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We expressed a bacterial glucan synthase (Agrobacterium GlgA) in the cytosol of developing endosperm cells in wheat grains, to discover whether it could generate a glucan from cytosolic ADP-glucose. Transgenic lines had high glucan synthase activity during grain filling, but did not accumulate glucan. Instead, grains accumulated very high concentrations of maltose. They had large volumes during development due to high water content, and very shrivelled grains at maturity. Starch synthesis was severely reduced. We propose that cytosolic glucan synthesized by the glucan synthase was immediately hydrolysed to maltose by cytosolic β-amylase(s). Maltose accumulation resulted in a high osmotic potential in developing grain, drawing in excess water that stretched the seed coat and pericarp. Loss of water during grain maturation then led to shrinkage when the grains matured. Maltose accumulation is likely to account for the reduced starch synthesis in transgenic grains, through signalling and toxic effects. Using bioinformatics, we identify an isoform of β-amylase likely to be responsible for maltose accumulation. Removal of this isoform through identification of TILLING mutants or genome editing, combined with co-expression of heterologous glucan synthase and a glucan branching enzyme, may in future enable elevated yields of carbohydrate through simultaneous accumulation of starch and cytosolic glucan.

A reduction in cytosolic invertase in Arabidopsis roots changes the sucrose to hexose ratio, resulting in profound alterations of metabolism, growth, development, and patterns of gene expression., Plant roots depend on sucrose imported from leaves as the substrate for metabolism and growth. Sucrose and hexoses derived from it are also signalling molecules that modulate growth and development, but the importance for signalling of endogenous changes in sugar levels is poorly understood. We report that reduced activity of cytosolic invertase, which converts sucrose to hexoses, leads to pronounced metabolic, growth, and developmental defects in roots of Arabidopsis (Arabidopsis thaliana) seedlings. In addition to altered sugar and downstream metabolite levels, roots of cinv1 cinv2 mutants have reduced elongation rates, cell and meristem size, abnormal meristematic cell division patterns, and altered expression of thousands of genes of diverse functions. Provision of exogenous glucose to mutant roots repairs relatively few of the defects. The extensive transcriptional differences between mutant and wild-type roots have hallmarks of both high sucrose and low hexose signalling. We conclude that the mutant phenotype reflects both low carbon availability for metabolism and growth and complex sugar signals derived from elevated sucrose and depressed hexose levels in the cytosol of mutant roots. Such reciprocal changes in endogenous sucrose and hexose levels potentially provide rich information about sugar status that translates into flexible adjustments of growth and development.

Starch granules contain two Glc polymers, amylopectin and amylose. Amylose makes up approximately 10% to 30% (w/w) of all natural starches thus far examined, but mutants of crop and model plants that produce amylose-free starch are generally indistinguishable from their wild-type counterparts with respect to growth, starch content, and granule morphology. Since the function and adaptive significance of amylose are unknown, we asked whether there is natural genetic variation in amylose synthesis within a wild, uncultivated species. We examined polymorphisms among the 1,135 sequenced accessions of Arabidopsis (Arabidopsis thaliana) in GRANULE-BOUND STARCH SYNTHASE (GBSS), encoding the enzyme responsible for amylose synthesis. We identified 18 accessions that are predicted to have polymorphisms in GBSS that affect protein function, and five of these accessions produced starch with no or extremely low amylose (< 0.5% [w/w]). Eight further accessions had amylose contents that were significantly lower or higher than that of Col-0 (9% [w/w]), ranging from 5% to 12% (w/w). We examined the effect of the polymorphisms on GBSS function and uncovered three mechanisms by which GBSS sequence variation led to different amylose contents: (1) altered GBSS abundance, (2) altered GBSS activity, and (3) altered affinity of GBSS for binding PROTEIN TARGETING TO STARCH1—a protein that targets GBSS to starch granules. These findings demonstrate that amylose in leaves is not essential for the viability of some naturally occurring Arabidopsis genotypes, at least over short timescales and under some environmental conditions and open an opportunity to explore the adaptive significance of amylose.

Large numbers of genes essential for embryogenesis in Arabidopsis encode enzymes of plastidial metabolism. Disruption of many of these genes results in embryo arrest at the globular stage of development. However, the cause of lethality is obscure. We examined the role of the plastidial oxidative pentose phosphate pathway (OPPP) in embryo development. In nonphotosynthetic plastids the OPPP produces reductant and metabolic intermediates for central biosynthetic processes. Embryos with defects in various steps in the oxidative part of the OPPP had cell division defects and arrested at the globular stage, revealing an absolute requirement for the production via these steps of ribulose-5-phosphate. In the nonoxidative part of the OPPP, ribulose-5-phosphate is converted to ribose-5-phosphate (R5P)—required for purine nucleotide and histidine synthesis—and subsequently to erythrose-4-phosphate, which is required for synthesis of aromatic amino acids. We show that embryo development through the globular stage specifically requires synthesis of R5P rather than erythrose-4-phosphate. Either a failure to convert ribulose-5-phosphate to R5P or a block in purine nucleotide biosynthesis beyond R5P perturbs normal patterning of the embryo, disrupts endosperm development, and causes early developmental arrest. We suggest that seed abortion in mutants unable to synthesize R5P via the oxidative part of the OPPP stems from a lack of substrate for synthesis of purine nucleotides, and hence nucleic acids. Our results show that the plastidial OPPP is essential for normal developmental progression as well as for growth in the embryo.

Summary 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

Plants accumulate reserves in the daytime to support growth at night. Circadian regulation of diel reserve turnover was investigated by profiling starch, sugars, glucose 6-phosphate, organic acids, and amino acids during a light-dark cycle and after transfer to continuous light in Arabidopsis wild types and in mutants lacking dawn (lhy cca1), morning (prr7 prr9), dusk (toc1, gi), or evening (elf3) clock components. The metabolite time series were integrated with published time series for circadian clock transcripts to identify circadian outputs that regulate central metabolism. (a) Starch accumulation was slower in elf3 and prr7 prr9. It is proposed that ELF3 positively regulates starch accumulation. (b) Reducing sugars were high early in the T-cycle in elf3, revealing that ELF3 negatively regulates sucrose recycling. (c) The pattern of starch mobilization was modified in all five mutants. A model is proposed in which dawn and dusk/evening components interact to pace degradation to anticipated dawn. (d) An endogenous oscillation of glucose 6-phosphate revealed that the clock buffers metabolism against the large influx of carbon from photosynthesis. (e) Low levels of organic and amino acids in lhy cca1 and high levels in prr7 prr9 provide evidence that the dawn components positively regulate the accumulation of amino acid reserves.

Starch, the major storage carbohydrate in plants, is synthesized in plastids as semi-crystalline, insoluble granules. Many organs and cell types accumulate starch at some point during their development and maturation. The biosynthesis of the starch polymers, amylopectin and amylose, is relatively well understood and mostly conserved between organs and species. However, we are only beginning to understand the mechanism by which starch granules are initiated, and the factors that control the number of granules per plastid and the size/shape of granules. Here, we review recent progress in understanding starch granule initiation and morphogenesis. In Arabidopsis, granule initiation requires several newly discovered proteins with specific locations within the chloroplast, and also on the availability of maltooligosaccharides which act as primers for initiation. We also describe progress in understanding granule biogenesis in the endosperm of cereal grains-within which there is large interspecies variation in granule initiation patterns and morphology. Investigating whether this diversity results from differences between species in the functions of known proteins, and/or from the presence of novel, unidentified proteins, is a promising area of future research. Expanding our knowledge in these areas will lead to new strategies for improving the quality of cereal crops by modifying starch granule size and shape in vivo.

Many plants accumulate transitory starch reserves in their leaves during the day to buffer their carbohydrate supply against fluctuating light conditions, and to provide carbon and energy for survival at night. It is universally accepted that transitory starch is synthesized from ADP-glucose (ADPG) in the chloroplasts. However, the consensus that ADPG is made in the chloroplasts by ADPG pyrophosphorylase has been challenged by a controversial proposal that ADPG is made primarily in the cytosol, probably by sucrose synthase (SUS), and then imported into the chloroplasts. To resolve this long-standing controversy, we critically re-examined the experimental evidence that appears to conflict with the consensus pathway. We show that when precautions are taken to avoid artefactual changes during leaf sampling, Arabidopsis thaliana mutants that lack SUS activity in mesophyll cells (quadruple sus1234) or have no SUS activity (sextuple sus123456) have wild-type levels of ADPG and starch, while ADPG is 20 times lower in the pgm and adg1 mutants that are blocked in the classical pathway of starch synthesis. We conclude that the ADPG needed for starch synthesis in leaves is synthesized primarily by ADPG pyrophosphorylase in the chloroplasts.Significance statementMutant analysis shows that sucrose synthase makes no significant contribution to transitory starch synthesis in Arabidopsis leaves, resolving a 20-year old controversy about one of the most important pathways of photosynthetic metabolism.

Research in the past decade has uncovered new and surprising information about the pathways of starch synthesis and degradation. This includes the discovery of previously unsuspected protein families required both for processes and for the long-sought mechanism of initiation of starch granules. There is also growing recognition of the central role of leaf starch turnover in making carbon available for growth across the day-night cycle. Sophisticated systems-level control mechanisms involving the circadian clock set rates of nighttime starch mobilization that maintain a steady supply of carbon until dawn and modulate partitioning of photosynthate into starch in the light, optimizing the fraction of assimilated carbon that can be used for growth. These discoveries also uncover complexities: Results from experiments with Arabidopsis leaves in conventional controlled environments are not necessarily applicable to other organs or species or to growth in natural, fluctuating environments.

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.

Starch is a major energy store in plants. It provides most of the calories in the human diet and, as a bulk commodity, it is used across broad industry sectors. Starch synthesis and degradation are not fully understood, owing to challenging biochemistry at the liquid/solid interface and relatively limited knowledge about the nature and control of starch degradation in plants. Increased societal and commercial demand for enhanced yield and quality in starch crops requires a better understanding of starch metabolism as a whole. Here we review recent advances in understanding the roles of carbohydrate-active enzymes in starch degradation in cereal grains through complementary chemical and molecular genetics. These approaches have allowed us to start dissecting aspects of starch degradation and the interplay with cell-wall polysaccharide hydrolysis during germination. With a view to improving and diversifying the properties and uses of cereal grains, it is possible that starch degradation may be amenable to manipulation through genetic or chemical intervention at the level of cell wall metabolism, rather than simply in the starch degradation pathway per se.

Since starch is by far the major component of the mature wheat grain, it has been assumed that variation in the capacity for starch synthesis during grain filling can influence final grain weight. We investigated this assumption by studying a total of 54 wheat genotypes including elite varieties and landraces that were grown in two successive years in fields in the east of England. The weight, water content, sugars, starch, and maximum catalytic activities of two enzymes of starch biosynthesis, ADP-glucose pyrophosphorylase and soluble starch synthase, were measured during grain filling. The relationships between these variables and the weights and starch contents of mature grains were analysed. Final grain weight showed few or no significant correlations with enzyme activities, sugar levels, or starch content during grain filling, or with starch content at maturity. We conclude that neither sugar availability nor enzymatic capacity for starch synthesis during grain filling significantly influenced final grain weight in our field conditions. We suggest that final grain weight may be largely determined by developmental processes prior to grain filling. Starch accumulation then fills the grain to a physical limit set by developmental processes. This conclusion is in accord with those from previous studies in which source or sink strength has been artificially manipulated., Journal of Experimental Botany, 69 (22), ISSN:1460-2431, ISSN:0022-0957

Arabidopsis (Arabidopsis thaliana) leaves synthesize starch faster in short days than in long days, but the mechanism that adjusts the rate of starch synthesis to daylength is unknown. To understand this mechanism, we first investigated whether adjustment occurs in mutants lacking components of the circadian clock or clock output pathways. Most mutants adjusted starch synthesis to daylength, but adjustment was compromised in plants lacking the GIGANTEA or FLAVIN-BINDING, KELCH REPEAT, F BOX1 components of the photoperiod-signaling pathway involved in flowering. We then examined whether the properties of the starch synthesis enzyme adenosine 5′-diphosphate-glucose pyrophosphorylase (AGPase) are important for adjustment of starch synthesis to daylength. Modulation of AGPase activity is known to bring about short-term adjustments of photosynthate partitioning between starch and sucrose (Suc) synthesis. We found that adjustment of starch synthesis to daylength was compromised in plants expressing a deregulated bacterial AGPase in place of the endogenous AGPase and in plants containing mutant forms of the endogenous AGPase with altered allosteric regulatory properties. We suggest that the rate of starch synthesis is in part determined by growth rate at the end of the preceding night. If growth at night is low, as in short days, there is a delay before growth recovers during the next day, leading to accumulation of Suc and stimulation of starch synthesis via activation of AGPase. If growth at night is fast, photosynthate is used for growth at the start of the day, Suc does not accumulate, and starch synthesis is not up-regulated.

Surface binding sites (SBSs) interact with carbohydrates outside of the enzyme active site. They are frequently situated on catalytic domains and are distinct from carbohydrate binding modules (CBMs). SBSs are found in a variety of enzymes and often seen in crystal structures. Notably about half of the > 45 enzymes (in 17 GH and two GT families) with an identified SBS are from GH13 and a few from GH77, both belonging to clan GH-H of carbohydrate active enzymes. The many enzymes of GH13 with SBSs provide an opportunity to analyse their distribution within this very large and diverse family. SBS containing enzymes in GH13 are spread among 15 subfamilies (two were not assigned a subfamily). Comparison of these SBSs reveals a complex evolutionary history with evidence of conservation of key residues and/or structural location between some SBSs, while others are found at entirely distinct structural locations, suggesting convergent evolution. An array of investigations of the two SBSs in barley α-amylase demonstrated they play different functional roles in binding and degradation of polysaccharides. MalQ from Escherichia coli is an α-1,4-glucanotransferase of GH77, a family that is known to have at least one member that contains an SBS. Whereas MalQ is a single domain enzyme lacking CBMs, its plant orthologue DPE2 contains two N-terminal CBM20s. Surface plasmon resonance binding studies showed that MalQ and DPE2 have a similar affinity for β-cyclodextrin and that MalQ binds malto-oligosaccharides of >DP4 at a second site in competition with β-cyclodextrin yielding a stoichiometry >1. This suggests that MalQ may have an SBS, though its structural location remains unknown.

The first step on the pathway of starch degradation in Arabidopsis (Arabidopsis thaliana) leaves at night is the phosphorylation of starch polymers, catalyzed by glucan, water dikinase (GWD). It has been suggested that GWD is important for the control of starch degradation, because its transcript levels undergo strong diel fluctuations, its activity is subject to redox regulation in vitro, and starch degradation is strongly decreased in gwd mutant plants. To test this suggestion, we analyzed changes in GWD protein abundance in relation to starch levels in wild-type plants, in transgenic plants in which GWD transcripts were strongly reduced by induction of RNA interference, and in transgenic plants overexpressing GWD. We found that GWD protein levels do not vary over the diel cycle and that the protein has a half-life of 2 d. Overexpression of GWD does not accelerate starch degradation in leaves, and starch degradation is not inhibited until GWD levels are reduced by 70%. Surprisingly, this degree of reduction also inhibits starch synthesis in the light. To discover the importance of redox regulation, we generated transgenic plants expressing constitutively active GWD. These plants retained normal control of degradation. We conclude that GWD exerts only a low level of control over starch degradation in Arabidopsis leaves.

The effects on barley starch and grain properties of four starch synthesis mutations were studied during the introgression of the mutations from diverse backgrounds into an elite variety. The lys5f (ADPglucose transporter), wax (granule-bound starch synthase), isa1 (debranching enzyme isoamylase 1) and sex6 (starch synthase IIa) mutations were introgressed into NFC Tipple to give mutant and wild-type BC2F4 families with different genomic contributions of the donor parent. Comparison of starch and grain properties between the donor parents, the BC2F4 families and NFC Tipple allowed the effects of the mutations to be distinguished from genetic background effects. The wax and sex6 mutations had marked effects on starch properties regardless of genetic background. The sex6 mutation conditioned low grain weight and starch content, but the wax mutation did not. The lys5 mutation conditioned low grain weight and starch content, but exceptionally high β-glucan contents. The isa1 mutation promotes synthesis of soluble α-glucan (phytoglycogen). Its introgression into NFC Tipple increased grain weight and total α-glucan content relative to the donor parent, but reduced the ratio of phytoglycogen to starch. This study shows that introgression of mutations into a common, commercial background provides new insights that could not be gained from the donor parent., Highlights • Four barley starch synthesis mutations were introgressed from diverse backgrounds into an elite variety. • wax and sex6 mutations had marked effects on starch properties regardless of genetic background. • sex6 and lys5 mutations reduced grain weight, but the lys5 mutation led to very high β-glucan contents. • Introgression reduced the phytoglycogen (soluble α-glucan) to starch ratio in isa1 mutants.

Background: Maltose metabolism during leaf starch degradation requires a multidomain glucanotransferase and a complex polysaccharide. Results: A conventional bacterial glucanotransferase rescues an Arabidopsis mutant lacking the multidomain glucanotransferase. Conclusion: Both the plant glucanotransferase-polysaccharide couple and the bacterial enzyme provide a glucosyl buffer in the starch degradation pathway. Significance: New light is shed on the regulation and evolution of maltose metabolism., Controlled conversion of leaf starch to sucrose at night is essential for the normal growth of Arabidopsis. The conversion involves the cytosolic metabolism of maltose to hexose phosphates via an unusual, multidomain protein with 4-glucanotransferase activity, DPE2, believed to transfer glucosyl moieties to a complex heteroglycan prior to their conversion to hexose phosphate via a cytosolic phosphorylase. The significance of this complex pathway is unclear; conversion of maltose to hexose phosphate in bacteria proceeds via a more typical 4-glucanotransferase that does not require a heteroglycan acceptor. It has recently been suggested that DPE2 generates a heterogeneous series of terminal glucan chains on the heteroglycan that acts as a “glucosyl buffer” to ensure a constant rate of sucrose synthesis in the leaf at night. Alternatively, DPE2 and/or the heteroglycan may have specific properties important for their function in the plant. To distinguish between these ideas, we compared the properties of DPE2 with those of the Escherichia coli glucanotransferase MalQ. We found that MalQ cannot use the plant heteroglycan as an acceptor for glucosyl transfer. However, experimental and modeling approaches suggested that it can potentially generate a glucosyl buffer between maltose and hexose phosphate because, unlike DPE2, it can generate polydisperse malto-oligosaccharides from maltose. Consistent with this suggestion, MalQ is capable of restoring an essentially wild-type phenotype when expressed in mutant Arabidopsis plants lacking DPE2. In light of these findings, we discuss the possible evolutionary origins of the complex DPE2-heteroglycan pathway.

Starch degradation in barley endosperm provides carbon for early seedling growth, but the control of this process is poorly understood. We investigated whether endosperm cell wall degradation is an important determinant of the rate of starch degradation. We identified iminosugar inhibitors of enzymes that degrade the cell wall component arabinoxylan. The iminosugar 1,4-dideoxy-1, 4-imino-l-arabinitol (LAB) inhibits arabinoxylan arabinofuranohydrolase (AXAH) but does not inhibit the main starch-degrading enzymes α- and β-amylase and limit dextrinase. AXAH activity in the endosperm appears soon after the onset of germination and resides in dimers putatively containing two isoforms, AXAH1 and AXAH2. Upon grain imbibition, mobilisation of arabinoxylan and starch spreads across the endosperm from the aleurone towards the crease. The front of arabinoxylan degradation precedes that of starch degradation. Incubation of grains with LAB decreases the rate of loss of both arabinoxylan and starch, and retards the spread of both degradation processes across the endosperm. We propose that starch degradation in the endosperm is dependent on cell wall degradation, which permeabilises the walls and thus permits rapid diffusion of amylolytic enzymes. AXAH may be of particular importance in this respect. These results provide new insights into the mobilization of endosperm reserves to support early seedling growth.

During germination and early seedling growth of barley (Hordeum vulgare), maltase is responsible for the conversion of maltose produced by starch degradation in the endosperm to glucose for seedling growth. Despite the potential relevance of this enzyme for malting and the production of alcoholic beverages, neither the nature nor the role of maltase is fully understood. Although only one gene encoding maltase has been identified with certainty, there is evidence for the existence of other genes and for multiple forms of the enzyme. It has been proposed that maltase may be involved directly in starch granule degradation as well as in maltose hydrolysis. The aim of our work was to discover the nature of maltase in barley endosperm. We used ion exchange chromatography to fractionate maltase activity from endosperm of young seedlings, and we partially purified activity for protein identification. We compared maltase activity in wild-type barley and transgenic lines with reduced expression of the previously-characterised maltase gene Agl97, and we used genomic and transcriptomic information to search for further maltase genes. We show that all of the maltase activity in the barley endosperm can be accounted for by a single gene, Agl97. Multiple forms of the enzyme most likely arise from proteolysis and other post-translational modifications.

Mature seeds of both the high-starch starch-excess1 (sex1) mutant and the almost starchless phosphoglucomutase1 mutant of Arabidopsis (Arabidopsis thaliana) have 30% to 40% less lipid than seeds of wild-type plants. We show that this is a maternal effect and is not attributable to the defects in starch metabolism in the embryo itself. Low lipid contents and consequent slow postgerminative growth are seen only in mutant embryos that develop on maternal plants with mutant phenotypes. Mutant embryos that develop on plants with wild-type starch metabolism have wild-type lipid contents and postgerminative growth. The maternal effect on seed lipid content is attributable to carbohydrate starvation in the mutant fruit at night. Fruits on sex1 plants grow more slowly than those on wild-type plants, particularly at night, and have low sugars and elevated expression of starvation genes at night. Transcript levels of the transcription factor WRINKLED1, implicated in lipid synthesis, are reduced at night in sex1 but not in wild-type seeds, and so are transcript levels of key enzymes of glycolysis and fatty acid synthesis. sex1 embryos develop more slowly than wild-type embryos. We conclude that the reduced capacity of mutant plants to convert starch to sugars in leaves at night results in low nighttime carbohydrate availability in the developing fruit. This in turn reduces the rate of development and expression of genes encoding enzymes of storage product accumulation in the embryo. Thus, the supply of carbohydrate from the maternal plant to the developing fruit at night can have an important influence on oilseed composition and on postgerminative growth.

The genetic and genomic resources available for the model plant Arabidopsis have allowed rapid progress in understanding the pathways of leaf starch synthesis and degradation. The pathway of starch synthesis is generally similar to that in other plants, but Arabidopsis research has permitted new insights into the mechanism of granule initiation. The pathway of starch degradation is very different from the ‘textbook’ version, largely derived from research on germinating cereal grains. The starch granule is attacked by β- rather than α-amylases, and this process is strongly dependent on a cycle of phosphorylation and dephosphorylation of the granule surface. The major product of granule degradation is maltose, which is exported to the cytosol where it is metabolised to hexose phosphate and then to sucrose. Cytosolic maltose metabolism requires a glucanotransferase and a glucosyl acceptor which is believed to be a complex heteroglycan. Plant productivity and yield are highly dependent on leaf starch turnover, and starch metabolism is coordinated with other factors and processes that determine growth rate. Research in Arabidopsis provides an important knowledge base for the study of starch metabolism in other, less tractable species.

The aim of this work was to investigate starch granule numbers in Arabidopsis (Arabidopsis thaliana) leaves. Lack of quantitative information on the extent of genetic, temporal, developmental, and environmental variation in granule numbers is an important limitation in understanding control of starch degradation and the mechanism of granule initiation. Two methods were developed for reliable estimation of numbers of granules per chloroplast. First, direct measurements were made on large series of consecutive sections of mesophyll tissue obtained by focused ion beam-scanning electron microscopy. Second, average numbers were calculated from the starch contents of leaves and chloroplasts and estimates of granule mass based on granule dimensions. Examination of wild-type plants and accumulation and regulation of chloroplast (arc) mutants with few, large chloroplasts provided the following new insights. There is wide variation in chloroplast volumes in cells of wild-type leaves. Granule numbers per chloroplast are correlated with chloroplast volume, i.e. large chloroplasts have more granules than small chloroplasts. Mature leaves of wild-type plants and arc mutants have approximately the same number of granules per unit volume of stroma, regardless of the size and number of chloroplasts per cell. Granule numbers per unit volume of stroma are also relatively constant in immature leaves but are greater than in mature leaves. Granule initiation occurs as chloroplasts divide in immature leaves, but relatively little initiation occurs in mature leaves. Changes in leaf starch content over the diurnal cycle are largely brought about by changes in the volume of a fixed number of granules.

Efforts to improve photosynthetic efficiency should result in increased rates of carbon assimilation in crop plants in the next few decades. Translation of increased assimilation into higher productivity will require a greater understanding of the relationship between assimilation and growth. In this review, we discuss new progress in understanding how carbon is provided for metabolism and growth at night. In Arabidopsis leaves, the circadian clock controls the rate of degradation of starch to ensure an optimal carbon supply and hence continued growth during the night. These discoveries shed new light on the integration of carbon assimilation and growth over the light–dark cycle. They reveal the importance of considering the carbon economy of the whole plant in attempting to increase crop productivity.

There are major issues regarding the proposed pathway for starch degradation in germinating cereal grain. Given the commercial importance but genetic intractability of the problem, we have embarked on a program of chemical genetics studies to identify and dissect the pathway and regulation of starch degradation in germinating barley grains. As a precursor to in vivo studies, here we report systematic analysis of the reversible and irreversible inhibition of the major β-amylase of the grain endosperm (BMY1). The molecular basis of inhibitor action was defined through high resolution X-ray crystallography studies of unliganded barley β-amylase, as well as its complexes with glycone site binder disaccharide iminosugar G1M, irreversible inhibitors α-epoxypropyl and α-epoxybutyl glucosides, which target the enzyme's catalytic residues, and the aglycone site binders acarbose and α-cyclodextrin.

The importance of α-glucosidase in the endosperm starch metabolism of barley (Hordeum vulgare) seedlings is poorly understood. The enzyme converts maltose to glucose (Glc), but in vitro studies indicate that it can also attack starch granules. To discover its role in vivo, we took complementary chemical-genetic and reverse-genetic approaches. We identified iminosugar inhibitors of a recombinant form of an α-glucosidase previously discovered in barley endosperm (ALPHA-GLUCOSIDASE97 [HvAGL97]), and applied four of them to germinating grains. All four decreased the Glc-to-maltose ratio in the endosperm 10 d after imbibition, implying inhibition of maltase activity. Three of the four inhibitors also reduced starch degradation and seedling growth, but the fourth did not affect these parameters. Inhibition of starch degradation was apparently not due to inhibition of amylases. Inhibition of seedling growth was primarily a direct effect of the inhibitors on roots and coleoptiles rather than an indirect effect of the inhibition of endosperm metabolism. It may reflect inhibition of glycoprotein-processing glucosidases in these organs. In transgenic seedlings carrying an RNA interference silencing cassette for HvAgl97, α-glucosidase activity was reduced by up to 50%. There was a large decrease in the Glc-to-maltose ratio in these lines but no effect on starch degradation or seedling growth. Our results suggest that the α-glucosidase HvAGL97 is the major endosperm enzyme catalyzing the conversion of maltose to Glc but is not required for starch degradation. However, the effects of three glucosidase inhibitors on starch degradation in the endosperm indicate the existence of unidentified glucosidase(s) required for this process.

The metabolism of starch is of central importance for many aspects of plant growth and development. Information on leaf starch metabolism other than in Arabidopsis (Arabidopsis thaliana) is scarce. Furthermore, its importance in several agronomically important traits exemplified by legumes remains to be investigated. To address this issue, we have provided detailed information on the genes involved in starch metabolism in Lotus japonicus and have characterized a comprehensive collection of forward and TILLING (for Targeting Induced Local Lesions IN Genomes) reverse genetics mutants affecting five enzymes of starch synthesis and two enzymes of starch degradation. The mutants provide new insights into the structure-function relationships of ADP-glucose pyrophosphorylase and glucan, water dikinase1 in particular. Analyses of the mutant phenotypes indicate that the pathways of leaf starch metabolism in L. japonicus and Arabidopsis are largely conserved. However, the importance of these pathways for plant growth and development differs substantially between the two species. Whereas essentially starchless Arabidopsis plants lacking plastidial phosphoglucomutase grow slowly relative to wild-type plants, the equivalent mutant of L. japonicus grows normally even in a 12-h photoperiod. In contrast, the loss of GLUCAN, WATER DIKINASE1, required for starch degradation, has a far greater effect on plant growth and fertility in L. japonicus than in Arabidopsis. Moreover, we have also identified several mutants likely to be affected in new components or regulators of the pathways of starch metabolism. This suite of mutants provides a substantial new resource for further investigations of the partitioning of carbon and its importance for symbiotic nitrogen fixation, legume seed development, and perenniality and vegetative regrowth.

Plant growth is driven by photosynthetic carbon fixation during the day. Some photosynthate is accumulated, often as starch, to support nocturnal metabolism and growth at night. The rate of starch degradation inArabidopsisleaves at night is essentially linear, and is such that almost all of the starch is used by dawn. We have investigated the timer that matches starch utilization to the duration of the night. The rate of degradation adjusted immediately and appropriately to an unexpected early onset of night. Starch was still degraded in an appropriate manner when the preceding light period was interrupted by a period of darkness. However, whenArabidopsiswas grown in abnormal day lengths (28 h or 17 h) starch was exhausted ∼24 h after the last dawn, irrespective of the actual dawn. A mutant lacking the LHY and CCA1 clock components exhausted its starch at the dawn anticipated by its fast-running circadian clock, rather than the actual dawn. Reduced growth of wild-type plants in 28-h days andlhy/cca1mutants in 24-h days was attributable to the inappropriate rate of starch degradation and the consequent carbon starvation at the end of night. Thus, starch degradation is under circadian control to ensure that carbohydrate availability is maintained until the next anticipated dawn, and this control is necessary for maintaining plant productivity.

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.

Only two mutations have been described in the literature, so far, regarding starch and root quality traits in cassava. This article reports on an induced mutation in this crop, first identified in 2006. Botanical seed from five different cassava families were irradiated with gamma rays. Seed was germinated, transplanted to the field (M1 plants), and self-pollinated to produce the M2 generation. Abnormal types regarding starch granule morphology were identified during the single plant evaluation of M2 genotypes. To confirm these characteristics, selected genotypes were cloned and a second evaluation, based on cloned plants obtained from vegetative multiplication, was completed in September 2007. Two M2 genotypes presented small starch granules, but only one could be fully characterized, presenting a granule size of 5.80 +/- 0.33 microm compared with three commercial clones with granule sizes ranging from 13.97 +/- 0.12 to 18.73 +/- 0.10 microm and higher-than-normal amylose content (up to 30.1% in cloned plants harvested in 2007, as compared with the typical values for "normal" cassava starch of around 19.8%). The gels produced by the starch of these plants did not show any viscosity when analyzed with the rapid viscoanalyzers (5% suspension), and the gels had low clarity. Low viscosity could be observed at higher concentrations (8 or 10% suspensions). Preliminary results suggest that the mutation may be due to a lesion in a gene encoding one of the isoforms of isoamylase (probably isa1 or isa2).

*† ‡ § – Summary SOB3, which encodes a plant-specific AT-hook motif containing protein, was identified from an activationtagging screen for suppressors of the long-hypocotyl phenotype of a weak phyB allele, phyB-4. sob3-D (suppressor of phyB-4#3 dominant) overexpressing seedlings have shorter hypocotyls, and as adults develop larger flowers and leaves, and are delayed in senescence compared with wild-type plants. At the nucleotide level, SOB3 is closely related to ESCAROLA (ESC), which was identified in an independent activation-tagging screen. ESC overexpression also suppresses the phyB-4 long-hypocotyl phenotype, and confers an adult morphology similar to sob3-D, suggesting similar functions. Analysis of transgenic plants harboring SOB3:SOB3-GUS or ESC:ESC-GUS translational fusions, driven by their endogenous promoter regions, showed GUS activity in the hypocotyl and vasculature tissue in light- and dark-grown seedlings. A lossof-function SOB3 allele (sob3-4) was generated through an ethyl methanesulfonate intragenic suppressor screen of sob3-D phyB-4 plants, and this allele was combined with a predicted null allele, disrupting ESC (esc-8), to examine potential genetic interactions. The sob3-4 esc-8 double mutant had a long hypocotyl in multiple fluence rates of continuous white, far-red, red and blue light. sob3-4 esc-8 phyB-9 and sob3-4 esc-8 cry-103 triple mutants also had longer hypocotyls than photoreceptor single mutants. In contrast, the sob3-4 esc-8 phyA-211 triple mutant was the same length as phyA-211 single mutants. Taken together, these data indicate that SOB3 and ESC act redundantly to modulate hypocotyl growth inhibition in response to light.

One of the objectives of the cassava-breeding project at CIAT is the identification of clones with special root quality characteristics. A large number of self-pollinations have been made in search of useful recessive traits. During 2006 harvests an S1 plant produced roots that stained brownish-red when treated with an iodine solution, suggesting that it had lower-than-normal levels of amylose in its starch. Colorimetric and DSC measurements indicated low levels (3.4%) and an absence of amylose in the starch, respectively. SDS-PAGE demonstrated the absence of GBSS enzyme in the starch from these roots. Pasting behavior was analyzed with a rapid visco-analyzer and resulted in larger values for peak viscosity, gel breakdown, and setback in the mutant compared with normal cassava starch. Solubility was considerably reduced, while the swelling index and volume fraction of the dispersed phase were higher in the mutant. No change in starch granule size or shape was observed. This is the first report of a natural mutation in cassava that drastically reduces amylose content in root starch.

The properties and expression patterns of the six isoforms of sucrose synthase in Arabidopsis are described, and their functions are explored through analysis of T-DNA insertion mutants. The isoforms have generally similar kinetic properties. Although there is variation in sensitivity to substrate inhibition by fructose this is unlikely to be of major physiological significance. No two isoforms have the same spatial and temporal expression patterns. Some are highly expressed in specific locations, whereas others are more generally expressed. More than one isoform is expressed in all organs examined. Mutant plants lacking individual isoforms have no obvious growth phenotypes, and are not significantly different from wild-type plants in starch, sugar and cellulose content, seed weight or seed composition under the growth conditions employed. Double mutants lacking the pairs of similar isoforms sus2 and sus3, and sus5 and sus6, are also not significantly different in these respects from wild-type plants. These results are surprising in the light of the marked phenotypes observed when individual isoforms are eliminated in crop plants including pea, maize, potato and cotton. A sus1/sus4 double mutant grows normally in well-aerated conditions, but shows marked growth retardation and accumulation of sugars when roots are subjected to hypoxia. The sucrose synthase activity in roots of this mutant is 3% or less of wild-type activity. Thus under well-aerated conditions sucrose mobilization in the root can proceed almost entirely via invertases without obvious detriment to the plant, but under hypoxia there is a specific requirement for sucrose synthase activity.

It has been proposed that delayed leaf senescence can extend grain filling duration and thus increase yields in cereal crops. We found that wheat (Triticum aestivum) NAM RNAi plants with delayed senescence carried out 40% more flag leaf photosynthesis after anthesis than control plants, but had the same rate and duration of starch accumulation during grain filling and the same final grain weight. The additional photosynthate available in NAM RNAi plants was in part stored as fructans in the stems, whereas stem fructans were remobilised during grain filling in control plants. In both genotypes, activity of starch synthase was limiting for starch synthesis in the later stages of grain filling. We suggest that in order to realise the potential yield gains offered by delayed leaf senescence, this trait should be combined with increased grain filling capacity.

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.

Nickel(II)-xylylbicyclam is a potent anti-HIV agent and binds strongly to the CXCR4 co-receptor. We have investigated configurational equilibria of Ni(II)-cyclam derivatives, since these are important for receptor recognition. Crystallographic studies show that both trans and cis configurations are readily formed: [Ni(cyclam)(OAc)(2)] x H(2)O adopts the trans-III configuration with axial monodentate acetates, as does [Ni(benzylcyclam)(NO(3))(2)] with axial nitrate ligands, whereas [Ni(benzylcyclam)(OAc)](OAc)2 x H(2)O has an unusual folded cis-V configuration with Ni(II) coordination to bidentate acetate. UV/Vis and NMR studies show that the octahedral trans-III configuration slowly converts to square-planar trans-I in aqueous solution. For Ni(II)-xylylbicyclam, a mixture of cis-V and trans-I configurations was detected in solution. X-ray diffraction studies showed that crystals of lysozyme soaked in Ni(II)-cyclam or Ni(II) (2)-xylylbicyclam contain two major binding sites, one involving Ni(II) coordination to Asp101 and hydrophobic interactions between the cyclam ring and Trp62 and Trp63, and the second hydrophobic interactions with Trp123. For Ni(II)-cyclam bound to Asp101, the cis-V configuration predominates.

This protocol describes a simple means of measuring the starch content of plant tissues by solubilizing the starch, converting it quantitatively to glucose and assaying the glucose. Plant tissue must initially be frozen rapidly to stop metabolism, then extracted to remove free glucose. Starch is solubilized by heating, then digested to glucose by adding glucan hydrolases. Glucose is assayed enzymatically. The method is more sensitive and accurate than iodine-based protocols, and is suitable for tissues that have a wide range of starch contents. Measurements on multiple samples can be completed within a day.

Gibberellic acid (GA) promotes germination, stem/hypocotyl elongation, and leaf expansion during seedling development. Using activation-tagging mutagenesis, we identified a mutation, sob2-D (for suppressor of phytochromeB-4 [phyB-4]#2 dominant), which suppresses the long-hypocotyl phenotype of a phyB missense allele, phyB-4. This mutant phenotype is caused by the overexpression of an APETALA2 transcription factor, SOB2, also called DRN-like. SOB2/DRN-like transcript is not detectable in wild-type seedling or adult tissues via RT-PCR analysis, suggesting that SOB2/DRN-like may not be involved in seedling development under normal conditions. Adult sob2-D phyB-4 plants have curled leaves and club-like siliques, resembling plants that overexpress a closely related gene, LEAFY PETIOLE (LEP). Hypocotyls of a LEP-null allele, lep-1, are shorter in the light and dark, suggesting LEP involvement in seedling development. This aberrant hypocotyl phenotype is due at least in part to a delay in germination. In addition, lep-1 is less responsive to GA and more sensitive to the GA biosynthesis inhibitor paclobutrazol, indicating that LEP is a positive regulator of GA-induced germination. RT-PCR shows that LEP transcript accumulates in wild-type seeds during imbibition and germination, and the transcript levels of REPRESSOR OF ga1-3-LIKE2 (RGL2), a negative regulator of GA signaling during germination, is unaffected in lep-1. These results suggest LEP is a positive regulator of GA-induced germination acting independently of RGL2. An alternative model places LEP downstream of RGL2 in the GA-signaling cascade.

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.

To study the role of the plastidial α-glucan phosphorylase in starch metabolism in the leaves of Arabidopsis, two independent mutant lines containing T-DNA insertions within the phosphorylase gene were identified. Both insertions eliminate the activity of the plastidial α-glucan phosphorylase. Measurement of other enzymes of starch metabolism reveals only minor changes compared with the wild type. The loss of plastidial α-glucan phosphorylase does not cause a significant change in the total accumulation of starch during the day or its remobilization at night. Starch structure and composition are unaltered. However, mutant plants display lesions on their leaves that are not seen on wild-type plants, and mesophyll cells bordering the lesions accumulate high levels of starch. Lesion formation is abolished by growing plants under 100% humidity in still air, but subsequent transfer to circulating air with lower humidity causes extensive wilting in the mutant leaves. Wilted sectors die, causing large lesions that are bordered by starch-accumulating cells. Similar lesions are caused by the application of acute salt stress to mature plants. We conclude that plastidial phosphorylase is not required for the degradation of starch, but that it plays a role in the capacity of the leaf lamina to endure a transient water deficit.

Maltose is exported from the Arabidopsis chloroplast as the main product of starch degradation at night. To investigate its fate in the cytosol, we characterised plants with mutations in a gene encoding a putative glucanotransferase (disproportionating enzyme; DPE2), a protein similar to the maltase Q (MalQ) gene product involved in maltose metabolism in bacteria. Use of a DPE2 antiserum revealed that the DPE2 protein is cytosolic. Four independent mutant lines lacked this protein and displayed a decreased capacity for both starch synthesis and starch degradation in leaves. They contained exceptionally high levels of maltose, and elevated levels of glucose, fructose and other malto-oligosaccharides. Sucrose levels were lower than those in wild-type plants, especially at the start of the dark period. A glucosyltransferase activity, capable of transferring one of the glucosyl units of maltose to glycogen or amylopectin and releasing the other, was identified in leaves of wild-type plants. Its activity was sufficient to account for the rate of starch degradation. This activity was absent from dpe2 mutant plants. Based on these results, we suggest that DPE2 is an essential component of the pathway from starch to sucrose and cellular metabolism in leaves at night. Its role is probably to metabolise maltose exported from the chloroplast. We propose a pathway for the conversion of starch to sucrose in an Arabidopsis leaf.

Study of mutants of the model plant Arabidopsis is providing new information about the nature and regulation of starch degradation in leaves. The onlyoc-amylase currently predicted to be in the chloroplast is not necessary for the initial attack on the starch granule, and the enzyme(s) responsible for this attack remain unknown. A starch-water dikinase that phosphorylates glucose residues in amylopectin is necessary for starch degradation, and it seems likely that the enzyme(s) responsible for the initial attack require either the dikinase itself or phosphorylated regions of amylopectin for their activity. At least four chloroplastic enzymes could potentially debranch glucans released by the initial attack on the granule: the relative importance of these enzymes is not yet known. The degradation of linear glucans to monomers is catalysed by β-amylasesrather than starch phosphorylase. Plants lacking chloroplastic starch phosphorylase have normal rates of starch degradation. The fate of the maltose produced by β-amylolysis of linear glucans is being investigated through study of maltose-accumulating mutants. Other malto-oligosaccharides too short to be attacked by β-amylase are metabolised by disproportionating enzyme to produce longer chains susceptible to further attack. The process of starch degradation is subject to strong diurnal regulation. The nature of this regulation is not understood, but new approaches to the problem are suggested.

We investigated the mechanism of amylose synthesis in Arabidopsis leaves using 14C-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[14C]glucose (ADP[14C]Glc) and MOS compared with granules supplied with ADP[14C]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[14C]Glc to isolated starch granules or14CO2 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.

Amyloses with distinct molecular masses are found in the starch of pea embryos compared with the starch of pea leaves. In pea embryos, a granule-bound starch synthase protein (GBSSIa) is required for the synthesis of a significant portion of the amylose. However, this protein seems to be insignificant in the synthesis of amylose in pea leaves. cDNA clones encoding a second isoform of GBSSI, GBSSIb, have been isolated from pea leaves. Comparison of GBSSIa and GBSSIb activities shows them to have distinct properties. These differences have been confirmed by the expression of GBSSIa and GBSSIb in the amylose-free mutant of potato. GBSSIa and GBSSIb make distinct forms of amylose that differ in their molecular mass. These differences in product specificity, coupled with differences in the tissues in which GBSSIa and GBSSIb are most active, explain the distinct forms of amylose found in different tissues of pea. The shorter form of amylose formed by GBSSIa confers less susceptibility to the retrogradation of starch pastes than the amylose formed by GBSSIb. The product specificity of GBSSIa could provide beneficial attributes to starches for food and nonfood uses.

International audience; The metabolism of starch and sucrose fuels all aspects of plant growth and development. Over the last decade, significant advances have been made in our understanding of the metabolism of these compounds through the use of model systems, mainly Arabidopsis. Legume species are characterised by their capacity to form symbioses with Rhizobium, a nitrogen-fixing bacterium, leading to up to half the carbon assimilated in photosynthesis being sequestered to their roots. Study of a legume model may therefore increase our knowledge about carbohydrate turnover. We review here the resources available and the contribution that research on Lotus japonicus has made to our knowledge of sucrose breakdown and starch metabolism in relation to plant growth and development processes, especially processes that are legume specific.

Perenniality and vegetative re-growth vigour represent key agronomic traits in forage legume (Fabaceae) species. The known determinants of perenniality include the conservation of the vegetative meristem during and after the flowering phase, and the separation of flowering from senescence. The ability of the plants to store nutrient resources in perennial organs and remobilize them may also play an important role in the perennial growth habit, and in determining the capacity of the plant to re-grow following grazing or from one season to the next. To examine the importance of stored starch, we examined the vegetative re-growth vigour following cutting back of a unique collection of Lotus japonicus mutants impaired in their ability to synthesize or degrade starch. Our results establish that starch stored in the roots is important for re-growth vigour in Lotus japonicus. We extended this analysis to a collection of Lotus (trefoil) species and two ecotypes of Lotus japonicus displaying a large variation in their carbohydrate resource allocation. There was a positive correlation between root starch content and re-growth vigour in these natural variants, and a good general correlation between high re-growth vigour and the perennial life-form. We discuss the relationship between perenniality and the availability of root carbohydrates for re-growth.

†These authors contributed equally to this work. Summary Disproportionating enzyme (D-enzyme) is a plastidial a-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 Denzyme 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 maltooligosaccharides 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 maltooligosaccharides generated during the dark period. We conclude that the primary function of D-enzyme

The chemical synthesis of the title compounds as maltose analogs, in which the non-reducing end is modified by acetylation of the 4′-OH group or by reversing its configuration, is reported. For synthesis of the 4′-O-acetylated analog, β-maltose was converted into its per-O-benzylated-4′,6′-O-benzylidene derivative followed by removal of the benzylidene acetal function and selective silylation at C-6′. Acetylation at C-4′ of the obtained silylated compound followed by removal of the benzyl ether protecting groups and subsequent desilylation afforded the desired analog. The other maltose analog was synthesized via the glycosidation reaction between the glycosyl donor, O-(2,3,4,6-tetra-O-benzyl-α/β- d -galactopyranosyl)trichloroacetimidate and the glycosyl acceptor, phenyl 2,3,6-tri-O-benzyl-1-thio-β- d -glucopyranoside followed by removal of the phenylthio group and debenzylation to provide the desired analog.

Summary Starch granules are composed of two types of glucose polymer, amylose and amylopectin, that differ in size and structure. One of the most intriguing challenges in understanding starch synthesis is to explain the apparently simultaneous synthesis of two such different polymers. One isoform of starch synthase, GBSSI, is responsible for amylose synthesis but can also contribute to amylopectin synthesis. The factors which determine the partitioning of GBSSI activity between these two processes are largely unknown. Understanding the properties of GBSSI and how these differ from the properties of the amylopectin-synthesising isoforms of starch synthase are important to the understanding of the control of amylose synthesis. In this review, we will describe how the synthesis of amylose and amylopectin are integrated and what factors may determine the relative amounts of these two polymers.