some of the newly discovered aspects of fine control of the biosynthetic pathway may well apply to many other proteins which are directly and indirectly involved in polymer synthesis and degradation.

Evidence from a number of plant tissues suggests that phosphoglucomutase (PGM) is present in both the cytosol and the plastid. The cytosolic and plastidic isoforms of PGM have been partially purified from wheat endosperm (Triticum aestivum L. cv. Axona). Both isoforms required glucose 1,6-bisphosphate for their activity with K(a) values of 4.5 micro M and 3.8 micro M for cytosolic and plastidic isoforms, respectively, and followed normal Michaelis-Menten kinetics with glucose 1-phosphate as the substrate with K(m) values of 0.1 mM and 0.12 mM for the cytosolic and plastidic isoforms, respectively. A cDNA clone was isolated from wheat endosperm that encodes the cytosolic isoform of PGM. The deduced amino acid sequence shows significant homology to PGMs from eukaryotic and prokaryotic sources. PGM activity was measured in whole cell extracts and in amyloplasts isolated during the development of wheat endosperm. Results indicate an approximate 80% reduction in measurable activity of plastidial and cytosolic PGM between 8 d and 30 d post-anthesis. Northern analysis showed a reduction in cytosolic PGM mRNA accumulation during the same period of development. The implications of the changes in PGM activity during the synthesis of starch in developing endosperm are discussed.

The biosynthesis of starch is the major determinant of yield in cereal grains. In this short review, attention is focused on the synthesis of the soluble substrate for starch synthesis, ADPglucose (ADPG). Consideration is given to the pathway of ADPG production, its subcellular compartmentation, and the role of metabolite transporters in mediating its delivery to the site of starch synthesis. As ADPG is an activated sugar, the dependence of its production on respiration, changes which occur during development, and the constraints which ATP production may place on carbon partitioning into different end-products are discussed.

The origin of second harmonic generation (SHG) in starch granules was investigated using ab initio quantum mechanical modeling and experimentally examined using polarization-in, polarization-out (PIPO) second harmonic generation microscopy. Ab initio calculations revealed that the largest contribution to the SHG signal from A- and B-type allomorphs of starch originates from the anisotropic organization of hydroxide and hydrogen bonds mediated by aligned water found in the polymers. The hypothesis was experimentally tested by imaging maize starch granules under various hydration and heat treatment conditions that alter the hydrogen bond network. The highest SHG intensity was found in fully hydrated starch granules, and heat treatment diminished the SHG intensity. The PIPO SHG imaging showed that dried starch granules have a much higher nonlinear optical susceptibility component ratio than fully hydrated granules. In contrast, deuterated starch granules showed a smaller susceptibility component ratio demonstrating that SHG is highly sensitive to the organization of the hydroxyl and hydrogen bond network. The polarization SHG imaging results of potato starch granules, representing starch allomorph B, were compared to those of maize starch granules representing allomorph A. The results showed that the amount of aligned water was higher in the maize granules. Nonlinear microscopy of starch granules provides evidence that varying hydration conditions leads to significant changes in the nonlinear susceptibility ratio as well as the SHG intensity, supporting the hypothesis from ab initio calculations that the dominant contribution to SHG is due to the ordered hydroxide and hydrogen bond network.