Biochemical and genetic analyses of physical associations among Zea mays starch biosynthetic enzymes

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 (SS), starch branching enzymes (BE), and starch debranching enzymes (DBE), however, the molecular explanation for these functional interactions is not known. One possibility is that specific SSs, BEs, and/or DBEs associate physically with each other in multisubunit complexes. To test this hypothesis, this study sought to identify stable associations between SSI, SSIIa, SSIII, SBEI, SBEIIa, and SBEIIb from maize amyloplasts. Three separate detection methods, yeast two-hybrid, co-immunoprecipitation, and affinity purification using recombinant proteins as the solid phase ligand were used to identify specific protein-protein interactions. Numerous instances were detected of specific pairs of proteins associating either directly or indirectly in the same multi-subunit complex.;Gel permeation chromatography of proteins extracted from maize amyloplasts revealed two high molecular weight complexes of approximately 600kDa (C600) and 300kDa (C300) containing either SSIIa, SSIII, SBEIIa, and SBEIIb, or SSIIa, SBEIIa, and SBEIIb, respectively. To further characterize these interactions, genetic analyses tested the interdependence of specific starch biosynthetic enzymes on each other for assembly into the complexes. Association of SSIIa, SBEIIa, and SBEIIb into C600 was found to require the presence of SSIII, however, loss of SSIII did not affect assembly of the C300 complex. Further purification of the complexes through successive chromatography steps demonstrated SSIII, SSIIa, SBEIIa, and SBEIIb co-purified with C600 and the latter three proteins co-purified with C300. These data support the hypothesis that all four enzymes are present in C600 and that SSIII mediates assembly of the other three proteins into that quaternary structure. Additional proteins that co-purified with each complex were identified, specifically pyruvate orthophosphate dikinase (PPDK) and sucrose synthase1. Both of these proteins bound to a specific conserved, non-catalytic fragment of SSIII expressed in E. coli, and co-immunoprecipitated with SSIII. Association of PPDK and starch biosynthetic enzymes suggests a means of global regulation of carbon partitioning between protein and starch in developing seeds.;Finally, the observed, specific interactions among the SSs and SBEs were further examined. Direct protein-protein interactions were reconstituted in a minimal system. Three full-length maize enzymes (SSI, SBEIIa, and SBEIIb) and one conserved domain known to participate in complex formation (SSIIIHD) were expressed in E. coli and purified. Pull-down experiments revealed direct binding between SSIIIHD and SSI, SBEIIa, and SBEIIb. This binding occurred in the absence of any other maize factors. SSIIIHD and SSI were phosphorylated after incubation with soluble extracts of maize amyloplasts. Phosphorylation of SSIIIHD enhanced its ability to bind SSI. These data provide novel information about the specificity of interactions between starch biosynthetic enzymes, and further demonstrate that phosphorylation is likely to play a regulatory role in assembly and activity of the complexes

Integrated functions among multiple starch synthases determine both amylopectin chain length and branch linkage location in Arabidopsis leaf starch

This study assessed the impact on starch metabolism in Arabidopsis leaves of simultaneously eliminating multiple soluble starch synthases (SS) from among SS1, SS2, and SS3. Double mutant ss1- ss2- or ss1- ss3- lines were generated using confirmed null mutations. These were compared to the wild type, each single mutant, and ss1- ss2- ss3- triple mutant lines grown in standardized environments. Double mutant plants developed similarly to the wild type, although they accumulated less leaf starch in both short-day and long-day diurnal cycles. Despite the reduced levels in the double mutants, lines containing only SS2 and SS4, or SS3 and SS4, are able to produce substantial amounts of starch granules. In both double mutants the residual starch was structurally modified including higher ratios of amylose:amylopectin, altered glucan chain length distribution within amylopectin, abnormal granule morphology, and altered placement of α(1→6) branch linkages relative to the reducing end of each linear chain. The data demonstrate that SS activity affects not only chain elongation but also the net result of branch placement accomplished by the balanced activities of starch branching enzymes and starch debranching enzymes. SS3 was shown partially to overlap in function with SS1 for the generation of short glucan chains within amylopectin. Compensatory functions that, in some instances, allow continued residual starch production in the absence of specific SS classes were identified, probaby accomplished by the granule bound starch synthase GBSS1.ANR Génoplante GPLA0611GEuropean Union-FEDER, Région Nord Pas de Calais ARCir PlantTEQ5National Science Foundation DBI-0209789Comisión Interministerial de Ciencia y Tecnología BIO2009-07040Junta de Andalucía P09-CVI-470

Direct Determination of the Site of Addition of Glucosyl Units to Maltooligosaccharide Acceptors Catalyzed by Maize Starch Synthase I

Starch synthase (SS) (ADP-glucose:1,4-α-D-glucan 4-α-D-glucosyltransferase) elongates α-(1→4)-linked linear glucans within plastids to generate the storage polymers that constitute starch granules. Multiple SS classes are conserved throughout the plant kingdom, indicating that each provides a unique function responsible for evolutionary selection. Evidence has been presented arguing for addition of glucosyl units from the ADPglucose donor to either the reducing end or the non-reducing end of the acceptor substrate, although until recently direct evidence addressing this question was not available. Characterization of newly incorporated glucosyl units determined that recombinant maize (Zea mays L.) SSIIa elongates its substrates at the non-reducing end. However, the possibility remained that other SSs might utilize distinct mechanisms, and that one or more of the conserved enzyme classes could elongate acceptors at the reducing end. This study characterized the reaction mechanism of recombinant maize SSI regarding its addition site. Newly incorporated residues were labeled with 13C, and reducing ends of the elongation products were labeled by chemical derivitization. Electrospray ionization-tandem mass spectroscopy traced the two parameters, i.e., the newly added residue and the reducing end. The results determined that SSI elongates glucans at the non-reducing end. The study also confirmed previous findings showing recombinant SSI can generate glucans of at least 25 units, that it is active using acceptors as short as maltotriose, that recombinant forms of the enzyme absolutely require an acceptor for activity, and that it is not saturable with maltooligosaccharide acceptor substrates

Functions of maize genes encoding pyruvate phosphate dikinase in developing endosperm

Maize opaque2 (o2) mutations are beneficial for endosperm nutritional quality but cause negative pleiotropic effects for reasons that are not fully understood. Direct targets of the bZIP transcriptional regulator encoded by o2 include pdk1 and pdk2 that specify pyruvate phosphate dikinase (PPDK). This enzyme reversibly converts AMP, pyrophosphate, and phosphoenolpyruvate to ATP, orthophosphate, and pyruvate and provides diverse functions in plants. This study addressed PPDK function in maize starchy endosperm where it is highly abundant during grain fill. pdk1 and pdk2 were inactivated individually by transposon insertions, and both genes were simultaneously targeted by endosperm-specific RNAi. pdk2 accounts for the large majority of endosperm PPDK, whereas pdk1 specifies the abundant mesophyll form. The pdk1- mutation is seedling-lethal, indicating that C4 photosynthesis is essential in maize. RNAi expression in transgenic endosperm eliminated detectable PPDK protein and enzyme activity. Transgenic kernels weighed the same on average as nontransgenic siblings, with normal endosperm starch and total N contents, indicating that PPDK is not required for net storage compound synthesis. An opaque phenotype resulted from complete PPDK knockout, including loss of vitreous endosperm character similar to the phenotype conditioned by o2-. Concentrations of multiple glycolytic intermediates were elevated in transgenic endosperm, energy charge was altered, and starch granules were more numerous but smaller on average than normal. The data indicate that PPDK modulates endosperm metabolism, potentially through reversible adjustments to energy charge, and reveal that o2- mutations can affect the opaque phenotype through regulation of PPDK in addition to their previously demonstrated effects on storage protein gene expression

Subcellular analysis of starch metabolism in developing barley seeds using a non-aqueous fractionation method

Compartmentation of metabolism in developing seeds is poorly understood due to the lack of data on metabolite distributions at the subcellular level. In this report, a non-aqueous fractionation method is described that allows subcellular concentrations of metabolites in developing barley endosperm to be calculated. (i) Analysis of subcellular volumes in developing endosperm using micrographs shows that plastids and cytosol occupy 50.5% and 49.9% of the total cell volume, respectively, while vacuoles and mitochondria can be neglected. (ii) By using non-aqueous fractionation, subcellular distribution between the cytosol and plastid of the levels of metabolites involved in sucrose degradation, starch synthesis, and respiration were determined. With the exception of ADP and AMP which were mainly located in the plastid, most other metabolites of carbon and energy metabolism were mainly located outside the plastid in the cytosolic compartment. (iii) In developing barley endosperm, the ultimate precursor of starch, ADPglucose (ADPGlc), was mainly located in the cytosol (80–90%), which was opposite to the situation in growing potato tubers where ADPGlc was almost exclusively located in the plastid (98%). This reflects the different subcellular distribution of ADPGlc pyrophosphorylase (AGPase) in these tissues. (iv) Cytosolic concentrations of ADPGlc were found to be close to the published Km values of AGPase and the ADPGlc/ADP transporter at the plastid envelope. Also the concentrations of the reaction partners glucose-1-phosphate, ATP, and inorganic pyrophosphate were close to the respective Km values of AGPase. (v) Knock-out of cytosolic AGPase in Riso16 mutants led to a strong decrease in ADPGlc level, in both the cytosol and plastid, whereas knock-down of the ADPGlc/ADP transporter led to a large shift in the intracellular distribution of ADPGlc. (v) The thermodynamic structure of the pathway of sucrose to starch was determined by calculating the mass–action ratios of all the steps in the pathway. The data show that AGPase is close to equilibrium, in both the cytosol and plastid, whereas the ADPGlc/ADP transporter is strongly displaced from equilibrium in vivo. This is in contrast to most other tissues, including leaves and potato tubers. (vi) Results indicate transport rather than synthesis of ADPGlc to be the major regulatory site of starch synthesis in barley endosperm. The reversibility of AGPase in the plastid has important implications for the regulation of carbon partitioning between different biosynthetic pathways

The barley amo1 locus is tightly linked to the starch synthase IIIa gene and negatively regulates expression of granule-bound starch synthetic genes

In this study of barley starch synthesis, the interaction between mutations at the sex6 locus and the amo1 locus has been characterized. Four barley genotypes, the wild type, sex6, amo1, and the amo1sex6 double mutant, were generated by backcrossing the sex6 mutation present in Himalaya292 into the amo1 ‘high amylose Glacier’. The wild type, amo1, and sex6 genotypes gave starch phenotypes consistent with previous studies. However, the amo1sex6 double mutant yielded an unexpected phenotype, a significant increase in starch content relative to the sex6 phenotype. Amylose content (as a percentage of starch) was not increased above the level observed for the sex6 mutation alone; however, on a per seed basis, grain from lines containing the amo1 mutation (amo1 mutants and amo1sex6 double mutants) synthesize significantly more amylose than the wild-type lines and sex6 mutants. The level of granule-bound starch synthase I (GBSSI) protein in starch granules is increased in lines containing the amo1 mutation (amo1 and amo1sex6). In the amo1 genotype, starch synthase I (SSI), SSIIa, starch branching enzyme IIa (SBEIIa), and SBEIIb also markedly increased in the starch granules. Genetic mapping studies indicate that the ssIIIa gene is tightly linked to the amo1 locus, and the SSIIIa protein from the amo1 mutant has a leucine to arginine residue substitution in a conserved domain. Zymogram analysis indicates that the amo1 phenotype is not a consequence of total loss of enzymatic activity although it remains possible that the amo1 phenotype is underpinned by a more subtle change. It is therefore proposed that amo1 may be a negative regulator of other genes of starch synthesis

Two carbon fluxes to reserve starch in potato (Solanum tuberosum L.) tuber cells are closely interconnected but differently modulated by temperature

Parenchyma cells from tubers of Solanum tuberosum L. convert several externally supplied sugars to starch but the rates vary largely. Conversion of glucose 1-phosphate to starch is exceptionally efficient. In this communication, tuber slices were incubated with either of four solutions containing equimolar [U-14C]glucose 1-phosphate, [U-14C]sucrose, [U-14C]glucose 1-phosphate plus unlabelled equimolar sucrose or [U-14C]sucrose plus unlabelled equimolar glucose 1-phosphate. 14C-incorporation into starch was monitored. In slices from freshly harvested tubers each unlabelled compound strongly enhanced 14C incorporation into starch indicating closely interacting paths of starch biosynthesis. However, enhancement disappeared when the tubers were stored. The two paths (and, consequently, the mutual enhancement effect) differ in temperature dependence. At lower temperatures, the glucose 1-phosphate-dependent path is functional, reaching maximal activity at approximately 20 °C but the flux of the sucrose-dependent route strongly increases above 20 °C. Results are confirmed by in vitro experiments using [U-14C]glucose 1-phosphate or adenosine-[U-14C]glucose and by quantitative zymograms of starch synthase or phosphorylase activity. In mutants almost completely lacking the plastidial phosphorylase isozyme(s), the glucose 1-phosphate-dependent path is largely impeded. Irrespective of the size of the granules, glucose 1-phosphate-dependent incorporation per granule surface area is essentially equal. Furthermore, within the granules no preference of distinct glucosyl acceptor sites was detectable. Thus, the path is integrated into the entire granule biosynthesis. In vitro 14C-incorporation into starch granules mediated by the recombinant plastidial phosphorylase isozyme clearly differed from the in situ results. Taken together, the data clearly demonstrate that two closely but flexibly interacting general paths of starch biosynthesis are functional in potato tuber cells

Starch biosynthesis in rice endosperm requires the presence of either starch synthase I or IIIa

Starch synthase (SS) I and IIIa are the first and second largest components of total soluble SS activity, respectively, in developing japonica rice (Oryza sativa L.) endosperm. To elucidate the distinct and overlapping functions of these enzymes, double mutants were created by crossing the ss1 null mutant with the ss3a null mutant. In the F2 generation, two opaque seed types were found to have either the ss1ss1/SS3ass3a or the SS1ss1/ss3ass3a genotype. Phenotypic analyses revealed lower SS activity in the endosperm of these lines than in those of the parent mutant lines since these seeds had different copies of SSI and SSIIIa genes in a heterozygous state. The endosperm of the two types of opaque seeds contained the unique starch with modified fine structure, round-shaped starch granules, high amylose content, and specific physicochemical properties. The seed weight was ∼90% of that of the wild type. The amount of granule-bound starch synthase I (GBSSI) and the activity of ADP-glucose pyrophosphorylase (AGPase) were higher than in the wild type and parent mutant lines. The double-recessive homozygous mutant prepared from both ss1 and ss3a null mutants was considered sterile, while the mutant produced by the leaky ss1 mutant×ss3a null mutant cross was fertile. This present study strongly suggests that at least SSI or SSIIIa is required for starch biosynthesis in rice endosperm

Genome assembly and population genomic analysis provide insights into the evolution of modern sweet corn.

Sweet corn is one of the most important vegetables in the United States and Canada. Here, we present a de novo assembly of a sweet corn inbred line Ia453 with the mutated shrunken2-reference allele (Ia453-sh2). This mutation accumulates more sugar and is present in most commercial hybrids developed for the processing and fresh markets. The ten pseudochromosomes cover 92% of the total assembly and 99% of the estimated genome size, with a scaffold N50 of 222.2 Mb. This reference genome completely assembles the large structural variation that created the mutant sh2-R allele. Furthermore, comparative genomics analysis with six field corn genomes highlights differences in single-nucleotide polymorphisms, structural variations, and transposon composition. Phylogenetic analysis of 5,381 diverse maize and teosinte accessions reveals genetic relationships between sweet corn and other types of maize. Our results show evidence for a common origin in northern Mexico for modern sweet corn in the U.S. Finally, population genomic analysis identifies regions of the genome under selection and candidate genes associated with sweet corn traits, such as early flowering, endosperm composition, plant and tassel architecture, and kernel row number. Our study provides a high-quality reference-genome sequence to facilitate comparative genomics, functional studies, and genomic-assisted breeding for sweet corn