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To uncover components of the mechanism that adjusts the rate of leaf starch degradation to the length of the night, we devised a screen for mutant Arabidopsis thaliana plants in which starch reserves are prematurely exhausted. The mutation in one such mutant, named early starvation1 (esv1), eliminates a previously uncharacterized protein. Starch in mutant leaves is degraded rapidly and in a nonlinear fashion, so that reserves are exhausted 2 h prior to dawn. The ESV1 protein and a similar uncharacterized Arabidopsis protein (named Like ESV1 [LESV]) are located in the chloroplast stroma and are also bound into starch granules. The region of highest similarity between the two proteins contains a series of near-repeated motifs rich in tryptophan. Both proteins are conserved throughout starch-synthesizing organisms, from angiosperms and monocots to green algae. Analysis of transgenic plants lacking or overexpressing ESV1 or LESV, and of double mutants lacking ESV1 and another protein necessary for starch degradation, leads us to propose that these proteins function in the organization of the starch granule matrix. We argue that their misexpression affects starch degradation indirectly, by altering matrix organization and, thus, accessibility of starch polymers to starch-degrading enzymes. [ABSTRACT FROM AUTHOR]

The branched glucans glycogen and starch are the most widespread storage carbohydrates in living organisms. The production of semicrystalline starch granules in plants is more complex than that of small, soluble glycogen particles in microbes and animals. However, the factors determining whether glycogen or starch is formed are not fully understood. The tropical tree Cecropia peltata is a rare example of an organism able to make either polymer type. Electron micrographs and quantitative measurements show that glycogen accumulates to very high levels in specialized myrmecophytic structures (Müllerian bodies), whereas starch accumulates in leaves. Compared with polymers comprising leaf starch, glycogen is more highly branched and has shorter branches—factors that prevent crystallization and explain its solubility. RNA sequencing and quantitative shotgun proteomics reveal that isoforms of all three classes of glucan biosynthetic enzyme (starch/glycogen synthases, branching enzymes, and debranching enzymes) are differentially expressed in Müllerian bodies and leaves, providing a system-wide view of the quantitative programming of storage carbohydrate metabolism. This work will prompt targeted analysis in model organisms and cross-species comparisons. Finally, as starch is the major carbohydrate used for food and industrial applications worldwide, these data provide a basis for manipulating starch biosynthesis in crops to synthesize tailor-made polyglucans. [ABSTRACT FROM AUTHOR]

Starch is the major storage carbohydrate in plants. It is comprised of glucans that form semicrystalline granules. Glucan phosphorylation is a prerequisite for normal starch breakdown, but phosphoglucan metabolism is not understood. A putative protein phosphatase encoded at the Starch Excess 4 (SEX4) locus of Arabidopsis thaliana was recently shown to be required for normal starch breakdown. Here, we show that SEX4 is a phosphoglucan phosphatase in vivo and define its role within the starch degradation pathway. SEX4 dephosphorylates both the starch granule surface and soluble phosphoglucans in vitro, and sex4 null mutants accumulate phosphorylated intermediates of starch breakdown. These compounds are linear α-1,4-glucans esterified with one or two phosphate groups. They are released from starch granules by the glucan hydrolases α-amylase and isoamylase. In vitro experiments show that the rate of starch granule degradation is increased upon simultaneous phosphorylation and dephosphorylation of starch. We propose that glucan phosphorylating enzymes and phosphoglucan phosphatases work in synergy with glucan hydrolases to mediate efficient starch catabolism. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

This work investigated the roles of 13-amylases in the breakdown of leaf starch. Of the nine β-amylase (BAM)-like proteins encoded in the Arabidopsis thaliana genome, at least four (BAM1, -2, -3, and -4) are chloroplastic. When expressed as recombinant proteins in Escherichia coli, BAM1, BAM2, and BAM3 had measurable β-amylase activity but BAM4 did not. BAM4 has multiple amino acid substitutions relative to characterized β-amylases, including one of the two catalytic residues. Modeling predicts major differences between the glucan binding site of BAM4 and those of active β-amylases. Thus, BAM4 probably lost its catalytic capacity during evolution. Total β-amylase activity was reduced in leaves of bam1 and bam3 mutants but not in bam2 and bam4 mutan's. The bam3 mutant had elevated starch levels and lower nighttime maltose levels than the wild type, whereas bam1 did not. However, the bam1 bam3 double mutant had a more severe phenotype than bam3, suggesting functional overlap between the two proteins. Surprisingly, bam4 mutants had elevated, starch levels. Introduction of the bam4 mutation into the bam3 and bam1 bam3 backgrounds further elevated the starch levels in both cases. These data suggest that BAM4 facilitates or regulates starch breakdown and operates independently of BAM1 and BAM3. Together, our findings are consistent with the proposal that β3-amylase is a major enzyme of starch breakdown in leaves, but they reveal unexpected complexity in terms of the specialization of protein function. [ABSTRACT FROM AUTHOR]

In Arabidopsis ( Arabidopsis thaliana ) leaves, starch is synthesized during the day and degraded at night to fuel growth and metabolism. Starch is degraded primarily by β-amylases, liberating maltose, but this activity is preceded by glucan phosphorylation and is accompanied by dephosphorylation. A glucan phosphatase family member, LIKE SEX4 1 (LSF1), binds starch and is required for normal starch degradation, but its exact role is unclear. Here, we show that LSF1 does not dephosphorylate glucans. The recombinant dual specificity phosphatase (DSP) domain of LSF1 had no detectable phosphatase activity. Furthermore, a variant of LSF1 mutated in the catalytic cysteine of the DSP domain complemented the starch-excess phenotype of the lsf1 mutant. By contrast, a variant of LSF1 with mutations in the carbohydrate binding module did not complement lsf1 Thus, glucan binding, but not phosphatase activity, is required for the function of LSF1 in starch degradation. LSF1 interacts with the β-amylases BAM1 and BAM3, and the BAM1-LSF1 complex shows amylolytic but not glucan phosphatase activity. Nighttime maltose levels are reduced in lsf1 , and genetic analysis indicated that the starch-excess phenotype of lsf1 is dependent on bam1 and bam3 We propose that LSF1 binds β-amylases at the starch granule surface, thereby promoting starch degradation., (© 2019 American Society of Plant Biologists. All rights reserved.)

This work investigated the roles of beta-amylases in the breakdown of leaf starch. Of the nine beta-amylase (BAM)-like proteins encoded in the Arabidopsis thaliana genome, at least four (BAM1, -2, -3, and -4) are chloroplastic. When expressed as recombinant proteins in Escherichia coli, BAM1, BAM2, and BAM3 had measurable beta-amylase activity but BAM4 did not. BAM4 has multiple amino acid substitutions relative to characterized beta-amylases, including one of the two catalytic residues. Modeling predicts major differences between the glucan binding site of BAM4 and those of active beta-amylases. Thus, BAM4 probably lost its catalytic capacity during evolution. Total beta-amylase activity was reduced in leaves of bam1 and bam3 mutants but not in bam2 and bam4 mutants. The bam3 mutant had elevated starch levels and lower nighttime maltose levels than the wild type, whereas bam1 did not. However, the bam1 bam3 double mutant had a more severe phenotype than bam3, suggesting functional overlap between the two proteins. Surprisingly, bam4 mutants had elevated starch levels. Introduction of the bam4 mutation into the bam3 and bam1 bam3 backgrounds further elevated the starch levels in both cases. These data suggest that BAM4 facilitates or regulates starch breakdown and operates independently of BAM1 and BAM3. Together, our findings are consistent with the proposal that beta-amylase is a major enzyme of starch breakdown in leaves, but they reveal unexpected complexity in terms of the specialization of protein function.