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.     

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.     

Up-regulation of sucrose phosphate synthase in rice grown under elevated CO2 and temperature

Rice (Oryza sativa L. cv. IR-30) was grown season-long in outdoor, controlled-environment chambers at 33 Pa CO₂ with day/night/paddy-water temperatures of 28/21/25 °C, and at 66 Pa CO₂ with five different day/night/paddy-water temperature regimes (25/18/21, 28/21/25, 31/24/28, 34/27/31 and 37/30/34 °C). Sucrose phosphate synthase (SPS) activities in leaf extracts at 21, 48 and 81 days after planting (DAP) were assayed under saturating and selective (limiting) conditions. Diel SPS activity data indicated that rice SPS was light regulated; with up to 2.2-fold higher rates during the day. Throughout the growth season, leaf SPS activities were up-regulated in the CO₂-enriched plants, averaging 20 and 12% higher than in ambient-CO₂ grown plants in selective and saturating assays, respectively. Similarly, SPS activities increased 2.4% for each 1 °C rise in growth temperature from 25 to 34 °C, but de creased 11.5% at 37 °C. Leaf sucrose content was higher, and mirrored SPS activity better, than starch, although starch was more responsive to CO₂ treatment. Leaf sucrose and starch contents were significantly higher throughout the season in plants at elevated CO₂, but the N content averaged 6.5% lower. Increasing growth temperatures from 25 to 37 °C caused a linear decrease (62%) in leaf starch content, but not in sucrose. Consequently, the starch:sucrose ratio declined with growth temperature. The data are consistent with the hypothesis that the up-regulation of leaf SPS may be an acclimation response of rice to optimize the utilization and export of organic-C with the increased rates of inorganic-C fixation in elevated CO₂ or temperature growth regimes.     

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.     

Sucrose metabolism in green algae I. The presence of sucrose synthetase and sucrose phosphate synthetase

Summary The presence of sucrose synthetase and sucrose phosphate synthetase has been demonstrated in two species of green algae:Chlorella vulgaris andScenedesmus obliquus. Partial purification from crude extracts allowed the determination of the kinetic constants of algae enzymes. They are very similar to the ones reported for enzymes from higher plants.Dedicated toLuis F. Leloir on his seventieth birthday.     

Overexpression of sucrose phosphate synthase increases sucrose unloading in transformed tomato fruit

Sucrose unloading and sink activity were examined in tomato plants (Lycopersicon esculentum) overexpression sucrose phosphate synthase (SPS; EC 2.3.1.14). Like the leaves, the fruit of the transformed tomato plants had elevated (2.4-fold) SPS activity. SPS over-expression in tomato fruit did not significantly change acid invertase, and only slightly reduced ADPglc ppase activity, but enhanced sucrose synthase activity by 27%. More importantly, the amount of sucrose unloaded into the fruit was considerably increased. Using [³H]- (fructosyl)-sucrose in in vitro unloading experiments with harvested 20-d-old fruit, 70% more sucrose was unloaded into the transformed fruits compared to the untransformed controls. Furthermore, the turnover of the sucrose unloaded into the fruit of transformed plants was 60% higher than that observed in the untransformed controls. Taken together, these results demonstrate that SPS overexpression increases the sink strength of transformed tomato fruit.     

Sucrose phosphate synthase, a key enzyme for sucrose biosynthesis in plants: protein purification from corn leaves and immunological detection

We have purified the protein for the enzyme sucrose phosphate synthase (SPS) from corn (Zea mays) leaves. Partially purified SPS protein was used to generate specific monoclonal antibodies. The following immunoaffinity chromatography allowed the isolation of pure SPS protein. The apparent molecular mass of the SPS polypeptide is 138 kilodaltons. By immunoblot, an SPS antigen was found to accumulate in mature leaves. SPS protein levels remain constant during the day/night cycle. The observed diurnal fluctuation of extractable enzyme activity, therefore, must be caused by modification of the specific activity of SPS in vivo.     

Sucrose synthase oligomerization and F-actin association are regulated by sucrose concentration and phosphorylation

Sucrose synthase (SUS) is a key enzyme in plant metabolism, as it serves to cleave the photosynthetic end-product sucrose into UDP-glucose and fructose. SUS is generally assumed to be a tetrameric protein, but results in the present study suggest that SUS can form dimers as well as tetramers and that sucrose may be a regulatory factor for the oligomerization status of SUS. The oligomerization of SUS may also affect the cellular localization of the protein. We show that sucrose concentration modulates the ability of SUS1 to associate with F-actin in vitro and that calcium-dependent protein kinase-mediated phosphorylation of recombinant SUS1 at the Ser15 site is a negative regulator of its association with actin. Although high sucrose concentrations and hyperphosphorylation have been shown to promote SUS association with the plasma membrane, we show that the opposite is true for the SUS-actin association. We also show that SUS1 has a unique 28 residue coiled-coil domain that does not appear to play a role in oligomerization, but may prove to be significant in the future for interactions of SUS with other proteins. Collectively, these results highlight the multifaceted nature of SUS association with cellular structures.     

Diurnal changes in sucrose phosphate synthase activity in leaves

Studies were conducted to identify and compare diurnal changes in sucrose phosphate synthase (EC 2.4.1.14) activity in leaves of different species, and the effect of nitrogen nutrition on the rhythm in soybean [ Glycine max (L). Merr] leaves. In recently expanded corn ( Zea mays L.) leaves, a single peak of enzyme activity was observed at the beginning of the photoperiod. A similar pattern was observed in older corn leaves, but activities (leaf fresh weight basis) were lower. In recently expanded pea ( Pisum sativum L.) and soybean leaves, two peaks of sucrose phosphate synthase activity were observed over a 24-h light:dark period, one at the beginning and one at the end of the photoperiod. A similar pattern was observed in older soybean leaves, but activities were generally lower and the amplitude of the changes was reduced. In a separate experiment, soybean plants were grown in the greenhouse with either 2 or 10 m M nitrate. The high-N plants had higher rates of photosynthesis and translocation, and greater activities of sucrose phosphate synthase in leaf extracts, compared to low-N plants. Over both experiments with soybeans, changes in sucrose phosphate synthase activity during the photoperiod were closely aligned with changes in translocation rate.     

Sucrose phosphate synthase activity and the co-ordination of carbon partitioning during sucrose and amino acid accumulation in desiccation-tolerant leaf material of the C4 resurrection plant Sporobolus stapfianus during dehydration

Both sucrose and amino acids accumulate in desiccation-tolerant leaf material of the C(4) resurrection plant, Sporobolus stapfianus Gandoger (Poaceae). The present investigation was aimed at examining sucrose phosphate synthase (SPS) activity and various metabolic checkpoints involved in the co-ordination of carbon partitioning between these competing pathways during dehydration. In the initial phase of dehydration, photosynthesis and starch content declined to immeasurable levels, whilst significant increases in hexose sugars, sucrose, and amino acids were associated with concomitant significant increases in SPS and pyruvate kinase (PK) activities, and maximal activity levels of phosphoenolpyruvate carboxylase (PEPCase), NADP-dependent isocitrate dehydrogenase (NADP-ICDH), and NADH-dependent glutamate synthase (NADH-GOGAT). The next phase of dehydration was characterized by changes in metabolism coinciding with net hexose sugar phosphorylation. This phase was characterized by a further significant increase in sucrose accumulation, with increased rates of net sucrose accumulation and maximum rates of SPS activity measured under both saturating and limiting (inhibitory) conditions. SPS protein was also increased. The stronger competitive edge of SPS for carbon entering glycolysis during hexose phosphorylation was also demonstrated by the further decrease in respiration and the simultaneous, significant decline in both PEPCase and PK activities. A decreased anabolic demand for 2-oxoglutarate (2OG), which remained constant, was shown by the co-ordinated decrease in GOGAT. It is proposed that the further increase in amino acids in this phase of dehydration may be in part attributable to the breakdown of insoluble proteins.     

Sucrose metabolism in green algae. I. The presence of sucrose synthetase and sucrose phosphate synthetase.

The presence of sucrose synthetase and sucrose phosphate synthetase has been demonstrated in two species of green algae: Chlorella vulgaris and Scenedesmus obliquus. Partial purification from crude extracts allowed the determination of the kinetic constants of algae enzymes. They are very similar to the ones reported for enzymes from higher plants.     

Evidence for the involvement of sucrose phosphate synthase in the pathway of sugar accumulation in sucrose-accumulating tomato fruits

To better understand the mechanism of sugar unloading and sugar concentration in hexose- and sucrose-accumulating tomato fruits (Lycopersicon chmielewskii and L. esculentum, respectively) and to determine the causes of the late accumulation of sucrose present in sucrose-accumulating tomato fruits, the assimilation of [³H](fructosyl)-sucrose was studied. Key enzymes involved in carbohydrate metabolism were also assayed. The results demonstrated that the low level of sucrose present in young fruits accumulates directly without undergoing hydrolysis, suggesting a symplastic pathway for sucrose unloading. By contrast, the large quantity of the sucrose present in ripe sucrose-accumulating fruits originates from hydrolysis and resynthesis, suggesting an apoplastic pathway for sucrose unloading. The increase in sucrose level observed in sucrose-accumulating fruits is associated with a gradual decline in invertase activity and an increase in sucrose phosphate synthase activity. This latter enzyme seems to play a key biochemical role in the accumulation of sucrose and the establishment of a high sugar content in tomato fruits.     

Relations between carbohydrate accumulation in leaves, sucrose phosphate synthase activity and photoassimilate transport in chilling treated maize seedlings

Sucrose and starch concentration, sucrose phosphate synthase (SPS) activity in leaves, and long distance transport were studied in maize seedlings treated with moderate chilling (14 °C/12 °C - day/night). Two inbred lines were tested: chilling-tolerant KW1074 and chilling-sensitive CM109. Seedlings were grown in phytotrone on water nutrient until the 4-th leaf appearance. The estimations were done on fully developed 2-nd leaf. Six days after the temperature was lowered, leaves of line KW 1074 plants contained 5-fold more sucrose and starch than the control ones. The same treatment of CM 109 seedlings resulted in accumulation of sucrose and starch by 2-fold and 8.5-fold, respectively. As the result of chilling-treatment, ¹⁴C assimilation rate (P_a), transport speed in the leaf blade (TS₁) and along the plant (TS_m) decreased by about 50 % in both lines. On the other hand, time necessary for radiolabel movement into the phloem loading region (AT) increased strongly, especially in chilling-sensitive line CM 109. It was also noted, that the radioactivity exported from leaves (R₁) and imported by roots (R_m) decreased in line CM 109, and increased slightly in line KW 1074. The activity of SPS extracted from leaves of both lines decreased by about 3.3 when temperature was lowered form 30°C to 10°C. There was no effect of 6 day treatment of chilling on SPS activity. Changes in sucrose and starch concentration, SPS activity as well as differences in transport parameters observed in KW1074 and CM109 seedlings at moderate low temperatures are discussed in terms of mechanism of maize chilling-sensitivity.     

Light/Dark profiles of sucrose phosphate synthase, sucrose synthase, and Acid invertase in leaves of sugar beets

The activity of sucrose phosphate synthase, sucrose synthase, and acid invertase was monitored in 1- to 2-month-old sugar beet (Beta vulgaris L.) leaves. Sugar beet leaves achieve full laminar length in 13 days. Therefore, leaves were harvested at 2-day intervals for 15 days. Sucrose phosphate synthase activity was not detectable for 6 days in the dark-grown leaves. Once activity was measurable, sucrose phosphate synthase activity never exceeded half that observed in the light-grown leaves. After 8 days in the dark, leaves which were illuminated for 30 minutes showed no significant change in sucrose phosphate synthase activity. Leaves illuminated for 24 hours after 8 days in darkness, however, recovered sucrose phosphate synthase activity to 80% of that of normally grown leaves. Sucrose synthase and acid invertase activity in the light-grown leaves both increased for the first 7 days and then decreased as the leaves matured. In contrast, the activity of sucrose synthase oscillated throughout the growth period in the dark-grown leaves. Acid invertase activity in the dark-grown leaves seemed to be the same as the activity found in the light-grown leaves.     

Phytochrome mediated regulation of sucrose phosphate synthase activity in maize

The extractable activity of sucrose phosphate synthase was determined in etiolated seedlings of maize (Zea mays L.), soybean (Glycine max [L.] Merr.), and sugar beet (Beta vulgaris L.) following treatments of changing light quality. A 30-minute illumination of 30 microeinsteins per square meter per second white light produced a three-fold increase in sucrose phosphate synthase activity at 2 hours postillumination when compared to seedlings maintained in total darkness. Etiolated maize seedlings treated with 3.6 microeinsteins per square meter per second of red and far-red light showed a 50% increase and a 50% decrease in sucrose phosphate synthase activity, respectively, when compared to etiolated maize seedlings treated with white light. Maize seedlings exposed for 30 minutes to red followed by 30 minutes to far-red showed an initial increase in sucrose phosphate synthase activity followed by a rapid decrease to control level. Neither soybean or sugar beet sucrose phosphate synthase responded to the 30-minute illumination of white light. Phytochrome is involved in sucrose phosphate synthase regulation in maize, whereas it is not responsible for changes in sucrose phosphate synthase activity in soybean or sugar beet.     

The equilibrium of the reaction catalyzed by sucrose phosphate synthase

The equilibrium constant for the reaction catalyzed by sucrose phosphate synthase was reported 25 years ago to be 3250 at pH 7.5. It has been redetermined and found to be about 2 in the direction of synthesis and 6 to 10 when measured in the opposite direction.