The function of the Waxy locus in starch synthesis in maize endosperm

The soluble adenosine diphosphate glucose-starch glucosyltransferase of maize (Zea mays L.) endosperm uses adenosine diphosphate glucose as a sole substrate, but the starch granule-bound nucleoside diphosphate glucose-starch glucosyltransferase utilizes both adenosine diphosphate glucose and uridine diphosphate glucose. The soluble glucosyltransferase can be bound to added amylose or to maize starch granules that contain amylose. However, binding of the soluble enzyme to the starch granules does not change its substrate specificity to that of the natural starch granule-bound glucosyltransferase. Furthermore, the soluble glucosyltransferase bound to starch granules can be removed by repeated washing without a change in specificity. The bound glucosyltransferase can be released by mechanical disruption of starch granules, and the released enzyme behaves in a manner similar to that of the bound enzyme in several respects. These observations suggest that the soluble and bound glucosyltransferases are different enzymes. The starch granule-bound glucosyltransferase activity is linearly proportional to the number of Wx alleles present in the endosperm. This is compatible with the hypothesis that the Wx allele is a structural gene coding for the bound glucosyltransferase, which is important for the normal synthesis of amylose.Journal Paper No. 4818 of the Purdue University Agricultural Experiment Station.     

Two Additional Phosphorylases in Developing Maize Seeds

Two additional phosphorylases (III and IV) have been detected in developing seeds of maize. Phosphorylase IV is found only in the embryo (with scutellum). It is also present in the embryo of the germinating seed where its activity is 90-fold greater than the activity in the developing embryo 22 days after pollination. Phosphorylase IV is eluted from a DEAE-cellulose column in the same fraction as phosphorylase I of the endosperm, and the 2 enzymes are similar in many respects. Phosphorylase IV is distinguished from phosphorylase I by electrophoretic mobility, by pH optimum, and because its properties are not affected by the shrunken-4 mutation.Phosphorylase III is found both in the endosperms and embryos of developing seeds. Activity for this enzyme is not detected in crude homogenates nor eluates from a DEAE-cellulose column apparently because it complexes with a non-dialyzable, heat-labile inhibitor. High activity is found after protamine sulfate fractionation. Phosphorylase III is bound to protamine sulfate and is then removed by washing with 0.3 m phosphate buffer. Phosphorylase III activity in the endosperm is not detectable 8 days after pollination but is present 12 days after pollination. Phosphorylase III differs from phosphorylases I, II, and IV in several respects-pH optimum, pH-independent ATP inhibition, time of appearance in the endosperm, and because purine and pyrimidine nucleotides are equally inhibitory. In common with phosphorylase II, phosphorylase III apparently does not require a primer to initiate the synthesis of an amylose-like polymer.     

Phosphorylases I and II of Maize Endosperm

Two phosphorylases have been found in the endosperm of Zea mays. Phosphorylase I is found through all stages of endosperm development and seed germination investigated. The other enzyme, phosphorylase II appears only at the stage of rapid starch biosynthesis and is not found during germination. At 22 days after pollination, the activity of phosphorylase II is 10 times that of phosphorylase I. These 2 phosphorylases are separable by column chromatography and behave differently in several respects.     

Starch-synthesizing Enzymes in the Endosperm and Pollen of Maize

Two mutations, amylose-extender and waxy, which affect the proportion of amylose and amylopectin of starch synthesized in the endosperm of maize (Zea mays L.) seeds, are also expressed in the pollen. However, most mutations that affect starch synthesis in the maize endosperm are not expressed in the pollen. In an attempt to understand the nonconcordance between the endosperm and pollen, extracts of mature pollen grains were assayed for a number of the enzymes possibly implicated in starch synthesis in the endosperm. Sucrose synthetase (sucrose-UDP glucosyl transferase, EC 2.4.1.13) activity was not detectable in either mature or immature pollen grains of nonmutant maize, but both bound and soluble invertase (EC 3.2.1.26) exhibited much greater specific activity (per milligram protein) in pollen extracts than in 22-day-old endosperm extracts. Phosphorylase (EC 2.4.1.1) activity was also higher in pollen than in endosperm extracts. ADP-Glucose pyrophosphorylase (EC 2.7.7.27) activity was much lower in pollen than endosperm extracts, but mutations that drastically reduced ADP-glucose pyrophosphorylase activity in the endosperm (brittle-2 and shrunken-2) did not markedly affect enzymic activity in the pollen. Specific activities of other enzymes implicated in starch synthesis were similar in endosperm and pollen extracts.     

EFFECT OF NUTRIENT DEPRIVATION AND RESUPPLY ON METABOLITES AND ENZYMES RELATED TO CARBON ALLOCATION IN GRACILARIA TENUISTIPITATA (RHODOPHYTA)1

The starch content of red algae normally increases during nitrogen limitation. Based on this we hypothesized that nutrient deprivation would result in an increased activity of starch‐synthesizing enzymes and a decrease in the activity of starch‐degrading enzymes, with the opposite scenario when nutrients were sufficient. We therefore examined the effect of the nutrient status of Gracilaria tenuistipitata Chang et Xia on the content of starch and floridoside and on the activity of enzymes involved in the allocation of carbon into starch, floridoside, and agar; floridoside phosphate synthase and α‐galactosidase involved in synthesis and degradation of floridoside; starch synthase and starch phosphorylase involved in the metabolism of starch; uridine 5′‐diphosphate (UDP)‐glucose pyrophosphorylase; adenosine 5′‐diphosphate‐glucose pyrophosphorylase; UDP‐glucose 4‐epimerase; and phosphoglucomutase. During the period of nutrient limitation the starch and floridoside content increased, as did dry weight and C/N ratio, whereas growth rate and protein content decreased. A general decrease in the enzyme activities during nutrient limitation was also observed, indicating a decrease in overall cellular metabolism. The addition of nutrients caused an increase in enzyme activities and a decrease in the contents of starch and floridoside. Of the enzymes examined, only the activity of UDP‐glucose pyrophosphorylase increased during nutrient limitation and decreased abruptly after nutrient addition. This implies a regulatory role for this enzyme in the supply of UDP‐glucose for starch synthesis. It also supports our suggestion that UDP‐glucose is the substrate for starch synthesis in red algae. This assertion is further strengthened by the observation that of the potential starch synthases only the UDP‐glucose starch synthase could support the observed rate of starch synthesis.     

Starch synthetase: Comparison of UDPG and ADPG as glucosyl donors in immature barley endosperm

Summary The activity of starch synthetase in developing barley endosperm was measured in amyloplasts and in the soluble endosperm fraction by incorporation of radioactively labelled glucose into starch. Both uridine diphosphate glucose (UDPG) and adenosine diphophate glucose (ADPG) were used as glucosyl donors. Enzyme activity was initially located in the soluble fraction, but increasing activity could be detected in the amyloplast fraction during endosperm maturation.     

Effects of abscisic acid (ABA) on grain filling processes in wheat

The effect of in situ water stress on the endogenous abscisic acid (ABA) content of the endosperm and the in vitro application of ABA on some important yield regulating processes in wheat have been studied. Water stress resulted in a marked increase in the ABA content of the endosperm at the time close to cessation of growth. Application of ABA to the culture medium of detached ears reduced grain weight. Exogenously applied ABA, at the highest concentration (0.1 mM) reduced transport of sucrose into the grains and lowered the starch synthesis ability of intact grains. In vitro sucrose uptake and conversion by isolated grains was stimulated by low ABA concentrations (0.001 mM) in the medium but was inhibited by higher concentrations. ABA application had no effect on sucrose synthase (SS) and uridine diphosphate glucose pyrophosphorylase (UDP-Gppase) activities, whereas adenosine diphosphate glucose pyrophosphorylase (ADP-Gppase), soluble starch synthase (SSS), and granule-bound starch synthase (GBSS) activities were reduced. These results raise the possibility that water stress-induced elevated levels of endogenous ABA contribute to reduced grain growth.     

Measurement of Metabolites Associated with Nonaqueously Isolated Starch Granules from Immature Zea mays L. Endosperm

Starch granules with associated metabolites were isolated from immature Zea mays L. endosperm by a nonaqueous procedure using glycerol and 3-chloro-1,2-propanediol. The soluble extract of the granule preparation contained varying amounts of neutral sugars, inorganic phosphate, hexose and triose phosphates, organic acids, adenosine and uridine nucleotides, sugar nucleotides, and amino acids. Based on the metabolites present and on information about translocators in chloroplast membranes, which function in transferring metabolites from the chloroplast stroma into the cytoplasm, it is suggested that sucrose is degraded in the cytoplasm, via glycolysis, to triose phosphates which cross the amyloplast membrane by means of a phosphate translocator. It is further postulated that hexose phosphates and sugars are produced from the triose phosphates in the amyloplast stroma by gluconeogenesis with starch being formed from glucose 1-phosphate via pyrophosphorylase and starch synthase enzymes. The glucose 1-phosphate to inorganic phosphate ratio in the granule preparation was such that starch synthesis by phosphorylase is highly unlikely in maize endosperm.     

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.     

Biosynthesis of Starch in Proplastids of Germinating Ricinus communis Endosperm Tissue

Electron photomicrographs of endosperm tissue from germinating seed of Ricinus communis L. cv. Hale show proplastids which contain prominent starch grains. The content of starch in endosperm tissue increased from 500 micrograms per seed, in imbibed seed, to 1,100 micrograms per seed in 5-day-old seedlings. The maximum net rate of starch deposition was 1.1 nanomoles glucose incorporated per minute per seed. About 200 micrograms of starch remained in the endosperm 9 days after imbibition. Starch content followed the same developmental pattern as the content of sucrose, free reducing sugars, and other metabolic processes found in this tissue. Two key enzymes of starch synthesis, adenosine diphosphoglucose (ADPG) pyrophosphorylase and ADPG-starch glucosyl transferase (starch synthetase) exhibited maximum activities at 4 and 5 days after germination, respectively. The maximum activity of ADPG pyrophosphorylase was 8.17 nanomoles ADPG formed per minute per seed, whereas starch synthetase exhibited an activity of 125 nanomoles glucose incorporated per minute per seed. These levels of enzyme activity are sufficient to account for the starch synthesis observed. Other enzymes which may be involved in starch synthesis include 3-phosphoglycerate kinase which showed an activity of 8.76 units per seed, triose-P isomerase (2.56 units per seed), fructose-1,6-bisphosphate aldolase (0.99 units per seed), fructose-1,6-bisphosphatase (0.23 units per seed), phosphoglucose isomerase (12.6 units per seed), and phosphoglucomutase (9.72 units per seed). The activities of these enzymes were similar to previously reported values.

Starch synthetase was found in association with the fraction containing proplastids isolated from endosperm tissue. Of the total starch synthetase activity in the endosperm, 38% was particulate. Forty-four% of the total particulate activity of starch synthetase placed on sucrose gradients was associated with the band containing proplastids. The proplastids contained 98% of the ribulose 1,5-bisphosphate carboxylase carboxylase activity placed on the gradient.

     

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.     

Sucrose and starch metabolism during Fargesia yunnanensis shoot growth

Bamboo is one of the fastest growing plants in the world, but their shoot buds develop very slowly. Information about the sugar storage and metabolism during the shoot growth is lacking. In the present study, we determined the activity of sucrose and starch metabolizing enzymes during the developmental period of Fargesia yunnanensis from shoot buds to the young culms that have achieved their full height. The soluble sugars and starch contents were also determined and analyzed in shoot buds and shoots at different developmental stages. The results showed that there were higher sucrose contents in shoot buds than shoots, which coincides with the sweeter taste of shoot buds. As the shoot buds sprouted out of the ground, the starch and sucrose were depleted sharply. Coupled with this, the activity of soluble acid invertase (SAI), cell wall-bound invertase (CWI), sucrose synthase at cleavage direction (SUSYC) and starch phosphorylase (STP) increased significantly in the rapidly elongating internodes. These enzymes dominated the rapid elongation of internodes. The activities of SAI, CWI, SUSYC and STP and adenosine diphosphate-glucose pyrophosphorylase were higher as compared to other enzymes in the shoot buds, but were far lower than those in the developing shoots. The slow growth of shoot buds was correlated with the low activity of these enzymes. These results complement our understanding of the physiological differences between shoot buds and elongating shoots and ascertain the physiological mechanism for the rapid growth of bamboo shoots.     

Regulation of starch biosynthesis in normal and opaque-2 maize during endosperm development

The absolute activities of sucrose-UDP glucosyltransferase, glucose-6-phosphate ketoisomerase and soluble and bound ADPG-starch glucosyltransferase have been studied in normal and Opaque-2 maize endosperms during development. In general, the activities of these enzymes except sucrose-UDP glucosyltransferase were higher up to 20 days post-pollination and lower at the 30 day stage in Opaque-2 than in normal maize endosperms. However, sucrose-UDP glucosyltransferase activity was higher in normal maize endosperm up to the 20 day stage while it was lower at subsequent stages than in Opaque-2. It is suggested that the lower level of these enzymes, except sucrose-UDP glucosyltransferase, might be responsible for the reduced accumulation of starch in Opaque-2 endosperm during later stages of endosperm development.     

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.     

The complexities of starch biosynthesis in cereal endosperms

Starch serves not only as an energy source for plants, animals, and humans but also as an environmentally friendly alternative for fossil fuels. Here, we describe recent findings concerning the synthesis of this important molecule in the cereal endosperm. Results from six separate transgenic reports point to the importance of adenosine diphosphate glucose pyrophosphorylase in controlling the amount of starch synthesized. The unexpected cause underlying the contrast in sequence divergence of its two subunits is also described. A major unresolved question concerning the synthesis of starch is the origin of nonrandom or clustered alpha-1,6 branch-points within the major component of starch, amylopectin. Developing evidence that several of the starch biosynthetic enzymes involved in amylopectin synthesis occur in complexes is reviewed. These complexes may provide the specificity for the formation of nonrandom branch-points.     

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.     

Spinach Leaf Intra and Extra Chloroplast Phosphorylase Activities during Growth

The amino terminal sequence of the spinach (Spinacia oleracea L. cv Bloomsdale Long Standing) leaf cytoplasmic phosphorylase was determined and shown to have little similarity to the known sequence of the potato tuber phosphorylase. The antigenic reaction of spinach chloroplast phosphorylase and rabbit muscle phosphorylase a to antiserum prepared against spinach leaf cytoplasmic phosphorylase was tested. Neither phosphorylase gave a positive reaction when tested by immunodiffusion or neutralization of enzyme activity. The two spinach phosphorylases were assayed throughout the growth of the plant. Activity of cytoplasmic phosphorylase increased 4- to 8-fold at 30 to 35 days from sowing. Enzyme protein levels, as measured by antibody neutralization, increased by a similar amount. There was no corresponding increase in chloroplast phosphorylase activity. The chloroplast phosphorylase varied in parallel with the chloroplast enzyme ADPglucose pyrophosphorylase. Starch levels were high during the earlier stages of growth and then fell to a constant low level just before the increase in cytoplasmic phosphorylase. The results are discussed with respect to the relationship and functions of the two phosphorylases.     

Complementation of subunits from glycogen phosphorylases of frog and rabbit skeletal muscle and rabbit liver.

Activity can be induced in potentially active rabbit skeletal muscle phosphorylase monomers covalently bound to Sepharose by noncovalent interaction with soluble subunits carrying inactive pyridoxal 5'-phosphate analogs or even salicyladlehyde. These analogs are themselves incapable of reconstituting active holophorphorylase from apophosphorylase. Phosphorylases with one intrinsically inactive and one potentially active subunit have about one half of the activity of the native phosphorylase dimer. The usefulness of this technique for subunit complementation was demonstrated by forming hybrid phosphorylases with inactive Sepharose-bound rabbit skeletal muscle subunits containing pyridoxal 5'-phosphate monomethylester and soluble activatable frog muscle and rabbit liver phosphorylase monomers. The inactive Sepharose-bound subunit induced in each case activity in the soluble subunit. But whereas the inactive rabbit muscle phosphorylase subunit even transmitted its characteristic temperature dependence of the rate of the reaction to the frog muscle subunit, it could not propagate its control properties to the liver enzyme. Differences of hybrid phosphorylases are related to immunological and amino acid divergencies among the component enzymes.