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.     

Analysis of carbohydrate metabolism enzymes and cellular contents of sugars and proteins during spruce somatic embryogenesis suggests a regulatory role of exogenous sucrose in embryo development.

Carbohydrate metabolism was investigated during spruce somatic embryogenesis. During the period of maintenance corresponding to the active phase of embryogenic tissue growth, activities of soluble acid invertase and alkaline invertase increased together with cellular glucose and fructose levels. During the same time, sucrose phosphate synthase (SPS) activity increased while sucrose synthase (SuSy) activity stayed constant together with the cellular sucrose level. Therefore, during maintenance, invertases were thought to generate the hexoses necessary for embryogenic tissue growth while SuSy and SPS would allow cellular sucrose to be kept at a constant level. During maturation on sucrose-containing medium, SuSy and SPS activities stayed constant whereas invertase activities were high during the early stage of maturation before declining markedly from the second to the fifth week. This decrease of invertase activities resulted in a decreased hexose:sucrose ratio accompanied by starch and protein deposition. Additionally, carbohydrate metabolism was strongly modified when sucrose in the maturation medium was replaced by equimolar concentrations of glucose and fructose. Essentially, during the first 2 weeks, invertase activities were low in tissues growing on hexose-containing medium while cellular glucose and fructose levels increased. During the same period, SuSy activity increased while the SPS activity stayed constant together with the cellular sucrose level. This metabolism reorganization on hexose-containing medium affected cellular protein and starch levels resulting in a decrease of embryo number and quality. These results provide new knowledge on carbohydrate metabolism during spruce somatic embryogenesis and suggest a regulatory role of exogenous sucrose in embryo development.     

Development of Sink Capacity of the "Storage Root" in a Radish Variety with a Low Ratio of "Storage Root" to Shoot

Controling mechanisms of sink capacity are poorly understood.Previously we suggested that sucrose synthase (SuSy), but notinvertase, plays an important role for sink capacity of theradish "storage root" in a variety, Raphanus sativus L. (cv.White Cherish) [plant Cell Physiol. (1999) 40: 369]. With thisvariety about 50% of the total dry weight (DW) was in the "storageroot" at 21 d after sowing (DAS). We investigated the sink capacityof another radish variety, R. sativus L. (cv. Kosena) with alow ratio of "storage root" to shoot. With the latter varietyonly 3% of the total DW was in the "storage root" at 21 DAS.Sink activity (increase in DW of the "storage root" per unitof DW present per unit of time) of the "storage root" in Kosenaas well as White Cherish was strongly related to the level andactivity of SuSy but not to the activity of invertase. Theseresults confirmed that SuSy rather than invertase may be criticalfor the development of the sink activity of the radish "storageroot" and that the reaction products of UDP-glucose and fructoseare utilized for sink growth including biosynthesis of the cellwall. In Kosena photosynthates seemed to be partitioned mainlyinto developing leaves and fibrous roots. Differences in partitioningof photosynthates among various sinks with these two varietiesare discussed including anatomical considerations. (Received July 19, 1999; Accepted September 30, 1999)     

A role for 'futile cycles' involving invertase and sucrose synthase in sucrose metabolism of tomato fruit.

Current concepts of the factors determining sink strength and the subsequent regulation of carbohydrate metabolism in tomato fruit are based upon an understanding of the relative roles of sucrose synthase, sucrose phosphate synthase and invertase, derived from studies in mutants and transformed plants. These enzymes participate in at least four futile cycles that involve sugar transport between the cytosol, vacuole and apoplast. Key reactions are (1) the continuous rapid degradation of sucrose in the cytosol by sucrose synthase (SuSy), (2) sucrose re-synthesis via either SuSy or sucrose phosphate synthase (SPS), (3) sucrose hydrolysis in the vacuole or apoplast by acid invertase, (4) subsequent transport of hexoses to the cytosol where they are once more converted into sucrose, and (5) rapid synthesis and breakdown of starch in the amyloplast. In this way futile cycles of sucrose/hexose interchange govern fruit sugar content and composition. The major function of the high and constant invertase activity in red tomato fruit is, therefore, to maintain high cellular hexose concentrations, the hydrolysis of sucrose in the vacuole and in the intercellular space allowing more efficient storage of sugar in these compartments. Vacuolar sugar storage may be important in sustaining fruit cell growth at times when less sucrose is available for the sink organs because of exhaustion of the carbohydrate pools in source leaves.     

Plant density influences fiber sucrose metabolism in relation to cotton fiber quality

Planting density plays an important role in improving cotton yield and regulating fiber quality. A 2-year experiment was conducted to investigate the effects of plant density on sucrose metabolism in relation to fiber quality of field-grown cotton. The results showed that lint yield increased with increasing plant density, fiber micronaire, fiber maturity ratio, and fiber fineness decreased with the increasing of plant density, whereas fiber length, fiber uniformity index, fiber strength, and fiber elongation were little affected by plant density. Increased plant density decreased sucrose synthase (SuSy) activity, sucrose content, and cellulose content in cotton fiber, but increased invertase activity. Increased invertase activity would restrain SuSy activity in cotton fiber: therefore, SuSy activity was the most severely affected enzyme in fiber sucrose metabolism by cotton plant density during fiber development. Abundant sucrose content in fiber after 24 days post anthesis (DPA) and high activities of SuSy and sucrose phosphate synthase (SPS) at 38 DPA were beneficial for cellulose synthesis, and were propitious to optimize the fiber maturity properties. The results also showed that fiber micronaire, maturity ratio, and fineness decreased 0.11, 0.02, and 5.89 mtex, respectively, with each increase of 10,000 plants per hectare. It was concluded that high plant density decreased SuSy activity, sucrose content, and cellulose content, but increased invertase activity in sucrose metabolism, resulting in low fiber micronaire, fiber maturity ratio, and fiber fineness.     

Sucrose utilization during somatic embryo development in black spruce: involvement of apoplastic invertase in the tissue and of extracellular invertase in the medium

The involvement of apoplastic invertase (Ap Inv) and sucrose synthase (SuSy) in the somatic embryo development of black spruce (Picea mariana) was investigated under different maturation conditions. Replacing 6% sucrose with 3% or 1% sucrose in the maturation medium drastically decreased Ap Inv activity and amount in embryogenic tissues. This was accompanied by a decrease in the hexose pool that resulted in a lower starch deposition and protein amount in embryogenic tissues together with a lower embryo production. Conversely, SuSy activity was stable during maturation regardless of the sucrose concentration used in the medium. The presence of an extracellular enzyme responsible for sucrose hydrolysis in the maturation medium was also verified. An immunodetection experiment with anti-acid invertase antibodies revealed the presence of an active 53 kDa polypeptide in the medium, which had a similar molecular mass to that of the Ap Inv polypeptide found in embryogenic tissues. Utilization of sucrose from the medium by the tissues was also studied using labelled 14C-sucrose. Distribution of the radioactivity between tissular sucrose, glucose, and fructose showed that sucrose was diffused into the cell wall of embryogenic tissues and partly hydrolyzed by Ap Inv. These results show that the utilization of sucrose from the medium, the Ap Inv activity in embryogenic tissues, and the release of an active invertase into the medium operate together for the utilization of the carbohydrates during somatic embryo development in black spruce.     

Comparative Enzymic Studies of Sucrose Metabolism in the Taproots and Fibrous Roots of Beta vulgaris L

Comparative enzymic studies of sugar beet (Beta vulgaris L.) taproots and fibrous roots revealed differences in invertase (EC 3.2.1.26) and sucrose synthetase (EC 2.4.1.13) activity. Invertase activity of the two root forms differs with respect to specific activity, pH optimum, and enzyme solubility. Acid invertase (pH 4.5) in the taproot was restricted to the peripheral meristematic tissue which produces cells for both taproot and fibrous root growth. This finding supports the hypothesis that the enzyme regulates sucrose partitioning between the taproot and fibrous roots. A distinct alkaline invertase (pH 8.0) was detected in sucrose storage tissues of the taproot.     

Extractable activities and protein content of sucrose-phosphate synthase, sucrose synthase and neutral invertase in trunk tissues of Robinia pseudoacacia L. are related to cambial wood production and heartwood formation

The presence of sucrose synthesizing and degrading enzymes and the correlation of their enzyme activity with cambial growth and heartwood formation are demonstrated in trunks of Robinia pseudoacacia L., black locust. Sucrose is formed by sucrose-phosphate synthase (SPS; EC 2.4.1.14), predominantly in the storage part of the sapwood. In the cambial differentiation zone and the sapwood-heartwood transition zone, both of which constitute carbohydrate sinks, sucrose is primarily cleaved by sucrose synthase (SuSy; EC 2.4.1.13) and a neutral invertase (NI; EC 3.2.1.26). In spring, enhanced activities of SuSy and NI were found in the differentiating xylem tissues. This coincided with elevated SPS rates at the sites of starch mobilization. Heartwood formation in autumn, a period of intense accumulation of phenolics in the innermost living wood tissues, was accompanied by high activities of SuSy and NI. Increased SPS and NI activities in all tissues of winter samples could be correlated with cold acclimation. Probing of SPS and SuSy protein from black locust with heterologous antibodies revealed a subunit size of 130 kDa for SPS and of 89 kDa for SuSy. Both SPS and SuSy exhibited a linear correlation between catalytic activity and amount of enzyme protein with respect to the radial profile from bark to inner core and with respect to the seasonal course. The highest amounts of SuSy-specific mRNA were detected in differentiating xylem in summer and the sapwood-heartwood transition zone in autumn. These data are taken as evidence for a pivotal role of SuSy in supplying carbon skeletons for the biosynthesis of secondary substances in woody axes. Received: 6 May 1998 / Accepted: 28 July 1998     

Sucrose Metabolism at Three Leaf Development Stages in Bean Plants

Sucrose metabolism was studied at three leaf development stages in two Phaseolus vulgaris L. cultivars, Tacarigua and Montalban. The changes of enzyme activities involved in sucrose metabolism at the leaf development stages were: (1) Sink (9-11 % full leaf expansion, FLE): low total sucrose phosphate synthase (SPS) activity, and higher acid invertase (AI) activity accompanied by low sucrose synthase (SuSy) synthetic and sucrolytic activities. (2) Sink to source transition (40-47 % FLE): increase in total SPS and SuSy activities, decrease in AI activity. (3) Source (96-97 % FLE): high total SPS activity, increased SuSy activities, decreased AI activity. The hexose/sucrose ratio decreased from sink to source leaves in both bean cultivars. The neutral invertase activity was lower than that of AI; it showed an insignificant decrease during the sink-source transition.     

Sucrose Metabolism at Three Leaf Development Stages in Bean Plants

Sucrose metabolism was studied at three leaf development stages in two Phaseolus vulgaris L. cultivars, Tacarigua and Montalban. The changes of enzyme activities involved in sucrose metabolism at the leaf development stages were: (1) Sink (9-11 % full leaf expansion, FLE): low total sucrose phosphate synthase (SPS) activity, and higher acid invertase (AI) activity accompanied by low sucrose synthase (SuSy) synthetic and sucrolytic activities. (2) Sink to source transition (40-47 % FLE): increase in total SPS and SuSy activities, decrease in AI activity. (3) Source (96-97 % FLE): high total SPS activity, increased SuSy activities, decreased AI activity. The hexose/sucrose ratio decreased from sink to source leaves in both bean cultivars. The neutral invertase activity was lower than that of AI; it showed an insignificant decrease during the sink-source transition. This revised version was published online in June 2006 with corrections to the Cover Date.     

Metabolism of carbohydrates during the development of seeds of the brazilian rubber tree [Hevea brasiliensis (Willd. Ex Adr. de Juss) Muell.-Arg.]

This work aimed at the assessment of the metabolism of carbohydrate during the development of the seeds of Brazilian rubber trees. The enzymatic activity of the acid invertase, neutral invertase and sucrose synthase (SuSy) and the levels of total soluble sugars (TSS), reducing sugars (RS) and sucrose were evaluated separately in each part of the fruit and seed—pericarp, seed coat, embryo and endosperm—on different days after the pollination (DAP). Based on the results obtained in this study, it is possible to conclude that in the beginning of the development of the rubber tree seeds, until 95 DAP, the endosperm presents high concentration of RS and low concentration of sucrose. After this period, the endosperm of the seed initiates starch accumulation and the concentration of RS decreases followed by the increase in the concentration of sucrose, presenting, after 120 DAP, an inversion of concentration of these two sugars. In the embryo, the levels of TSS, RS and sucrose show significant increase with the progress of the seed development. In the endosperm, the transition of the division phase and cell expansion for the storage of reserve material seem to occur around 120 DAP and is to be controlled mainly by the enzymes acid invertase and SuSy, while in the embryo, such transition seems to occur around 135 DAP and is to be controlled mainly by the enzymes acid and neutral invertases.     

Antisense inhibition of tomato fruit sucrose synthase decreases fruit setting and the sucrose unloading capacity of young fruit

The role of sucrose synthase (SuSy) in tomato fruit was studied in transgenic tomato (Lycopersicon esculentum) plants expressing an antisense fragment of fruit-specific SuSy RNA (TOMSSF) under the control of the cauliflower mosaic virus 35S promoter. Constitutive expression of the antisense RNA markedly inhibited SuSy activity in flowers and fruit pericarp tissues. However, inhibition was only slight in the endosperm and was undetectable in the embryo, shoot, petiole, and leaf tissues. The activity of sucrose phosphate synthase decreased in parallel with that of SuSy, but acid invertase activity did not increase in response to the reduced SuSy activity. The only effect on the carbohydrate content of young fruit was a slight reduction in starch accumulation. The in vitro sucrose import capacity of fruits was not reduced by SuSy inhibition at 23 days after anthesis, and the rate of starch synthesized from the imported sucrose was not lessened even when SuSy activity was decreased by 98%. However, the sucrose unloading capacity of 7-day-old fruit was substantially decreased in lines with low SuSy activity. In addition, the SuSy antisense fruit from the first week of flowering had a slower growth rate. A reduced fruit set, leading to markedly less fruit per plant at maturity, was observed for the plants with the least SuSy activity. These results suggest that SuSy participates in the control of sucrose import capacity of young tomato fruit, which is a determinant for fruit set and development.     

Decreased sucrose content triggers starch breakdown and respiration in stored potato tubers (Solanum tuberosum)

To change the hexose-to-sucrose ratio within phloem cells, yeast-derived cytosolic invertase was expressed in transgenic potato (Solanum tuberosum cv. Desirée) plants under control of the rolC promoter. Vascular tissue specific expression of the transgene was verified by histochemical detection of invertase activity in tuber cross-sections. Vegetative growth and tuber yield of transgenic plants was unaltered as compared to wild-type plants. However, the sprout growth of stored tubers was much delayed, indicating impaired phloem-transport of sucrose towards the developing bud. Biochemical analysis of growing tubers revealed that, in contrast to sucrose levels, which rapidly declined in growing invertase-expressing tubers, hexose and starch levels remained unchanged as compared to wild-type controls. During storage, sucrose and starch content declined in wild-type tubers, whereas glucose and fructose levels remained unchanged. A similar response was found in transgenic tubers with the exception that starch degradation was accelerated and fructose levels increased slightly. Furthermore, changes in carbohydrate metabolism were accompanied by an elevated level of phosphorylated intermediates, and a stimulated rate of respiration. Considering that sucrose breakdown was restricted to phloem cells it is concluded that, in response to phloem-associated sucrose depletion or hexose elevation, starch degradation and respiration is triggered in parenchyma cells. To study further whether elevated hexose and/or hexose-phosphates or decreased sucrose levels are responsible for the metabolic changes observed, sucrose content was decreased by tuber-specific expression of a bacterial sucrose isomerase. Sucrose isomerase catalyses the reversible conversion of sucrose into palatinose, which is not further metabolizable by plant cells. Tubers harvested from these plants were found to accumulate high levels of palatinose at the expense of sucrose. In addition, starch content decreased slightly, while hexose levels remained unaltered, compared with the wild-type controls. Similar to low sucrose-containing invertase tubers, respiration and starch breakdown were found to be accelerated during storage in palatinose-accumulating potato tubers. In contrast to invertase transgenics, however, no accumulation of phosphorylated intermediates was observed. Therefore, it is concluded that sucrose depletion rather than increased hexose metabolism triggers reserve mobilization and respiration in stored potato tubers.     

Absolute quantification of Medicago truncatula sucrose synthase isoforms and N-metabolism enzymes in symbiotic root nodules and the detection of novel nodule phosphoproteins by mass spectrometry

Mass spectrometry (MS) has become increasingly important for tissue specific protein quantification at the isoform level, as well as for the analysis of protein post-translational regulation mechanisms and turnover rates. Thanks to the development of high accuracy mass spectrometers, peptide sequencing without prior knowledge of the amino acid sequence--de novo sequencing--can be performed. In this work, absolute quantification of a set of key enzymes involved in carbon and nitrogen metabolism in Medicago truncatula 'Jemalong A17' root nodules is presented. Among them, sucrose synthase (SuSy; EC 2.4.1.13), one of the central enzymes in sucrose cleavage in root nodules, has been further characterized and the relative phosphorylation state of the three most abundant isoforms has been quantified. De novo sequencing provided sequence information of a so far unidentified peptide, most probably belonging to SuSy2, the second most abundant isoform in M. truncatula root nodules. TiO(2)-phosphopeptide enrichment led to the identification of not only a phosphorylation site at Ser11 in SuSy1, but also of several novel phosphorylation sites present in other root nodule proteins such as alkaline invertase (AI; EC 3.2.1.26) and an RNA-binding protein.     

The Differential Expression of Sucrose Synthase in Relation to Diverse Patterns of Carbon Partitioning in Developing Cotton Seed

Developing cotton (Gossypium hirsutum L.) seed exhibits complex patterns of carbon allocation in which incoming sucrose (Suc) is partitioned to three major sinks: the fibers, seed coat, and cotyledons, which synthesize cellulose, starch, and storage proteins or oils, respectively. In this study we investigated the role of Suc synthase (SuSy) in the mobilization of Suc into such sinks. Assessments of SuSy gene expression at various levels led to the surprising conclusion that, in contrast to that found for other plants, SuSy does not appear to play a role in starch synthesis in the cotton seed. However, our demonstration of functional symplastic connections between the phloem-unloading area and the fiber cells, as well as the SuSy expression pattern in fibers, indicates a major role of SuSy in partitioning carbon to fiber cellulose synthesis. SuSy expression is also high in transfer cells of the seed coat facing the cotyledons. Such high levels of SuSy could contribute to the synthesis of the thickened cell walls and to the energy generation for Suc efflux to the seed apoplast. The expression of SuSy in cotyledons also suggests a role in protein and lipid synthesis. In summary, the developing cotton seed provides an excellent example of the diverse roles played by SuSy in carbon metabolism.     

Biochemical and genetic analysis of carbohydrate accumulation in Allium cepa L

Onion and shallot (Allium cepa L.) exhibit wide variation in bulb fructan content, and the Frc locus on chromosome 8 conditions much of this variation. To understand the biochemical basis of Frc, we conducted biochemical and genetic analyses of Allium fistulosum (FF)-shallot (A. cepa Aggregatum group) alien monosomic addition lines (AALs; FF+1A-FF+8A) and onion mapping populations. Sucrose and fructan levels in leaves of FF+2A were significantly lower than in FF throughout the year, and the springtime activity of acid invertase was also lower. FF+8A showed significantly higher winter sucrose accumulation and sucrose phosphate synthase (SPS) activity. Inbred high fructan (Frc_) lines from the 'W202Ax Texas Grano 438' onion population exhibited significantly higher sucrose levels prior to bulbing than low fructan (frcfrc) lines. Sucrose synthase (SuSy) activity in these lines was correlated with leaf hexose content but not with Frc phenotype. Markers for additional candidate genes for sucrose metabolism were obtained by cloning a major SPS expressed in onion leaf and exhaustively mining onion expressed sequence tag resources. SPS and SuSy loci were assigned to chromosome 8 and 6, respectively, using AALs and linkage mapping. Further loci were assigned, using AALs, to chromosomes 1 (sucrose phosphate phosphatase), 2 (SuSy and three invertases) and 8 (neutral invertase). The concordance between chromosome 8 localization of SPS and elevated leaf sucrose levels conditioned by high fructan alleles at the Frc locus in bulb onion or alien monosomic additions of chromosome 8 in A. fistulosum suggest that the Frc locus may condition variation in SPS activity.     

Sucrose Utilization via Invertase and Sucrose Synthase with Respect to Accumulation of Cellulose and Callose Synthesis in Wheat Roots under Oxygen Deficiency

The sucrose cleavage by sucrose synthase (SuSy) and neutral invertase was studied in wheat roots (Triticum aestivum L.) subjected to hypoxia or anoxia for 4 days. By in situ activity staining, increased SuSy activity was observed in the tip region and stele of root axes while the activity of invertase decreased. Cellulose content significantly increased in hypoxically treated roots. The cellulose deposition was correlated with regions of high SuSy activity, being mainly located in the pericycle and endodermis. Invertase activity was distributed along the root without clear difference between cortex and stele. Under root hypoxia, a significant increase in the structural carbohydrates, callose and especially cellulose, was shown. Increasing levels of soluble carbohydrates were partially used to synthesize cellulose for secondary wall thickening and callose to counteract the tissue injury following low-oxygen stress. Under strict anoxia, the roots were much more injured but sustained a high level of cellulose and callose while the soluble carbohydrates almost disappeared.     

The developmental and organ specific expression of sucrose cleaving enzymes in sugar beet suggests a transition between apoplasmic and symplasmic phloem unloading in the tap roots

Sucrose utilisation in sink tissues depend on its cleavage and is mediated by two different classes of enzymes, invertase and sucrose synthase, which determine the mechanism of phloem unloading. Cloning of two extracellular (BIN35 and BIN46) and one vacuolar invertase (BIN44) provided the basis for a detailed molecular analysis of the relative contribution of the sucrose cleaving enzymes to the sink metabolism of sugar beets (Beta vulgaris) during development. The determination of the steady state levels of mRNAs has been complemented by the analysis of the corresponding enzyme activities. The present study demonstrates an inverse regulation of extracellular invertase and sucrose synthase during tap root development indicating a transition between functional unloading pathways. Extracellular cleavage by invertase is the dominating mechanism to supply hexoses via an apoplasmic pathway at early stages of storage root development. Only at later stages sucrose synthase takes over the function of the key sink enzyme to contribute to the sink strength of the tap root via symplasmic phloem unloading. Whereas mRNAs for both extracellular invertase BIN35 and sucrose synthase were shown to be induced by mechanical wounding of mature leaves of adult plants, only sucrose synthase mRNA was metabolically induced by glucose in this source organ supporting the metabolic flexibility of this species.