Sucrose Phosphate Synthase Protein Research Articles

Analysis of sugar components and identification of SPS genes in citrus fruit development.

Sugar is a primary determinant of citrus fruit flavour, but undergoes varied accumulation processes across different citrus varieties owing to high genetic variability. Sucrose phosphate synthase (SPS), a key enzyme in glucose metabolism, plays a crucial role in this context. Despite its significance, there is limited research on sugar component quality and the expression and regulatory prediction of SPS genes during citrus fruit development. Therefore, we analysed the sugar quality formation process in 'Kiyomi' and 'Succosa', two citrus varieties, and performed a comprehensive genome-wide analysis of citrus CsSPSs. We observed that the accumulation of sugar components significantly differs between the two varieties, with the identification of four CsSPSs in citrus. CsSPS sequences were highly conserved, featuring typical SPS protein domains. Expression analysis revealed a positive correlation between CsSPS expression and sugar accumulation in citrus fruits. However, CsSPS expression displays specificity to different citrus tissues and varieties. Transcriptome co-expression network analysis suggests the involvement of multiple transcription factors in shaping citrus fruit sugar quality through the regulation of CsSPSs. Notably, the expression levels of four CsWRKYs (CsWRKY2, CsWRKY20, CsWRKY28, CsWRKY32), were significantly positively correlated with CsSPSs and CsWRKY20 might can activate sugar accumulation in citrus fruit through CsSPS2. Collectively, we further emphasize the potential importance of CsWRKYs in citrus sugar metabolism, our findings serve as a reference for understanding sugar component formation and predicting CsSPS expression and regulation during citrus fruit development.

Genome-wide identification and expression profiling analysis of sucrose synthase (SUS) and sucrose phosphate synthase (SPS) genes family in Actinidia chinensis and A. eriantha

Sucrose synthase (SUS) is a common sugar-base transfer enzyme in plants, and sucrose phosphate synthase (SPS) is one of the major enzymes in higher plants that regulates sucrose synthesis. However, information of the SPS and SUS gene families in Actinidia, as well as their evolutionary and functional properties, is limited. According to the SPS and SUS proteins conserved domain of Arabidopsis thaliana, we found 6 SPS genes and 6 SUS genes from A. chinensis (cultivar: ‘Hongyang’), and 3 SPS genes and 6 SUS genes from A. eriantha (cultivar: ‘White’). The novel CDC50 conserved domains were discovered on AcSUS2, and all members of the gene family contain similar distinctive conserved domains. The majority of SUS and SPS proteins were hydrophilic, lipid-soluble enzymes that were expected to be found in the cytoplasm. The tertiary structure of SPS and SUS protein indicated that there were many tertiary structures in SPS, and there were windmill-type and spider-type tertiary structures in SUS. The phylogenetic tree was created using the neighbor-joining method, and members of the SPS and SUS gene families are grouped into three subgroups. Genes with comparable intron counts, conserved motifs, and phosphorylation sites were clustered together first. SPS and SUS were formed through replication among their own family members. AcSPS1, AcSPS2, AcSPS4, AcSPS5, AcSUS5, AcSUS6, AeSPS3, AeSUS3 and AeSUS4 were the important genes in regulating the synthesis and accumulation of sucrose for Actinidia during the fruit growth stages.

Distinct nodule and leaf functions of two different sucrose phosphate synthases in alfalfa.

In alfalfa, the B form of Sucrose phosphate synthase synthesizes sucrose in the leaves while the A form participates in regulatory cycles of synthesis/breakdown of sucrose/starch in the root nodules. Sucrose (Suc) is the major stable product of photosynthesis that is transported to all heterotrophic organs as a source of energy and carbon. The enzyme sucrose phosphate synthase (SPS) catalyzes the synthesis of Suc. Besides the leaves, SPS is also found in heterotrophic organs. There are two isoforms of SPS in alfalfa (Medicago sativa): SPSA and SPSB. While SPSA is expressed in the vasculature of all the organs and in the N2-fixing zone in the nodules, SPSB is exclusively expressed in the photosynthetic cells. Two classes of alfalfa transformants were produced, one with a gene construct consisting of the alfalfa SPSA promoter and the other with the SPSB promoter-both driving the maize SPS coding region-referred to as SPSA-ZmSPS and SPSB-ZmSPS, respectively. Both classes of transformants showed increased growth compared to control plants. The SPSB-ZmSPS transformants showed increased SPS protein levels and activity along with a significant increase in the Suc levels in the leaves. The SPSA-ZmSPS transformants showed an increase in the SPS protein level and enzyme activity both in the leaves and the nodules with no increase in Suc content in the leaves but a substantial increase in the nodules. Both SPSA and SPSB have unique roles in the nodules (sink) and leaves (source). SPSB is responsible for the synthesis of Suc in the photosynthetic cells and SPSA participates in a regulatory cycle in which Suc is simultaneously degraded and re-synthesized; both these functions contribute to plant growth in rhizobia nodulated alfalfa plants.

A dual‐targeted purple acid phosphatase in Arabidopsis thaliana moderates carbon metabolism and its overexpression leads to faster plant growth and higher seed yield

• Overexpression of AtPAP2, a purple acid phosphatase (PAP) with a unique C-terminal hydrophobic motif in Arabidopsis, resulted in earlier bolting and a higher seed yield. Metabolite analysis showed that the shoots of AtPAP2 overexpression lines contained higher levels of sugars and tricarboxylic acid (TCA) metabolites. Enzyme assays showed that sucrose phosphate synthase (SPS) activity was significantly upregulated in the overexpression lines. The higher SPS activity arose from a higher level of SPS protein, and was independent of SnRK1. • AtPAP2 was found to be targeted to both plastids and mitochondria via its C-terminal hydrophobic motif. Ectopic expression of a truncated AtPAP2 without this C-terminal motif in Arabidopsis indicated that the subcellular localization of AtPAP2 is essential for its biological actions. • Plant PAPs are generally considered to mediate phosphorus acquisition and redistribution. AtPAP2 is the first PAP shown to modulate carbon metabolism and the first shown to be dual-targeted to both plastids and mitochondria by a C-terminal targeting signal. • One PAP-like sequence carrying a hydrophobic C-terminal motif could be identified in the genome of the smallest free-living photosynthetic eukaryote, Ostreococcus tauri. This might reflect a common ancestral function of AtPAP2-like sequences in the regulation of carbon metabolism.

If the antibody fails – a mass Western approach

Sucrose-phosphate synthase (SPS) has attracted the interest of plant scientists for decades. It is the key enzyme in sucrose metabolism and is under investigation in various plant species, e.g. spinach, tobacco, poplar, resurrection plants, maize, rice, kiwi and Arabidopsis thaliana. In A. thaliana, there are four distinct SPS isoforms. Their expression is thought to depend on environmental conditions and plant tissue. However, these data were derived from mRNA expression levels only. No data on SPS protein identification from crude extracts have been available until now. An antibody approach failed to distinguish the four isoforms. Therefore, we developed a method for SPS quantification and isoform-specific identification in A. thaliana complex protein samples. Samples were separated on SDS-PAGE, digested and directly applied to liquid chromatography/triple-stage quadrupole mass spectrometry (LC/TSQ-MS). In this approach, known as mass Western, samples were analysed in multi-reaction monitoring (MRM) mode, so that all four SPS isoforms could be measured in one experiment. In addition to the relative quantification, stable isotope-labelled internal peptide standards allowed absolute quantification of SPS proteins. Protein extracts from various plant tissues, samples harvested during the day or the night, and cold-stressed plants were analysed. The stress-specific SPS5a isoform showed increased concentrations in cold-stressed leaf material.

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

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

Low temperature regulates sucrose-phosphate synthase activity in Colobanthus quitensis (Kunth) Bartl. by decreasing its sensitivity to Pi and increased activation by glucose-6-phosphate

Colobanthus quitensis (Kunth) Bartl. is widely distributed from Mexico to the Antarctic. C. quitensis is a freezing resistant species that accumulates sucrose in response to cold. We tested the hypothesis that low temperature modifies the kinetic properties of C. quitensis sucrose phosphate synthase (SPS) to increase its activity and ability to synthesize sucrose during cold acclimation. Cold acclimation caused a fourfold increment in sucrose concentration and a 100% increase in SPS activity, without changes in the level of SPS protein. Cold acclimation did not affect the optimal temperature and pH for SPS activity. However, it caused a tenfold increase in the inhibition constant (K i) for inorganic phosphate (Pi) calculated as a function of fructose-6-phosphate (Fruc-6-P). SPS from cold acclimated plants also exhibited a higher reduction of its Michaelis constant (K m) for glucose-6-phosphate (Gluc-6-P) with respect to non-acclimated plants. We suggest that the increase in C. quitensis SPS K i for Pi and the increase in activation by Gluc-6-P in response to cold keep SPS activated, leading to high sucrose accumulation. This may be an important adaptation that allows efficient accumulation of sucrose during the harsh Antarctic summer.

Expression of a cyanobacterial sucrose-phosphate synthase from Synechocystis sp. PCC 6803 in transgenic plants.

Sucrose-phosphate synthase (SPS) from the cyanobacterium Synechocystis sp. PCC 6803 lacks all of the Ser residues known to be involved in the regulation of higher plant SPS by protein phosphorylation. The Synechocystis SPS is also not allosterically regulated by glucose 6-phosphate or orthophosphate. To investigate the effects of expressing a potentially unregulated SPS in plants, the Synechocystis sps gene was introduced into tobacco, rice and tomato under the control of constitutive promoters. The Synechocystis SPS protein was expressed at a high level in the plants, which should have been sufficient to increase overall SPS activity 2-8-fold in the leaves. However, SPS activities and carbon partitioning in leaves from transgenic and wild-type plants were not significantly different. The maximal light-saturated rates of photosynthesis in leaves from tomato plants expressing the Synechocystis SPS were the same as those from wild-type plants. Tomato plants expressing the maize SPS showed 2-3-fold increases in SPS activity, increased partitioning of photoassimilate to sucrose and up to 58% higher maximal rates of photosynthesis. To investigate the apparent inactivity of the Synechocystis SPS the enzyme was purified from transgenic tobacco and rice plants. Surprisingly, the purified enzyme was found to have full catalytic activity. It is proposed that some other protein in plant cells binds to the Synechocystis SPS resulting in inhibition of the enzyme.

A comparison of the carbohydrate composition and kinetic properties of sucrose phosphate synthase (SPS) in transgenic tobacco (Nicotiana tabacum) leaves expressing maize SPS protein with untransformed controls

SummaryTobacco plants, transformed with a maize sucrose phosphate synthase (SPS) cDNA clone, had threefold increased SPS activity compared to wild‐type tobacco. Measurement of SPS maximal activity and protein abundance using specific antibodies to the maize protein showed that the specific activity of the maize SPS protein was maintained when expressed in tobacco. Comparison of the kinetic properties of SPS in the transgenic lines compared to either wild‐type maize or tobacco revealed that the heterologously expressed protein had reduced affinity for both substrates (fructose‐6‐phosphate and UDP‐glucose) and reduced sensitivity to allosteric inhibition by inorganic phosphate. Moreover, the extent of light‐induced activation was reduced in the transgenic lines, with smaller changes observed in the Km for both F6P compared to maize and tobacco wild‐type plants. Increased sucrose concentrations were observed in the transgenic lines at the end of the photoperiod and this was linearly related to SPS activity and associated with a parallel decrease in starch content. This suggests that SPS is a major control point for carbohydrate partitioning between starch and sucrose during photosynthesis.

Potential for manipulating carbon metabolism in wheat

SummaryThe transformation of wheat opens up opportunities for manipulation of key steps in plant metabolic pathways. Attempts to alter the expression of sucrose phosphate synthase (SPS) and glycine decarboxylase (GDC) in wheat leaves have been made.Using a maize SPS gene under the control of either a constitutive promoter (in p35SMSPS) or a green tissue specific promoter (in pRGMSPS), transformation of wheat (Triticum aestivum L. cv. Cadenza) led to the production of 22 independent transformed lines that contained the SPS transgene. Expression of the transgene, analysed by RT‐PCR, revealed that the transcript was present in nine of the 12 p35SMSPS plants and six of the 10 pRGMSPS plants. The maize SPS protein could not, however, be detected in any of these lines.The expression of GDC, and in particular that of the P subunit, has been shown to be altered in plant species with the intermediate C3/C4 photosynthetic phenotype. Attempts have been made to make similar changes in the expression of GDC in wheat leaves by transforming them with full length cDNA sequences for the P subunit of GDC in the sense and antisense orientation. More than 70 transformed lines were produced. Of these, 11 contained the gene in the sense, and 31 in the antisense, orientation, as determined by PCR. Results with Tt generation plants suggested that the activity of GDC had been reduced in some lines transformed with antisense constructs. However, there was no evidence for the expression of the sense constructs. GDC mRNA could not be detected by RT‐PCR in any of the 10 lines produced in the sense transformation experiments.Taken together, these results demonstrate the effectiveness of the technology to deliver large numbers of transgenic wheat plants. However, clearly this alone is not sufficient to guarantee the correct expression of the transgenes at sufficient levels to establish the presence of the gene product.

Ser162-Dependent inactivation of overproduced sucrose-phosphate synthase protein of maize leaf in transgenic rice plants.

To investigate the role of Ser162 in phosphorylation-dependent regulation of maize sucrose-phosphate synthase (SPS) activities in rice, transgenic rice plants expressing wild-type or mutagenized maize SPS were produced. Our results indicate that Ser162 was responsible for overproduction-induced inactivation of SPS protein and for light/ dark modulation of this protein in vivo.

Characterization of a Maize Sucrose–phosphate Synthase Protein and Its Effect on Carbon Partitioning in Transgenic Rice Plants

We obtained transgenic rice (Oryza sativa L. cv. Nipponbare) plants with the gene for maize sucrose-phosphate synthase (EC 2.4.1.14, SPS). Some of the transgenic plants over-expressed maize SPS (over-expressing plants) and some had reduced levels of native SPS protein (co-suppressed plants). There was a positive correlation between the amounts of maize SPS protein and SPS activities. However, apparent Km values for uridine diphosphoglucose (UDPG) were higher in over-expressing plants than in control rice plants. These results suggest that overproduced maize SPS protein was not fully activated. The sucrose contents did not differ significantly between control and over-expressing rice plants, but they were lower in co-suppressed plants than in control plants. The starch contents were negatively and the sucrose/starch ratios were positively correlated with SPS activities. Thus, carbon partitioning in the transgenic rice was changed, even though rice is predominantly a sucrose-former.

Changes in Sugars and Activities of Sucrose Phosphate Synthase, Sucrose Synthase, and Invertase during Potato Tuber (Russet Burbank) Reconditioning at 10 °C in Air and 2.53 kPa Oxygen after Storage for 28 Days at 1 °C

Potato tubers (Solanum tuberosum `Russet Burbank') were stored at 1 °C in air for 28 days and then transferred to 10 °C in either air or 2.53 kPa O2. During cold storage there was an increase in sucrose, glucose, and fructose. The activities of extractable sucrose phosphate synthase (SPS) and invertase increased by 2.2- and 7.7-fold, respectively, during 28 days at 1 °C. The activity of sucrose synthase (SS) remained constant at 1 °C and was similar to that found in tubers kept continuously at 10 °C. With the transfer of tubers from 1 to 10 °C, there was an initial sharp rise in respiration which peaked at ≈7 days, followed by a gradual decline. Sucrose declined rapidly during reconditioning, while glucose and fructose declined more slowly. With the transfer of tubers from 1 to 10 °C, the activity of SS increased sharply after 7 days at 10 °C, to be followed by a decline to the levels found in control tubers. The activities of both extractable SPS and invertase decreased during reconditioning, reaching the values of the control tubers within ≈15 days. Low O2 inhibited the decrease in sugars and suppressed the rise in SS activity, but it did not alter the decrease in SPS and invertase. Western blot analysis showed that the amount of SPS protein remained unchanged at 1 and 10 °C. These results indicate that the activity of SPS is regulated by factors other than the amount of its protein. The activities of the above three enzymes showed no changes in tubers kept at 10 °C continuously. In control tubers SPS showed the highest activity, followed by SS and invertase.

Similar temperature requirement for sugar accumulation and for the induction of new forms of sucrose phosphate synthase and amylase in cold‐stored potato tubers

The onset of sugar accumulation in cold‐stored potato tubers coincides with an activation of sucrose phosphate synthase (SPS) and the appearance of a new form of amylase (Hill et al. 1996; Nielsen et al. 1997). To provide more evidence that these changes are involved in the regulation of cold‐induced sugar accumulation we have compared the temperature dependence of sugar accumulation with the temperature dependence of changes in SPS and amylase activity. To do this, we investigated: sugars and metabolites; SPS activity, protein and transcript; invertase activity; and amylolytic activity and amylase forms, during the first 10 d after transferring tubers from room temperature to 3, 5, 7, 9 and 11 °C. After 10 d there was negligible accumulation of sugars at 11, 9 and 7 °C, and a large accumulation at 5 and 3 °C. Activation of SPS was assayed by comparing activity in an assay with limiting substrates and phosphate and an assay with saturating substrate concentrations. The activation state increased slightly at 7 °C, 3‐ to 4‐fold at 5 °C and 5‐ to 6‐fold at 3 °C. The cold‐induced change in the kinetic properties of SPS was accompanied by the appearance of a new form of SPS with a slightly higher apparent molecular weight, and by an increase of the SPS transcript. The changes in SPS protein and transcript showed the same temperature dependence as the changes in the kinetic properties. Total starch hydrolysing enzyme activity was unaltered at 7°C, increased at 5°C and increased further (up to 7‐ to 8‐fold) at 3 °C. The increase in amylolytic activity correlated with the appearance of a new amylase band on zymograms. Acid invertase activity showed a similar increase at 3, 5, and 7°C, and it did not correlate with the total sugar accumulation. The cold‐induced accumulation of sugar can be reversed by transferring tubers back to warmer temperatures. We compared the decline of sugar levels with the changes of SPS, amylolytic activity and metabolites at various times (up to 14 d) after transfer of tubers from a 4 °C storage (for 14 d) back to 20°C. SPS activation state is reversed and the cold‐induced form of SPS virtually disappears during the first 2–4 d at 20 °C. The cold‐induced amylase activity also vanishes within 2–4 d.

Protein phosphorylation as a mechanism for osmotic-stress activation of sucrose-phosphate synthase in spinach leaves.

Experiments were performed to investigated 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 Ca(2+)-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, Ca(2+)-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 Ca(2+)-dependent, 150-kD protein kinase.

Circadian Regulation of Sucrose Phosphate Synthase Activity in Tomato by Protein Phosphatase Activity.

Sucrose phosphate synthase (SPS), a key enzyme in sucrose biosynthesis, is regulated by protein phosphorylation and shows a circadian pattern of activity in tomato. SPS is most active in its dephosphorylated state, which normally coincides with daytime. Applying okadaic acid, a potent protein phosphatase inhibitor, prevents SPS activation. More interesting is that a brief treatment with cycloheximide, a cytoplasmic translation inhibitor, also prevents the light activation of SPS without any effect on the amount of SPS protein. Cordycepin, an inhibitor of transcript synthesis and processing, has the same effect. Both of these inhibitors also prevent the activation phase of the circadian rhythm in SPS activity. Conversely, cycloheximide and cordycepin do not prevent the decline in circadian SPS activity that normally occurs at night. These observations indicate that SPS phosphatase activity but not SPS kinase activity is controlled, directly or indirectly, at the level of gene expression. Taken together, these data imply that there is a circadian rhythm controlling the transcription of a protein phosphatase that subsequently dictates the circadian rhythm in SPS activity via effects on this enzyme's phosphorylation state.

The onset of sucrose accumulation in cold‐stored potato tubers is caused by an increased rate of sucrose synthesis and coincides with low levels of hexose‐phosphates, an activation of sucrose phosphate synthase and the appearance of a new form of amylase

ABSTRACTThese experiments investigate events involved in triggering sugar accumulation in the cold in tubers of Solanum tuberosum L. cv. Desirée. Sugar content, 14C‐glucose metabolism, metabolite levels and activities of sucrose phosphate synthase (SPS) and starch‐degrading enzymes were followed after transfer to 4°C. (i) Net sucrose accumulation began between 2 and 4 d. By 10 d, reducing sugars were also increasing. From 20 d onwards, sugar accumulation slowed. Sucrose fell, but reducing sugars continued to increase. (ii) To measure unidirectional sucrose synthesis, U‐[14C]glucose was injected into tubers after various times at 4°C. The tubers were then incubated for 6 h. After 1 d at 4°C, both the absolute and the relative (expressed as a percentage of the metabolized label) rates of sucrose synthesis decreased compared to those at 20°C. Between 2 and 4 d at 4°C, labelling of sucrose increased 3‐fold, to over 60% of the metabolized label. This high rate was maintained for up to 50 d in cold storage. When tissue slices were incubated with 2.5 mol m−3 U‐[14C]glucose, the rate of labelling of sucrose in slices from 6 d cold‐stored material was higher than in slices from warm‐stored material, irrespective of whether the incubation occurred at 4°C or at 20°C. (iii) Hexose‐phosphates increased during the first day after transfer to 4°C. Their levels fell during the next 3 d, as sucrose synthesis increased. They then rose (until 20 d) and fell, in parallel with the rise and decline of sucrose levels. UDPglucose remained unaltered during the first 4 d, and then increased and decreased in parallel with sucrose. (iv) SPS activity assayed in optimal conditions and the total amount of SPS protein did not change. However, when assayed in the presence of phosphate and limiting substrate concentrations, activity rose 3–5‐fold between 2 and 4 d. (v) Amylases and phosphorylases were investigated using zymograms to separate isoforms. Phosphorylases did not change. Between 2 and 4 d at 4°C, a new amylolytic activity appeared. (vi) Estimates of the specific activity of the phosphorylated intermediates and the absolute rate of sucrose synthesis (calculated from the 14C‐labelling data and metabolite analysis) showed that changed kinetic properties of SPS and decreased levels of hexose‐phosphate are accompanied by a 6–8‐fold stimulation of sucrose synthesis. They also show that the final level of sugar is partly determined by a cycle of sugar synthesis and degradation. (vii) It is concluded that the onset of sugar accumulation in cold‐stored tubers is initiated by a change in the kinetic properties of SPS and the appearance of a new amylolytic activity. It is discussed how other factors, including hexose‐phosphate levels and subcellular compartmentalization, could also influence the final levels of sugars by altering the balance of sugar synthesis and remobilization.

Coarse and Fine Control and Annual Changes of Sucrose-Phosphate Synthase in Norway Spruce Needles.

Annual changes of activity of sucrose-phosphate synthase (SPS) from spruce (Picea abies [L.] Karst.) needles were studied with respect to three regulatory levels: metabolic fine control, covalent modification (phosphorylation), and protein amount. Glucose-6-phosphate served as an allosteric activator of spruce SPS by shifting the Michaelis constant for the substrate fructose-6-phosphate from 4.2 to 0.59 mM, whereas inorganic phosphate competitively inhibited this activation. The affinity for the other substrate, UDP-glucose, was unaffected. Incubation of the crude extract with ATP resulted in a time- and concentration-dependent decrease of the maximal velocity of SPS. This inactivation was sensitive to staurosporine, a potent protein kinase inhibitor, indicating the participation of a protein kinase. Probing SPS protein with heterologous antibodies showed that the subunit of spruce SPS is an approximately 139-kD protein and that changes in the extractable activity during the course of a year were correlated with the amount of SPS protein. High SPS activities in winter were paralleled by increased levels of the activator glucose-6-phosphate and the substrate fructose-6-phosphate, indicating a high capacity for sucrose synthesis that may be necessary to maintain photosynthetic CO2 fixation in cold-hardened spruce needles.

Sucrose Phosphate Synthase Expression at the Cell and Tissue Level Is Coordinated with Sucrose Sink-to-Source Transitions in Maize Leaf.

Immunohistological analyses for sucrose phosphate synthase (SPS) show that the protein is localized in both bundle-sheath cells (BS) and mesophyll cells (M) in maize (Zea mays) leaves. In young leaves, SPS protein was predominantly in the BS, whereas mature leaves showed nearly equal levels of signal in both BS and M. A cell-type-specific response was also seen in light and dark treatments. Dark treatments led to reduced signal in M; however, little or no change was detected in BS. We suggest that SPS in BS is engaged in sucrose biosynthesis by both photoassimilatory and starch turnover reactions in maize leaves. In addition, we suggest that the enzyme in BS may play a major role in the early biosynthesis of sucrose in young leaves. These cell-specific changes in expression in situ were in agreement with the estimates of extractable enzyme activity from isolated BS and M of mature leaves (R. Ohsugi, S.C. Huber [1987] Plant Physiol 84: 1096-1101). In contrast, western blot analyses did not show any significant changes in the levels of SPS protein in either young or mature leaves subsequent to similar dark treatments. It is interesting that the northern blot analyses indicate that the steady-state levels of SPS transcripts were markedly reduced after dark treatments of > 12 h. Overall, our results indicate that Sps gene expression in maize leaf is modulated at multiple levels of controls by both developmental and environmental factors.

Phosphate Deficiency in Maize. IV. Changes in Amounts of Sucrose Phosphate Synthase during the Course of Phosphate Deprivation

With low-P treatment of maize, the level of sucrose phosphate synthase (SPS) protein decreased to 15% of the control, while the “Vmax” activity stayed relatively high (100–80% of the control) and the substrate limiting activity increased about 2 fold in the leaves. These results suggest that leaf phosphate status has dual effects on the amount of SPS protein and the activation state of SPS.