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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.

Arabidopsis proton gradient regulation (pgr) mutants have high chlorophyll fluorescence and reduced non-photochemical quenching (NPQ) caused by defects in photosynthetic electron transport. Here, we identify PGR6 as the chloroplast lipid droplet (plastoglobule, PG) kinase ABC1K1 (activity of bc1 complex kinase 1). The members of the ABC1/ADCK/UbiB family of atypical kinases regulate ubiquinone synthesis in bacteria and mitochondria, and impact various metabolic pathways in plant chloroplasts. Here, we demonstrate that abc1k1 has a unique photosynthetic and metabolic phenotype that is distinct from that of the abc1k3 homolog. The abc1k1/pgr6 single mutant is specifically deficient in the electron carrier plastoquinone, as well as in ß–carotene and the xanthophyll lutein, and is defective in membrane antioxidant tocopherol metabolism. After 2 days of continuous high light stress, abc1k1/pgr6 plants suffer extensive photosynthetic and metabolic perturbations, strongly affecting carbohydrate metabolism. Remarkably, however, the mutant acclimates to high light after 7 days together with a recovery of carotenoid levels and a drastic alteration in the starch-to-sucrose ratio. Moreover, ABC1K1 behaves as an active kinase and phosphorylates VTE1, a key enzyme of tocopherol (vitamin E) metabolism in vitro. Our results indicate that the ABC1K1 kinase constitutes a new type of regulatory link between photosynthetic activity and chloroplast metabolism.

A putative phosphatase, LSF1 (for LIKE SEX4; previously PTPKIS2), is closely related in sequence and structure to STARCH-EXCESS4 (SEX4), an enzyme necessary for the removal of phosphate groups from starch polymers during starch degradation in Arabidopsis (Arabidopsis thaliana) leaves at night. We show that LSF1 is also required for starch degradation: lsf1 mutants, like sex4 mutants, have substantially more starch in their leaves than wild-type plants throughout the diurnal cycle. LSF1 is chloroplastic and is located on the surface of starch granules. lsf1 and sex4 mutants show similar, extensive changes relative to wild-type plants in the expression of sugar-sensitive genes. However, although LSF1 and SEX4 are probably both involved in the early stages of starch degradation, we show that LSF1 neither catalyzes the same reaction as SEX4 nor mediates a sequential step in the pathway. Evidence includes the contents and metabolism of phosphorylated glucans in the single mutants. The sex4 mutant accumulates soluble phospho-oligosaccharides undetectable in wild-type plants and is deficient in a starch granule-dephosphorylating activity present in wild-type plants. The lsf1 mutant displays neither of these phenotypes. The phenotype of the lsf1/sex4 double mutant also differs from that of both single mutants in several respects. We discuss the possible role of the LSF1 protein in starch degradation.

In most plants, a large fraction of photo-assimilated carbon is stored in the chloroplasts during the day as starch and remobilized during the subsequent night to support metabolism. Mutations blocking either starch synthesis or starch breakdown in Arabidopsis thaliana reduce plant growth. Maltose is the major product of starch breakdown exported from the chloroplast at night. The maltose excess 1 mutant (mex1), which lacks the chloroplast envelope maltose transporter, accumulates high levels of maltose and starch in chloroplasts and develops a distinctive but previously unexplained chlorotic phenotype as leaves mature. The introduction of additional mutations that prevent starch synthesis, or that block maltose production from starch, also prevent chlorosis of mex1. In contrast, introduction of mutations in disproportionating enzyme (DPE1) results in the accumulation of maltotriose in addition to maltose, and greatly increases chlorosis. These data suggest a link between maltose accumulation and chloroplast homeostasis. Microscopic analyses show that the mesophyll cells in chlorotic mex1 leaves have fewer than half the number of chloroplasts than wild-type cells. Transmission electron microscopy reveals autophagy-like chloroplast degradation in both mex1 and the dpe1/mex1 double mutant. Microarray analyses reveal substantial reprogramming of metabolic and cellular processes, suggesting that organellar protein turnover is increased in mex1, though leaf senescence and senescence-related chlorophyll catabolism are not induced. We propose that the accumulation of maltose and malto-oligosaccharides causes chloroplast dysfunction, which may by signaled via a form of retrograde signaling and trigger chloroplast degradation.

This work investigated the roles of β-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 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 β-amylase is a major enzyme of starch breakdown in leaves, but they reveal unexpected complexity in terms of the specialization of protein function.

The aim of this article is to provide an overview of current models of starch breakdown in leaves. We summarise the results of our recent work focusing on Arabidopsis, relating them to other work in the field. Early biochemical studies of starch containing tissues identified numerous enzymes capable of participating in starch degradation. In the non-living endosperms of germinated cereal seeds, starch breakdown proceeds by the combined actions of α-amylase, limit dextrinase (debranching enzyme), β-amylase and α-glucosidase. The activities of these enzymes and the regulation of some of the respective genes on germination have been extensively studied. In living plant cells, additional enzymes are present, such as α-glucan phosphorylase and disproportionating enzyme, and the major pathway of starch breakdown appears to differ from that in the cereal endosperm in some important aspects. For example, reverse-genetic studies of Arabidopsis show that α-amylase and limit-dextrinase play minor roles and are dispensable for starch breakdown in leaves. Current data also casts doubt on the involvement of α-glucosidase. In contrast, several lines of evidence point towards a major role for β-amylase in leaves, which functions together with disproportionating enzyme and isoamylase (debranching enzyme) to produce maltose and glucose. Furthermore, the characterisation of Arabidopsis mutants with elevated leaf starch has contributed to the discovery of previously unknown proteins and metabolic steps in the pathway. In particular, it is now apparent that glucan phosphorylation is required for normal rates of starch mobilisation to occur, although a detailed understanding of this step is still lacking. We use this review to give a background to some of the classical genetic mutants that have contributed to our current knowledge.