Sucrose Synthase,Starch Accumulation,and Tomato Fruit Sink Strength

Contrasting evidence has accumulated regarding the role of acid invertase and sucrose synthase in tomato fruit sink establishment and maintenance. In this work the relationships among the activities of sucrose synthase and acid invertase, Lycopersicon esculentum Mill cv UC-82B fruit growth, and starch accumulation were analyzed in fruit at 0 to 39 d after anthesis. Sucrose synthase, but not acid invertase, was found to be positively correlated with tomato fruit relative growth rate and with starch content in the pericarp tissue. A similar association between sucrose synthase activity and starch accumulation was also evident in the basal portion of the stem. Heat-shock treatments, which inhibited the increase in sucrose synthase activity at the beginning of the light period and had no effect on acid invertase activity, were used to examine the importance of sucrose synthase in relation to sucrose metabolism and starch synthesis. After the heat-shock treatment, concomitantly with the suppressed sucrose synthase activity relative to the controls, there was a reduction in sucrose cleavage and starch accumulation. These data substantiate the conclusion that, during the early phases of tomato fruit development, sucrose synthase rather than acid invertase is the dominant enzyme in metabolizing imported sucrose, which in turn plays a part in regulating the import of sucrose into the fruit.     

Companion-Cell Specific Localization of Sucrose Synthase in Zones of Phloem Loading and Unloading

An immunohistochemical approach was used in maize (Zea mays) and citrus (Citrus paradisi) to address the previously noted association between sucrose synthase and vascular bundles and to determine the localization of the low but detectable levels of sucrose synthase that remain in leaves after the import-export transition. Sucrose synthase protein was immunolocalized at the light microscope level using paraffin sections reacted with rabbit sucrose synthase polyclonal antisera and gold-conjugated goat anti-rabbit immunoglobulin G. Immunolabel was specifically observed in phloem companion cells of minor and intermediate veins in mature leaves of both species. Similar localization was apparent in the midrib of mature citrus leaves, with additional labeling in selected files of phloem parenchyma cells. A clear companion-cell specificity was evident in the phloem unloading zone of citrus fruit, where high activity of sucrose synthase has been demonstrated in vascular bundles during periods of rapid import. Sucrose synthase protein was not associated with adjacent cells surrounding the vascular strands in this tissue. The companion-cell specificity of sucrose synthase in phloem of both importing and exporting structures of these diverse species implies that this may be a widespread association and underscores its potential importance to the physiology of vascular bundles.     

Inhibition of fructokinase and sucrose synthase by cytosolic levels of fructose in young tomato fruit undergoing transient starch synthesis

Immature tomato fruit are characterized by a transient period of starch accumulation. Sucrose synthase (EC 2.4,1.13) and fructokinase (EC 2.7,1.4) are two of the initial enzymes in the sucrose to starch synthetic pathway. Both enzymes in tomato fruit are significantly inhibited by fructose at concentrations physiological to young tomato fruit. Compartmental analysis of immature fruit pericarp indicates that fructose is not specifically compartmentalized in the vacuole and that physiological cytosolic concentrations of fructose in young tomato fruit are above 30 m M . Such physiological levels of fructose significantly inhibit sucrose synthase cleavage activity as well as the activity of a partially purified fructokinase. These data suggest a mechanism of a coordinated, in vivo regulation of tomato sucrose synthase and fructokinase activity, which may be potentially limiting to starch accumulation in young tomato fruit.     

Response of sucrose metabolizing enzyme activity to CPPU and p-CPA treatments in excised discs of muskmelon

We investigated the effects of 1-(2-chloro-4-pyridyl)-3-phenylurea(CPPU) and para-chlorophenoxyacetic acid(p-CPA) treatments on the sucrose metabolism-relatedenzymeactivities in excised mesocarp discs of muskmelon fruit at different growthstages. Both a CPPU and a p-CPA treatment applied to discsprepared at 5 and at 20 days after anthesis (DAA) increased acid invertase (AI)activity and neutral invertase (NI) activity, but neither treatment affectedthese activities in the discs prepared at 45 DAA. Both plant growth substancesincreased the activity of sucrose phosphate synthase (SPS) in the discs at 5and20 DAA, but neither affected it in the 45 DAA discs. The sucrose synthase (SS)activity was markedly increased by p-CPA treatment in the20 and 45 DAA discs, but was not affected at 5 DAA. CPPU treatment did notactivate SS of discs throughout the growth stage.     

Transgenic tomato plants with decreased sucrose synthase are unaltered in starch and sugar accumulation in the fruit

Sucrose is the photoassimilate transported from the leaves to the fruit of tomato yet the fruit accumulates predominantly glucose and fructose. Hydrolysis of sucrose entering the fruit can be accomplished by invertase or sucrose synthase. Early in tomato fruit development there is a transient increase in sucrose synthase activity and starch which is correlated with fruit growth and sink strength suggesting a regulatory role for sucrose synthase in sugar import. Using an antisense sucrose synthase cDNA under the control of a fruit-specific promoter we show that sucrose synthase activity can be reduced by up to 99% in young fruit without affecting starch or sugar accumulation. This result calls into question the importance of sucrose synthase in regulating sink strength in tomato fruit.     

Developmental regulation of two tomato lipoxygenase promoters in transgenic tobacco and tomato

Two lipoxygenase (LOX) genes (tomloxA and tomloxB) are expressed in ripening tomato fruit, and tomloxA is also expressed in germinating seedlings [12]. The 5'-upstream regions of these genes were isolated to study the regulatory elements involved in coordinating tomlox gene expression. Sequence analysis of the promoters did not reveal any previously characterized regulatory elements except for TATA and CAAT boxes. However, the sequence motif GATAcAnnAAtnTGATG was found in both promoters. Chimeric gene fusions of each tomlox promoter with the -glucuronidase reporter gene (gus) were introduced into tobacco and tomato plants via Agrobacterium-mediated transformation. GUS activity in tomloxA-gus plants during seed germination peaked at day 5 and was enhanced by methyl jasmonate (MeJa) treatment. No GUS activity was detected in tomloxB-gus seedlings. Neither wounding nor abscisic acid (ABA) treatment of transgenic seedlings modified the activity of either promoter. During fruit development, GUS expression in tomloxA-gus tobacco fruit increased 5 days after anthesis (DAA) and peaked at 20 DAA. In tomloxB-gus tobacco fruit, GUS activity increased at 10 DAA and peaked at 20 DAA. In transgenic tomato fruit, tomloxA-gus expression was localized to the outer pericarp during fruit ripening, while tomloxB-gus expression was localized in the outer pericarp and columella. These data demonstrate that the promoter regions used in these experiments contain cis-acting regulatory elements required for proper regulation of tomlox expression during development and for MeJa-responsiveness.     

Sugar uptake by protoplasts isolated from tomato fruit tissues during various stages of fruit growth

The uptake of radioactive glucose and sucrose by protoplasts isolated from pericarp and placenta tissues of tomato ( Lycopersicon esculentum cv. Counter) fruit was investigated in relation to the dry matter accumulation rates of these tissues. Uptake of glucose by protoplasts isolated from pericarp tissue was highest in fruit of around 20 g fresh weight or 25 days after anthesis. Sucrose uptake by pericarp protoplasts was lower than that of glucose and did not show a peak of uptake. The maximum rate of glucose uptake by protoplasts from the pericarp was at the time when the tomato fruit was accumulating dry matter at the highest rate. Glucose uptake by placenta protoplasts was lower and at a similar level as sucrose.
Protoplast uptake of glucose, but not of sucrose, was partially inhibited by (1) p -chloromercuribenzene sulphonic acid, a sulphydryl group modifier; (2) erythrosin B, an H⁺-ATPase inhibitor; and (3) valinomycin, a K⁺-ionophore, suggesting that membrane transport of glucose by tomato fruit sink cells may be a carrier-mediated, energy-dependent process.
The main route of carbohydrate accumulation by tomato fruit during the period of rapid fruit growth may be by cleavage of sucrose by apoplastic acid invertase prior to hexose transport across the plasma membrane.     

Regulation of Tomato Fruit Polygalacturonase mRNA Accumulation by Ethylene: A Re-Examination

Polygalacturonase (PG) is the major enzyme responsible for pectin disassembly in ripening fruit. Despite extensive research on the factors regulating PG gene expression in fruit, there is conflicting evidence regarding the role of ethylene in mediating its expression. Transgenic tomato (Lycopersicon esculentum) fruits in which endogenous ethylene production was suppressed by the expression of an antisense 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene were used to re-examine the role of ethylene in regulating the accumulation of PG mRNA, enzyme activity, and protein during fruit ripening. Treatment of transgenic antisense ACC synthase mature green fruit with ethylene at concentrations as low as 0.1 to 1 μL/L for 24 h induced PG mRNA accumulation, and this accumulation was higher at concentrations of ethylene up to 100 μL/L. Neither PG enzyme activity nor PG protein accumulated during this 24-h period of ethylene treatment, indicating that translation lags at least 24 h behind the accumulation of PG mRNA, even at high ethylene concentrations. When examined at concentrations of 10 μL/L, PG mRNA accumulated within 6 h of ethylene treatment, indicating that the PG gene responds rapidly to ethylene. Treatment of transgenic tomato fruit with a low level of ethylene (0.1 μL/L) for up to 6 d induced levels of PG mRNA, enzyme activity, and protein after 6 d, which were comparable to levels observed in ripening wild-type fruit. A similar level of internal ethylene (0.15 μL/L) was measured in transgenic antisense ACC synthase fruit that were held for 28 d after harvest. In these fruit PG mRNA, enzyme activity, and protein were detected. Collectively, these results suggest that PG mRNA accumulation is ethylene regulated, and that the low threshold levels of ethylene required to promote PG mRNA accumulation may be exceeded, even in transgenic antisense ACC synthase tomato fruit.     

Sucrose accumulation in developing peach fruit

Uptake of ¹⁴C-sugars and activities of sucrose metabolizing enzymes were determined in order to study the mechanism(s) of sucrose accumulation in developing peach fruit. Mesocarp of young peach fruit contained glucose and fructose but little sucrose. Starting 88 days after anthesis (DAA) the sucrose concentration increased greatly. The mechanism of sucrose accumulation was studied by measuring ¹⁴C-sucrose and ¹⁴C-glucose uptake rates at three different stages of fruit development, and by assaying weekly the activity of enzymes involved in the hydrolysis and/or synthesis of the soluble sugars. Uptake of 0.5–100 m M ¹⁴C-sucrose and ¹⁴C-glucose by mesocarp tissue slices showed a complex pattern at the first stage of fruit development (62 DAA). During the subsequent growth stages the pattern of sugar uptake changed and was approximately monophasic at the third stage of fruit development.
At 10 m M , glucose was taken up more rapidly than sucrose at the first and second stage of fruit development. Uptake was partially inhibited by the uncoupler carbonylcyanide m -chlorophenylhydrazone (CCCP) at 25 μ M. These results, together with the presence of a putative extracellular invertase, suggest an apoplastic route for sucrose uptake which is dependent, at least in part, on energy supply.
Activities of sucrose hydrolyzing enzymes (insoluble acid invertase, soluble acid invertase, neutral invertase, sucrose synthase) were high in young fruits and declined sharply with fruit development concomitantly with accumulation of sucrose. The storage of the sugar was not accompanied by a rise in synthetic activities (sucrose synthase, sucrose phosphate synthase), suggesting that sucrose could, at least in part enter the carbohydrate pool directly.     

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.     

Sucrose metabolism and IAA and ethylene production in muskmelon ovaries

Changes induced by the pollination of ovaries may be mediated by phytohormones and involve sudar-mediated by phytohormones and involve sugar-metabolizing enzymes. In order to further explore these relationships, soluble sugars, sucrose-phosphate synthase (EC 2.4.1.14), sucrose synthase (EC 2.4.1.13), acid and neutral invertases (EC 3.2.1.26), indole-3-acetic acid (IAA), and ethylene were investigated in muskmelon (Cucumis melo L.) ovaries sampled before, during, and after anthesis. The fresh weight of ovaries increased 100% within 48 h after pollination, but did not change significantly in the absence of pollination. While sugar content per ovary increased after pollination, sugar content per mg protein was unaffected. Sucrose was not detected in nonpollinated ovaries 48 h after anthesis. Free IAA content was highest in ovaries sampled 48h before anthesis. Pollination had no immediate effect on IAA content per mg protein in postanthesis ovaries. Although detected in all ovaries sampled, ethylene production increased significantly only in nonpollinated ovaries. Activity of sucrose-phosphate synthase was the same at all stages. The specific activities of sucrose synthase and the invertases were highest in nonpollinated ovaries. The increase in rate of sugar import into ovaries following pollination was not accompanied by an increase in the specific activity of any enzyme assayed, but was coincident with an increase in the total activity per ovary of surcose synthase and acid invertase. There appears to be no direct relationship between sucrose-metabolizing enzymes, IAA or ethylene in developing pollinated ovaries but the increase in sucrose cleavage activity in nonpollinated ovaries may be related to the increase in ethylene production.     

Carbohydrate metabolism during early fruit development of sweet melon (Cucumis melo)

In sink tissues of cucurbits, including sweet melon fruits, the galactosyl-sucrose oligosaccharides, stachyose and raffinose, together with sucrose, are the major translocated carbohydrates. In the present study we investigated the carbohydrate metabolism of young melon ( Cucumis melo L. cv. C-8) fruit during the period of initial fruit set and development, from 3 days prior to anthesis until 20 days after anthesis (DAA), prior to the onset of sucrose accumulation. The enzymes assayed could be classified into two categories according to developmental patterns. Two of the enzymes, alkaline α -galactosidase I [EC 3.2.1.22], which hydrolyzes both raffinose and stachyose, and acid invertase [EC 3.2.1.26] either increased or remained stable during the first 10 DAA. The remaining measured enzymes (the stachyose-specific alkaline α -galactosidase form II, acid α -galactosidase, alkaline invertase, sucrose synthase [EC 2.4.1.13], galactokinase [EC 2.7.1.6], UDP-Gal PPase [EC 2.7.7.10], UDP-Glc-4 epimerase [EC 5.1.3.2], UDP-Glc PPase [EC 2.7.7.9], phosphoglucomutase [EC 5.4.2.2] and phosphoglucoisomerase [EC 5.3.1.9]) all showed a similar developmental pattern of steady decrease in activity following anthesis. We also compared the saccharide metabolism of pollinated and non-pollinated ovaries during the initial days following anthesis. In the absence of pollination, ovary growth dramatically decreased by the first DAA and was accompanied by a sharp decrease in the activity of UDP-Glc PPase. Other enzymes in the pathway, including the enzymes of stachyose and raffinose hydrolysis, did not decrease in activity until 2 or 4 DAA, after ovary growth was affected. These results provide information to assess the possible regulating enzymes in cucurbit ovary development and fruit set.     

Sucrose Synthase in Wild Tomato, Lycopersicon chmielewskii, and Tomato Fruit Sink Strength

Here it is reported that sucrose synthase can be readily measured in growing wild tomato fruits (Lycopersicon chmielewskii) when suitable methods are adopted during fruit extraction. The enzyme also was present in fruit pericarp tissues, in seeds, and in flowers. To check for novel characteristics, the wild tomato fruit sucrose synthase was purified, by (NH₄)₂SO₄ fraction and chromatography with DE-32, Sephadex G-200, and PBA-60, to one major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The following characteristics were obtained: native protein relative molecular weight 380,000; subunit relative molecular weight 89,000; K_m values with: sucrose 53 millimolar, UDP 18.9 micromolar, UDP-glucose 88 micromolar, fructose 8.4 millimolar; pH optima between 6.2 to 7.3 for sucrose breakdown and 7 to 9 for synthesis; and temperature optima near 50°C. The enzyme exhibited a high affinity and a preference for uridylates. The enzyme showed more sensitivity to divalent cations in the synthesis of sucrose than in its breakdown. Sink strength in tomato fruits also was investigated in regard to sucrose breakdown enzyme activities versus fruit weight gain. Sucrose synthase activity was consistently related to increases in fruit weight (sink strength) in both wild and commercial tomatoes. Acid and neutral invertases were not, because the published invertase activity values were too variable for quantitative analyses regarding the roles of invertases in tomato fruit development. In rapidly growing fruits of both wild and commercially developed tomato plants, the activity of sucrose synthase per growing fruit, i.e. sucrose synthase peak activity X fruit size, was linearly related to final fruit size; and the activity exceeded fruit growth and carbon import rates by at least 10-fold. In mature, nongrowing fruits, sucrose synthase activities approached nil values. Therefore, sucrose synthase can serve as an indicator of sink strength in growing tomato fruits.     

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 metabolism and IAA and ethylene production in muskmelon ovaries

Changes induced by the pollination of ovaries may be mediated by phytohormones and involve sudar-mediated by phytohormones and involve sugar-metabolizing enzymes. In order to further explore these relationships, soluble sugars, sucrose-phosphate synthase (EC 2.4.1.14), sucrose synthase (EC 2.4.1.13), acid and neutral invertases (EC 3.2.1.26), indole-3-acetic acid (IAA), and ethylene were investigated in muskmelon (Cucumis melo L.) ovaries sampled before, during, and after anthesis. The fresh weight of ovaries increased 100% within 48 h after pollination, but did not change significantly in the absence of pollination. While sugar content per ovary increased after pollination, sugar content per mg protein was unaffected. Sucrose was not detected in nonpollinated ovaries 48 h after anthesis. Free IAA content was highest in ovaries sampled 48h before anthesis. Pollination had no immediate effect on IAA content per mg protein in postanthesis ovaries. Although detected in all ovaries sampled, ethylene production increased significantly only in nonpollinated ovaries. Activity of sucrose-phosphate synthase was the same at all stages. The specific activities of sucrose synthase and the invertases were highest in nonpollinated ovaries. The increase in rate of sugar import into ovaries following pollination was not accompanied by an increase in the specific activity of any enzyme assayed, but was coincident with an increase in the total activity per ovary of surcose synthase and acid invertase. There appears to be no direct relationship between sucrose-metabolizing enzymes, IAA or ethylene in developing pollinated ovaries but the increase in sucrose cleavage activity in nonpollinated ovaries may be related to the increase in ethylene production.Mention of a specfic product does not imply an endorsement by the United States Depertment of Agriculture or Texas Aricultural Experiment Station over other suitable products.     

The control of single-celled cotton fiber elongation by developmentally reversible gating of plasmodesmata and coordinated expression of sucrose and K+ transporters and expansin

Each cotton fiber is a single cell that elongates to 2.5 to 3.0 cm from the seed coat epidermis within approximately 16 days after anthesis (DAA). To elucidate the mechanisms controlling this rapid elongation, we studied the gating of fiber plasmodesmata and the expression of the cell wall-loosening gene expansin and plasma membrane transporters for sucrose and K(+), the major osmotic solutes imported into fibers. Confocal imaging of the membrane-impermeant fluorescent solute carboxyfluorescein (CF) revealed that the fiber plasmodesmata were initially permeable to CF (0 to 9 DAA), but closed at approximately 10 DAA and re-opened at 16 DAA. A developmental switch from simple to branched plasmodesmata was also observed in fibers at 10 DAA. Coincident with the transient closure of the plasmodesmata, the sucrose and K(+) transporter genes were expressed maximally in fibers at 10 DAA with sucrose transporter proteins predominately localized at the fiber base. Consequently, fiber osmotic and turgor potentials were elevated, driving the rapid phase of elongation. The level of expansin mRNA, however, was high at the early phase of elongation (6 to 8 DAA) and decreased rapidly afterwards. The fiber turgor was similar to the underlying seed coat cells at 6 to 10 DAA and after 16 DAA. These results suggest that fiber elongation is initially achieved largely by cell wall loosening and finally terminated by increased wall rigidity and loss of higher turgor. To our knowledge, this study provides an unprecedented demonstration that the gating of plasmodesmata in a given cell is developmentally reversible and is coordinated with the expression of solute transporters and the cell wall-loosening gene. This integration of plasmodesmatal gating and gene expression appears to control fiber cell elongation.