Light-induced pH changes in leaves of C4 plants

Light-induced changes in the fluorescence of the pH-indicating dyes pyranine or 5-(and 6-)carboxy-2, 7-dichlorofluorescein (CDCF) which had been fed to leaves were examined to monitor cellular pH changes. After short-term feeding of pyranine (pK 7.3) to leaves of Amaranthus caudatus L., a NAD-malic-enzyme-type C₄ plant, vascular bundles and surrounding cells became fluorescent. Fluorescence emission from mesophyll cells required longer feeding times. In CO₂-free air, pyranine fluorescence increased much more on illumination after mesophyll cells had become fluorescent than when only the vascular bundles and the bundle sheath of Amaranthus leaves had been stained. After short feeding times and in the absence of actinic illumination, CO₂ decreased pyranine fluorescence very slowly in Amaranthus and rapidly in C₃ leaves. After prolonged feeding times, the extent of the light-dependent increase in pyranine fluorescence was several times greater in different C₄ plants than in C₃ species. The kinetics of the fluorescence changes were also remarkably different in C₃ and C₄ plants. Carbon dioxide (500 l · l^–1) suppressed the light-induced increase in pyranine fluorescence more in C₄ than in C₃ leaves. Light-dependent changes in light scattering, which are indicative of chloroplast energization, and in 410-nm transmission, which indicate chloroplast movement, differed kinetically from those of the changes in pyranine fluorescence. Available evidence indicated that light-dependent changes in pyranine fluorescence did not originate from the apoplast of leaf cells. Microscopic observation led to the conclusion that, after prolonged feeding times or prolonged incubation, changes in pyranine fluorescence emitted from C₄ leaves reflect pH changes mainly in the cytosol of mesophyll cells. A transient acidification reaction indicated by quenching of pyranine fluorescence in the dark-light transient and not observed in C₃ species is attributed to the carboxylation of phosphoenolpyruvate. After short feeding times and in the absence of actinic illumination, CO₂ (250 l l^–1) decreased pyranine fluorescence very slowly in Amaranthus and more rapidly in C₃ leaves. After prolonged feeding times, both the rate and the extent of CO₂-dependent quenching of pyranine fluorescence increased, but the increase was insufficient to indicate the presence of highly active carbonic anhydrase in the compartment from which pyranine fluorescence was emitted. In contrast to pyranine, CDCF (pK 4.8) did not increase but rather decreased its fluorescence on illumination of an Amaranthus leaf, indicating acidification of an acidic compartment, most probably the vacuole of green leaf cells. The pattern of the acidification reaction was similar in C₄ and C₃ leaves. The remarkably large extent of the light-dependent increase in pyranine fluorescence from leaves of C₄ species and its slow kinetics are proposed to be caused by an alkalization of the cytosol which in the absence of CO₂ is larger in the mesophyll than in the bundle sheath. It gives rise to deprotonation of dye originally located in the mesophyll and, in addition, of dye which diffuses from the bundle sheath into the mesophyll following a pH gradient. Implications of slow diffusional transport of pyranine and CO₂ between mesophyll and bundle-sheath cells and the fast metabolite transport required in C₄ photosynthesis are discussed.Abbreviations CDCF 5-(and 6-)carboxy-2,7-dichlorofluorescein - DHAP dihydroxyacetone phosphate - PGA 3-phosphoglycerate This work was supported by the Sonderforschungsbereiche 176 and 251 of the University of Würzburg and by the Gottfried-Wilhelm-Leibniz Program of the Deutsche Forschungsgemeinschaft. A.S.R. was the recipient of a fellowship of the Alexander-von-Humboldt Foundation. We are grateful to Mrs. S. Neimanis for cooperation.     

Light-dependent pH changes in leaves of C4 plants Comparison of the pH response to carbon dioxide and oxygen with that of C3 plants

Cytosolic and vacuolar pH changes caused by illumination or a changed composition of the gas phase were monitored in leaves of the NAD malic-enzyme-type C₄ plant Amaranthus caudatus L. and the C₃ plant Vicia faba L. by recording changes in the fluorescence of pH-indicating dyes which had been fed to the leaves. Light-dependent cytosolic alkalization and vacuolar acidification were maximal in the mesophyll cells under high-fluence-rate illumination and in the absence of CO₂. Under the same conditions, measurements of light scattering and electrochromic absorption changes at 518 nm revealed maximum thylakoid energization. The results show an intimate relationship between the energization of the photosynthetic apparatus by light, an increase in cytosolic pH and a decrease in vacuolar pH. This was true for both the C₄ and the C₃ plant, although kinetics, extent and even direction of cytosolic pH changes differed considerably in these plants, reflecting the differences in photosynthetic carbon metabolism. Darkening produced rapid acidification in Vicia, but not in Amaranthus. Continued alkalization in Amaranthus is interpreted to be the result of the decarboxylation of a C₄ intermediate and the release of liberated CO₂. In the presence of CO₂, energy consumption by carbon reduction decreased thylakoid energization, cytosolic alkalization and vacuolar acidification. Under low-fluence-rate illumination, thylakoid energization and light-dependent cytosolic and vacuolar pH changes were decreased in CO₂-free air compared with thylakoid energization and pH changes in 1% oxygen/99% nitrogen not only in the C₃ plant, but also in Amaranthus. Since oxygenation of ribulose bisphosphate initiates energy-consuming photorespiratory reactions in 21% oxygen, but not in 1% oxygen, this shows that photorespiratory reactions are active not only in the C₃ but also in the C₄ plant in the absence of external CO₂. Photorespiratory conditions appeared to decrease energization not only in the chloroplasts, but also in the cytosol. This is indicated by decreased transfer of protons from the cytosol into the vacuole, a process which is energy-dependent.Abbreviations CDCF 5-(and 6-)carboxy-2,7-dichlorofluorescein - P₇₀₀ electron-donor pigment in the reaction center of photosystem I - RuBP ribulose-1,5-bisphosphate This work was supported, within the framework of the Sonderforschungsbereiche 176 and 251 of the University of Würzburg, by the Gottfried-Wilhelm-Leibniz Program of the Deutsche Forschungsgemeinschaft. A.S.R. was the recipient of a fellowship from the Alexander-von-Humboldt-Foundation. We are grateful to Mr. Carsten Werner and Mrs. Spidola Neimanis for cooperation.     

Gas-exchange of ears of cereals in response to carbon dioxide and light

Data for the maximum carboxylation velocity of ribulose-1,5-biosphosphate carboxylase, V_m, and the maximum rate of whole-chain electron transport, J_m, were calculated according to a photosynthesis model from the CO₂ response and the light response of CO₂ uptake measured on ears of wheat (Triticum aestivum L. cv. Arkas), oat (Avena sativa L. cv. Lorenz), and barley (Hordeum vulgare L. cv. Aramir). The ratio J_m/V_m is lower in glumes of oat and awns of barley than it is in the bracts of wheat and in the lemmas and paleae of oat and barley. Light-microscopy studies revealed, in glumes and lemmas of wheat and in the lemmas of oat and barley, a second type of photosynthesizing cell which, in analogy to the Kranz anatomy of C₄ plants, can be designated as a bundle-sheath cell. In wheat ears, the CO₂-compensation point (in the absence of dissimilative respiration) is between those that are typical for C₃ and C₄ plants.A model of the CO₂ uptake in C₃–C₄ intermediate plants proposed by Peisker (1986, Plant Cell Environ. 9, 627–635) is applied to recalculate the initial slopes of the A(p^c) curves (net photosynthesis rate versus intercellular partial pressure of CO₂) under the assumptions that the J_m/V_m ratio for all organs investigated equals the value found in glumes of oat and awns of barley, and that ribulose-1,5-bisphosphate carboxylase is redistributed from mesophyll to bundle-sheath cells. The results closely match the measured values. As a consequence, all bracts of wheat ears and the inner bracts of oat and barley ears are likely to represent a C₃–C₄ intermediate type, while glumes of oat and awns of barley represent the C₃ type.Abbreviations A net photosynthesis rate (mol·m^-2·s^-1) - J_m maximum rate of whole-chain electron transport (mol·e^-·m^-2·s^-1) - p^c (bar) intercellular partial pressure of CO₂ - PEP phosphoenolpyruvate - PPFD photosynthetic photon flux density (mol quanta·m^-2·s^-1) - RuBPCase ribulose bisphosphate carboxylase/oxygenase - RuBP ribulose bisphosphate - V_m maximum carboxylation velocity of RuBPCase (mol·m^-2·s^-1) - T^* CO₂ compensation point in the absence of dissimilative respiration (bar)     

The roles of malate and aspartate in C4 photosynthetic metabolism of Flaveria bidentis (L.)

In C₄ grasses belonging to the NADP-malic enzyme-type subgroup, malate is considered to be the predominant C₄ acid metabolized during C₄ photosynthesis, and the bundle sheath cell chloroplasts contain very little photosystem-II (PSII) activity. The present studies showed that Flaveria bidentis (L.), an NADP-malic enzyme-type C₄ dicotyledon, had substantial PSII activity in bundle sheath cells and that malate and aspartate apparently contributed about equally to the transfer of CO₂ to bundle sheath cells. Preparations of bundle sheath cells and chloroplasts isolated from these cells evolved O₂ at rates between 1.5 and 2 mol · min^–1 · mg^–1 chlorophyll (Chl) in the light in response to adding either 3-phosphoglycerate plus HCO ₃ ^– or aspartate plus 2-oxoglutarate. Rates of more than 2 mol O₂ · min^–1 · mg^–1 Chl were recorded for cells provided with both sets of these substrates. With bundle sheath cell preparations the maximum rates of light-dependent CO₂ fixation and malate decarboxylation to pyruvate recorded were about 1.7 mol · min^–1 · mg^–1 Chl. Compared with NADP-malic enzyme-type grass species, F. bidentis bundle sheath cells contained much higher activities of NADP-malate dehydrogenase and of aspartate and alanine aminotransferases. Time-course and pulse-chase studies following the kinetics of radiolabelling of the C-4 carboxyl of C₄ acids from ¹⁴CO₂ indicated that the photosynthetically active pool of malate was about twice the size of the aspartate pool. However, there was strong evidence for a rapid flux of carbon through both these pools. Possible routes of aspartate metabolism and the relationship between this metabolism and PSII activity in bundle sheath cells are considered.Abbreviations DHAP dihydroxyacetone phosphate - NADP-ME(-type) NADP-malic enzyme (type) - NADP-MDH NADP-malate dehydrogenase - OAA oxaloacetic acid - 2-OG 2-oxoglutarate - PEP phosphoenolpyruvate - PGA 3-phosphoglycerate - Pi orthophosphate - Ru5P ribulose 5-phosphate     

Interspecific variation in assimilation of 14CO2 into C4 acids by leaves of C3, C4 and C3−C4 intermediate Flaveria species near the CO2 compensation concentration

The assimilation of ¹⁴CO₂ into the C₄ acids malate and aspartate by leaves of C₃, C₄ and C₃–C₄ intermediate Flaveria species was investigated near the CO₂ compensation concentration ^* in order to determine the potential role of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) in reducing photorespiration in the intermediates. Relative to air concentrations of CO₂, the proportion of CO₂ fixed by PEP carboxylase at ^* increased in all six C₃–C₄ intermediate species examined. However, F. floridana J.R. Johnston and F. ramosissima Klatt were shown to be markedly less responsive to reduced external CO₂, with only about a 1.6-fold enhancement of CO₂ assimilation by PEP carboxylase, as compared to a 3.0- to 3.7-fold increase for the other C₃–C₄ species examined, namely, F. linearis Lag., F. anomala B.L. Robinson, F. chloraefolia A. Gray and F. pubescens Rydb. The C₃ species F. pringlei Gandoger and F. cronquistii A.M. Powell exhibited a 1.5- and 2.9-fold increase in labeled malate and aspartate, respectively, at ^*. Assimilation of CO₂ by PEP carboxylase in the C₄ species F. trinervia (Spreng.) C. Mohr, F. australasica Hook., and the C₄-like species F. brownii A.M. Powell was relatively insensitive to subatmospheric levels of CO₂. The interspecific variation among the intermediate Flaverias may signify that F. floridana and F. ramosissima possess a more C₄-like compartmentation of PEP carboxylase and ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) between the mesophyll and bundle-sheath cells. Chasing recently labeled malate and aspartate with ¹²CO₂ for 5 min at ^* resulted in an apparent turnover of 25% and 30% of the radiocarbon in these C₄ acids for F. ramosissima and F. floridana, respectively. No substantial turnover was detected for F. linearis, F. anomala, F. chloraefolia or F. pubescens. With the exception of F. floridana and F. ramosissima, it is unlikely that enhanced CO₂ fixation by PEP carboxylase at the CO₂ compensation concentration is a major mechanism for reducing photorespiration in the intermediate Flaveria species. Moreover, these findings support previous related ¹⁴CO₂-labeling studies at air-levels of CO₂ which indicated that F. floridana and F. ramosissima were more C₄-like intermediate species. This is further substantiated by the demonstration that F. floridana PEP carboxylase, like the enzyme in C₄ plants, undergoes a substantial activation (2.2-fold) upon illuminating dark-adapted green leaves. In contrast, light activation was not observed for the enzyme in F. linearis or F. chloraefolia.Abbreviations and symbols PEP phosphoenolpyruvate - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - CO₂ compensation concentration - ^* a subatmospheric level of CO₂ approximating Published as Paper No. 8832, Journal Series, Nebraska Agricultural Research Division     

Incorporation of 14CO2 into C4 acids by leaves of C3-C4 intermediate and C3 species of Moricandia and Panicum at the CO2 compensation concentration

Comparative ¹⁴CO₂ pulse-¹²CO₂ chase studies performed at CO₂ compensation ()-versus air-concentrations of CO₂ demonstrated a four-to eightfold increase in assimilation of ¹⁴CO₂ into the C₄ acids malate and aspartate by leaves of the C₃-C₄ intermediate species Panicum milioides Nees ex Trin., P. decipiens Nees ex Trin., Moricandia arvensis (L.) DC., and M. spinosa Pomel at . Specifically, the distribution of ¹⁴C in malate and aspartate following a 10-s pulse with ¹⁴CO₂ increases from 2% to 17% (P. milioides) and 4% to 16% (M. arvensis) when leaves are illuminated at the CO₂ compensation concentration (20 l CO₂/l, 21% O₂) versus air (340 l CO₂/l, 21% O₂). Chasing recently incorporated ¹⁴C for up to 5 min with ¹²CO₂ failed to show any substantial turnover of label in the C₄ acids or in carbon-4 of malate. The C₄-acid labeling patterns of leaves of the closely related C₃ species, P. laxum Sw. and M. moricandioides (Boiss.) Heywood, were found to be relatively unresponsive to changes in pCO₂ from air to . These data demonstrate that the C₃-C₄ intermediate species of Panicum and Moricandia possess an inherently greater capacity for CO₂ assimilation via phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) at the CO₂ compensation concentration than closely related C₃ species. However, even at , CO₂ fixation by PEP carboxylase is minor compared to that via ribulosebisphosphate carboxylase (EC 4.1.1.39) and the C₃ cycle, and it is, therefore, unlikely to contribute in a major way to the mechanism(s) facilitating reduced photorespiration in the C₃-C₄ intermediate species of Panicum and Moricandia.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - PEP phosphoenolpyruvate - CO₂ compensation concentration - 3PGA 3-phosphoglycerate - SuP sugar monophosphates - SuP₂ sugar bisphosphates Published as Paper No. 8249, Journal Series, Nebraska Agricultural Research Division     

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     

Carbon-isotope discrimination by leaves of Flaveria species exhibiting different amounts of C3-and C4-cycle co-function

Carbon-isotope ratios were examined as ¹³C values in several C₃, C₄, and C₃–C₄ Flaveria species, and compared to predicted ¹³C, values generated from theoretical models. The measured ¹³C values were within 4 of those predicted from the models. The models were used to identify factors that contribute to C₃-like ¹³C values in C₃–C₄ species that exhibit considerable C₄-cycle activity. Two of the factors contributing to C₃-like ¹³C values are high CO₂ leakiness from the C₄ pathway and pi/pa values that were higher than C₄ congeners. A marked break occurred in the relationship between the percentage of atmospheric CO₂ assimilated through the C₄ cycle and the ¹³C value. Below 50% C₄-cycle assimialtion there was no significant relationship between the variables, but above 50% the ¹³C values became less negative. These results demonstrate that the level of C₄-cycle expression can increase from, 0 to 50% with little integration of carbon transfer from the C₄ to the C₃ cycle. As expression increaces above 50%, however, increased integration of C₃- and C₄-cycle co-function occurs.Abbreviations and symbols RuBP carboxylase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) - PEP carboxylase phosphoenolpyruvate carboxylase (EC 4.1.1.31) - pa atmospheric CO₂ partial pressure - pi intercellular CO₂ partial pressure - isotope ratio - quantum yield for CO₂ uptake     

Photon yield of O2 evolution and chlorophyll fluorescence characteristics at 77 K among vascular plants of diverse origins

Photon yields of oxygen evolution at saturating CO₂ were determined for 44 species of vascular plants, representing widely diverse taxa, habitats, life forms and growth conditions. The photonyield values on the basis of absorbed light ( ^a) were remarkably constant among plants possessing the same pathway of photosynthetic CO₂ fixation, provided the plants had not been subjected to environmental stress. The mean ^a value ±SE for 37 C₃ species was 0.106±0.001 O₂·photon^-1. The five C₄ species exhibited lower photon yields and greater variation than the C₃ species ( ^a=0.0692±0.004). The ^a values for the two Crassulaceanacid-metabolism species were similar to those of C₃ species. Leaf chlorophyll content had little influence on ^a over the range found in normal, healthy leaves. Chlorophyll fluorescence characteristics at 77 K were determined for the same leaves as used for the photon-yield measurements. Considerable variation in fluorescence emission both at 692 nm and at 734 nm, was found 1) among the different species; 2) between the upper and lower surfaces of the same leaves; and 3) between sun and shade leaves of the same species. By contrast, the ratio of variable to maximum fluorescence emission at 692 nm (F_v/F_M, 692) remained remarkably constant (The mean value for the C₃ species was 0.832±0.004). High-light treatments of shade leaves resulted in a reduction in both ^a and the F_v/F_M, 692 ratio. The extent of the reductions increased with time of exposure to bright light. A linear relationship was obtained when ^a was plotted against F_v/F_M, 692. The results show that determinations of the photon yield of O₂ evolution and the F_v/F_M, 692 ratio can serve as excellent quantitative measures of photoinhibition of overall photosynthetic energy-conversion system and of photochemistry of photosystem II, respectively. This is especially valuable in field work where it is often impossible to obtain appropriate controls.Abbreviations and symbols CAM Crassulacean acid metabolism - PFD photon flux density (photon fluence rate) - PSI, PSII photosystem I, II - F_o, F_M, F_v instantaneous, maximum, variable fluorescence emission - absorptance - ^a photon yield (absorbed light) - ⁱ photon yield (incident light) C.I.W.-D.P.B. Publication No. 923     

Evidence for a light-dependent system for reassimilation of photorespiratory CO2, which does not include a C4 cycle,in the C3−C4 intermediate species Moricandia arvensis

Three methods of estimating photorespiratory rate in leaves of the C₃–C₄ intermediate species Moricandia arvensis and the related C₃ species Moricandia moricandioides were compared. The results indicated that the photorespiratory rate in M. arvensis is less than in M. moricandioides, and that this is caused partly by reduced carbon flux through the photorespiratory pathway, and partly by the presence of a mechanism for enhanced photorespiratory CO₂ reassimilation in the intermediate species. Measurements of the CO₂ compensation point () in the two species supported this conclusion. A functional C₄ pathway is unlikely to be involved in the reduction of photorespiratory rate in M. arvensis since pulse-chase experiments showed that carbon did not move from C₄ acids to the reductive pentose-phosphate pathway in attached leaves under steady-state conditions at .Abbreviations and symbols APR apparent photosynthetic rate - C_i, C_e intercellular, external CO₂ concentration - CO₂ compensation point - PAR photosynthetically active radiation - PFD photon flux density     

Stomatal responses to carbon dioxide of isolated epidermis from a C3 plant,the Argenteum mutant of Pisum sativum L., and a crassulacean-acid-metabolism plant Kalanchoë daigremontiana Hamet et Perr

The response of stomata in isolated epidermis to the concentration of CO₂ in the gaseous phase was examined in a C₃ species, the Argenteum mutant of Pisum sativum, and a crassulacean-acid-metabolism (CAM) species, Kalanchoë daigremontiana. Epidermis from leaves of both species was incubated on buffer solutions in the presence of air containing various volume fractions of CO₂ (0 to 10000·10^–6). In both species and in the light and in darkness, the effect of CO₂ was to inhibit stomatal opening, the maximum inhibition of opening occurring in the range 0 to 360·10^–6. The inhibition of opening per unit change in concentration was greatest between volume fractions of 0 and 240·10^–6. There was little further closure above the volume fraction of 360·10^–6, i.e. approximately ambient concentration of CO₂. Thus, although leaves of CAM species may experience much higher internal concentrations of CO₂ in the light than those of C₃ plants, this does not affect the sensitivity of their stomata to CO₂ concentration or the range over which they respond. Stomatal responses to CO₂ were similar in both the light and the dark, indicating that effects of CO₂ on stomata occur via mechanisms which are independent of light. The responses of stomata to CO₂ in the gaseous phase took place without the treatments changing the pH of the buffered solutions. Thus it is unlikely that CO₂ elicited stomatal movement by changing either the pH or the HCO ₃ ^– /CO ₃ ^2- equilibria. It is suggested that the concentration of dissolved unhydrated CO₂ may be the effector of stomatal movement and that its activity is related to its reactivity with amines.     

The kinetics of ribulose-1,5-bisphosphate carboxylase/oxygenase in vivo inferred from measurements of photosynthesis in leaves of transgenic tobacco

Transgenic tobacco (Nicotiana tabacum L. cv. W38) with an antisense gene directed against the mRNA of the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit was used to determine the kinetic properties of Rubisco in vivo. The leaves of these plants contained only 34% as much Rubisco as those of the wild type, but other photosynthetic components were not significantly affected. Consequently, the rate of CO₂ assimilation by the antisense plants was limited by Rubisco activity over a wide range of CO₂ partial pressures. Unlike in the wild-type leaves, where the rate of regeneration of ribulose bisphosphate limited CO₂ assimilation at intercellular partial pressures above 400 ubar, photosynthesis in the leaves of the antisense plants responded hyperbolically to CO₂, allowing the kinetic parameters of Rubisco in vivo to be inferred. We calculated a maximal catalytic turnover rate, k_cat, of 3.5+0.2 mol CO₂·(mol sites)^–1·s^–1 at 25° C in vivo. By comparison, we measured a value of 2.9 mol CO₂·(mol sites)^–1·^–1 in vitro with leaf extracts. To estimate the Michaelis-Menten constants for CO₂ and O₂, the rate of CO₂ assimilation was measured at 25° C at different intercellular partial pressures of CO₂ and O₂. These measurements were combined with carbon-isotope analysis (¹³C/¹²C) of CO₂ in the air passing over the leaf to estimate the conductance for transfer of CO₂ from the substomatal cavities to the sites of carboxylation (0.3 mol·m^–2·s^–1·bar^–1) and thus the partial pressure of CO₂ at the sites of carboxylation. The calculated Michaelis-Menten constants for CO₂ and O₂ were 259 ±57 bar (8.6±1.9M) and 179 mbar (226 M), respectively, and the effective Michaelis-Menten constant for CO₂ in 200 mbar O₂ was 549 bar (18.3 M). From measurements of the photocompensation point (_* = 38.6 ubar) we estimated Rubisco's relative specificity for CO₂, as opposed to O₂ to be 97.5 in vivo. These values were dependent on the size of the estimated CO₂-transfer conductance.Abbreviations and Symbols A CO₂-assimilation rate - g_w conductance for CO₂ transfer from the substomatal cavities to the sites of carboxylation - K_c, K_o Michaelis-Menten constants for carboxylation, oxygenation of Rubisco - k_cat V_cmax/[active site] - O partial pressure of O₂ at the site of carboxylation - p_c partial pressure of CO₂ at the site of carboxylation - p_i intercellular CO₂ partial pressure - R_d day respiration (non-photorespiratory CO₂ evolution) - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - S_c/o relative specificity factor for Rubisco - SSu small subunit of Rubisco - V_cmax, V_omax maximum rates of Rubisco carboxylation, oxygenation - _* partial pressure of CO₂ in the chloroplast at which photorespiratory CO₂ evolution equals the rate of carboxylation     

Novel characteristics of cassava,Manihot esculenta Crantz,a reputed C3-C4 intermediate photosynthesis species

The cassava plant, Manihot esculenta, grows exceptionally well in low fertility and drought prone environments, but the mechanisms that allow this growth are unknown. Earlier, and sometimes contradictory, work speculated about the presence of a C₄-type photosynthesis in cassava leaves. In the present work we found no evidence for a C₄ metabolism in mature attached cassava leaves as indicated i) by the low, 2 to 8%, incorporation of ¹⁴CO₂ into C₄ organic acids in short time periods, 10 s, and the lack of ¹⁴C transfer from C₄ acids to other compounds in ¹²CO₂, ii) by the lack of C₄ enzyme activity changes during leaf development and the inability to detect C₄ acid decarboxylases, and iii) by leaf CO₂ compensation values between 49 and 65 l of CO₂ 1^–1 and by other infrared gas exchange photosynthetic measurements. It is concluded that the leaf biochemistry of cassava follows the C₃ pathway of photosynthesis with no indication of a C₃-C₄ mechanism.However, cassava leaves exhibit several novel characteristics. Attached leaves have the ability to effectively partition carbon into sucrose with nearly 45% of the label in sucrose in about one min of ¹⁴CO₂ photosynthesis, contrasting with 34% in soybean (C₃) and 25% in pigweed (C₄). Cassava leaves displayed a strong preference for the synthesis of sucrose versus starch. Field grown cassava leaves exhibited high rates of photosynthesis and curvilinear responses to increasing sunlight irradiances with a tendency to saturate only at high irradiances, above 1500 mol m^–2 s^–1. Morphologically, the cassava leaf has papillose epidermal cells on its lower mesophyll surface that form fence-like arrangements encircling guard cells. It is proposed that the active synthesis of sugars has osmotic functions in the cassava plant and that the papillose epidermal cells function to maintain a healthy leaf water status in various environments.Abbreviations ADP adenosine diphosphate - Asp aspartate - BSA bovine serum albumin - CoA coenzyme A - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - FBP fructose-1,6-biphosphate - Gly glycine - HEPES N-2-hydroxyethylpiperazine-N-2-ethansulfonic acid - Mal malate - NAD nicotinamide adenine dinucleotide (oxidized form) - NADH nicotinamide adenine dinucleotide (reduced form) - NADP nicotinamide adenine dinucleotide phosphate (oxidized form) - PAR photosynthetic active radiation (400–700 nm) - PEP phosphenolpyruvate carboxylase - p-FBPase plastid fructose-1,6-biphosphatase - PGA 3-phosphoglyceric acid - PMSF phenylmethylsulfonyl fluoride - PVP polyvinylpyrrolidone - Rubisco ribulose-1,5-biphosphate carboxylase/oxygenase - RuBP ribulose-1,5-biphosphate - Ser serine - sugar-P sugar-phosphates     

Comparative effects of growth irradiance on photosynthesis and leaf anatomy of Flaveria brownii (C4-like), Flaveria linearis (C3–C4) and their F1 hybrid

Photosynthetic rates and related anatomical characteristics of leaves developed at three levels of irradiance (1200, 300 and 80 umol · m^–2 · s^–1) were determined in the C₄-like species Flaveria brownii A.M. Powell, the C₃–C₄-intermediate species F. linearis Lag., and the F₁ hybrid between them (F. brownii × F. linearis). In the C₃–C₄ and F₁ plants, increases in photosynthetic capacity per unit leaf area were strongly correlated with changes in mesophyll area per unit leaf area. The C₄-like plant F. brownii, however, showed a much lower correlation between photosynthetic capacity and mesophyll area per unit leaf area. Plants of F. brownii developed at high irradiance showed photosynthetic rates per unit of mesophyll cell area 50% higher than those plants developed at medium irradiance. These results along with an increase in water-use efficiency are consistent with an increase of C₄ photosynthesis in high-irradiance-grown F. brownii plants, whereas in the other two genotypes such plasticity seems to be absent. Photosynthetic discrimination against ¹³C in the three genotypes was less at high than at low irradiance, with the greatest change occurring in F. brownii. Discrimination against ¹³C expressed as ¹³C was linearly correlated (r ² = 0.81; P<0.001) with the ratio of bundle-sheath volume to mesophyll cell area when all samples from the three genotypes were combined. This tissue ratio increased for F. brownii and the F₁ hybrid as growth irradiance increased, indicating a greater tendency towards Kranz anatomy. The results indicated that F. brownii had plasticity in its C₄-related anatomical and physiological characteristics as a function of growth irradiance, whereas plasticity was less evident in the F₁ hybrid and absent in F. linearis.Abbreviations A leaf surface area - A_ma, A_mn, A_lm total ma, mn or lm cell surface area - bs vascular bundle sheath - lm large spongy-mesophyll cells - ma mesophyll cells adjacent to bundle sheath - mn mesophyll cells not adjacent to bundle sheath - P_n net photosynthesis - (H, M, L) PPFD (high, medium, low) photosynthetic photon flux density - SLDW specific leaf dry wight - V_bs bs volume - V_{(ma + mn + bs)} total photosynthetic tissue volume - ¹³C ¹³C discrimination We thank Mrs. Lisa Smith for technical assistance in light microscopy and Dr. Ned Friedman (Department of Botany, University of Georgia, Athens, GA, USA) for the use of digitizing equipment. Participation of Dr. J.L. Araus in this work was supported by a grant Beca de Especialización para Doctores y Tecnólogos en el Extranjero, from Ministerio de Educatión y Ciencia, Spain.     

Glycine decarboxylase is confined to the bundle-sheath cells of leaves of C3−C4 intermediate species

Immunogold labelling has been used to determine the cellular distribution of glycine decarboxylase in leaves of C₃, C₃–C₄ intermediate and C₄ species in the genera Moricandia, Panicum, Flaveria and Mollugo. In the C₃ species Moricandia foleyi and Panicum laxum, glycine decarboxylase was present in the mitochondria of both mesophyll and bundle-sheath cells. However, in all the C₃–C₄ intermediate (M. arvensis var. garamatum, M. nitens, M. sinaica, M. spinosa, M. suffruticosa, P. milioides, Flaveria floridana, F. linearis, Mollugo verticillata) and C₄ (P. prionitis, F. trinervia) species studied glycine decarboxylase was present in the mitochondria of only the bundle-sheath cells. The bundle-sheath cells of all the C₃–C₄ intermediate species have on their centripetal faces numerous mitochondria which are larger in profile area than those in mesophyll cells and are in close association with chloroplasts and peroxisomes. Confinement of glycine decarboxylase to the bundle-sheath cells is likely to improve the potential for recapture of photorespired CO₂ via the Calvin cycle and could account for the low rate of photorespiration in all C₃–C₄ intermediate species.Abbreviation and symbol kDa kilodaltons - CO₂ compensation point     

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     

The relationship between the redox state of Q A and photosynthesis in leaves at various carbon-dioxide,oxygen and light regimes

The response of chlorophyll fluorescence elicited by a low-fluence-rate modulated measuring beam to actinic light and to superimposed 1-s pulses from a high-fluence-rate light source was used to measure the redox state of the primary acceptor Q _A of photosystem II in leaves which were photosynthesizing under steady-state conditions. The leaves were exposed to various O₂ and CO₂ concentrations and to different energy fluence rates of actinic light to assess the relationship between rates of photosynthesis and the redox state of Q _A. Both at low and high fluence rates, the redox state of Q _A was little altered when the CO₂ concentration was reduced from saturation to about 600 l·l^-1 although photosynthesis was decreased particularly at high fluence rates. Upon further reduction in CO₂ content the amount of reduced Q _A increased appreciably even at low fluence rates where light limited CO₂ reduction. Both in the presence and in the absence of CO₂, a more reduced Q _A was observed when the O₂ concentration was below 2%. Q _A was almost fully reduced when leaves were exposed to high fluence rates under nitrogen. Even at low fluence rates, Q _A was more reduced in shade leaves of Asarum europaeum and Fagus sylvatica than in leaves of Helianthus annuus and Fagus sylvatica grown under high light. Also, in shade leaves the redox state of Q _A changed more during a transition from air containing 350 l·l^-1 CO₂ to CO₂-free air than in sun leaves. The results are discussed with respect to the energy status and the CO₂-fixation rate of the leaves.Abbreviations and symbols L 1,2 first and second actinic light beam - Q _A primary acceptor of photosystem II - q _Q Q-quenching     

Photosynthetic metabolism of cyanate by the cyanobacterium Synechococcus UTEX 625

Intact cells of the unicellular cyanobacterium Synechococcus UTEX 625 degraded exogenously supplied cyanate (as KOCN) to CO₂ and NH₃ in a light-dependent reaction. NH₃ release to the medium was as high as 80 mol(mgChl)^-1h^-1 and increased 1.7-fold in the presence of methionine sulfoximine, a glutamine synthetase inhibitor. Cyanate also supporte photosynthetic O₂ evolution to a maximum rate of 188 mol O₂(mgChl)^-1h^-1 at pH 8 and 30°C. Cyanate decomposition in cell-free extracts, measured by mass spectrometry as ¹³CO₂ production from KO¹³CN, occurred in the soluble enzyme fraction, but not in the thylakoid/carboxysome fraction, and was enhanced by HCO₃ ^– and inhibited by the dianion oxalate. CO₂, rather, than HCO₃ ^–, was a product of cyanate decomposition. The ability to decompose cyanate was not dependent upon pre-exposure of cells to cyanate to induce activity. The collective results indicate that Synechococcus UTEX 625 possesses a constitutive, cytosolic cyanase (EC 4.3.99.1), similar in mechanism to that found in some species of heterotrophic bacteria. The reaction catalyzed was: OCN+HCO₃+2H⁺2CO₂+NH₃. In intact cells, the CO₂ produced by the action of cyanase on OCN^- was either directly fixed by the Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase, leading to O₂ evolution, or leaked into the medium where it was returned to the cell by the active CO₂/HCO₃ ^– transport systems for fixation. However, leakage of CO₂ from air-grown cells was only observed when the active CO₂ transport system was inhibited by darkness or the CO₂ analogue carbon oxysulfide.Abbreviations BTP bistrispropane - C _i inorganic carbon (=CO₂+HCO₃ ^-+CO₃ ^2-) - CA carbonic anhydrase - Chl chlorophyll - COS carbon oxysulfide - MSX methionine sulfoximine - PAR photosynthetically active radiation - Rubisco ribulose bisphosphate carboxylase/oxygenase     

Aspartic-acid synthesis in C3 plants

In a previous study (Melzer and O'Leary, 1987, Plant Physiol. 84, 58–60), we used isotopic methods to show that a substantial fraction of protein-bound aspartic acid in tobacco is derived from anaplerotic synthesis via phosphoenolpyruvate (PEP) carboxylase. Similar studies in soybean (Glycine max L.) and spinach (Spinacia oleracea L.) showed a similar pattern, and this pattern persists with age because of slow protein turnover. A more quantitative analysis indicates that about 40% of protein-bound aspartate is derived in this manner. Analyses of free aspartic and malic acids show that contribution of PEP carboxylase to the synthesis of these acids decreases with increasing age. The C₄ plant Zea mays L. did not show this pattern.Abbreviations and Symbols RuBP ribulose bisphosphate - PEP phosphoenolpyruvate - OAA oxaloacetic acid - PGA 3-phosphoglyceric acid - ¹³C carbon-13 - isotopic content [R(sample)/R(standard)-1] × 1000, where R = [¹³CO₂]/[¹²CO₂] This work was supported by contract DE-ACO₂-83ER 13076 and grant DE-FGO₂-86ER13534 from the U.S. Department of Energy. E. M. was supported by a fellowship from Deutsche Forschungsgemeinschaft. We are grateful to Isabel Treichel for assistance with isotopic analyses.