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Starch, a plant-derived insoluble carbohydrate composed of glucose polymers, is the principal carbohydrate in our diet and a valuable raw material for industry. The properties of starch depend on the arrangement of glucose units within the constituent polymers. However, key aspects of starch structure and the underlying biosynthetic processes are not well understood, limiting progress towards targeted improvement of our starch crops. In particular, the major component of starch, amylopectin, has a complex three-dimensional, branched architecture. This architecture stems from the combined actions of a multitude of enzymes, each having broad specificities that are difficult to capture experimentally. In this review, we reflect on experimental approaches and limitations to decipher the enzymes' specificities and explore possibilities for in silico simulations of these activities. We believe that the synergy between experimentation and simulation is needed for the correct interpretation of experimental data and holds the potential to greatly advance our understanding of the overall starch biosynthetic process. We furthermore propose that the formation of glucan secondary structures, concomitant with its synthesis, is a previously overlooked factor that directly affects amylopectin architecture through its impact on enzyme function.

Performing accurate measurements of photosynthetic and respiration rates is vital to a large proportion of plant-based studies. While several commercial systems exist to perform such measurements, few are ideal for whole-plant measurements of small herbaceous plants such as Arabidopsis and none offer the capacity for simultaneous analysis of multiple plants. We, therefore, designed a multi-chamber, computer-controlled, infrared gas analyzer-coupled system for the continuous measurement of gas exchange in whole-plant shoots or rosettes. This system was called ETH Gas Exchange System-1 (EGES-1). We have subsequently expanded the device to accommodate a wider variety of species while providing precise control over environmental parameters. Critically, we have (1) increased the flow rates through each of the eight chambers, (2) introduced a computer-controlled feedback loop for the precise introduction of CO 2 , and (3) added an additional feedback loop for the introduction and control of humidity. The advantages of this new system (EGES-2) are illustrated here in the context of a variety of physiological experiments.

The formation of normal starch granules in Arabidopsis ( Arabidopsis thaliana ) leaf chloroplasts requires STARCH SYNTHASE 4 (SS4). In plants lacking SS4, chloroplasts typically produce only one round granule rather than multiple lenticular granules. The mechanisms by which SS4 determines granule number and morphology are not understood. The N-terminal region of SS4 is unique among SS isoforms and contains several long coiled-coil motifs, typically implicated in protein-protein interactions. The C-terminal region contains the catalytic glucosyltransferase domains, which are widely conserved in plant SS and bacterial glycogen synthase (GS) isoforms. We investigated the specific roles of the N- and C-terminal regions of SS4 by expressing truncated versions of SS4 and a fusion between the N-terminal region of SS4 and GS in the Arabidopsis ss4 mutant. Expression of the N-terminal region of SS4 alone did not alter the ss4 mutant phenotype. Expression of the C-terminal region of SS4 alone increased granule initiation but did not rescue their aberrant round morphology. Expression of a self-priming GS from Agrobacterium tumefaciens also increased the number of round granules. Remarkably, fusion of the N-terminal region of SS4 to A. tumefaciens GS restored the development of wild-type-like lenticular starch granules. Interestingly, the N-terminal region of SS4 alone or when fused to GS conferred a patchy subchloroplastic localization similar to that of the full-length SS4 protein. Considered together, these data suggest that, while the glucosyltransferase activity of SS4 is important for granule initiation, the N-terminal part of SS4 serves to establish the correct granule morphology by properly localizing this activity., (© 2018 American Society of Plant Biologists. All Rights Reserved.)

Starch-rich crops form the basis of our nutrition, but plants have still to yield all their secrets as to how they make this vital substance. Great progress has been made by studying both crop and model systems, and we approach the point of knowing the enzymatic machinery responsible for creating the massive, insoluble starch granules found in plant tissues. Here, we summarize our current understanding of these biosynthetic enzymes, highlighting recent progress in elucidating their specific functions. Yet, in many ways we have only scratched the surface: much uncertainty remains about how these components function together and are controlled. We flag-up recent observations suggesting a significant degree of flexibility during the synthesis of starch and that previously unsuspected non-enzymatic proteins may have a role. We conclude that starch research is not yet a mature subject and that novel experimental and theoretical approaches will be important to advance the field.

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., (© 2016 American Society of Plant Biologists. All rights reserved.)

The major component of starch is the branched glucan amylopectin, the branching pattern of which is one of the key factors determining its ability to form semicrystalline starch granules. Here, we investigated the functions of different branching enzyme (BE) types by expressing proteins from maize (Zea mays BE2a), potato (Solanum tuberosum BE1), and Escherichia coli (glycogen BE [EcGLGB]) in Arabidopsis (Arabidopsis thaliana) mutant plants that are deficient in their endogenous BEs and therefore, cannot make starch. The expression of each of these three BE types restored starch biosynthesis to differing degrees. Full complementation was achieved using the class II BE ZmBE2a, which is most similar to the two endogenous Arabidopsis isoforms. Expression of the class I BE from potato, StBE1, resulted in partial complementation and high amylose starch. Expression of the glycogen BE EcGLGB restored only minimal amounts of starch production, which had unusual chain length distribution, branch point distribution, and granule morphology. Nevertheless, each type of BE together with the starch synthases and debranching enyzmes were able to create crystallization-competent amylopectin polymers. These data add to the knowledge of how the properties of the BE influence the final composition of starch and fine structure of amylopectin., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

Plant BZR1-BAM transcription factors contain a β-amylase (BAM)-like domain, characteristic of proteins involved in starch breakdown. The enzyme-derived domains appear to be noncatalytic, but they determine the function of the two Arabidopsis thaliana BZR1-BAM isoforms (BAM7 and BAM8) during transcriptional initiation. Removal or swapping of the BAM domains demonstrates that the BAM7 BAM domain restricts DNA binding and transcriptional activation, while the BAM8 BAM domain allows both activities. Furthermore, we demonstrate that BAM7 and BAM8 interact on the protein level and cooperate during transcriptional regulation. Site-directed mutagenesis of residues in the BAM domain of BAM8 shows that its function as a transcriptional activator is independent of catalysis but requires an intact substrate binding site, suggesting it may bind a ligand. Microarray experiments with plants overexpressing truncated versions lacking the BAM domain indicate that the pseudo-enzymatic domain increases selectivity for the preferred cis-regulatory element BBRE (BZR1-BAM Responsive Element). Side specificity toward the G-box may allow crosstalk to other signaling networks. This work highlights the importance of the enzyme-derived domain of BZR1-BAMs, supporting their potential role as metabolic sensors., (© 2014 American Society of Plant Biologists. All rights reserved.)

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.

Starch is a primary product of photosynthesis in leaves. In most plants, a large fraction of the carbon assimilated during the day is stored transiently in the chloroplast as starch for use during the subsequent night. Photosynthetic partitioning into starch is finely regulated, and the amount of carbohydrate stored is dependent on the environmental conditions, particularly day length. This regulation is applied at several levels to control the flux of carbon from the Calvin cycle into starch biosynthesis. Starch is composed primarily of branched glucans with an architecture that allows the formation of a semi-crystalline insoluble granule. Biosynthesis has been most intensively studied in non-photosynthetic starch-storing organs, such as developing seeds and tubers. Biosynthesis in leaves has received less attention, but recent reverse-genetic studies of Arabidopsis (thale cress) have produced data generally consistent with what is known for storage tissues. The pathway involves starch synthases, which elongate the glucan chains, and branching enzymes. Remarkably, enzymes that partially debranch glucans are also required for normal amylopectin synthesis. In the last decade, our understanding of starch breakdown in leaves has advanced considerably. Starch is hydrolysed to maltose and glucose at night via a pathway that requires recently discovered proteins in addition to well-known enzymes. These sugars are exported from the plastid to support sucrose synthesis, respiration and growth. In the present review we provide an overview of starch biosynthesis, starch structure and starch degradation in the leaves of plants. We focus on recent advances in each area and highlight outstanding questions.

The Arabidopsis thaliana genome encodes three alpha-amylase-like proteins (AtAMY1, AtAMY2, and AtAMY3). Only AtAMY3 has a predicted N-terminal transit peptide for plastidial localization. AtAMY3 is an unusually large alpha-amylase (93.5 kDa) with the C-terminal half showing similarity to other known alpha-amylases. When expressed in Escherichia coli, both the whole AtAMY3 protein and the C-terminal half alone show alpha-amylase activity. We show that AtAMY3 is localized in chloroplasts. The starch-excess mutant of Arabidopsis sex4, previously shown to have reduced plastidial alpha-amylase activity, is deficient in AtAMY3 protein. Unexpectedly, T-DNA knock-out mutants of AtAMY3 have the same diurnal pattern of transitory starch metabolism as the wild type. These results show that AtAMY3 is not required for transitory starch breakdown and that the starch-excess phenotype of the sex4 mutant is not caused simply by deficiency of AtAMY3 protein. Knock-out mutants in the predicted non-plastidial alpha-amylases AtAMY1 and AtAMY2 were also isolated, and these displayed normal starch breakdown in the dark as expected for extraplastidial amylases. Furthermore, all three AtAMY double knock-out mutant combinations and the triple knock-out degraded their leaf starch normally. We conclude that alpha-amylase is not necessary for transitory starch breakdown in Arabidopsis leaves.

To gain insight into the synthesis and functions of enzymes of starch metabolism in leaves of Arabidopsis L. Heynth, Affymetrix microarrays were used to analyze the transcriptome throughout the diurnal cycle. Under the conditions employed, transitory leaf starch is degraded progressively during a 12-h dark period, and then accumulates during the following 12-h light period. Transcripts encoding enzymes of starch synthesis changed relatively little in amount over 24 h except for two starch synthases, granule bound starch synthase and starch synthase II, which increased appreciably during the transition from dark to light. The increase in RNA encoding granule-bound starch synthase may reflect the extensive destruction of starch granules in the dark. Transcripts encoding several enzymes putatively involved in starch breakdown showed a coordinated decline in the dark followed by rapid accumulation in the light. Despite marked changes in their transcript levels, the amounts of some enzymes of starch metabolism do not change appreciably through the diurnal cycle. Posttranscriptional regulation is essential in the maintenance of amounts of enzymes and the control of their activities in vivo. Even though the relationships between transcript levels, enzyme activity, and diurnal metabolism of starch metabolism are complex, the presence of some distinctive diurnal patterns of transcripts for enzymes known to be involved in starch metabolism facilitates the identification of other proteins that may participate in this process.

This review describes recent progress in discovering the pathway of starch breakdown in leaves. The synthesis of starch from photo-assimilated carbon is one of the major biochemical fluxes in plants. Despite this, the pathway through which this starch is remobilized has not been defined. Numerous enzymes that could participate in starch breakdown are present in leaves, but until recently, the relative importance of each had not been determined. Through studies using model species such as Arabidopsis and potato, significant progress has now been made in determining the roles of known enzymes, and in the discovery of novel proteins necessary for breakdown. These data allow a tentative pathway for starch breakdown to be mapped out, involving hydrolysis primarily to maltose and subsequent maltose export to the cytosol. This provides a framework for complete discovery of the pathway and for the analysis of its regulation. Contents Summary 247 I. Introduction 247 II. Structure of the starch granule 248 III. Initial attack on the granule and the role of glucan, water dikinase 249 IV. Debranching of branched glucans 250 V. The metabolism of linear glucans 251 VI. Export of starch catabolites 254 VII. Metabolism of glucose and maltose 255 VIII. The emerging pathway of starch breakdown and its regulation 256 Acknowledgements 258 References 258.

Starch mobilization is well understood in cereal endosperms, but both the pathway and the regulation of the process are poorly characterized in other types of plant organs. Arabidopsis leaves offer the opportunity for rapid progress in this area, because of the genomic resources available in this species and the ease with which starch synthesis and degradation can be monitored and manipulated. Progress in understanding three aspects of starch degradation is described: the role of disproportionating enzyme, the importance of phosphorolytic degradation, and new evidence about the involvement of a starch-phosphorylating enzyme in the degradative process. Major areas requiring further research are outlined.

The aim of this work was to characterize starch synthesis, composition, and granule structure in Arabidopsis leaves. First, the potential role of starch-degrading enzymes during starch accumulation was investigated. To discover whether simultaneous synthesis and degradation of starch occurred during net accumulation, starch was labeled by supplying (14)CO(2) to intact, photosynthesizing plants. Release of this label from starch was monitored during a chase period in air, using different light intensities to vary the net rate of starch synthesis. No release of label was detected unless there was net degradation of starch during the chase. Similar experiments were performed on a mutant line (dbe1) that accumulates the soluble polysaccharide, phytoglycogen. Label was not released from phytoglycogen during the chase indicating that, even when in a soluble form, glucan is not appreciably degraded during accumulation. Second, the effect on starch composition of growth conditions and mutations causing starch accumulation was studied. An increase in starch content correlated with an increased amylose content of the starch and with an increase in the ratio of granule-bound starch synthase to soluble starch synthase activity. Third, the structural organization and morphology of Arabidopsis starch granules was studied. The starch granules were birefringent, indicating a radial organization of the polymers, and x-ray scatter analyses revealed that granules contained alternating crystalline and amorphous lamellae with a periodicity of 9 nm. Granules from the wild type and the high-starch mutant sex1 were flattened and discoid, whereas those of the high-starch mutant sex4 were larger and more rounded. These larger granules contained "growth rings" with a periodicity of 200 to 300 nm. We conclude that leaf starch is synthesized without appreciable turnover and comprises similar polymers and contains similar levels of molecular organization to storage starches, making Arabidopsis an excellent model system for studying granule biosynthesis.

We investigated the mechanism of amylose synthesis in Arabidopsis leaves using (14)C-labeling techniques. First, we tested the hypothesis that short malto-oligosaccharides (MOS) may act as primers for granule-bound starch synthase I. We found increased amylose synthesis in isolated starch granules supplied with ADP[(14)C]glucose (ADP[(14)C]Glc) and MOS compared with granules supplied with ADP[(14)C]Glc but no MOS. Furthermore, using a MOS-accumulating mutant (dpe1), we found that more amylose was synthesized than in the wild type, correlating with the amount of MOS in vivo. When wild-type and mutant plants were tested in conditions where both lines had similar MOS contents, no difference in amylose synthesis was observed. We also tested the hypothesis that branches of amylopectin might serve as the primers for granule-bound starch synthase I. In this model, elongated branches of amylopectin are subsequently cleaved to form amylose. We conducted pulse-chase experiments, supplying a pulse of ADP[(14)C]Glc to isolated starch granules or (14)CO(2) to intact plants, followed by a chase period in unlabeled substrate. We detected no transfer of label from the amylopectin fraction to the amylose fraction of starch either in isolated starch granules or in intact leaves, despite varying the time course of the experiments and using a mutant line (sex4) in which high-amylose starch is synthesized. We therefore find no evidence for amylopectin-primed amylose synthesis in Arabidopsis. We propose that MOS are the primers for amylose synthesis in Arabidopsis leaves.