Partial purification and characterisation of sucrose synthase in sugarcane

Three sucrose synthase (SuSy) (EC 2.4.1.13) forms were isolated from sugarcane leaf roll tissue. During anion exchange chromatography, one peak of activity (SuSyA) eluted during the wash step and the other peak (SuSyB) during the salt gradient phase at 180mM KCl concentration. A third form of activity (SuSyC), which also eluted at 180mM KCl, was also present in the leaf roll and replaced SuSyB depending on the season of the year. Substrate Km values, as well as sucrose breakdown/synthesis ratios, differed between these forms. For SuSyA, SuSyB, and SuSyC, respectively, Km values+/-SE (mM) were: 41.8+/-3.4, 109+/-23, and 35.9+/-2.3 for sucrose, 1.07+/-0.08, 0.214+/-0.039, and 0.00191+/-0.00019 for UDP, 6.62+/-1.55, 11.7+/-2.6, and 6.49+/-0.61 for fructose, and 3.59+/-0.37, 0.530+/-0.142, and 0.234+/-0.025 for UDP-glucose. Sucrose breakdown/synthesis ratios+/-SE were 0.0791+/-0.0199, 0.330+/-0.180, and 0.426+/-0.069 for SuSyA, SuSyB, and SuSyC, respectively. The ratio of the area of peak 1 (low breakdown/synthesis ratio) to the area of peak 2 (high breakdown/synthesis ratio) in sucrose accumulating tissue (internode 9) was 0.88, while in non-accumulating (leaf roll) tissue it was 14.5 at the same time of year. The molecular mass of the denatured subunits of all three forms was 94kDa by SDS-PAGE. A polyclonal antiserum raised against SuSyB cross-reacted with all three forms on an immunoblot, but only SuSyA and SuSyB were immunoinactivated by this serum.     

Invertase,sucrose synthase and sucrose phosphate synthase in lyophilized spruce needles; microplate reader assays

Summary Data on changes of apparent activities of enzymes involved in sucrose metabolism of developing spruce needles are presented. Extractable activities of sucrose phosphate synthase (SPS, sucrose synthesis), and sucrose synthase (SS) and acid invertase (both sucrolysis) were determined in small volumes using a novel microplate reader system which combined high rates of activity with good reproducibility and high sample throughput. During a developmental period of up to 18 months after bud break characteristic changes in SPS and SS occurred. During the first 4 months of needle development SS declined while SPS increased which is indicative of a transition from net import to net export of photoassimilates (sink/source transition). After needle maturation both enzymes exhibited parallel annual changes with increasing rates towards autumn, which was mirrored by the pool sizes of sucrose (possibly due to the acquisition of frost hardiness). Acid invertase activity was comparable to that of SS but showed only marginal seasonal changes. Approximately 70% of its total activity was found to be soluble.     

Over-expression of an arabidopsis family A sucrose phosphate synthase (SPS) gene alters plant growth and fibre development

The objective of this study was to manipulate the intracellular pools of sucrose by differentially expressing exogenous sucrose phosphate synthase (SPS) and investigating its role in regulating plant growth and fibre development. Tobacco (Nicotiana tabacum cv. Xanthi) plants were transformed with an arabidopsis SPS gene under the regulation of the ubiquitously expressed tandem repeat of the 35S cauliflower mosaic virus promoter, and subject to growth trials and fibre characterization. It was apparent that over-expression of SPS resulted in substantially elevated concentrations of sink sucrose pools compared to wild-type plants, while source tissue sucrose pools remained the same. All transformed plants had significantly increased stem height, which was ascribed to internode elongation, and greater stem diameters, longer fibers and increased total dry biomass relative to the control plants. Difference in the chemical composition of either the storage or structural carbohydrates of the wild-type and SPS transgenic lines were only minor. The correlation between increased stem sucrose content and plant phenotypes with elevated SPS gene expression confirm a role for sucrose availability in controlling plant growth and fibre elongation.     

Regulation of sucrose storage by amino acid arginine in sugarcane cell suspension cultures

Sugarcane cell cultures were obtained from callus formed on explants derived from young expanding leaves of two early maturing sugarcane varieties viz “CoJ83” and “CoJ86”. The cell cultures were varied with different arginine concentrations in the culture medium. For each cultivar, sucrose content with 20 μM arginine in the culture medium decreased from 3 to 5 days and then increased to 10 days after subculturing. Higher concentration of arginine in the culture medium (60 μM) decreased the sucrose content at different days after subculturing and thus significantly stimulated sucrose mobilization. The activity of sucrose synthase and sucrose phosphate synthase reached maximum while the activity of acid and neutral invertase was minimal in the culture medium with 20 μM arginine. Thus arginine at low concentration (20 μM) enables the cells to accumulate the higher level of sucrose. The optimum level of amino acids can be utilized to regulate the in vivo activity of sucrose synthase, sucrose phosphate synthase and invertase to achieve maximum sucrose accumulation in sugarcane storage tissue.     

Carbon metabolism in leaves of micropropagated sugarcane during acclimatization phase

The activity of the main enzymes related to the sucrose metabolism, photosynthesis, and sucrose concentration were studied in sugarcane (Saccharum spp hybrid) plantlets. Acclimatization was developed in two steps. (1) Light intensity of 1,000 μmol m⁻² s⁻¹ and 90% relative humidity during the first 21 d; followed by 2,000 μmol m⁻² s⁻¹ and approximately 80% of relative humidity. All measurements were carried out at the end of rooting phase concomitant with day 0 of acclimatization and at 7-d intervals thereafter (0, 7, 14, 21, 28, 35, 42 d). As the in vitro plantlets were transferred to the acclimatization phase, photosynthesis increased significantly during the first 7 d. After this period, the increase was constant with only a small but nonsignificant decline after being transferred to the uncontrolled external conditions. The activity of the sucrose synthase began to show a decrease, starting from day 7, and was related to the changes that began to happen in these plants from its adaptation to new ex vitro conditions. Due to the increase of fresh weight favored by the high light intensity and lower relative humidity, an increase of the sucrose phosphate synthase activity was observed. The maximum activity of the acid and neutral invertases was reached at 14 and 21 d, respectively, after 21 d of acclimatization period. There was a marked tendency for the activity of both enzymes to decrease. The sucrose content was decreased only in the first 7 d. The metabolism of sugarcane plantlets seemed to be susceptible to the environmental changes during the acclimatization phase but did not contribute to inhibitory factors for normal development.     

Sucrose partitioning between vascular bundles and storage parenchyma in the sugarcane stem: a potential role for the ShSUT1 sucrose transporter

A transporter with homology to the SUT/SUC family of plant sucrose transporters was isolated from a sugarcane (Saccharum hybrid) stem cDNA library. The gene, designated ShSUT1, encodes a protein of 517 amino acids, including 12 predicted membrane-spanning domains and a large central cytoplasmic loop. ShSUT1 was demonstrated to be a functional sucrose transporter by expression in yeast. The estimated K_m for sucrose of the ShSUT1 transporter was 2 mM at pH 5.5. ShSUT1 was expressed predominantly in mature leaves of sugarcane that were exporting sucrose and in stem internodes that were actively accumulating sucrose. Immunolocalization with a ShSUT1-specific antiserum identified the protein in cells at the periphery of the vascular bundles in the stem. These cells became lignified and suberized as stem development proceeded, forming a barrier to apoplasmic solute movement. However, the movement of the tracer dye, carboxyfluorescein from phloem to storage parenchyma cells suggested that symplasmic connections are present. ShSUT1 may have a role in partitioning of sucrose between the vascular tissue and sites of storage in the parenchyma cells of sugarcane stem internodes.     

Sucrose phosphate synthase and other sucrose metabolizing enzymes in fruits of various species

Recent reports have suggested that sucrose phosphate synthase (EC 2.4.1.14), a key enzyme in sucrose biosynthesis in photosynthetic “source” tissues, may also be important in some sucrose accumulating “sink” tissues. These experiments were conducted to determine if sucrose phosphate synthase is involved in sucrose accumulation in fruits of several species. Peach (Prunus persica NCT 516) and strawberry (Fragaria x ananassa cv. Chandler) fruits were harvested directly from the plant at various stages of fruit development. Kiwi (Actinidia chinensis), papaya (Carica papaya), pineapple (Ananas comosus) and mango (Mangifera indica) were sampled in postharvest storage over a period of several days. Carbohydrate concentrations and activities of sucrose phosphate synthase, sucrose synthase (EC 2.4.1.13), and acid and neutral invertases (EC 3.2.1.26) were measured. All fruits contained significant activities of sucrose phosphate synthase. Moreover, in fruits from all species except pineapple and papaya, there was an increase in sucrose phosphate synthase activity associated with the accumulation of sucrose in situ. The increase in sucrose concentration in peaches was also associated with an increase in sucrose synthase activity and, in strawberries, with increased activity of both sucrose synthase and neutral invertase. The hexose pools in all fruits were comprised of equimolar concentrations of fructose and glucose, except in the mango. In mango, the fructose to glucose ratio increased from 2 to 41 during ripening as sucrose concentration more than doubled. The results of this study indicate that activities of the sucrose metabolizing enzymes, including sucrose phosphate synthase, within the fruit itself, are important in determining the soluble sugar content of fruits of many species. This appears to be true for fruits which sweeten from a starch reserve and in fruits from sorbitol translocating species, raffinose saccharide translocating species, and sucrose translocating species.     

Analysis of the sucrose synthase gene family in Arabidopsis

The properties and expression patterns of the six isoforms of sucrose synthase in Arabidopsis are described, and their functions are explored through analysis of T-DNA insertion mutants. The isoforms have generally similar kinetic properties. Although there is variation in sensitivity to substrate inhibition by fructose this is unlikely to be of major physiological significance. No two isoforms have the same spatial and temporal expression patterns. Some are highly expressed in specific locations, whereas others are more generally expressed. More than one isoform is expressed in all organs examined. Mutant plants lacking individual isoforms have no obvious growth phenotypes, and are not significantly different from wild-type plants in starch, sugar and cellulose content, seed weight or seed composition under the growth conditions employed. Double mutants lacking the pairs of similar isoforms sus2 and sus3, and sus5 and sus6, are also not significantly different in these respects from wild-type plants. These results are surprising in the light of the marked phenotypes observed when individual isoforms are eliminated in crop plants including pea, maize, potato and cotton. A sus1/sus4 double mutant grows normally in well-aerated conditions, but shows marked growth retardation and accumulation of sugars when roots are subjected to hypoxia. The sucrose synthase activity in roots of this mutant is 3% or less of wild-type activity. Thus under well-aerated conditions sucrose mobilization in the root can proceed almost entirely via invertases without obvious detriment to the plant, but under hypoxia there is a specific requirement for sucrose synthase activity.     

Ultrastructural detection of sucrose synthase distribution in developing maize leaves

Summary A developing maize leaf grows by the activity of a basal meristematic region and an adjacent elongating zone, resulting in a morphological and functional gradient along the leaf. We have used this system to detect the spatial and temporal expression of an enzyme, sucrose synthase, which plays a pivotal role in the sucrose import-export transition which occurs along a monocotyledon leaf. Immunogold labeling was used to detect the cellular and sub-cellular distribution of sucrose synthase (SS) at the electron microscopical level; the protein was visualized using a polyclonal antiserum on embedded tissue sections. Immunolabel was observed in the cytosol of dividing meristematic cells, expanding cells of the elongation zone, and in differentiating cells of young photosynthetic tissue. In fully differentiated leaf tissue, however, the protein was no longer immuno-detectable in photosynthetic cells, but was present in the guard and subsidiary cells of stomata and in companion cells within the phloem tissue of vascular bundles. The tissue- and cell-specific localization of sucrose synthase changes along the growing leaf as a function of the developmental state and the associated need for sucrose import or export.     

Sugars regulate sucrose transporter gene expression in citrus

We report the isolation and characterization of two sucrose transporter cDNAs (CitSUT1 and CitSUT2) from citrus. CitSUT1 and CitSUT2 encode putative proteins (CitSUT1 and CitSUT2) of 528 and 607 amino acids, respectively. CitSUT1 and CitSUT2 share high similarities with sucrose transporters isolated from other plants. The expression of CitSUT1 in mature leaf discs is repressed by exogenous sucrose, glucose, mannose, and the glucose analog 2-deoxyglucose but not by another glucose analog 3-O-methylglucose, indicating a hexokinase (HXK)-mediated signaling pathway. CitSUT2 expression is not affected by exogenous sugars. Whereas CitSUT1 expresses strongly in source, sugar exporting organs, CitSUT2 expresses more strongly in sink, sugar importing organs, suggesting different physiological roles for these sucrose transporters.     

Minimization of post-harvest sucrose losses in drought affected sugarcane using chemical formulation

Sugarcane is a significant crop for production of sugar and ethanol in the world. In present perspective, drought is one of the frequently occurring abiotic stresses hampering the productivity of sugarcane causing heavy losses in sugar recovery. Post-harvest sugarcane deterioration attains more importance. Measures have been recommended in harvested canes to prevent these losses in general and under drought conditions but application of chemical formulation has not yet been tested over drought effected ones. Thus, we tried to investigate the efficacy of chemical formulation [Benzalkonium chloride (BKC) + Sodium metasilicate (SMS)] on sucrose losses occurring in harvested canes grown under drought and normal conditions. Results showed that application of chemical formulation had higher effect on drought canes in comparison to normal grown canes. Loss in cane weight was reduced to 8.25% and 11% in drought treated and normal treated grown canes, respectively, after 240 h of harvest in comparison to their respective control. In sucrose content and Commercial cane sugars %, drought treated canes showed an effect of BKC + SMS by reducing the losses to 1.26 units and 1.42 units, respectively, whereas in normal ones, reduction was of 0.38 units and 0.10 units, respectively. Biochemical analysis revealed that in reducing sugars, reduction in increase were of 44.51% and 25.50% in drought and normal grown canes, respectively, after 240 h of harvest. Dextran and soluble acid invertase estimations revealed that after application of BKC + SMS, reduction of dextran and invertase activity were of 49.74%, 66.84%, respectively, and 33.92%, 42.75%, respectively, in drought and normal grown canes, respectively. Total microbial load, showed effectiveness of 25.01% in drought grown canes while 14.41% in normal grown ones after 240 h of harvest. Our study was planned to use the anti-bacterial efficiency of both the chemicals over harvested canes so that the major sucrose losses occurring due to microbial deterioration could be inhibited. The use of this chemical formulation proves to be an effective one over post-harvest sucrose losses, particularly in drought grown canes.     

Sucrose metabolism in cold-stored potato tubers with decreased expression of sucrose phosphate synthase

Transfer of potato tubers to low temperature leads after 2–4 d to a stimulation of sucrose synthesis, a decline of hexose-phosphates and a change in the kinetic properties, and the appearance of a new form of sucrose phosphate synthase (SPS). Antisense and co-suppression transformants with a 70–80% reduction in SPS expression have been used to analyse the contribution of SPS to the control of cold sweetening. The rate of sucrose synthesis in cold-stored tubers was investigated by measuring the accumulation of sugars, by injecting labelled glucose of high specific activity into intact tubers, and by providing 50 mol m^–3 labelled glucose to fresh tuber slices from cold-stored tubers. A 70–80% decrease of SPS expression resulted in a reproducible but non-proportional (10–40%) decrease of soluble sugars in cold-stored tubers, and a non-proportional (about 25%) inhibition of label incorporation into sucrose, increased labelling of respiratory intermediates and carbon dioxide, and increased labelling of glucans. The maximum activity of SPS is 50-fold higher than the net rate of sugar accumulation in wild-type tubers, and decreased expression of SPS in the transformants was partly compensated for increased levels of hexose-phosphates. It is concluded that SPS expression per se does not control sugar synthesis. Rather, a comparison of the in vitro properties of SPS with the estimated in vivo concentrations of effectors shows that SPS is strongly substrate limited in vivo . Alterations in the kinetic properties of SPS, such as occur in response to low temperature, will provide a more effective way to stimulate sucrose synthesis than changes of SPS expression.     

蔗糖磷酸合酶功能、结构与催化机制的研究进展

蔗糖是自然界中广泛存在的一种天然产物.在植物等生命体中,蔗糖磷酸合酶(Sucrose phosphate synthase,SPS)是蔗糖合成的限速酶.SPS催化合成蔗糖-6-磷酸;蔗糖磷酸酶(Sucrose Phosphatase,SPP)进一步把蔗糖-6-磷酸上的磷酸根水解下来而形成蔗糖.近几十年来关于SPS的研究...     

Identification of sucrose synthase as an actin-binding protein

Several lines of evidence indicate that sucrose synthase (SuSy) binds both G- and F-actin: (i) presence of SuSy in the Triton X-100-insoluble fraction of microsomal membranes (i.e. crude cytoskeleton fraction); (ii) co-immunoprecipitation of actin with anti-SuSy monoclonal antibodies; (iii) association of SuSy with in situ phalloidin-stabilized F-actin filaments; and (iv) direct binding to F-actin, polymerized in vitro. Aldolase, well known to interact with F-actin, interfered with binding of SuSy, suggesting that a common or overlapping binding site may be involved. We postulate that some of the soluble SuSy in the cytosol may be associated with the actin cytoskeleton in vivo.     

Expression of the Arabidopsis thaliana invertase gene family

Two new loci have been found to be clustered with five other genes for the nitrate assimilation pathway in the Chlamydomonas reinhardtii genome. One gene, located close to the 3′-end of the high-affinity nitrate transporter (HANT) gene Nrt2;2, corresponds to the nitrite reductase (NiR) structural gene Nii1. This is supported by a number of experimental findings: (i) NiR-deficient mutants have lost Nii1 gene expression; (ii) Nii1 mRNA accumulation is co-regulated with the expression of other structural genes of the nitrate assimilation pathway; (iii) nitrite (nitrate) utilization ability is recovered in the NiR mutants by functional complementation with a wild-type Nii1 gene; (iv) the elucidated NII1 amino acid sequence is highly similar to that of the cyanobacterial and higher-plant enzyme, and contains the predicted domains for plastidic ferredoxin-NiRs. Thus, the mutant phenotype and the mRNA sequence and expression of the Nii1 gene have been unequivocally related. Accumulation of mRNA for the second locus identified, Lde1 (light-dependent expression), was not regulated by nitrogen, but like nitrate-assimilation clustered genes, its expression was down-regulated in the dark. Received: 27 November 1997 / Accepted: 19 January 1998