Véronique Planchot

Starch granules: structure and biosynthesis

The emphasis of this review is on starch structure and its biosynthesis. Improvements in understanding have been brought about during the last decade through the development of new physicochemical and biological techniques, leading to real scientific progress. All this literature needs to be kept inside the general literature about biopolymers, despite some confusing results or discrepancies arising from the biological variability of starch. However, a coherent picture of starch over all the different structural levels can be presented, in order to obtain some generalizations about its structure. In this review we will focus first on our present understanding of the structures of amylose and amylopectin and their organization within the granule, and we will then give insights on the biosynthetic mechanisms explaining the biogenesis of starch in plants.

Starch Granule Initiation in Arabidopsis Requires the Presence of Either Class IV or Class III Starch Synthases

The mechanisms underlying starch granule initiation remain unknown. We have recently reported that mutation of soluble starch synthase IV (SSIV) in Arabidopsis thaliana results in restriction of the number of starch granules to a single, large, particle per plastid, thereby defining an important component of the starch priming machinery. In this work, we provide further evidence for the function of SSIV in the priming process of starch granule formation and show that SSIV is necessary and sufficient to establish the correct number of starch granules observed in wild-type chloroplasts. The role of SSIV in granule seeding can be replaced, in part, by the phylogenetically related SSIII. Indeed, the simultaneous elimination of both proteins prevents Arabidopsis from synthesizing starch, thus demonstrating that other starch synthases cannot support starch synthesis despite remaining enzymatically active. Herein, we describe the substrate specificity and kinetic properties of SSIV and its subchloroplastic localization in specific regions associated with the edges of starch granules. The data presented in this work point to a complex mechanism for starch granule formation and to the different abilities of SSIV and SSIII to support this process in Arabidopsis leaves.

Proteins from Multiple Metabolic Pathways Associate with Starch Biosynthetic Enzymes in High Molecular Weight Complexes: A Model for Regulation of Carbon Allocation in Maize Amyloplasts

Starch biosynthetic enzymes from maize (Zea mays) and wheat (Triticum aestivum) amyloplasts exist in cell extracts in high molecular weight complexes; however, the nature of those assemblies remains to be defined. This study tested the interdependence of the maize enzymes starch synthase IIa (SSIIa), SSIII, starch branching enzyme IIb (SBEIIb), and SBEIIa for assembly into multisubunit complexes. Mutations that eliminated any one of those proteins also prevented the others from assembling into a high molecular mass form of approximately 670 kD, so that SSIII, SSIIa, SBEIIa, and SBEIIb most likely all exist together in the same complex. SSIIa, SBEIIb, and SBEIIa, but not SSIII, were also interdependent for assembly into a complex of approximately 300 kD. SSIII, SSIIa, SBEIIa, and SBEIIb copurified through successive chromatography steps, and SBEIIa, SBEIIb, and SSIIa coimmunoprecipitated with SSIII in a phosphorylation-dependent manner. SBEIIa and SBEIIb also were retained on an affinity column bearing a specific conserved fragment of SSIII located outside of the SS catalytic domain. Additional proteins that copurified with SSIII in multiple biochemical methods included the two known isoforms of pyruvate orthophosphate dikinase (PPDK), large and small subunits of ADP-glucose pyrophosphorylase, and the sucrose synthase isoform SUS-SH1. PPDK and SUS-SH1 required SSIII, SSIIa, SBEIIa, and SBEIIb for assembly into the 670-kD complex. These complexes may function in global regulation of carbon partitioning between metabolic pathways in developing seeds.

Mutants of Arabidopsis Lacking a Chloroplastic Isoamylase Accumulate Phytoglycogen and an Abnormal Form of Amylopectin

Mutant lines defective for each of the four starch debranching enzyme (DBE) genes (AtISA1, AtISA2, AtISA3, and AtPU1) detected in the nuclear genome of Arabidopsis (Arabidopsis thaliana) were produced and analyzed. Our results indicate that both AtISA1 and AtISA2 are required for the production of a functional isoamylase-type of DBE named Iso1, the major isoamylase activity found in leaves. The absence of Iso1 leads to an 80% decrease in the starch content in both lines and to the accumulation of water-soluble polysaccharides whose structure is similar to glycogen. In addition, the residual amylopectin structure in the corresponding mutant lines displays a strong modification when compared to the wild type, suggesting a direct, rather than an indirect, function of Iso1 during the synthesis of amylopectin. Mutant lines carrying a defect in AtISA3 display a strong starch-excess phenotype at the end of both the light and the dark phases accompanied by a small modification of the amylopectin structure. This result suggests that this isoamylase-type of DBE plays a major role during starch mobilization. The analysis of the Atpu1 single-mutant lines did not lead to a distinctive phenotype. However, Atisa2/Atpu1 double-mutant lines display a 92% decrease in starch content. This suggests that the function of pullulanase partly overlaps that of Iso1, although its implication remains negligible when Iso1 is present within the cell.

Extensive degradation of native starch granules by alpha-amylase from aspergillus fumigatus

Abstract Starch granules of various botanical origins were subjected to enzymic degradation by purified alpha -amylases from pig pancreas, Bacillus sp. and Aspergillus fumigatus ( Aspergillus sp. K-27). With the A. fumigatus enzyme, glucose in alpha -anomeric configuration was the sole end degradation product regardless of the starch tested. The efficiency of this enzyme was very high on all native starch granules. Starches from normal and waxy maize, smooth pea and wheat were completely solubilised within 30 h using 1·34 nKat/mg of substrate. High-amylose maize, wrinkled pea and potato starches were degraded to lower extents (50, 70 and 45%, respectively). Such high enzymic efficiency was not observed with alpha -amylases from pig pancreas or Bacillus sp. With alpha -amylase from A. fumigatus , normal and waxy maize starches displayed highly eroded layered structures when observed by scanning or transmission electron microscopy during degradation. In contrast, potato and high-amylose maize starches produced a minor fraction of endo-eroded granules, whereas the rest of the granules exhibited superficial porosity.

Proteome and phosphoproteome analysis of starch granule-associated proteins from normal maize and mutants affected in starch biosynthesis

In addition to the exclusively granule-bound starch synthase GBSSI, starch granules also bind significant proportions of other starch biosynthetic enzymes, particularly starch synthases (SS) SSI and SSIIa, and starch branching enzyme (BE) BEIIb. Whether this association is a functional aspect of starch biosynthesis, or results from non-specific entrapment during amylopectin crystallization, is not known. This study utilized genetic, immunological, and proteomic approaches to investigate comprehensively the proteome and phosphoproteome of Zea mays endosperm starch granules. SSIII, BEI, BEIIa, and starch phosphorylase were identified as internal granule-associated proteins in maize endosperm, along with the previously identified proteins GBSS, SSI, SSIIa, and BEIIb. Genetic analyses revealed three instances in which granule association of one protein is affected by the absence of another biosynthetic enzyme. First, eliminating SSIIa caused reduced granule association of SSI and BEIIb, without affecting GBSS abundance. Second, eliminating SSIII caused the appearance of two distinct electrophoretic mobility forms of BEIIb, whereas only a single migration form of BEIIb was observed in wild type or any other mutant granules examined. Third, eliminating BEIIb caused significant increases in the abundance of BEI, BEIIa, SSIII, and starch phosphorylase in the granule, without affecting SSI or SSIIa. Analysis of the granule phosphoproteome with a phosphorylation-specific dye indicated that GBSS, BEIIb, and starch phosphorylase are all phosphorylated as they occur in the granule. These results suggest the possibility that starch metabolic enzymes located in granules are regulated by post-translational modification and/or protein–protein interactions.

Amylosucrase from Neisseria polysaccharea: novel catalytic properties.

Amylosucrase is a glucosyltransferase that synthesises an insoluble α‐glucan from sucrose. The catalytic properties of the highly purified amylosucrase from Neisseria polysaccharea were characterised. Contrary to previously published results, it was demonstrated that in the presence of sucrose alone, several reactions are catalysed, in addition to polymer synthesis: sucrose hydrolysis, maltose and maltotriose synthesis by successive transfers of the glucosyl moiety of sucrose onto the released glucose, and finally turanose and trehalulose synthesis – these two sucrose isomers being obtained by glucosyl transfer onto fructose. The effect of initial sucrose concentration on initial activity demonstrated a non‐Michaelian profile never previously described.

Amylose determination in genetically modified starches

Abstract The amylose contents of starches from various botanical origins (potato, smooth pea, wrinkled pea, wheat, maize) and from maize mutants [waxy (wx), amylose extender (ae), dull (du), sugary-2 (su2), aewx, aedu, dusu2 and wxdu] were determined by size exclusion chromatography (SEC), iodine-binding capacity (IBC) measurements, differential scanning calorimetry (DSC) and complexation with concanavalin A. SEC (with a 2.6×200 cm column) on HW75 S gel was used as the reference method for analyzing the macromolecular composition of starches. Variations in the fine structure of amylopectin affected its reactivity in classical methods such as IBC and were probably responsible for erroneous values in determinations when this polymer was the only starch component studied. When starches were composed of two macromolecules, all methods gave similar results, but with some discrepancies in DSC. The elution volume for a third class of α-glucans detected in some maize mutant starches was between that of amylopectin and amylose. Only SEC gave accurate results in this case since all other tested methods showed higher apparent amylose contents.

Enzymatic hydrolysis of α-glucan crystallites

Abstract Crystalline starchy substrates (lintners, i.e. parts of granules resistant to mild acid hydrolysis, and amylose spherocrystals) were subjected to enzymatic degradation by α-amylase from Bacillus sp. The crystallinity of these substrates before and after amylolysis was studied by X-ray diffractometry and differential scanning calorimetry. Chain length distribution was investigated by high-performance size-exclusion chromatography and anion-exchange chromatography. Regardless of the initial morphology, particles with A-type crystallinity were more susceptible to amylolysis than those with B-type. The final rate of hydrolysis after 30 h was less than 10% for high-amylose starch lintners, between 20 and 37% for potato and C-type lintners, and greater than 62% for pure A-type lintners. For C-type lintners, the final rate of hydrolysis increased in relation to the proportion of A-type crystallinity as evaluated by X-ray diffraction. A progression towards B-type was observed during amylolysis of both A- and C-type lintners, confirming the greater susceptibility of the A-type structure. However, this dependence on crystalline type cannot account for the behavior of all studied lintners during enzymatic action. The effect of other parameters, such as morphology and crystal defects and the interrelations of the crystals, should also be taken into consideration.

Mutants of Arabidopsis Lacking Starch Branching Enzyme II Substitute Plastidial Starch Synthesis by Cytoplasmic Maltose Accumulation

Three genes, BE1, BE2, and BE3, which potentially encode isoforms of starch branching enzymes, have been found in the genome of Arabidopsis thaliana. Although no impact on starch structure was observed in null be1 mutants, modifications in amylopectin structure analogous to those of other branching enzyme II mutants were detected in be2 and be3. No impact on starch content was found in any of the single mutant lines. Moreover, three double mutant combinations were produced (be1 be2, be1 be3, and be2 be3), and the impact of the mutations on starch content and structure was analyzed. Our results suggest that BE1 has no apparent function for the synthesis of starch in the leaves, as both be1 be2 and be1 be3 double mutants display the same phenotype as be2 and be3 separately. However, starch synthesis was abolished in be2 be3, while high levels of α-maltose were assayed in the cytosol. This result indicates that the functions of both BE2 and BE3, which belong to class II starch branching enzymes, are largely redundant in Arabidopsis. Moreover, we demonstrate that maltose accumulation depends on the presence of an active ADP-glucose pyrophosphorylase and that the cytosolic transglucosidase DISPROPORTIONATING ENZYME2, required for maltose metabolization, is specific for β-maltose.