The role of enzyme activation state in limiting carbon assimilation under variable light conditions

The mechanisms regulating transient photosynthesis by soybean (Glycine max) leaves were examined by comparing photosynthetic rates and carbon reduction cycle enzyme activities under flashing (saturating 1 s lightflecks separated by low photon flux density (PFD) periods of different durations) and continuous PFD. At the same mean PFD, the mean photosynthetic rates were reduced under flashing as compared to continuous light. However, as the duration of the low PFD period lengthened, the CO₂ assimilation attributable to a lightfleck increased. This enhanced lightfleck CO₂ assimilation was accounted for by a greater postillumination CO₂ fixation occurring after the lightfleck. The induction state of photosynthesis, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco), fructose 1,6-bisphosphatase (FBPase) and ribulose 5-phosphate kinase (Ru5P kinase) activities all responded similarly and were all lower under flashing as compared to constant PFD of the same integrated mean value. However, the fast phase of induction and FBPase and Ru5P kinase activities were reduced more than were the slow phase of induction and rubisco activity. This was consistent with the role of the former enzymes in the fast induction component that limited RuBP regeneration. Competition for reducing power between carbon metabolism and thioredoxin-mediated enzyme activation may have resulted in lower enzyme activation states and hence lower induction states under flashing than continuous PFD, especially at low lightfleck frequencies (low mean PFD).Abbreviations FBPase fructose 1,6-bisphosphatase (EC 3.1.3.11) - LUE lightfleck use efficiency - P-glycerate 3-phosphoglycerate - PICF post-illumination CO₂ fixation - Ru5P kinase ribulose 5-phosphate kinase (EC 2.7.1.19) - RuBP ribulose 1,5-bisphosphate - rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) - SBpase sedoheptulose 1,7-bisphosphatase (EC 3.1.3.37)     

Regulation of photosynthetic carbon metabolism during phosphate limitation of photosynthesis in isolated spinach chloroplasts

Intact chloroplasts isolated from spinach were illuminated in the absence of inorganic phosphate (Pi) or with optimum concentrations of Pi added to the reaction medium. In the absence of Pi photosynthesis declined after the first 1–2 min and was less than 10% of the maximum rate after 5 min. Export from the chloroplast was inhibited, with up to 60% of the ¹⁴C fixed being retained in the chloroplast, compared to less than 20% in the presence of Pi. Despite the decreased export, chloroplasts depleted of Pi had lower levels of triose phosphate while the percentage of total phosphate in 3-phosphoglycerate was increased. Chloroplast ATP declined during Pi depletion and reached dark levels after 3–4 min in the light without added Pi. At this point, stromal Pi concentration was 0.2 mM, which would be limiting to ATP synthesis. Addition of Pi resulted in a rapid burst of oxygen evolution which was not initially accompanied by net CO₂ fixation. There was a large decrease in 3-phosphoglycerate and hexose plus pentose monophosphates in the chloroplast stroma and a lesser decrease in fructose-1,6-bisphosphate. Stromal levels of triose phosphate, ribulose-1,5-bisphosphate and ATP increased after resupply of Pi. There was an increased export of ¹⁴-labelled compounds into the medium, mostly as triose phosphate. Light activation of both fructose-1,6-bisphosphatase and ribulose-1,5-bisphosphate carboxylase was decreased in the absence of Pi but increased following Pi addition.It is concluded that limitation of Pi supply to isolated chloroplasts reduced stromal Pi to the point where it limits ATP synthesis. The resulting decrease in ATP inhibits reduction of 3-phosphoglycerate to triose phosphate via mass action effects on 3-phosphoglycerate kinase. The lack of Pi in the medium also inhibits export of triose phosphate from the chloroplast via the phosphate transporter. Other sites of inhibition of photosynthesis during Pi limitation may be located in the regeneratige phase of the reductive pentose phosphate pathway.Abbreviations FBP Fructose-1,6-bisphosphate - FBPase Fructose-1,6-bisphosphatase - MP Hexose plus pentose monophosphates - PGA 3-phosphoglycerate - Pi inorganic orthophosphate - RuBP ribulose-1,5-bisphosphate - RuBPCase ribulose-1,5-bisphosphate carboxylase - TP Triose Phosphate     

Regulation of sedoheptulose-1,7-bisphosphatase by sedoheptulose-7-phosphate and glycerate,and of fructose-1,6-bisphosphatase by glycerate in spinach chloroplasts

Using partially purified sedoheptulose-1,7-bisphosphatase from spinach (Spinacia oleracea L.) chloroplasts the effects of metabolites on the dithiothreitoland Mg²⁺-activated enzyme were investigated. A screening of most of the intermediates of the Calvin cycle and the photorespiratory pathway showed that physiological concentrations of sedoheptulose-7-phosphate and glycerate specifically inhibited the enzyme by decreasing its maximal velocity. An inhibition by ribulose-1,5-bisphosphate was also found. The inhibitory effect of sedoheptulose-7-phosphate on the enzyme is discussed in terms of allowing a control of sedoheptulose-1,7-bisphosphate hydrolysis by the demand of the product of this reaction. Subsequent studies with partially purified fructose-1,6-bisphosphatase from spinach chloroplasts showed that glycerate also inhibited this enzyme. With isolated chloroplasts, glycerate was found to inhibit CO₂ fixation by blocking the stromal fructose-1,6-bisphosphatase. It is therefore possible that the inhibition of the two phosphatases by glycerate is an important regulatory factor for adjusting the activity of the Calvin cycle to the ATP supply by the light reaction.Abbreviations DTT dithiothreitol - FBPase fructose-1,6-bisphosphatase - Fru-1,6-P₂ fructose-1,6-bisphosphate - Fru-6-P fructose-6-phosphate - 3-PGA 3-phosphoglycerate - Ru-1,5-P₂ ribulose-1,5-bisphosphate - Ru-5-P ribulose-5-phosphate - SBPase sedoheptulose-1,7-bisphosphatase - Sed-1,7-P₂ sedoheptulose-1,7-bisphosphate - Sed-7-P sedoheptulose-7-phosphate This work was supported by the Deutsche Forschungsgemein-schaft.     

Synthesis and degradation of fructose 2,6-bisphosphate in endosperm of castor bean seedlings

The aim of this work was to examine the possibility that fructose 2,6-bisphosphate (Fru-2,6-P₂) plays a role in the regulation of gluconeogenesis from fat. Fru-2,6-P₂ is known to inhibit cytoplasmic fructose 1,6-bisphosphatase and stimulate pyrophosphate:fructose 6-phosphate phosphotransferase from the endosperm of seedlings of castor bean (Ricinus communis). Fru-2,6-P₂ was present throughout the seven-day period in amounts from 30 to 200 picomoles per endosperm. Inhibition of gluconeogenesis by anoxia or treatment with 3-mercaptopicolinic acid doubled the amount of Fru-2,6-P₂ in detached endosperm. The maximum activities of fructose 6-phosphate,2-kinase and fructose 2,6-bisphosphatase (enzymes that synthesize and degrade Fru-2,6-P₂, respectively) were sufficient to account for the highest observed rates of Fru-2,6-P₂ metabolism. Fructose 6-phosphate,2-kinase exhibited sigmoid kinetics with respect to fructose 6-phosphate. These kinetics became hyperbolic in the presence of inorganic phosphate, which also relieved a strong inhibition of the enzyme by 3-phosphoglycerate. Fructose 2,6-bisphosphatase was inhibited by both phosphate and fructose 6-phosphate, the products of the reaction. The properties of the two enzymes suggest that in vivo the amounts of fructose-6-phosphate, 3-phosphoglycerate, and phosphate could each contribute to the control of Fru-2,6-P₂ level. Variation in the level of Fru-2,6-P₂ in response to changes in the levels of these metabolites is considered to be important in regulating flux between fructose 1,6-bisphosphate and fructose 6-phosphate during germination.     

Ribose 1,5-bisphosphate is a putative regulator of fructose 6-phosphate/fructose 1,6-bisphosphate cycle in liver

6-Phosphofructo-1-kinase and fructose-1,6-bisphosphatase are rate-limiting enzymes for glycolysis and gluconeogenesis respectively, in the fructose 6-phosphate/fructose 1,6-bisphosphate cycle in the liver. The effect of ribose 1,5-bisphosphate on the enzymes was investigated. Ribose 1,5-bisphosphate synergistically relieved the ATP inhibition and increased the affinity of liver 6-phosphofructo-1-kinase for fructose 6-phosphate in the presence of AMP. Ribose 1,5-bisphosphate synergistically inhibited fructose-1,6-bisphosphatase in the presence of AMP. The activating effect on 6-phosphofructo-1-kinase and the inhibitory effect on fructose-1,6-bisphosphatase suggest ribose 1,5-bisphosphate is a potent regulator of the fructose 6-phosphate/fructose 1,6-bisphosphate cycle in the liver.     

The in-vivo response of the ribulose-1,5-bisphosphate carboxylase activation state and the pool sizes of photosynthetic metabolites to elevated CO2 inPhaseolus vulgaris L.

The short-term, in-vivo response to elevated CO₂ of ribulose-1,5-bisphosphate carboxylase (RuBPCase, EC 4.1.1.39) activity, and the pool sizes of ribulose 1,5-bisphosphate, 3-phosphoglyceric acid, triose phosphates, fructose 1,6-bisphosphate, glucose 6-phosphate and fructose 6-phosphate in bean were studied. Increasing CO₂ from an ambient partial pressure of 360–1600 bar induced a substantial deactivation of RuBPCase at both saturating and subsaturating photon flux densities. Activation of RuBPCase declined for 30 min following the CO₂ increase. However, the rate of photosynthesis re-equilibrated within 6 min of the switch to high CO₂, indicating that RuBPCase activity did not limit photosynthesis at high CO₂. Following a return to low CO₂, RuBPCase activation increased to control levels within 10 min. The photosynthetic rate fell immediately after the return to low CO₂, and then increased in parallel with the increase in RuBPCase activation to the initial rate observed prior to the CO₂ increase. This indicated that RuBPCase activity limited photosynthesis while RuBPCase activation increased. Metabolite pools were temporarily affected during the first 10 min after either a CO₂ increase or decrease. However, they returned to their original level as the change in the activation state of RuBPCase neared completion. This result indicates that one role for changes in the activation state of RuBPCase is to regulate the pool sizes of photosynthetic intermediates.Abbreviations and symbols A net CO₂ assimilation rate - C_a ambient CO₂ partial pressure - C_i intercellular CO₂ partial pressure - CABP 2-carboxyarabinitol 1,5-bisphosphate - k_cat catalytic turnover rate per RuBPCase molecule - PFD photon flux density (400 to 700 nm on an area basis) - PGA 3-phosphoglyceric acid - P_i orthophosphate - RuBP ribulose 1,5-bisphosphate - RuBPCase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39)     

Variation in photosynthetic electron transport capacity in vivo and its effects on the light modulation of ribulose bisphosphate carboxylase

Photosynthetic electron transport capacity was varied in vivo in sugar beets using iron deficiency, and its effects on the light modulation of ribulose bisphosphate carboxylase (RuBPCase) studied. Three treatment groups corresponding to decreasing amounts of thylakoids per leaf area were examined: iron sufficient (control), moderately iron-stressed, and severely iron-stressed. Reduction in electron transport capacity in vivo was correlated with a substantial decrease in the level of RuBPCase activation, even at saturating irradiances. These results indicate a direct relationship between RuBPCase activation and photosynthetic electron transport. In addition, our data suggest that the activation of RuBPCase could not solely account for the increases in the photosynthetic rate at high irradiances; RuBPCase reached maximal activation at irradiances well below light saturation for net photosynthesis.Abbreviations Chl chlorophyll - FeCN ferricyanide - FBPase fructose 1,6-bisphosphatase - RuBP ribulose 1,5-bisphosphate - RuBPCase ribulose 1,5-bisphosphate carboxylase - SBPase sedoheptulose 1,7-bisphosphatase     

Modelling C3 photosynthesis: A sensitivity analysis of the photosynthetic carbon-reduction cycle

A model of the C ₃ photosynthetic system is developed which describes the sensitivity of the steadystate rate of carbon dioxide assimilation to changes in the activity of several enzymes of the system. The model requires measurements of the steady-state rate of carbon dioxide assimilation, the concentrations of several intermediates in the photosynthetic system, and the concentration of the active site of ribulose 1,5-bisphosphate carboxyalse/oxygenase (Rubisco). It is shown that in sunflowers (Helianthus annuus L.) at photon flux densities that are largely saturating for the rate of photosynthesis, the steady-stete rate of carbon dioxide assimilation is most sensitive to Rubisco activity and, to a lesser degree, to the activities of the stromal fructose, 6-bisphosphatase and the enzymes catalysing sucrose synthesis. The activities of sedoheptulose 1,7-bisphosphatase, ribulose 5-phosphate kinase, ATP synthase and the ADP-glucose pyrophosphorylase are calculated to have a negligible effect on the flux under the high-light conditions. The utility of this analysis in developing simpler models of photosynthesis is also discussed.Abbreviations c _i intercellular CO₂ concentration - C _infP ^supJ control coefficient for enzyme P with respect to flux J - DHAP dihydroxyacetonephosphate - E4P erythrose 4-phosphate - F6P fructose 6-phosphate - FBP fructose 1,6-bisphosphate - FBPase fructose 1,6-bisphosphatase - G3P glyceraldehyde 3-phosphate - G1P glucose 1-phosphate - G6P glucose 6-phosphate - Pi inorganic phosphate - PCR photosynthetic carbon reduction - PGA 3-phosphoglyceric acid - PPFD photosynthetically active photon flux density - R _n ^J response coefficient for effector n with respect to flux J - R5P ribose 5-phosphate - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - Ru5P ribulose 5-phosphate - RuBP ribulose 1,5-bisphosphate - S7P sedoheptulose 7-phosphate - SBP sedoheptulose 1,7-bisphosphate - SBPase sedoheptulose 1,7-bisphosphatase - SPS sucrose-phosphate synthase - Xu5P xylulose 5-phosphate - _n ^P elasticity coefficient for effector n with respect to the catalytic velocity of enzyme P This research was funded by an Australian Research Council grant to I.E.W. and was undertaken during a visity by K.A.M. to the James Cook University of North Queensland. The expert help of Glenys Hanley and Mick Kelly is greatly appreciated.     

Control of Photosynthetic Sucrose Synthesis by Fructose 2,6-Bisphosphate : III. Properties of the Cytosolic Fructose 1,6-Bisphosphatase

The cytosolic fructose 1,6-bisphosphatase from spinach (Spinacia oleracea U.S. hybrid 424) leaves has been partially purified and its response to fructose 2,6-bisphosphate, AMP, and fructose 1,6-bisphosphate studied, using concentrations present in the cytosol during photosynthesis. In the presence of fructose 2,6-bisphosphate, the substrate saturation kinetics for fructose 1,6-bisphosphate are sigmoidal, with half-maximal activity being attained in 0.1 to 1 millimolar concentration range. The inhibition is enhanced by AMP. Using these results, and information published elsewhere on metabolite concentrations, it is discussed how fructose 1,6-bisphosphatase activity will vary in vivo in response to alterations in the availability of triose phosphate and AMP, and the accumulation of the product, fructose 6-phosphate.     

Rate-limiting factors in leaf photosynthesis. I. Carbon fluxes in the calvin cycle

Rates of photosynthesis of spinach leaves were varied by varying light intensity and CO₂ concentration. Metabolism of the leaves was then arrested by freezing them in liquid nitrogen. Chloroplasts were isolated by a nonaqueous procedure. In the chloroplast fractions, levels of intermediates of the carbon reduction cycle were determined and considered in relation to the photosynthetic flux situation of the leaves at the time before freezing. During induction of photosynthesis, ribulose 1,5-bisphosphate levels increased in parallel with CO₂ fixation. In the steady state, a similar relation between ribulose 1,5-bisphosphate levels and CO₂ uptake was observed at light intensities between 0 and 50 W·m⁻². A further increase in light intensity increased CO₂ fixation rates but not ribulose 1,5-bisphosphate levels. Increasing the CO₂ concentration resulted in increased CO₂ uptake, whereas ribulose 1,5-bisphosphate levels decreased. Even under CO₂ saturation, ribulose 1,5-bisphosphate levels were about 100 nmol/mg chlorophyll corresponding to about 3.5 mM ribulose 1,5-bisphosphate in the chloroplast stroma. This suggests that even under CO₂ saturation, ribulose-1,5-bisphosphate carboxylase limits photosynhetic CO₂ uptake. Mass action ratios calculated from measured metabolite levels demonstrated that the thermodynamic gradient required for the regeneration of ribulose 1,5-bisphosphate from hexosephosphate and triosephosphate increased considerably as photosynthetic flux increased. Similar calculations revealed that the enzymatic apparatus responsible for the reduction of 3-phosphoglycerate to dihydroxyacetone phosphate is not displaced much from equilibrium even under maximum rates of photosynthesis at saturating CO₂. The same is true for aldolase. Fructose-1,6-bisphosphatase also did not limit Calvin cycle turnover. Only at very low light intensities and during the first minutes of the induction period was the ratio of fructose 1,6-bisphosphate to fructose 6-phosphate high. This observation was more readily explained in terms of fructose 1,6-bisphosphate binding to ribulose-1,5-bisphosphate carboxylase than by a rate limitation imposed by insufficient activation of fructose-1,6-bisphosphatase.     

Autocatalysis and light activation of enzymes in relation to photosynthetic induction in wheat chloroplasts

In order to study the relative contributions of the autocatalytic increase in the level of substrates and the light activation of enzymes to the control of the induction phase or “lag” in wheat chloroplasts, we measured the light-induced reductive activation of fructose 1,6-bisphosphatase, phosphoglycerate kinase, NADP⁺-dependent glyceraldehyde-phosphate dehydrogenase, ribulose 1,5-bisphosphate carboxylase, and phosphoribulokinase in isolated chloroplasts. Each was rapidly activated to levels more than adequate to support the maximum rate of photosynthesis. Induction in wheat chloroplasts is characterized by a period of about 1 min during which no O₂ is evolved. If small quantities of intermediates such as dihydroxyacetone phosphate (DHAP) or 3-phosphoglycerate (PGA) are added, maximum rates of photosynthesis are achieved within the first minute of illumination. The presence of PGA did not affect the activation of any of the above-mentioned enzymes. Each of the enzymes was therefore capable of sustaining maximum rates of photosynthesis in the presence of PGA, even though there was no O₂ evolution from those chloroplasts incubated with CO₂ alone as substrate. The inclusion of PGA did not give rise to abnormally high levels of DHAP, FBP, or fructose 6-phosphate in the stroma. We conclude that the levels of substrates or cofactors are the principal, if not the sole, determinants of the rate of photosynthetic carbon assimilation during induction in wheat chloroplasts.     

The photosynthetic induction response in wheat leaves: net CO2 uptake,enzyme activation,and leaf metabolites

The photosynthetic induction response was studied in whole leaves of wheat (Triticum aestivum L.) following 5-min, 30-min and 10-h dark periods. After the 5-min dark treatment there was a rapid burst in the rate of photosynthesis upon illumination (half of maximum after 30s), followed by a slight decrease after 1.5 more min and then a gradual rise to the maximum rate. During this initial burst in photosynthesis, there was a rapid rise in the level of 3-phosphoglycerate (PGA) and a high PGA/triose-phosphate (triose-P) ratio was obtained. In addition, after the 5-min dark treatment, ribulose-1,5-bisphosphate carboxylase (Rubisco, EC 4.1.1.39), ribulose-5-phosphate kinase (EC 2.7.1.19) and chloroplastic fructose-1,6-bisphosphatase (EC 3.1.3.11) maintained a relatively high state of activation, and maximum activation occurred within 1 min of illumination. The results indicate there is a high capacity for CO₂ fixation in the cycle upon illumination but attaining maximum rates requires an increase in the ribulose-1,5-bisphosphate (RuBP) pool (adjustment in triose-P utilization for carbohydrate synthesis versus RuBP synthesis). With both the 30-min and 10-h dark pretreatments there was only a slight rise in photosynthesis upon illumination, followed by a lag, then a gradual increase to steady-state (half-maximum rate after 6 min). In contrast to the 5-min dark treatment, the level of PGA was low and actually decreased initially, whereas the level of RuBP increased and was high during induction, indicating that Rubisco is limiting. This regulation via the carboxylase was not reflected in the initial extractable activity, which reached a maximum by 1 min after illumination. The light activation of chloroplastic fructose-1,6-bisphosphatase in leaves darkened for 30 min and 10 h prior to illumination was relatively slow (reaching a maximum after 8 min). However, this was not considered to limit carbon flux through the carbon-fixation cycle during induction since RuBP was not limiting. When photosynthesis approached the maximum steady-state rate, a high PGA/triose-P ratio and a high PGA/RuBP ratio were obtained. This may allow a high rate of photosynthesis by producing a favorable mass-action ratio for the reductive phase (the conversion of PGA to triose phosphate) while stimulating starch and sucrose synthesis.Abbreviations Chl chlorophyll - FBP fructose-1,6-bisphosphate - FBPase fructose-1,6-bisphosphatase - Fru6P fructose-6-phosphate - Glc6P glucose-6-phosphate - PGA 3-phosphoglycerate - Pi inoganic phosphate - Rubisco RuBP carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - Ru5P ribulose-5-phosphate - triose-P triose phosphates (dihydroxyacetone phosphate+glyceraldehyde-3-phosphate)     

Leaf phosphate status, photosynthesis, and carbon partitioning in sugar beet: I. Changes in growth, gas exchange, and calvin cycle enzymes

Sugar beets (Beta vulgaris L. cv F58-554H1) were cultured hydroponically for 2 weeks in growth chambers with two levels of orthophosphate (Pi) supplied in half strength Hoagland solution. Low-P plants were supplied with 1/20th of the Pi supplied to control plants. With low-P treatment, the acid soluble leaf phosphate and total leaf P decreased by about 88%. Low-P treatment had a much greater effect on leaf area than on photosynthesis. Low-P decreased total leaf area by 76%, dry weight per plant by 60%, and the rate of photosynthesis per area at light saturation by 35%. Low-P treatment significantly decreased the total extractable activity of phosphoglycerate kinase (by 18%) and NADP-glyceraldehyde-3-phosphate dehydrogenase (by 16%), but did not decrease the total activities of ribulose-1,5-bisphosphate (RuBP) carboxylase (RuBPCase) and ribulose-5-phosphate kinase. Low-P treatment decreased the initial activities of three rate-limiting Calvin cycle enzymes, but had no effect on the initial activity of RuBPCase. Furthermore, low-P treatment significantly increased the total extractable activities of fructose-1,6-bisphosphatase (by 61%), fructose-1,6-bisphosphate aldolase (by 53%), and transketolase (by 46%). The results suggest that low-P treatment affected photosynthetic rate through an effect on RuBP regeneration rather than through RuBPCase activity and that the changes in Calvin cycle enzymes with low-P resulted in an increased flow of carbon to starch.     

Regulation of 2-carboxyarabinitol 1-phosphatase

The regulation of 2-carboxyarabinitol 1-phosphatase (CA 1-Pase) by phosphorylated effectors was studied with enzyme purified from tobacco (Nicotiana tabacum) leaves. CA 1-Pase activity was most stimulated by fructose 1,6-bisphosphate, exhibiting an A_0.5 value of 1.9 millimolar and a 10-fold enhancement of catalysis. With ribulose-1,5-bisphosphate, the A_0.5 was 0.6 millimolar, and maximal stimulation of activity was 5.3-fold. Among the monophosphates, 3-phosphoglycerate and phosphoglycolate were more potent positive effectors than glyceraldehyde 3-phosphate, glucose 1-phosphate, glucose 6-phosphate, and dihydroxyacetone phosphate. Stimulation of CA 1-Pase by ribulose-1,5-bisphosphate and fructose 1,6-bisphosphate increased V_max but did not appreciably alter K_m (2-carboxyarabinitol 1-phosphate) values. Inorganic phosphate appeared to inhibit CA 1-Pase noncompetitively with respect to 2-carboxyarabinitol 1-phosphate, exhibiting a K_i of 0.3 millimolar. The results suggest that these positive and negative effectors bind to a regulatory site on CA 1-Pase and may have a physiologial role in the light regulation of this enzyme. Related experiments with CA 1-Pase inactivated by dialysis in the absence of dithiothreitol show that partial reactivation can be achieved in the presence of a range of reducing reagents, including dithiothreitol, cysteine, and reduced glutathione. This could imply an ancillary involvement of sulfhydryl reduction during light activation of CA 1-Pase in vivo. The enzyme was thermally stable up to 35°C, in contrast to ribulose-1,5-bisphosphate carboxylase/oxygenase activase which lost activity above 30°C. The activation energy for CA 1-Pase was calculated to be 56.14 kilojoules per mole.     

Reduced-activity mutants of phosphoglucose isomerase in the cytosol and chloroplast of Clarkia xantiana

(i) We have studied the influence of reduced phosphoglucose-isomerase (PGI) activity on photosynthetic carbon metabolism in mutants of Clarkia xantiana Gray (Onagraceae). The mutants had reduced plastid (75% or 50% of wildtype) or reduced cytosolic (64%, 36% or 18% of wildtype) PGI activity. (ii) Reduced plastid PGI had no significant effect on metabolism in low light. In high light, starch synthesis decreased by 50%. There was no corresponding increase of sucrose synthesis. Instead glycerate-3-phosphate, ribulose-1,5-bisphosphate, reduction of Q_A (the acceptor for photosystem II) and energy-dependent chlorophyll-fluorescence quenching increased, and O₂ evolution was inhibited by 25%. (iii) Decreased cytosolic PGI led to lower rates of sucrose synthesis, increased fructose-2,6-bisphosphate, glycerate-3-phosphate and ribulose-1,5-bisphosphate, and a stimulation of starch synthesis, but without a significant inhibition of O₂ evolution. Partitioning was most affected in low light, while the metabolite levels changed more at saturating irradiances. (iv) These results provide decisive evidence that fructose-2,6-bisphosphate can mediate a feedback inhibition of sucrose synthesis in response to accumulating hexose phosphates. They also provide evidence that the ensuing stimulation of starch synthesis is due to activation of ADP-glucose pyrophosphorylase by a rising glycerate-3-phosphate: inorganic phosphate ratio, and that this can occur without any loss of photosynthetic rate. However the effectiveness of these mechanisms varies, depending on the conditions. (v) These results are analysed using the approach of Kacser and Burns (1973, Trends Biochem. Sci. 7, 1149–1161) to provide estimates for the elasticities and flux-control coefficient of the cytosolic fructose-1,6-bisphosphatase, and to estimate the gain in the fructose-2,6-bisphosphate regulator cycle during feedback inhibition of sucrose synthesis.Abbreviations and symbols Chl chlorophyll - Fru6P fructose-6-phosphate - Frul,6bisP fructose-1,6-bisphosphate - Fru-1,6Pase fructose-1,6-bisphosphatase - Fru2,6bisP fructose-2,6-bisphosphate - Fru2,6Pase fructose-2,6-bisphosphatase - Glc6P glucose-6-phosphate - PGI phosphoglucose isomerase - Pi inorganic phosphate - Q_A acceptor for photosystem II - Ru1,5bisP ributose-1,5-bisphosphate - SPS sucrose-phosphate synthase     

Limitation of CO(2) Assimilation and Regulation of Benson-Calvin Cycle Activity in Barley Leaves in Response to Changes in Irradiance, Photoinhibition, and Recovery

The response of the Benson-Calvin cycle to changes in irradiance and photoinhibition was measured in low-light grown barley (Hordeum vulgare) leaves. Upon the transition from the growth irradiance (280 micromoles per square meter per second) to a high photoinhibitory irradiance (1400 micromoles per square meter per second), the CO₂ assimilation rate of the leaves doubled within minutes but high irradiance rapidly caused a reduction in quantum efficiency. Following exposure to high light the activities of NADP-malate dehydrogenase and fructose-1,6-bisphosphatase obtained near maximum values and the activation state of ribulose-1,5-bisphosphate carboxylase increased. The activity of the latter remained constant throughout the period of photoinhibitory irradiance, but the increase in the activities of fructose-1,6-bisphosphatase and NADP-malate dehydrogenase was transient decreasing once more to much lower values. This suggests that immediately following the transition to high light reduction and activation of redox-modulated enzymes occurred, but then the stroma became relatively oxidized as a result of photoinhibition. The leaf contents of glucose 6-phosphate and fructose 6-phosphate increased following exposure to high light but subsequently decreased, suggesting that following photoinhibition sucrose synthesis exceeded the rate of carbon assimilation. The ATP content attained a constant value much higher than that in low light. During photoinhibition the glycerate 3-phosphate content greatly increased while ribulose-1,5-bisphosphate decreased. The fructose-1,6-bisphosphate and triose phosphate contents increased initially and then remained constant. During photoinhibition CO₂ assimilation was not limited by ribulose-1,5-bisphosphate carboxylase activity but rather by the regeneration of the substrate, ribulose-1,5-bisphosphate, related to a restriction on the supply of reducing equivalents.     

Regulation of fructose 2,6-bisphosphate concentration in spinach leaves

Fructose-6-phosphate 2-kinase and fructose-2,6-bisphosphatase have been partially purified from spinach leaves and their regulatory properties studied. Fructose-6-phosphate 2-kinase was activated by phosphate and fructose 6-phosphate, and inhibited by 3-phosphoglycerate and dihydroxyacetone phosphate. Fructose-2,6-bisphosphatase was inhibited by fructose 6-phosphate and phosphate. The interaction between these effectors was studied when they were varied, alone or in combination, over a range of concentrations representative of those in the cytosol of spinach leaf cells. In conditions when dihydroxyacetone phosphate or 3-phosphoglycerate rise, as is typical during photosynthesis, the fructose 2,6-bisphosphate level will decrease, which will favour sucrose synthesis. In conditions when fructose 6-phosphate accumulates, fructose 2,6-bisphosphate should rise, which will favour a restriction of sucrose synthesis and promotion of starch synthesis.     

Feedback-limited photosynthesis and regulation of sucrose-starch accumulation during cold acclimation and low-temperature stress in a spring and winter wheat

Regulation of sucrose-starch accumulation and its effect on CO₂ gas exchange and electron transport were studied in low-temperature-stressed and cold-acclimated spring (Katepwa) and winter (Monopol) cultivars of wheat (Triticum aestivum L.). Low-temperature stress of either the spring or winter cultivar was associated with feedback-limited photosynthesis as indicated by a 50–60% reduction in CO₂ assimilation rates, twofold lower ATP/ADP ratio, and threefold lower electron transport rate than 20°C-grown control plants. However, no limitations were evident at the level of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) in low-temperature-stressed plants. Cold acclimation of the spring cultivar resulted in similar feedback-limited photosynthesis observed during low-temperature stress. In contrast, cold acclimation of the winter cultivar resulted in an adjustment of CO₂ assimilation rates to that of control plants. However, we show, for the first time, that this capacity to adjust CO₂ assimilation still appeared to be associated with limited triose phosphate utilisation, a twofold lower ATP/ADP ratio, a reduction in electron transport rates but no restriction at the level of Rubisco compared to controls grown at 20°C. Thus, contrary to previous suggestions, we conclude that cold-acclimated Monopol appears to exhibit feedback limitations at the level of electron transport characteristic of cold-stressed plants despite the maintenance of high rates of CO₂ assimilation. Furthermore, the differential capacity of the winter cultivar to adjust CO₂ assimilation rates was associated with higher levels of sucrose accumulation and a threefold higher sucrose-phosphate synthase activity despite an apparent limitation in triose phosphate utilisation.Abbreviations AGPase ADP-glucose pyrophosphorylase - FBPase fructose-1,6-bisphosphatase - Fru 6-P fructose 6-phosphate - Fru 1,6-BP fructose 1,6-bisphosphate - Glc 6-P glucose 6-phosphate - PGA 3-phosphoglyceric acid - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - RuBP ribulose 1,5-bisphosphate - SPS sucrose-phosphate synthase - Triose-P triose phosphate     

Regulation of ribulose 1,5-bisphosphate carboxylase-oxygenase activities by temperature pretreatment and chloroplast metabolites

Crystalline ribulose 1,5-bisphosphate carboxylase-oxygenase (EC 4.1.1.39) was isolated from tobacco (Nicotiana tabacum L.) leaf homogenates and the two competing reactions were examined for differential regulation in vitro by temperature pretreatment and chloroplast metabolites. Both the carboxylase and oxygenase activities were inactivated 50% by storing the dissolved protein at 0 °C and fully reactivated by heating the solution at 50 °C in the absence of Mg²⁺ and a sulfhydryl reagent. When the heat-activated enzyme was preincubated with physiological levels of various chloroplast metabolites and CO₂ and the two reactions were assayed simultaneously in the same reaction vessel upon initiation with ribulose 1,5-bisphosphate, three classes of effectors were observed: (a) those which stimulated both activities (NADPH, 6-phosphogluco-bisphosphate gluconate, fructose 1,6-bisphosphate, 3-phosphoglycerate glycerate), (b) those which essentially had no effect (fructose 6-phosphate, glucose 6-phosphate), and (c) one, ribose 5-phosphate, which inhibited the two reactions. However, within the limits of experimental error, none of the metabolites examined produced a differential regulation of the ribulose 1,5-bisphosphate carboxylase-oxygenase activities. The similar response of the two competing activities to temperature pretreatment and various chloroplast metabolites is consistent with the notion that both reactions are associated with the same or adjacent catalytic sites on this bifunctional enzyme.     

Control of photosynthetic sucrose synthesis by fructose-2,6-bisphosphate

The metabolite levels in the mesophyll of leaves of Zea mays L. have been compared with the regulatory properties of the cytosolic fructose-1,6-bisphosphatase from the mesophyll to show how withdrawal of triose phosphate for sucrose synthesis is reconciled with generation of the high concentrations of triose phosphate which are needed to allow intercellular diffusion of carbon during photosynthesis. i) A new technique is presented for measuring the intercellular distribution of metabolites in maize. The bundle-sheath and mesophyll tissues are partially separated by differential homogenization and filtration through nylon nets under liquid nitrogen. ii) considerable gradients of 3-phosphoglycerate, triose phosphate, malate and phosphoenolpyruvate exist between the mesophyll and bundle sheath which would allow intercellular shuttles to be driven by diffusion. These gradients could result from the distribution of electron transport and the Calvin cycle in maize leaves. iii) consequently, the mesophyll contains high concentrations of triose phosphate and fructose-1,6-bisphosphate. iv) Most of the regulator metabolite fructose-2,6-bisphosphate, is present in the mesophyll. v) The cytosolic fructose-1,6-bisphosphatase has a lower substrate affinity than that found for the enzyme from C₃ species, especially in the presence of inhibitors like fructose-2,6-bisphosphate. vi) This lowered affinity for substrate makes it possible to reconcile use of triose phosphate for sucrose synthesis with the maintenance of the high concentration of triose phosphate in the mesophyll needed for operation of photosynthesis in this species.Abbreviations DHAP Dihydroxyacetonephosphate - Fru1,6-bisP fructose-1,6-bisphosphate - Fru2,6bisP fructose-2,6-bisphosphate - PEP(Case) phosphoenolpyruvate (carboxylase) - PGA 3-phosphoglycerate - Rubisco ribulose-1,5-bisphosphate carboxylase