Involvement of sucrose synthase in sucrose catabolism

Maize scutellum slices incubated in water utilized sucrose at a maximum rate of 0.12,μmol/min per g fr. wt of slices. When slices were incubated in DNP, there was a three-fold increase in the rate of sucrose utilization. Sucrose breakdown in higher plants can be achieved by pathways starting with either invertase or sucrose synthase (SS). Invertase activity in scutellum homogenates was found only in the cell wall fraction, indicating that SS was responsible for sucrose breakdown in vivo. SS in crude scutellum extracts broke down sucrose to fructose and UDPG at 0.39,μmol/min per g fresh wt of slices. The UDPG formed was not converted to UDP + glucose, UMP + glucose-1-P, UDP + glucose-1-P or broken down by any other means by the crude extract in the absence of PPi. In the presence of PPi, UDPG was broken down by UDPG pyrophosphorylase which had a maximum activity of 26 μmol/min per g fr. wt of slices. Levels of PPi in the scutellum could not be measured using the UDPG pyrophosphorylase: phosphoglucomutase: glucose-6-P dehydrogenase assay because they were too low relative to glucose-6-P which interferes in the assay. An active inorganic pyrophosphatase was present in the scutellum extract which could prevent the accumulation of PPi in the cytoplasm. ATP pyrophosphohydrolase, which hydrolyses ATP to AMP and PPi, was found in the soluble portion of the scutellum extract. The enzyme activity was increased by fructose-2,6-bisP and Ca²⁺. In the presence of both activators, enzyme activity was 1.1 μmol/min per g fr. wt of slices, a rate sufficient to supply PPi for the breakdown of UDPG. These results indicate that sucrose breakdown in maize scutellum cells occurs via the SS: UDPG pyrophosphorylase pathway.     

No evidence for the occurrence of substrate inhibition of Arabidopsis thaliana sucrose synthase-1 (AtSUS1) by fructose and UDP-glucose

Sucrose synthase (SuSy) catalyzes the reversible conversion of sucrose and NDP into the corresponding nucleotide-sugars and fructose. The Arabidopsis genome possesses six SUS genes (AtSUS1–6) that code for proteins with SuSy activity. As a first step to investigate optimum fructose and UDP-glucose (UDPG) concentrations necessary to measure maximum sucrose-producing SuSy activity in crude extracts of Arabidopsis, in this work we performed kinetic analyses of recombinant AtSUS1 in two steps: (1) SuSy reaction at pH 7.5, and (2) chromatographic measurement of sucrose produced in step 1. These analyses revealed a typical Michaelis-Menten behavior with respect to both UDPG and fructose, with Km values of 50 μM and 25 mM, respectively. Unlike earlier studies showing the occurrence of substrate inhibition of UDP-producing AtSUS1 by fructose and UDP-glucose, these analyses also revealed no substrate inhibition of AtSUS1 at any UDPG and fructose concentration. By including 200 mM fructose and 1 mM UDPG in the SuSy reaction assay mixture, we found that sucrose-producing SuSy activity in leaves and stems of Arabidopsis were exceedingly higher than previously reported activities. Furthermore, we found that SuSy activities in organs of the sus1/sus2/sus3/sus4 mutant were ca. 80–90% of those found in WT plants.     

Enzymatic activities of starch synthesis in potato tubers of different sizes

The objective of this study was to determine the relationship between tuber weight and enzymatic activities involved in tuber starch synthesis. As tuber weight increased, the activities of sucrose synthetase, UDPG pyrophosphorylase, and granular starch synthetase escalated, whereas the activities of soluble starch synthetase and ADPG pyrophosphorylase stayed constant and that of phosphorylase declined. This suggests that when samples are taken to determine specific enzymatic activities, the sampling procedure should ensure that results do not vary because of differences in the tuber weight or size distribution.     

Sucrose synthetase from triploid quaking aspen callus

Sucrose synthetase and UDPG pyrophosphorylase were found in crude extracts of dark-grown triploid quaking aspen callus. The sucrose synthetase was like that from other plants, but it appeared to be membrane-bound. The activity also occurred in extracts prepared from callus of other tree species.     

Carbon partitioning to cellulose synthesis

This article discusses the importance and implications of regulating carbon partitioning to cellulose synthesis, the characteristics of cells that serve as major sinks for cellulose deposition, and enzymes that participate in the conversion of supplied carbon to cellulose. Cotton fibers, which deposit almost pure cellulose into their secondary cell walls, are referred to as a primary model system. For sucrose synthase, we discuss its proposed role in channeling UDP-Glc to cellulose synthase during secondary wall deposition, its gene family, its manipulation in transgenic plants, and mechanisms that may regulate its association with sites of polysaccharide synthesis. For cellulose synthase, we discuss the organization of the gene family and how protein diversity could relate to control of carbon partitioning to cellulose synthesis. Other enzymes emphasized include UDP-Glc pyrophosphorylase and sucrose phosphate synthase. New data are included on phosphorylation of cotton fiber sucrose synthase, possible regulation by Ca²⁺ of sucrose synthase localization, electron microscopic immunolocalization of sucrose synthase in cotton fibers, and phylogenetic relationships between cellulose synthase proteins, including three new ones identified in differentiating tracheary elements of Zinnia elegans. We develop a model for metabolism related to cellulose synthesis that implicates the changing intracellular localization of sucrose synthase as a molecular switch between survival metabolism and growth and/or differentiation processes involving cellulose synthesis. Abbreviations: CesA, cellulose synthase; Csl, cellulose-like synthase (genes); DCB, dichlobenil; DPA, days after anthesis; SPS, sucrose phosphate synthase; SuSy, sucrose synthase; P-SuSy, particulate SuSy; S-SuSy, soluble SuSy     

New enzymes,new pathways and an alternative view on starch biosynthesis in both photosynthetic and heterotrophic tissues of plants

Since the initial discovery showing that ADPglucose (ADPG) serves as the universal glucosyl donor in the reaction catalyzed by starch synthase, the mechanism of starch biosynthesis in both leaves and heterotrophic organs has generally been considered to be an unidirectional process wherein ADPG pyrophosphorylase (AGPase) exclusively catalyzes the synthesis of ADPG and acts as the major limiting step of the gluconeogenic process. There is however mounting evidence that ADPG linked to starch biosynthesis is produced de novo in the cytosol by means of sucrose synthase (SuSy). In this review we show and discuss the numerous pitfalls of the ‘classic’ view of starch biosynthesis. In addition, we describe many overlooked aspects of both ADPG and starch metabolism. With the overall data we propose an ‘alternative’ model of starch biosynthesis, applicable to both photosynthetic and heterotrophic tissues, according to which both sucrose and starch biosynthetic processes are tightly interconnected by means of an ADPG synthesizing SuSy activity. According to this new view, starch metabolism embodies catabolic and anabolic reactions taking place simultaneously in which AGPase plays a vital role in the scavenging of starch breakdown products.     

Regulation of Sucrose Metabolism in Higher Plants: Localization and Regulation of Activity of Key Enzymes

ABSTRACT

Sucrose (Sue) plays a central role in plant growth and development. It is a major end product of photosynthesis and functions as a primary transport sugar and in some cases as a direct or indirect regulator of gene expression. Research during the last 2 decades has identified the pathways involved and which enzymes contribute to the control of flux. Availability of metabolites for Sue synthesis and 'demand' for products of sucrose degradation are important factors, but this review specifically focuses on the biosynthetic enzyme sucrose-phosphate synthase (SPS), and the degradative enzymes, sucrose synthase (SuSy), and the invertases. Recent progress has included the cloning of genes encoding these enzymes and the elucidation of posttranslational regulatory mechanisms. Protein phosphorylation is emerging as an important mechanism controlling SPS activity in response to various environmental and endogenous signals. In terms of Sue degradation, invertase-catalyzed hydrolysis generally has been associated with cell expansion, whereas SuSy-catalyzed metabolism has been linked with biosynthetic processes (e.g., cell wall or storage products). Recent results indicate that SuSy may be localized in multiple cellular compartments: (1) as a soluble enzyme in the cytosol (as traditionally assumed); (2) associated with the plasma membrane; and (3) associated with the actin cytoskeleton. Phosphorylation of SuSy has been shown to occur and may be one of the factors controlling localization of the enzyme. The purpose of this review is to summarize some of the recent developments relating to regulation of activity and localization of key enzymes involved in sucrose metabolism in plants.     

Sucrose accumulation in sweet sorghum stem internodes in relation to growth

Sweet sorghum (Sorghum bicolor L. Moench) stems of different cultivars (NK 405. Keller and Tracy) reveal a different pattern of sucrose accumulation with respect to in-ternodal sugar content and distribution. The onset of sucrose storage is not necessarily associated with the reproductive stage of the plant, as was hitherto assumed, but obviously occurs after cessation of internodai elongation as was postulated for the sugarcane stem. For at least two of the three cultivars, ripening is an internode to internode process beginning at the lowermost culm parts. Intensive growth of the internodes, combined with a high hexose content in stern parenchyma, shows a strong positive correlation (r |Mg 0.94) to the activity of sucrose synthase (SuSy; EC 2.4.13), but not to invertase (EC 3.2.1.26) which is not present as soluble (neutral and acid) or cell wall-bound, salt-extractable enzyme in the three culsivars investigated. Sucrose synthase measured in sucrose cleavage and synthesis direction reveals divergent activity rates and sensitivity towards exogenously applied Mg²⁺ ions and pH. SuSy activity is connected to the increase of internodai sucrose content in so far as (1) its decline is a prerequisite for the onset of sucrose accumulation and (2) it remains at a constant low level during sucrose storage. Sucrose phosphate synthase (SPS; EC 2.4.1.14) activity in the sorghum stem is low compared to SuSy and uniformly distributed over all inter-nodes. Only source leaves of sorghum show a considerable SPS activity, but neither stem nor leaf SPS reveal a positive correlation to the increase of internodai sucrose content. Sucrose phosphate phosphatase (SPP; EC 3.1.3.24) amounts lo only 24–30% of the respective SPS activity but follows the same distribution pattern. None of the enzymes under study proves to be responsible for the extent of sucrose storage in the stem, so other phenomena such as transport processes within the stern tissue require further investigation.     

Regulation of Sucrose Metabolism in Higher Plants: Localization and regulation of Activity of Key Enzymes

Sucrose (Suc) plays a central role in plant growth and development. It is a major end product of photosynthesis and functions as a primary transport sugar and in some cases as a direct or indirect regulator of gene expression. Research during the last 2 decades has identified the pathways involved and which enzymes contribute to the control of flux. Availability of metabolites for Suc synthesis and ‘demand’ for products of sucrose degradation are important factors, but this review specifically focuses on the biosynthetic enzyme sucrose-phosphate synthase (SPS), and the degradative enzymes, sucrose synthase (SuSy), and the invertases. Recent progress has included the cloning of genes encoding these enzymes and the elucidation of posttranslational regulatory mechanisms. Protein phosphorylation is emerging as an important mechanism controlling SPS activity in response to various environmental and endogenous signals. In terms of Suc degradation, invertase-catalyzed hydrolysis generally has been associated with cell expansion, whereas SuSy-catalyzed metabolism has been linked with biosynthetic processes (e.g., cell wall or storage products). Recent results indicate that SuSy may be localized in multiple cellular compartments: (1) as a soluble enzyme in the cytosol (as traditionally assumed); (2) associated with the plasma membrane; and (3) associated with the actin cytoskel-eton. Phosphorylation of SuSy has been shown to occur and may be one of the factors controlling localization of the enzyme. The purpose of this review is to summarize some of the recent developments relating to regulation of activity and localization of key enzymes involved in sucrose metabolism in plants.     

Changes in enzyme activities involved in formation and interconversion of UDP-sugars during the cell cycle in a synchronous culture of Catharanthus roseus

Changes in the activities of enzymes involved in UDP-sugar formation [UDP-glucose pyrophosphorylase (EC 2.7.7.9), sucrose synthase (EC 2.4.1.13) and UDP-glucuronic acid pyrophosphorylase (EC 2.7.7.44)], and interconversion [UDP-glucuse 4-epimerase (EC 5.1.3.2), UDP-glucose dehydrogenase (EC 1.1.1.22), UDP-glucuronic acid decarboxylase (EC 4.1.1.35) and UDP-xylose 4-epimerase (EC 5.1.3.5)] were investigated during the cell cycle in a synchronous culture of Catharanthus roseus (L.) G. Don. The specific activities of UDP-glucose pyrophosphorylase and UDP-glucose 4-epimerase increased in the G2 phase before the first cell division, and those of sucrose synthase, UDP-glucose dehydrogenase and UDP-glucuronic acid pyrophosphorylase increased in the G1 phase after the first cell division. However, during the cell cycle, UDP-glucuronic acid decarboxylase and UDP-xylose 4-epimerase did not change significantly in their specific activities. Changes in enzyme activities are discussed in relation to those reported previously for cell wall composition (S. Amino et al. 1984. Physiologia Plantarum 60: 326–332).     

Toward a blueprint for UDP-glucose pyrophosphorylase structure/function properties: homology-modeling analyses

UDP-glucose pyrophosphorylase (UGPase) is an important enzyme of synthesis of sucrose, cellulose, and several other polysaccharides in all plants. The protein is evolutionarily conserved among eukaryotes, but has little relation, aside from its catalytic reaction, to UGPases of prokaryotic origin. Using protein homology modeling strategy, 3D structures for barley, poplar, and Arabidopsis UGPases have been derived, based on recently published crystal structure of human UDP-N-acetylglucosamine pyrophosphorylase. The derived 3D structures correspond to a bowl-shaped protein with the active site at a central groove, and a C-terminal domain that includes a loop (I-loop) possibly involved in dimerization. Data on a plethora of earlier described UGPase mutants from a variety of eukaryotic organisms have been revisited, and we have, in most cases, verified the role of each mutation in enzyme catalysis/regulation/structural integrity. We have also found that one of two alternatively spliced forms of poplar UGPase has a very short I-loop, suggesting differences in oligomerization ability of the two isozymes. The derivation of the structural model for plant UGPase should serve as a useful blueprint for further function/structure studies on this protein.     

Production of high-starch, low-glucose potatoes through over-expression of the metabolic regulator SnRK1

Transgenic potato ( Solanum tuberosum cv. Prairie) lines were produced over-expressing a sucrose non-fermenting-1-related protein kinase-1 gene ( SnRK1 ) under the control of a patatin (tuber-specific) promoter. SnRK1 activity in the tubers of three independent transgenic lines was increased by 55%−167% compared with that in the wild-type. Glucose levels were decreased, at 17%−56% of the levels of the wild-type, and the starch content showed an increase of 23%−30%. Sucrose and fructose levels in the tubers of the transgenic plants did not show a significant change. Northern analyses of genes encoding sucrose synthase and ADP-glucose pyrophosphorylase, two key enzymes involved in the biosynthetic pathway from sucrose to starch, showed that the expression of both was increased in tubers of the transgenic lines compared with the wild-type. In contrast, the expression of genes encoding two other enzymes of carbohydrate metabolism, α-amylase and sucrose phosphate synthase, showed no change. The activity of sucrose synthase and ADP-glucose pyrophosphorylase was also increased, by approximately 20%–60% and three- to five-fold, respectively, whereas the activity of hexokinase was unchanged. The results are consistent with a role for SnRK1 in regulating carbon flux through the storage pathway to starch biosynthesis. They emphasize the importance of SnRK1 in the regulation of carbohydrate metabolism and resource partitioning, and indicate a specific role for SnRK1 in the control of starch accumulation in potato tubers.     

Evidence for a tonoplast-associated form of sucrose synthase and its potential involvement in sucrose mobilization from the vacuole

The following work presents new evidence for a tonoplast localization of sucrose synthase and its functional role during the mobilization of vacuolar sucrose. Highly purified tonoplast vesicles were associated with significant SuSy levels as determined by immuno-recognition, enzymatic activity, and by-product measurements. Total tonoplast-bound SuSy was estimated to be approximately 7% of the total tissue activity. SuSy affinity to the tonoplast was confirmed by the lack of SuSy displacement by ionic washes and also by the tonoplast ability to bind to exogenously added SuSy as compared to the cytosolic marker alcohol dehydrogenase. UL-[(14)C]sucrose-loaded vesicles incubated with ATP and UDP produced [(14)C]UDP-Glc as determined by UDP-Glc dehydrogenase and by the ability of the product to bind to DEAE-cellulose and to co-migrate with authentic UDP-Glc on TLC. ATP alone induced sucrose efflux but not the production of [(14)C]ADP-Glc. Kinetic analysis of [(14)C]UDPG formation under conditions of low sucrose availability suggests sucrose channelling between the ATP-dependent sucrose transporter and SuSy, thus corroborating the association of SuSy with the tonoplast and its involvement in sucrose mobilization from the vacuole.     

Regulation and roles for alternative pathways of hexose metabolism in plants

Plant cells have two cytoplasmic pathways of glycolysis and gluconeogenesis for the reversible interconversion of fructose 6-phosphate (F-6-P) and fructose 1,6-bisphosphate (F-1,6-P₂). One pathway is described as a maintenance pathway that is catalyzed by a nucleotide triphosphate-dependent phosphofructokinase (EC 2.7.1.11; ATP-PFK) glycolytically and a F-1,6 bisphosphatase (EC 3.1.3.11) gluconeogenically. These are non-equilibrium reactions that are energy consuming. The second pathway, described as an adaptive pathway, is catalyzed by a readily reversible pyrophosphate-dependent phosphofructokinase (EC 2.7.1.90; PP-PFK) in an equilibrium reaction that conserves energy through the utilization and the synthesis of pyrophosphate. A constitutive regulator cycle is also present for the synthesis and hydrolysis of fructose 2,6-bisphosphate (F-2,6-P₂) via a 2-kinase and a 2-phosphatase, respectively. The pathway catalyzed by the ATP-PFK and F-1,6-bisphosphatase, the maintenance pathway, is fairly constant in maximum activity in various plant tissues and shows less regulation by F-2,6-P₂. Plants use F-2,6-P₂ initially to regulate the adaptive pathway at the reversible PP_i-PFK step. The adaptive pathway, catalyzed by PP_i-PFK, varies in maximum activity with a variety of phenomena such as plant development or changing biological and physical environments. Plants can change F-2,6-P₂ levels rapidly, in less than 1 min when subjected to rapid environmental change, or change levels slowly over periods of hours and days as tissues develop. Both types of change enable plants to cope with the environmental and developmental changes that occur during their lifetimes. The two pathways of sugar metabolism can be efficiently linked by the cycling of uridylates and pyrophosphate required for sucrose breakdown via a proposed sucrose synthase pathway. The breakdown of sucrose via the sucrose synthase pathway requires half the net energy of breakdown via the invertase pathway. Pyrophosphate occurs in plant tissues as a substrate pool for biosynthetic reactions such as the PP_i-PFK or uridine diphosphate glucose pyrophosphorylase (EC 2.7.7.9; UDPG pyrophosphorylase) that function in the breakdown of imported sucrose. Also, pyrophosphate links the two glycolytic/gluco-neogenic pathways; and in a reciprocal manner pyrophosphate is produced as an energy source during gluconeogenic carbon flow from F-1,6-P₂ toward sucrose synthesis.     

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.     

Gene expression studies on developing kernels of maize sucrose synthase (SuSy) mutants show evidence for a third SuSy gene

Previous studies have identified two tissue- and cell-specific, yet functionally redundant, sucrose synthase (SuSy) genes, Sh1 and Sus1, which encode biochemically similar isozymes, SH1 and SUS1 (previously referred to as SS1 and SS2, respectively). Here we report evidence for a third SuSy gene in maize, Sus3, which is more similar to dicot than to monocot SuSys. RNA and/or protein blot analyses on developing kernels and other tissues show evidence of expression of Sus3, although at the lowest steady-state levels of the three SuSy gene products and without a unique pattern of tissue specificity. Immunoblots of sh1sus1-1 embryos that are either lacking or deficient for the embryo-specific SUS1 protein have shown a protein band which we attribute to the Sus3 gene, and may contribute to the residual enzyme activity seen in embryos of the double mutant. We also studied developing seeds of the double mutant sh1sus1-1, which is missing 99.5% of SuSy enzyme activity, for evidence of co-regulation of several genes of sugar metabolism. We found a significant reduction in the steady-state levels of Miniature-1 encoded cell wall invertase2, and Sucrose transporter (Sut) mRNAs in the double mutant, relative to the lineage-related sh1Sus1 and sh1Sus1 kernels. Down-regulation of the Mn1 gene was also reflected in significant reductions in cell wall invertase activity. Co-regulatory changes were not seen in the expression of Sucrose phosphate synthase, UDP-glucose pyrophosphorylase, and ADP-glucose pyrophosphorylase.     

Role of enzymes of sucrose-starch conversion in seed sink strength in mung bean

Changes in the activities of sucrose synthase (SuSy), ADP-glucose pyrophosphorylase (AGPase), UDP-glucose pyrophosphorylase (UGPase), alkaline inorganic pyrophosphatase, 3-phosphoglycerate (3-PGA) phosphatase and amylases were monitored in relation to accumulation of starch in developing pods of mung bean (Vigna radiata L.). With the advancement in the seed development, the contents of starch rose with a concomitant fall in the branch of inflorescence and podwall after 10 d after flowering. The activity of UDPase in all the three pod tissues remained higher than the activity of AGPase showing it to be an important enzyme controlling carbon flux. The activity of alkaline inorganic pyrophosphatase in developing seed in contrast to 3-PGA phosphatase correlated with starch accumulation rate. Activity of β-amylase increased in all the pod tissues till maturity. It appears that the cooperative action of SuSy, UGPase and AGPase controls the efficient partitioning of sucrose into ADP glucose and thereby regulate the seed sink strength of the mung bean.