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Steven C. Huber - One of the best experts on this subject based on the ideXlab platform.
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site specific regulatory interaction between spinach leaf sucrose phosphate Synthase and 14 3 3 proteins
FEBS Letters, 1998Co-Authors: Dikran Toroser, Gurdeep S Athwal, Steven C. HuberAbstract:We report an Mg2+-dependent interaction between spinach leaf Sucrose-Phosphate Synthase (SPS) and endogenous 14-3-3 proteins, as evidenced by co-elution during gel filtration and co-immunoprecipitation. The content of 14-3-3s associated with an SPS immunoprecipitate was inversely related to activity, and was specifically reduced when tissue was pretreated with 5-aminoimidazole-4-carboxamide riboside, suggesting metabolite control in vivo. A synthetic phosphopeptide based on Ser-229 was shown by surface plasmon resonance to bind a recombinant plant 14-3-3, and addition of the phosphorylated SPS-229 peptide was found to stimulate the SPS activity of an SPS:14-3-3 complex. Taken together, the results suggest a regulatory interaction of 14-3-3 proteins with Ser-229 of SPS.
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3 hydroxy 3 methylglutaryl coenzyme a reductase kinase and sucrose phosphate Synthase kinase activities in cauliflower florets ca2 dependence and substrate specificities
Archives of Biochemistry and Biophysics, 1998Co-Authors: Dikran Toroser, Steven C. HuberAbstract:Abstract Plant 3-hydroxy-3-methylglutaryl-CoA reductase(HMGR; EC 1.1.1.34) and sucrose–phosphate Synthase (SPS; EC 2.4.1.14) and synthetic peptides designed from the known phosphorylation sites of plant HMGR (SAMS*: KSHMKYNR S TKDVK), rat acetyl-CoA carboxylase (SAMS: HMRSAM S GLHLVKRR), spinach SPS (SP2: GRRJRRIS S VEJJDKK), and spinach NADH:nitrate reductase (NR6: GPTLKRTA S TPFJNTTSK) were used to characterize kinase activities from cauliflower ( Brassica oleracea L.) inflorescences. The three major peaks of protein kinase activity resolved by anion-exchange FPLC are homologs of those observed previously in spinach leaves and thus are designated PK I , PK IV , and PK III , listed in order of elution. PK IV was the most active in terms of phosphorylation and inactivation of recombinant Nicotiana HMGR and was also strictly Ca 2+ dependent. The novel aspects are that PK III has not been detected in previous cauliflower studies, that SAMS* is a more specific peptide substrate to identify potential HMGR kinases, and that the major HMGR kinase in cauliflower is Ca 2+ dependent. Of the three major kinases that phosphorylated the SP2 peptide only PK I (partially Ca 2+ sensitive) and PK III (Ca 2+ insensitive) inactivated native spinach leaf SPS. Cauliflower extracts contained endogenous SPS that was inactivated by endogenous kinase(s) in an ATP-dependent manner and this may be one of the substrate target proteins for PK I and/or PK III . The substrate specificity of the three kinase peaks was studied using synthetic peptide variants of the SP2 sequence. All three kinases had a strong preference for peptides with a basic residue at P-6 (as in SP2 and SAMS*; SAMS has a free amino terminus at this position) or a Pro at P-7 (as in NR6). This requirement for certain residues at P-6 or P-7 was not recognized in earlier studies but appears to be a general requirement. In plant HMGR, a conserved His residue at P-6 is involved directly in catalysis and this may explain why substrates reduced HMGR phosphorylation in vitro.
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protein phosphorylation as a mechanism for osmotic stress activation of sucrose phosphate Synthase in spinach leaves
Plant Physiology, 1997Co-Authors: Dikran Toroser, Steven C. HuberAbstract:Experiments were performed to investigate the mechanism of Sucrose-Phosphate Synthase (SPS) activation by osmotic stress in darkened spinach (Spinacia oleracea L.) leaves. The activation was stable through immunopurification and was not the result of an increased SPS protein level. The previously described Ca2+, independent peak III kinase, obtained by ion-exchange chromatography, is confirmed to be the predominant enzyme catalyzing phosphorylation and inactivation of dephosphoserine-158-SPS. A new, Ca2+-dependent SPS-protein kinase activity (peak IV kinase) was also resolved and shown to phosphorylate and activate phosphoserine-158-SPS in vitro. The peak IV kinase also phosphorylated a synthetic peptide (SP29) based on the amino acid sequence surrounding serine-424, which also contains the motif described for the serine-158 regulatory phosphorylation site; i.e. basic residues at P-3 and P-6 and a hydrophobic residue at P-5. Peak IV kinase had a native molecular weight of approximately 150,000 as shown by gel filtration. The SP29 peptide was not phosphorylated by the inactivating peak III kinase. Osmotically stressed leaves showed increased peak IV kinase activity with the SP29 peptide as a substrate. Tryptic 32P-phosphopeptide analysis of SPS from excised spinach leaves fed [32P]inorganic P showed increased phosphorylation of the tryptic peptide containing serine-424. Therefore, at least part of the osmotic stress activation of SPS in dark leaves results from phosphorylation of serine-424 catalyzed by a Ca2+-dependent, 150-kD protein kinase.
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role and regulation of sucrose phosphate Synthase in higher plants
Annual review of plant physiology and plant molecular biology, 1996Co-Authors: Steven C. Huber, Joan HuberAbstract:▪ Abstract Sucrose-Phosphate Synthase (SPS; E.C. 2.4.1.14) is the plant enzyme thought to play a major role in sucrose biosynthesis. In photosynthetic and nonphotosynthetic tissues, SPS is regulated by metabolites and by reversible protein phosphorylation. In leaves, phosphorylation modulates SPS activity in response to light/dark signals and end-product accumulation. SPS is phosphorylated on multiple seryl residues in vivo, and the major regulatory phosphorylation site involved is Ser158 in spinach leaves and Ser162 in maize leaves. Regulation of the enzymatic activity of SPS appears to involve calcium, metabolites, and novel “coarse” control of the protein phosphatase that activates SPS. Activation of SPS also occurs during osmotic stress of leaf tissue in darkness, which may function to facilitate sucrose formation for osmoregulation. Manipulation of SPS expression in vivo confirms the role of this enzyme in the control of sucrose biosynthesis.
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effects of elevated sucrose phosphate Synthase activity on photosynthesis assimilate partitioning and growth in tomato lycopersicon esculentum var uc82b
Plant Physiology, 1993Co-Authors: Nathalie Galtier, Joan L Huber, Christine H. Foyer, Toni Voelker, Steven C. HuberAbstract:The expression of a Sucrose-Phosphate Synthase (SPS) gene from maize (Zea mays, a monocotyledon) in tomato (Lycopersicon esculentum, a dicotyledon) resulted in marked increases in extractable SPS activity in the light and the dark. Diurnal modulation of the native tomato SPS activity was found. However, when the maize enzyme was present the tomato leaf cells were unable to regulate its activation state. No detrimental effects were observed and total dry matter production was unchanged. However, carbon allocation within the plants was modified such that in shoots it increased, whereas in roots it decreased. There was, therefore, a change in the shoot:root dry weight ratio favoring the shoot. This was positively correlated with increased SPS activity in leaves. SPS was a major determinant of the amount of starch in leaves as well as sucrose. There was a strong positive correlation between the ratio of sucrose to starch and SPS activity in leaves. Therefore, SPS activity is a major determinant of the partitioning of photosynthetically fixed carbon in the leaf and in the whole plant. The photosynthetic rate in air was not significantly increased as a result of elevated leaf SPS activity. However, the light- and CO2-saturated rate of photosynthesis was increased by about 20% in leaves expressing high SPS. In addition, the temporary enhancement of the photosynthetic rate following brief exposures to low light was increased in the high SPS plants relative to controls. We conclude that the level of SPS in the leaves plays a pivotal role in carbon partitioning. Furthermore, high SPS levels have the potential to boost photosynthetic rates under favorable conditions.
Mark Stitt - One of the best experts on this subject based on the ideXlab platform.
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diurnal changes in sucrose nucleotides starch synthesis and agps transcript in growing potato tubers that are suppressed by decreased expression of sucrose phosphate Synthase
Plant Journal, 2000Co-Authors: Peter Geigenberger, Mark StittAbstract:Sucrose export from leaves is high during the day and lower at night, when it depends on starch remobilisation. We have investigated the consequences of diurnal changes of photoassimilate supply for starch synthesis and other metabolic processes in growing potato tubers. Sucrose, the levels of the transcripts for SUS and AGPS, the levels of key metabolites, and the rate of synthesis of starch and other major end products, including protein and cell wall polysaccharides, increased twofold or more between the start and end of the light period. The stimulation of starch synthesis was accompanied by an increase of UDPglucose and ADPglucose, whereas glycolytic intermediates remained unaltered, revealing that sucrose Synthase and ADP-glucose pyrophosphorylase are being co-ordinately regulated. Sucrose Synthase is stimulated via an increase of its substrates, UDP and sucrose. UDP increases due to an increase of the overall uridine nucleotide pool, and a decrease of the UTP/UDP ratio that occurs in parallel with a decrease of the ATP/ADP ratio and adenylate energy charge when biosynthetic fluxes are high at the end of the day. Within the time frame of the diurnal changes, the changes in the SUS and AGPS transcript levels do not lead to significant changes in the encoded enzymes. Transformants with a progressive decrease of sucrose phosphate Synthase expression, where diurnal changes in leaf sugar levels were damped, exhibited a progressive attenuation of the diurnal changes of sucrose, nucleotide sugars and nucleotides, and fluxes in their tubers. It is concluded that metabolic processes in tubers are tightly linked to the momentary supply of sucrose.
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decreased expression of two key enzymes in the sucrose biosynthesis pathway cytosolic fructose 1 6 bisphosphatase and sucrose phosphate Synthase has remarkably different consequences for photosynthetic carbon metabolism in transgenic arabidopsis thaliana
Plant Journal, 2000Co-Authors: Asa Strand, Mark Stitt, Rita Zrenner, Stephen J Trevanion, Petter Gustafsson, Per GardestromAbstract:Photosynthetic carbon metabolism was investigated in antisense Arabidopsis lines with decreased expression of sucrose phosphate Synthase (SPS) and cytosolic fructose-1,6-bisphosphatase (cFBPase). I ...
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decreased expression of sucrose phosphate Synthase strongly inhibits the water stress induced synthesis of sucrose in growing potato tubers
Plant Journal, 1999Co-Authors: Peter Geigenberger, R Reimholz, Uwe Sonnewald, Uta Deiting, Mark StittAbstract:Summary Water stress stimulates sucrose synthesis and inhibits starch synthesis in wild-type tubers. Antisense and co-suppression potato transformants with decreased expression of sucrose–phosphate Synthase (SPS) have been used to analyse the importance of SPS for the regulation of this water-stress induced change in partitioning. (i) In the absence of water stress, a 70–80% decrease in SPS activity led to a 30–50% inhibition of sucrose synthesis and a slight (10–20%) increase of starch synthesis in tuber discs in short-term labelling experiments with low concentrations of labelled glucose. Similar changes were seen in short-term labelling experiments with intact tubers attached to well-watered plants. Provided plants were grown with ample light and water, transformant tubers had a slightly lower water and sucrose content and a similar or even marginally higher starch content than wild-type tubers. (ii) When wild-type tuber slices were incubated with labelled glucose in the presence of mannitol to generate a moderate water deficit (between –0.12 and –0.72 MPa), there was a marked stimulation of sucrose synthesis and inhibition of starch synthesis. A similar stimulation was seen in labelling experiments with wild-type tubers that were attached to water-stressed wild-type plants. These changes were almost completely suppressed in transformants with a 70–80% reduction of SPS activity. (iii) Decreased irrigation led to an increase in the fraction of the dry-matter allocated to tubers in wild-type plants. This shift in allocation was prevented in transformants with reduced expression of SPS. (iv) The results show that operation of SPS and the sucrose cycle in growing potato tubers may lead to a marginal decrease in starch accumulation in non-stressed plants. However, SPS becomes a crucial factor in water-stressed plants because it is required for adaptive changes in tuber metabolism and whole plant allocation.
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tuber specific expression of a yeast invertase and a bacterial glucokinase in potato leads to an activation of sucrose phosphate Synthase and the creation of a sucrose futile cycle
Planta, 1999Co-Authors: Richard N Trethewey, Mark Stitt, Jorg W Riesmeier, Lothar Willmitzer, Peter GeigenbergerAbstract:Fluxes were investigated in growing tubers from wild-type potato (Solanum tuberosum L. cv. Desiree) and from transformants expressing a yeast invertase in the cytosol under the control of the tuber-specific patatin promoter either alone (EC 3.2.1.26; U-IN2-30) or in combination with a Zymomonas mobilis glucokinase (EC 2.7.1.2; GK3-38) by supplying radiolabelled [14C]sucrose, [14C]glucose or [14C]fructose to tuber discs for a 90-min pulse and subsequent chase incubations of 4 and 12 h, and by supplying [14C]fructose for 2 h and 4 h to intact tubers attached to the mother plant. Contrary to the expectation that this novel route for sucrose degradation would promote starch synthesis, the starch content decreased in the transgenic lines. Labelling kinetics did not reveal whether this was due to changes in the fluxes into or out of starch. However, they demonstrated that glycolysis is enhanced in the transgenic lines in comparison to the wild type. There was also a significant stimulation of sucrose synthesis, leading to a rapid cycle of sucrose degradation and resynthesis. The labelling pattern indicated that sucrose phosphate Synthase (SPS; EC 2.4.1.14) was responsible for the enhanced recycling of label into sucrose. In agreement, there was a 4-fold and 6-fold increase in the activation status of SPS in U-IN2-30 and GK3-38, respectively, and experiments with protein phosphatase inhibitors indicated that this activation involves enhanced dephosphorylation of SPS. It is proposed that this activation of SPS is promoted by the elevated glucose 6-phosphate levels in the transgenic tubers. These results indicate the pitfalls of metabolic engineering without a full appreciation of the metabolic system and regulatory circuits present in the tissue under investigation.
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sucrose metabolism in cold stored potato tubers with decreased expression of sucrose phosphate Synthase
Plant Cell and Environment, 1998Co-Authors: K P Krause, L M Hill, R Reimholz, Tom Hamborg Nielsen, Uwe Sonnewald, Mark StittAbstract: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.
Christine H. Foyer - One of the best experts on this subject based on the ideXlab platform.
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evolution and function of the sucrose phosphate Synthase gene families in wheat and other grasses
Plant Physiology, 2004Co-Authors: C K Castleden, Peter Buchner, Christine H. Foyer, N Aoki, V J Gillespie, Elspeth A Macrae, W P Quick, Robert T Furbank, John E LunnAbstract:Suc-phosphate Synthase (SPS) is a key regulatory enzyme in the pathway of Suc biosynthesis and has been linked to quantitative trait loci controlling plant growth and yield. In dicotyledonous plants there are three SPS gene families: A, B, and C. Here we report the finding of five families of SPS genes in wheat (Triticum aestivum) and other monocotyledonous plants from the family Poaceae (grasses). Three of these form separate subfamilies within the previously described A, B, and C gene families, but the other two form a novel and distinctive D family, which on present evidence is only found in the Poaceae. The D-type SPS proteins lack the phosphorylation sites associated with 14-3-3 protein binding and osmotic stress activation, and the linker region between the N-terminal catalytic glucosyltransferase domain and the C-terminal Suc-phosphatase-like domain is 80 to 90 amino acid residues shorter than in the A, B, or C types. The D family appears to have arisen after the divergence of mono- and dicotyledonous plants, with a later duplication event resulting in the two D-type subfamilies. Each of the SPS gene families in wheat showed different, but overlapping, spatial and temporal expression patterns, and in most organs at least two different SPS genes are expressed. Analysis of expressed sequence tags indicated similar expression patterns to wheat for each SPS gene family in barley (Hordeum vulgare) but not in more distantly related grasses. We identified an expressed sequence tag from rice (Oryza sativa) that appears to be derived from an endogenous antisense SPS gene, and this might account for the apparently low level of expression of the related OsSPS11 sense gene, adding to the already extensive list of mechanisms for regulating the activity of SPS in plants.
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elevated sucrose phosphate Synthase activity in transgenic tobacco sustains photosynthesis in older leaves and alters development
Journal of Experimental Botany, 2003Co-Authors: Charles J Baxter, Christine H. Foyer, J C Turner, Stephen A Rolfe, Paul W QuickAbstract:Constitutive over-expression of a maize Sucrose-Phosphate Synthase (SPS) gene in tobacco (Nicotiana tabacum) had major effects on leaf carbohydrate budgets with consequences for whole plant development. Transgenic tobacco plants flowered earlier and had greater flower numbers than wild-type plants. These changes were not linked to modified source leaf carbon assimilation or carbon export, although sucrose to starch ratios were significantly higher in leaves expressing the transgene. The youngest and oldest leaves of plants over-expressing SPS had up to 10-fold wild-type maximal extractable SPS activity, but source leaf SPS activities were only 2-3 times greater in these lines than in the wild type. In the oldest leaves, where the expression of the transgene led to the most marked enhancement in SPS activity, photosynthesis was also increased. It was concluded that these increases in the capacity for sucrose synthesis and carbon assimilation, particularly in older leaves, accelerate the whole plant development and increase the abundance of flowers without substantial changes in the overall shoot biomass.
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A role for ‘futile cycles’ involving invertase and sucrose Synthase in sucrose metabolism of tomato fruit
Journal of Experimental Botany, 2001Co-Authors: Binh Nguyen-quoc, Christine H. FoyerAbstract:: 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.
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effects of light and atmospheric carbon dioxide enrichment on photosynthesis and carbon partitioning in the leaves of tomato lycopersicon esculentum l plants over expressing sucrose phosphate Synthase
Journal of Experimental Botany, 1995Co-Authors: Nathalie Galtier, Christine H. Foyer, Paul Quick, Toni Voelker, Erik H Murchie, Rhu Aired, Catherine Thepenier, Gerard Lasceve, Thomas BetscheAbstract:Photosynthetic carbon assimilation, carbon parti tioning and foliar carbon budgets were measured in the leaves of transformed tomato plants expressing a maize Sucrose-Phosphate Synthase (SPS) gene in addi tion to the native enzyme, and in untransformed con trols. The maize SPS gene was expressed under control of either the promoter of the small subunit of ribulose 1,5-bisphosphate carboxylase (rbcS promoter; lines 2, 9 and 18) or the 35S promoter from cauliflower mosaic virus (CaMV promoter; line 13). The rate of sucrose synthesis was increased relative to that of starch and sucrose/starch ratios were higher through out the photoperiod in the leaves of all plants expressing high SPS activity. The leaf carbon budget over the day/night cycle in air at low irradiance (180//mol photon m~2 s1) was similar in all plants. Net photosynthesis measured in air and at elevated C02 (800-1500 //I r1) on whole plants grown in air at 400//mol m~2 s-1 irradiance was significantly increased in the high SPS expressors compared to the untransformed controls and was highest where SPS activity was greatest. At high C02 the stimulation of photosynthesis was more pronounced. We conclude that SPS activity is a major point of control of photo synthesis particularly under saturating light and C02.
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effects of elevated sucrose phosphate Synthase activity on photosynthesis assimilate partitioning and growth in tomato lycopersicon esculentum var uc82b
Plant Physiology, 1993Co-Authors: Nathalie Galtier, Joan L Huber, Christine H. Foyer, Toni Voelker, Steven C. HuberAbstract:The expression of a Sucrose-Phosphate Synthase (SPS) gene from maize (Zea mays, a monocotyledon) in tomato (Lycopersicon esculentum, a dicotyledon) resulted in marked increases in extractable SPS activity in the light and the dark. Diurnal modulation of the native tomato SPS activity was found. However, when the maize enzyme was present the tomato leaf cells were unable to regulate its activation state. No detrimental effects were observed and total dry matter production was unchanged. However, carbon allocation within the plants was modified such that in shoots it increased, whereas in roots it decreased. There was, therefore, a change in the shoot:root dry weight ratio favoring the shoot. This was positively correlated with increased SPS activity in leaves. SPS was a major determinant of the amount of starch in leaves as well as sucrose. There was a strong positive correlation between the ratio of sucrose to starch and SPS activity in leaves. Therefore, SPS activity is a major determinant of the partitioning of photosynthetically fixed carbon in the leaf and in the whole plant. The photosynthetic rate in air was not significantly increased as a result of elevated leaf SPS activity. However, the light- and CO2-saturated rate of photosynthesis was increased by about 20% in leaves expressing high SPS. In addition, the temporary enhancement of the photosynthetic rate following brief exposures to low light was increased in the high SPS plants relative to controls. We conclude that the level of SPS in the leaves plays a pivotal role in carbon partitioning. Furthermore, high SPS levels have the potential to boost photosynthetic rates under favorable conditions.
Dabing Zhang - One of the best experts on this subject based on the ideXlab platform.
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international collaborative study of the endogenous reference gene sucrose phosphate Synthase sps used for qualitative and quantitative analysis of genetically modified rice
Journal of Agricultural and Food Chemistry, 2009Co-Authors: Lingxi Jiang, Litao Yang, Haibo Zhang, Jinchao Guo, Marco Mazzara, Guy Van Den Eede, Dabing ZhangAbstract:One rice (Oryza sativa) gene, sucrose phosphate Synthase (SPS), has been proven to be a suitable endogenous reference gene for genetically modified (GM) rice detection in a previous study. Herein are the reported results of an international collaborative ring trial for validation of the SPS gene as an endogenous reference gene and its optimized qualitative and quantitative polymerase chain reaction (PCR) systems. A total of 12 genetically modified organism (GMO) detection laboratories from seven countries participated in the ring trial and returned their results. The validated results confirmed the species specificity of the method through testing 10 plant genomic DNAs, low heterogeneity, and a stable single-copy number of the rice SPS gene among 7 indica varieties and 5 japonica varieties. The SPS qualitative PCR assay was validated with a limit of detection (LOD) of 0.1%, which corresponded to about 230 copies of haploid rice genomic DNA, while the limit of quantification (LOQ) for the quantitative PCR ...
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validation of a rice specific gene sucrose phosphate Synthase used as the endogenous reference gene for qualitative and real time quantitative pcr detection of transgenes
Journal of Agricultural and Food Chemistry, 2004Co-Authors: Jiayu Ding, Litao Yang, Junwei Jia, Haibo Wen, Chengmei Zhang, Wenxuan Liu, Dabing ZhangAbstract:With the development of transgenic crops, many countries have issued regulations to label the genetically modified organisms (GMOs) and their derived products. Polymerase Chain Reaction (PCR) methods are thought to be reliable and useful techniques for qualitative and quantitative detection of GMOs. These methods generally need to amplify the transgene and compare the amplified result with that of the corresponding reference gene to obtain reliable results. In this article, we reported the development of specific primers and probe for the rice (Oryza sativa) sucrose phosphate Synthase (SPS) gene and PCR cycling conditions suitable for the use of this sequence as an endogenous reference gene in both qualitative and quantitative PCR assays. Both methods were assayed with 13 different rice varieties, and identical amplification products were obtained with all of them. No amplification products were observed when DNA samples from other species, such as wheat, maize, barley, tobacco, soybean, rapeseed, tomato,...
Ryu Ohsugi - One of the best experts on this subject based on the ideXlab platform.
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a reduction of sucrose phosphate Synthase sps activity affects sucrose starch ratio in leaves but does not inhibit normal plant growth in rice
Plant Science, 2016Co-Authors: Yoichi Hashida, Ryu Ohsugi, Tatsuro Hirose, Masaki Okamura, Kenichiro Hibara, Naohiro AokiAbstract:Abstract Sucrose phosphate Synthase (SPS) has been shown to mediate sucrose/starch ratio in plant leaves through studies of ‘starch leaf’ species that mainly accumulate starch in their leaves. However, the contribution of SPS to sucrose/starch ratio in rice leaves, which mainly accumulate sucrose (i.e., ‘sugar leaf’), has not been confirmed due to inconsistencies in the results of previous studies. In this study, we analyzed mutant lines with reduced SPS activity, which were generated using Tos17 insertion, RNAi, and the CRISPR/Cas9 system. The knockdown and knockout mutants of OsSPS1 showed a 29–46% reduction in SPS activity in the leaves, but the carbohydrate content in the leaves and plant growth were not significantly different from those of wild-type plants. In a double knockout mutant of OsSPS1 and OsSPS11 ( sps1/sps11 ), an 84% reduction in leaf SPS activity resulted in higher starch accumulation in the leaves than in the wild-type leaves. However, the sps1/sps11 plants grew normally, which is in contrast to the inhibited growth of SPS mutants of Arabidopsis thaliana , a typical starch leaf plant. These results suggest that SPS has a smaller effect on the sucrose/starch ratio in leaves and growth of rice than on starch leaf species.
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analysis of gene disruption mutants of a sucrose phosphate Synthase gene in rice ossps1 shows the importance of sucrose synthesis in pollen germination
Plant Science, 2014Co-Authors: Tatsuro Hirose, Yoichi Hashida, Naohiro Aoki, Masaki Okamura, Madoka Yonekura, Chikara Ohto, Tomio Terao, Ryu OhsugiAbstract:The molecular function of an isoform of sucrose phosphate Synthase (SPS) in rice, OsSPS1, was investigated using gene-disruption mutant lines generated by retrotransposon insertion. The progeny of the heterozygote of disrupted OsSPS1 (SPS1(+/-)) segregated into SPS1(+/+), SPS1(+/-), and SPS1(-/-) at a ratio of 1:1:0. This distorted segregation ratio, together with the expression of OsSPS1 in the developing pollen revealed by quantitative RT-PCR analysis and promoter-beta-glucuronidase (GUS) fusion assay, suggested that the disruption of OsSPS1 results in sterile pollen. This hypothesis was reinforced by reciprocal crosses of SPS1(+/-) plants with wild-type plants in which the disrupted OsSPS1 was not paternally transmitted to the progeny. While the pollen grains of SPS(+/-) plants normally accumulated starch during their development, pollen germination on the artificial media was reduced to half of that observed in the wild-type control. Overall, our data suggests that sucrose synthesis via OsSPS1 is essential in pollen germination in rice.
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tissue specificity and diurnal change in gene expression of the sucrose phosphate Synthase gene family in rice
Plant Science, 2011Co-Authors: Masaki Okamura, Tatsuro Hirose, Naohiro Aoki, Madoka Yonekura, Chikara Ohto, Ryu OhsugiAbstract:The rice genome contains 5 isogenes for sucrose phosphate Synthase (SPS), the key enzyme in sucrose synthesis; however, little is known about their transcriptional regulation. In order to determine the expression patterns of the SPS gene family in rice plants, we conducted an expression analysis in various tissues and developmental stages by real-time quantitative RT-PCR. At the transcript level, the rice SPS genes, particularly SPS1, were preferentially expressed in source tissues, whereas SPS2, SPS6, and SPS8 were expressed equally in source and sink tissues. We also investigated diurnal changes in SPS gene expression, SPS activity, and soluble sugar content in leaf blades. Interestingly, the expression of all the SPS genes, particularly that of SPS1 and SPS11, tended to be higher at night when the activation state of the SPS proteins was low, and the mRNA levels of SPS1 and SPS6 were negatively correlated with sucrose content. Furthermore, the temporal patterns of SPS gene expression and sugar content under continuous light conditions suggested the involvement of endogenous rhythm and/or sucrose sensing in the transcriptional regulation of SPS genes. Our data revealed differential expression patterns in the rice SPS gene family and part of the complex mechanisms of their transcriptional control.
