Starch Synthesis Pathway Research Articles

Starch synthesis in amyloplasts purified from developing potato tubers

Amyloplasts have been purified from potato tubers by mechanical homogenization and gravity sedimentation through Nycodenz. Based on the recovery and latency of organelle‐specific marker enzymes, the recovery of amyloplasts is approximately 13%, exhibiting 65% intactness, with less than 1% contamination by other subcellular fractions. These preparations were able to synthesize starch from glucose‐1‐phosphate plus ATP, or ADP‐glucose but not from glucose‐6‐phosphate. Rates of starch synthesis from glucose‐1‐phosphate plus ATP were linear for up to 1 h and sensitive to the inhibitor 4,4‐diisothiocyanato‐stilbene 2,2‐disulphonic acid (DIDS). Starch synthesis was optimal at pH 7.0 and was saturated by 5–10 mM glucose‐1‐phosphate and by 1 mM ADP‐glucose. The results are discussed in the context of the pathway of starch synthesis and the transport of metabolites across the amyloplast envelope.

Evaluation of Root Sink Ability of Sweet Potato (Ipomoea batatas Lam.) Cultivars on the Basis of Enzymatic Activity in the Starch Synthesis Pathway

AbstractActivities of five enzymes, sucrose synthase (SUS), uridine 5′‐diphosphate glucose pyrophosphorylase (UDPGPPase), fructose‐1,6 bisphosphatase (FBPase), adenosine 5’diphosphate glucose pyrophosphorylase (ADPGPPase) and starch synthase (STS), in the metabolic pathway of starch synthesis, were compared between two sweet potato (Ipomoea batatas Lam.) cultivars, Koganesengan (C.V.KOG, a recently released high yield cultivar) and Tsurunasigenji (C.V.TSU, an old, local cultivar with poor yield). The measurements were carried out using the root samples (tuberous, thick and fibrous roots) harvested at the fast tubering stage. Of the five enzymes, SUS, ADPGPPase and STS showed high activities in the tuberous root, particularly in that of C.V.KOG, and a similar trend was observed for activities of these enzymes on a protein basis. The increased activity of the three enzymes is considered to be one of the characteristics in a high yield cultivar, allowing the root to function effectively as a starch synthesis and storage organ.

What Controls the Amount and Structure of Starch in Storage Organs?

The regulation of starch synthesis in the starch-storing organs of higher plants-organs such as tubers, the embryos of grain legumes, and the endosperms of cereals, in which starch constitutes 50 to 80% of the dry weight at maturity-is complex and poorly understood. Our ignorance of this process has been highlighted by the recent upsurge of interest in starch synthesis, fueled largely by the possibility of producing novel starches by genetic modification. Details of the metabolic pathway itself remain the subject of controversy, and remarkably little is known of the factors that determine either the rate of synthesis or the structure of the starch in storage organs. Some widely accepted views about the regulation of starch synthesis are no longer tenable or require revision in the light of recent findings. We shall discuss briefly the nature of the pathway of starch synthesis, then examine critically the current ideas about factors that determine the flux through the pathway and two important aspects of starch structure: the ratio of amylose to amylopectin and the branching of amylopectin.

Effect of Temperature on Enzymes in the Pathway of Starch Biosynthesis in Developing Wheat and Maize Grain

Soluble starch synthase (SSS) is shown to be a major site of control of flux through the pathway of starch synthesis in developing wheat and maize grain. Temperatures above 25�C adversely affect flux, and therefore, limit yield. This process is linked to SSS which is heat sensitive. Two apparently different properties of SSS can be identified which differ in the period required before full activity is restored after heat treatment. First, enzyme rate is adversely affected by elevated temperature, an effect which is reversible on returning to a lower temperature. The effect on enzyme rate was quantified using enzyme Q10 which was found to begin to be sub-optimal above 20�C. Second, with a prolonged period of exposu;e to elevated temperature there is a loss of enzyme activity which is not freely reversible which we have termed thermal inactivation. Although this occurs at temperatures in excess of 20�C in wheat, higher temperatures of more than 30�C are needed in maize SSS. Elevated temperature did not affect the inherent stability or Q10 characteristics of other enzymes in the pathway of starch synthesis except for branching enzyme which we believe has minimal flux-control strength. SSS thermal inactivation may not be a major problem in field conditions for developing maize grain, because temperatures rarely are high enough. However, we suggest that the effect on enzyme Q10 is more physiologically relevant, since maize SSS is operating sub-optimally as temperatures exceed 20�C. Calculations of the reductions in maize US corn-belt yield showed that significant yield improvement might be obtained by a 5�C shift in the temperature optimum. Thus selections for a more temperature tolerant form of maize SSS were conducted using enzyme Q10 as a selection tool. Of several hundred maize specimens screened, two were found to be significantly different. However, attempts to use backcross breeding to transfer this trait from the tropical donor to another line have not yet succeeded. Transgenic approaches to altering relations of starch deposition are now underway.

The Effect of High Temperature on Starch Synthesis and the Activity of Starch Synthase

The decrease in yield which is observed when developing storage organs such as cereal grains or potato tubers are exposed to high temperatures is due to a lower final starch content. The rate of starch synthesis during the development of these storage organs at high temperature, is either reduced or fails to increase sufficiently to compensate for the shorter developmental period. This effect on the rate of starch synthesis does not seem to be due to a reduction in the supply of photosynthate. One of the enzymes in the pathway of starch synthesis, soluble starch synthase, is susceptible to heat inactivation at unusually low temperatures and may also have a low optimum temperature for maximum activity. In some storage organs, the maximum catalytic activity of soluble starch synthase is not very much greater than the rate of starch synthesis. A decrease in the activity of this enzyme is therefore, likely to affect the rate of starch synthesis. Thus, the effect of high temperature on the rate of starch synthesis may be due, at least in part, to the properties of this enzyme. This review discusses the unusual heat-sensitivity of starch synthase in the context ofthe effects of high temperature on starch synthesis in storage organs.

Elevated temperature reduces starch deposition in wheat endosperm by reducing the activity of soluble starch synthase

Temperatures of more than 25° C adversely affect the activity of soluble starch synthase (SSS), an amyloplastic enzyme, in endosperm of wheat (Triticum aestivum L. cv. Mardler). Enzyme rate was found to have a temperature optimum between 20 and 25°C. This effect was apparently reversible after a short period of exposure to elevated temperature. We also found that with a prolonged period of exposure to elevated temperature there was another temperature-related phenomenon which caused a loss of enzyme activity that appeared to be much slower to reverse. We have termed this effect of temperature on SSS activity “knockdown”. The knockdown in SSS activity also occurred in-vivo. However, elevated temperature did not affect the activities of several other enzymes in the pathway of starch synthesis (ADP-glucose pyrophosphorylase, UDP-glucose pyrophosphorylase, sucrose synthase, phosphoglucomutase, phosphoglucose isomerase, bound starch synthase or hexokinase). Because the knockdown effect appeared to be specific to the enzyme SSS, we quantified the effect of knockdown on flux of carbon into starch and used these data to calculate the flux-control coefficient for SSS. Using data at 10–20°C the flux-control coefficient was CStarch10–20C = 0.50, whereas at 20–30° C the flux-control coefficient was CStarch20–30C = 1.38, and between 30–40°C the flux-control coefficient was CStarch30–40C = 0.69. Using data at 10–30°C the flux-control coefficient was CStarch10–30C = 1.15, and at 10–40°C the flux-control coefficient was CStarch10–40C = 0.82. In conclusion, we suggest that SSS is a major site of regulation of starch synthesis in developing wheat grain. During periods of high temperature the control point in the pathway of starch synthesis is apparently not associated exclusively with ADP-glucose pyrophosphorylase. In field conditions, in which temperatures are fluctuating, there will likely be periods of control of starch synthesis being exerted predominantly by SSS. During periods at lower temperature, control of flux may be exerted by SSS, perhaps in combination with other flux-controlling enzymes in the pathway. Our data point-out a crucial new aspect of quantifying control strengths of enzymes in plants: the determination of enzyme control strengths should be done in carefully regulated temperature conditions. Thus, since temperature is a major determinant of real flux through a pathway and the individual enzymes can respond differently to changing temperature conditions, the control strengths of individual steps in a pathway may vary with changing environmental conditions. This is particularly pronounced in starch deposition, because of the temperature instability of SSS.

Metabolism of glucose 6-phosphate by plastids from developing pea embryos

The aim of this work was to investigate the metabolism of glucose 6-phosphate by plastids isolated from developing pea (Pisum sativum L.) embryos. Plastids metabolise exogenously supplied glucose 6-phosphate via the pathway of starch synthesis and the oxidative pentose-phosphate pathway. The flux through the latter pathway is greatly stimulated by the provision of glutamine and 2-oxoglutarate — the substrates of glutamate synthase — indicating that it is regulated by the demand for reductant within the plastid. Flux in the presence of glutamine and 2-oxoglutarate is about 20% of the maximum flux through the pathway of starch synthesis. There is no competition for glucose 6-phosphate between the two pathways at concentrations which are saturating for both. Isolated plastids do not convert glucose 6-phosphate to amino acids or fatty acids at significant rates under the conditins of our experiments.

High Temperature Affects the Activity of Enzymes in the Committed Pathway of Starch Synthesis in Developing Wheat Endosperm

Ears of wheat were exposed for up to 10 days during the grain-filling stage to high temperature (35�C) and activities of five enzymes in the sucrose to starch pathway were compared to those in ears maintained at lower temperature (21�C day/16�C night). Two cultivars of wheat known to differ in their post-anthesis tolerance of high temperature were compared. On a per grain basis, the activity of sucrose synthase and of ADPglucose pyrophosphorylase in ears maintained at 21/16�C throughout did not change greatly between days 16 and 32 after anthesis, whereas UDPglucose pyrophosphorylase and soluble starch synthase activities declined with advancing development. Soluble starch synthase activity in grains of heated ears was decreased within 1 day to about one- half of the value in unheated grains, and 3 days' additional heating did not reduce the activity much further. Insoluble starch synthase activity was not significantly reduced by heating. Compared to soluble starch synthase, ADPglucose pyrophosphorylase activity was more slowly affected and decreased to a lesser extent by heat. Sucrose synthase and UDPglucose pyrophosphorylase activities were either not affected or only slightly reduced; part of this reduction could be due to advanced development at the higher temperature. In recovery experiments ears were heated for brief periods and then returned to 21/16�C for a few days. ADPglucose pyrophosphorylase and soluble starch synthase activities recovered in the cooler conditions but the other two enzymes generally only maintained or lost further activity. From a comparison of the activities of these enzymes with the rate of starch deposition, and by taking into account the effects of heating, it is proposed that the influence of heating on final grain dry weight is attributable to the observed reductions of soluble starch synthase activity.

Is There an Alternative Pathway for Starch Synthesis?

In leaf tissue, carbon enters starch via the gluconeogenesis pathway where d-glycerate 3-phosphate formed from CO(2) fixation is converted into hexose monophosphates within the chloroplast stroma. In starch-containing sink organs, evidence has been obtained indicating that the flow of carbon into starch follows a different pathway whereby hexose monophosphates formed from sucrose are transported into the amyloplast, a plastid specialized in starch accumulation. In both chloroplasts and amyloplasts, the formation of ADPglucose, the substrate for starch synthase, is controlled by the activity of ADPglucose pyrophosphorylase, a key regulatory enzyme of starch synthesis localized in the plastid. Recently, an alternative pathway of starch synthesis has been proposed in which ADPglucose is synthesized from sucrose and transported directly into the plastid compartment, where it is used for starch synthesis. On the basis of the biochemical phenotypes exhibited by various plant mutants with defined genetic lesions, it is concluded that ADPglucose pyrophosphorylase is essential for starch synthesis, whereas the alternative pathway has only a minor role in this process.

Starch synthesis in developing pea embryos.

The pea embryo stores about half of its carbon as starch and has proved to be an excellent system on which to study the nature and regulation of the pathway of starch synthesis. The developing embryo receives its carbon as sucrose, which is metabolized via glycolysis in the cytosol of cotyledonary cells. Glucose 6-phosphate enters the amyloplast - probably via a phosphate-exchange translocator - where it is converted to ADPglucose via phosphoglucomutase and ADPglucose pyrophosphorylase. ADPglucose pyrophosphorylase is the site of action of a mutation at the rb locus, which reduces activity by more than 90 % and the rate of starch synthesis by about 50 %. Study of mutant and wildtype embryos reveals that one of four putative subunits of the enzyme is eliminated by the mutation. Three distinct isoforms of starch synthase catalyze the incorporation of the glucosyl moiety of ADPglucose into starch. Two of these are probably active in the soluble phase of the amyloplast and become incorporated into the granule as it grows, while the third is almost exclusively granule-bound. Analysis of cDNA clones for starch synthases shows that the exclusively granule-bound form is very similar to the 'waxy' gene product believed to be responsible for amylose synthesis in cereal endosperms. The soluble starch synthases show some similarities to the 'waxy' proteins, but clearly belong to a different and previously undescribed class of starch synthases. The pea embryo contains two forms of starch branching enzyme, which are encoded by different genes, are maximally expressed at different times in development, and have different kinetic properties. It is likely that they play different roles in the synthesis of the granule. A mutation at the r locus, which reduces the rate of starch synthesis by about 50% and increases the amylose content of the starch from 30% to 70%, consists of a transposon-like insertion in the gene encoding starch-branching enzyme I. Activity of this isoform is abolished by the mutation. CONTENTS Summary 21 I. Introduction 21 II. The supply of sucrose to the embryo 22 III. The timing and location of starch synthesis 23 IV. The supply of carbon to the amyloplast 23 V. Mutations affecting the committed pathway of starch synthesis 26 VI. The committed pathway 28 Acknowledgements 31 References 31.

Photosynthate Metabolism in the Source Leaves of N2-Fixing Soybean Plants

Soybean plants (Glycine max [L.] Merr. cv Williams), which were symbiotic with Bradyrhizobium japonicum, and which grew well upon reduced nitrogen supplied solely through N(2) fixation processes, often exhibited excess accumulation of starch and sucrose and diminished soluble protein in their source leaves. Nitrate and ammonia, when supplied to the nodulated roots of N(2)-fixing plants, mediated a reduction of foliar starch accumulation and a corresponding increase in soluble protein in the source leaves. This provided an opportunity to examine the potential metabolic adjustments by which NO(3) (-) and NH(4) (+) (N) sufficiency or deficiency exerted an influence upon soybean leaf starch synthesis. When compared with soybean plants supplied with N, elevated starch accumulation was focused in leaf palisade parenchyma tissue of N(2)-fixing plants. Foliar activities of starch synthesis pathway enzymes including fructose-1,6-bisphosphate phosphatase, phosphohexoisomerase, phosphoglucomutase (PGM), as well as adenosine diphosphate glucose pyrophosphorylase (in some leaves) exhibited highest activities in leaf extracts of N(2)-fixing plants when expressed on a leaf protein basis. This was interpreted to mean that there was an adaptation of these enzyme activities in the leaves of N(2)-fixing plants, and this contributed to an increase in starch accumulation. Another major causal factor associated with increased starch accumulation was the elevation in foliar levels of fructose-6-phosphate, glucose-6-phosphate, and glucose-1-phosphate (G1P), which had risen to chloroplast concentrations considerably in excess of the K(m) values for their respective target enzymes associated with starch synthesis, e.g. elevated G1P with respect to adenosine diphosphate glucose pyrophosphorylase (ADPG-PPiase) binding sites. The cofactor glucose-1,6-bisphosphate (G1,6BP) was found to be obligate for maximal PGM activity in soybean leaf extracts of N(2)-fixing as well as N-supplemented plants, and G1,6BP levels in N(2)-fixing plant leaves was twice that of levels in N-supplied treatments. However the concentration of chloroplastic G1,6BP in illuminated leaves was computed to be saturating with respect to PGM in both N(2)-fixing and N-supplemented plants. This suggested that the higher level of this cofactor in N(2)-fixing plant leaves did not confer any higher PGM activation and was not a factor in higher starch synthesis rates. Relative to plants supplied with NO(3) (-) and NH(4) (+), the source leaf glycerate-3-phosphate (3-PGA) and orthophosphate (Pi) concentrations in leaves of N(2)-fixing plants were two to four times higher. Although Pi is a physiological competitive inhibitor of leaf chloroplast ADPG-PPiase, and hence, starch synthesis, elevated chloroplast 3-PGA levels in N(2)-fixing plant leaves apparently prevented interference of Pi with ADPG-PPiase catalysis and starch synthesis.

Influence of Water Deficit on Maize Endosperm Development

In maize (Zea mays L.), drought during the post-pollination stage decreases kernel growth and often leads to grain yield losses. Kernels in the apical region of the ear are more severely affected than basally positioned kernels. We hypothesized that water deficit during early endosperm development might inhibit kernel growth by decreasing endosperm cell division, and that this response might be mediated by changes in endosperm abscisic acid (ABA) levels. Greenhouse-grown maize, cultivar Pioneer 3925, was subjected to water limitation from 1 to 15 days after pollination (DAP), spanning the period of endosperm cell division and induction of storage product accumulation. Water deficit decreased the number of endosperm nuclei during the treatment period; the most substantial effect was in the apical region of ears. Correspondingly, endosperm fresh weight, starch accumulation and dry mass at maturity were decreased by water limitation. Abscisic acid concentrations in endosperm were quantified by enzyme-linked immunosorbent assay. Water deficit increased ABA concentration in apical-region endosperm by four-fold compared to controls. ABA concentrations were also increased in middle and basal regions of the ear, but to a lesser extent. Two key enzymes in the starch synthesis pathway, sucrose synthase and granule-bound ADP-glucose starch synthase, and zein, the major storage protein in maize endosperm, were studied as markers of storage product synthesis. Water deficit did not affect sucrose synthase enzyme activity or RNA transcript abundance relative to total RNA. However, ADP-glucose starch synthase activity and RNA transcript abundance decreased slightly in apical-region endosperm of water-limited plants by 15 DAP, compared with well-watered controls. In contrast to starch, there was no treatment effect on the accumulation of zein, evaluated at either the polypeptide or RNA level. We conclude that under the conditions tested, the establishment of starch and zein synthetic potential in endosperm was only slightly affected by plant water deficit during the early phase of kernel growth, and that capacity for growth and starch accumulation was affected by the extent to which cell division was inhibited. Based on correlative changes in ABA concentration and cell division we suggest that ABA may play a role in inhibiting endosperm cell division during water limitation.

The pathway of starch synthesis in developing pea embryos.

Conference Article| August 01 1991 The pathway of starch synthesis in developing pea embryos Alison M. Smith; Alison M. Smith 1John Innes Institute, Colney Lane, Norwich NR4 7UH, U.K. Search for other works by this author on: This Site PubMed Google Scholar Cathie Martin; Cathie Martin 1John Innes Institute, Colney Lane, Norwich NR4 7UH, U.K. Search for other works by this author on: This Site PubMed Google Scholar Kay Denyer Kay Denyer 1John Innes Institute, Colney Lane, Norwich NR4 7UH, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1991) 19 (3): 547–550. https://doi.org/10.1042/bst0190547 Article history Received: April 12 1991 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation Alison M. Smith, Cathie Martin, Kay Denyer; The pathway of starch synthesis in developing pea embryos. Biochem Soc Trans 1 August 1991; 19 (3): 547–550. doi: https://doi.org/10.1042/bst0190547 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: SBE, starch-branching enzyme This content is only available as a PDF. © 1991 Biochemical Society1991 Article PDF first page preview Close Modal You do not currently have access to this content.

A mathematical model of the carbon metabolism in photosynthesis. Difficulties in explaining oscillations by fructose 2, 6-bisphosphate regulation

A mathematical model of the pentosephosphate carbon reduction (PCR) cycle is presented. The internal structure of the model is consistent with and complements the known biochemical pathways in the PCR cycle, together with starch and sucrose synthesis. Individual enzymes are described by maximum rate ( V m ), standard free energy change (Δ G´ 0 ) and Michaelis constant ( K m ) values as parameters and rate-equations, sym­metrical for the direct and reverse reactions. Enzymic control is included in the starch synthesis pathway (activation by phosphoglycerate (PGA)), inhibition by inorganic phosphate) and in the reactions of sucrose synthesis based on fructose 2, 6-bisphosphate (F2, 6BP) as a metabolite con­trolling the cytosolic fructose bisphosphatase (FBPase) activity. The phosphate translocator carries out the exchange of triose phosphates, orthophosphate and PGA. Ionic forms of metabolites are calculated in relation to pH and assumed to be the actual reacting substances. The significant concentration of the active sites of ribulose 1, 5-bisphosphate (RuBP) carboxylase is taken into account. Light reactions are included only in the form of an ATPase the Δ G´ 0 of which is shifted towards ATP synthesis by the existing proton gradient. The behaviour of the model was studied with the aim of reproducing oscillations in photosynthesis. It is concluded that oscillations in photosynthesis cannot be caused by the fructose 2, 6-bisphosphate control of sucrose synthesis alone, but that an additional control of photosynthetic rate must also be involved.