Products of photosynthetic 14CO2 fixation and related enzyme activities in fruiting structures of chickpea

Fruiting structures of a number of legumes including chickpea are known to carry out photosynthetic CO₂ assimilation, but the pathway of CO₂ fixation and particularly the role of phosphoenolpyruvate carboxylase (EC 4.1.1.31) in these tissues is not clear. Activities of some key enzymes of the Calvin cycle and C₄ metabolism, rates of ¹⁴CO₂ fixation in light and dark, and initial products of photosynthetic ¹⁴CO₂ fixation were determined in podwall and seedcoat (fruiting structures) and their subtending leaf in chickpea (Cicer arietinum L.). Compared to activities of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) and other Calvin cycle enzyme, viz. NADP⁺-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13), NAD⁺-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) and ribulose-5-phosphate kinase (EC 2.7.1.19), the levels of phosphoenolpyruvate carboxylase and other enzymes of C₄ metabolism viz. NADP⁺-malate dehydrogenase (EC 1.1.1.82), NAD⁺-malate dehydrogenase (EC 1.1.1.37), NADP⁺ malic enzyme (EC 1.1.1.40), NAD⁺-malic enzyme (EC 1.1.1.39), glutamate oxaloacetate transaminase (EC 2.6.1.1) and glutamate pyruvate transaminase (EC 2.6.1.2), were generally much higher in podwall and seedcoat than in the leaf. Podwall and seedcoat fixed ¹⁴CO₂ in light and dark at much higher rates than the leaf. Short-term assimilation of ¹⁴CO₂ by illuminated fruiting structures produced malate as the major labelled product with less labelling in 3-phosphoglycerate, whereas the leaf showed a major incorporation into 3-phosphoglycerate. It seems likely that the fruiting structures of chickpea utilize phosphoenolpyruvate carboxylase for recapturing the respired carbon dioxide.     

Effects of temperature and CO2 enrichment on kinetic properties of NADP+-malate dehydrogenase in two ecotypes of Barnyard grass (Echinochloa crus-galli (L.) Beauv.) from contrasting climates

Summary The apparent energy of activation (E _a), Michaelis-Menten constant (K _mfor oxaloacetate), V _max/K _mratios and specific activities of NADP⁺-malate dehydrogenase (NADP⁺-MDH; EC 1.1.1.82) were analyzed in plants of Barnyard grass from Québec (QUE) and Mississippi (MISS) acclimated to two thermoperiods 28/22°C, 21/15°C, and grown under two CO₂ concentrations, 350 l l^-1 and 675 l l^-1. E _avalues of NADP⁺-MDH extracted from QUE plants were significantly lower than those of MISS plants. K _mvalues and V _max/K _mratios of the enzyme from both ecotypes were similar over the range of 10–30°C but reduced V _max/K _mratios were found for the enzyme of QUE plants at 30 and 40°C assays. MISS plants had higher enzyme activities when measured on a chlorophyll basis but this trend was reversed when activities were expressed per fresh weight leaf or per leaf surface area. Activities were significantly higher in plants of both populations acclimated to 22/28°C. CO₂ enrichment did not modify appreciably the catalytic properties of NADP⁺-MDH and did not have a compensatory effect upon catalysis or enzyme activity under cool acclimatory conditions. NADP⁺-MDH activities were always in excess of the amount required to support observed rates of CO₂ assimilation and these two parameters were significantly correlated. The enhanced photosynthetic performance of QUE plants under cold temperature conditions, as compared to that of MISS plants, cannot be attributed to kinetic differences of NADP⁺-malate dehydrogenase among these ecotypes.     

Pod photosynthesis and seed dark CO2 fixation support oil synthesis in developingBrassica seeds

Rate of photosynthesis and activities of photosynthetic carbon reduction cycle enzymes were determined in pods (siliqua), whereas rate of dark CO₂ fixation, oil content and activities of enzymes involved in dark CO₂ metabolism were measured in seeds ofBrassica campestris L. cv. Toria at different stages of pod/seed development. The period between 14 and 35 days after anthesis corresponded to active phase of seed development during which period, seed dry weight and oil content increased sharply. Rate of pod photosynthesis and activities of photosynthetic carbon reduction cycle enzymes were maximum in younger pods but sufficiently high levels were retained up to 40 days after anthesis. The rate of dark¹⁴CO₂ fixation in seeds increased up to 21 days after anthesis and declined thereafter but maintaining sufficiently high rates till 35 days after anthesis. Similarly various enzymes viz., phosphoenolpyruvate carboxylase, NAD⁺-malate dehydrogenase and NADP⁺-malic enzyme, involved in dark CO₂ metabolism retained sufficient activities during the above period. These enzyme activities were more than adequate to maintain the desired supply of malate which mainly arises from dark CO₂ fixation in seeds and further translocated to leucoplasts for onward synthesis of fatty acids. Enzyme localization experiments revealed phosphoenolpyruvate carboxylase and enzymes of sucrose metabolism to be present only in cytosol, whereas enzymes of glycolysis were present both in cytosolic and leucoplastic fractions. These results indicated that oil synthesis in developingBrassica seeds is supported by pod photosynthesis and dark CO₂ fixation in seeds as the former serves as the source of sucrose and the latter as a source of malate     

Photosynthetic carbon metabolism in isolated maize bundle sheath strands

Photosynthetically active bundle sheath strands capable of assimilating up to 8 micromoles CO₂ per milligram chlorophyll per hour have been isolated from fully expanded leaves of Zea mays L. Mesophyll cell contamination of the preparations was negligible, as evidenced by light and electron microscopy and by a high ratio of chlorophyll a to chlorophyll b in the strands. Ribose 5-phosphate markedly stimulated the rate of photosynthetic ¹⁴CO₂ fixation by the isolated strands. In contrast, both pyruvate and phosphoenolpyruvate had a comparatively small stimulatory effect on bundle sheath ¹⁴CO₂ fixation. After 5 minutes of photosynthesis in ¹⁴C-bicarbonate, 95% of the incorporated ¹⁴C was found in compounds other than C₄-dicarboxylic acids, most notably in 3-phosphoglycerate and sugar phosphates. A similar distribution of ¹⁴C was observed in the presence of exogenous ribose 5-phosphate. Extracts of bundle sheath strands contained high specific activities of “malic” enzyme, phosphoglycolate phosphatase, hydroxypyruvate reductase, and ribulose 1,5-diphosphate carboxylase, whereas the specific activities of NADP⁺-malate dehydrogenase and phosphopyruvate carboxylase were extremely low. These results indicate that the Calvin cycle occurs in the bundle sheath cells of maize.     

Photosynthetic Plasticity in Flaveria brownii: Growth Irradiance and the Expression of C(4) Photosynthesis

Photosynthesis was examined in leaves of Flaveria brownii A. M. Powell, grown under either 14% or 100% full sunlight. In leaves of high light grown plants, the CO₂ compensation point and the inhibition of photosynthesis by 21% O₂ were significantly lower, while activities of ribulose 1,5-bisphosphate carboxylase/oxygenase and various C₄ cycle enzymes were considerably higher than those in leaves grown in low light. Both the CO₂ compensation point and the degree of O₂ inhibition of apparent photosynthesis were relatively insensitive to the light intensity used during measurements with plants from either growth conditions. Partitioning of atmospheric CO₂ between Rubisco of the C₃ pathway and phosphoenolpyruvate carboxylase of the C₄ cycle was determined by exposing leaves to ¹⁴CO₂ for 3 to 16 seconds, and extrapolating the labeling curves of initial products to zero time. Results indicated that ~94% of the CO₂ was fixed by the C₄ cycle in high light grown plants, versus ~78% in low light grown plants. Thus, growth of F. brownii in high light increased the expressed level of C₄ photosynthesis. Consistent with the carbon partitioning patterns, photosynthetic enzyme activities (on a chlorophyll basis) in protoplasts from leaves of high light grown plants showed a more C₄-like pattern of compartmentation. Pyruvate, Pi dikinase and phosphoenolpyruvate carboxylase were more enriched in the mesophyll cells, while NADP-malic enzyme and ribulose 1,5-bisphosphate carboxylase/oxygenase were relatively more abundant in the bundle sheath cells of high light than of low light grown plants. Thus, these results indicate that F. brownii has plasticity in its utilization of different pathways of carbon assimilation, depending on the light conditions during growth.     

Photosynthetic CO(2) Fixation Products and Activities of Enzymes Related to Photosynthesis in Bermudagrass and Other Plants

After a 5-second exposure of illuminated bermudagrass (Cynodon dactylon L. var. `Coastal') leaves to ¹⁴CO₂, 84% of the incorporated ¹⁴C was recovered as aspartate and malate. After transfer from ¹⁴CO₂-air to ¹²CO₂-air under continuous illumination, total radioactivity decreased in aspartate, increased in 3-phosphoglyceric acid and alanine, and remained relatively constant in malate. Carbon atom 1 of alanine was labeled predominantly, which was interpreted to indicate that alanine was derived from 3-phosphoglyceric acid. The activity of phosphoenolpyruvate carboxylase, alkaline pyrophosphatase, adenylate kinase, pyruvate-phosphate dikinase, and malic enzyme in bermudagrass leaf extracts was distinctly higher than those in fescue (Festuca arundinacea Schreb.), a reductive pentose phosphate cycle plant. Assays of malic enzyme activity indicated that the decarboxylation of malate was favored. Both malic enzyme and NADP⁺-specific malic dehydrogenase activity were low in bermudagrass compared to sugarcane (Saccharum officinarum L.). The activities of NAD⁺-specific malic dehydrogenase and acidic pyrophosphatase in leaf extracts were similar among the plant species examined, irrespective of the predominant cycle of photosynthesis. Ribulose-1, 5-diphosphate carboxylase in C₄-dicarboxylic acid cycle plant leaf extracts was about 60%, on a chlorophyll basis, of that in reductive pentose phosphate cycle plants.     

Effects of temperature and CO2 enrichment on kinetic properties of phospho-enol-pyruvate carboxylase in two ecotypes of Echinochloa crus-galli (L.) Beauv., a C4 weed grass species

Summary Two populations of Echinochloa crus-galli (Québec, Mississippi) were grown at the Duke University Phytotron under 2 thermoperiods (28°/22°C, 21°/15°C day/night) and 2 CO₂ regimes (350 and 675 l l^-1). Thermostability, energy of activation (E _a ),K _m (PEP), K _m (Mg⁺⁺), and specific activity of phospho-enol-pyruvate carboxylase (PEP_c) were analyzed in partially purified enzyme preparations of plants grown for 5 weeks. Thermostability of PEP_c from extracts (in vitro) and leaves (in situ) was significantly higher in Mississippi plants. In vitro denaturation was not appreciably modified by thermal acclimation but CO₂ enrichment elicited higher thermostability of PEP_c. In situ thermostability was significantly higher than that of in vitro assays and was higher in Mississippi plants acclimated at 28°/22°C and in plants of the two ecotypes grown at 675 l l^-1 CO₂. E _a (Q ₁₀ 30°/20°C) for PEP_c was significantly lower in Québec plants as compared to Mississippi and no acclimatory shifts were observed. Significantly higher K _m's (PEP) in 20°C assays were obtained for Mississippi as compared to Québec plants but values were similar at 30°C and 40°C assays. K _m (Mg⁺⁺) decreased at higher assay temperatures and were significantly lower for PEP_c of the Québec ecotype. No significant changes in K _m (Mg⁺⁺) values were associated with modifications in temperature on CO₂ regimes. PEP_c activity measured at 30°C was significantly higher for Québec plants when measured on a leaf fresh weight, leaf area or protein basis but not on a chlorophyll basis. Significantly higher PEP_c activity for both genotypes was observed for plants acclimated at 21°/15°C or grown at 675 l l^-1 CO₂. Net photosynthesis (Ps) and net assimilation rates (NAR) were higher in Québec plants and were enhanced by CO₂ enrichment. NAR was higher in plants acclimated at low temperature, while an opposite trend was observed for Ps. PEP_c activities were always in excess of the amounts required to support observed rates of CO₂ assimilation.     

Thermal stability and kinetic properties of NADP+-malate dehydrogenase isomorphs in two populations of the C4 weed species Echinohloa crus-galli (Barnyard grass) from sites of contrasting climates

The thermal stability and kinetic properties of purified NADP⁺-malate dehydrogenase (NADP⁺-MDH; EC 1.1.1.82) isomorphs were analyzed from plants of two populations of Barnyard grass from contrasting thermal environments. Plants from Québec (QUE) and Mississippi (MISS) were acclimated under controlled conditions at 26/20°C and 14/8°C (day/night). While the enzyme from QUE showed one isomorph, 3 isomorphs were detected in all plants from MISS, suggesting the presence of gene duplication and fixed heterozygosity for the expression of this dimeric enzyme. This findig raises the possibility that the enhanced acclimatory potential of NADP⁺-MDH from MISS plants, as found from previous studies with the partially purified and unfractioned enzyme, may result from differential kinetic properties of isomorphs which would allow for the proper modulation of catalysis over a wide temperature range. The thermal stability of the QUE isomorph was significantly higher than that of any of the MISS isomorphs. The apparent activation energy of the QUE isomorph was within the range of values found for the 3 MISS isomorphs which were similar to each other. The Michaelis-Menten constants (K_m) for oxalacetic acid were not significantly different among isomorphs or between thermoperiods, but K_m (NADP⁺) values for the QUE isomorph were significantly higher than those of two of the MISS isomorphs over the 15–25°C assay range V_max/K_m ratios for OAA and NADP⁺ were not significantly different among isomorphs or between thermoperiods. Our data indicate that, under highly purified conditions, the single NADP⁺-MDH isomorph of QUE plants possesses good acclimatory potential for maintaining catalytic efficiency under a wide range of temperature conditions. In vitro thermal and kinetic data do not support the hypothesis that the the multiple NADP⁺-MDH isomorphs found in MISS plants may have been selected to optimize the thermal and catalytic efficiency of NADP⁺-MDH under warm temperature conditions.     

Low night temperature effect on photosynthate translocation of two C4 grasses

Summary Translocation of assimilates in plants of Echinochloa crus-galli, from Quebec and Mississippi, and of Eleusine indica from Mississippi was monitored, before and after night chilling, using radioactive tracing with the short-life isotope ¹¹C. Plants were grown at 28°/22°C (day/night temperatures) under either 350 or 675 l·l^-1 CO₂. Low night temperature reduced translocation mainly by increasing the turn-over times of the export pool. E. crus-galli plants from Mississippi were the most susceptible to chilling; translocation being completely inhibited by exposure for one night to 7°C at 350 l·l^-1 CO₂. Overall, plants from Quebec were the most tolerant to chilling-stress. For plants of all three populations, growth under CO₂ enrichment resulted in higher ¹¹C activity in the leaf phloem. High CO₂ concentrations also seemed to buffer the transport system against chilling injuries.     

Carbon isotope fractionation in plants

Plants with the C₃, C₄, and crassulacean acid metabolism (CAM) photosynthetic pathways show characteristically different discriminations against ¹³C during photosynthesis. For each photosynthetic type, no more than slight variations are observed within or among species. CAM plants show large variations in isotope fractionation with temperature, but other plants do not. Different plant organs, subcellular fractions and metabolises can show widely varying isotopic compositions. The isotopic composition of respired carbon is often different from that of plant carbon, but it is not currently possible to describe this effect in detail. The principal components which will affect the overall isotope discrimination during photosynthesis are diffusion of CO₂, interconversion of CO₂ and HCO^?₃, incorporation of CO₂ by phosphoenolpyruvate carboxylase or ribulose bisphosphate carboxylase, and respiration. Theisotope fractionations associated with these processes are summarized. Mathematical models are presented which permit prediction of the overall isotope discrimination in terms of these components. These models also permit a correlation of isotope fractionations with internal CO₂ concentrations. Analysis of existing data in terms of these models reveals that CO₂ incorporation in C₃ plants is limited principally by ribulose bisphosphate carboxylase, but CO₂ diffusion also contributes. In C₄ plants, carbon fixation is principally limited by the rate of CO₂ diffusion into the leaf. There is probably a small fractionation in C₄ plants due to ribulose bisphosphate carboxylase.     

Metabolism of epidermal tissues, mesophyll cells, and bundle sheath strands resolved from mature nutsedge leaves

Mesophyll cells and bundle sheath strands were isolated from Cyperus rotundus L. leaf sections infiltrated with a mixture of cellulase and pectinase followed by a gentle mortar and pestle grind. The leaf suspension was filtered through a filter assembly and mesophyll cells and bundle sheath strands were collected on 20-μm and 80-μm nylon nets, respectively. For the isolation of leaf epidermal strips longer leaf cross sections were incubated with the enzymes and gently ground as above. Loosely attached epidermal strips were peeled off with forceps. The upper epidermis, which lacks stomata, could be clearly distinguished from the lower epidermis which contains stomata. Microscopic evidence for identification and assessment of purity is provided for each isolated tissue.Enzymes related to the C₄-dicarboxylic acid cycle such as phosphoenolpyruvate carboxylase, malate dehydrogenase (NADP⁺), pyruvate, P_i dikinase were found to be localized, ≥98%, in mesophyll cells. Enzymes related to operating the reductive pentose phosphate cycle such as RuDP carboxylase, phosphoribulose kinase, and malic enzyme are distributed, ≥99%, in bundle sheath strands. Other photosynthetic enzymes such as aspartate aminotransferase, pyrophosphatase, adenylate kinase, and glyceraldehyde 3-P dehydrogenase (NADP⁺) are quite active in both mesophyll and bundle sheath tissues.Enzymes involved in photorespiration such as RuDP oxygenase, catalase, glycolate oxidase, hydroxypyruvate reductase (NAD⁺), and phosphoglycolate phosphatase are preferentially localized, ≥84%, in bundle sheath strands.Nitrate and nitrite reductase can be found only in mesophyll cells, while glutamate dehydrogenase is present, ≥96%, in bundle sheath strands.Starch- and sucrose-synthesizing enzymes are about equally distributed between the mesophyll and bundle sheath tissues, except that the less active phosphorylase was found mainly in bundle sheath strands. Fructose-1,6-diP aldolase, which is a key enzyme in photosynthesis and glycolysis leading to sucrose and starch synthesis, is localized, ≥90%, in bundle sheath strands. The glycolytic enzymes, phosphoglyceromutase and enolase, have the highest activity in mesophyll cells, while the mitochondrial enzyme, cytochrome c oxidase, is more active in bundle sheath strands.The distribution of total nutsedge leaf chlorophyll, protein, and PEP carboxylase activity, using the resolved leaf components, is presented. ¹⁴CO₂ Fixation experiments with the intact nutsedge leaves and isolated mesophyll and bundle sheath tissues show that complete C₄ photosynthesis is compartmentalized into mesophyll CO₂ fixation via PEP carboxylase and bundle sheath CO₂ fixation via RuDP carboxylase. These results were used to support the proposed pathway of carbon assimilation in C₄-dicarboxylic acid photosynthesis and to discuss the individual metabolic characteristics of intact mesophyll cells, bundle sheath cells, and epidermal tissues.     

Light-dependent net CO-evolution by C3 and C4 plants

Light-dependent CO-evolution by the green leaves of C₃ and C₄ plants depends on the CO₂/O₂ ratio in the ambient atmosphere. This and other physiological responses suggest that CO-evolution is a byproduct of photorespiration. At CO₂/O₂ ratios up to 10^-3, the ratio of CO evolved: CO₂ fixed in photosynthesis is significantly higher in C₃ than in C₄ plants. This discrepancy disappears when a correction is made for the CO₂-concentrating mechanism in C₄ photosynthesis, by which CO₂-concentration at the site of ribulose-bis-phosphate carboxylase/oxygenase in the bundle sheaths is raised significantly as compared to the ambient atmosphere. Since the oxygenase function of this enzyme is responsible for glycolate synthesis, i.e., the substrate of photorespiration, this result seems to support the conclusion that CO-evolution is a consequence of photorespiration. CO-evolution may turn out to be a useful and rather straightforward indicator for photorespiration in ecophysiological studies.Abbreviations CAM crassulacean acid metabolism - CO net CO-evolution - CO₂ net CO₂-fixation - PEP-C phosphoenolpyruvate carboxylase - RubP-C ribulose-bisphosphate carboxylase/oxygenase Dedicated to Professor André Pirson on the occasion of his 70th birthday     

Carbonic anhydrase activity in leaves and its role in the first step of c(4) photosynthesis

In C₄ plants carbonic anhydrase catalyzes the critical first step of C₄ photosynthesis, the hydration of CO₂ to bicarbonate. The maximum activity of this enzyme in C₄ leaf extracts, measured by H⁺ production with saturating CO₂ and extrapolated to 25°C, was found to be 3,000 to 10,000 times the maximum photosynthesis rate for these leaves. Similar activities were found in C₃ leaf extracts. However, the calculated effective activity of this enzyme at in vivo CO₂ concentrations was apparently just sufficient to prevent the rate of conversion of CO₂ to HCO₃⁻ from limiting C₄ photosynthesis. This conclusion was supported by the mass spectrometric determination of leaf carbonic anhydrase activities.     

NADP-Utilizing Enzymes in the Matrix of Plant Mitochondria

Purified potato tuber (Solanum tuberosum L. cv Bintie) mitochondria contain soluble, highly latent NAD⁺- and NADP⁺-isocitrate dehydrogenases, NAD⁺- and NADP⁺-malate dehydrogenases, as well as an NADPH-specific glutathione reductase (160, 25, 7200, 160, and 16 nanomoles NAD(P)H per minute and milligram protein, respectively). The two isocitrate dehydrogenase activities, but not the two malate dehydrogenase activities, could be separated by ammonium sulfate precipitation. Thus, the NADP⁺-isocitrate dehydrogenase activity is due to a separate matrix enzyme, whereas the NADP⁺-malate dehydrogenase activity is probably due to unspecificity of the NAD⁺-malate dehydrogenase. NADP⁺-specific isocitrate dehydrogenase had much lower K_ms for NADP⁺ and isocitrate (5.1 and 10.7 micromolar, respectively) than the NAD⁺-specific enzyme (101 micromolar for NAD⁺ and 184 micromolar for isocitrate). A broad activity optimum at pH 7.4 to 9.0 was found for the NADP⁺-specific isocitrate dehydrogenase whereas the NAD⁺-specific enzyme had a sharp optimum at pH 7.8. Externally added NADP⁺ stimulated both isocitrate and malate oxidation by intact mitochondria under conditions where external NADPH oxidation was inhibited. This shows that (a) NADP⁺ is taken up by the mitochondria across the inner membrane and into the matrix, and (b) NADP⁺-reducing activities of malate dehydrogenase and the NADP⁺-specific isocitrate dehydrogenase in the matrix can contribute to electron transport in intact plant mitochondria. The physiological relevance of mitochondrial NADP(H) and soluble NADP(H)-consuming enzymes is discussed in relation to other known mitochondrial NADP(H)-utilizing enzymes.     

Inorganic Carbon Diffusion between C(4) Mesophyll and Bundle Sheath Cells: Direct Bundle Sheath CO(2) Assimilation in Intact Leaves in the Presence of an Inhibitor of the C(4) Pathway

Photosynthesis rates of detached Panicum miliaceum leaves were measured, by either CO₂ assimilation or oxygen evolution, over a wide range of CO₂ concentrations before and after supplying the phosphoenolpyruvate (PEP) carboxylase inhibitor, 3,3-dichloro-2-(dihydroxyphosphinoyl-methyl)-propenoate (DCDP). At a concentration of CO₂ near ambient, net photosynthesis was completely inhibited by DCDP, but could be largely restored by elevating the CO₂ concentration to about 0.8% (v/v) and above. Inhibition of isolated PEP carboxylase by DCDP was not competitive with respect to HCO₃⁻, indicating that the recovery was not due to reversal of enzyme inhibition. The kinetics of ¹⁴C-incorporation from ¹⁴CO₂ into early labeled products indicated that photosynthesis in DCDP-treated P. miliaceum leaves at 1% (v/v) CO₂ occurs predominantly by direct CO₂ fixation by ribulose 1,5-bisphosphate carboxylase. From the photosynthesis rates of DCDP-treated leaves at elevated CO₂ concentrations, permeability coefficients for CO₂ flux into bundle sheath cells were determined for a range of C₄ species. These values (6-21 micromoles per minute per milligram chlorophyll per millimolar, or 0.0016-0.0056 centimeter per second) were found to be about 100-fold lower than published values for mesophyll cells of C₃ plants. These results support the concept that a CO₂ permeability barrier exists to allow the development of high CO₂ concentrations in bundle sheath cells during C₄ photosynthesis.     

Anapleurotic CO(2) Fixation by Phosphoenolpyruvate Carboxylase in C(3) Plants

The role of phosphoenolpyruvate carboxylase in photosynthesis in the C₃ plant Nicotiana tabacum has been probed by measurement of the ¹³C content of various materials. Whole leaf and purified ribulose bisphosphate carboxylase are within the range expected for C₃ plants. Aspartic acid purified following acid hydrolysis of this ribulose bisphosphate carboxylase is enriched in ¹³C compared to whole protein. Carbons 1-3 of this aspartic acid are in the normal C₃ range, but carbon-4 (obtained by treatment of the aspartic acid with aspartate β-decarboxylase) has an isotopic composition in the range expected for products of C₄ photosynthesis (−5‰), and it appears that more than half of the aspartic acid is synthesized by phosphoenolpyruvate carboxylase using atmospheric CO₂/HCO₃⁻. Thus, a primary role of phosphoenolpyruvate carboxylase in C₃ plants appears to be the anapleurotic synthesis of four-carbon acids.     

Effect of phosphinothricin (glufosinate) on photosynthesis and photorespiration of C3 and C4 plants

Phosphinothricin (glufosinate), an irreversible inhibitor of glutamine synthetase, causes an inhibition of photosynthesis in C₃ (Sinapis alba) and C₄ (Zea mays) plants under atmospheric conditions (400 ppm CO₂, 21% O₂). This photosynthesis inhibition is proceeding slower in C₄ leaves. Under non-photorespiratory conditions (1000 ppm CO₂, 2% O₂) there is no inhibition of photosynthesis. The inhibition of glutamine synthetase by phosphinothricin results in an accumulation of NH₄ ⁺. The NH₄ ⁺-accumulation is lower in C₄ plants than in C₃ plants. The inhibition of glutamine synthetase through phosphinothricin in mustard leaves results in a decrease in glutamine, glutamate, aspartate, asparagine, serine, and glycine. In contrast to this, a considerable increase in leucine and valine following phosphinothricin treatment is measured. With the addition of either glutamine, glutamate, aspartate, glycine or serine, photosynthesis inhibition by phosphinothricin can be reduced, although the NH₄ ⁺-accumulation is greatly increased. This indicates that NH₄ ⁺-accumulation cannot be the primary cause for photosynthesis inhibition by phosphinothricin. The investigations demonstrate the inhibition of transmination of glyoxylate to glycine in photorespiration through the total lack of amino donors. This could result in a glyoxylate accumulation inhibiting ribulose-1,5-bisphosphate-carboxylase and consequently CO₂-fixation.Abbreviations GOGAT glutamine-2-oxoglutarate-amidotransferase - GS glutamine synthetase - PPT phosphinothricin - MSO methionine sulfoximine - RuBP ribulose-1,5-bisphosphate     

Photosynthetic characteristics of mesophyll eells isolated from sunflower (helianthus annuus L.) leaves

Mesophyll cells were isolated from sunflower leaves by an enzymic procedure. The cell suspensions possessed high photosynthesis rates. The products of cell photosynthesis were similar to the products of leaf disc photosynthesis. The relatively high radioactivity incorporated into malate after ¹⁴CO₂ feeding suggests that PEP carboxylase might participate in CO₂ fixation. Sunflower leaf extracts possessed a PEP carboxylase activity slightly higher than that of other C₃ species. Inhibition of PEP carboxylase by maleate decreased cell photosynthesis by only 15% and the first products of cell photosynthesis were phosphorylated compounds. It is concluded that the high photosynthesis rates displayed by sunflower are not due to a parallel C₄ pathway of photosynthesis but are rather dependent, at least in part, on the activity, or the amount, of RuBP carboxylase.Abbreviations PVP polyvinylpyrrolidone - PDS potassium dextran sulfate - DTT dithiothreitol - PEG polyethyleneglycol - RuBP ribulose 1,5-bisphosphate - PEP phosphoenolpyruvate - Mes 2-(N-morpholino) ethanesulfonic acid - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid     

On the significance of C3—C4 intermediate photosynthesis to the evolution of C4 photosynthesis

Abstract Evidence is drawn from previous studies to argue that C₃—C₄ intermediate plants are evolutionary intermediates, evolving from fully-expressed C₃ plants towards fully-expressed C₄ plants. On the basis of this conclusion, C₃—C₄ intermediates are examined to elucidate possible patterns that have been followed during the evolution of C₄ photosynthesis. An hypothesis is proposed that the initial step in C₄-evolution was the development of bundle-sheath metabolism that reduced apparent photorespiration by an efficient recycling of CO₂ using RuBP carboxylase. The CO₂-recycling mechanism appears to involve the differential compartmentation of glycine decarboxylase between mesophyll and bundle-sheath cells, such that most of the activity is in the bundlesheath cells. Subsequently, elevated phosphoenolpyruvate (PEP) carboxylase activities are proposed to have evolved as a means of enhancing the recycling of photorespired CO₂. As the activity of PEP carboxylase increased to higher values, other enzymes in the C₄-pathway are proposed to have increased in activity to facilitate the processing of the products of C₄-assimilation and provide PEP substrate to PEP carboxylase with greater efficiency. Initially, such a ‘C₄-cycle’ would not have been differentially compartmentalized between mesophyll and bundlesheath cells as is typical of fully-expressed C₄ plants. Such metabolism would have limited benefit in terms of concentrating CO₂ at RuBP carboxylase and, therefore, also be of little benefit for improving water- and nitrogen-use efficiencies. However, the development of such a limited C₄-cycle would have represented a preadaptation capable of evolving into the leaf biochemistry typical of fully-expressed C₄ plants. Thus, during the initial stages of C₄-evolution it is proposed that improvements in photorespiratory CO₂-loss and their influence on increasing the rate of net CO₂ assimilation per unit leaf area represented the evolutionary ‘driving-force’. Improved resourceuse efficiency resulting from an efficient CO₂-concentrating mechanism is proposed as the driving force during the later stages.     

Role of Photorespiration and Cyclic Electron Transport in C<Subscript>4</Subscript> Photosynthesis Evolution in the C<Subscript>3</Subscript>–C<Subscript>4</Subscript> Intermediate Species <Emphasis Type="Italic">Sedobassia sedoides</Emphasis>

Plants from two Sedobassia sedoides (Pall.) Aschers populations (Makan and Valitovo) (Chenopodiaceae) with C₂ photosynthesis (precursor of C₄ photosynthesis in phylogenesis) and photorespiratory CO₂-concentrating mechanism were studied. Genetic polymorphism and isotope discrimination (δ¹³С) levels of the plants were determined under natural conditions, and their morpho-physiological parameters such as fresh and dry biomass of the above ground parts of plants, functioning of photosystem I (PSI) and photosystem II (PSII), intensity of net photosynthesis (A), transpiration (E), photorespiration and water use efficiency (WUE) of plants were calculated under control and salinine conditions (0 and 200 mM NaCl). Results of the population-genetic analysis showed that the Makan population is polymorphic (plastic) and the Valitovo population is monomorphic (narrowly specialized). There were no significant differences between the populations based on δ¹³С values or growth parameters, PSII, A, E and WUE under control conditions. Under saline conditions, dry biomass accumulation decreased in the Makan population by 15% and by more than 2- fold in the Valitovo population. Population differences were revealed in terms of photorespiration intensity and P700 oxidation kinetics under control and saline conditions. Under control conditions, Makan plants were characterized by a higher photorespiration intensity, which decreased by 2-fold under saline conditions to the photorespiration level of Valitovo plants. Cyclic electron transport activity was minimal in the control Makan plants, and it increased by almost 2-fold under saline conditions to the level of that in Valitovo plants under control and saline conditions. Under control conditions, photosynthesis in Makan plants can be specified as the proto-Kranz type (transitional type from C₃ to C₂) and that in Valitovo plants can be specified as the C₂ type (C₄ photosynthesis with photorespiratory CO₂-concentrating mechanism), based on their photorespiration level and cyclic electron transport activity. Under saline conditions, Makan plants exhibited features of C₂ photosynthesis. Intraspecific functional differences of photosynthesis were revealed in different populations of intermediate C₃–C₄ plant species S. sedoides which reflect the initial stages of formation of a photorespiratory CO₂-concentrating mechanism during C₄ photosynthesis evolution, accompanied by decrease in salt tolerance.