Starch Synthesis in Developing Potato Tubers

The activities of enzymes involved in starch metabolism were measured at intervals during tuberization and the early stages of tuber growth in Solanum tubersum grown in water culture under controlled environmental conditions. Starch synthase, ADPglucose pyrophosphorylase, UDPglucose pyrophosphorylase and phosphorylase activities all increased during tuber development, the most pronounced increases occurring in the activities of ADP-glucose pyrophosphorylase and phosphorylase. The activity ratio ADPglucose pyrophosphorylase/phosphorylase was lowest in slow growing tubers and hightest in fast growing tubers. In addition, high sugar concentrations in fast growing tubers and low sugar concentrations in slow growing tubers suggested that enzyme levels might be influenced by sugar concentration. The activities of starch synthase, phosphorylase and ADPglucose pyrophosphorylase were increased 2–2.5 fold by the presence of 100 mM K⁺. It is concluded that the major enzyme changes occur as a consequence of tuber initiation and that starch accumulation is controlled, at least in part, by the activities of ADPglucose pyrophosphorylase and phosphorylase.     

Enzymes of starch metabolism in leaves and berries of Vitis vinifera

Leaves of Vitis vinifera L., cv. Cabernet Sauvignon contained 2.0 mg of starch per g fresh weight, whereas young green berries and maturing grape berries contained less than 0.03 mg of starch, despite the presence of abundant substrates (reducing sugars and sucrose) in berries for starch synthesis. the activities of several enzymes likely to be involved in starch synthesis were determined in extracts of berries and leaves. Fractionation procedures resulted in final recoverable ADPglucose-starch glucosyltransferase activity which was 2–3 times the activity measured in crude extracts of leaves. Compared to leaves, berries contained low activities of ADPglucose-starch glucosyltransferase and ADPglucose pyrophosphorylase. These enzymes increased only 2- to 3-fold from young to maturing berries. ADPglucose-starch glucosyltransferase activity in the absence of added primer was found in leaf extracts but not in berry extracts. The activities of UDP-glucose pyrophosphorylase, phosphorylase and amylase were comparable in both leaves and berries and increased 6- to 7-fold during berry development. The low activities of ADPglucose-starch glucosyltransferase and ADPglucose pyrophosphorylase probably account for the paucity of starch in grape berries.     

Sucrose-to-Starch Metabolism in Tomato Fruit Undergoing Transient Starch Accumulation

Immature green tomato (Lycopersicon esculentum) fruits undergo a period of transient starch accumulation characterized by developmental changes in the activities of key enzymes in the sucrose (Suc)-to-starch metabolic pathway. Activities of Suc synthase, fructokinase, ADP-glucose (Glc) pyrophosphorylase, and soluble and insoluble starch synthases decline dramatically in parallel to the decrease in starch levels in the developing fruit. Comparison of "maximal" in vitro activities of the enzymes in the Suc-to-starch pathway suggests that these same enzymes are limiting to the rate of starch accumulation. In contrast, activities of invertase, UDP-Glc pyrophosphorylase, nucleoside diphosphate kinase, phosphoglucoisomerase, and phosphoglucomutase do not exhibit dramatic decreases in activity and appear to be in excess of starch accumulation rates. Starch accumulation is spatially localized in the inner and radial pericarp and columella, whereas the outer pericarp and seed locule contain little starch. The seed locule is characterized by lower activities of Suc synthase, UDP-Glc pyrophosphorylase, phosphoglucomutase, ADP-Glc pyrophosphorylase, and soluble and insoluble starch synthases. The outer pericarp exhibits comparatively lower activities of ADP-Glc pyrophosphorylase and insoluble starch synthase only. These data are discussed in terms of the developmental and tissue-specific coordinated control of Suc-to-starch metabolism.     

Regulation of Starch Synthesis in the Bundle Sheath and Mesophyll of Zea mays L. : Intercellular Compartmentalization of Enzymes of Starch Metabolism and the Properties of the ADPglucose Pyrophosphorylases

The intercellular localization of enzymes involved in starch metabolism and the kinetic properties of ADPglucose pyrophosphorylase were studied in mesophyll protoplasts and bundle sheath strands separated by cellulase digestion of Zea mays L. leaves. Activities of starch synthase, branching enzyme, and ADPglucose pyrophosphorylase were higher in the bundle sheath, whereas the degradative enzymes, starch phosphorylase, and amylase were more evenly distributed and slightly higher in the mesophyll. ADPglucose pyrophosphorylase partially purified from the mesophyll and bundle sheath showed similar apparent affinities for Mg²⁺, ATP, and glucose-1-phosphate. The pH optimum of the bundle sheath enzyme (7.0-7.8) was lower than that of the mesophyll enzyme (7.8-8.2). The bundle sheath enzyme showed greater activation by 3-phosphoglycerate than did the mesophyll enzyme, and also showed somewhat higher apparent affinity for 3-phosphoglycerate and lower apparent affinity for the inhibitor, orthophosphate. The observed activities of starch metabolism pathway enzymes and the allosteric properties of the ADPglucose pyrophosphorylases appear to favor the synthesis of starch in the bundle sheath while restricting it in the mesophyll.     

Enzymes involved in starch synthesis in the developing mung bean seed

The levels of free sugars, starch and enzymes involved in starch metabolism—sucrose synthetase, UDP and ADP glucose pyrophosphorylase, phosphorylase and starch synthetase—were assayed during seed development of three cultivars of mung bean (Vigna radiata). Free sugars and starch increased with increasing seed weight. Changes in levels of sucrose synthetase, UDP- and ADP-glucose pyrophosphorylases, and phosphorylase were paralleled by changes in starch accumulation. After the maximum activity levels of these enzymes had been reached, maximum activities of soluble starch synthetase and starch granule-bound starch synthetase occurred. There were high activities of sucrose synthetase and phosphorylase at maximum rates of starch accumulation. Thus, starch could be synthesized via the ADP glucose pathway in mung bean seeds. However, phosphorylase may account for the starch accumulation in the early stages of mung bean seed development.     

Evaluation of breeding utility of a chromosomal segment from Lycopersicon chmielewskii that enhances cultivated tomato soluble solids

A chromosomal segment from the wild tomato species, Lycopersicon chmielewskii, when backcrossed into the tomato cultivar VF145B-7879, increases fruit soluble solids concentration. In the investigation reported here, the near-isoline (VF145B-7M) homozygous for the chmielewskii (chm) chromosomal segment was crossed to the cultivar to study allelic interactions and then hybridized to three commercial cultivars (UC204c, E6203, and ChicoIII) to investigate the effect of this segment when heterozygous in different genetic backgrounds. Parents, isogenic hybrids, and unmodified hybrid controls were evaluated in three consecutive years of field study. The chm segment, when either homozygous or heterozygous, significantly increased soluble solids concentration by 13% and 12%, respectively, over VF145B-7879 (esc/esc), suggesting dominant gene action. Averaged across the three isohybrids and one isoline, one dose of this segment significantly increased soluble solids concentration by 6.0% compared to the unmodified hybrids and VF145B-7879. Other than increasing fruit total and soluble solids in mature-green and ripe-red tomatoes, no consistent negative effects of the chmielewskii segment were observed in the various genetic backgrounds on plant yield, fruit weight, or fruit pH. These results favor the use of this segment in breeding programs to develop fresh market and processing tomato cultivars with enhanced quality and reduced processed product dehydration costs. Received: 15 April 2000 / Accepted: 13 January 2001     

Enzymes of starch and sucrose metabolism in Zea mays leaves

Mesophyll and bundle sheath cells of maize leaves were separated and enzymes of starch and sucrose metabolism assayed. The starch content and activities of ADPglucose (ADPG) starch synthetase and phosphorylase expressed both on a chlorophyll and a protein basis were much lower in mesophyll cells compared to bundle sheath preparations. Exposure of the leaves to continuous illumination for 2·5 days caused the starch content of mesophyll cells to rise greatly and led to considerable increases in ADPG starch synthetase and phosphorylase activity. In glasshouse grown leaves the bulk of invertase, sucrose phosphate synthetase, sucrose phosphatase, UDPglucose pyrophosphorylase and amylase was situated in the mesophyll layer. Sucrose synthetase, ADPG starch synthetase and phosphorylase were largely confined to the bundle sheath. No enzyme could be completely assigned to one particular cell layer. Upon continuous illumination both ADPG starch synthetase and phosphorylase increased in the mesophyll bythe same relative amount. The mesophyll is likely to be a major site for sucrose synthesis in maize leaves.     

Subcellular localization of the starch degradative and biosynthetic enzymes of spinach leaves

The subcellular localization of the starch biosynthetic and degradative enzymes of spinach leaves was carried out by measuring the distribution of the enzymes in a crude chloroplast pellet and soluble protein fraction, and by the separation on sucrose density gradients of intact organelles, chloroplasts, peroxisomes, and mitochondria of a protoplast lysate. ADP-Glucose pyrophosphorylase, starch synthase, and starch-branching enzymes are quantitatively associated with the chloroplasts. The starch degradative enzymes amylase, R-enzyme (debranching activity), phosphorylase, and D-enzyme (transglycosylase) are observed both in the chloroplast and soluble protein fractions, the bulk of the degradative enzyme activities reside in the latter fraction. Chromatography of a chloroplast extract on diethylaminoethyl-cellulose resolves the R- and D-enzymes from amylase and phosphorylase activities although the two latter enzyme activities coeluted. The digestion pattern of amylase with amylopectin as a substrate indicates an endolytic activity but displays properties unlike the typical α-amylase as isolated from endosperm tissue.     

‘台农1号’芒果果实发育过程中的糖分积累与相关酶活性研究

以‘台农1号’芒果为材料,测定了果实生长发育过程中淀粉、蔗糖、葡萄糖和果糖含量以及淀粉酶、蔗糖代谢相关酶———酸性转化酶(AI)、中性转化酶(NI)、蔗糖合成酶(SS)和蔗糖磷酸合成酶(SPS)的活性,并对果实中糖组分与酶活性的关系进行了分析.结果显示,(1)台农1号芒果果实属于单S型生长曲线,发育前期主要积累淀粉、葡萄糖和果糖,果实成熟软化时,淀粉酶活性降至最低,淀粉水解,蔗糖快速积累.(2)酸性转化酶活性在果实整个发育过程中维持最高,完熟时略有降低;蔗糖磷酸合成酶在果实发育前期略有降低,完熟时升至最高;蔗糖合成酶和中性转化酶活性在整个发育期一直很低且较稳定.(3)淀粉含量与淀粉酶活性呈显著正相关,与SPS活性呈极显著负相关,蔗糖、葡萄糖含量均与SPS、SS呈显著、极显著的正相关;果糖含量与SS呈极显著的正相关.研究表明,芒果成熟时淀粉分解、酸性转化酶活性的降低,且蔗糖合成酶和蔗糖磷酸合成酶活性的增加是引起果实蔗糖积累的主要因子.     

Light Regulation of Sink Metabolism in Tomato Fruit : II. Carbohydrate Metabolizing Enzymes

Effects of light on carbohydrate levels and certain carbon metabolizing enzyme activities were studied during the early development of tomato (Lycopersicon esculentum) fruit. Sucrose levels were low and continued to decline during development and were unaffected by light. Starch was significantly greater in light. Invertase activity was similar in both light- and dark-grown fruit. Sucrose synthase activity was much lower than invertase and showed a slight decrease in light-grown fruit between days 21 and 28. Light-grown fruit also had higher ADP glucose pyrophosphorylase activity than dark-grown fruit, which was correlated with higher starch levels. The rapidly decreasing activity of ADP glucose pyrophosphorylase during early fruit development in the dark in conjunction with reduced starch levels and rates of accumulation indicates that ADP glucose pyrophosphorylase is crucial for carbon import and storage in tomato. The differential stimulation of ADP glucose pyrophosphorylase activity from light- and dark-grown tissue by 3-phosphoglycerate suggests that this enzyme may be allosterically altered by light.     

Enzymes of starch and sugar phosphate metabolism in achlorophyllous ribosome-deficient plastids from high-temperature-grown rye leaves

Several enzymes of non–photosynthetic sugar phosphate and starch metabolism were measured in gradient–purified chloroplasts from normal rye leaves ( Secale cereale L. cv. Halo) grown at 22°C and in the non-photosynthetic plastids isolated from 70S ribosome-deficient rye leaves grown at a non–permissive elevated temperature of 32°C. Activities of the enzymes phosphoglycerate kinase (EC 2.7.2.3), hexokinase (EC 2.7.1.1), phosphoglucose isomerase (EC 5.3.1.9), phosphoglucomutase (EC 2.7.5.1), glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate de-hydrogenase (EC 1.1.1.46), ADPglucose pyrophosphorylase (EC 2.7.7.27), starch synthase (EC 2.4.1.21), and phosphorylase (EC 2.4.1.1) were present in ribosome-deficient plastids from 32°C-grown leaves indicating a cytoplasmic origin of the plastid-specific forms of these enzymes. While the photosynthetic marker enzyme NADP⁺-dependent glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) was considerably diminished, both the specific activities and the total activities per leaf of the plastid-specific forms of hexokinase, phosphoglucose isomerase and phosphoglucomutase were markedly increased in the ribosome–deficient plastids, relative to normal chloroplasts. The results demonstrate that after elimination of functional protein synthesis in the chloroplasts the supply of chloroplast–specific enzymes by the cytoplasm is not generally suppressed as observed for many enzymes and proteins involved in photosynthesis, but may even be increased in accord with changed metabolic demands.     

Nucleotide sugars and starch synthesis in spadix of Arum maculatum and suspension cultures of Glycine max

The aim of this work was to determine the relative contributions of ADPglucose and UDPglucose to starch synthesis in two non-photosynthetic tissues, the developing club of the spadix of Arum maculatum and suspension cultures of Glycine max. Rates of starch accumulation during growth are compared with estimates of the maximum catalytic activities in vitro of ADPglucose starch synthase, ADPglucose pyrophosphorylase, UDPglucose pyrophosphorylase and UDPglucose starch synthase. The latter could only be measured at high concentrations (10–30 mM) of UDPglucose. Clubs of Arum and cells of Glycine contained 292 and 6.8 nmol UDPglucose per gram fresh weight, respectively. The corresponding figures for ADPglucose were 29 and 0.4. From the above data it is argued that in both Arum club and Glycine cells the activity of UDPglucose starch synthase is too low to make any quantitatively significant contribution to starch synthesis. The activities of ADPglucose starch synthase and pyrophosphorylase were high enough to mediate the observed rates of starch accumulation. It is suggested that starch synthesis in these tissues is via ADPglucose.     

Girdling affects carbohydrate-related gene expression in leaves,bark and roots of alternate-bearing citrus trees

Effects of girdling on carbohydrate status and carbohydrate-related gene expression in citrus trees were investigated. Alternate-bearing 'Murcott' (a Citrus reticulata hybrid of unknown origin) trees were girdled during autumn (25 Sep. 2001) and examined 10 weeks later. Girdling brought about carbohydrate (soluble sugar and starch) accumulation in leaves and shoot bark above the girdle, in trees during their fruitless, 'off' year. Trees during their heavy fruit load, 'on' year did not accumulate carbohydrates above the girdle due to the high demand for carbohydrates by the developing fruit. Girdling caused a strong decline in soluble sugar and starch concentrations in organs below the girdle (roots), in both 'on' and 'off' trees. Expression of STPH-L and STPH-H (two isoforms of starch phosphorylase), Agps (ADP-glucose pyrophosphorylase, small subunit), AATP (plastidic ADP/ATP transporter), PGM-C (phosphoglucomutase) and CitSuS1 (sucrose synthase), all of which are associated with starch accumulation, was studied. It was found that gene expression is related to starch accumulation in all 'off' tree organs. RNA levels of all the genes examined were high in leaves and bark that accumulated high concentrations of starch, and low in roots with declining starch concentrations. It may be hypothesized that changes in specific sugars signal the up- and down-regulation of genes involved in starch synthesis.     

Leaf Starch Metabolism during the Growth of Pepper (Capsicum annuum) Plants

Levels of starch and sugars, and the activities of amylase and starch phosphorylase were measured in expanding leaves harvested in early morning and early evening during the growth of pepper (Capsicum annuum L.; cv `Bellboy') plants. The differences between starch levels 1 hour after dawn and 1 hour after dusk increased during the period of initial fruit expansion. This diurnal starch difference was strongly correlated with post-dusk amylase activities in leaves at both stages of expansion. There was also a strong correlation between levels of amylase in immature and those in mature leaves throughout the experiment. Phosphorylase activity showed no direct relationship to leaf starch levels, and there was no similarity between activities in immature and mature leaves. An increase in photosynthesis during plant development was observed which could account for the increased starch synthesis at initial fruit expansion.     

Contrasted Responses to Root Hypoxia in Tomato Fruit at Two Stages of Development

The biochemical consequences of root hypoxia have been documented in many sink organs, but not extensively in fruit. Therefore, in the present study, the response to root hypoxia in tomato fruit (Solanum lycopersicum L.) was investigated at two developmental stages, during the cell division and the cell expansion phases. Our results showed that in dividing fruit, root hypoxia caused an exhaustion of carbon reserves and proteins. However, ammonium and major amino acids (glutamine, asparagine and γ–aminobutyric acid (GABA)) significantly accumulated. In expanding fruit, root hypoxia had no effect on soluble sugar, protein and glutamine contents, whereas starch content was significantly decreased, and asparagine and GABA contents slightly increased. Metabolite contents were well correlated with activities of the corresponding metabolising enzymes. Contrary to nitrogen metabolising enzymes (glutamine synthetase, asparagine synthetase and glutamate decraboxylase), the activities of enzymes involved in sugar metabolism (invertase, sucrose synthase, sucrose phosphate synthase and ADP glucose pyrophosphorylase) were significantly reduced by root hypoxia, in diving fruit. In expanding fruit, only a slight decrease in ADP glucose pyrophosphorylase and an increase in asparagine synthetase and glutamate decarboxylase activities were observed. Taken together, the present data revealed that the effects of root hypoxia are more pronounced in the youngest fruits as it is probably controlled by the relative sink strength of the fruit and by the global disturbance in plant functioning.     

Genes from Lycopersicon chmielewskii affecting tomato quality during fruit ripening

Three chromosomal segments from the wild tomato, L. chmielewskii, introgressed into the L. esculentum genome have been previously mapped to the middle and terminal regions of chromosome 7 (7M, 7T respectively), and to the terminal region of chromosome 10 (10T). The present study was designed to investigate the physiological mechanisms controlled by the 7M and 7T segments on tomato soluble solids (SS) and pH, and their genetic regulation during fruit development. The effects of 7M and 7T were studied in 64 BC₂F₅ backcross inbred lines (BILs) developed from a cross between LA 1501 (an L. esculentum line containing the 7M and 7T fragments from L. chmielewskii), and VF145B-7879 (a processing cultivar). BILs were classified into four homozygous genotypes with respect to the introgressed segments based on RFLP analysis, and evaluated for fruit chemical characteristics at different harvest stages. Gene(s) in the 7M fragment reduce fruit water uptake during ripening increasing pH, sugars, and SS concentration. Gene(s) in the 7T fragment were found to be associated with higher mature green fruit starch concentration and red ripe fruit weight. Comparisons between tomatoes ripened on or off the vine suggest that the physiological mechanisms influenced by the L. chmielewskii alleles are dependent on the translocation of photosynthates and water during fruit ripening.     

Inhibition of fructokinase and sucrose synthase by cytosolic levels of fructose in young tomato fruit undergoing transient starch synthesis

Immature tomato fruit are characterized by a transient period of starch accumulation. Sucrose synthase (EC 2.4,1.13) and fructokinase (EC 2.7,1.4) are two of the initial enzymes in the sucrose to starch synthetic pathway. Both enzymes in tomato fruit are significantly inhibited by fructose at concentrations physiological to young tomato fruit. Compartmental analysis of immature fruit pericarp indicates that fructose is not specifically compartmentalized in the vacuole and that physiological cytosolic concentrations of fructose in young tomato fruit are above 30 m M . Such physiological levels of fructose significantly inhibit sucrose synthase cleavage activity as well as the activity of a partially purified fructokinase. These data suggest a mechanism of a coordinated, in vivo regulation of tomato sucrose synthase and fructokinase activity, which may be potentially limiting to starch accumulation in young tomato fruit.     

Starch metabolism in developing strawberry (Fragaria × ananassa) fruits

Fruit starch reserves can be an important contributor to the sugar content of some ripe fruit, and despite the relatively high financial premiums (compared to other fruit) commanded by ripe strawberries, neither their starch or sugar biochemistry has been examined in detail. This study assessed the rate of starch biosynthesis and breakdown in developing strawberry and sought to determine the temporal changes in the activities of selected enzymes known to be involved in sucrose-starch interconversions. Scanning electron microscopy revealed that starch levels appeared greatest in immature strawberry ( Fragaria × ananassa , cv. Elsanta) at 7 days postanthesis, as evidenced by a decrease in the number of cells containing starch granules as ripening progressed. Levels of key enzymes of starch and sugar metabolism estimated using Western blotting and enzyme activity analysis showed that activities did not correlate with antigen levels. In particular, enzyme activity recovery experiments indicated that losses were due to non-proteinaceous inhibitors, and in particular protein binding: highlighting the potential for misinterpretation of enzyme activity data gathered from ripening (strawberry) fruit tissue extracts. Consequently, in vitro experiments using [U-¹⁴C] glucose revealed that incorporation to starch is low (11%) at the earliest developmental stages when starch content is greatest. Starch synthesis rate then declines to non-detectable levels as fruit expand and ripen. These results show that starch accumulates extremely early in the fruit formation process and that starch degradation predominates during fruit growth and development. We estimate that breakdown of transient starch can contribute up to 3% of the sugar accumulated in ripe fruit.     

Identification of restriction fragment length polymorphisms linked to genes controlling soluble solids content in tomato fruit

Summary Gene(s) conferring high soluble solids (SS) in tomato fruit had been backcrossed previously from a wild tomato species, Lycopersicon chmielewskii LA1028 ( 10% SS), into a L. esculentum cultivar, VF36 ( 5% SS), to derive a BC₅S₅ line, LA1563, similar to VF 36 but with 7–8% SS. DNAs from these lines and a tomato breeding line, H2038, were screened for restriction fragment length polymorphisms (RFLPs) using four restriction endonucleases and sixty clones chosen at random from a tomato cDNA library. Most of the cDNA clones (56) identified the same RFLP in VF 36 and LA1563 and a different RFLP in LA1028. However, two cDNA clones identified the same RFLP in LA1563 and LA1028 and a different RFLP in VF36. To determine whether RFLPs identified by these two cDNA clones were linked to SS genes, a H2038 x LA1563 F₂ population was screened for segregation of the RFLPs and for SS content. The segregation ratios of these RFLPs were consistent with ratios expected for codominant alleles at unlinked loci. Analysis of variance of SS content for different RFLP genotypic classes indicated that RFLP alleles at one of the loci were linked to genes controlling SS content. The RFLP allele from the high SS tomato line, LA1563, was associated with significantly higher SS content and, therefore, could be useful in selecting for high SS gene(s) in a tomato breeding program.     

A Starch Deficient Mutant of Arabidopsis thaliana with Low ADPglucose Pyrophosphorylase Activity Lacks One of the Two Subunits of the Enzyme

A starch deficient mutant of Arabidopsis thaliana (L.) Heynh. has been isolated in which leaf extracts contain only about 5% as much activity of ADPglucose pyrophosphorylase (EC 2.7.7.27) as the wild type. A single, nuclear mutation at a previously undescribed locus designated adg2 is responsible for the mutant phenotype. Although the mutant contained only 5% as much ADPglucose pyrophosphorylase activity as the wild type, it accumulated 40% as much starch when grown in a 12 hour photoperiod. The mutant also contained about 40% as much starch as the wild type when grown in continuous light, suggesting that the rate of synthesis regulates its steady state accumulation. Immunological analysis of leaf extracts using antibodies against the spinach 54 and 51 kilodalton (kD) ADPglucose pyrophosphorylase subunits indicated that the mutant is deficient in a cross-reactive 54 kD polypeptide and has only about 4% as much as the wild type of a cross-reactive 51 kD polypeptide. This result and genetic studies suggested that adg2 is a structural gene which codes for the 54 kD polypeptide, and provides the first functional evidence that the 54 kD polypeptide is a required component of the native ADPglucose pyrophosphorylase enzyme.