Enzyme In Sucrose Biosynthesis Research Articles

Molecular cloning, sequence analyses, and expression studies of sucrose-phosphate synthase in the potato (Solanum tuberosum L.) cultivars

Sucrose-phosphate synthase (SPS, EC 2.4.1.14) refers to a key enzyme in sucrose biosynthesis in both photosynthetic and nonphotosynthetic tissues of plants. It is encoded by different gene families. SPS exists in multiple forms which show differential distributions and functional specializations in the plant tissues. SPS activity is highly regulated by hierarchy of mechanisms including transcriptional control. Here, we report an isolation of a cDNA clone (3,591 bp) encoding full-length SPS A form consisting of 1,054 amino acids (designated KC-SPS1) from a commercially important Indian potato (Solanum tuberosum L.) cultivar, Kufri Chipsona-1 by RT-PCR approach from tuber total RNA. KC-SPS1 shared 99 % sequence identity with 1,053-amino acid SPS from potato cv. Desiree. Apart from some prominent amino acid substitutions, one extra Met residue at position 235 made KC-SPS1 a distinct member of SPS A family in potato. Full-length SPS sequences from taxonomically different plant species were used in making a phylogenetic tree which showed both evolutionary relatedness, and also their grouping into different SPS families. Hydropathy characters and secondary structures were predicted in various SPS forms. Sequence analyses and comparison of the SPS sequences from the Solanaceae family members revealed many distinct features within and between the SPS gene families which were not documented earlier. SPS A form-specific expression patterns were studied in the leaves and tubers of different potato cultivars based on semi-quantitative RT-PCR and protein blot analyses. SPS activities particularly in the cold-stored tubers were probably due to altered kinetic properties. This report would be useful for in-depth studies on various SPS isoforms in potato and other Solanaceae family members.

Phylogenetic and expression analysis of sucrose phosphate synthase isozymes in plants

In plants and microbes, sucrose phosphate synthase (SPS) is an important enzyme in sucrose biosynthesis. Several different isozymes of SPS exist in plants. Genomic and EST sequence data from Arabidopsis, rice and maize has been analyzed. This analysis has revealed that the Arabidopsis genome contains four unique SPS genes. The rice databases (Monsanto proprietary, and public databases) contain five unique full-length SPS genes. Using the Monsanto maize EST and genomic sequence databases, we have identified five full length and two partial SPS sequences, bringing the total number of presently known maize SPS genes to at least seven. Phylogenetic analysis of all known SPS sequences revealed several putative evolutionary branches of SPS. We have classified SPS genes into three major groups in higher plants, all with distinct features from the known microbial SPS genes. Furthermore, this analysis suggests evolutionary divergence of monocotyledonous (monocot) and dicotyledonous (dicot) SPS sequences. The evidence suggests that several gene duplication events occurred at various points during evolution, both before and after the monocot/dicot split. It appears that at least one of the major forms of SPS genes may have evolved after the divergence of monocots and dicots. In addition, several more recent gene duplication events may have occurred after maize/rice speciation, giving rise to additional SPS genes in maize. Some of the variants lack one or more of the presently known regulatory sites, implying that this evolutionary divergence may have given rise to enzymes with functional differences. We present evidence from transcript distribution studies using cDNA libraries as well as transcriptional profiling experiments and propose that specific SPS genes have diverse patterns of expression that are sometimes responsive to environmental signals. Our data suggests that higher plant SPS isozymes differ with respect to their patterns of expression and regulation and that our proposed phylogenetic classification reflects specific functional categories for higher plant SPS isozymes.

The Expression of a Cytosolic Fructose-1,6-Bisphosphatase, a Key Enzyme in Sucrose Biosynthesis, Gene was Diurnally Fluctuated and Increased in Cold Acclimated Leaves of Chinese Cabbage

Chinese cabbage (Brassica rapa ssp. pekinesis) is one of the most important vegetable crops in korea and other East Asian countries. Cytosolic fructose-1,6-bisphospha-tase (cytFBPase) is a key enzyme in sucrose biosyn-thesis, which controls the sucrose levels as well as the productivity at plants. The Chinese cabbage cytFBPase gene, BrFBPase, encodes the 340 amino acid polypep-tide, giving a theoretical molecular weight of 37.2 kD and a isolectric point of 5.4. BrFBPase showed high sequence identity with Brassica homologs and its functional domains, such as 12,6P binding site or active site and F6P binding site, were highly conserved in diverse sources of organisms. Although the genome of Chinese cabbage seemed to be triplicated, BrFBPase appears to be a single copy gene. The expression of BrFBPase was examined at transcript and protein levels under various conditions. BrFBPase expression was observed only in photosynthetic source tissue, not in sink tissue. The expression was slightly higher during the day than at night, and it showed a diurnal cycle with circadian rhythmicity. Short-term exposure to low temperature inhibited the expression of the BrFBPase, while long-term exposure increased the expression, supporting that sugar levels are high in late autumn when temperature are low.

Light regulation of sucrose-phosphate synthase activity in the freezing-tolerant grass Deschampsia antarctica

Deschampsia antarctica, a freezing-tolerant grass that has colonized the Maritime Antarctic, has an unusually high content of sucrose (Suc) in leaves, reaching up to 36% of dry weight. Suc accumulation has often been linked with increased activity of sucrose phosphate synthase (SPS; EC: 2.4.1.1.14). SPS, a key enzyme in sucrose biosynthesis, is controlled by an intricate hierarchy of regulatory mechanisms including allosteric modulators, reversible covalent modification in response to illumination, and transcriptional regulation. We hypothesized that during long day conditions in the Antarctic summer D. antarctica can maintain high SPS activity longer by indirect light regulation, thereby leading to a high sucrose accumulation in the leaves. The objectives of this study were to investigate a possible indirect light regulation of SPS activity and the effect of cold and day length on transcriptional and protein level of SPS in D. antarctica. Although SPS activity did not display an endogenous rhythm of activity in continuous light, activation of SPS at the end of the dark period was observed in D. antarctica. This activation of SPS is possibly controlled by covalent modification, because it was inhibited by okadaic acid while the SPS protein level did not significantly change. The highest SPS activity increase was observed after 21 days of cold-acclimation under long day conditions. This increased activity was not related to an increase in SPS gene expression or protein content. High SPS activity in cold long days leading to hyper accumulation of Suc appears to be among the features that permit D. antarctica to survive in the harsh Antarctic conditions.

Circadian Regulation of Sucrose Phosphate Synthase Activity in Tomato by Protein Phosphatase Activity.

Sucrose phosphate synthase (SPS), a key enzyme in sucrose biosynthesis, is regulated by protein phosphorylation and shows a circadian pattern of activity in tomato. SPS is most active in its dephosphorylated state, which normally coincides with daytime. Applying okadaic acid, a potent protein phosphatase inhibitor, prevents SPS activation. More interesting is that a brief treatment with cycloheximide, a cytoplasmic translation inhibitor, also prevents the light activation of SPS without any effect on the amount of SPS protein. Cordycepin, an inhibitor of transcript synthesis and processing, has the same effect. Both of these inhibitors also prevent the activation phase of the circadian rhythm in SPS activity. Conversely, cycloheximide and cordycepin do not prevent the decline in circadian SPS activity that normally occurs at night. These observations indicate that SPS phosphatase activity but not SPS kinase activity is controlled, directly or indirectly, at the level of gene expression. Taken together, these data imply that there is a circadian rhythm controlling the transcription of a protein phosphatase that subsequently dictates the circadian rhythm in SPS activity via effects on this enzyme's phosphorylation state.

Sucrose metabolism and cell elongation in developing sunflower hypocotyls

The relationships between cell elongation and changes in specific activities of enzymes of sucrose metabolism were investigated in the growing region of hypocotyls of sunflower seedlings (Helianthus annuus L.) that were grown either in darkness or irradiated with continuous white light (WL). After transfer of dark-grown seedlings into WL an inhibition of cell elongation was observed. In etiolated stems, changes in enzymes of sucrose breakdown (acid invertase, sucrose synthase) were closely correlated with the rate of cell elongation. Irradiation with WL induced a large drop in acid invertase and a significant decrease in sucrose synthase. The changes in concentration of sucrose were inversely correlated with the activities of the sucrose breakdown enzymes. A short-term experiment revealed that the effect of WL on growth was more rapid than the inhibitory effect on invertase activity. In dark-grown stems the activities of enzymes of sucrose biosynthesis (sucrose-phosphate synthase, ribulose 1,5 bisphosphate carboxylase/oxygenase) were very low. After WL irradiation significant enhancements were measured. However, activities of enzymes of sucrose breakdown were still much larger than those of sucrose biosynthesis, indicating that the green (de-etiolated) stem remains a sink for sucrose, We suggest that the relative maintenance of cell osmotic pressure and turgor during rapid cell elongation in darkness is due to enhanced hydrolysis of imported sucrose, which is cleaved by two enzymes (invertase, sucrose synthase). This process is regulated by light and hence is under environmental control

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.