Proteasome activity and the post-translational control of sucrose synthase stability in maize leaves.

The serine-170 (S170) calcium-dependent protein kinase phosphorylation site of maize (Zea mays L.) sucrose synthase (SUS) (EC 2.4.1.13) has been implicated in the post-translational regulation of SUS protein stability. To clarify the proteolytic process and the role of phosphorylation, SUS degradation and proteasome activities were studied in the maize leaf elongation zone. Size-exclusion chromatography resolved two peaks of proteasome-like proteolytic activity. The large molecular mass ( approximately 1350 kDa) peak required Mg(2+) and ATP for maximal activity and was inhibited by the proteasome inhibitors MG132 and NLVS. Anion-exchange chromatography resolved a similar proteolytic activity that was activated by ATP, characteristics that are consistent with those of a 26S-proteasome. Appropriately, immunoblotting revealed the presence of a 26S-proteasome subunit and highly ubiquitinated proteins within the active fractions eluted from both columns. The smaller molecular mass ( approximately 600 kDa) peak represented only 40% of the total proteasome-like activity and is likely a maize 20S-proteasome as it was activated in vitro by low levels of sodium dodecyl sulfate (SDS). S170 phosphorylated SUS (pS170-SUS) was detected as both high molecular mass (HMM) forms and proteolytic fragments that co-eluted with 26S-proteasome activities on both size-exclusion and anion-exchange columns. Conditions that maintained maximal 26S-proteasome activity reduced the amounts of pS170-SUS recovered. In vitro, the 26S-proteasome degraded SUS and proteasome-specific inhibitors reduced SUS proteolysis. HMM-SUS conjugates were produced in vitro and immunoprecipitations suggested that some SUS might be ubiquitinated in vivo. The results suggest that S170 phosphorylation promotes the formation of HMM, ubiquitin-SUS conjugates that can be targeted for 26S-proteasome-dependent degradation.     

Glucose control of sucrose synthase in the maize scutellum

The in vivo amounts of UDPG, UTP, UDP and UMP, metabolites known to influence the activity of sucrose phosphate synthase (SPS) and sucrose synthase (SS), were measured throughout 5 hr incubations of scutellum slices in fructose or water, i.e. under conditions of sucrose synthesis or breakdown. Cytosolic concentrations were estimated assuming that these metabolites were confined to the cytosol. Within the estimated in vivo concentration ranges, UDPG, UTP and UDP had little effect on the in vitro SS activity, but glucose (100 mM) inhibited SS in the synthesis direction by 63–70% and in the breakdown direction by 86–93%. Glucose inhibition of SS was considerably less when saturating levels of substrates were used. Sucrose did not inhibit SS. It is concluded that during germination the glucose produced from starch breakdown in the maize endosperm enters the scutellum and inhibits SS, preventing a futile cycle and limiting SS participation in sucrose synthesis.     

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.     

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.     

Role of sucrose-phosphate synthase in sucrose metabolism in leaves

Sucrose is formed in the cytoplasm of leaf cells from triose phosphates exported from the chloroplast. Flux control is shared among key enzymes of the pathway, one of which is sucrose-phosphate synthase (SPS). Regulation of SPS by protein phosphorylation is important in vivo and may explain diurnal changes in SPS activity and carbon partitioning. The signal transduction pathway mediating the light activation of SPS in vivo appears to involve metabolites and novel “coarse” control of the protein phosphatase that dephosphorylates and activates SPS. Regulation of the phosphorylation of SPS may provide a general mechanism whereby sucrose formation is coordinated with the rate of photosynthesis and the rate of nitrate assimilation. There are apparent differences among species in the properties of SPS that may reflect different strategies for the control of carbon partitioning. The SPS gene has recently been cloned from maize; results of preliminary studies with transgenic tomato plants expressing high levels of maize SPS support the postulate that SPS activity can influence the partitioning of carbon between starch and sucrose.     

Coordinate control of sucrose formation in soybean leaves by sucrose-phosphate synthase and fructose-2,6-bisphosphate

Net photosynthesis (CER), assimilate-export rate, sucrose-phosphate-synthase (EC 2.4.1.14) activity, fructose-2,6-bisphosphate content, and 6-phosphofructo-2-kinase (EC 2.7.1.105) activity were monitored in leaves of soybean (Glycine max (L.) Merr.) plants during a 12:12 h day-night cycle, and in plants transferred, at regular intervals throughout the diurnal cycle, to an illuminated chamber for 3 h. In the control plants, assimilate-export rate decreased progressively during the day whereas in transferred plants, a strongly rhythmic fluctuation in both CER and export rate was observed over the 24-h test period. Two maxima during the 24-h period for both processes were observed: one when plants were transferred during the middle of the normal light period, and a second when plants were transferred during the middle of the normal dark period. Overall, the results indicated that export rate was correlated positively with photosynthetic rate and sucrose-phosphate-synthase activity, and correlated negatively with fructose-2,6-bisphosphate levels, and that coarse control and fine control of the sucrose-formation pathway are coordinated during the diurnal cycle. Diurnal changes in sucrose-phosphate-synthase activity were not associated with changes in regulatory properties (phosphate inhibition) or substrate affinities. The biochemical basis for the diurnal rhythm in sucrose-phosphate-synthase activity in the soybean leaf thus appears to involve changes in the amount of the enzyme or a post-translational modification that affects only the maximum velocity.Abbreviations FBPase fructose-1,6-bisphosphatase - SPS sucrose-phosphate synthase - F26BPase fructose-2,6-bisphosphatase - PGI glucose-6-phosphate isomerase - F6P fructose-6-phosphate - F26BP fructose-2,6-bisphosphate - G6P glucose-6-phosphate - CER net carbon exchange rate - Pi inorganic phosphate - DHAP dihydroxyacetone phosphate - PGA glycerate 3-phosphate - F6P,2-kinase 6-phosphofructo-2-kinase Cooperative investigations of the U.S. Department of Agriculture, Agricultural Research Service, and the North Carolina Agricultural Research Service, Raleigh. Paper No. 10503 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601     

Purification and properties of sucrose synthase from maize kernels

Sucrose synthase was purified from 22-day-old maize (Zea mays L.) kernels to homogeneity by the successive steps of ammonium sulfate fractionation, gel filtration through a Sephadex G-200 column, and affinity chromatography on a UDP-hexanol-amino-agarose column. The degree of purification is 42-fold and the yield is over 80%. Polyacrylamide gel electrophoretic techniques, sedimentation velocity, and gel filtration studies revealed that the enzyme has identical subunits and could assume tetrameric, octameric, and other higher aggregated forms which are dependent on the ionic species and ionic strength of the solution. All of the enzyme forms exhibit catalytic activity but show differences in their specific activities. In most cases, the tetramer is the predominant form and has the highest specific activity. It is thus concluded that the tetramer could be the native form of the enzyme. The subunit protein has a molecular weight of 88,000 and a blocked NH₂ terminus which is not available to Edman degradation. Some general properties and the amino acid composition of the enzyme are also reported.     

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.     

Induction of sucrose synthase in the phloem of phytoplasma infected maize

In this study, we have analyzed the expression of the low oxygen inducible sucrose synthase isozyme SH1 (SUS-SH1) in the phloem of maize (Zea mays L.) infected with maize bushy stunt phytoplasma. Immunolocalization and Western blot analysis revealed several fold induction of SUS-SH1 in companion cells of phytoplasma inhabited phloem of leaf sheaths and stems. The results imply higher rates of sucrose metabolism and intensified hypoxia in the phloem.     

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.     

A novel sucrose synthase pathway for sucrose degradation in cultured sycamore cells

Enzymes of sucrose degradation and glycolysis in cultured sycamore (Acer pseudoplatanus L.) cells were assayed and characterized in crude extracts and after partial purification, in an attempt to identify pathways for sucrose catabolism. Desalted cell extracts contained similar activities (20-40 nanomoles per milligram protein per minute) of sucrose synthase, neutral invertase, glucokinase, fructokinase, phosphofructokinase, and UDPglucose pyrophosphorylase (assayed with 2 micromolar pyrophosphate (PPi). PPi-linked phosphofructokinase activity was virtually dependent upon fructose 2,6-bisphosphate, and the maximum activity exceeded that of ATP-linked phosphofructokinase. Hexokinase activity, with glucose as substrate, was highly specific for ATP, whereas fructokinase activity was relatively nonspecific. At 1 millimolar nucleoside triphosphate, fructokinase activity decreased in the order: UTP > ATP > CTP > GTP. We propose two pathways for sucrose degradation. One involves invertase action, followed by classical glycolysis of hexose sugars, and the other is a novel pathway initiated by sucrose synthase. The K_m for sucrose of sucrose synthase was severalfold lower than that of neutral invertase (15 versus 65 millimolar), which may determine carbon partitioning between the two pathways. The sucrose synthase pathway proposed involves cycling of uridylates and PPi. UDPglucose pyrophosphorylase, which is shown to be an effective `PPi-scavenger,' would consume PPi and form UTP. The UTP could be then utilized in the UTP-linked fructokinase reaction, thereby forming UDP for sucrose synthase. The source of PPi is postulated to arise from the back reaction of PPi-linked phosphofructokinase. Sycamore cells contained a substantial endogenous pool of PPi (about 3 nanomoles per gram fresh weight, roughly 1/10 the amount of ATP in these cells), and sufficient fructose 2,6-bisphosphate (0.09 nanomole per gram fresh weight) to activate the PPi-linked phosphofructokinase. Possible regulation and energetic differences between the sucrose synthase and invertase pathways are discussed.     

Mitochondrial localization and putative signaling function of sucrose synthase in maize

In many organisms, an increasing number of proteins seem to play two or more unrelated roles. Here we report that maize sucrose synthase (SUS) is distributed in organelles not involved in sucrose metabolism and may have novel roles beyond sucrose degradation. Bioinformatics analysis predicts that among the three maize SUS isoforms, SH1 protein has a putative mitochondrial targeting peptide (mTP). We validated this prediction by the immunodetection of SUS in mitochondria. Analysis with isoform-specific antisera revealed that both SH1 and SUS1 are represented in mitochondria, although the latter lacks a canonical mTP. The SUS2 isoform is not detectable in mitochondria, despite its presence in the cytosol. In maize primary roots, the mitochondrion-associated SUS (mtSUS; which includes SH1 and SUS1) is present mostly in the root tip, indicating tissue-specific regulation of SUS compartmentation. Unlike the glycolytic enzymes that occur attached to the outside of mitochondria, SH1 and SUS1 are intramitochondrial. The low abundance of SUS in mitochondria, its high Km value for sucrose, and the lack of sucrose in mitochondria suggest that mtSUS plays a non-sucrolytic role. Co-immunoprecipitation studies indicate that SUS interacts with the voltage-dependent anion channel in an isoform-specific and anoxia-enhanced manner and may be involved in the regulation of solute fluxes into and out of mitochondria. In several plant species, at least one of the SUS proteins possesses a putative mTP, indicating the conservation of the noncatalytic function across plant species. Taken together, these observations suggest that SUS has a novel noncatalytic function in plant cells.     

Shrunken-1 encoded sucrose synthase is not required for sucrose synthesis in the maize endosperm

Kernels of wild-type maize (Zea mays L.) shrunken-1 (sh1), deficient in the predominant form of endosperm sucrose synthase and shrunken-2 (sh2), deficient in 95% of the endosperm ADP-glucose pyrophosphorylase were grown in culture on sucrose, glucose, or fructose as the carbon source. Analysis of the endosperm extracts by gas-liquid chromatography revealed that sucrose was present in the endosperms of all genotypes, regardless of carbon supply, indicating that all three genotypes are capable of synthesizing sucrose from reducing sugars. The finding that sucrose was present in sh1 kernels grown on reducing sugars is evidence that shrunken-1 encoded sucrose synthase is not necessary for sucrose synthesis. Shrunken-1 kernels developed to maturity and produced viable seeds on all carbon sources, but unlike wild-type and sh2 kernels grown in vitro, sucrose was not the superior carbon source. This latter result provides further evidence that the role of sucrose synthase in maize endosperm is primarily that of sucrose degradation.     

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.     

Evidence for plasma membrane-associated forms of sucrose synthase in maize

Plasma membrane fractions were isolated from maize (Zea mays L.) endosperms and etiolated kernels to investigate the possible membrane location of the sucrose synthase (SS) protein. Endosperms from seedlings at both 12 and 21 days after pollination (DAP), representing early and mid-developmental stages, were used, in addition to etiolated leaf and elongation zones from seedlings. Plasma membrane fractions were isolated from this material using differential centrifugation and aqueous two-phase partitioning. The plasma membrane-enriched fraction obtained was then analyzed for the presence of sucrose synthase using protein blots and activity measurements. Both isozymes SS1 and SS2, encoded by the lociSh1 andSus1, respectively, were detected in the plasma membrane-enriched fraction using polyclonal and monoclonal antisera to SS1 and SS2 isozymes. In addition, measurements of sucrose synthase activity in plasma membrane fractions of endosperm revealed high levels of specific activity. The sucrose synthase enzyme is tightly associated with the membrane, as shown by Triton X-100 treatment of the plasma membrane-enriched fraction. It is noteworthy that the gene products of bothSh1 andSus1 were detectable as both soluble and plasma membrane-associated forms.     

Genetic control of sucrose synthetase in maize endosperm

Summary Sucrose synthetase activity in endosperm extracts of seven shrunken(sh) mutants of spontaneous origin and three similar mutants due to the association of the controlling element Ds with the Sh locus is examined. A residual level of 3 to 5% as compared to the normal (Sh) endosperm is seen in all the mutants. The residual activity is similar to that of the Sh locus encoded endosperm sucrose synthetase by several criteria including an identical size of polypeptides and a similarity in antigenic properties. These two enzymes are, however, distinguishable by a slight difference in electrophoretic mobility in native gels and a difference in the relative abundance of enzyme molecules. The latter property is a reflection of a marked difference seen in the developmental profile of enzyme activity in the two genotypes. The earlier hypothesis (Chourey and Nelson 1976) that these two sucrose synthetases are encoded by two separate genes is strengthened by: (a) the presence of the residual enzyme in a sh deletion mutant and (b) an electrophoretic demonstration of two proteins, corresponding to the major and minor sucrose synthetase proteins, in the wild type (Sh) genotype. The two sucrose synthetase genes seem to provide a model system in plants for studying the molecular basis of temporal specificity of genes.Cooperative Investigation, United States Department of Agriculture and Institute of Food and Agricultural Sciences, University of Florida, Florida Agricultural Experiment Station Journal Series No. 3288. Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable     

Anaerobiosis induces transcription but not translation of sucrose synthase in maize

This report examines the effect of anaerobic stress on the expression of sucrose synthase in maize (Zea mays L.). Following 24 hours of anaerobic treatment, alcohol dehydrogenase displayed the classical characteristics of induction: increased mRNA and protein levels. However, there was no detectable increase in sucrose synthase specific proteins by either native or denaturing Western blot analysis nor was there an increase in sucrose synthase activity. Anaerobic treatment did induce significantly higher steady state levels of sucrose synthase mRNA. Even though previous work has implicated sucrose synthase as an anaerobically induced protein, the data in this report suggest that sucrose synthase is not inducible at the protein level by anaerobic treatment.     

Influence of light on the ferredoxin-dependent glutamate synthase in maize leaves

The ferredoxin (Fd)-dependent glutamate synthase (EC 1.4.7.1) and NADH-dependent glutamate synthase (EC 1.4.1.14) activities are carried out by two immunochemically distinct enzyme proteins in maize leaves (Zea mays W64A and W182E). Continuous irradiation of etiolated tissue at 75 micro einsteins per square meter per second for 24 hours resulted in a 3-fold increase on a fresh weight basis in the activity of the Fd-dependent glutamate synthase and a slight decrease in the activity of the NADH-dependent enzyme. There was also a significant increase of the Fd-glutamate synthase protein during greening of etiolated tissue.     

Tissue- and cell-specific expression of the two sucrose synthase isoenzymes in developing maize kernels

Summary The localization of the two known sucrose synthase isoenzymes of Zea mays L., sucrose synthase 1 and sucrose synthase 2, was studied during kernel development by indirect immunohistochemistry. These enzymes are encoded by the Sh and Sus genes, respectively. Since the antiserum used cross-reacts with both enzymes, tissue sections of Sh and sh kernels were compared. In the latter tissue no sucrose synthase 1 is expressed and thus the signal obtained was ascribed to sucrose synthase 2. We found that the isoenzymes are differentially expressed. While sucrose synthase 1 is expressed only in the endosperm, sucrose synthase 2 is found in almost all tissues of the kernel with cxpression levels specific for cell type and developmental stage. Sucrose synthase 2 is expressed strongly in the aleurone and subaleurone cell layers, where the signal detected is as strong as or even stronger than the sucrose synthase 1 signal in the inner endosperm. The distribution of the enzymes changes characteristically during development.