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 F(2) 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.     

Function and characterization of starch synthase I using mutants in rice

Four starch synthase I (SSI)-deficient rice (Oryza sativa) mutant lines were generated using retrotransposon Tos17 insertion. The mutants exhibited different levels of SSI activities and produced significantly lower amounts of SSI protein ranging from 0% to 20% of the wild type. The mutant endosperm amylopectin showed a decrease in chains with degree of polymerization (DP) 8 to 12 and an increase in chains with DP 6 to 7 and DP 16 to 19. The degree of change in amylopectin chain-length distribution was positively correlated with the extent of decrease in SSI activity in the mutants. The structural changes in the amylopectin increased the gelatinization temperature of endosperm starch. Chain-length analysis of amylopectin in the SSI band excised from native-polyacrylamide gel electrophoresis/SS activity staining gel showed that SSI preferentially synthesized DP 7 to 11 chains by elongating DP 4 to 7 short chains of glycogen or amylopectin. These results show that SSI distinctly generates DP 8 to 12 chains from short DP 6 to 7 chains emerging from the branch point in the A or B(1) chain of amylopectin. SSI seemingly functions from the very early through the late stage of endosperm development. Yet, the complete absence of SSI, despite being a major SS isozyme in the developing endosperm, had no effect on the size and shape of seeds and starch granules and the crystallinity of endosperm starch, suggesting that other SS enzymes are probably capable of partly compensating SSI function. In summary, this study strongly suggested that amylopectin chains are synthesized by the coordinated actions of SSI, SSIIa, and SSIIIa isoforms.     

Effects of starch synthase IIa gene dosage on grain, protein and starch in endosperm of wheat

Starch synthases (SS) are responsible for elongating the alpha-1,4 glucan chains of starch. A doubled haploid population was generated by crossing a line of wheat, which lacks functional ssIIa genes on each genome (abd), and an Australian wheat cultivar, Sunco, with wild type ssIIa alleles on each genome (ABD). Evidence has been presented previously indicating that the SGP-1 (starch granule protein-1) proteins present in the starch granule in wheat are products of the ssIIa genes. Analysis of 100 progeny lines demonstrated co-segregation of the ssIIa alleles from the three genomes with the SGP-1 proteins, providing further evidence that the SGP-1 proteins are the products of the ssIIa genes. From the progeny lines, 40 doubled haploid lines representing the eight possible genotypes for SSIIa (ABD, aBD, AbD, ABd, abD, aBd, Abd, abd) were characterized for their grain weight, protein content, total starch content and starch properties. For some properties (chain length distribution, pasting properties, swelling power, and gelatinization properties), a progressive change was observed across the four classes of genotypes (wild type, single nulls, double nulls and triple nulls). However, for other grain properties (seed weight and protein content) and starch properties (total starch content, granule morphology and crystallinity, granule size distribution, amylose content, amylose-lipid dissociation properties), a statistically significant change only occurred for the triple nulls, indicating that all three genes had to be missing or inactive for a change to occur. These results illustrate the importance of SSIIa in controlling grain and starch properties and the importance of amylopectin fine structure in controlling starch granule properties in wheat.     

Evidence for distinct mechanisms of starch granule breakdown in plants

The aim of this work was to understand the initial steps of starch breakdown inside chloroplasts. In the non-living endosperm of germinating cereal grains, starch breakdown is initiated by alpha-amylase secreted from surrounding cells. However, loss of alpha-amylase from Arabidopsis does not prevent chloroplastic starch breakdown (Yu, T.-S., Zeeman, S. C., Thorneycroft, D., Fulton, D. C., Dunstan, H., Lue, W.-L., Hegemann, B., Tung, S.-Y., Umemoto, T., Chapple, A., Tsai, D.-L., Wang, S.-M, Smith, A. M., Chen, J., and Smith, S. M. (2005) J. Biol. Chem. 280, 9773-9779), implying that other enzymes must attack the starch granule. Here, we present evidence that the debranching enzyme isoamylase 3 (ISA3) acts at the surface of the starch granule. Atisa3 mutants have more leaf starch and a slower rate of starch breakdown than wild-type plants. The amylopectin of Atisa3 contains many very short branches and ISA3-GFP localizes to granule-like structures inside chloroplasts. We suggest that ISA3 removes short branches from the granule surface. To understand how some starch is still degraded in Atisa3 mutants we eliminated a second debranching enzyme, limit dextrinase (pullulanase-type). Atlda mutants are indistinguishable from the wild type. However, the Atisa3/Atlda double mutant has a more severe starch-excess phenotype and a slower rate of starch breakdown than Atisa3 single mutants. The double mutant accumulates soluble branched oligosaccharides (limit dextrins) that are undetectable in the wild-type and the single mutants. Together these results suggest that glucan debranching occurs primarily at the granule surface via ISA3, but in its absence soluble branched glucans are debranched in the stroma via limit dextrinase. Consistent with this model, chloroplastic alpha-amylase AtAMY3, which could release soluble branched glucans, is induced in Atisa3 and in the Atisa3/Atlda double mutant.     

Chain-length specificities of maize starch synthase I enzyme: studies of glucan affinity and catalytic properties

It is widely known that some of the starch synthases and starch-branching enzymes are trapped inside the starch granule matrix during the course of starch deposition in amyloplasts. The objective of this study was to use maize SSI to further our understanding of the protein domains involved in starch granule entrapment and identify the chain-length specificities of the enzyme. Using affinity gel electrophoresis, we measured the dissociation constants of maize SSI and its truncated forms using various glucans. The enzyme has a high degree of specificity in terms of its substrate-enzyme dissociation constant, but has a greatly elevated affinity for increasing chain lengths of alpha-1, 4 glucans. Deletion of the N-terminal arm of SSI did not affect the Kd value. Further small deletions of either N- or C-terminal domains resulted in a complete loss of any measurable affinity for its substrate, suggesting that the starch-affinity domain of SSI is not discrete from the catalytic domain. Greater affinity was displayed for the amylopectin fraction of starch as compared to amylose, whereas glycogen revealed the lowest affinity. However, when the outer chain lengths (OCL) of glycogen were extended using the phosphorylase enzyme, we found an increase in affinity for SSI between an average OCL of 7 and 14, and then an apparently exponential increase to an average OCL of 21. On the other hand, the catalytic ability of SSI was reduced several-fold using these glucans with extended chain lengths as substrates, and most of the label from [14C]ADPG was incorporated into shorter chains of dp < 10. We conclude that the rate of catalysis of SSI enzyme decreases with the OCL of its glucan substrate, and it has a very high affinity for the longer glucan chains of dp approximately 20, rendering the enzyme catalytically incapable at longer chain lengths. Based on the observations in this study, we propose that during amylopectin synthesis shorter A and B1 chains are extended by SSI up to a critical chain length that soon becomes unsuitable for catalysis by SSI and hence cannot be elongated further by this enzyme. Instead, SSI is likely to become entrapped as a relatively inactive protein within the starch granule. Further glucan extension for continuation of amylopectin synthesis must require a handover to other SS enzymes which can extend the glucan chains further or for branching by branching enzymes. If this is correct, this proposal provides a biochemical basis to explain how the specificities of various SS enzymes determine and set the limitations on the length of A, B, C chains in the starch granule.     

Degradation of Glucan Primers in the Absence of Starch Synthase 4 Disrupts Starch Granule Initiation in Arabidopsis

Arabidopsis leaf chloroplasts typically contain five to seven semicrystalline starch granules. It is not understood how the synthesis of each granule is initiated or how starch granule number is determined within each chloroplast. An Arabidopsis mutant lacking the glucosyl-transferase, STARCH SYNTHASE 4 (SS4) is impaired in its ability to initiate starch granules; its chloroplasts rarely contain more than one large granule, and the plants have a pale appearance and reduced growth. Here we report that the chloroplastic α-amylase AMY3, a starch-degrading enzyme, interferes with granule initiation in the ss4 mutant background. The amy3 single mutant is similar in phenotype to the wild type under normal growth conditions, with comparable numbers of starch granules per chloroplast. Interestingly, the ss4 mutant displays a pleiotropic reduction in the activity of AMY3. Remarkably, complete abolition of AMY3 (in the amy3 ss4 double mutant) increases the number of starch granules produced in each chloroplast, suppresses the pale phenotype of ss4, and nearly restores normal growth. The amy3 mutation also restores starch synthesis in the ss3 ss4 double mutant, which lacks STARCH SYNTHASE 3 (SS3) in addition to SS4. The ss3 ss4 line is unable to initiate any starch granules and is thus starchless. We suggest that SS4 plays a key role in granule initiation, allowing it to proceed in a way that avoids premature degradation of primers by starch hydrolases, such as AMY3.     

Physico-chemical and degradative properties of in-planta re-structured potato starch

Starch re-structured directly in potato tubers by antisense suppression of starch branching enzyme (SBE), granule bound starch synthase (GBSS) or glucan water dikinase (GWD) genes was studied with the aim at disclosing the effects on resulting physico-chemical and enzyme degradative properties. The starches were selected to provide a combined system with specific and extensive alterations in amylose and covalently esterified glucose-6-phosphate (G6P) contents. As an effect of the altered chemical composition of the starches their hydrothermal characteristics varied significantly. Despite of the extreme alterations in phosphate content, the amylose content had a major affect on swelling power, enthalpy for starch gelatinization and pasting parameters as assessed by Rapid Visco Analysis (RVA). However, a combined influence of the starch phosphate and long glucan chains as represented by high amylose or long amylopectin chain length was indicated by their positive correlation to the final viscosity and set back (RVA) demonstrating the formation of a highly hydrated and gel-forming system during re-structuring of the starch pastes. Clear inverse correlations between glucoamylase-catalyzed digestibility and amylopectin chain length and starch phosphate and lack of such correlation with amylose content indicates a combined structuring role of the phosphate groups and amylopectin chains on the starch glucan matrix.     

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

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

Starch biosynthesis in cereal endosperm

Stored starch generally consists of two d-glucose homopolymers, the linear polymer amylose and a highly branched glucan amylopectin that connects linear chains. Amylopectin structurally contributes to the crystalline organization of the starch granule in cereals. In the endosperm, amylopectin biosynthesis requires the proper execution of a coordinated series of enzymatic reactions involving ADP glucose pyrophosphorylase (AGPase), soluble starch synthase (SS), starch branching enzyme (BE), and starch debranching enzyme (DBE), whereas amylose is synthesized by AGPase and granule-bound starch synthase (GBSS). It is highly possible that plastidial starch phosphorylase (Pho1) plays an important role in the formation of primers for starch biosynthesis in the endosperm. Recent advances in our understanding of the functions of individual enzyme isoforms have provided new insights into how linear polymer chains and branch linkages are synthesized in cereals. In particular, genetic analyses of a suite of mutants have formed the basis of a new model outlining the role of various enzyme isoforms in cereal starch production. In our current review, we summarize the recent research findings related to starch biosynthesis in cereal endosperm, with a particular focus on rice.     

Cas9‐mediated mutagenesis of potato starch‐branching enzymes generates a range of tuber starch phenotypes

We investigated whether Cas9‐mediated mutagenesis of starch‐branching enzymes (SBEs) in tetraploid potatoes could generate tuber starches with a range of distinct properties. Constructs containing the Cas9 gene and sgRNAs targeting SBE1, SBE2 or both genes were introduced by Agrobacterium‐mediated transformation or by PEG‐mediated delivery into protoplasts. Outcomes included lines with mutations in all or only some of the homoeoalleles of SBE genes and lines in which homoeoalleles carried several different mutations. DNA delivery into protoplasts resulted in mutants with no detectable Cas9 gene, suggesting the absence of foreign DNA. Selected mutants with starch granule abnormalities had reductions in tuber SBE1 and/or SBE2 protein that were broadly in line with expectations from genotype analysis. Strong reduction in both SBE isoforms created an extreme starch phenotype, as reported previously for low‐SBE potato tubers. HPLC‐SEC and ¹H NMR revealed a decrease in short amylopectin chains, an increase in long chains and a large reduction in branching frequency relative to wild‐type starch. Mutants with strong reductions in SBE2 protein alone had near‐normal amylopectin chain‐length distributions and only small reductions in branching frequency. However, starch granule initiation was enormously increased: cells contained many granules of <4 μm and granules with multiple hila. Thus, large reductions in both SBEs reduce amylopectin branching during granule growth, whereas reduction in SBE2 alone primarily affects numbers of starch granule initiations. Our results demonstrate that Cas9‐mediated mutagenesis of SBE genes has the potential to generate new, potentially valuable starch properties without integration of foreign DNA into the genome.     

Mutations affecting starch synthase III in Arabidopsis alter leaf starch structure and increase the rate of starch synthesis

The role of starch synthase (SS) III (SSIII) in the synthesis of transient starch in Arabidopsis (Arabidopsis thaliana) was investigated by characterizing the effects of two insertion mutations at the AtSS3 gene locus. Both mutations, termed Atss3-1 and Atss3-2, condition complete loss of SSIII activity and prevent normal gene expression at both the mRNA and protein levels. The mutations cause a starch excess phenotype in leaves during the light period of the growth cycle due to an apparent increase in the rate of starch synthesis. In addition, both mutations alter the physical structure of leaf starch. Significant increases were noted in the mutants in the frequency of linear chains in amylopectin with a degree of polymerization greater than approximately 60, and relatively small changes were observed in chains of degree of polymerization 4 to 50. Furthermore, starch in the Atss3-1 and Atss3-2 mutants has a higher phosphate content, approximately two times that of wild-type leaf starch. Total SS activity is increased in both Atss3 mutants and a specific SS activity appears to be up-regulated. The data indicate that, in addition to its expected direct role in starch assembly, SSIII also has a negative regulatory function in the biosynthesis of transient starch in Arabidopsis.     

Role of granule-bound starch synthase in determination of amylopectin structure and starch granule morphology in potato.

Reductions in activity of SSIII, the major isoform of starch synthase responsible for amylopectin synthesis in the potato tuber, result in fissuring of the starch granules. To discover the causes of the fissuring, and thus to shed light on factors that influence starch granule morphology in general, SSIII antisense lines were compared with lines with reductions in the major granule-bound isoform of starch synthase (GBSS) and lines with reductions in activity of both SSIII and GBSS (SSIII/GBSS antisense lines). This revealed that fissuring resulted from the activity of GBSS in the SSIII antisense background. Control (untransformed) lines and GBSS and SSIII/GBSS antisense lines had unfissured granules. Starch analyses showed that granules from SSIII antisense tubers had a greater number of long glucan chains than did granules from the other lines, in the form of larger amylose molecules and a unique fraction of very long amylopectin chains. These are likely to result from increased flux through GBSS in SSIII antisense tubers, in response to the elevated content of ADP-glucose in these tubers. It is proposed that the long glucan chains disrupt organization of the semi-crystalline parts of the matrix, setting up stresses in the matrix that lead to fissuring.     

The phosphoglucan phosphatase like sex Four2 dephosphorylates starch at the C3-position in Arabidopsis

Starch contains phosphate covalently bound to the C6-position (70 to 80% of total bound phosphate) and the C3-position (20 to 30%) of the glucosyl residues of the amylopectin fraction. In plants, the transient phosphorylation of starch renders the granule surface more accessible to glucan hydrolyzing enzymes and is required for proper starch degradation. Phosphate also confers desired properties to starch-derived pastes for industrial applications. In Arabidopsis thaliana, the removal of phosphate by the glucan phosphatase Starch Excess4 (SEX4) is essential for starch breakdown. We identified a homolog of SEX4, LSF2 (Like Sex Four2), as a novel enzyme involved in starch metabolism in Arabidopsis chloroplasts. Unlike SEX4, LSF2 does not have a carbohydrate binding module. Nevertheless, it binds to starch and specifically hydrolyzes phosphate from the C3-position. As a consequence, lsf2 mutant starch has elevated levels of C3-bound phosphate. SEX4 can release phosphate from both the C6- and the C3-positions, resulting in partial functional overlap with LSF2. However, compared with sex4 single mutants, the lsf2 sex4 double mutants have a more severe starch-excess phenotype, impaired growth, and a further change in the proportion of C3- and C6-bound phosphate. These findings significantly advance our understanding of the metabolism of phosphate in starch and provide innovative options for tailoring novel starches with improved functionality for industry.     

A critical role for disproportionating enzyme in starch breakdown is revealed by a knock-out mutation in Arabidopsis

Disproportionating enzyme (D-enzyme) is a plastidial alpha-1,4-glucanotransferase but its role in starch metabolism is unclear. Using a reverse genetics approach we have isolated a mutant of Arabidopsis thaliana in which the gene encoding this enzyme (DPE1) is disrupted by a T-DNA insertion. While D-enzyme activity is eliminated in the homozygous dpe1-1 mutant, changes in activities of other enzymes of starch metabolism are relatively small. During the diurnal cycle, the amount of leaf starch is higher in dpe1-1 than in wild type and the amylose to amylopectin ratio is increased, but amylopectin structure is unaltered. The amounts of starch synthesised and degraded are lower in dpe1-1 than in wild type. However, the lower amount of starch synthesised and the higher proportion of amylose are both eliminated when plants are completely de-starched by a period of prolonged darkness prior to the light period. During starch degradation, a large accumulation of malto-oligosaccharides occurs in dpe1-1 but not in wild type. These data show that D-enzyme is required for malto-oligosaccharide metabolism during starch degradation. The slower rate of starch degradation in dpe1-1 suggests that malto-oligosaccharides affect an enzyme that attacks the starch granule, or that D-enzyme itself can act directly on starch. The effects on starch synthesis and composition in dpe1-1 under normal diurnal conditions are probably a consequence of metabolism at the start of the light period, of the high levels of malto-oligosaccharides generated during the dark period. We conclude that the primary function of D-enzyme is in starch degradation.     

Intermediary glucan structures formed during starch granule biosynthesis are enriched in short side chains, a dynamic pulse labeling approach

The formation of intermediary glucans, mature starch, and phytoglycogen was studied using leaves of Arabidopsis thaliana wild type and dbe mutant, which lacks plastidic isoamylase (Zeeman, S. C., Umemoto, T., Lue, W. L., Au-Yeung, P., Martin, C., Smith, A. M., and Chen, J. (1998) Plant Cell 10, 1699-1711). A new approach to the study of starch biosynthesis was developed based on "very short pulse" labeling of leaf starch through photosynthetic fixation of (14)CO(2). This allowed selective analysis of the structure of starch formed within a 30-s period. This time frame is shorter than the period required for the formation of a single crystalline amylopectin lamella and consequently permits a direct analysis of intermediary structures during granule formation. Analysis of chain length distribution showed that the most recently formed outer layer of the granules has a structure different from the mature starch. The outer layer is enriched in short chains that are 6-11 glucose residues long. Side chains with 6 glucose residues are the shortest abundant chains formed, and they are formed exclusively by transfer from donor chains of 12 glucose residues or longer. The labeling pattern shows that chain transfer resulting in branching is a rapid and efficient process, and the preferential labeling of shorter chains in the intermediary granule bound glucan is suggested to be a direct consequence of efficient branching. Although similar, the short chain intermediary structure is not identical to phytoglycogen, which is an even more highly branched molecule with very few longer chains (more than 40 glucose residues). Pulse and chase labeling profiles for the dbe mutant showed that the final structure is more highly branched than the intermediary structures, which implies that branching of phytoglycogen occurs over a longer time period than branching of starch.     

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.     

Starch Granule Biosynthesis in Arabidopsis Is Abolished by Removal of All Debranching Enzymes but Restored by the Subsequent Removal of an Endoamylase

Several studies have suggested that debranching enzymes (DBEs) are involved in the biosynthesis of amylopectin, the major constituent of starch granules. Our systematic analysis of all DBE mutants of Arabidopsis thaliana demonstrates that when any DBE activity remains, starch granules are still synthesized, albeit with altered amylopectin structure. Quadruple mutants lacking all four DBE proteins (Isoamylase1 [ISA1], ISA2, and ISA3, and Limit-Dextrinase) are devoid of starch granules and instead accumulate highly branched glucans, distinct from amylopectin and from previously described phytoglycogen. A fraction of these glucans are present as discrete, insoluble, nanometer-scale particles, but the structure and properties of this material are radically altered compared with wild-type amylopectin. Superficially, these data support the hypothesis that debranching is required for amylopectin synthesis. However, our analyses show that soluble glucans in the quadruple DBE mutant are degraded by α- and β-amylases during periods of net accumulation, giving rise to maltose and branched malto-oligosaccharides. The additional loss of the chloroplastic α-amylase AMY3 partially reverts the phenotype of the quadruple DBE mutant, restoring starch granule biosynthesis. We propose that DBEs function in normal amylopectin synthesis by promoting amylopectin crystallization but conclude that they are not mandatory for starch granule synthesis.     

Loss of Starch Granule Initiation Has a Deleterious Effect on the Growth of Arabidopsis Plants Due to an Accumulation of ADP-Glucose

STARCH SYNTHASE4 (SS4) is required for proper starch granule initiation in Arabidopsis (Arabidopsis thaliana), although SS3 can partially replace its function. Unlike other starch-deficient mutants, ss4 and ss3/ss4 mutants grow poorly even under long-day conditions. They have less chlorophyll and carotenoids than the wild type and lower maximal rates of photosynthesis. There is evidence of photooxidative damage of the photosynthetic apparatus in the mutants from chlorophyll a fluorescence parameters and their high levels of malondialdehyde. Metabolite profiling revealed that ss3/ss4 accumulates over 170 times more ADP-glucose (Glc) than wild-type plants. Restricting ADP-Glc synthesis, by introducing mutations in the plastidial phosphoglucomutase (pgm1) or the small subunit of ADP-Glc pyrophosphorylase (aps1), largely restored photosynthetic capacity and growth in pgm1/ss3/ss4 and aps1/ss3/ss4 triple mutants. It is proposed that the accumulation of ADP-Glc in the ss3/ss4 mutant sequesters a large part of the plastidial pools of adenine nucleotides, which limits photophosphorylation, leading to photooxidative stress, causing the chlorotic and stunted growth phenotypes of the plants.The metabolism of starch plays an essential role in the physiology of plants. Starch breakdown provides the plant with carbon skeletons and energy when the photosynthetic machinery is inactive (transitory starch) or in the processes of germination and sprouting (storage starch). Deficiencies in the accumulation of transitory starch in Arabidopsis (Arabidopsis thaliana) have been described previously, specifically in mutants affected in the plastidial phosphoglucomutase (PGM1) or the small subunit (APS1) of the ADP-Glc pyrophosphorylase (AGPase). While they are described as “starchless,” they actually contain small amounts of starch (1%–2% of the wild-type levels; Streb et al., 2009) and share similar phenotypic alterations, such as growth retardation when cultivated under a short-day photoregime and increased levels of soluble sugars during the light phase and reduced levels during the night (Caspar et al., 1985; Lin et al., 1988b; Schulze et al., 1991). Carbon partitioning is altered in these plants. As photosynthate cannot be accumulated as starch, it is diverted via hexose phosphates in the cytosol to the synthesis of Suc, which accumulates together with the hexose sugars, Glc and Fru (Caspar et al., 1985). In Arabidopsis, there are five starch synthase isoforms: one granule-bound starch synthase and four soluble starch synthases: SS1, SS2, SS3, and SS4. We have described previously an Arabidopsis mutant plant lacking SS3 and SS4 that is also severely affected in the accumulation of starch (Szydlowski et al., 2009). SS4 is involved in the initiation of the starch granule and controls the number of granules per chloroplast (Roldán et al., 2007). The elimination of SS3 in an ss4 background leads to an absence of starch in most of the chloroplasts, despite the fact that SS1 and SS2 are still present and total starch synthase activity is only reduced by 35% (Szydlowski et al., 2009). However, a very small proportion of chloroplasts of this mutant plant contain a single huge starch granule, which is also a characteristic of chloroplasts in the ss4 single mutant (D’Hulst and Mérida, 2012). Thus, like aps1 and pgm1, ss3/ss4 plants contain only small amounts of starch. However, unlike aps1 or pgm1 plants, most of the cells of this mutant have empty chloroplasts, without starch (Szydlowski et al., 2009).In this work, we have analyzed the phenotypic effects of the impaired starch accumulation of ss3/ss4 plants. We show that this mutant displays phenotypic changes that are not found in other mutants with very low levels of starch, such as aps1 or pgm1 plants. We provide evidence that extremely high levels of ADP-Glc accumulate in the ss3/ss4 plants. Using reverse genetics to block the pathway of starch synthesis upstream of the starch synthases reduced the level of ADP-Glc in ss3/ss4 plants and reverted the other phenotypic traits. This suggests that ADP-Glc accumulation is the causal factor behind the chlorotic and stunted growth phenotypes of the ss3/ss4 mutant.