Identification of granule-bound starch synthase in potato tubers

Starch granules isolated from potato (Solanum tuberosum L.) tubers were extracted with sodium dodecyl sulfate and the extract was analyzed. A major protein with a molecular weight of 60,000 daltons was detected. This protein was purified by preparative sodium dodecyl sulfate-gel electrophoresis and specific antibodies were prepared. The anti-60-kilodalton antibodies obtained (a) cross-reacted with the waxy proteins of both maize (Zea mays L.) and grain amaranth (Amaranthus hypochondriacus L.), and (b) inhibited starch synthase activity in partially digested starch granules of the grain amaranth. This evidence strongly suggests that the major 60-kilodalton protein present in potato starch granules represents the granule-bound starch synthase.     

Biochemical and molecular characterization of a novel starch synthase from potato tubers

An isoform of starch synthase from potato tubers which is present both in the stroma of the plastid and tightly bound to starch granules has been identified biochemically and a cDNA has been isolated. The protein encoded by the cDNA is 79.9 kDa and has a putative transit peptide and a distinct N-terminal domain which is predicted to be highly flexible. It is similar in both amino acid sequence and predicted structure to the granule-bound starch synthase II (GBSSII) of pea embryos. When expressed in Escherichia coli, the mature protein has starch synthase activity. The importance of the isoform has been assessed by biochemical measurements and antisense transformation experiments in which the amount of the isoform in the tuber is severely and specifically reduced. Both approaches indicate that the isoform contributes a maximum of 15% of the total starch synthase activity of the tuber. It is suggested that this isoform and the GBSSII of pea embryos represent a widely distributed class of isoforms of starch synthase. The contribution to total starch synthase activity of members of this class probably varies considerably from one type of storage organ to another.     

Large-scale isolation, fractionation, and purification of soluble starch-synthesizing enzymes: starch synthase and branching enzyme from potato tubers

Soluble starch-synthesizing enzymes, starch synthase (SSS) and starch-branching enzyme (SBE), were isolated, fractionated, and purified from white potato tubers (Solanum tuberosum) on a large scale. Five steps were used: potato tuber extract from 2 kg of peeled potatoes, two acetone precipitations, and two fractionations on a large ultrafiltration polysulfone hollow fiber 100 kDa cartridge. Three kinds of fractions were obtained: (1) mixtures of SSS and SBE; (2) SSS, free of SBE; and (3) SBE, free of SSS. Contaminating enzymes (amylase, phosphorylase, and disproportionating enzyme) and carbohydrates were absent from the 2nd acetone precipitate and from the column fractions, as judged by the Molisch test and starch triiodide test. Activity yields of 122% (300,000-400,000 units) of SSS fractions and 187% (40,000-50,000 units) of SBE fractions were routinely obtained from the cartridge. Addition of 0.04% (w/v) polyvinyl alcohol 50 K and 1 mM dithiothreitol to the glycine buffer (pH 8.4) gave long-term stability and higher yields of SSS and SBE, due to activation of inactive enzymes. Several SSS and SBE fractions from the two fractionations had very high specific activities, indicating high degrees of purification. Polyacrylamide gel electrophoresis of selected SSS and SBE fractions gave two to five SSS and/or SBE activity bands, corresponding to the one to five protein bands present in the 2nd acetone precipitate.     

The catecholamine potentiates starch mobilization in transgenic potato tubers.

In human and animal cells, the catecholamines are involved in glycogen mobilization. Since the compounds are found in a potato, their function in starch mobilization was hypothesized. In order to verify this hypothesis, the transgenic potato plants Solanum tuberosum L. cv. Desiree overexpressing tyrosine decarboxylase (TD EC 4.1.1.25) cDNA from parsley has been generated. The cDNA expression was judged by the northern blot analysis and the enzyme activity measurements. Four independent transgenic lines with the highest TD mRNA expression were selected and analyzed. The expected substantial decrease in tyrosine content was followed by significant increase in tyramine and dramatic enhancement of norepinephrine synthesis was detected. The level of L-3,4-dihydroxyphenylalanin (L-Dopa) was only slightly increased and dopamine significantly decreased in most cases in these plants. The increase in norepinephrine was accompanied by changes in carbohydrate metabolism. The significant increase in glucose and sucrose and the decrease in starch content were characteristic features of TD overexpressed transgenic potato tubers. The features mentioned above indicate that catecholamines potentiate starch mobilization in potato plants in common with animal cells. The decrease in tyrosine content in transgenic plants is also compensated by significant increase in chlorogenic acid synthesis thus potentially increasing the antioxidant capacity of transgenic tubers. The glycoalkaloids content is changed in the transformants. This may originate from glucose accumulation and glycolysis activation. The obtained transgenic potato provides material for further detailed studies of the physiological function of catecholamines in plants.     

Growth ring formation in the starch granules of potato tubers

Starch granules from higher plants contain alternating zones of semicrystalline and amorphous material known as growth rings. The regulation of growth ring formation is not understood. We provide several independent lines of evidence that growth ring formation in the starch granules of potato (Solanum tuberosum) tubers is not under diurnal control. Ring formation is not abolished by growth in constant conditions, and ring periodicity and appearance are relatively unaffected by a change from a 24-h to a 40-h photoperiod, and by alterations in substrate supply to the tuber that are known to affect the diurnal pattern of tuber starch synthesis. Some, but not all, of the features of ring formation are consistent with the involvement of a circadian rhythm. Such a rhythm might operate by changing the relative activities of starch-synthesizing enzymes: Growth ring formation is disrupted in tubers with reduced activity of a major isoform of starch synthase. We suggest that physical as well as biological mechanisms may contribute to the control of ring formation, and that a complex interplay of several factors may by involved.     

Specificity of starch synthase isoforms from potato.

In higher plants several isoforms of starch synthase contribute to the extension of glucan chains in the synthesis of starch. Different isoforms are responsible for the synthesis of essentially linear amylose chains and branched, amylopectin chains. The activity of granule-bound starch synthase I from potato has been compared with that of starch synthase II from potato following expression of both isoforms in Escherichia coli. Significant differences in their activities are apparent which may be important in determining their specificities in vivo. These differences include affinities for ADPglucose and glucan substrates, activation by amylopectin, response to citrate, thermosensitivity and the processivity of glucan chain extension. To define regions of the isoforms determining these characteristic traits, chimeric proteins have been produced by expression in E. coli. These experiments reveal that the C-terminal region of granule-bound starch synthase I confers most of the specific properties of this isoform, except its processive elongation of glucan chains. This region of granule-bound starch synthase I is distinct from the C-terminal region of other starch synthases. The specific properties it confers may be important in defining the specificity of granule-bound starch synthase I in producing amylose in vivo.     

Formation of a soluble amylopectin-like polysaccharide in potato tubers

When potato sprouts or potato tuber slices were incubated with 0.1 m glucose 1-phosphate, a soluble amylopectin-like polysaccharide was excreted to the medium. This polysaccharide was found to be a very good primer for phosphorylase and a poor one for starch synthetase. Beside the formation of this extracellular polysaccharide, a more branched intracellular polysaccharide could be isolated. This polysaccharide was an excellent primer for starch synthetase. Fructose 6-phosphate, glucose 6-phosphate, fructose 1,6-diphosphate, glucose or sucrose could not substitute for glucose 1-phosphate. 2,4-Dinitrophenol or nitrogen did not affect the excretion of the polysaccharide. Some properties of these 2 polysaccharides are described.     

Identification of the regulatory steps in glycolysis in potato tubers

The aim of this work was to identify the regulatory reactions of glycolysis in potato tubers. The amounts of glycolytic intermediates in aerobic and anoxic tubers were measured in freeze-clamped samples of tissue. Comparison of mass—action ratios with apparent equilibrium constants showed that in vivo the reactions catalysed by glucosephosphate isomerase, phosphoglycerate mutase and enolase were close to equilibrium. The ratios fructose-1,6-bisphosphate:fructose 6-phosphate, and pyruvate:phosphoenolpyruvate, respectively, showed that the reactions catalysed by phosphofructokinase and pyruvate kinase were considerably displaced from equilibrium. Stimulation of glycolysis by placing tubers in an atmosphere of nitrogen led to significant declines in their contents of fructose-6-phosphate and phosphoenolpyruvate. It is concluded that phosphofructokinase plays a dominant role in regulating entry into glycolysis, and that pyruvate kinase may regulate exit from glycolysis and the oxidative pentose phosphate pathway. Cold-induced sweetening of the tubers is discussed in the light of the above conclusions.     

Enzymatic activities of starch synthesis in potato tubers of different sizes

The objective of this study was to determine the relationship between tuber weight and enzymatic activities involved in tuber starch synthesis. As tuber weight increased, the activities of sucrose synthetase, UDPG pyrophosphorylase, and granular starch synthetase escalated, whereas the activities of soluble starch synthetase and ADPG pyrophosphorylase stayed constant and that of phosphorylase declined. This suggests that when samples are taken to determine specific enzymatic activities, the sampling procedure should ensure that results do not vary because of differences in the tuber weight or size distribution.     

Identification of multiple isoforms of soluble and granule-bound starch synthase in developing wheat endosperm

We have investigated the nature and locations of isoforms of starch synthase in the developing endosperm of wheat (Triticum aestivum L.). There are three distinct granule-bound isoforms of 60 kDa (the Waxy gene product), 77 kDa and 100–105 kDa. One of these isoforms, the 77-kDa protein, is also present in the soluble fraction of the endosperm but it contributes only a small proportion of the total soluble activity. Most of the soluble activity is contributed by isoforms which are apparently not also granule-bound. The 60-kDa and 77kDa isoforms of wheat are antigenically related to isoforms of very similar size in the developing pea embryo, but the other isoforms in the endosperm appear to have no counterparts in the pea embryo. The significance of these results in terms of the diversity of isoforms of starch synthase and their locations is discussed.Abbreviations DEAE diethylaminoethyl - GBSS granule-bound starch synthase - NT nullisomictetrasomic We are grateful to the late John Hawker (University of Adelaide, Australia) and to John Snape (John Innes Centre, UK) for useful discussions during the course of this work, to John Snape and Catherine Chinoy (John Innes Centre, UK) for the gift of the NT lines and to Richard Batt (University of Adelaide, Australia) for technical assistance.     

Granule-bound starch synthase I. A major enzyme involved in the biogenesis of B-crystallites in starch granules.

Starch defines a semicrystalline polymer made of two different polysaccharide fractions. The A- and B-type crystalline lattices define the distinct structures reported in cereal and tuber starches, respectively. Amylopectin, the major fraction of starch, is thought to be chiefly responsible for this semicrystalline organization while amylose is generally considered as an amorphous polymer with little or no impact on the overall crystalline organization. STA2 represents a Chlamydomonas reinhardtii gene required for both amylose biosynthesis and the presence of significant granule-bound starch synthase I (GBSSI) activity. We show that this locus encodes a 69 kDa starch synthase and report the organization of the corresponding STA2 locus. This enzyme displays a specific activity an order of magnitude higher than those reported for most vascular plants. This property enables us to report a detailed characterization of amylose synthesis both in vivo and in vitro. We show that GBSSI is capable of synthesizing a significant number of crystalline structures within starch. Quantifications of amount and type of crystals synthesized under these conditions show that GBSSI induces the formation of B-type crystals either in close association with pre-existing amorphous amylopectin or by crystallization of entirely de novo synthesized material.     

Role of granule-bound starch synthase in determination of amylopectin structure and starch granule morphology in potato.

Reductions in activity of SSIII, the major isoform of starch synthase responsible for amylopectin synthesis in the potato tuber, result in fissuring of the starch granules. To discover the causes of the fissuring, and thus to shed light on factors that influence starch granule morphology in general, SSIII antisense lines were compared with lines with reductions in the major granule-bound isoform of starch synthase (GBSS) and lines with reductions in activity of both SSIII and GBSS (SSIII/GBSS antisense lines). This revealed that fissuring resulted from the activity of GBSS in the SSIII antisense background. Control (untransformed) lines and GBSS and SSIII/GBSS antisense lines had unfissured granules. Starch analyses showed that granules from SSIII antisense tubers had a greater number of long glucan chains than did granules from the other lines, in the form of larger amylose molecules and a unique fraction of very long amylopectin chains. These are likely to result from increased flux through GBSS in SSIII antisense tubers, in response to the elevated content of ADP-glucose in these tubers. It is proposed that the long glucan chains disrupt organization of the semi-crystalline parts of the matrix, setting up stresses in the matrix that lead to fissuring.     

Immunological comparison of the starch branching enzymes from potato tubers and maize kernels

Starch branching enzyme was purified from potato (Solanum tuberosum L.) tubers as a single species of 79 kilodaltons and specific antibodies were prepared against both the native enzyme and against the gel-purified, denatured enzyme. The activity of potato branching enzyme could only be neutralized by antinative potato branching enzyme, whereas both types of antibodies reacted with denatured potato branching enzyme. Starch branching enzymes were also isolated from maize (Zea mays L.) kernels. All of the denatured forms of the maize enzyme reacted with antidenatured potato branching enzyme, whereas recognition by antinative potato branching enzyme was limited to maize branching enzymes I and IIb. Antibodies directed against the denatured potato enzyme were unable to neutralize the activity of any of the maize branching enzymes. Antinative potato branching enzyme fully inhibited the activity of maize branching enzyme I; the neutralized maize enzyme was identified as a 82 kilodalton protein. It is concluded that potato branching enzyme (M_r = 79,000) shares a high degree of similarity with maize branching enzyme I (M_r = 82,000), in the native as well as the denatured form. Cross-reactivity between potato branching enzyme and the other forms of maize branching enzyme was observed only after denaturation, which suggests mutual sequence similarities between these species.     

Evidence that amylose synthesis occurs within the matrix of the starch granule in potato tubers

Transgenic potatoes expressing reduced levels of granule-bound starch synthase I (GBSSI) have been used to investigate whether the synthesis of amylose occurs at the surface of the starch granule or within the matrix formed by the synthesis and organization of amylopectin. Amylose in these potatoes is wholly or largely confined to a central region of the granule. Consequently this core region stains blue with iodine whereas the peripheral zone stains red. By making extensive measurements of the relative sizes of the granules and their blue-staining cores in tubers over a range of stages of development, we have established that the blue core increases in size as the granule grows. The extent of the increase in size of the blue core is greater in potatoes with higher levels of GBSSI. These data show that amylose synthesis occurs within the matrix of the granule, and are consistent with the idea that the space available in the matrix may be an important determinant of the amylose content of storage starches.     

Growth of14C-labelled starch granules in potato tubers as revealed by autoradiographs

Summary Experiments have been presented which indicate that the layers of potato starch granules are built up by a gradual process of apposition. This process is dependent upon the supply of carbohydrates to the amyloplasts. The stripping film technique has made it possible for the first time to make starch granule growth more directly visible.     

Manipulation of starch granule size distribution in potato tubers by modulation of plastid division

Starch granule size is an important parameter for starch applications in industry. Starch granules are formed in amyloplasts, which are, like chloroplasts, derived from proplastids. Division processes and associated machinery are likely to be similar for all plastids. Essential roles for FtsZ proteins in plastid division in land plants have been revealed. FtsZ forms the so-called Z ring which, together with inner and outer plastid division rings, brings about constriction of the plastid. It has been shown that modulation of the expression level of FtsZ may result in altered chloroplast size and number. To test whether FtsZ is also involved in amyloplast division and whether this, in turn, may affect the starch granule size in crop plants, FtsZ protein levels were either reduced or increased in potato. As shown previously in other plant species, decreased StFtsZ1 protein levels in leaves resulted in a decrease in the number of chloroplasts in guard cells. More interestingly, plants with increased StFtsZ1 protein levels in tubers resulted in less, but larger, starch granules. This suggests that the stoichiometry between StFtsZ1 and other components of the plastid division machinery is important for its function. Starch from these tubers also had altered pasting properties and phosphate content. The importance of our results for the starch industry is discussed.     

Sucrose metabolism in cold-stored potato tubers with decreased expression of sucrose phosphate synthase

Transfer of potato tubers to low temperature leads after 2–4 d to a stimulation of sucrose synthesis, a decline of hexose-phosphates and a change in the kinetic properties, and the appearance of a new form of sucrose phosphate synthase (SPS). Antisense and co-suppression transformants with a 70–80% reduction in SPS expression have been used to analyse the contribution of SPS to the control of cold sweetening. The rate of sucrose synthesis in cold-stored tubers was investigated by measuring the accumulation of sugars, by injecting labelled glucose of high specific activity into intact tubers, and by providing 50 mol m^–3 labelled glucose to fresh tuber slices from cold-stored tubers. A 70–80% decrease of SPS expression resulted in a reproducible but non-proportional (10–40%) decrease of soluble sugars in cold-stored tubers, and a non-proportional (about 25%) inhibition of label incorporation into sucrose, increased labelling of respiratory intermediates and carbon dioxide, and increased labelling of glucans. The maximum activity of SPS is 50-fold higher than the net rate of sugar accumulation in wild-type tubers, and decreased expression of SPS in the transformants was partly compensated for increased levels of hexose-phosphates. It is concluded that SPS expression per se does not control sugar synthesis. Rather, a comparison of the in vitro properties of SPS with the estimated in vivo concentrations of effectors shows that SPS is strongly substrate limited in vivo . Alterations in the kinetic properties of SPS, such as occur in response to low temperature, will provide a more effective way to stimulate sucrose synthesis than changes of SPS expression.     

Particulate and soluble forms of o-diphenol oxidase from potato tubers

A soluble and two different particulate forms of o-diphenol oxidase have been obtained from aged or fresh potato slices by differential and density gradient centrifugation. The particulate enzymes were shown to sediment with microsomes and peroxisomes, respectively. Over half the enzyme activity of aged slices was found to be particle bound, with approximately twice as much enzyme in the microsomes as in the peroxisomal fraction. Very similar distribution patterns have been obtained with fresh potatoes, which have an o-diphenol oxidase activity approximately one-third that of aged slices.     

Tissue distribution and change in potato starch phosphorylase mRNA levels in wounded tissue and sprouting tubers.

Starch phosphorylase has been cloned from a lambda gt10 cDNA library of potato tuber mRNA. Selected recombinants have been used to demonstrate that phosphorylase mRNA is most abundant in tubers but is also detectable in stolon, root, stem and leaf tissue. The level of phosphorylase mRNA was greatly reduced in wounded stem and tuber tissue. The wounding-induced decrease in phosphorylase mRNA levels is not reversed in the presence of sucrose or mannitol. Regional differences are described in the levels of phosphorylase and patatin mRNA in different parts of the tuber and in the shoot of sprouting potatoes.