Maize Sucrose-phosphate Synthase Research Articles

Expression of a truncated maize SPS increases source capacity in maize

AbstractIn an attempt to increase source capacity, transgenic corn was generated by expressing a truncated maize sucrose phosphate synthase (ZmSPSΔ482) under two leaf mesophyll cellspecific promoters (CAB and PPDK). The endogenous and truncated SPS proteins from transgenic leaf extracts were distinguishable by protein immunoblot analysis. The expression of transgenic SPS protein across events varied from very low to very high and included several cosuppressed events. SPS activity showed a diurnal pattern in both transgenic and wild-type maize leaves. In greenhouse experiments, transgenic maize had higher leaf sucrose and lower leaf starch, suggesting a shift in carbon partitioning from starch to sucrose. Conversely, cosuppressed events had lower leaf sucrose and higher leaf starch. A field test was performed to compare sucrose and starch in positive and negative isolines of hybrid maize CAB and PPDK ZmSPSΔ482 events. In the field, many positive isolines had higher levels of both leaf sucrose and starch than the negative isolines. This suggests that in the field, with higher light intensity the shift in carbon partitioning from starch to sucrose, observed under greenhouse conditions did not occur. This in turn suggests that the environment affects the phenotype of the transgenics and that in the field, there was an overall increase in carbon assimilation. Six events from each construct were tested in a pilot multi-density yield trial but overall, no effect on yield was observed. Therefore, although the transgenic plants had more source capacity, this did not translate into higher seed yield.

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.

Overexpression of a Maize SPS Gene Improves Yield Characters of Potato under Field Conditions

We analyzed the yield characters of field-grown transgenic potato plants (Solanum tuberosum) carrying a maize gene for sucrose-phosphate synthase (SPS), the key enzyme in sucrose synthesis. The SPS activity in the leaves of transgenic plants (line Ag1203) was 2 times that of the control (cv. May Queen). There was no difference in the photosynthetic CO2 uptake rates between Ag1203 and May Queen plants, and the leaf starch content of Ag1203 was lower. These observations indicate that the introduction of a foreign SPS gene improved the supply of photosynthate from source (leaves) to sink (tubers). Additionally, leaf senescence of the transgenic potato plants was delayed relative to that of May Queen. The average tuber weight and total yield of Ag1203 plants were at least 20% higher, and the tuber sucrose content, which is related to eating quality, was also higher. Increased translocation of photosynthate and longer period of photosynthetic activity in the leaves may have increased the yield of Ag1203. These results suggest that introduction of the SPS gene improved the yield characters and quality of potato tubers under field conditions.

Phylogenetic and expression analysis of sucrose phosphate synthase isozymes in plants

In plants and microbes, sucrose phosphate synthase (SPS) is an important enzyme in sucrose biosynthesis. Several different isozymes of SPS exist in plants. Genomic and EST sequence data from Arabidopsis, rice and maize has been analyzed. This analysis has revealed that the Arabidopsis genome contains four unique SPS genes. The rice databases (Monsanto proprietary, and public databases) contain five unique full-length SPS genes. Using the Monsanto maize EST and genomic sequence databases, we have identified five full length and two partial SPS sequences, bringing the total number of presently known maize SPS genes to at least seven. Phylogenetic analysis of all known SPS sequences revealed several putative evolutionary branches of SPS. We have classified SPS genes into three major groups in higher plants, all with distinct features from the known microbial SPS genes. Furthermore, this analysis suggests evolutionary divergence of monocotyledonous (monocot) and dicotyledonous (dicot) SPS sequences. The evidence suggests that several gene duplication events occurred at various points during evolution, both before and after the monocot/dicot split. It appears that at least one of the major forms of SPS genes may have evolved after the divergence of monocots and dicots. In addition, several more recent gene duplication events may have occurred after maize/rice speciation, giving rise to additional SPS genes in maize. Some of the variants lack one or more of the presently known regulatory sites, implying that this evolutionary divergence may have given rise to enzymes with functional differences. We present evidence from transcript distribution studies using cDNA libraries as well as transcriptional profiling experiments and propose that specific SPS genes have diverse patterns of expression that are sometimes responsive to environmental signals. Our data suggests that higher plant SPS isozymes differ with respect to their patterns of expression and regulation and that our proposed phylogenetic classification reflects specific functional categories for higher plant SPS isozymes.

Elevated sucrose-phosphate synthase activity in transgenic tobacco sustains photosynthesis in older leaves and alters development.

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.

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.

Changes in Photosynthetic Activity and Export of Carbon by Overexpressing a Maize Sucrose-Phosphate Synthase Gene under Elevated C02 in Transgenic Rice

To investigate whether increased sucrose-phosphate synthase (SPS) activity alters photosynthetic activity and/or the export of carbon from leaves under elevated C02 partial pressure ([C02]), we raised two lines of transgenic rice (H54-9 and H69-7), each overexpressing a maize SPS gene, and wild-type rice under ambient [C02] (35 Pa) and elevated [0O2] (100 Pa). Under ambient [C02], no significant difference was observed between the transgenic and wild-type plants in the levels of sucrose or starch in leaves or the photosynthetic activity; but the carbon export rate was higher in H69-7 than in the wild-type. Under elevated [C02], photosynthetic activity increased in all plants, but the accumulation of starch was significantly repressed in H54-9, whose SPS activity was about 12.5 times higher than that of the wild-type. The carbon export rate was higher in both transgenic lines than the wild-type. We considered that increased SPS activity in rice plants would promote the export of carbon from leaves and, as a result, starch accumulation in the leaves would be suppressed and/or photosynthetic activity would be promoted under elevated [CO2].

Promoter strength and tissue specificity effects on growth of tomato plants transformed with maize sucrose-phosphate synthase

When sucrose-phosphate synthase (SPS; EC 2.4.1.14) is expressed in tomato (Lycopersicon esculentum Mill.) from a ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) small subunit (rbcS) promoter, yields are often unchanged but when SPS is expressed from a Cauliflower Mosaic Virus 35S promoter, yield is enhanced up to 80%. Two explanations for this phenomenon are (i) that expression of SPS in tissues other than leaves accounts for the increased yield or (ii) that the lower level of expression directed by the 35S promoter is more beneficial than the high level of expression directed by the rbcS promoter. To test the first hypothesis, we conducted a reciprocal graft experiment, which showed that root SPS activity did not substantially affect growth. To test the second hypothesis, we conducted a field trial using a backcrossed, segregating, population of SPS-transformed plants derived from 35S and rbcS lines. The optimal dose of SPS activity for growth was approximately twice that of the wild type regardless of which promoter was used. The effect of SPS on growth was the result of a shift in partitioning of carbon among starch, sucrose, and ionic compounds (primarily amino acids), rather than of an increase in net photosynthesis. Excessive SPS activity resulted in a decreased rate of amino acid synthesis, which could explain the non-linear response of plant growth to the level of SPS expression.

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.

Effects of elevated sucrose-phosphate synthase activity on photosynthesis, carbohydrate partitioning and growth rate in hybrid aspen (Populus tremula x P. tremuloides)

Starch and sucrose are synthesised from CO2 assimilated during photosynthesis. In rapidly growing plants, sucrose is the major end product of photosynthesis and the principal form of carbohydrate translocated throughout the plant. One of the key points for the regulation of the sucrose synthesis pathway is the reaction catalysed by sucrose-phosphate synthase (SPS). This enzyme occupies a strategic site downstream of the point where the pathways for sucrose synthesis and starch mobilisation converge. In herbaceous plant leaves, an increase in SPS activity often correlates with changes in the rate of sucrose synthesis and export. The consequences of this increased SPS expression on the sugar phloem transport and the sugar flows between source and sink tissues have not been investigated because of the morphological difficulties encountered with herbaceous plants. To investigate this further, hybrid aspen were transformed with the gene encoding for the maize SPS. Preliminary results show an increase in total leaf carbohydrates and surprisingly leaf starch content. The increase observed in SPS activity is correlated with a significant increase in photosynthetic activity, but no significant changes in sugar leaf partitioning. Diurnal carbohydrate content of leaves of transgenic trees is also modified, soluble sugar (glucose, fructose and sucrose) largely increase during the first part of the light period compared to wild type. Moreover, this sugar accumulation appears to improve the growth rate of the trees. Thus, genetic modification of SPS expression in trees can be used to modify traits important for the forest and paper industries.

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.

Overexpression of sucrose-phosphate synthase in tomato plants grown with CO 2 enrichment leads to decreased foliar carbohydrate accumulation relative to untransformed controls

Tomato plants expressing the maize sucrose-phosphate synthase (SPS) cDNA under the control of the promoterof the small subunit of ribulose-1,5-bisphosphate carboxylase oxygenase ( rbcS) promoter were grown 5 weeks in air (450 μmol.m –2.s –1 irradiance, 350 ppm CO 2) and then either maintained in air or exposed to CO 2 enrichment (1 000 ppm CO 2) for 8 d. A linear relationship between the foliar sucrose to starch ratio and maximal extractable SPS activity was found both in air and high CO 2. Starch accumulation was dramatically increased in all plants subjected to CO 2 enrichment but the CO 2-dependent increase in foliar starch accumulation was much lower in the leaves of the SPS transformants than in those of the untransformed controls in the same conditions. Maximal extractable ribulose-1,5-bisphosphate carboxylase/oxygenase activity was reduced by growth at high CO 2 to a similar extent in both plant types. The carbon/nitrogen ratios were similar in both plant lines in both growth conditions after 20 d exposure to high CO 2. A small (5 %) increase in carbon export capacity was observed at high CO 2 in the leaves of transformed plants compared to leaves from untransformed controls. Increased foliar SPS activity did not, however, prevent acclimation of photosynthesis in plants grown with long-term CO 2 enrichment.

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.

Over-expression of sucrose phosphate synthase in Arabidopsis thaliana results in increased foliar sucrose/starch ratios and favours decreased foliar carbohydrate accumulation in plants after prolonged growth with CO2 enrichment

Arabidopsis thaliana ecotype Columbia was transformed with a maize sucrose phosphate synthase (SPS) cDNA under the control of the promoter for the small subunit of ribulose-1 ,5-bisphosphate carboxylase from tobacco (rbcS). The effects of SPS over-expression were compared in plants of the T 2 and T 3 generations grown either in air or with CO 2 enrichment (700 μl l -1 ) for either 4 or 10 weeks. Maximal extractable foliar SPS activities were three times those of the untransformed controls in the highest rbcS-SPS expressing line. In untransformed Arabidopsis leaves SPS activity was not subject to light/dark regulation, but was modified by incubation with either the inhibitor, orthophosphate, or the activator, mannose. Photosynthesis (A max ) values were similar in all lines grown in air. After 10 weeks of CO 2 enrichment a decrease in A max in the untransformed controls, but not in the high SPS expressors, was observed. There was a strong correlation between the sucrose-to- starch ratio of the leaves and their SPS activity in both growth conditions. The total foliar carbohydrate contents of 4-week-old plants was similar in all lines whether plants were grown in air or with CO 2 enrichment. After 10 weeks growth the leaves of the high rbcS-SPS expressors accumulated much less total carbohydrate than untransformed control leaves in both growth conditions. It was concluded that SPS over-expression causes increased foliar sucrose/starch ratios in Arabidopsis leaves and favours decreased foliar carbohydrate contents when plants are grown for long periods with CO 2 enrichment.

Sucrose-phosphate synthase activity and yield analysis of tomato plants transformed with maize sucrose-phosphate synthase

Sucrose synthesis is a major element of the interactions between photosynthesis and plant growth and development. Tomato (Lycopersicon esculentum Mill. cv. UC82B) plants transformed with maize sucrose-phosphate synthase (SPS; EC 2.3.1.14) expressed from either a ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) small subunit promoter (SSU) or the cauliflower mosaic virus 35S promoter (35S) were used to study effects of increased sucrose synthesis rates on plant growth. The plants were grown in growth chambers, field plots, and open-top chambers. The 35S plants had a 2 to 3-fold increase in young-leaf SPS activity, a 10 to 20-fold increase in young-root SPS activity and no increase in young-fruit SPS activity. The leaf SPS activity in one of the 35S lines fell to control levels by two months of age. The SSU plants had a 4 to 5-fold increase in leaf SPS activity and no significant increase in root or young-fruit SPS activity. One 35S line, which maintained high leaf SPS activity throughout development, yielded 70–80% more than controls at both normal and elevated CO2 in open-top chambers in the field and 20–30% more than controls in two additional field trials. The other 35S line and the two SSU lines either yielded less or did not differ from controls under several growth conditions. Since only one of four transformed lines showed an increase in yield, we can not yet conclude that increased leaf SPS activity leads to increased yield. However, increased leaf SPS activity appears to result in increased fruit sugar content since all three lines with increased leaf SPS usually also had increased fruit sugars.

Characterization of a rice sucrose-phosphate synthase-encoding gene

A rice genomic clone ( spsl) coding for sucrose phosphate synthase (SPS) was isolated and sequenced. Rice spsl contains 13 exons and 12 introns, an unusually long 366-bp leader region with a highly organized primary structure and a promoter region with no obvious homology with eukaryotic promoter consensus sequences. Southern blot analysis showed that SPS is encoded by a single-copy gene in the rice genome. Comparison of the rice, maize, potato and spinach SPS deduced amino acid (aa) sequences showed that these enzymes have a well conserved region comprising their first 700 aa, and a variable C-terminal region. Analysis of rice spsl expression showed that mRNA levels change during leaf development. SPS activity and mRNA were undetectable in roots.

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

Abstract Photosynthetic carbon assimilation, carbon partitioning and foliar carbon budgets were measured in the leaves of transformed tomato plants expressing a maize sucrose-phosphate synthase (SPS) gene in addition to the native enzyme, and in untransformed controls. 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 throughout 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 (180μmol photon m−2 s−1) was similar in all plants. Net photosynthesis measured in air and at elevated CO2 (800–1500 μl l−1) on whole plants grown in air at 400μmol 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 CO2 the stimulation of photosynthesis was more pronounced. We conclude that SPS activity is a major point of control of photosynthesis particularly under saturating light and CO2.

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.

Expression of a Maize Sucrose Phosphate Synthase in Tomato Alters Leaf Carbohydrate Partitioning

Expression of a Maize Sucrose Phosphate Synthase in Tomato Alters Leaf Carbohydrate Partitioning