Enzymes of sucrose and hexose metabolism in developing kernels of two inbreds of maize

Tissue distribution and activity of enzymes involved in sucrose and hexose metabolism were examined in kernels of two inbreds of maize (Zea mays L.) at progressive stages of development. Levels of sugars and starch were also quantitated throughout development. Enzyme activities studied were: ATP-linked fructokinase, UTP-linked fructokinase, ATP-linked glucokinase, sucrose synthase, UDP-Glc pyrophosphorylase, UDP-Glc dehydrogenase, PPi-linked phosphofructokinase, ATP-linked phosphofructokinase, NAD-dependent sorbitol dehydrogenase, NADP-dependent 6-P-gluconate dehydrogenase, NADP-dependent Glc-6-P dehydrogenase, aldolase, phosphoglucoisomerase, and phosphoglucomutase. Distribution of invertase activity was examined histochemically. Hexokinase and ATP-linked phosphofructokinase activities were the lowest among these enzymes and it is likely that these enzymes may regulate the utilization of sucrose in developing maize kernels. Most of the hexokinase activity was found in the endosperm, but the embryo had high activity on a dry weight basis. The endosperm, which stores primarily starch, contained high PPi-linked phosphofructokinase and low ATP-linked phosphofructokinase activities, whereas the embryo, which stores primarily lipids, had much higher ATP-linked phosphofructokinase activity than did the endosperm. It is suggested that PPi required by UDP-Glc pyrophosphorylase and PPi-linked phosphofructokinase in the endosperm may be supplied by starch synthesis. Sorbitol dehydrogenase activity was largely restricted to the endosperm, whereas 6-P-gluconate and Glc-6-P dehydrogenase activities were highest in the base and pericarp. A possible metabolic pathway by which sucrose is converted into starch is proposed.     

Selective measurement of starch synthesizing enzymes in permeabilized potato tuber slices

Osmotically permeabilized potato (Solanum tuberosum L.) tuber slices were used to study the biosynthesis of starch under semi in vivo conditions. Criteria to distinguish the various enzymes involved in starch biosynthesis were developed based on the characteristics of the enzymes in in vitro experiments. Branching enzyme activity was inhibited at pH 8.5 or higher, while the starch synthases functioned optimally between pH 8.8 and 9.1. Unprimed soluble starch synthase activity was only apparent in the presence of sodium citrate (0.4 molar or higher). Granulebound and primed soluble starch synthase were active in the absence of sodium citrate. Primed soluble starch synthase activity was susceptible to inhibition by 10 millimolar zinc sulfate, while granule-bound starch synthase activity was not. The incorporation of the Glc moiety of ADP-Glc into starch in tissue slices by the various starch synthases was consistent with in vitro data with respect to the affinity of the enzymes for substrate, the pH profile, the stimulation by citrate, and the inhibition by zinc sulfate. These data were used to determine the activity of each of the starch synthases in tissue slices: granule-bound and soluble starch synthase transferred 37 and 55 picomoles ADP-Glc per hour per milligram fresh weight into starch of permeabilized tissue slices at 30°C and pH 9.1. In the presence of 0.5 molar sodium citrate, at least 40 picomoles ADP-Glc per hour per milligram fresh weight as transferred into starch by unprimed soluble starch synthase activity.     

Carbohydrate Content and Enzyme Metabolism in Developing Canola Siliques

Little biochemical information is available on carbohydrate metabolism in developing canola (Brassica napus L.) silique (pod) wall and seed tissues. This research examines the carbohydrate contents and sucrose (Suc) metabolic enzyme activities in different aged silique wall and seed tissues during oil filling. The silique wall partitioned photosynthate into Suc over starch and predominantly accumulated hexose. The silique wall hexose content and soluble acid invertase activity rapidly fell as embryos progressed from the early- to late-cotyledon developmental stages. A similar trend was not evident for alkaline invertase, Suc synthase (SuSy), and Suc-phosphate synthase. Silique wall SuSy activities were much higher than source leaves at all times and may serve to supply the substrate for secondary cell wall thickening. In young seeds starch was the predominant accumulated carbohydrate over the sampled developmental range. Seed hexose levels dropped as embryos developed from the early- to midcotyledon stage. Hexose and starch were localized to the testa or liquid endosperm, whereas Suc was evenly distributed among seed components. With the switch to oil accumulation, seed SuSy activity increased by 3.6-fold and soluble acid invertase activity decreased by 76%. These data provide valuable baseline knowledge for the genetic manipulation of canola seed carbon partitioning.     

Activities of key enzymes in sucrose-to-starch conversion in wheat grains subjected to water deficit during grain filling

This study tested the hypothesis that a controlled water deficit during grain filling of wheat (Triticum aestivum) could accelerate grain-filling rate through regulating the key enzymes involved in Suc-to-starch pathway in the grains. Two high lodging-resistant wheat cultivars were field grown. Well-watered and water-deficit (WD) treatments were imposed from 9 DPA until maturity. The WD promoted the reallocation of prefixed 14C from the stems to grains, shortened the grain-filling period, and increased grain-filling rate or starch accumulation rate (SAR) in the grains. Activities of Suc synthase (SuSase), soluble starch synthase (SSS), and starch branching enzyme (SBE) in the grains were substantially enhanced by WD and positively correlated with the SAR. ADP Glc pyrophosphorylase activity was also enhanced in WD grains initially and correlated with SAR with a smaller coefficient. Activities of granule-bound starch synthase and soluble and insoluble acid invertase in the grains were less affected by WD. Abscisic acid (ABA) content in the grains was remarkably enhanced by WD and very significantly correlated with activities of SuSase, SSS, and SBE. Application of ABA on well-watered plants showed similar results as those by WD. Spraying with fluridone, an ABA synthesis inhibitor, had the opposite effect. The results suggest that increased grain-filling rate is mainly attributed to the enhanced sink activity by regulating key enzymes involved in Suc-to-starch conversion, especially SuSase, SSS, and SBE, in wheat grains when subjected to a mild water deficit during grain filling, and ABA plays a vital role in the regulation of this process.     

Starch Synthesis and Programmed Cell Death during Endosperm Development in Triticale (x Triticosecale Wittmack)

Triticale (x Triticosecale Wittmack) grains synthesize and accumulate starch as their main energy source.Starch accumulation rate and synthesis activities of ADP-glucose pyrophosphorylase, soluble starch synthases, granule-bound starch synthase and starch-branching enzyme showed similar pattern of unimodal curves during endosperm development. There was no significant difference in activity of the starch granule-bound protein isolated from total and separated starch granules at different developmental stages after anthesis in triticale. Evans Blue staining and analysis of DNA fragmentation indicated that cells of triticale endosperm undergo programmed cell death during its development. Dead cells within the endosperm were detected at 6 d post anthesis (DPA), and evidence of DNA fragmentation was first observed at 21 DPA. The period between initial detection of PCD to its rapid increase overlapped with the key stages of rapid starch accumulation during endosperm development. Cell death occurred stochastically throughout the whole endosperm, meanwhile, the activities of starch biosynthetic enzymes and the starch accumulation rate decreased in the late stages of grain filling. These results suggested that the timing and progression of PCD in triticale endosperm may interfere with starch synthesis and accumulation.     

Starch Synthesis and Programmed Cell Death during Endosperm Development in Triticale(× Triticosecale Wittmack)

Triticale(× Triticosecale Wittmack) grains synthesize and accumulate starch as their main energy source.Starch accumulation rate and synthesis activities of ADP-glucose pyrophosphorylase,soluble starch synthases,granule-bound starch synthase and starch-branching enzyme showed similar pattern of unimodal curves during endosperm development.There was no significant difference in activity of the starch granule-bound protein isolated from total and separated starch granules at different developmental stages after anthesis in triticale.Evans Blue staining and analysis of DNA fragmentation indicated that cells of triticale endosperm undergo programmed cell death during its development.Dead cells within the endosperm were detected at 6 d post anthesis(DPA),and evidence of DNA fragmentation was first observed at 21 DPA.The period between initial detection of PCD to its rapid increase overlapped with the key stages of rapid starch accumulation during endosperm development.Cell death occurred stochastically throughout the whole endosperm,meanwhile,the activities of starch biosynthetic enzymes and the starch accumulation rate decreased in the late stages of grain filling.These results suggested that the timing and progression of PCD in triticale endosperm may interfere with starch synthesis and accumulation.     

Embryo-specific reduction of ADP-Glc pyrophosphorylase leads to an inhibition of starch synthesis and a delay in oil accumulation in developing seeds of oilseed rape

In oil-storing Brassica napus (rape) seeds, starch deposition occurs only transiently in the early stages of development, and starch is absent from mature seeds. This work investigates the influence of a reduction of ADP-Glc pyrophosphorylase (AGPase) on storage metabolism in these seeds. To manipulate the activity of AGPase in a seed-specific manner, a cDNA encoding the small subunit of AGPase was expressed in the sense or antisense orientation under the control of an embryo-specific thioesterase promoter. Lines were selected showing an embryo-specific decrease in AGPase due to antisense and cosuppression at different stages of development. At early developmental stages (25 days after flowering), a 50% decrease in AGPase activity was accompanied by similar decreases in starch content and the rate of starch synthesis measured by injecting (14)C-Suc into seeds in planta. In parallel to inhibition of starch synthesis, the level of ADP-Glc decreased, whereas Glc 1-phosphate levels increased, providing biochemical evidence that inhibition of starch synthesis was due to repression of AGPase. At 25 days after flowering, repression of starch synthesis also led to a decrease in the rate of (14)C-Suc degradation and its further metabolism via other metabolic pathways. This was not accompanied by an increase in the levels of soluble sugars, indicating that Suc import was inhibited in parallel. Flux through glycolysis, the activities of hexokinase, and inorganic pyrophosphate-dependent phosphofructokinase, and the adenylate energy state (ATP to ADP ratio) of the transgenic seeds decreased, indicating inhibition of glycolysis and respiration compared to wild type. This was accompanied by a marked decrease in the rate of storage lipid (triacylglycerol) synthesis and in the fatty acid content of seeds. In mature seeds, glycolytic enzyme activities, metabolite levels, and ATP levels remained unchanged, and the fatty acid content was only marginally lower compared to wild type, indicating that the influence of AGPase on carbon metabolism and oil accumulation was largely compensated for in the later stages of seed development. Results indicate that AGPase exerts high control over starch synthesis at early stages of seed development where it is involved in establishing the sink activity of the embryo and the onset of oil accumulation.     

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.     

Sink metabolism in tomato fruit : I. Developmental changes in carbohydrate metabolizing enzymes

In developing tomato (Lycopersicon esculentum Mill.) fruit, starch levels reach a peak early in development with soluble sugars (hexoses) gradually increasing in concert with starch degradation. To determine the enzymic basis of this transient partitioning of carbon to starch, the activities of key carbohydrate-metabolizing enzymes were investigated in extracts from developing fruits of three varieties (cv VF145-7879, cv LA1563, and cv UC82B), differing in final soluble sugar accumulation. Of the enzymes analyzed, ADPglucose pyrophosphorylase and sucrose synthase levels were temporally correlated with the transient accumulation of starch, having highest activities in cv LA1563, the high soluble sugar accumulator. Of the starch-degrading enzymes, phosphorylase levels were fivefold higher than those of amylase, and these activities did not increase during the period of starch degradation. Fiften percent of the amylase activity and 45 to 60% of the phosphorylase activity was localized in the chloroplast in cv VF145-7879. These results suggest that starch degradation in tomato fruit is predominantly phosphorolytic. The results suggest that starch biosynthetic capacity, as determined by levels of ADPglucose pyrophosphorylase rather than starch degradative capacity, regulate the transient accumulation of starch that occurs early in tomato fruit development. The results also suggest that ADPglucose pyrophosphorylase and sucrose synthase levels correlated positively with soluble sugar accumulation in the three varieties examined.     

Nucleotides and Nucleotide Sugars in Developing Maize Endosperms (Synthesis of ADP-Glucose in brittle-1)

As part of an in vivo study of carbohydrate metabolism during development of Zea mays L. kernels, quantities of nucleotides and nucleotide sugars were measured in endosperm extracts from normal, the single-mutant genotypes shrunken-1 (sh1), shrunken-2 (sh2), and brittle-1 (btl}, and the multiple-mutant genotypes sh1bt1, sh2bt1, and sh1sh2bt1. Results showed that bt1 kernels accumulated more than 13 times as much adenosine 5[prime] diphospho-glucose (ADP-Glc) as normal kernels. Activity of starch synthase in bt1 endosperm was equal to that in endosperm extracts from normal kernels. Thus the ADP-Glc accumulation in bt1 endosperm cells was not due to a deficiency in starch synthase. ADP-Glc content in extracts of sh1bt1 endosperms was similar to that in bt1, but in extracts of the sh2bt1 mutant kernels ADP-Glc content was much reduced compared to bt1 (about 3 times higher than that in normal). Endosperm extracts from sh1sh2bt1, kernels that are deficient in both ADP-Glc pyrophosphorylase (AGPase) and sucrose synthase, had quantities of ADP-Glc much lower than in normal kernels. These results clearly indicate that AGPase is the predominant enzyme responsible for the in vivo synthesis of ADP-Glc in bt1 mutant kernels, but Suc synthase may also contribute to the synthesis of ADP-Glc in kernels deficient in AGPase.     

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.     

Molecular dissection of variation in carbohydrate metabolism related to water-soluble carbohydrate accumulation in stems of wheat

Water-soluble carbohydrates (WSCs; composed of mainly fructans, sucrose [Suc], glucose [Glc], and fructose) deposited in wheat (Triticum aestivum) stems are important carbon sources for grain filling. Variation in stem WSC concentrations among wheat genotypes is one of the genetic factors influencing grain weight and yield under water-limited environments. Here, we describe the molecular dissection of carbohydrate metabolism in stems, at the WSC accumulation phase, of recombinant inbred Seri/Babax lines of wheat differing in stem WSC concentrations. Affymetrix GeneChip analysis of carbohydrate metabolic enzymes revealed that the mRNA levels of two fructan synthetic enzyme families (Suc:Suc 1-fructosyltransferase and Suc:fructan 6-fructosyltransferase) in the stem were positively correlated with stem WSC and fructan concentrations, whereas the mRNA levels of enzyme families involved in Suc hydrolysis (Suc synthase and soluble acid invertase) were inversely correlated with WSC concentrations. Differential regulation of the mRNA levels of these Suc hydrolytic enzymes in Seri/Babax lines resulted in genotypic differences in these enzyme activities. Down-regulation of Suc synthase and soluble acid invertase in high WSC lines was accompanied by significant decreases in the mRNA levels of enzyme families related to sugar catabolic pathways (fructokinase and mitochondrion pyruvate dehydrogenase complex) and enzyme families involved in diverting UDP-Glc to cell wall synthesis (UDP-Glc 6-dehydrogenase, UDP-glucuronate decarboxylase, and cellulose synthase), resulting in a reduction in cell wall polysaccharide contents (mainly hemicellulose) in the stem of high WSC lines. These data suggest that differential carbon partitioning in the wheat stem is one mechanism that contributes to genotypic variation in WSC accumulation.     

Characterization of recombinant UDP- and ADP-glucose pyrophosphorylases and glycogen synthase to elucidate glucose-1-phosphate partitioning into oligo- and polysaccharides in Streptomyces coelicolor

Streptomyces coelicolor exhibits a major secondary metabolism, deriving important amounts of glucose to synthesize pigmented antibiotics. Understanding the pathways occurring in the bacterium with respect to synthesis of oligo- and polysaccharides is of relevance to determine a plausible scenario for the partitioning of glucose-1-phosphate into different metabolic fates. We report the molecular cloning of the genes coding for UDP- and ADP-glucose pyrophosphorylases as well as for glycogen synthase from genomic DNA of S. coelicolor A3(2). Each gene was heterologously expressed in Escherichia coli cells to produce and purify to electrophoretic homogeneity the respective enzymes. UDP-glucose pyrophosphorylase (UDP-Glc PPase) was characterized as a dimer exhibiting a relatively high V(max) in catalyzing UDP-glucose synthesis (270 units/mg) and with respect to dTDP-glucose (94 units/mg). ADP-glucose pyrophosphorylase (ADP-Glc PPase) was found to be tetrameric in structure and specific in utilizing ATP as a substrate, reaching similar activities in the directions of ADP-glucose synthesis or pyrophosphorolysis (V(max) of 0.15 and 0.27 units/mg, respectively). Glycogen synthase was arranged as a dimer and exhibited specificity in the use of ADP-glucose to elongate α-1,4-glucan chains in the polysaccharide. ADP-Glc PPase was the only of the three enzymes exhibiting sensitivity to allosteric regulation by different metabolites. Mannose-6-phosphate, phosphoenolpyruvate, fructose-6-phosphate, and glucose-6-phosphate behaved as major activators, whereas NADPH was a main inhibitor of ADP-Glc PPase. The results support a metabolic picture where glycogen synthesis occurs via ADP-glucose in S. coelicolor, with the pathway being strictly regulated in connection with other routes involved with oligo- and polysaccharides, as well as with antibiotic synthesis in the bacterium.     

Purification and properties of the adenosine diphosphate-glucose and uridine diphosphate-glucose pyrophosphorylases of Mycobacterium smegmatis: inhibition and activation of the adenosine diphosphate-glucose pyrophosphorylase

Crude extracts of Mycobacterium smegmatis catalyzed the synthesis of adenosine diphosphate-glucose (ADP-Glc), cytidine diphosphate-glucose, guanosine diphosphate-glucose (GDP-Glc), thymidine diphosphate-glucose (TDP-Glc), and uridine diphosphate-glucose (UDP-Glc). In these crude enzyme fractions, high concentrations of trehalose-P inhibited the ADP-Glc and GDP-Glc pyrophosphorylases but did not effect the UDP-Glc or TDP-Glc pyrophosphorylases. Both the ADP-Glc pyrophosphorylase and the UDP-Glc pyrophosphorylase were partially purified (about 140-fold and 60-fold, respectively), and their properties were compared. For the ADP-Glc pyrophosphorylase, the K(m) for adenosine triphosphate was 6 x 10(-4)m, whereas that for glucose-1-P was 8 x 10(-4)m. The optimal concentration of Mg(2+) was 1 x 10(-3)m, and the pH optimum was 8.5. For the UDP-Glc pyrophosphorylase, the K(m) for uridine triphosphate was 1 x 10(-3)m and for glucose-1-P was 2 x 10(-3)m. The optimal Mg(2+) concentration was 1 x 10(-3)m, and the pH optimum was about 8.0. The purified ADP-Glc pyrophosphorylase was inhibited by fructose-6-P, fructose-1, 6-diphosphate, glucose-6-P, and phosphoenolpyruvate. On the other hand, trehalose, trehalose diphosphate, sodium pyruvate, and ribose-5-P did not effect the ADP-Glc pyrophosphorylase. None of these compounds, including trehalose-P, had any effect on the UDP-Glc pyrophosphorylase.     

Activities of enzymes involved in starch synthesis in wheat grains differing in starch content

This work was carried out to characterize starch accumulation and activities of key enzymes during grain filling in two wheat cultivars differing in starch content. The results showed that the starch accumulation rate (SAR) and activities of sucrose synthase, ADP-glucose pyrophosphorylase, soluble starch synthase, granule-bound starch synthase, and starch branching enzyme in the cultivar with a high starch content were significantly higher than those in the cultivar with a low starch content. The simulation with Richards’ equation showed that it was average starch accumulation rate but not active starch accumulation duration that determined starch accumulation. As compared with the cultivar with a low starch content, plants of the cultivar with a high starch content maintained the higher SAR and greater activities of related enzymes during mid and late grain filling stages. Consequently, the cultivar with a high starch content had advantages over that with a low starch content in terms of the amount of starch accumulation at mid and late grain filling stages.     

Effects of the activities of key enzymes involved in starch biosynthesis on the fine structure of amylopectin in developing rice (Oryza sativa L.) endosperms

The dynamic changes of the activities of enzymes involving in starch biosynthesis, including ADP-glucose pyrophosphorylase (AGPase), soluble starch synthases (SSS), starch branching enzyme (SBE) and starch debranching enzymes (DBE) were studied, and changes of fine structure of amy- lopectin were characterized by isoamylase treatment during rice grain development, using trans anti-waxy gene rice plants. The relationships between the activities of those key enzymes were also analyzed. The amylose synthesis was significantly inhibited in transgenic Wanjing 9522, but the total starch content and final grain weight were less affected as compared with those of non-transgenic Wanjing 9522 rice cultivar. Analyses on the changes of activities of enzymes involving in starch bio- synthesis showed that different enzyme activities were expressed differently during rice endosperm development. Soluble starch synthase is relatively highly expressed in earlier stage of endosperm de- velopment, whilst maximal expression of granule-bound starch synthase (GBSS) occurred in mid-stage of endosperm development. No obvious differences in changes of the activities of AGPase and SBE between two rice cultivars investigated, except the DBEs. Distribution patterns of branches of amy- lopectin changed continually during the development of rice grains and varied between two rice culti- vars. It was suggested that amylopectin synthesis be prior to the synthesis of amylose and different enzymes have different roles in controlling syntheses of branches of amylopectin.     

A possible role for pyrophosphate in the coordination of cytosolic and plastidial carbon metabolism within the potato tuber

The early stages of tuber development are characterized by cell division, high metabolic activity, and the predominance of invertase as the sucrose (Suc) cleaving activity. However, during the subsequent phase of starch accumulation the cleavage of Suc occurs primarily by the action of Suc synthase. The mechanism that is responsible for this switch in Suc cleaving activities is currently unknown. One striking difference between the invertase and Suc synthase mediated cleavage of Suc is the direct involvement of inorganic pyrophosphate (PPi) in the latter case. There is presently no convincing explanation of how the PPi required to support this process is generated in potato (Solanum tuberosum) tubers. The major site of PPi production in a maturing potato tubers is likely to be the reaction catalyzed by ADP-glucose pyrophosphorylase, the first committed step of starch biosynthesis in amyloplasts. We present data based on the analysis of the PPi levels in various transgenic plants altered in starch and Suc metabolism that support the hypothesis that PPi produced in the plastid is used to support cytosolic Suc breakdown and that PPi is an important coordinator of cytosolic and plastidial metabolism in potato tubers.     

Short-term effects of experimental warming and enhanced ultraviolet-B radiation on photosynthesis and antioxidant defense of <Emphasis Type="Italic">Picea asperata</Emphasis> seedlings

It is generally accepted that sucrose synthase (SuSy), ADP-glucose pyrophosphorylase (AGPase), soluble starch synthase (SSS), granule-bound starch synthases (GBSS) and starch branching enzyme (SBE) play a key role in starch synthesis in wheat grains. Starch synthesis in wheat grains is influenced by genotype and environment. However, what is not known is the degree of variation in enzyme activity during starch accumulation of wheat cultivars differing in kernel types. The present study was carried out to characterize the changing activities of key enzymes during grain filling in two kernel type winter wheat cultivars. Results showed that starch accumulation rate (SAR) and activities of SuSy, AGPase, SSS, GBSS and SBE in large kernel types were significantly higher than those in small kernel types. The soil water deficit experienced during the course of the experiment led to an increase at early grain-filling period and decrease during late grain-filling, respectively, in SAR and activities of key enzymes involved in starch synthesis, especially SuSy, AGPase, SSS, and SBE. Water deficit enhanced grain starch accumulation in small kernel types. It suggests that rainfed treatment increase physiological activities during early grain-filling and promote starch accumulation in small kernel types. The simulation with Richards’ equation showed that it was accumulation duration and SAR that determined the starch accumulation in large kernel types. Compared with small kernel types, plants of large kernel types maintained longer filling duration, higher SAR and greater activities of related enzymes during mid and late grain-filling. These observations suggest stronger sink activities in large kernel types at a later stage of development. Consequently, large kernel types have advantages over the small kernel types in terms of the amount of starch accumulation at mid and late stage, but are sensitive to water deficit.     

Differentiation of legume cotyledons as related to metabolic gradients and assimilate transport into seeds

Legume seed development is closely related to metabolism and nutrient transport. To analyse this relationship, a combination of biochemical, histological and transgenic approaches was used. Sugars within tissue sections have been quantitatively measured by metabolic imaging. During cotyledon differentiation glucose gradients emerge related to a particular cell type, with higher concentrations in non-differentiated premature regions. Sucrose in creases at the beginning of maturation in a layer underneath the outer epidermis expressing a sucrose transporter. Sucrose distribution is initially controlled by uptake activity and the permeability within the parenchyma and, later on, also by differences in growth and starch accumulation. Increased sucrose levels are accompanied by increased levels of sucrose synthase and ADP-Glc pyrophosphorylase mRNAs, but carbon flux into starch is initially still low. Rates increase at a stage when hexose concentrations become low, allowing increased flux through the sucrose synthase pathway. Transfer cell formation represents a regional specification of the cotyledonary epidermis for embryo nutrition characterized by increased transport-active cell surfaces and up-regulated expression of transport-related genes. The E2748 pea seed mutation blocks epidermal differentiation into transfer cells and leads to the loss of epidermal cell identity. Embryos with impaired epidermis cannot tolerate elevated levels of sucrose and respond with disorganized growth. The E2748 gene product is required for transfer cell formation in developing cotyledons with no other function during plant growth. Seed coat permeability provides a hypoxic environment for embryo development. However, at maturity, seed energy supply is not limited indicating fundamental developmental and metabolic adaptations. Results from transgenic seeds show that altered expression of single genes induces complex and unexpected changes. In AGP-antisense seeds the block in starch synthesis leads to pleiotropic effects of water and nitrogen content and induces temporal changes in seed development.     

Sucrose synthase catalyzes the de novo production of ADPglucose linked to starch biosynthesis in heterotrophic tissues of plants

By using barley seeds, developmental changes of ADPglucose (ADPG)-producing sucrose synthase (SS) and ADPG pyrophosphorylase (AGPase) have been compared with those of UDPglucose (UDPG), ADPG, sucrose (Suc) and starch contents. Both ADPG-synthesizing SS and AGPase activity patterns were found to correlate well with those of ADPG and starch contents. Remarkably, however, maximal activities of ADPG-synthesizing SS were found to be several fold higher than those of AGPase throughout seed development, the highest rate of starch accumulation being well accounted for by SS. Kinetic analyses of SS from barley endosperms and potato tubers in the Suc cleavage direction showed similar K(m) values for ADP and UDP, whereas apparent affinity for Suc was shown to be higher in the presence of UDP than with ADP. Moreover, measurements of transglucosylation activities in starch granules incubated with purified SS, ADP and [U-(14)C]Suc revealed a low inhibitory effect of UDP. The ADPG and UDPG contents in the transgenic S-112 SS and starch deficient potato mutant [Zrenner et al. (1995) Plant J. 7: 97] were found to be 35% and 30% of those measured in wild-type plants, whereas both glucose-1-phosphate and glucose-6-phosphate contents were found to be normal as compared with those of wild-type plants. The overall results thus strongly support a novel gluconeogenic mechanism reported previously [Pozueta-Romero et al. (1999) CRIT: Rev. Plant Sci. 18: 489] wherein SS catalyses directly the de novo production of ADPG linked to starch biosynthesis in heterotrophic tissues of plants.