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.     

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.     

A role for 'futile cycles' involving invertase and sucrose synthase in sucrose metabolism of tomato fruit.

Current concepts of the factors determining sink strength and the subsequent regulation of carbohydrate metabolism in tomato fruit are based upon an understanding of the relative roles of sucrose synthase, sucrose phosphate synthase and invertase, derived from studies in mutants and transformed plants. These enzymes participate in at least four futile cycles that involve sugar transport between the cytosol, vacuole and apoplast. Key reactions are (1) the continuous rapid degradation of sucrose in the cytosol by sucrose synthase (SuSy), (2) sucrose re-synthesis via either SuSy or sucrose phosphate synthase (SPS), (3) sucrose hydrolysis in the vacuole or apoplast by acid invertase, (4) subsequent transport of hexoses to the cytosol where they are once more converted into sucrose, and (5) rapid synthesis and breakdown of starch in the amyloplast. In this way futile cycles of sucrose/hexose interchange govern fruit sugar content and composition. The major function of the high and constant invertase activity in red tomato fruit is, therefore, to maintain high cellular hexose concentrations, the hydrolysis of sucrose in the vacuole and in the intercellular space allowing more efficient storage of sugar in these compartments. Vacuolar sugar storage may be important in sustaining fruit cell growth at times when less sucrose is available for the sink organs because of exhaustion of the carbohydrate pools in source leaves.     

Starch synthesis in tomato remains constant throughout fruit development and is dependent on sucrose supply and sucrose synthase activity

Studies designed to investigate the cellular pathway of phloem unloading were conducted on two tomato lines with either high or low fruit invertase activities. Experiments were based on determination of the degree to which ³H label from [³H]-(fructosyl)-sucrose was randomized between fructose and glucose following exposure of excised fruit to a pulse of labelled sucrose delivered through pedicels. Fruit from the low invertase line harvested 10, 20 and 40 d after anthesis had similar sucrose uptake kinetics to the high invertase line. A positive correlation was found between sucrose synthase activity and sucrose uptake in both low and high invertase lines. In contrast, no correlation was observed between acid or neutral invertase activities and sucrose uptake. Within the putative apoplasmic sap collected from fruit, label in [³H]-(fructosyl)-sucrose was randomized between the free hexoses and sucrose hexose moieties. Label asymmetry was retained in sucrose on arrival within the tissues. Randomization patterns were similar in both the low and high acid invertase lines. These data support the view that sucrose imported into the fruit was not exposed to extracellular hydrolysis. This suggests that movement from the phloem is likely to occur predominantly through a symplastic pathway. About 25% of the sucrose taken up by the fruit was converted into starch regardless of fruit age, suggesting that starch turnover remains constant throughout fruit development and that starch synthesis was dependent on sucrose supply.     

Cucumber fruit sucrose synthase isozymes

Two forms of sucrose synthase (SSI and SSII) were resolved from cucumber (Cucumis sativus) fruit pericarp and fruit peduncle tissue using DEAE-cell     

Changes in the phosphorylation state of sucrose synthase during development of Japanese pear fruit

Changes in the protein level and phosphorylation state of sucrose synthase (SS) were studied throughout the development of Japanese pear fruit. The level of SS protein was high at the young stage, dropped with fruit enlargement and increased again with fruit maturation. Antibody against phospho-Ser reacted with SS from young fruit, but did not react with SS that had been dephosphorylated by alkaline phosphatase (AP). The activities of SS isozymes were separated by ion-exchange chromatography. It was found that the fluctuation in SS activity was caused by two SS isozymes (SSI and SSII); (SSI reacted with antibody against phospho-Ser, while SSII did not. Phosphorylation of SS affected its kinetic parameters, that is, the affinity of phosphorylated SS for UDP was higher than that of dephosphorylated SS, while it was the contrary for UDP-glucose. The reaction of dephosphorylated SS was inclined toward sucrose synthesis more than that of phosphorylated SS. Phosphorylated SS protein was most abundant in young fruit, but decreased with fruit development, while non-phosphorylated SS protein increased in mature fruit. These results suggest that SS isoforms may be affected by post-translational modifications such as phosphorylation, and that the regulation of phosphorylation may potentially control the properties and functions of SS throughout the development of Japanese pear fruit.     

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.     

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.     

In vivo and in vitro phosphorylation of membrane and soluble forms of soybean nodule sucrose synthase

Sucrose synthase (SS) is a known phosphoserine (SerP)-containing enzyme in a variety of plant "sink" organs, including legume root nodules, where it is phosphorylated primarily at Ser-11. Using immunofluorescence confocal microscopy, we documented that part of the total SS (nodulin-100) pool in mature soybean (Glycine max) nodules is apparently associated with the plasma membrane in situ, and we report that this association is very "tight," as evidenced by a variety of chemical and enzymatic pretreatments of the isolated microsomal fraction. To investigate the in situ and in planta phosphorylation state of the membrane (m) and soluble (s) forms of nodule SS, three complementary approaches were used. First, excised nodules were radiolabeled in situ with [(32)P]Pi for subsequent analysis of phosphorylated m- and s-SS; second, immunopurified s- and m-SS were used as substrate in "on-bead" assays of phosphorylation by nodule Ca(2+)-dependent protein kinase; and third, SS-Ser-11(P) phosphopeptide-specific antibodies were developed and used. The collective results provide convincing evidence that microsomal nodulin-100 is phosphorylated in mature nodules, and that it is hypophosphorylated relative to s-SS (on an equivalent SS protein basis) in attached, unstressed nodules. Moreover, the immunological data and related phosphopeptide mapping analyses indicate that a homologous N-terminal seryl-phosphorylation domain and site reside in microsomal nodulin-100. We also observed that mild, short-term inorganic nitrogen and salt stresses have a significant negative impact on the content and N-terminal phosphorylation state of nodule m- and s-SS, with the former being the more sensitive of the two SS forms.     

Membrane association of sucrose synthase: changes during the graviresponse and possible control by protein phosphorylation

Sucrose synthase (SuSy) plays an important role in sucrose degradation and occurs both as a soluble and as a membrane-associated enzyme in higher plants. We show that membrane association can vary in vivo in response to gravistimulation, apparently involving SuSy dephosphorylation, and is a reversible process in vitro. Phosphorylation of SuSy has little effect on its activity but decreases its surface hydrophobicity as reported with the fluorescent probe bis-ANS. We postulate that phosphorylation of SuSy (and perhaps other membrane proteins) is involved in the release of the membrane-bound enzyme in part as a result of decreased surface hydrophobicity.     

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.     

Phosphorylation of the amino terminus of maize sucrose synthase in relation to membrane association and enzyme activity

Sucrose synthase (SUS) is phosphorylated on a major, amino-terminal site located at Ser-15 (S15) in the maize (Zea mays) SUS1 protein. Site- and phospho-specific antibodies against a phosphorylated S15 (pS15) peptide allowed direct analysis of S15 phosphorylation in relation to membrane association. Immunoblots of the maize leaf elongation zone, divided into 4-cm segments, demonstrated that the abundance of soluble (s-SUS) and membrane (m-SUS) SUS protein showed distinct positional profiles. The content of m-SUS was maximal in the 4- to 8-cm segment where it represented 9% of total SUS and occurred as a peripheral membrane protein. In contrast, s-SUS was highest in the 12- to 16-cm segment. Relative to s-SUS, m-SUS was hypophosphorylated at S15 in the basal 4 cm but hyperphosphorylated in apical segments. Differing capabilities of the anti-pS15 and anti-S15 peptide antibodies to immunoprecipitate SUS suggested that phosphorylation of S15, or exposure of unphosphorylated SUS to slightly acidic pH, altered the structure of the amino terminus. These structural changes were generally coincident with the increased sucrose cleavage activity that occurs at pH values below 7.5. In vitro S15 phosphorylation of the S170A SUS protein by a maize calcium-dependent protein kinase (CDPK) significantly increased sucrose cleavage activity at low pH. Collectively, the results suggest that (1) SUS membrane binding is controlled in vivo; (2) relative pS15 content of m-SUS depends on the developmental state of the organ; and (3) phosphorylation of S15 affects amino-terminal conformation in a way that may stimulate the catalytic activity of SUS and influence membrane association.     

Expression of a maize sucrose phosphate synthase in tomato alters leaf carbohydrate partitioning.

We isolated a complementary DNA sequence for the enzyme sucrose phosphate synthase (SPS) from maize utilizing a limited amino acid sequence. The 3509-bp cDNA encodes a 1068-amino acid polypeptide. The identity of the cDNA was confirmed by the ability of the cloned sequence to direct sucrose phosphate synthesis in Escherichia coli. Because no plant-specific factors were necessary for enzymatic activity, we can conclude that SPS enzyme activity is conferred by a single gene product. Sequence comparisons showed that SPS is distantly related to the enzyme sucrose synthase. When expressed from a ribulose bisphosphate carboxylase small subunit promoter in transgenic tomatoes, total SPS activity was boosted up to sixfold in leaves and appeared to be physiologically uncoupled from the tomato regulation mechanism. The elevated SPS activity caused a reduction of starch and increase of sucrose in the tomato leaves. This result clearly demonstrates that SPS is involved in the regulation of carbon partitioning in the leaves.     

Determination of structural requirements and probable regulatory effectors for membrane association of maize sucrose synthase 1

Sucrose (Suc) synthase (SUS) cleaves Suc to form UDP glucose and fructose, and exists in soluble and membrane-associated forms, with the latter proposed to channel UDP glucose to the cellulose-synthase complex on the plasma membrane of plant cells during synthesis of cellulose. However, the structural features responsible for membrane localization and the mechanisms regulating its dual intracellular localization are unknown. The maize (Zea mays) SUS1 isoform is likely to have the intrinsic ability to interact directly with membranes because we show: (1) partial membrane localization when expressed in Escherichia coli, and (2) binding to carbonate-stripped plant microsomes in vitro. We have undertaken mutational analyses (truncations and alanine substitutions) and in vitro microsome-binding assays with the SUS1 protein to define intrinsic membrane-binding regions and potential regulatory factors that could be provided by cellular microenvironment. The results suggest that two regions of SUS1 contribute to membrane affinity: (1) the amino-terminal noncatalytic domain, and (2) a region with sequence similarity to the C-terminal pleckstrin homology domain of human pleckstrin. Alanine substitutions within the pleckstrin homology-like domain of SUS1 reduced membrane association in E. coli and with plant microsomes in vitro without reducing enzymatic activity. Microsomal association of wild-type SUS1 displayed cooperativity with SUS1 protein concentration and was stimulated by both lowering the pH and adding Suc. These studies offer insight into the molecular level regulation of SUS1 localization and its participation in carbon partitioning in plants. Moreover, transgenics with active SUS mutants altered in membrane affinity may be of technological utility.     

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.     

Activity of sucrose hydrolysing enzymes and sugar accumulation during tomato fruit development

Relationships between the assimilate import rate and the activity of acid invertase and/or sucrose synthase have been investigated in the pericarp, locule and placenta of tomato fruit during development to establish the possible role of sucrose cleavage as the control step for the import of sucrose into these sink tissues. The rate of sucrose cleavage was estimated from the activities of these two enzymes as well as the ratio of hexoses to sucrose (i.e. the sucrose degradation index, SDI) in the tissues of the fruit, based on the assumption that the accumulation of hexoses is the consequence of imported sucrose being degraded by either or both of these two enzymes. The results showed that the change of sucrose synthase activity during fruit development was positively related to both the rate of dry matter accumulation in the fruit tissue and SDI. Although the role of acid invertase in regulating the rate of import during development remains uncertain, the actions of sucrose synthase on sucrose cleavage may regulate the import and compartmentation of sucrose in the early stage of tomato fruit development.     

Protein phosphorylation in developing and regenerating rat kidney

Renal cytosolic extracts from rats of different ages and mononephrectomized rats were incubated with gamma-[32P]ATP and analysed by high resolution two-dimensional electrophoresis and autoradiography. Extracts from new-born and young rats showed a great number of phosphorylated proteins migrating between the origin and Mr 52,000. Among these proteins, the group co-migrating with phosphorylase b (Mr 97,000) was particularly evident in new-born and days-old rats. In extracts from mature rats, other proteins of lower molecular weight, particularly those migrating between Mr 60,000 and 44,000, became intensely phosphorylated. The number and intensity of phosphorylated proteins from extracts of normal and nephrectomized rats, however, did not vary. Activity of cAMP-dependent protein kinase and [3H]cAMP binding was also modified during neonatal development but not in compensatory renal growth. Since cAMP-PK and protein phosphorylation are known to be regulated in response to hormonal stimulations, these results may provide good indications for the understanding of hormonal involvement in kidney growth.     

Protein phosphorylation in developing and regenerating rat kidney

Abstract. Renal cytosolic extracts from rats of different ages and mononephrectomized rats were incubated with γ-[³²P]ATP and analysed by high resolution two-dimensional electrophoresis and autoradiography. Extracts from new-born and young rats showed a great number of phosphorylated proteins migrating between the origin and Mr 52,000. Among these proteins, the group co-migrating with phosphorylase b (Mr 97,000) was particularly evident in new-born and days-old rats. In extracts from mature rats, other proteins of lower molecular weight, particularly those migrating between Mr 60,000 and 44,000, became intensely phosphorylated. The number and intensity of phosphorylated proteins from extracts of normal and nephrectomized rats, however, did not vary. Activity of cAMP-dependent protein kinase and [³H]cAMP binding was also modified during neonatal development but not in compensatory renal growth. Since cAMP-PK and protein phosphorylation are known to be regulated in response to hormonal stimulations, these results may provide good indications for the understanding of hormonal involvement in kidney growth.     

Purification and characterization of two ribonucleases from developing tomato fruit

Two neutral ribonucleases have been purified from developing tomato fruit. Their activity is maximal 5 days after anthesis, declines during maturation, and then increases slightly in the mature green through breaker stages. The ribonucleases Tf1 and Tf2 have molecular weights of 59 and 29 K, respectively, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and are glycoproteins. The reduced and denatured Tf1 is composed of two subunits, 30 and 29 K, of which only the 30-K subunit displays ribonuclease activity after renaturation. Reduced and denatured Tf2 is a single 29-K polypeptide that is renaturable to an active ribonuclease. Only the 30-K, active subunit of Tf1 is immunologically cross-reactive with Tf2. Both ribonucleases are cyclyzing endoribonucleases with a strong preference for cleavage at pyrimidine residues, thus generating oligonucleotide products ending with pyrimidine 2',3'-cyclic phosphate. These tomato fruit ribonucleases share a number of properties in common with the S-glycoprotein ribonucleases that are involved in self-incompatibility reactions in some solanaceous plants.