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

Tobacco 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 K_m 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.     

In vitro and in vivo inhibition of sucrose synthesis by sucrose

The inhibitory effects of sucrose on rates of sucrose synthesis by sucrose phosphate synthase (SPS) from the maize scutellum and on net rates of sucrose production in maize scutellum slices from added glucose or fructose were studied. Scutellum extracts were prepared by freezing and thawing scutellum slices in buffer. The extracts contained SPS and sucrose phosphate phosphatase, but were free of sucrose synthase. SPS activity was calculated from measurement of UDP formation in the presence of UDPG, fructose-6-P and sucrose. The ranges of metabolite concentrations used were those estimated to be in scutellum slices after incubation in water or fructose for periods up to 5 hr. UDPG and fructose-6-P also were added at concentrations that saturated SPS. At saturating substrate levels, sucrose inhibition of SPS was less than that when tissue levels of substrates were used. With tissue levels of substrates and sucrose concentrations up to ca 166 mM, sucrose inhibitions of sucrose synthesis in vitro by SPS were similar to those observed in vivo. However, as the sucrose concentration rose above 166 mM, SPS activity was not inhibited further, whereas there was a further sharp decline in sucrose production by the slices. It is concluded that sucrose synthesis in vivo is controlled by sucrose inhibition of SPS over a considerable range of internal sucrose concentrations.     

Characterization of diurnal changes in activities of enzymes involved in sucrose biosynthesis

Experiments were conducted with vegetative soybean plants (Glycine max [L.] Merr., `Ransom') to determine whether the activities in leaf extracts of key enzymes in sucrose metabolism changed during the daily light/dark cycle. The activity of sucrose-phosphate synthase (SPS) exhibited a distinct diurnal rhythm, whereas the activities of UDP-glucose pyrophosphorylase, cytoplasmic fructose-1,6-bisphosphatase, and sucrose synthase did not. The changes in extractable SPS activity were not related directly to photosynthetic rates or light/dark changes. Hence, it was postulated that the oscillations were under the control of an endogenous clock. During the light period, the activity of SPS was similar to the estimated rate of sucrose formation. In the dark, however, SPS activity declined sharply and then increased even though degradation of starch was linear. The activity of SPS always exceeded the estimated maximum rate of sucrose formation in the dark. Transfer of plants into light during the normal dark period (when SPS activity was low) resulted in increased partitioning of photosynthate into starch compared to partitioning observed during the normal light period. These results were consistent with the hypothesis that SPS activity in situ was a factor regulating the rate of sucrose synthesis and partitioning of fixed carbon between starch and sucrose in the light.     

Identification of factors regulating the phosphorylation status of sucrose-phosphate synthase in vivo

The purpose of this study was to identify the factors that control sucrose-phosphate synthase (SPS)-kinase and SPS-protein phosphatase (SPS-PP) activity in situ, and thereby mediate the activation of SPS by light or mannose. Feeding mannose to excised spinach (Spinacia oleracea) leaves in darkness resulted in a general sequestration of cellular phosphate (as evidenced by accumulation of mannose-6-P and depletion of glucose-6-P [Glc-6-P] and fructose-6-P [Fru-6-P]) and a relatively slow activation of SPS (maximum activation achieved within 90 min). Supplying exogenous inorganic phosphate (Pi) with mannose reduced sequestration of cellular Pi (as evidenced by mannose-6-P accumulation without depletion of hexose-P) and substantially reduced mannose activation of SPS. Thus, depletion of cytoplasmic Pi may be required for SPS activation; accumulation of mannose-6-P alone is clearly not sufficient. It was verified that Glc-6-P, but not mannose-6-P, was an inhibitor of partially purified SPS-kinase, and that Pi was an inhibitor of partially purified SPS-PP. Total extractable activity of SPS-kinase did not vary diurnally, whereas a pronounced light activation of SPS-PP activity was observed. Pretreatment of leaves in the dark with cycloheximide blocked the light activation of SPS-PP (assayed in vitro) and dramatically reduced the rate of SPS activation in situ (in saturating light and carbon dioxide). We conclude that rapid activation of SPS by light involves reduction in cytosolic Pi, an inhibitor of SPS-PP, and light activation of SPS-PP, by a novel mechanism that may involve (directly or indirectly) a protein synthesis step. An increase in cytosolic Glc-6-P, an inhibitor of SPS-kinase, would also favor SPS activation. Thus, the signal transduction pathway mediating the light activation of SPS involves elements of “fine” and “coarse” control.     

Biochemical Basis for Partitioning of Photosynthetically Fixed Carbon between Starch and Sucrose in Soybean (Glycine max Merr.) Leaves

The control of photosynthetic starch/sucrose formation in leaves of soybean (Glycine max L. Merr.) cultivars was studied in relation to stage of plant development, photosynthetic photoperiod, and nitrogen source. At each sampling, leaf tissue was analyzed for starch content, activities of sucrose-metabolizing enzymes, and labeling of starch and sucrose (by ¹⁴CO₂ assimilation) in isolated cells. In three of the four varieties tested, nodulated plants had lower leaf starch levels and higher activities of sucrose phosphate synthetase (SPS), and isolated mesophyll cells incorporated more carbon (percentage of total ¹⁴CO₂ fixed) into sucrose and less into starch as compared to nonnodulated (nitrate-dependent) plants. The variation among cultivars and nitrogen treatments observed in the activity of SPS in leaf extracts was positively correlated with labeling of sucrose in isolated cells (r = 0.81) and negatively correlated with whole leaf starch content (r = −0.66). The results suggested that increased demand for assimilates by nodulated roots may be accommodated by greater partitioning of carbon into sucrose in the mesophyll cells. We have also confirmed the earlier report (Chatterton, Silvius 1979 Plant Physiol 64: 749-753) that photoperiod affects partitioning of fixed carbon into starch. Within two days of transfer of nodulated soybean Ransom plants from a 14-hour to a 7-hour photoperiod, leaf starch accumulation rates doubled, and this effect was associated with increased labeling of starch and decreased labeling of sucrose in isolated cells. Concurrently, activities of SPS, sucrose synthase, and uridine diphosphatase in leaves were decreased.     

Light Modulation and Localization of Sucrose Phosphate Synthase Activity between Mesophyll Cells and Bundle Sheath Cells in C(4) Species

Experiments were conducted with several Panicum species (representing the different C₄ subtypes) to examine the light modulation of sucrose phosphate synthase (SPS) activity and the effect of illumination on the distribution of SPS activity between mesophyll cells (MC) and bundle sheath cells (BSC). Activity of SPS in the light decreased in the order: C₄ > C₃-C₄ intermediate > C₃. In illuminated leaves, SPS activities were similar among the three C₄ subtypes, but SPS activity was higher for NAD-malic enzyme (NAD-ME) species with centripetal chloroplasts in BSC (NAD-ME(P) species) than for NAD-ME species with centrifugal chloroplasts in BSC (NAD-ME(F) species). Transfer of plants into darkness for 30 minutes resulted in decreased SPS activity for all species tested except Panicum bisulcatum (C₃ species) and Panicum virgatum (NAD-ME(P) species) which showed little or no change. All C₄ subtypes had some SPS activity both in MC and BSC. In the light, SPS activity was mainly in the MC for NADP-ME, NAD-ME(F) and phosphoenolpyruvate carboxykinase species, while it was mainly in the BSC for NAD-ME(P) species. In the dark, for all C₄ subtypes, SPS activity in the MC was decreased to a greater extent than that in the BSC. It is intriguing that NAD-ME(F) and NAD-ME(P) species differed in the activity and distribution of SPS activity between MC and BSC, although they are otherwise identical in the photosynthetic carbon assimilation pathway. Diurnal changes in SPS activity in the MC and BSC were also examined in maize leaves. SPS activity in the MC in maize leaves was high and relatively constant throughout the middle of the light period, dropped rapidly after sunset and increased again prior to the light period. On the other hand, SPS activity in the BSC was lower and changed more coincidently with light intensity than that in the MC. The results suggested that light activation of SPS activity located in the BSC may require higher irradiance for saturation than the SPS in the MC. We conclude that SPS may function in both MC and BSC for sucrose synthesis in the light, particularly at high light intensity, while in the dark, the major function may be in the BSC during starch degradation.     

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

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

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.     

Possible control of maize leaf sucrose-phosphate synthase activity by light modulation

Sucrose phosphate synthase (SPS) activity was measured in extracts of maize (Zea mays L.) and soybean (Glycine max L. [Merr.]) leaves over a single day/night cycle. There was a 2- to 3-fold postillumination increase in extractable enzyme activity in maize leaves, whereas the activity of soybean SPS was only about 30% higher in extracts prepared from light- compared to dark-adapted leaves. Alterations in extractable maize leaf SPS activity correlated with light/dark transitions suggesting that the enzyme may be light modulated. Diurnal variations of extractable maize leaf SPS activity were also observed in a greenhouse experiment. A transition from high (light) to low (dark) extractable SPS activity occurred near the light compensation point for photosynthesis (about 20 micromole photons per square meter per second). Further increases in irradiance did not increase extractable SPS activity. Substrate affinities for uridine 5′-diphosphoglucose (Michaelis constant = 3.5 and 5.1 millimolar) and fructose-6 phosphate (half maximal concentration = 1.0 and 2.5 millimolar) were lower for partially purified SPS obtained from light compared to dark acclimated maize leaves. Light-induced changes in extractable SPS activity were stable for at least one column chromatography step. The above results indicate that light-induced changes in SPS activity may be important in controlling the photosynthetic production of sucrose.     

Effect of Light and Chilling Temperatures on Chilling-sensitive and Chilling-resistant Plants. Pretreatment of Cucumber and Spinach Thylakoids in Vivo and in Vitro

The effects of chilling temperatures, in light or dark, on the isolated thylakoids and leaf discs of cucumber (Cucumis sativa L. “Marketer”) and spinach (Spinacia oleracea L. “Bloomsdale”) were studied. The pretreatment of isolated thylakoids and leaf discs at 4 C in the dark did not affect the phenazine methosulfate-dependent phosphorylation, proton uptake, osmotic response to sucrose, Ca²⁺-dependent ATPase activity, or chlorophyll content. Exposure of cucumber cotyledon discs and isolated thylakoids of cucumber and spinach to 4 C in light resulted in a rapid inactivation of the thylakoids. The sequence of activities or components lost during inactivation (starting with the most sensitive) are: phenazine methosulfate-dependent cyclic phosphorylation, proton uptake, osmotic response to sucrose, Ca²⁺-dependent ATPase activity, and chlorophyll. The rate of loss of proton uptake, osmotic response to sucrose, Ca²⁺-dependent ATPase activity and chlorophyll is similar for isolated cucumber and spinach thylakoids, whereas spinach thylakoids are more resistant to the loss of phenazine methosulfate-dependent phosphorylation. The thylakoids of spinach leaf discs were unaffected by exposure to 4 C in light. The results question whether the extreme resistance of spinach thylakoids treated in vivo is solely a function of the chloroplast thylakoid membranes and establish the validity of using in vitro results to make inferences about cucumber thylakoids treated in vivo at 4 C in light.     

Nitrate activation of cytosolic protein kinases diverts photosynthetic carbon from sucrose to amino Acid biosynthesis: basis for a new concept

The regulation of carbon partitioning between carbohydrates (principally sucrose) and amino acids has been only poorly characterized in higher plants. The hypothesis that the pathway of sucrose and amino acid biosynthesis compete for carbon skeletons and energy is widely accepted. In this review, we suggest a mechanism involving the regulation of cytosolic protein kinases whereby the flow of carbon is regulated at the level of partitioning between the pathways of carbohydrate and nitrogen metabolism via the covalent modulation of component enzymes. The addition of nitrate to wheat seedlings (Triticum aestivum) grown in the absence of exogenous nitrogen has a dramatic, if transient, impact on sucrose formation and on the activities of sucrose phosphate synthase (which is inactivated) and phosphoenolpyruvate carboxylase (which is activated). The activities of these two enzymes are modulated by protein phosphorylation in response to the addition of nitrate, but they respond in an inverse fashion. Sucrose phosphate synthase in inactivated and phosphoenolpyruvate carboxylase is activated. Nitrate functions as a signal metabolite activating the cytosolic protein kinase, thereby modulating the activities of at least two of the key enzymes in assimilate partitioning and redirecting the flow of carbon away from sucrose biosynthesis toward amino acid synthesis.     

Effects of Elevated Sucrose-Phosphate Synthase Activity on Photosynthesis,Assimilate Partitioning,and Growth in Tomato (Lycopersicon esculentum var UC82B)

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.     

Regulation of key enzymes of sucrose biosynthesis in soybean leaves : effect of dark and light conditions and role of gibberellins and abscisic Acid

An important part in the understanding of the regulation of carbon partitioning within the leaf is to investigate the endogenous variations of parameters related to carbon metabolism. This study of diurnal changes in the activities of sucrose-synthesizing enzymes and levels of nonstructural carbohydrates in intact leaves of field-grown soybean plants (Glycine max [L.]) showed pronounced diurnal fluctuations in sucrose phosphate synthase (SPS) activity. However, there was no distinct diurnal change in the activity of fructose-1,6-bisphosphatase (F1,6BPase). SPS activity in leaves from plants grown in controlled environments presented two peaks during the light period. In contrast to field-grown plants, F1,6BPase activity in leaves from growth chamber-grown plants manifested one peak during the first half of the light period. In plants grown under both conditions, sucrose and starch accumulation rates were highest during early hours of the light period. By the end of the dark period, most of the starch was depleted. A pattern of diurnal fluctuations of abscisic acid (ABA) levels in leaves was also observed under all growing conditions. Either imposition of water stress or exogenous applications of ABA inhibited F1,6BPase activity. However, SPS-extractable activity increased following water deficit but did not change in response to ABA treatment. Gibberellin application to intact soybean leaves increased levels of both starch and sucrose. Both gibberellic acid (10⁻⁶m) and gibberellins 4 and 7 (10⁻⁵m) increased the activity of SPS but had an inconsistent effect on F1,6BPase. Correlation studies between the activities of SPS and F1,6BPase suggest that these two enzymes are coordinated in their function, but the factors that regulate them may be distinct because they respond differently to certain environmental and physiological changes.     

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

Photosynthetic carbon metabolism was investigated in antisense Arabidopsis lines with decreased expression of sucrose phosphate synthase (SPS) and cytosolic fructose-1,6-bisphosphatase (cFBPase). In the light, triose phosphates are exported from the chloroplast and converted to sucrose via cFBPase and SPS. At night, starch is degraded to glucose, exported and converted to sucrose via SPS. cFBPase therefore lies upstream and SPS downstream of the point at which the pathways for sucrose synthesis in the day and night converge. Decreased cFBPase expression led to inhibition of sucrose synthesis; accumulation of phosphorylated intermediates; Pi-limitation of photosynthesis; and stimulation of starch synthesis. The starch was degraded to maintain higher levels of sugars and a higher rate of sucrose export during the night. This resembles the response in other species when expression of enzymes in the upper part of the sucrose biosynthesis pathway is reduced. Decreased expression of SPS inhibited sucrose synthesis, but phosphorylated intermediates did not accumulate and carbon partitioning was not redirected towards starch. Sugar levels and sucrose export was decreased during the night as well as during the day. Although ribulose-1,5-bisphosphate regeneration and photosynthesis were inhibited, the PGA/triose-P ratio remained low and the ATP/ADP ratio high, showing that photosynthesis was not limited by the rate at which Pi was recycled during end-product synthesis. Two novel responses counteracted the decrease in SPS expression and explain why phosphorylated intermediates did not accumulate, and why allocation was not altered in the antisense SPS lines. Firstly, a threefold decrease of PPi and a shift of the UDP-glucose/hexose phosphate ratio favoured sucrose synthesis and prevented the accumulation of phosphorylated intermediates. Secondly, there was no increase of AGPase activity relative to cFBPase activity, which would prevent a shift in carbon allocation towards starch synthesis. These responses are presumably triggered when sucrose synthesis is decreased in the night, as well as by day.     

Glucose control of sucrose synthase in the maize scutellum

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

Carbon Partitioning in Eelgrass (Regulation by Photosynthesis and the Response to Daily Light-Dark Cycles)

Diel variations in rates of C export, sucrose-phosphate synthase (SPS) and sucrose synthase (SS) activity, and C reserves were investigated in Zostera marina L. (eelgrass) to elucidate the environmental regulation of sucrose formation and partitioning in this ecologically important species. Rates of C flux and SPS activity increased with leaf age, consistent with the ontogenic transition from sink to source status. Rates of C export and photosynthesis were low but quantitatively consistent with those of many terrestrial plant species. The Vmax activity of SPS approached that of maize, but substrate-limited rates were 20 to 25% of Vmax, indicating a large pool of inactive SPS. SPS was unresponsive to the day/night transition or to a 3-fold increase in photosynthesis generated by high [CO2] and showed little sensitivity to inorganic phosphate. Consequently, regulation of eelgrass SPS appeared similar to starch- rather than to sugar-accumulating species even though eelgrass accumulates sucrose. Leaf [sucrose] was constant and high throughout the diel cycle, which may contribute to the down-regulation of SPS. Root sucrose synthase activity was high but showed no response to nocturnal anoxia. Root [sucrose] also showed no diel cycle. The temporal stability of [sucrose] confers an ability for eelgrass to buffer the effects of prolonged light limitation that may be key to its survival and ecological success in environments subject to periods of extreme light limitation and chaotic daily variation in light availability.     

Studies on Genetic Male-Sterile Soybeans : II. Effect of Nodulation on Photosynthesis and Carbon Partitioning in Leaves

Soybean (Glycine max L. Merr.) germplasm, essentially isogenic except for loci controlling male sterility (ms₁) and nodulation (rj₁), were developed to study the effects of reproductive development and nitrogen source on certain aspects of photosynthesis. Plants were sampled from flowering (77 days after transplanting) until maturity (150 days after transplanting). With all four genotypes, net carbon exchange rates were highest at flowering and declined thereafter. Photosynthetic rates of the sterile genotypes (nodulated and non-nodulated) declined more rapidly than the fertile genotypes, and after 105 days, both sterile genotypes maintained low but relatively constant carbon exchange rates (<3 milligrams CO₂/gram fresh weight per hour). Photosynthetic rates and starch accumulation (difference between afternoon and morning levels) declined with time. The sterile genotypes attained the highest morning starch levels, which reflected reduced starch mobilization. After 92 days, the proportion of photosynthetically fixed carbon that was partitioning into starch (relative leaf starch accumulation) in the sterile genotypes increased dramatically. In contrast, relative leaf starch accumulation in the fertile genotypes remained relatively constant with time. Throughout the test period, all four genotypes maintained leaf sucrose levels between 5 and 15 micromoles glucose equivalents per gram fresh weight.

The activities of sucrose phosphate synthase (SPS) in leaf extracts of the four genotypes declined from 77 to 147 days. Nodulated genotypes tended to maintain higher activities (leaf fresh weight basis) than did the non-nodulated genotypes. In general, relative leaf starch accumulation was correlated negatively with the activity of SPS (normalized with leaf net carbon exchange rate) in leaf extracts for all four genotypes during early reproductive development, and for the fertile genotypes at all sampling dates. In contrast, leaf sucrose content was correlated positively with SPS activity during early reproductive development. These results suggested that a direct relation existed between the activity of SPS and starch/sucrose levels in soybean leaves. However, the interaction between these processes also may be influenced by other factors, particularly when leaf photosynthetic rates and plant demand for assimilates is low, as in the sterile genotypes.

     

Transgenic cotton over-producing spinach sucrose phosphate synthase showed enhanced leaf sucrose synthesis and improved fiber quality under controlled environmental conditions

Prior data indicated that enhanced availability of sucrose, a major product of photosynthesis in source leaves and the carbon source for secondary wall cellulose synthesis in fiber sinks, might improve fiber quality under abiotic stress conditions. To test this hypothesis, a family of transgenic cotton plants (Gossypium hirsutum cv. Coker 312 elite) was produced that over-expressed spinach sucrose-phosphate synthase (SPS) because of its role in regulation of sucrose synthesis in photosynthetic and heterotrophic tissues. A family of 12 independent transgenic lines was characterized in terms of foreign gene insertion, expression of spinach SPS, production of spinach SPS protein, and development of enhanced extractable V _max SPS activity in leaf and fiber. Lines with the highest V _max SPS activity were further characterized in terms of carbon partitioning and fiber quality compared to wild-type and transgenic null controls. Leaves of transgenic SPS over-expressing lines showed higher sucrose:starch ratio and partitioning of ¹⁴C to sucrose in preference to starch. In two growth chamber experiments with cool nights, ambient CO₂ concentration, and limited light below the canopy, the transgenic line with the highest SPS activity in leaf and fiber had higher fiber micronaire and maturity ratio associated with greater thickness of the cellulosic secondary wall.