Gerald E. Edwards

Biochemical and cytological relationships in C4 plants

SummaryC4 plants can be divided into three groups based on differences in activities of three decarboxylating enzymes: NADP-malic enzyme, NAD-malic enzyme, and phosphopyruvate carboxykinase.In the Gramineae the three C4 groups are distinguished by anatomical and ultrastructural characteristics of bundle-sheath chloroplasts. NADP-malic enzyme species lack well-developed grana in bundle-sheath chloroplasts (grana reduced) and the bundle-sheath chloroplasts are in the centrifugal position. NAD-malic enzyme species have bundle-sheath chloroplasts in the centripetal position and contain grana. Phosphopyruvate carboxykinase species have bundle-sheath chloroplasts in the centrifugal position and they contain grana. NADP-malic enzyme species of the Gramineae have only been found in the subfamilies Aristidoideae and Panicoideae. With the exception of the genera Panicum, and Urochloa, NAD-malic enzyme species and phosphopyruvate carboxykinase species have only been found in the subfamily Eragrostoideae. C4 species of the genus Panicum are found among all three of the C4 groups.The dicotyledonous C4 species examined fall into two groups: those having high NADP-malic enzyme and those having high NAD-malic enzyme. No phosphopyruvate carboxykinase C4 species have been found among the dicotyledons. The NADP-malic enzyme C4 species of the dicotyledons like NADP-malic enzyme species of the Gramineae have bundle-sheath chloroplasts with reduced grana but in contrast to NADP-malic enzyme species of the Gramineae the bundle-sheath chloroplasts are in the centripetal position. The NAD-malic enzyme species of the dicotyledons like the NAD-malic enzyme species of the Gramineae have bundlesheath chloroplasts in the centripetal position with well developed grana.The results are discussed in terms of evolutionary and functional diversification of C4 plants.

Oxygen inhibition of photosynthesis

The effect of leaf temperature, O2 and calculated O2/CO2 solubility ratio in the leaf on the quantum yield of photosynthesis was studied for the C4 species, Zea mays L., and the C3 species, Triticum aestivum L. Over a range of leaf temperatures of 16 to 35° C, the quantum yield of Z. mays was relatively constant and was similar under 1.5 and 21% O2, being ca. 0.059 mol CO2 mol-1 quanta absorbed. Under 1.5% O2 and atmospheric levels of CO2, the quantum yield of T. aestivum was relatively constant (0.083 mol CO2 mol-1 quanta absorbed) at leaf temperatures from 15 to 35° C. Atmospheric levels of O2 (21%) reduced the quantum yield of photosynthesis in T. aestivum and as leaf temperature increased, the quantum yield decreased from 0.062 at 15°C to 0.046 mol CO2 mol-1 quanta absorbed at 35°C. Increasing temperature decreases the solubility of CO2 relatively more than the solubility of O2, resulting in an increased solubility ratio of O2/CO2. Experimentally manipulating the atmospheric levels of O2 or CO2 to maintain a near-constant solubility ratio of O2/CO2 at varying leaf temperatures largely prevented the temperature-dependent decrease in quantum yield in t. aestivum. Thus, the decrease in quantum yield with increasing leaf temperature in C3 species may be largely caused by a temperaturedependent change in the solubility ratio of O2/CO2.

Microstegium vimineum, a shade adapted C4 grass☆

Abstract Microstegium vimineum, a C4 plant of the family Poaceae, is shown to be extremely adaptable to growing under shade conditions. The capacity for dry matter production of the species was similar from 18% to 100% full sunlight. Even at 5% full sunlight substantial growth occurred (17% of that at full sunlight). In comparison, growth of the C4 species Digitaria sanguinalis and Sporobolus airoides declined rapidly below full sunlight with no growth occurring at 5% full sunlight. A number of measurements on leaves of M. vimineum grown under 5% of full sunlight versus full sunlight reflects the ability of this species to adapt to low light. Under low light there was a 2-fold increase in leaf area per leaf with an approx. 2-fold decrease in leaf thickness. The soluble protein/ chlorophyll ratio increased about 3.5-fold in the low light-grown plants due to an increase in chlorophyll/leaf area and a decrease in soluble protein/leaf area. Under both light regimes the plants had Kranz anatomy and high levels of phosphoenolpyruvate (PEP) carboxylase which are known features of C4 plants. The results indicate that there are no inherent limitations in the C4 mechanism or in the efficiency of energy utilization for carbon assimilation which prevents C4 plants from adapting to low light conditions.

Transport in C4 mesophyll chloroplasts. Characterization of the pyruvate carrier

Abstract 1. Evidence is presented for high rates of carrier-mediated uptake of pyruvate into the stroma of intact mesophyll chloroplasts of the C 4 plant Digitaria sanguinalis , but not the chloroplasts of the C 3 plant Spinacea oleracea . Uptake of pyruvate in the dark with the C 4 mesophyll chloroplasts was followed using two techniques: uptake of [ 14 C]pyruvate as determined by silicon oil centrifugal filtration and uptake as indicated by absorbance changes at 535 nm (shrinkage/swelling) after addition of 0.1 M pyruvate salts. 2. Uptake of the pyruvate anion by an electrogenic carrier is suggested to be the major mode of transport. Chloroplast swelling was observed in potassium pyruvate plus valinomycin and uptake of [ 14 C]pyruvate was inhibited by membrane-permeant anions. Valinomycin reduced uptake in the absence of external potassium and the inhibition could be reversed by addition of external potassium. 3. Uptake of pyruvic acid (or a pyruvate − /OH − antiport) is ruled unlikely since [ 14 C]pyruvate uptake was relatively independent of the pH gradient across the envelope and addition of pyruvate to chloroplasts did not result in an alkalization of the medium. The low rate of swelling observed in ammonium pyruvate may be due to non-mediated permeation of pyruvic acid, which is possible only at high pyruvate concentrations. 4. The concentration of pyruvate in the stroma increased with external concentration over the range tested (up to 40 mM) but the concentration ratio (internal/external) was always less than one. The steady-state concentration of [ 14 C]pyruvate in the stroma was dependent on the ionic strength of the medium, with saturation at roughly I = 0.04 M, while accumulation of the membrane-permeant cation tetraphenylmethylphosphonium decreased with increasing ionic strength. This suggests that ionic strength modifies a membrane potential (inside negative) across the envelope and that pyruvate uptake responds to the magnitude and direction of that potential (−80 mV at low ionic strength). 5. Chloride and inorganic phosphate were potent inhibitors of [ 14 C]pyruvate uptake. Of the sulfhydryl reagents tested, N -ethylmaleimide was not inhibitory while mersalyl completely blocked [ 14 C]pyruvate uptake and swelling in potassium pyruvate plus valinomycin. Pyruvate uptake, as measured by valinomycin induced swelling in potassium pyruvate, was highly temperature sensitive, with an energy of activation of 39 kcal/mol above 9 °C. 6. Phenylpyruvate, α-ketoisovalerate, α-ketoisocaproate, α-cyano-4-hydroxycinnamic acid and α-cyanocinnamic acid inhibited [ 14 C]pyruvate but not [ 14 C]-acetate uptake in the dark and also reduced pyruvate metabolism by the chloroplasts in the light.

Transport in C4 mesophyll chloroplasts. Evidence for an exchange of inorganic phosphate and phosphoenolpyruvate

1. Mesophyll chloroplasts of the C4 plant Digitaria sanguinalis contain endogenous phosphoenolpyruvate which appears to distribute across the envelope according to the existing pH gradient. The phosphoenolpyruvate remaining in the stroma can be rapidly released by external inorganic phosphate or 3-phosphoglycerate while external pyruvate did not affect the distribution. 2. Phosphoenolpyruvate (PEP) was a competitive inhibitor (Ki (PEP) = 450 micrometer) of 32Pi uptake (Km(Pi)=200 micrometer) by chloroplasts in the dark and also reduced the steady-state internal concentration of 32Pi, which is consistent with phosphate and phosphoenolpyruvate sharing a common carrier. 3. Phosphoenolpyruvate formation by chloroplasts in the light in the presence of pyruvate but in the absence of inorganic phosphate was slow and the concentration ratio of phosphoenolpyruvate (internal/external) was high. Addition of 0.1 mM phosphate induced a high rate of phosphoenolpyruvate formation and the concentration ratio (internal/external) decreased 15-fold. It is proposed that external phosphate is required both for phosphoenolpyruvate formation and efflux from the chloroplast.

Intracellular localization of certain photosynthetic enzymes in bundle sheath cells of plants possessing the C4 pathway of photosynthesis

Bundle sheath cells were enzymatically isolated from representatives of three groups of C4 plants: Zea mays (NADP malic enzyme type), Panicum miliaceum (NAD malic enzyme type), and Panicum maximum (phosphoenolpyruvate (PEP) carboxykinase type). Cellular organelles from bundle sheath homogenates were partially resolved by differential centrifugation and on isopycnic sucrose density gradients in order to study compartmentation of photosynthetic enzymes. A 48-h-dark pretreatment of the leaves allowed the isolation of relatively intact chloroplasts. Enzymes that decarboxylate C4 acids and furnish CO2 to the Calvin cycle are localized as follows: NADP malic enzyme, chloroplastic in Z. mays; NAD malic enzyme, mitochondrial in all three species; PEP carboxykinase, chloroplastic in P. maximum. The activity of NAD malic enzyme in the three species was in the order of P. miliaceum > P. maximum > Z. mays. There were high levels of aspartate and alanine aminotransferases in bundle sheath extracts of P. miliaceum and P. maximum and substantial activity in Z. mays. In all three species, aspartate aminotransferase was mitochondrial whereas alanine aminotransferase was cytoplasmic. Based on the activity and localization of certain enzymes, the concept for aspartate and malate as transport metabolites from mesophyll to bundle sheath cells in C4 species of the three C4 groups is discussed.

Intracellular localization of phosphoenolpyruvate carboxykinase in leaves of C4 and CAM plants

Abstract Three C 4 species of the genera Brachiaria and Urochloa from the family Gramineae and 4 CAM species from the families Asclepiadaceae, Bromeliaceae, and Liliaceae were studied for the intracellular localization of the decarboxylating enzyme phosphoenolpyruvate (PEP) carboxykinase. Bundle sheath protoplasts of the C 4 plants and mesophyll protoplasts of the CAM plants were isolated, purified, mechanically ruptured, and organelles separated by differential centrifugation. The chloroplasts were about 70 95% intact based on the retention of chloroplast marker enzymes. Most of the PEP carboxykinase was in the non-chloroplastic fraction (98 100% in C 4 and 80% or more in CAM). The enzyme was still largely non-particulate after centrifugation of protoplast extracts of Urochloa mosambicensis (C 4 ) and Ananas comosus (CAM) to pellet chloroplasts, mitochondria, and microbodies. These results suggest that PEP carboxykinase is localized mainly in the cytoplasm in both C 4 and CAM plants.

Activity and quantity of ribulose bisphosphate carboxylase- and phosphoenolpyruvate carboxylase-protein in two Crassulacean acid metabolism plants in relation to leaf age, nitrogen nutrition, and point in time during a day/night cycle

Activity of ribulose 1,5-bisphosphate (RuBP) carboxylase in leaf extracts of the constitutive Crassulacean acid metabolism (CAM) plant Kalanchoe pinnata (Lam.) Pers. decreased with increasing leaf age, whereas the activity of phosphoenolpyruvate (PEP) carboxylase increased. Changes in enzyme activities were associated with changes in the amount of enzyme proteins as determined by immunochemical analysis, sucrose density gradient centrifugation, and SDS gel electrophoresis of leaf extracts. Young developing leaves of plants which received high amounts of NO3-during growth contained about 30% of the total soluble protein in the form of RuBP carboxylase; this value declined to about 17% in mature leaves. The level of PEP carboxylase in young leaves of plants at high NO3-was an estimated 1% of the total soluble protein and increased to approximately 10% in mature leaves, which showed maximum capacity for dark CO2 fixation. The growth of plants at low levels of NO3-decreased the content of soluble protein per unit leaf area as well as the extractable activity and the percentage contribution of both RUBP carboxylase and PEP carboxylase to total soluble leaf protein. There was no definite change in the ratio of RuBP carboxylase to PEP carboxylase activity with a varying supply of NO3-during growth. It has been suggested (e.g., Planta 144, 143–151, 1978) that a rhythmic pattern of synthesis and degradation of PEP carboxylase protein is involved in the regulation of β-carboxylation during a day/night cycle in CAM. No such changes in the quantity of PEP carboxylase protein were observed in the leaves of Kalanchoe pinnata (Lam.) Pers. or in the leaves of the inducible CAM plant Mesembryanthemum crystallinum L.

Localization of the C4 and C3 pathways of photosynthesis in the leaves of Pennisetum purpureum and other C4 species. Insignificance of phenol oxidase

SummaryMesophyll protoplasts and bundle-sheath cells of Pennisetum purpureum Schum., a C4 plant with low phenol-oxidase activity, were enzymatically separated according to methods recently developed with sugarcane (Saccharum officinarum L.), maize (Zea mays L.), and sorghum (Sorghum bicolor L.). The phosphoenolpyruvate carboxylase and NADP-malic dehydrogenase of the C4 pathway were found to be localized in the mesophyll protoplasts while ribulose-1,5-diphosphate (RuDP) carboxylase, phosphoribulokinase and NADP-malic enzyme were localized in the bundle-sheath cells. The levels of these enzyme activities in the leaf extracts and in certain cellular preparations of P. purpureum are sufficient to account for the rate of photosynthesis in the leaf. These results on the activities and distribution of photosynthetic enzymes with P. purpureum preparations are consistent with our previous evidence for cellular separation of the C4 and the reductive pentose-phosphate pathways in C4 species.With chlorogenic acid as the substrate, P. purpureum, Setaria lutescens (Weigel) Hubb. and Panicum texanum Buckl. have relatively low phenol-oxidase activity, similar to that found in spinach (Spinacia oleracea L.); while sorghum, sugarcane, maize, Panicum capillare L. and P. miliaceum L. have relatively high phenoloxidase activity, similar to that in tobacco (Nicotiana tabacum L.). C4 species having high phenol-oxidase activity have substantial activity of the enzyme in both mesophyll and bundle-sheath extracts. Since phenol oxidase is found in both cell types it is not logical to expect preferential inhibition of RuDP carboxylase or other photosynthetic enzymes through phenol oxidation in mesophyll extracts, as has been previously suggested. When dithiothreitol and polyvinylpyrrolidone were included in the enzyme extraction medium, the activity of RuDP carboxylase increased 10% in P. purpureum and 59% in sugarcane leaf extracts.

C4 acid decarboxylation and CO2 donation to photosynthesis in bundle sheath strands and chloroplasts from species representing three groups of C4 plants

Abstract The mechanism of decarboxylation of C4 acids and carboxyl donation to the Calvin pathway was studied in bundle sheath strands and chloroplasts isolated from leaves of species representing three groups of C4 plants: Digitaria sanguinalis (NADP-malic enzyme type),Panicum miliaceum (NAD-malic enzyme type), and Eriochloa borumensis (phosphoenolpyruvate-carboxykinase type). High rates of 14CO2 fixation and HCO3− dependent O2 evolution were exhibited by bundle sheath strands and chloroplasts of P. miliaceum and E. borumensis, while those of D. sanguinalis (the NADP-malic enzyme type) required the addition of ribose 5-phosphate and malate for maximum activity. In all three species, 3-(3-4-dichlorophenyl)-1,1-dimethylurea inhibition of 14CO2 fixation was partially overcome by the addition of malate, suggesting that malate can serve as a source of reductive power for carbon assimilation. Decarboxylation of 4-14C-labeled C4 acids was followed in the presence of glyceraldehyde, an inhibitor of the Calvin pathway. A large light-dependent stimulation of malate decarboxylation occurred by the addition of 3-phosphoglycerate (6 m m ) with bundle sheath cells of all three groups and with bundle sheath chloroplasts of D. sanguinalis and E. borumensis. Although malate is suggested to be decarboxylated through the respective primary decarboxylase of each species, the 3-phosphoglycerate enhancement suggests that pyridine nucleotide reduction from malate oxidation is used during the reductive phase of the Calvin pathway. Bundle sheath strands from all three species decarboxylated aspartate with severalfold stimulation by the addition of either α-ketoglutarate (10 m m ) or pyruvate (1 m m ), with the lowest rate in D. sanguinalis. Bundle sheath chloroplasts from either of the three species could not decarboxylate aspartate. A light-independent decarboxylation of aspartate is suggested through NAD-malic enzyme in bundle sheath mitochondria in all three types of C4 species, the rates being highest in P. miliaceum. A light-dependent portion of aspartate decarboxylation in E. borumensis is suggested to be through phosphoenolpyruvate carboxykinase. C4. acid decarboxylation in all three species was inhibited by HCO3− (CO2 may be the active species). Consistent with the C4 acid decarboxylation studies, the bundle sheath strands of the three species gave light-dependent O2 evolution with aspartate or malate, the bundle sheath chloroplasts of D. sanguinalis and E. borumensis exhibited light-dependent O2 evolution only with malate, and the bundle sheath chloroplasts of P. miliaceum showed no C4 acid-dependent O2 evolution. In all three C4 types, malate decarboxylation, though by different decarboxylases, interacted with the Calvin pathway by providing both CO2 and reducing power, while aspartate decarboxylation provided only CO2.