Gas-exchange analysis of chloroplastic fructose-1,6-bisphosphatase antisense potatoes at different air humidities and at elevated CO2

Gas-exchange measurements were performed to analyze the leaf conductances and assimilation rates of potato (Solanum tuberosum L. cv. Desireé) plants expressing an antisense construct against chloroplastic fructose-1,6-bisphosphatase (FBPase, EC 3.1.3.11) in response to increasing photon flux densities, different relative air humidities and elevated CO₂ concentrations. Assimilation rates (A) and transpiration rates (E) were observed during a stepwise increase of photon flux density. These experiments were carried out under atmospheric conditions and in air containing 500 μmol mol⁻¹ CO₂. In both gas atmospheres, two levels of relative air humidity (60–70% and 70–80%) were applied in different sets of measurements. Intercellular CO₂ concentration, leaf conductance, air-to-leaf vapour pressure deficit, and instantaneous water-use efficiency (A/E) were determined. As expected, assimilation rates of the FBPase antisense plants were significantly reduced as compared to the wild type. Saturation of assimilation rates in transgenic plants occurred at a photon flux density of 200 μmol m⁻² s⁻¹, whereas saturation in wild type plants was observed at 600 μmol m⁻² s⁻¹. Elevated ambient CO₂ levels did not effect assimilation rates of transgenic plants. At 70–80% relative humidity and atmospheric CO₂ concentration the FBPase antisense plants had significantly higher leaf conductances than wild-type plants while no difference emerged at 60–70%. These differences in leaf conductance vanished at elevated levels of ambient CO₂. Stomatal response to different relative air humidities was not affected by mesophyll photosynthetic activity. It is suggested that the regulation of stomatal opening upon changes in photon flux density is merely mediated by a signal transmitted from mesophyll cells, whereas the intercellular CO₂ concentration plays a minor role in this kind of stomatal response. The results are discussed with respect to stomatal control by environmental parameters and mesophyll photosynthesis. Received: 24 September 1998 / Accepted: 9 February 1999     

Dark chilling increases glucose-6-phosphate dehydrogenase activity in soybean leaves

Available evidence suggests that the stress‐induced increase in the activity of glucose‐6‐phosphate dehydrogenase (G6PDH, EC 1.1.1.49), the key regulatory enzyme of the oxidative pentose phosphate pathway, might often be related to the presence of plant water deficit. The response of G6PDH to dark chilling in chilling sensitive plant species is still unknown. In this communication we report on this response and its dependence on the presence of chill‐induced drought stress. A chilling sensitive soybean (Glycine max L. Merr.) genotype was exposed to dark chilling of the entire plant (whole‐chilled) or only the shoots and leaves (shoot‐chilled). The development of chill‐induced drought stress upon illumination was quantified by measurement of proline and relative water content (RWC). Chill‐induced drought stress (decrease in RWC and increase in proline content) developed with time in whole‐chilled plants, but not in shoot‐chilled plants. The response of the above‐mentioned treatments on G6PDH activity in fully expanded leaves was assessed. In parallel, the effects on CO₂ assimilation, PSII activity and chloroplast fructose‐1,6‐bisphosphatase (FBPase EC 3.1.3.11) and ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco EC 4.1.1.39) activity were quantified. A decrease in CO₂ assimilation rate, FBPase activity and ribulose‐1,5‐bisphosphate (RuBP) content was observed in whole‐chilled but not in shoot‐chilled plants. However, in shoot‐chilled plants regulation of diurnal PSII activity was altered. The increase in the activation state of NADP‐dependent malate dehydrogenase (NADP‐MDH EC 1.1.1.82) in shoot‐chilled plants suggests an increase in stromal redox state. Although the two different dark chilling treatments resulted in distinct physiological and biochemical effects, both induced an increase in foliar G6PDH activity, suggesting an important role of this enzyme during and following dark chilling stress, irrespective of the presence of chill‐induced drought stress.     

Reduction of the chloroplastic fructose-1,6-bisphosphatase in transgenic potato plants impairs photosynthesis and plant growth

Transgenic potato plants expressing reduced levels of the chloroplastic isoform of fructose-1,6-bisphosphatase (cp-FBPase) were created via the antisense RNA technique. Transformants with different levels of FBPase activity were selected and analysed with respect to photosynthesis, carbon metabolism, and growth. FBPase activity of less than 15% of wild-type levels led to reduced growth rates, probably due to the reduction of photosynthetic activity. A significant decrease in tuber yield is observed in plants with a FBPase activity below 15% of wild-type levels, whereas plants with 36% of wild-type enzyme activity still give normal tuber yields, even though they demonstrate a lowered photosynthetic capacity. Decreased photosynthesis also results in a reduction of total carbohydrate contents in leaves. Interestingly, increased carbohydrate partitioning towards soluble sugars is observed in plants displaying less than 15% of the wild-type FBPase activity. When excised leaf discs are placed on sucrose-containing media in darkness, discs derived from plants with a reduced FBPase activity accumulate higher amounts of starch. Possible implications are discussed.     

Antisense inhibition of sorbitol synthesis leads to up-regulation of starch synthesis without altering CO<Subscript>2</Subscript> assimilation in apple leaves

Sorbitol is a primary end-product of photosynthesis in apple (Malus domestica Borkh.) and many other tree fruit species of the Rosaceae family. Sorbitol synthesis shares a common hexose phosphate pool with sucrose synthesis in the cytosol. In this study, Greensleeves apple was transformed with a cDNA encoding aldose 6-phosphate reductase (A6PR, EC 1.1.1.200) in the antisense orientation. Antisense expression of A6PR decreased A6PR activity in mature leaves to approximately 15–30% of the untransformed control. The antisense plants had lower concentrations of sorbitol but higher concentrations of sucrose and starch in mature leaves at both dusk and predawn. ¹⁴CO₂ pulse-chase labeling at ambient CO₂ demonstrated that partitioning of the newly fixed carbon to starch was significantly increased, whereas that to sucrose remained unchanged in the antisense lines with decreased sorbitol synthesis. Total activities of ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), sucrose-phosphate synthase (EC 2.4.1.14), and ADP-glucose pyrophosphorylase (EC 2.7.7.27) were not significantly altered in the antisense lines, whereas both stromal and cytosolic fructose-1,6-bisphosphatase (EC 3.1.3.11) activities were higher in the antisense lines with 15% of the control A6PR activity. Concentrations of glucose 6-phosphate and fructose 6-phosphate (F6P) were higher in the antisense plants than in the control, but the 3-phosphoglycerate concentration was lower in the antisense plants with 15% of the control A6PR activity. Fructose 2, 6-bisphosphate concentration increased in the antisense plants, but not to the extent expected from the increase in F6P, comparing sucrose-synthesizing species. There was no significant difference in CO₂ assimilation in response to photon flux density or intercellular CO₂ concentration. We concluded that cytosolic FBPase activity in vivo was down-regulated and starch synthesis was up-regulated in response to decreased sorbitol synthesis. As a result, CO₂ assimilation in source leaves was sustained at both ambient CO₂ and saturating CO₂.     

Reduction of the cytosolic fructose-1,6-bisphosphatase in transgenic potato plants limits photosynthetic sucrose biosynthesis with no impact on plant growth and tuber yield

Sucrose produced in source leaves is the predominant carbon source for developing sink tissues in most higher plants. Consequently the rate of sucrose synthesis is likely to be important for sink development and final crop yield. Two sucrose biosynthetic enzymes are believed to possess regulatory properties with respect to the rate of sucrose synthesis: (i) cytosolic FBPase and (ii) sucrose phosphate synthase. To study the impact of reduced photosynthetic sucrose biosynthesis on plant growth and crop yield a cDNA clone encoding cytosolic FBPase was isolated from a potato leaf cDNA library and used for antisense experiments in transgenic potato plants. The cDNA clone cy-F1, containing an open reading frame of 1020 bp highly homologous (85%) to other known sequences of plant cytosolic FBPases, was cloned in reversed orientation between the 35S CaMV promoter and the octopine synthase polyadenylation signal. Out of 75 independent transformants five transgenic lines having 9 to 55% of the wild-type FBPase activity were chosen for further analysis. A 45% reduction of the cytosolic FBPase activity did not cause any measurable change in metabolite concentrations, growth behaviour or photosynthetic parameters of the transgenic plants. Inhibition of cytosolic FBPase activity below 20% of the wild-type activity led to an accumulation of 3-PGA, triose-phosphates and fructose-1,6bisphosphate in source leaves. This resulted in a reduced light-saturated rate of assimilation measured via gas exchange and a decreased photosynthetic rate under conditions of the leaf disc electrode with saturating light and CO₂. Measuring photosynthetic carbon fluxes by labelling leaf discs with ¹⁴CO₂ revealed a 53–65% reduction of sucrose synthesis whereas starch synthesis decreased only by 18–24%. The flux into the anionic and cationic fraction was not altered. Despite these changes steadystate sucrose concentrations were not effected in source leaves from transgenic plants. Starch accumulated by more than a factor of 3 compared with wild-type leaves and was degraded during the night. This provides strong evidence for the hypothesis that hexoses and/or hexosephosphates are exported out of the chloroplasts, thereby circumventing the limitation of sucrose biosynthesis caused by the inhibition of cytosolic FBPase in the dark. Accordingly, plant growth and potato tuber yield remained unaltered. From these data it can be concluded that a reduced photosynthetic sucrose biosynthetic capacity can be efficiently compensated without any reduction in crop yield under greenhouse or growth chamber conditions by changing carbon export strategy. Whether the same holds true for field conditions remains to be elucidated.     

Reduced sedoheptulose-1,7-bisphosphatase levels in transgenic tobacco lead to decreased photosynthetic capacity and altered carbohydrate accumulation

Transgenic tobacco (Nicotiana tabacum L. cv. Samsun) plants with reduced levels of the Calvin cycle enzyme sedoheptulose-1,7-bisphosphatase (SBPase; EC 3.1.3.37) were produced using an antisense construct in which the expression of a tobacco SBPase cDNA clone was driven by the cauliflower mosaic virus (CaMV) promoter. The reduction in SBPase protein levels observed in the primary transformants correlated with the presence of the antisense construct and lower levels of the endogenous SBPase mRNA. No changes in the amounts of other Calvin cycle enzymes were detected using Western blot analysis. The SBPase antisense plants with less than 20% of wild-type SBPase activity were observed to display a range of phenotypes, including chlorosis and reduced growth rates. Measurements of photosynthesis, using both light-dosage response and CO₂ response curves, of T1 plants revealed a reduction in carbon assimilation rates, which was apparent in plants retaining 57% of wild-type SBPase activity. Reductions were also observed in the quantum efficiency of photosystem II. This decrease in photosynthetic capacity was reflected in a reduction in the carbohydrate content of leaves. Analysis of carbohydrate status in fully expanded source leaves showed a shift in carbon allocation away from starch, whilst sucrose levels were maintained in all but the most severely affected plants. Plants with less than 15% of wild-type SBPase activity were found to contain less than 5% of wild-type starch levels. The results of this preliminary analysis indicate that SBPase activity may limit the rate of carbon assimilation. Received: 23 February 1997 / Accepted: 2 May 1997     

Contribution of fructose-1,6-bisphosphatase and sedoheptulose-1,7-bisphosphatase to the photosynthetic rate and carbon flow in the Calvin cycle in transgenic plants

To clarify the contributions of fructose-1,6-bisphosphatase (FBPase) and sedoheptulose-1,7-bisphosphatase (SBPase) separately to the carbon flux in the Calvin cycle, we generated transgenic tobacco plants expressing cyanobacterial FBPase-II in chloroplasts (TpF) or Chlamydomonas SBPase in chloroplasts (TpS). In TpF-11 plants with 2.3-fold higher FBPase activity and in TpS-11 and TpS-10 plants with 1.6- and 4.3-fold higher SBPase activity in chloroplasts compared with the wild-type plants, the amount of final dry matter was approximately 1.3-, 1.5- and 1.5-fold higher, respectively, than that of the wild-type plants. At 1,500 micromol m(-2) s(-1), the photosynthetic activities of TpF-11, TpS-11 and TpS-10 were 1.15-, 1.27- and 1.23-fold higher, respectively, than that of the wild-type plants. The in vivo activation state of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and the level of ribulose-1,5-bisphosphate (RuBP) in TpF-11, TpS-10 and TpS-11 were significantly higher than those in the wild-type plants. However, the transgenic plant TpF-9 which had a 1.7-fold higher level of FBPase activity showed the same phenotype as the wild-type plant, except for the increase of starch content in the source leaves. TpS-11 and TpS-10 plants with 1.6- and 4.3-fold higher SBPase activity, respectively, showed an increase in the photosynthetic CO(2) fixation, growth rate, RuBP contents and Rubisco activation state, while TpS-2 plants with 1.3-fold higher SBPase showed the same phenotype as the wild-type plants. These data indicated that the enhancement of either a >1.7-fold increase of FBPase or a 1.3-fold increase of SBPase in the chloroplasts had a marked positive effect on photosynthesis, that SBPase is the most important factor for the RuBP regeneration in the Calvin cycle and that FBPase contributes to the partitioning of the fixed carbon for RuBP regeneration or starch synthesis.     

Modification of carbonic anhydrase activity by antisense and over-expression constructs in transgenic tobacco

The activity and location of carbonic anhydrase has been modified by transformation of tobacco with antisense and over-expression constructs. Antisense expression resulted in the inhibition of up to 99% of carbonic anhydrase activity but had no significant impact on net CO₂ assimilation. Stomatal conductance and susceptibility to water stress appeared to increase in response to the decline in carbonic anhydrase activity. An over-expression construct designed to increase cytosolic carbonic anhydrase abundance resulted in a significant increase in net activity, a small increase in stomatal conductance but little impact on CO₂ assimilation. Chloroplastic carbonic anhydrase activity was enhanced by the expression of an additional construct which targeted the polypeptide to the organelle. The increase in chloroplastic carbonic anhydrase appeared to be accompanied by a concomitant increase in Rubisco activity.     

The Calvin cycle revisited

The sequence of reactions in the Calvin cycle, and the biochemical characteristics of the enzymes involved, have been known for some time. However, the extent to which any individual enzyme controls the rate of carbon fixation has been a long standing question. Over the last 10 years, antisense transgenic plants have been used as tools to address this and have revealed some unexpected findings about the Calvin cycle. It was shown that under a range of environmental conditions, the level of Rubisco protein had little impact on the control of carbon fixation. In addition, three of the four thioredoxin regulated enzymes, FBPase, PRKase and GAPDH, had negligible control of the cycle. Unexpectedly, non-regulated enzymes catalysing reversible reactions, aldolase and transketolase, both exerted significant control over carbon flux. Furthermore, under a range of growth conditions SBPase was shown to have a significant level of control over the Calvin cycle. These data led to the hypothesis that increasing the amounts of these enzymes may lead to an increase in photosynthetic carbon assimilation. Remarkably, photosynthetic capacity and growth were increased in tobacco plants expressing a bifunctional SBPase/FBPase enzyme. Future work is discussed which will further our understanding of this complex and important pathway, particularly in relation to the mechanisms that regulate and co-ordinate enzyme activity.This revised version was published online in October 2005 with corrections to the Cover Date.     

Increased glucose production in mice overexpressing human fructose-1,6-bisphosphatase in the liver

Increased endogenous glucose production (EGP) predominantly from the liver is a characteristic feature of type 2 diabetes, which positively correlates with fasting hyperglycemia. Gluconeogenesis is the biochemical pathway shown to significantly contribute to increased EGP in diabetes. Fructose-1,6-bisphosphatase (FBPase) is a regulated enzyme in gluconeogenesis that is increased in animal models of obesity and insulin resistance. However, whether a specific increase in liver FBPase can result in increased EGP has not been shown. The objective of this study was to determine the role of upregulated liver FBPase in glucose homeostasis. To achieve this goal, we generated human liver FBPase transgenic mice under the control of the transthyretin promoter, using insulator sequences to flank the transgene and protect it from site-of-integration effects. This resulted in a liver-specific model, as transgene expression was not detected in other tissues. Mice were studied under the following conditions: 1) at two ages (24 wk and 1 yr old), 2) after a 60% high-fat diet, and 3) when bred to homozygosity. Hemizygous transgenic mice had an approximately threefold increase in total liver FBPase mRNA with concomitant increases in FBPase protein and enzyme activity levels. After high-fat feeding, hemizygous transgenics were glucose intolerant compared with negative littermates (P < 0.02). Furthermore, when bred to homozygosity, chow-fed transgenic mice showed a 5.5-fold increase in liver FBPase levels and were glucose intolerant compared with negative littermates, with a significantly higher rate of EGP (P < 0.006). This is the first study to show that FBPase regulates EGP and whole body glucose homeostasis in a liver-specific transgenic model. Our homozygous transgenic model may be useful for testing human FBPase inhibitor compounds with the potential to treat patients with type 2 diabetes.     

Reduction of ribulose-1,5-bisphosphate carboxylase/oxygenase content by antisense RNA reduces photosynthesis in transgenic tobacco plants

A complementary DNA for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was cloned from tobacco (Nicotiana tabacum) and fused in the antisense orientation to the cauliflower mosaic virus 35S promoter. This antisense gene was introduced into the tobacco genome, and the resulting transgenic plants were analyzed to assess the effect of the antisense RNA on Rubisco activity and photosynthesis. The mean content of extractable Rubisco activity from the leaves of 10 antisense plants was 18% of the mean level of activity of control plants. The soluble protein content of the leaves of anti-small subunit plants was reduced by the amount equivalent to the reduction in Rubisco. There was little change in phosphoribulokinase activity, electron transport, and chlorophyll content, indicating that the loss of Rubisco did not affect these other components of photosynthesis. However, there was a significant reduction in carbonic anhydrase activity. The rate of CO₂ assimilation measured at 1000 micromoles quanta per square meter per second, 350 microbars CO₂, and 25°C was reduced by 63% (mean value) in the antisense plants and was limited by Rubisco activity over a wide range of intercellular CO₂ partial pressures (p_i). In control leaves, Rubisco activity only limited the rate of CO₂ assimilation below a p_i of 400 microbars. Despite the decrease in photosynthesis, there was no reduction in stomatal conductance in the antisense plants, and the stomata still responded to changes in p_i. The unchanged conductance and lower CO₂ assimilation resulted in a higher p_i, which was reflected in greater carbon isotope discrimination in the leaves of the antisense plants. These results suggest that stomatal function is independent of total leaf Rubisco activity.     

Antisense inhibition of NADH glutamate synthase impairs carbon/nitrogen assimilation in nodules of alfalfa (Medicago sativa L.)

Legumes acquire significant amounts of nitrogen for growth from symbiotic nitrogen fixation. The glutamine synthetase (GS)/NADH-dependent glutamate synthase (NADH-GOGAT) cycle catalyzes initial nitrogen assimilation. This report describes the impact of specifically reducing nodule NADH-GOGAT activity on symbiotic performance of alfalfa (Medicago sativa L.). Four independent transgenic alfalfa lines, designated GA89, GA87, GA88, and GA82 (for GOGATantisense), containing an antisense NADH-GOGAT cDNA fragment under the control of the soybean leghemoglobin (lbc3) promoter were evaluated. The GA plants were fertile and showed normal growth in non-symbiotic conditions. The NADH-GOGAT antisense transgene was heritable and the T1 plants showed phenotypic alterations - similar to primary transformants. Clonally propagated plants were inoculated with Sinorhizobium meliloti after rooting and the symbiotic phenotype was analyzed 21 days post-inoculation. Nodules of each GA line had reduced NADH-GOGAT activity, ranging from 33 to 87% of control plants, that was accompanied by comparable decreases in RNA and protein. Plants from the GA89 line, with the lowest NADH-GOGAT activity (c. 30%), presented a strikingly altered symbiotic phenotype: concomitantly activities of key enzyme for carbon and nitrogen assimilation decreased; nodule amino acids and amides were reduced while sucrose accumulated. Antisense GOGAT plants were chlorotic, reduced in fresh weight, and had a lower N content than control plants. Photosynthesis was also impaired in antisense plants. Specifically, reducing NADH-GOGAT in nodules resulted in plants having impaired nitrogen assimilation and altered carbon/nitrogen metabolic flux.     

Dietary high protein and vitamin C mitigate stress due to chelate claw ablation in Macrobrachium rosenbergii males

Stress due to claw ablation was tested in Macrobrachium rosenbergii males. Dietary high protein and vitamin C were supplemented for amelioration of stress. We used four different treatments: fed with 25% protein and a normal dose (0.12%) of vitamin C (T(1)); 35% protein and a normal dose (0.12%) of vitamin C (T(2)); 25% protein and a high dose (0.24%) of vitamin C (T(3)); and high protein 35% and a high dose (0.24%) of vitamin C (T(4)) for 30 days. All test prawns (T(1) to T(4)) were subjected to ablation of their second chelate legs after the 15th day of the feeding trial. A control treatment was maintained without claw ablation and fed with 25% protein. Haemolymph glucose, hepatopancreatic glycogen, muscle ascorbate and enzyme activities (glucose 6 phosphatase (G6Pase), fructose-1,6-bisphosphatase (FBPase), lactate dehydrogenase (LDH), Alanine aminotransferase (ALT) in hepatopancreas) were tested at different recovery periods (0, 6, 24 h, 7 and 14 days). Results indicate a high glucose level immediately after claw ablation and a concomitant increase in gluconeogenic enzymes (G6Pase and FBPase). However, glycogen reserves were regained in the treatments due to claw ablation stress after 24 h. LDH and ALT activity decreased in the hepatopancreas of M. rosenbergii up to 24 h after claw ablation. Overall results indicate that claw ablation is stressful to M. rosenbergii and high protein and vitamin C diet may mitigate stress due to claw ablation.     

Reciprocal regulation of fructose 1,6-bisphosphatase and phosphofructokinase by fructose 2,6-bisphosphate in swine kidney

The effect of fructose 2,6-P₂, AMP and substrates on the coordinate inhibition of FBPase and activation of PFK in swine kidney has been examined. Fructose 2,6-P₂ inhibits the activity of FBPase and stimulates the activity of PFK in the presence of inhibitory concentrations of ATP. Under similar conditions 2.2 μM fructose 2,6-P₂ was required for 50% inhibition of FBPase and 0.04 μM fructose 2,6-P₂ restored 50% of the activity of PFK. Fructose 2,6-P₂ also enhanced the allosteric activation of PFK by AMP and it increased the extent of inhibition of FBPase by AMP. Fructose 2,6-P₂, AMP and fructose 6-P act cooperatively to stimulate the activity of PFK whereas the same latter two effectors and fructose 1,6-P₂ inhibit the activity of FBPase. Taken collectively, these results suggest that an increase in the intracellular level of fructose 2,6-P₂ during gluconeogenesis could effectively overcome the inhibition of PFK by ATP and simulataneously inactivate FBPase. When the level of fructose 2,6-P₂ is low, a glycolytic state would be restored, since under these conditions PFK would be inhibited by ATP and FBPase would be active.     

Rabbit muscle fructose-1,6-bisphosphatase is phosphorylatedin vivo

Phosphorylated fructose-1,6-bisphosphatase (FBPase) was isolated from rabbit muscle in an SDS/PAGE homogeneous form. Its dephosphorylation with alkaline phosphatase revealed 2.8 moles of inorganic phosphate per mole of FBPase. The phosphorylated FBPase (P-FBPase) differs from the dephosphorylated enzyme in terms of its kinetic properties like K(m) and k(cat), which are two times higher for the phosphorylated FBPase, and in the affinity for aldolase, which is three times lower for the dephosphorylated enzyme. Dephosphorylated FBPase can be a substrate for protein kinase A and the amount of phosphate incorporated per FBPase monomer can reach 2-3 molecules. Since interaction of muscle aldolase with muscle FBPase results in desensitisation of the latter toward AMP inhibition (Rakus & Dzugaj, 2000, Biochem. Biophys. Res. Commun. 275, 611-616), phosphorylation may be considered as a way of muscle FBPase activity regulation.     

The inhibition of fructose 1,6-bisphosphatase by fructose 2,6-bisphosphate is enhanced by EDTA and diminished by zinc(II)

The sensitivity of the Mg(II)-dependent activity of rabbit liver fructose 1,6-bisphosphatase (FBPase, EC 3.1.3.11) to inhibition by fructose 2,6-bisphosphate (Fru-2,6-P2) was enhanced by EDTA and diminished to negligible levels by 0.5-2 microM Zn(II) added as another FBPase inhibitor. Fru-2,6-P2 was more efficient in the presence of the synergistic effector AMP: still, the Fru-2,6-P2 concentration inhibiting 50% changed from 3 microM (with EDTA) to higher than 50 microM (with Zn(II]. On the other hand, the Zn(II)-dependent FBPase activity was inhibited by Fru-2,6-P2 to a much lesser extent than the Mg(II)-dependent activity.     

Reduction of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase by Antisense RNA in the C4 Plant Flaveria bidentis Leads to Reduced Assimilation Rates and Increased Carbon Isotope Discrimination

Transgenic Flaveria bidentis (a C4 species) plants with an antisense gene directed against the mRNA of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) were used to examine the relationship between the CO2 assimilation rate, Rubisco content, and carbon isotope discrimination. Reduction in the amount of Rubisco in the transgenic plants resulted in reduced CO2 assimilation rates and increased carbon isotope discrimination of leaf dry matter. The H2O exchange was similar in transgenic and wild-type plants, resulting in higher ratios of intercellular to ambient CO2 partial pressures. Carbon isotope discrimination was measured concurrently with CO2 and H2O exchange on leaves of the control plants and T1 progeny with a 40% reduction in Rubisco. From the theory of carbon isotope discrimination in the C4 species, we conclude that the reduction in the Rubisco content in the transgenic plants has led to an increase in bundle-sheath CO2 concentration and CO2 leakage from the bundle sheath; however, some down-regulation of the C4 cycle also occurred.     

Decreased SBPase activity alters growth and development in transgenic tobacco plants

The effects of reduced SBPase activity on growth and development were examined in a set of transgenic tobacco plants produced using an antisense construct driven by the ribulose bisphosphate carboxylase, small subunit promoter. Photosynthetic carbon assimilation rates and carbohydrate levels in source leaves were decreased in the antisense plants. Growth rate and total shoot biomass were reduced in the SBPase antisense plants, even in plants where SBPase activity was reduced by only 25%. Floral biomass also decreased in response to reductions in SBPase activity and the onset of flowering was delayed by 5-10 d. This is the first demonstration of a link between reproductive biomass and reductions in Calvin cycle enzyme activity using antisense plants. Furthermore, unexpected changes in the growth and development of the antisense plants were evident. Small reductions in SBPase activity (above 50% wild type) resulted in shorter plants with only a small decrease in stem biomass and specific leaf area. In contrast, plants with larger reductions in SBPase activity had an increase in specific leaf area and attained heights similar to that of the wild-type plants but with a much reduced stem biomass, largely due to a decrease in xylem tissue. This bi-modal response of growth to reductions in SBPase activity has similarities to changes in leaf and stem anatomy and morphology that accompany light acclimation.