The priming of amylose synthesis in Arabidopsis leaves

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.     

Normal starch content and composition in tubers of antisense potato plants lacking D-enzyme (4-α-glucanotransferase)

Transgenic potato (Solanum tuberosum L.) plants were created with sense and antisense copies of the potato D-enzyme (disproportionating enzyme; EC␣2.4.1.25) cDNA linked to patatin and cauliflower mosaic virus 35 S promoters, and screened for D-enzyme activity in tubers. Transformants with sense constructs mostly had wild type D-enzyme activity but two plants had only about 1% wild-type activity. Transformants with antisense constructs had activity ranging from 90% to about 1% of wild type. Three 35 S antisense plants with very low activity were analysed in detail. Western blot analysis showed that D-enzyme was present in greatly reduced amounts in tubers and in leaves, whereas plastidic starch phosphorylase (EC 2.4.1.1) was unaffected. The lack of D-enzyme resulted in slow plant growth but development was otherwise apparently normal. Furthermore, the starch content of tubers was not appreciably altered in amount, proportion of amylose, molecular weight of debranched amylopectin, or branch chain length, despite the lack of D-enzyme. These results do not indicate a direct requirement for D-enzyme in the synthesis and accumulation of storage starch in tubers. The results are discussed in terms of the known reactions catalysed by D-enzyme and possible involvement of D-enzyme in starch metabolism. Received: 12 November 1997 / Accepted: 23 December 1997     

Genetic and Biochemical Evidence for the Involvement of α-1,4 Glucanotransferases in Amylopectin Synthesis

We describe a novel mutation in the Chlamydomonas reinhardtii STA11 gene, which results in significantly reduced granular starch deposition and major modifications in amylopectin structure and granule shape. This defect simultaneously leads to the accumulation of linear malto-oligosaccharides. The sta11-1 mutation causes the absence of an α-1,4 glucanotransferase known as disproportionating enzyme (D-enzyme). D-enzyme activity was found to be correlated with the amount of wild-type allele doses in gene dosage experiments. All other enzymes involved in starch biosynthesis, including ADP-glucose pyrophosphorylase, debranching enzymes, soluble and granule-bound starch synthases, branching enzymes, phosphorylases, α-glucosidases (maltases), and amylases, were unaffected by the mutation. These data indicate that the D-enzyme is required for normal starch granule biogenesis in the monocellular alga C. reinhardtii.     

A cytosolic glucosyltransferase is required for conversion of starch to sucrose in Arabidopsis leaves at night

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.     

Biochemical Characterization of the Chlamydomonas reinhardtii α-1,4 Glucanotransferase Supports a Direct Function in Amylopectin Biosynthesis

Plant α-1,4 glucanotransferases (disproportionating enzymes, or D-enzymes) transfer glucan chains among oligosaccharides with the concomitant release of glucose (Glc). Analysis of Chlamydomonas reinhardtii sta11-1 mutants revealed a correlation between a D-enzyme deficiency and specific alterations in amylopectin structure and starch biosynthesis, thereby suggesting previously unknown biosynthetic functions. This study characterized the biochemical activities of the α-1,4 glucanotransferase that is deficient in sta11-1 mutants. The enzyme exhibited the glucan transfer and Glc production activities that define D-enzymes. D-enzyme also transferred glucans among the outer chains of amylopectin (using the polysaccharide chains as both donor and acceptor) and from malto-oligosaccharides into the outer chains of either amylopectin or glycogen. In contrast to transfer among oligosaccharides, which occurs readily with maltotriose, transfer into polysaccharide required longer donor molecules. All three enzymatic activities, evolution of Glc from oligosaccharides, glucan transfer from oligosaccharides into polysaccharides, and transfer among polysaccharide outer chains, were evident in a single 62-kD band. Absence of all three activities co-segregated with the sta11-1 mutation, which is known to cause abnormal accumulation of oligosaccharides at the expense of starch. To explain these data we propose that D-enzymes function directly in building the amylopectin structure.     

Integrated functions among multiple starch synthases determine both amylopectin chain length and branch linkage location in Arabidopsis leaf starch

This study assessed the impact on starch metabolism in Arabidopsis leaves of simultaneously eliminating multiple soluble starch synthases (SS) from among SS1, SS2, and SS3. Double mutant ss1- ss2- or ss1- ss3- lines were generated using confirmed null mutations. These were compared to the wild type, each single mutant, and ss1- ss2- ss3- triple mutant lines grown in standardized environments. Double mutant plants developed similarly to the wild type, although they accumulated less leaf starch in both short-day and long-day diurnal cycles. Despite the reduced levels in the double mutants, lines containing only SS2 and SS4, or SS3 and SS4, are able to produce substantial amounts of starch granules. In both double mutants the residual starch was structurally modified including higher ratios of amylose:amylopectin, altered glucan chain length distribution within amylopectin, abnormal granule morphology, and altered placement of α(1→6) branch linkages relative to the reducing end of each linear chain. The data demonstrate that SS activity affects not only chain elongation but also the net result of branch placement accomplished by the balanced activities of starch branching enzymes and starch debranching enzymes. SS3 was shown partially to overlap in function with SS1 for the generation of short glucan chains within amylopectin. Compensatory functions that, in some instances, allow continued residual starch production in the absence of specific SS classes were identified, probaby accomplished by the granule bound starch synthase GBSS1.     

The elongation of amylose and amylopectin chains in isolated starch granules

The aim of this work was to investigate the conditions required for amylose synthesis in starch granules. Although the major granule-bound isoform of starch synthase - GBSSI - catalyses the synthesis of amylose in vivo, ¹⁴C from ADP[¹⁴C]glucose was incorporated primarily into a specific subset of amylopectin chains when supplied to starch granules isolated from pea (Pisum sativum L.) embryos and potato (Solanum tuberosum L.) tubers. Incubation of granules with soluble extracts of these organs revealed that the extracts contained compounds that increased the incorporation of ¹⁴C into amylose. These compounds were rendered inactive by treatment of the extracts with α-glucosidase, suggesting that they were malto-oligosaccharides. Consistent with this idea, provision of pure malto-oligosaccharides to isolated granules resulted in a dramatic shift in the pattern of incorporation of ¹⁴C, from amylopectin chains to amylose molecules. Comparison of the pattern of incorporation in granules from wild-type peas and lam mutant peas which lack GBSSI showed that this effect of malto-oligosaccharides was specifically on GBSSI. The significance of these results for understanding of the synthesis of amylose and amylopectin in storage organs is discussed.     

A starch-accumulating mutant of Arabidopsis thaliana deficient in a chloroplastic starch-hydrolysing enzyme

The aim of this work was to identify enzymes that participate in the degradation of transitory starch in Arabidopsis . A mutant line was isolated by screening leaves at the end of the night for the presence of starch. The mutant had a higher starch content than the wild-type throughout the diurnal cycle. This accumulation was due to a reduction in starch breakdown, leading to an imbalance between the rates of synthesis and degradation. No reduction in the activity of endo-amylase (α-amylase), β-amylase, starch phosphorylase, maltase, pullulanase or D-enzyme could be detected in crude extracts of leaves of the mutant. However, native PAGE in gels containing amylopectin revealed that a starch-hydrolysing activity, putatively identified as an endo-amylase and present in wild-type chloroplasts, was absent or appreciably reduced in the mutant. This is the first time that a specific enzyme required for starch degradation has been identified in leaves.     

Starch degradation by the mould Trichoderma viride I. The mechanism of starch degradation

The mechanism of starch degradation by the fungus Trichoderma viride was studied in strain CBS 354.44, which utilizes glucose, starch and dextrins but is unable to assimilate maltose. It was shown that the amylolytic enzyme system is completely extracellular, equally well induced by starch, amylose or amylopectin and that it consists mainly of enzymes of the glucoamylase type which yield glucose as the main product of starch hydrolysis. Small amounts of -amylase are produced also. The enzymes produced in starch cultures degrade starch, amylose and amylopectin equally well.Enzyme synthesis in starch media takes place to a considerable extent after exhaustion of the carbon source when maximum growth has been attained.Low-molecular dextrins are degraded by extracellular enzymes of the glucoamylase type. These enzymes are produced in media containing starch or dextrins. Maltotriose is consumed for only one third leaving maltose in the culture filtrate. Maltose is hardly attacked and hardly induces any amylolytic enzyme activity. No stable -glucosidase appears to be produced.     

Activation of particulate starch synthetase from Zea mays embryo

The effect of spermine on particulate ADP-glucose: starch synthetase from the developing embryo of sweet corn has been studied. Spermine induces a considerable increase of glucose incorporation from ADP-glucose into the starch granules. The change in kinetic constants, the distribution of incorporated glucose between amylose and amylopectin and the pattern of incorporation into starch granules or malto-oligosaccharides has been studied. The data were compared with those obtained with citrate ions.     

Tuber physiology and properties of starch from tubers of transgenic potato plants with altered plastidic adenylate transporter activity

We showed recently that antisense plants with decreased activity of the plastidic ATP/ADP-transporter protein exhibit drastically reduced levels of starch and a decreased amylose/amylopectin ratio, whereas sense plants with increased activity of the transporter possessed more starch than wild-type plants and an increased amylose/amylopectin ratio. In this paper we investigate the effect of altered plastidic ATP/ADP-transporter protein expression on primary metabolism and granule morphology in more detail. Tuber tissues from antisense and sense plants exhibited substantially increased respiratory activity compared with the wild type. Tubers from antisense plants contained markedly increased levels of free sugars, UDP-Glc, and hexose phosphates, whereas phosphoenolpyruvate, isocitrate, ATP, ADP, AMP, UTP, UDP, and inorganic pyrophosphate levels were slightly decreased. In contrast, tubers from sense plants revealed a slight increase in adenine and uridine nucleotides and in the levels of inorganic pyrophosphate, whereas no significant changes in the levels of soluble sugars and metabolites were observed. Antisense tubers contained 50% reduced levels of ADP-Glc, whereas sense tubers contained up to 2-fold increased levels of this sole precursor for starch biosynthesis. Microscopic examination of starch grain morphology revealed that the size of starch grains from antisense tubers was substantially smaller (50%) compared with the wild type. The large starch grains from sense tubers appeared of a more angular morphology, which differed to the more ellipsoid shape of wild type grains. The results suggest a close interaction between plastidial adenylate transport and starch biosynthesis, indicating that ADP-Glc pyrophosphorylase is ATP-limited in vivo and that changes in ADP-Glc concentration determine starch yield, as well as granule morphology. Possible factors linking starch synthesis and respiration are discussed.     

淀粉含量不同的玉米自交系籽粒淀粉积累及其关键酶活性

以2个高淀粉和2个低淀粉玉米自交系为材料,分析了玉米籽粒淀粉的动态积累规律,同时对高低淀粉玉米籽粒灌浆过程中淀粉生物合成关键酶活性的动态变化及其与淀粉积累动态的相关性进行讨论分析。研究结果表明:灌浆过程中4个自交系淀粉含量变化趋势均呈sigmoid型曲线。灌浆过程中ADPG-PPase(腺苷二磷酸葡萄糖焦磷酸化酶)、SSS(可溶性淀粉合成酶)、GBSS(颗粒结合淀粉合成酶)活性均呈单峰曲线变化,峰值都出现在20～30DAP(授粉后天数)。2个高淀粉自交系的Q酶(淀粉分支酶)活性也呈单峰曲线变化,峰值也出现在20DAP,而2个低淀粉自交系的Q酶活性则呈双峰曲线变化,2个峰值分别出现在15～20DAP和30～35DAP。4个自交系籽粒淀粉的积累速率与各自交系ADPG-PPase、SSS和GBSS的活性变化呈极显著正相关。各自交系关键酶活性之间,ADPG-PPase、SSS和GBSS三者间活性变化呈极显著正相关,这3种酶活性变化与Q酶活性变化也呈不同程度的正相关。     

The role of amylomaltase in maltose metabolism in the cytosol of photosynthetic cells

Transitory starch is stored during the day inside chloroplasts and then broken down at night for export. Recent data indicate that maltose is the major form of carbon exported from the chloroplast at night but its fate in the cytosol is unknown. An amylomaltase gene (malQ) cloned from Escherichia coli is necessary for maltose metabolism in E. coli. We investigated whether there is an amylomaltase in the cytosol of plant leaves and the role of this enzyme in plants. Two mutants of Arabidopsis thaliana (L) Heynh. were identified in which the gene encoding a putative amylomaltase enzyme [disproportionating enzyme 2, DPE2 (DPE1 refers to the plastid version of this enzyme)] was disrupted by a T-DNA insertion. Both dpe2-1 and dpe2-2 plants exhibited a dwarf phenotype and accumulated a large amount of maltose. In addition, dpe2 mutants accumulated starch and a water-soluble, ethanol/KCl-insoluble maltodextrin in their chloroplasts. At night, the amount of sucrose in dpe2 plants was lower than that in wild-type plants. These results show that Arabidopsis has an amylomaltase that is involved in the conversion of maltose to sucrose in the cytosol. We hypothesize that knocking out amylomaltase blocks the conversion from maltose to sucrose, and that the higher amount of maltose feeds back to limit starch degradation reactions in chloroplasts. As a result, dpe2 plants have higher maltose, higher starch, and higher maltodextrin but lower nighttime sucrose than wild-type plants. Finally, we propose that maltose metabolism in the cytosol of Arabidopsis leaves is similar to that in the cytoplasm of E. coli.Abbreviations F6P fructose 6-phosphate - G1P glucose 1-phosphate - G6P glucose 6-phosphate - GTase glucanotransferase     

STA11, a Chlamydomonas reinhardtii locus required for normal starch granule biogenesis,encodes disproportionating enzyme. Further evidence for a function of alpha-1,4 glucanotransferases during starch granule biosynthesis in green algae

In Chlamydomonas reinhardtii, the presence of a defective STA11 locus results in significantly reduced granular starch deposition displaying major modifications in shape and structure. This defect simultaneously leads to the accumulation of linear malto-oligosaccharides (MOS). The mutants of STA11 were showed to lack D-enzyme, a plant alpha-1,4 glucanotransferase analogous to the Escherichia coli amylomaltase. We have cloned and characterized both the cDNA and gDNA corresponding to the C. reinhardtii D-enzyme. We now report allele-specific modifications of the D-enzyme gene in the mutants of STA11. These allele-specific modifications cosegregate with the corresponding sta11 mutations, thereby demonstrating that STA11 encodes D-enzyme. MOS production and starch accumulation were investigated during day and night cycles in wild-type and mutant C. reinhardtii cells. We demonstrate that in the algae MOS are produced during starch biosynthesis and degraded during the phases of net polysaccharide catabolism.     

Mutations in leaf starch metabolism modulate the diurnal root growth profiles of Arabidopsis thaliana

Roots of Arabidopsis thaliana exhibit stable diurnal growth profiles that are controlled by the circadian clock. Here we describe the effects of mutations in leaf starch metabolism on the diurnal root growth characteristics of Arabidopsis thaliana. High temporal and spatial resolution video imaging was performed to quantify the growth kinetics of Arabidopsis wild-type as well as pgm, sex1, mex1, dpe1 and dpe2 starch metabolism mutants grown in three different photoperiods. As a result, root growth patterns of all genotypes displayed characteristic modifications in their diurnal kinetics that were also affected by the photoperiod. To further investigate the role of starch derived substrate deficiency on root growth, the effect of 0.05% extracellular sucrose was studied in 12 h-12 h light-dark cycles.Key words: diurnal root growth kinetics, dpe1, dpe2, mex1, pgm, sex1, starch metabolism, video imagingRoot growth of Arabidopsis thaliana is highly rhythmic with respect to the time of the day.^1–3 In general, root growth rates increase at night while most of the light period is characterized by declining elongation rates. Since a slow oscillation in root growth rate with a periodicity of approximately 24 h persists in free running conditions it was demonstrated that the circadian clock mediates these daily fluctuations.¹ Root growth at night is fueled by the degradation of starch within the leaves. Thus, a correspondence between the time taken to degrade starch reserves and the length of the night is important to optimize growth in C-limiting conditions. Gibon et al. observed a strong correlation between the rate of starch degradation and the relative growth rate when Arabidopsis Col-0 was grown in a range of different photoperiods.⁴ Therefore, to avoid periods of C starvation at the end of the night the circadian clock was postulated to function as a timer that adjusts degradation of starch to the prevailing length of the night.^1,5Root growth strongly depends on the supply of sucrose from the leaves. To investigate the effects of substrate depletion on root elongation at night, 12-day-old seedlings of Col-0, pgm and sex1 growing in a 16 h photoperiod were previously investigated by digital time resolved video imaging.¹ As a result, the diel growth response was strongly modified in pgm and sex1 as compared to the wild-type. Both mutants showed a pronounced inhibition of growth during the night and a gradual recovery of growth during the light period. To substantiate these findings, we here report on the root elongation patterns of additional mutants in starch metabolism, e.g., mex1, dpe1 and dpe2 detected at different photoperiods and elevated external sucrose supply.     

Scanning Electron Microscopy and Fermentation Studies on Selected Known Maize Starch Mutants Using STARGEN? Enzyme Blends

The conversion of maize (corn) kernels to bio-ethanol is an energy-intensive process involving many stages. One step typically required is the liquefaction of the ground kernel to enable enzyme hydrolysation of the starch to glucose. The enzyme blends STARGEN? (Genencor) are capable of hydrolysing starch granules without liquefaction, reducing energy inputs and increasing efficiency. Studies were conducted on maize starch mutants amylose extender 1 (ae1), dull 1 (du1) and waxy 1 (wx1) in the inbred line Oh43 to determine whether different maize starches affected hydrolysation rates by STARGEN? 001 and STARGEN? 002. All mutants contained similar proportions of starch in the kernel but varied in the amylose to amylopectin ratio. Ground maize kernels were incubated with STARGEN? 001 and viewed using scanning electron microscopy to examine the hydrolysis action of STARGEN? 001 on the starch granules. The ae1 mutant exhibited noticeably less enzymic hydrolysis action, on the granules visualised, than wx1 and background line Oh43. Kernels were batch-fermented with STARGEN? 001 and STARGEN? 002. The ae1 mutant exhibited a 50% lower ethanol yield compared to the wx1 mutant and background line. A final study compared hydrolysation rates of STARGEN? 001 and STARGEN? 002 on purified maize starch, amylopectin and amylose. Though almost twice the amylopectin was hydrolysed using STARGEN? 002 than STARGEN? 001 in this trial, fermentations using STARGEN? 002 resulted in lower ethanol yields than fermentations using STARGEN? 001. Both STARGEN? enzyme blends were more suitable for the fermentation of high amylopectin maize starches than high amylose starches.     

Exoenzymic activity of alpha-amylase immobilized on a phenol-formaldehyde resin

Amylose and amylopectin from two starch sources were partially degraded by alpha-amylase immobilized on a phenol-formaldehyde resin. The degradation products were fractionated by gel-permeation chromatography and high-pressure, liquid chromatography. Two distinct fractions were obtained from tapioca amylose. One is a fragment having a molecular weight exceeding 200,000, and the other consists of oligosaccharides of low molecular weight with a degree of polymerization of 1–8. In contrast, treatment of tapioca amylose with soluble alpha-amylase produces a single fraction, nearly all of which has a molecular weight of <35,000, with only traces of small oligosaccharides detectable by high-pressure, liquid chromatography. Even wider differences were observed in degradation products from tapioca amylopectin. Similar activity-patterns were obtained with immobilized and soluble enzymes, using corn amylose and corn amylopectin as substrates. Immobilization of alpha-amylase on the resin apparently restricts the activity of the enzyme to the ends of the starch molecules, making it appear to be limited to exoenzymic activity.     

New look at starch degradation in Arabidopsis thaliana L. chloroplasts

Transitory starch is accumulated during the day and is the main source of energy for the cell metabolism during the night. The observed periodical starch degradation has become a model often used by scientist in their experiments. Starch granule degradation could be divided into 2 periods: initiation of degradation and digestion of amylopectin and amylose into maltooligosaccharide and their derivative. Key meaning is attributed in this process to beta-amylaze, product of its activity beta-maltose is transported to the cytosole and there it subjects farthest conversions. It has been demonstrated that a number of enzymes take part in the starch degradation process. However, the way of regulating their activity is still not fully explained. There is most important elements effecting rate of starch decomposition: day cycle, starch phosphorylation and regulation of enzyme activity. It proceeds through redox potential, pH changes and phosphorylation of protein involved in starch degradation due specific phosphatases. The purpose of the current work is to systematize the knowledge of the Arabidopsis thaliana L. leaf starch degradation. The results of the recent research cast a new light on the starch degradation process as well as on its control.     

Starch depletion and sugars in developing cotton leaves

Cotton plants (cv. Coker 100) were exposed to a 14-hour dark period. Starch degradation occurred with no accumulation of sugars due mainly to translocation. Considerable amounts of starch degradation products however were detected from leaves after phloem transport was blocked. A minor component (10 to 25% of total starch) with a linear structure, amylose, was preferentially degraded, whereas the major multiple-branched component (about 80%), amylopectin, showed an increasing resistance to degradation with leaf age. This relationship was also shown by the decreasing iodine-binding capacity of unit starch with increasing leaf age. The structural resistance of amylopectin to enzymic dark degradation was one of the barriers to starch dissolution in cotton.