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.     

Sugar-responsive gene expression, invertase activity, and senescence in aborting maize ovaries at low water potentials

• Background and Aims Ovary abortion can occur in maize(Zea mays) if water deficits lower the water potential (_w) sufficientlyto inhibit photosynthesis around the time of pollination. Theabortion decreases kernel number. The present work exploredthe activity of ovary acid invertases and their genes, togetherwith other genes for sucrose-processing enzymes, when this kindof abortion occurred. Cytological evidence suggested that senescencemay have been initiated after 2 or 3 d of low _w, and the expressionof some likely senescence genes was also determined. • Methods Ovary abortion was assessed at kernel maturity.Acid invertase activities were localized in vivo and in situ.Time courses for mRNA abundance were measured with real timePCR. Sucrose was fed to the stems to vary the sugar flux. • Key Results Many kernels developed in controls but mostaborted when _w became low. Ovary invertase was active in controlsbut severely inhibited at low _w for cell wall-bound forms invivo and soluble forms in situ. All ovary genes for sucroseprocessing enzymes were rapidly down-regulated at low _w exceptfor a gene for invertase inhibitor peptide that appeared tobe constitutively expressed. Some ovary genes for senescencewere subsequently up-regulated (RIP2 and PLD1). In some genes,these regulatory changes were reversed by feeding sucrose tothe stems. Abortion was partially prevented by feeding sucrose. • Conclusions A general response to low _w in maize ovarieswas an early down-regulation of genes for sucrose processingenzymes followed by up-regulation of some genes involved insenescence. Because some of these genes were sucrose responsive,the partial prevention of abortion with sucrose feeding mayhave been caused in part by the differential sugar-responsivenessof these genes. The late up-regulation of senescence genes mayhave caused the irreversibility of abortion.     

Imaging and quantifying carbohydrate transport to the developing ovaries of maize

BACKGROUND AND AIMS: Shade or inadequate water can inhibit photosynthesis and limit the development of maize (Zea mays) ovaries around the time of pollination, potentially reducing the number of kernels at harvest. This study investigated whether the decreased photosynthesis diminished only the sugar supply or also altered the transport path to the ovaries. METHODS: Photosynthesis and water potentials (Psiw) were measured in the leaves while dry matter delivery was monitored in the ovaries. Ovary glucose, starch and acid invertase activities were measured in situ. Stems were fed xylem-mobile safranin or phloem-mobile carboxyfluorescein (CF), and the dye transport to the ovaries was determined. KEY RESULTS: Under normal conditions, the ovaries gained in dry mass, and starch accumulated in the pedicel and ovary wall. Glucose accumulated in the pedicel, apparently in the apoplast where insoluble (cell-wall-bound) acid invertase acted on the arriving sucrose. A glucose gradient developed from pedicel to nucellus. Safranin moved in the xylem and did not reach the ovary, but CF moved in the phloem and arrived at the ovary. CF also spread into the pedicel but unlike glucose it did not enter the nucellus. Low Psiw or shade decreased leaf photosynthesis, ovary dry mass accumulation, invertase activities, pedicel glucose, starch accumulation and CF delivery. Removal of these treatments reversed the effects. CONCLUSIONS: The success of CF in tracing the general path and rate of carbohydrate transport gave visual evidence that phloem transport to the ovary decreased at low Psiw or in the shade but otherwise remained functional. The decreases indicated that losses in carbohydrate delivery are central features of failed ovary development under these conditions. The selectivity of transport into the nucellus resembled the situation later when embryo and endosperm are present and selective uptake occurs from the apoplast.     

The sucrose analog palatinose leads to a stimulation of sucrose degradation and starch synthesis when supplied to discs of growing potato tubers

In the present paper we investigated the effect of the sucrose (Suc) analog palatinose on potato (Solanum tuberosum) tuber metabolism. In freshly cut discs of growing potato tubers, addition of 5 mM palatinose altered the metabolism of exogenously supplied [U-14C]Suc. There was slight inhibition of the rate of 14C-Suc uptake, a 1.5-fold increase in the rate at which 14C-Suc was subsequently metabolized, and a shift in the allocation of the metabolized label in favor of starch synthesis. The sum result of these changes was a 2-fold increase in the absolute rate of starch synthesis. The increased rate of starch synthesis was accompanied by a 3-fold increase in inorganic pyrophosphate, a 2-fold increase in UDP, decreased UTP/UDP, ATP/ADP, and ATP/AMP ratios, and decreased adenylate energy charge, whereas glycolytic and Krebs cycle intermediates were unchanged. In addition, feeding palatinose to potato discs also stimulated the metabolism of exogenous 14C-glucose in favor of starch synthesis. In vitro studies revealed that palatinose is not metabolized by Suc synthases or invertases within potato tuber extracts. Enzyme kinetics revealed different effects of palatinose on Suc synthase and invertase activities, implicating palatinose as an allosteric effector leading to an inhibition of Suc synthase and (surprisingly) to an activation of invertase in vitro. However, measurement of tissue palatinose levels revealed that these were too low to have significant effects on Suc degrading activities in vivo. These results suggest that supplying palatinose to potato tubers represents a novel way to increase starch synthesis.     

Sucrose feeding reverses shade-induced kernel losses in maize

Background and Aims

Water limitations can inhibit photosynthesis and change gene expression in ways that diminish or prevent reproductive development in plants. Sucrose fed to the plants can reverse the effects. To test whether the reversal acts generally by replacing the losses from photosynthesis, sucrose was fed to the stems of shaded maize plants (Zea mays) during reproductive development.

Methods

Shading was adjusted to mimic the inhibition of photosynthesis around the time of pollination in water-limited plants. Glucose and starch were imaged and quantified in the female florets. Sucrose was infused into the stems to vary the sugar flux to the ovaries.

Key Results

Ovaries normally grew rapidly and contained large amounts of glucose and starch, with a glucose gradient favouring glucose movement into the developing ovary. Shade inhibited photosynthesis and diminished ovary and kernel size, weight, and glucose and starch contents compared with controls. The glucose gradient became small. Sucrose fed to the stem reversed these losses, and kernels were as large as the controls.

Conclusions

Despite similar inhibition of photosynthesis, the depletion of ovary glucose and starch was not as severe in shade as during a comparable water deficit. Ovary abortion prevalent during water deficits did not occur in the shade. It is suggested that this difference may have been caused by more translocation in shade than during the water deficit, which prevented low sugar contents necessary to trigger an up-regulation of senescence genes known to be involved in abortion. Nevertheless, sucrose feeding reversed kernel size losses and it is concluded that feeding acted generally to replace diminished photosynthetic activity.     

Effect of pollination on carbohydrate metabolism,in young fruits of Citrullus lanatus and Capsicum annuum

During a 9 day period after anthesis the concentration of reducing sugars showed a 6-fold increase in fruits of Citrullus lanatus, and a 2-fold increase in those of Capsicum annuum. These increases were associated with acid invertase, the specific activity of which was high in ovaries at anthesis and which increased 5-fold in watermelon and 1.5-fold in pepper during the same period. Sucrose synthase apparently plays only a minor role in sucrose hydrolysis. Changes in sugar concentrations and both acid invertase and sucrose synthase activities were similar in fruits developed both after pollination or hormone (NAA) treatment of ovaries. In non-pollinated ovaries of watermelon there was also an increase in invertase activity up to 6 days after anthesis which paralleled the increase in activity in seeded and parthenocarpic fruits. However, there was no increase in either reducing sugars or sucrose, indicating that sucrose is not imported into non-pollinated ovaries. Utilisation of reserve starch may help prolong the life of non-pollinated ovaries for up to one week after anthesis.     

Low Water Potential Disrupts Carbohydrate Metabolism in Maize (Zea mays L.) Ovaries

Water deficit during pollination increases the frequency of kernel abortion in maize (Zea mays L.). Much of the kernel loss is attributable to lack of current photosynthate, but a large number of kernels fail to develop on water-deficient plants even when assimilate supply is increased. We examined the possibility that assimilate utilization by developing ovaries might be impaired at low water potential ([Psi]w). Plants were grown in the greenhouse in 20-L pots containing 22 kg of amended soil. Water was withheld on the first day silks emerged, and plants were hand-pollinated 4 d later when leaf [Psi]w decreased to approximately - 1.8 MPa and silk [Psi]w was approximately -1.0 MPa. Plants were rehydrated 2 d after pollination. The brief water deficit inhibited ovary growth (dry matter accumulation) and decreased kernel number per ear by 60%, compared to controls. Inhibition of ovary growth was associated with a decrease in the level of reducing sugars, depletion of starch, a 75-fold increase in sucrose concentration (dry weight basis), and inhibition of acid invertase (EC 3.2.1.26) activity. These results indicate that water deficits during pollination disrupt carbohydrate metabolism in maize ovaries. They suggest that acid invertase activity is important for establishing and maintaining reproductive sink strength during pollination and early kernel development.     

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.     

Genetic and transgenic perturbations of carbon reserve production in Arabidopsis seeds reveal metabolic interactions of biochemical pathways

The biosynthesis of seed oil and starch both depend on the supply of carbon from the maternal plant. The biochemical interactions between these two pathways are not fully understood. In the Arabidopsis mutant shrunken seed 1 (sse1)/pex16, a reduced rate of fatty acid synthesis leads to starch accumulation. To further understand the metabolic impact of the decrease in oil synthesis, we compared soluble metabolites in sse1 and wild type (WT) seeds. Sugars, sugar phosphates, alcohols, pyruvate, and many other organic acids accumulated in sse1 seeds as a likely consequence of the reduced carbon demand for lipid synthesis. The enlarged pool size of hexose-P, the metabolites at the crossroad of sugar metabolism, glycolysis, and starch synthesis, was likely a direct cause of the increased flow into starch. Downstream of glycolysis, more carbon entered the TCA cycle as an alternative to the fatty acid pathway, causing the total amount of TCA cycle intermediates to rise while moving the steady state of the cycle away from fumarate. To convert the excess carbon metabolites into starch, we introduced the Escherichia coli starch synthetic enzyme ADP-glucose pyrophosphorylase (AGPase) into sse1 seeds. Expression of AGPase enhanced net starch biosynthesis in the mutant, resulting in starch levels that reached 37% of seed weight. However, further increases above this level were not achieved and most of the carbon intermediates remained high in comparison with the WT, indicating that additional mechanisms limit starch deposition in Arabidopsis seeds.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

套袋对梨果实发育过程中糖组分及其相关酶活性的影响

以翠冠和黄金梨为试材,测定套袋和未套袋(对照)梨果实发育时期果实中蔗糖、葡萄糖、果糖和山梨醇含量以及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,并对果实中糖组分积累与酶活性的关系进行了分析.结果表明:(1)两梨品种套袋果实在发育过程中蔗糖、葡萄糖、果糖、山梨醇和糖代谢相关酶活性变化趋势与对照基本一致,套袋果实糖含量均低于对照但差异不显著,而各相关酶活性在两类果实间差异表现各异.(2)在梨果实发育早期,果实中以分解酶类为主,糖分积累低;发育后期以合成酶类为主,糖分积累多.(3)两品种套袋和对照果实AI活性与葡萄糖含量均呈显著或极显著正相关,SS合成方向活性与蔗糖含量均为极显著正相关,且翠冠对照果SPS活性与蔗糖含量呈极显著正相关.可见,套袋通过提高果实发育早期转化酶(Inv)活性,降低果实后期蔗糖磷酸合成酶(SPS)、蔗糖合成酶(SS)的活性来影响糖分积累,从而影响梨果品质.     

The transport of sugars to developing embryos is not via the bulk endosperm in oilseed rape seeds

The fate of sucrose (Suc) supplied via the phloem to developing oilseed rape (Brassica napus) seeds has been investigated by supplying [(14)C]Suc to pedicels of detached, developing siliques. The method gives high, sustained rates of lipid synthesis in developing embryos within the silique comparable with those on the intact plant. At very early developmental stages (3 d after anthesis), the liquid fraction that occupies most of the interior of the seed has a very high hexose-to-Suc ratio and [(14)C]Suc entering the seeds is rapidly converted to hexoses. Between 3 and 12 d after anthesis, the hexose-to-Suc ratio of the liquid fraction of the seed remains high, but the fraction of [(14)C]Suc converted to hexose falls dramatically. Instead, most of the [(14)C]Suc entering the seed is rapidly converted to products in the growing embryo. These data, together with light and nuclear magnetic resonance microscopy, reveal complex compartmentation of sugar metabolism and transport within the seed during development. The bulk of the sugar in the liquid fraction of the seed is probably contained within the central vacuole of the endosperm. This sugar is not in contact with the embryo and is not on the path taken by carbon from the phloem to the embryo. These findings have important implications for the sugar switch model of embryo development and for understanding the relationship between the embryo and the surrounding endosperm.     

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.     

Carbohydrates and carbohydrate enzymes in developing cotton ovules

Patterns of carbohydrates and carbohydrate enzymes were investigated in developing cotton ovules to establish which of these might be related to sink strength in developing bolls. Enzymatic analysis of extracted tissue indicated that beginning 1 week following anthesis, immature cotton seeds (Gossypium hirsutum L. cv. Coker 100A glandless) accumulated starch in the tissues which surround the embryo. Starting at 15 days post anthesis (DPA), this starch was depleted and starch simultaneously appeared in the embryo. Sucrose entering the tissues surrounding the embryo was rapidly degraded, apparently by sucrose synthase; the free hexose content of these tissues reached a peak at about 20 DPA. During the first few weeks of development these tissues contained substantial amounts of hexose but little sucrose; the reverse was true for cotton embryos. Embryo sucrose content rose sharply from the end of the first week until about 20 DPA; it then remained roughly constant during seed maturation. Galactinol synthase (EC 2.4.1.x) appeared in the embryos approximately 25 days after flowering. Subsequently, starch disappeared and the galactosides raffinose and stachyose appeared in the embryo. Except near maturity, sucrose synthase (EC 2.4.1.13) activity in the embryos predominated over that of both sucrose phosphate synthase (EC 2.4.1.14) and acid invertase (EC 3.2.1.26). Activities of the latter enzymes increased during the final stages of embryo maturation. The ratio of sucrose synthase to sucrose phosphate synthase was found to be high in young cotton embryos but the ratio reversed about 45 DPA, when developing ovules cease being assimilate sinks. Insoluble acid invertase was present in developing cotton embryos, but at very low activities; soluble acid invertase was present at significant activities only in nearly mature embryos. From these data it appears that sucrose synthase plays an important role in young cotton ovule carbohydrate partitioning and that sucrose phosphate synthase and the galactoside synthesizing enzymes assume the dominant roles in carbohydrate partitioning in nearly mature cotton seeds. Starch was found to be an important carbohydrate intermediate during the middle stages of cotton ovule development and raffinose and stachyose were found to be important carbohydrate pools in mature cotton seeds.     

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.     

Pistil Starch Reserves at Anthesis Correlate with Final Flower Fate in Avocado (Persea americana)

A common observation in different plant species is a massive abscission of flowers and fruitlets even after adequate pollination, but little is known as to the reason for this drop. Previous research has shown the importance of nutritive reserves accumulated in the flower on fertilization success and initial fruit development but direct evidence has been elusive. Avocado (Persea americana) is an extreme case of a species with a very low fruit to flower ratio. In this work, the implications of starch content in the avocado flower on the subsequent fruit set are explored. Firstly, starch content in individual ovaries was analysed from two populations of flowers with a different fruit set capacity showing that the flowers from the population that resulted in a higher percentage of fruit set contained significantly more starch. Secondly, in a different set of flowers, the style of each flower was excised one day after pollination, once the pollen tubes had reached the base of the style, and individually fixed for starch content analysis under the microscope once the fate of its corresponding ovary (that remained in the tree) was known. A high variability in starch content in the style was found among flowers, with some flowers having starch content up to 1,000 times higher than others, and the flowers that successfully developed into fruits presented significantly higher starch content in the style at anthesis than those that abscised. The relationship between starch content in the ovary and the capacity of set of the flower together with the correlation found between the starch content in the style and the fate of the ovary support the hypothesis that the carbohydrate reserves accumulated in the flower at anthesis are related to subsequent abscission or retention of the developing fruit.     

Functional reversion to identify controlling genes in multigenic responses: analysis of floral abortion

In many situations, organisms respond to stimuli by altering the activity of large numbers of genes. Among these, certain ones are likely to control the phenotype while others play a secondary role or are passively altered without directly affecting the phenotype. Identifying the controlling genes has proven difficult. However, in a few instances, it has been possible to reverse the phenotype by physiological or biochemical means without altering the genetics of the organism. During this functional reversion, only a few genes may respond, thus identifying those likely to be controlling the phenotype. Floral abortion during a water shortage in maize is an example because the response is inherently multigenic, and the phenotype can be reversed by physiological/biochemical means. A recent analysis used this reversal to reveal that only a few genes are likely to control the abortion phenotype. In maize, these genes coded for a cell wall invertase (Incw2), a soluble invertase (Ivr2), a ribosome-inactivating protein (RIP2), and phospholipase D (PLD1). The invertases appeared to control the normal sugar uptake by the ovaries. Their down-regulation depleted ovary sugar pools and resulted in an up-regulation of the genes for ribosome-inactivating protein and for phospholipase. The latter changes appeared to initiate senescence that degraded cell membranes, thus causing irreversible abortion. With these findings, these genes have become targets for preventing abortion. This approach might have value in other contexts with some additional methods.