Relative Contribution of Ribulose 1,5-Diphosphate Carboxylase and Phosphoenolpyruvate Carboxylase to CO2 Fixation Activity in Roots of Dark-grown or Light-grown Lens culinaris Seedlings

The pathway of carbon assimilation in greening roots was compared to the pathway in leaves of Lens culinaris seedlings by means of labelling distribution analysis among the products of ¹⁴CO₂ fixation in vivo, and in vitro with ribulose 1,5-diphosphate as the substrate. In green leaves, CO₂ fixation via ribulose 1,5-diphosphate carboxylase predominated largely while, in green roots, this carboxylase activity and the phosphoenolpyruvate carboxylase contributed almost equally to the whole in vivo CO₂ fixation. A participation of the activities of both carboxylases according to the double carboxylation pathway in the synthesis of dicarboxylic acids (malate and aspartate) was demonstrated in vitro after 48 h of greening in roots but seemed to be absent in in vivo experiments.     

The Effect of Sodium Chloride on the Balance between the C3- and C4-Carbon Fixation Pathways

Young leaves of salt-depleted Aeluropus litoralis Parl. plants show CO₂ fixation by the C₃-carbon fixation pathway. No detectable activity of phosphoenol pyruvate (PEP) carboxylase was found. When A. litoralis plants were exposed to a NaCl solution, the leaves showed a high activity of PEP carboxylase as well as a significant CO₂ fixation by the C₄-pathway. — Also in Zea mays L. and Chloris gayana Kunth., the presence of NaCl in the medium influences the balance between phosphoenol pyruvate carboxylase and ribulose-1,5-diphosphate carboxylase.     

Crassulacean acid metabolism (CAM) in Kalanchoë daigremontiana: Temperature response of phosphoenolpyruvate (PEP)-carboxylase in relation to allosteric effectors

Net CO₂ dark fixation of Kalanchoë daigremontiana varies with night temperature. We found an optimum of fixation at about 15° C; with increasing night temperature fixation decreased. We studied the temperature dependence of the activity of phosphoenolpyruvate (PEP)-carboxylase, the key enzyme for CO₂ dark fixation. We varied the pH, the substrate concentration (PEP), and the L-malate and glucose-6-phosphate (G-6-P) concentration in the assay. Generally, lowering the pH and reducing the amount of substrate resulted in an increase in activation by G-6-P and in an increase in malate inhibition of the enzyme. Furthermore, malate inhibition and G-6-P activation increased with increasing temperature. Activity measurements between 10° C and 45°C at a given concentration of the effectors revealed that the temperature optimum and maximum activities at that optimum varied with the effector applied. Under the influence of 5 mol m^-3 L-malate the temperature optimum and maximum activity dropped drastically, especially when the substrate level was low (at 0.5 mol m^-3 PEP from 32° C to 20° C). G-6-P raised the temperature optimum and maximum activity when the substrate level was low. If both malate and G-6-P were present, intermediate values were measured. We suggest that changes in metabolite levels in K. daigremontiana leaves can alter the temperature features of PEP-carboxylase so that the observed in vivo CO₂ dark fixation can be explained on the basis of PEP-carboxylase activity.Abbreviations PEP-c phosphoenolpyruvate carboxylase - CAM crassulacean acid metabolism - PEP phosphoenolpyruvate - G-6-P glucose-6-phosphate     

Carbon Dioxide Fixation by Lupin Root Nodules: I. Characterization, Association with Phosphoenolpyruvate Carboxylase, and Correlation with Nitrogen Fixation during Nodule Development

In vivo CO₂ fixation and in vitro phosphoenolpyruvate (PEP) carboxylase levels have been measured in lupin (Lupinus angustifolius L.) root nodules of various ages. Both activities were greater in nodule tissue than in either primary or secondary root tissue, and increased about 3-fold with the onset of N₂ fixation. PEP carboxylase activity was predominantly located in the bacteroid-containing zone of mature nodules, but purified bacteroids contained no activity. Partially purified PEP carboxylases from nodules, roots, and leaves were identical in a number of kinetic parameters. Both in vivo CO₂ fixation activity and in vitro PEP carboxylase activity were significantly correlated with nodule acetylene reduction activity during nodule development. The maximum rate of in vivo CO₂ fixation in mature nodules was 7.9 nmol hour⁻¹ mg fresh weight⁻¹, similar to rates of N₂ fixation and reported values for amino acid translocation.     

Reduction in chlorophyll content without a corresponding reduction in photosynthesis and carbon assimilation enzymes in yellow-green oil yellow mutants of maize

The photosynthetic properties of a yellow lethal mutant, Oy/oy, and two yellow-green mutants of maize which are allelic (a homozygous recessive oy/oy and a heterozygous dominant Oy/+) were examined. Although Oy/oy had little or no chlorophyll or capacity for CO₂ fixation compared to normal siblings, it had 28% as much ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) activity, and from 40% to near normal activities of C₄ cycle enzymes.Both yellow-green mutants had only half as much chlorophyll per leaf area as normal green seedlings in greenhouse-grown plants in winter and spring. However, the absorbance of light by the mutants was relatively high, as their transmittance was only 5 to 8% greater than normal leaves. In winter-grown greenhouse plants, the activities of Rubisco and several C₄ cycle enzymes in the mutants were unaffected and similar to those of normal seedlings on a leaf area basis. After allowing for small differences in leaf absorbance, the light response curves for photosynthesis in the mutants were similar on a leaf area basis but much higher on a chlorophyll basis than those of the normal seedlings. In spring-grown greenhouse plants the enzyme activities and photosynthesis rates were about 30% lower per leaf area in the yellow-green mutant leaves compared to the wild type. The maximum carboxylation efficiency (measured under low CO₂ and 1000 mol quanta m^-2 s^-1) in the mutants and normal leaves was similar on a Rubisco protein basis. The results indicate that maize can undergo a 50% reduction in chlorophyll content without a corresponding reduction in enzymes of carbon assimilation, and still maintain a high capacity for photosynthesis.Abbreviations Chl chlorophyll - PEP phosphoenolypruvate - Rubisco ribulose-1,5-bisphosphate carboxylase oxygenase This research was supported by CSIRO and by USDA Competitive Grant 86-CRCR-1-2036.     

Inhibition of the circadian rhythm of CO2 metabolism in Bryophyllum leaves by cycloheximide and dinitrophenol

The circadian rhythm of CO₂ output in darkened leaves of Bryophyllum fedtschenkoi R. Hamet and Perrier can be inhibited by cycloheximide (10^-6 mol) and 2,4-dinitrophenol (10^-5 mol) applied via the transpiration stream. After having been suppressed by 10^-6 M cycloheximide, the rhythm can be reinitiated with a 12-h exposure to light. Experiments using ¹⁴CO₂ show that cycloheximide abolishes the rhythm by inhibiting the dark fixation of CO₂. Cycloheximide inhibits malate accumulation and acidification of the leaves, but does not affect the amount of the CO₂-fixing enzyme phosphoenol-pyruvate carboxylase (PEP-C, EC 4.1.1.31) which can be extracted from the leaves during the 45 h of the experiment. Cycloheximide has no direct effect on the activity of the enzyme as measured in the assay. PEP-C from desalted leaf extracts was inhibited by L-malate (K_i=0.4 mmol). The most likely explanation for the inhibitory effect of cycloheximide and dinitrophenol is that they cause changes in tonoplast properties which result in a redistribution of malate from the vacuole to the cytoplasm. An increase in malate concentration in the cytoplasm will lead to inhibition of PEP-carboxylase, and hence the suppression of the rhythm of CO₂ output.Abbreviations CAM crassulacean acid metabolism - PEP-C phosphoenol-pyruvate carboxylase - MDH malate dehydrogenase - CHM cycloheximide - DNP 2,4-dinitrophenol - LD light-dark-cycle - DD continuous darkness     

Alfalfa root nodule carbon dioxide fixation : I. Association with nitrogen fixation and incorporation into amino acids

In vivo CO₂ fixation activity and in vitro phosphoenolpyruvate carboxylase activity were demonstrated in effective and ineffective nodules of alfalfa (Medicago sativa L.) and in the nodules of four other legume species. Phosphoenolpyruvate carboxylase activity was greatly reduced in nodules from both host and bacterially conditioned ineffective alfalfa nodules as compared to effective alfalfa nodules.

Forage harvest and nitrate application reduced both in vivo and in vitro CO₂ fixation activity. By day 11, forage harvest resulted in a 42% decline in in vitro nodule phosphoenolpyruvate carboxylase activity while treatment with either 40 or 80 kilograms nitrogen per hectare reduced activity by 65%. In vitro specific activity of phosphoenolpyruvate carboxylase and glutamate synthase were positively correlated with each other and both were positively correlated with acetylene reduction activity.

The distribution of radioactivity in the nodules of control plants (unharvested, 0 kilograms nitrogen per hectare) averaged 73% into the organic acid and 27% into the amino acid fraction. In nodules from harvested plants treated with nitrate, near equal distribution of radioactivity was observed in the organic acid (52%) and amino acid (48%) fractions by day 8. Recovery to control distribution occurred only in those nodules whose in vitro phosphoenolpyruvate carboxylase activity recovered.

The results demonstrate that CO₂ fixation is correlated with nitrogen fixation in alfalfa nodules. The maximum rate of CO₂ fixation for attached and detached alfalfa nodules at low CO₂ concentrations (0.13-0.38% CO₂) were 18.3 and 4.9 nanomoles per hour per milligram dry weight, respectively. Nodule CO₂ fixation was estimated to provide 25% of the carbon required for assimilation of symbiotically fixed nitrogen in alfalfa.

     

The Effects of Shoot Water Status on Some Photosynthetic Partial Processes in Sitka Spruce

Clonal cuttings of Picea sitchensis (Bong.) Carr. were grown in a controlled environment and, after completion of shoot extension and maturation, subjected to a drying cycle. Photosynthesis and stomatal conductance were measured in situ using ¹⁴CO₂ and a porometer, respectively. Shoot water potential was measured with the pressure chamber. Photosystem and carboxylase activities of chloroplast preparations were measured in vitro. A considerable fall in photosynthetic rate occurred at low water potential. This was associated with stomatal closure and a decrease in CO₂ transfer or fixation processes in the mesophyll. Little change in activity of photosystem I, photosystem II, and ribulose 1,5-diphosphate carboxylase was detected during the drying cycle. Any decline in activity of the photosynthetic partial processes in vivo under severe water stress (Ψ < – 30 bar) was probably masked in vitro as a result of rehydration prior to assay.     

CO(2) Assimilation and Activities of Photosynthetic Enzymes in High Chlorophyll Fluorescence Mutants of Maize Having Low Levels of Ribulose 1,5-Bisphosphate Carboxylase

Photosynthetic properties were examined in several hcf (high chlorophyll fluorescence 11, 21, 42 and 45) nuclear recessive mutants of maize which were previously found to have normal photochemistry and low CO₂ fixation. Mutants usually either died after depletion of seed reserves (about 18 days after planting), or survived with slow growth up to 7 or 8 weeks. Both the activity and quantity of ribulose 1,5-bisphosphate carboxylase (Rubisco) were low in the mutants (5-25% of the normal siblings on a leaf area basis) and the loss of Rubisco tended to parallel the reduction in photosynthetic capacity. The Rubisco content in the mutants was often marginal for photosynthetic carbon gain, with some leaves and positions along a leaf having no net photosynthesis, while other leaves had a low carbon gain. Conversely, the activities of C₄ cycle enzymes, phosphoenolpyruvate carboxylase, pyruvate, Pi dikinase, NADP-malate dehydrogenase, and NADP-malic enzyme, were the same or only slightly reduced compared to the normal siblings. The mutants had about half as much chlorophyll content per leaf area as the normal green plants. However, the Rubisco activity in the mutants was low on both a leaf area and chlorophyll basis. Low Rubisco activity and lower chlorophyll content may both contribute to the low rates of photosynthesis in the mutants on a leaf area basis.     

Diurnal modulation of phosphoenolpyruvate carboxylation in pea leaves and roots as related to tissue malate concentrations and to the nitrogen source

Phosphoenolpyruvate (PEP) carboxylation was measured as dark ¹⁴CO₂ fixation in leaves and roots (in vivo) or as PEP carboxylase (PEPCase) activity in desalted leaf and roof extracts (in vitro) from Pisum sativum L. cv. Kleine Rheinländerin. Its relation to the malate content and to the nitrogen source (nitrate or ammonium) was investigated. In tissue from nitrate-grown plants, PEP carboxylation varied diurnally, showing an increase upon illumination and a decrease upon darkening. Diurnal variations in roots were much lower than in leaves. Fixation rates in leaves remained constantly low in continuous darkness or high in continuous light. Dark CO₂ fixation of leaf slices also decreased when leaves were preilluminated for 1 h in CO₂-free air, suggesting that the modulation of dark CO2 fixation was related to assimilate availability in leaves and roots. Phosphoenolpyruvate carboxylase activity was also measured in vitro. However, no difference in maximum enzyme activity was found in extracts from illuminated or darkened leaves, and the response to substrate and effectors (PEP, malate, glucose-6-phosphate, pH) was also identical. The serine/threonine protein kinase inhibitors K252b, H7 and staurosporine, and the protein phosphatase 2A inhibitors okadaic acid and cantharidin, fed through the leaf petiole, did not have the effects on dark CO₂ fixation predicted by a regulatory system in which PEPCase is modulated via reversible protein phosphorylation. Therefore, it is suggested that the diurnal modulation of PEP carboxylation in vivo in leaves and roots of pea is not caused by protein phosphorylation, but rather by direct allosteric effects. Upon transfer of plants to ammonium-N or to an N-free nutrient solution, mean daily malate levels in leaves decreased drastically within 4–5 d. At that time, the diurnal oscillations of PEP carboxylation in vivo disappeared and rates remained at the high light-level. The coincidence of the two events suggests that PEPCase was de-regulated because malate levels became very low. The drastic decrease of leaf malate contents upon transfer of plants from nitrate to ammonium nutrition was apparently not caused by increased amino acid or protein synthesis, but probably by higher decarboxylation rates.Abbreviations CAM crassulacean acid metabolism - PEP Phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase - PP protein phosphatase - PK protein kinase This work was supported by the Deutsche Forschungsgemeinschaft. B. Baur was a recipient of a doctoral grant, and L. Leport recipient of a post-doctoral grant of the DFG. The skilled technical assistance of Eva Wirth and Maria Lesch is gratefully acknowledged.     

Regulation of Soybean Net Photosynthetic CO(2) Fixation by the Interaction of CO(2), O(2), and Ribulose 1,5-Diphosphate Carboxylase

Kinetic properties of soybean net photosynthetic CO₂ fixation and of the carboxylase and oxygenase activities of purified soybean (Glycine max [L.] Merr.) ribulose 1, 5-diphosphate carboxylase (EC 4.1.1.39) were examined as functions of temperature, CO₂ concentration, and O₂ concentration. With leaves, O₂ inhibition of net photosynthetic CO₂ fixation increased when the ambient leaf temperature was increased. The increased inhibition of CO₂ fixation at higher temperatures was caused by a reduced affinity of the leaf for CO₂ and an increased affinity of the leaf for O₂. With purified ribulose 1,5-diphosphate carboxylase, O₂ inhibition of CO₂ incorporation and the ratio of oxygenase activity to carboxylase activity increased with increased temperature. The increased O₂ sensitivity of the enzyme at higher temperature was caused by a reduced affinity of the enzyme for CO₂ and a slightly increased affinity of the enzyme for O₂. The similarity of the effect of temperature on the affinity of intact leaves and of ribulose 1,5-diphosphate carboxylase for CO₂ and O₂ provides further evidence that the carboxylase regulates the O₂ response of photosynthetic CO₂ fixation in soybean leaves. Based on results reported here and in the literature, a scheme outlining the stoichiometry between CO₂ and O₂ fixation in vivo is proposed.     

Ribulose-1,5-bisphosphate carboxylase and phosphoenolpyruvate carboxylase activities of photoautotrophic callus of Platycerium coronarium (Koenig ex O.F. Muell.) Desv. under CO2 enrichment

The in vitro activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC) were measured in cell-free extracts of Platycerium coronarium callus cultured for up to 42 days under photoautotrophic conditions with CO₂ enrichment. With an increase in CO₂ in the culture environment to 10% (v/v) at low light, the apparent photoautotrophic fixation of CO₂ by Rubisco declined, whereas the non-photoautotrophic CO₂ fixation by PEPC activity was enhanced. Hence, photosynthesis appears to play a lesser role in providing carbon skeletons and energy with prolonged culture in a CO₂-enriched environment. Instead, the anaplerotic supply of C-skeletons by PEPC may be important under such a situation. Short-term H¹⁴CO₃-fixation experiments indicated that photoautotrophic callus cultured for 3 weeks with 10% CO₂ enrichment assimilated less ¹⁴CO₂ than the control (0.03% CO₂). Analyses of ¹⁴C-metabolites indicated that about 50% of the total soluble ¹⁴CO₂ fixed was in the organic acid fraction and 35% in the amino acid fraction. Despite the changes in the in vitro Rubisco/PEPC activity-ratio, no significant change in the ¹⁴C distribution pattern was apparent in response to increasing sucrose or CO₂ concentrations. The suppression of Rubisco activity and total chlorophyll content in high sucrose or elevated CO₂ concentrations suggests an inhibition of the capacity for photoautotrophic callus growth under these conditions. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of temperature on the activity of phosphoenolpyruvate carboxylase and on the control of CO2 fixation in Bryophyllum fedtschenkoi

The phosphorylation state and the malate sensitivity of phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) in Bryophyllum fedtschenkoi Hamet et Perrier are altered by changes in the ambient temperature. These effects, in turn alter the in-vivo activity of the enzyme. Low temperature (3 °C or less), stabilizes the phosphorylated form of the enzyme, while high temperature (30 °C) promotes its dephosphorylation. The catalytic activity of the phosphorylated and dephosphorylated forms of PEPCase increases with temperature, but the apparent K _i values for malate of both forms of the enzyme decrease. Results of experiments with detached leaves maintained in darkness in normal air indicate that the changes in malate sensitivity and phosphorylation state of PEPCase with temperature are of physiological significance. When the phosphorylated form of PEPCase is stabilized by reducing the temperature of leaves 9 h after transfer to constant darkness at 15 °C, a prolonged period of CO₂ fixation follows. When leaves are maintained in constant darkness at 15 °C until CO₂ output reaches a low steady-state level and the PEPCase is dephosphorylated, reducing the temperature to 3 °C results in a further period of CO₂ fixation even though the phosphorylation state of PEPCase does not change.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase We thank the Agricultural and Food Research Council for financial support for this work.     

Photosynthetic carbon metabolism during leaf ontogeny in Zea mays L.: Enzyme studies

The activities of several enzymes, including ribulose-1,5-diphosphate (RuDP) carboxylase (EC 4.1.1.39) and phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) were measured as a function of leaf age in Z. mays. Mature leaf tissue had a RuDP-carboxylase activity of 296.7 mol CO₂ g^-1 fresh weight h^-1 and a PEP-carboxylase activity of 660.6 mol CO₂ g^-1 fresh weight h^-1. In young corn leaves the activity of the two enzymes was 11 and 29%, respectively, of the mature leaves. In senescent leaf tissue, RuDP carboxylase activity declined more rapidly than that of any of the other enzymes assayed. On a relative basis the activities of NADP malic enzyme (EC 1.1.1.40), aspartate (EC 2.6.1.1) and alanine aminotransferase (EC 2.6.1.2), and NAD malate dehydrogenase (EC 1.1.1.37) exceeded those of both PEP and RuDP carboxylase in young and senescent leaf tissue. Pulse-chase labeling experiments with mature and senescent leaf tissue show that the predominant C₄ acid differs between the two leaf ages. Labeling of alanine in senescent tissue never exceeded 4% of the total ¹⁴C remaining during the chase period, while in mature leaf tissue alanine accounted for 20% of the total after 60 s in ¹²CO₂. The activity of RuDP carboxylase during leaf ontogeny in Z. mays parallels the development of the activity of this enzyme in C₃ plants.Abbreviations RuDP ribulose-1,5-diphosphate - PEP phosphoenol pyruvate - PGA 3-phosphoglycerate     

Acclimation to High CO(2) in Bean : Carbonic Anhydrase and Ribulose Bisphosphate Carboxylase

Young bean plants (Phaseolus vulgaris L. cv Seafarer) grew faster in air enriched with CO₂ (1200 microliters per liter) than in ambient CO₂ (330 microliters per liter). However, by 7 days when increases in overall growth (dry weight, leaf area) were visible, there was a significant decline (about 25%) in the leaf mineral content (N, P, K, Ca, Mg) and a drop in the activity of two enzymes of carbon fixation, carbonic anhydrase and ribulose 1,5-bisphosphate (RuBP) carboxylase under high CO₂. Although the activity of neither enzyme was altered in young, expanding leaves during the acclimation period, in mature leaves the activity of carbonic anhydrase was reduced 95% compared with a decline of 50% in ambient CO₂. The drop in RuBP carboxylase was less extreme with 40% of the initial activity retained in the high CO₂ compared with 50% in the ambient atmosphere. While CO₂ enrichment might alter the flow of carbon into the glycolate pathway by modifying the activities of carbonic anhydrase or RuBP carboxylase, there is no early change in the ability of photosynthetic tissue to oxidize glycolate to CO₂.     

Effect of sulphite on phosphoenolpyruvate carboxylase and malate formation in extracts of Zea mays

The effect of SO₃^2? on the activity of PEP-carboxylase and on subsequent malate formation has been studied in leaf extracts of Zea mays. PEP-carboxylase was assayed by incorporation of H¹⁴CO₃ - into oxaloacetate dinitrophenylhydrazone and by a spectrophotometric method. In contrast to ribulose diphosphate carboxylase, PEP-carboxylase was not inhibited by 10 mM SO₃^2? with respect to PEP. As was the case with ribulose diphosphate carboxylase, the activity of PEP-carboxylase was inhibited non-competitively with respect to Mg²⁺. However, the K_i value (84.5 mM) was found to be very high. With respect to HCO₃^?, like ribulose diphosphate carboxylase, PEP-carboxylase was inhibited competitively, but the K_i value (27 mM SO₃^2?) increased by about the same factor (× 9) as the K_m, (0·5 mM HCO₃^?) is decreased. This indicates that the replacement of HCO₃^? by SO₃^2?, common to both enzymes, is facilitated by decreasing the affinity of the enzyme for HCO₃^?. At substrate saturating conditions malate formation by the combined action of PEP-carboxylase and endogenous NADH-dependent malate dehydrogenase in leaf extracts was not inhibited by 10 mM SO₃^2?. Although the malate dehydrogenase is inhibited at this SO₃^2? concentration to about 85%, malate formation is unaffected, as PEP-carboxylase is the rate limiting step its turnover rate being only about 8% of NADH-dependent malate dehydrogenase.     

Photosynthesis and Activity of Ribulose Bisphosphate Carboxylase of Wheat and Maize Seedlings during and following Exposure to O(2)-Low, CO(2)-Free N(2)

Seven day old wheat and maize seedlings were exposed to 1300 or 2000 microeinsteins per square meter per second photosynthetically active radiation in CO₂-free air for 3 hours with either 1% O₂ in N₂ or N₂-only and then returned to normal air of 340 microliters per liter CO₂, 21% O₂ in N₂. Activity of the ribulose bisphosphate carboxylase and amount of the substrate, ribulose 1,5-bisphosphate, were measured during and following the CO₂-free treatments as was photosynthetic CO₂ fixation. Photoinhibition of photosynthesis was observed only with wheat seedlings following the N₂ only treatment. During the CO₂-free treatments, the levels of RuBP rose during all experiments except when wheat was photoinhibited. The activity of the ribulose bisphophate carboxylase, measured directly upon grinding the leaves, declined during the CO₂-free conditions. The carboxylase total activity increased in minutes in the leaf during and following the CO₂-free treatments. The specific activities of the wheat carboxylase went from 0.16 to 1.06 micromoles CO₂ fixed per milligram protein per minute while the maize carboxylase varied from 0.05 to 0.36 micromole CO₂ fixed per millogram protein per minute. This suggests that in these seedlings considerable inactive carboxylase must be stored in a form not activatable in extracts by CO₂ and Mg²⁺. Possible mechanisms of regulation of photosynthesis by the ribulose bisphosphate carboxylase must consider not only the amount of active enzyme, but the amount of enzyme which the plant can make activatable upon demand.     

Isolation and Characterization of High CO(2)-Requiring-Mutants of the Cyanobacterium Synechococcus PCC7942 : Two Phenotypes that Accumulate Inorganic Carbon but Are Apparently Unable to Generate CO(2) within the Carboxysome

A total of 24 high CO₂-requiring-mutants of the cyanobacterium Synechococcus PCC7942 have been isolated and partially characterized. These chemically induced mutants are able to grow at 1% CO₂, on agar media, but are incapable of growth at air levels of CO₂. All the mutants were able to accumulate inorganic carbon (C_i) to levels similar to or higher than wild type cells, but were apparently unable to generate intracellular CO₂. On the basis of the rate of C_i release following a light (5 minutes) → dark transition two extreme phenotypes (fast and slow release mutants) and a number of `intermediate' mutants (normal release) were identified. Compared to wild-type cells, Type I mutants had the following characteristics: fast C_i release, normal internal C_i pool, normal carbonic anhydrase (CA) activity in crude extracts, reduced internal exchange of ¹⁸O from ¹⁸O-labeled CO₂, 1% CO₂ requirement for growth in liquid media, normal affinity of carboxylase for CO₂, and long, rod-like carboxysomes. Type II mutants had the following characteristics: slow C_i release, increased internal C_i pool, normal CA activity in crude extracts, normal internal ¹⁸O exchange, a 3% CO₂ requirement for growth in liquid media, high carboxylase activity, normal affinity of carboxylase for CO₂, and normal carboxysome structure but increased in numbers per cell. Both mutant phenotypes appear to have genetic lesions that result in an inability to convert intracellular HCO₃⁻ to CO₂ inside the carboxysome. The features of the type I mutants are consistent with a scenario where carboxysomal CA has been mistargeted to the cytosol. The characteristics of the type II phenotype appear to be most consistent with a scenario where CA activity is totally missing from the cell except for the fact that cell extracts have normal CA activity. Alternatively the type II mutants may have a lesion in their capacity for H⁺ import during photosynthesis.     

Veränderliche Markierungsmuster bei 14CO2-Fütterung von Bryophyllum tubiflorum zu verschiedenen Zeitpunkten der Hell-Dunkelperiode

Summary The distribution of radioactivity between the products of ¹⁴CO₂ light fixation in phyllodia of Bryophyllum tubiflorum could be influenced experimentally by manipulating the malic acid content of the cells. Accelerating the deacidification of the tissue during the light period by application of higher light intensities accelerated the increase of malate labelling and the decrease of the sucrose labelling after ¹⁴CO₂ light fixation under our standard conditions (10 min preillumination, 15 min ¹⁴CO₂ light fixation, 8000 lux).In other experiments different malate contents of the tissues were induced by treating the phyllodia with different temperatures during the night period. In the morning, phyllodia with low malate content transferred most of the label into malate, and phyllodia with high malate content incorporated most of the ¹⁴C radioactivity into sugars. However, this was true only after preillumination of 1 hour. When the phyllodia fixed ¹⁴CO₂ without preillumination, no differences in the labelling patterns between acidified and non-acidified phyllodia could be observed.In experiments using leaf tissue slices of Bryophyllum daigremontianum we could again observe that malate was labelled more heavily in the deacidified tissue than in the acidified controls, with less radioactivity being transferred into phosphate esters and sugars. The rates of ¹⁴CO₂ light fixation were identical in tissue slices with high and low malate content. However, the rates of CO₂ dark fixation in the acidified samples were clearly lower than those in the deacidified ones. The low rate of CO₂ dark fixation in acidified samples could not be inhibited by an inhibitor of PEP-carboxylase as the high CO₂ dark fixation rate of the deacidified tissue could be inhibited.The results are discussed in relation to the feed back inhibition of PEP-carboxylase in vivo by malate. Compartmentation also seemed to be involved in controlling the flow of carbon during CO₂ light fixation in succulent tissue.     

Higher plant phosphoenolpyruvate carboxylase kinase is regulated at the level of translatable mRNA in response to light or a circadian rhythm

Phosphoenolpyruvate carboxylase is regulated by reversible phosphorylation in response to light in C₃ and C₄ plants and to a circadian oscillator in CAM plants. Increases in phosphoenolpyruvate carboxylase kinase activity require protein synthesis. This requirement has been analysed by quantifying translatable mRNA for this protein kinase using in vitro translation of isolated RNA followed by direct assay of kinase activity. In leaves of the CAM plant Bryophyllum (Kalanchoë) fedtschenkoi, in normal diurnal conditions, kinase mRNA was 20-fold more abundant at night than in the day. In constant environmental conditions (continuous darkness, CO₂-free air, 15°C) kinase mRNA exhibited circadian oscillations. The circadian disappearance of kinase mRNA and kinase activity was delayed by lowering the temperature to 4°C and accelerated by raising the temperature to 30°C. The appearance of kinase mRNA and activity was blocked by cordycepin and puromycin. In maize and barley, kinase mRNA increased in response to light. For all three plants, the phosphoenolpyruvate carboxylase kinase activity generated during in vitro translation was Ca²⁺-independent. These results demonstrate that phosphoenolpyruvate carboxylase kinase activity is regulated at the level of translatable mRNA in C₃, C₄ and CAM plants.