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     

C4 photosynthesis in Spartina townsendii at low and high temperatures

The metabolism of ¹⁴CO₂ in the cool temperate saltmarsh grass Spartina townsendii was investigated in plants grown in their natural habitats at two temperatures. Both in the spring at 10°C and in the late summer at 25°C radioactivity was initially incorporated into the organic acids malate and aspartate and then transferred to 3-phosphoglycerate in the manner characteristic of the C₄ pathway of photosynthesis. Metabolism was not disrupted at the lower temperature as in some C₄ plants. Radioactivity was transferred more slowly from malate into alanine, glycine and serine at 10°C, but sugars were labelled equally at both temperatures.     

Dark fixation of CO2 during gluconeogenesis by the cotyledons of Cucurbita pepo L.

We did this work to discover the pathway of CO₂ fixation into sugars in the dark during gluconeogenesis by the cotyledons of 5-day-old seedlings of Cucurbita pepo L. We paid particular attention to the possibility of a contribution from ribulosebisphosphate carboxylase. The detailed distribution of ¹⁴C after exposure of excised cotyledons to ¹⁴CO₂ in the dark was determined in a series of pulse and chase experiments. After 4s in ¹⁴CO₂, 89% of the ¹⁴C fixed was in malate and aspartate. In longer exposures, and in chases in ¹²CO₂, label appeared in alanine, phosphoenolpyruvate, 3-phosphoglycerate and sugar phosphates, and accumulated in sugars. The transfer of label from C-4 acids to sugars was restricted by inhibition of phosphoenolpyruvate carboxykinase in vivo by 3-mercaptopicolinic acid. We conclude as follows. Initial fixation of CO₂ in the dark is almost entirely into phosphoenolpyruvate, probably via phosphoenolpyruvate carboxylase (EC 4.1.1.31) which we showed to be present in appreciable amounts. Incorporation into sugars occurs chiefly, if not completely, as a result of randomization of the carboxyl groups of the C-4 acids and subsequent conversion of the oxaloacetate to sugars via the accepted sequence for gluconeogenesis. Ribulosebisphosphate carboxylase appears to make very little contribution to sugar synthesis from fat.     

Photoautotrophic growth of soybean cells in suspension culture III. Characterization of carbon fixation products under high and low CO2 levels

A photoautotrophic soybean suspension culture (SB-P) was used to study CO₂ assimilation while exposed to elevated or ambient CO₂ levels. These studies showed that under elevated CO₂ (5% v/v) malate is the dominant fixation product, strongly suggesting that phosphoenolpyruvate carboxylase (PEPCase) is the primary enzyme involved in carbon fixation in these cells under their normal growth conditions. Citrate and [aspartate + glutamate] were also significant fixation products during fifteen minutes of exposure to ¹⁴CO₂. During the ten minute unlabeled CO₂ chase however, ¹⁴C-malate continued to increase while citrate and [aspartate + glutamate] declined. Fixation of ¹⁴CO₂ under ambient CO₂ levels (0.037%) showed a very different product pattern as 3-phosphoglycerate was very high in the first one to two minutes followed by increases in [serine + glycine] and [aspartate + glutamate]. Hexose phosphates were also quite high initially but then declined relatively rapidly. Thus, the carbon fixation pattern at ambient CO₂ levels resembles somewhat that seen in C3 leaf cells while that seen at elevated CO₂ levels more closely resembles that of a C₄ plant. The initial fixation product of C₃ plants, 3-PGA, was never detectable under high CO₂ conditions. These data suggest that an in vitro photoautotrophic system would be suitable for studying carbon fixation physiology during photosynthetic and non-photosynthetic growth.Abbreviations SB-P photoautotrophic soybean cells - PEPCase phosphoenol-pyruvate carboxylase - RuBPCase ribulose bisphosphate carboxylase/oxygenase - 3-PGA 3-phosphoglycerate     

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     

C4 photosynthetic carbon metabolism in the leaves of aromatic tropical grasses — I. Leaf anatomy,CO2 compensation point and CO2 assimilation

A few species of Cymbopogon and Vetiveria are potentially important tropical grasses producing essential oils. In the present study, we report on the leaf anatomy and photosynthetic carbon assimilation in five species of Cymbopogon and Vetiveria zizanioides. Kranz-type leaf anatomy with a centrifugal distribution of chloroplasts and exclusive localization of starch in the bundle sheath cells were common among the test plants. Besides the Kranz leaf anatomy, these grasses displayed other typical C₄ characteristics including a low (0–5 µl/l) CO₂ compensation point, lack of light saturation of CO₂ uptake at high photon flux densities, high temperature (35°C) optimum of net photosynthesis, high rates of net photosynthesis (55–67 mg CO₂ dm^-2 leaf area h^-1), little or no response of net photosynthesis to atmospheric levels of O₂ and high leaf ¹³C/¹²C ratios. The biochemical studies with ¹⁴CO₂ indicated that the leaves of the above plant species synthesize predominantly malate during short term (5 s) photosynthesis. In pulse-chase experiments it was shown that the synthesis of 3-phosphoglycerate proceeds at the expense of malate, the major first formed product of photosynthesis in these plant species.     

Autotrophic growth and CO2 fixation of Chloroflexus aurantiacus

Chlorofluexus aurantiacus OK-70 fl was grown photoautotrophically with hydrogen as the electron source. The lowest doubling time observed was 26 h.The mechanism of CO₂ fixation in autotrophically grown cells was studied. The presence of ribulose-1,5-bis-phosphate carboxylase and phosphoribulokinase could not be demonstrated. Carbon isotope fractionation (¹³C) was small, and alanine and aspartate but not 3-phosphoglycerate were the major labelled compounds in short term ¹⁴CO₂ labelling. Thus CO₂ is not fixed by the Calvin cycle.Fluoroacetate (FAc) completely inhibited protein synthesis in cultures and caused a slight citrate accumulation. However, CO₂ fixation continued and increased polyglucose formation occurred. Under these conditions added acetate was metabolized to polyglucose, as were glycine, serine, glyoxylate and succinate, but to a lesser extent; little or no formate or CO was utilised.Glyoxylate inhibited CO₂ fixation in vivo, indicating that pyruvate is formed from acetyl-CoA and CO₂ by pyruvate synthase. Two key enzymes of the reductive TCA cycle, citrate lyase and -ketoglutarate synthase were not detected in cell free extracts, but pyruvate synthase and phosphoenolpyruvate carboxylase were demonstrated. It is concluded that acetyl-CoA is a central intermediate in the CO₂ fixation process, but the mechanism of its synthesis is not clear.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase - TCA cycle tricarboxylic acid cycle - FAc monofluoroacetate - PEP phosphoenolpyruvate - MV methyl viologen - TTC triphenyltetrazolium chloride - PMS phenazine methosulfate     

Co-function of C3-and C4-photosynthetic pathways in C3, C4 and C3-C4 intermediate Flaveria species

The potential for C₄ photosynthesis was investigated in five C₃-C₄ intermediate species, one C₃ species, and one C₄ species in the genus Flaveria, using ¹⁴CO₂ pulse-¹²CO₂ chase techniques and quantum-yield measurements. All five intermediate species were capable of incorporating ¹⁴CO₂ into the C₄ acids malate and aspartate, following an 8-s pulse. The proportion of ¹⁴C label in these C₄ products ranged from 50–55% to 20–26% in the C₃-C₄ intermediates F. floridana Johnston and F. linearis Lag. respectively. All of the intermediate species incorporated as much, or more, ¹⁴CO₂ into aspartate as into malate. Generally, about 5–15% of the initial label in these species appeared as other organic acids. There was variation in the capacity for C₄ photosynthesis among the intermediate species based on the apparent rate of conversion of ¹⁴C label from the C₄ cycle to the C₃ cycle. In intermediate species such as F. pubescens Rydb., F. ramosissima Klatt., and F. floridana we observed a substantial decrease in label of C₄-cycle products and an increase in percentage label in C₃-cycle products during chase periods with ¹²CO₂, although the rate of change was slower than in the C₄ species, F. palmeri. In these C₃-C₄ intermediates both sucrose and fumarate were predominant products after a 20-min chase period. In the C₃-C₄ intermediates, F. anomala Robinson and f. linearis we observed no significant decrease in the label of C₄-cycle products during a 3-min chase period and a slow turnover during a 20-min chase, indicating a lower level of functional integration between the C₄ and C₃ cycles in these species, relative to the other intermediates. Although F. cronquistii Powell was previously identified as a C₃ species, 7–18% of the initial label was in malate+aspartate. However, only 40–50% of this label was in the C-4 position, indicating C₄-acid formation as secondary products of photosynthesis in F. cronquistii. In 21% O₂, the absorbed quantum yields for CO₂ uptake (in mol CO₂·[mol quanta]^-1) averaged 0.053 in F. cronquistii (C₃), 0.051 in F. trinervia (Spreng.) Mohr (C₄), 0.052 in F. ramosissima (C₃-C₄), 0.051 in F. anomala (C₃-C₄), 0.050 in F. linearis (C₃-C₄), 0.046 in F. floridana (C₃-C₄), and 0.044 in F. pubescens (C₃-C₄). In 2% O₂ an enhancement of the quantum yield was observed in all of the C₃-C₄ intermediate species, ranging from 21% in F. ramosissima to 43% in F. pubescens. In all intermediates the quantum yields in 2% O₂ were intermediate in value to the C₃ and C₄ species, indicating a co-function of the C₃ and C₄ cycles in CO₂ assimilation. The low quantum-yield values for F. pubescens and F. floridana in 21% O₂ presumably reflect an ineffcient transfer of carbon from the C₄ to the C₃ cycle. The response of the quantum yield to four increasing O₂ concentrations (2–35%) showed lower levels of O₂ inhibition in the C₃-C₄ intermediate F. ramosissima, relative to the C₃ species. This indicates that the co-function of the C₃ and C₄ cycles in this intermediate species leads to an increased CO₂ concentration at the site of ribulose-1,5-bisphosphate carboxylase/oxygenase and a concomitant decrease in the competitive inhibition by O₂.Abbreviations PEP phosphoenolpyruvate - PGA 3-phosphoglycerate - RuBP ribulose-1,5-bisphosphate     

The CO2 assimilation via the reductive tricarboxylic acid cycle in an obligately autotrophic,aerobic hydrogen-oxidizing bacterium,Hydrogenobacter thermophilus

The incorporation of ¹⁴CO₂ by the cell suspensions of an extremely thermophilic, aerobic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus was studied. After short time incubation of the cell suspensions with ¹⁴CO₂, the radiactivity was initially present in aspartate, glutamate, succinate, phosphorylated compounds, citrate, malate and fumarate. All of these compounds except phosphorylated compounds were related to the members of the tricarboxylic acid cycle. The proportion of labelled aspartate onglutamate in total radioactivity on each chromatogram decreased with incubation time, while the percentage of the radioactivity incorporated in phosphorylated compounds increased with time up to 10 s. These indicated that aspartate and glutamate is derived from primary products of CO₂ fixation.In cell-free extracts of Hydrogenobacter thermophilus, the two key enzymes in the Calvin cycle, ribulose-1,5-bisphosphate carboxylase and phosphoribulokinase could not be detected. The key enzymes of the reductive tricarboxylic acid cycle, fumarate reductase and ATP citrate lyase were present. Activities of phosphoenolpyruvate synthetase and pyruvate carboxylase were also detected. The referse reactions (dehydrogenase reactions) of -ketoglutarate synthase and pyruvate synthase could be detected by using methyl viologen as an electron acceptor.These findings strongly suggested that a new type of the reductive tricarboxylic acid cycle operated as the CO₂ fixation pathway in Hydrogenobacter thermophilus.     

Demonstration of C3-photosynthesis in a bluegreen alga,Coccochloris peniocystis

Air-grown cells of the cyanobacterium, Coccochloris peniocystis Kutz were exposed to [¹⁴C] bicarbonate in the light for periods of 0.5 to 2.0 s followed by longer exposures to unlabelled bicarbonate. Although C₄ acids are among the initial products of photosynthesis, the kinetics of tracer movement during the pulse-chase experiments demonstrate that the principal mechanism of CO₂ fixation in this alga is the C₃-pathway.Abbreviations PGA 3-phophoglyceric acid - PEP phosphoenolpyruvate - RuBP ribulose-1,5-bisphosphate     

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     

Carboxylating enzymes and pathway of photosynthetic carbon assmilation in different marine algae—Evidence for the C4-pathway?

Experiments on short-term photosynthesis in H¹⁴CO₃ ^- (2–5 s) using various species of different algal classes resulted in predominant ¹⁴C-labelling (>90% of total ¹⁴C-incorporation) of phosphorylated compounds. The percentage of malate and aspartate usually accounts for distinctly less than 10% of the total ¹⁴C-labelling. These findings are consistent with data from enzymatic analyses, since 97–100% of the carboxylation capacity is due to ribulose-1.5-biphosphate carboxylase (EC 4.1.1.39) in Rhodophyceae and Chlorophyceae. Phaeophyceae are generally characterized by considerable activity of phosphoenolpyruvate carboxykinase (EC 4.1.1.32): at least 10% of carboxylation is confined to this enzyme. Similar ratios are obtained when rates of photosynthesis and of light-independent CO₂-fixation are compared. Activity of phosphoenolpyruvate carboxylase (EC 4.1.1.31) could not be detected in the species investigated. The results are discussed with emphasis on the pathway of photosynthetic carbon assimilation in marine algae.Abbreviations PEP-CK phosphoenolpyruvate carboxykinase (EC 4.1.1.32) - PEP-C phosphoenolpyruvate carboxylase (EC 4.1.1.31) - RubP-C ribulose-1.5-biphosphate carboxylase (EC 4.1.1.39) Dedicated to Professor H. Fischer, Bonn, on his 65th birthday     

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 characteristics of photomixotrophic and photoautotrophic cell suspension cultures ofChenopodium rubrum

Summary Cell suspension cultures ofChenopodium rubrum have been grown for more than 2 years photoautotrophically with CO₂ as sole carbon source. Average increase in fresh weight is appr. 600% within 14 days. The chlorophyll content of photoautotrophic cells (200 g/g fresh weight) is much higher than of photomixotrophic cells (50 g/g fresh weight). The photosynthetic activity of the cells (190 moles CO₂×mg^–1 chlorophyllXh^–1) is comparable to the values found with intact leaves. As shown by short-term¹⁴CO₂ photosynthesis, both, the photomixotrophic and the photoautotrophic cell suspension cultures assimilate CO₂ predominantly via the Calvin pathway.Major differences were found with cells from either exponential or stationary phase of growth with regard to differential labelling of 3-phosphoglyceric acid, malate, sucrose and glucose/fructose.In vitro measurements of carboxylation reactions only partially corroborate our findings with¹⁴CO₂ incorporation. The ratio of ribulosebisphosphate to phosphoenolpyruvate carboxylase activity is 4.7 for leaves of C.rubrum, 1.2 for photoautotrophic cells during stationary growth and 0.5 for cells during exponential growth phase, however, 0.18 was found for photomixotrophic cells. Though the¹⁴CO₂ incorporation into 3-phosphoglyceric acid is clearly higher than into malate, thein vitro activity of phosphoenolpyruvatecarboxylase is 2–6 fold higher than that of ribulosebisphosphate carboxylase. We postulate that anaplerotic reactions of the tricarboxylic acid cycle are involved in the regulation of phosphoenolpyruvate carboxylase.Abbreviations 2,4-D didilorophenoxyacetic acid - EDTA ethylene-diamine-tetraacetic acid - fr. w. fresh weight - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - PGA 3-phosphoglyceric acid - PPO 2,5-diphenyloxazole - PEP phosphoenolpyruvate - RuBP nbulosebisphosphate     

Reassimilation of carbon dioxide by Flaveria (Asteraceae) species representing different types of photosynthesis

The capability to reassimilate CO₂ originating from intracellular decarboxylating processes connected with the photorespiratory glycolate pathway and-or decarboxylation of C₄ acids during C₄ photosynthesis has been investigated with four species of the genus Flaveria (Asteraceae). The C₃-C₄ intermediate species F. pubescens and F. anomala reassimilated CO₂ much more efficiently than the C₃ species F. cronquistii and, with respect to this feature, behaved similarly to the C₄ species F. trinervia. Therefore, under atmospheric conditions the intermediate species photorespired with rates only between 10–20% of that measured with F. cronquistii. At low oxygen concentrations (1,5%) the reassimilation potential of F. anomala approached that of F. trinervia and was distinct from that found with F. pubescens. The data are discussed with respect to a possible sequence of events during evolution of C₄ photosynthesis. If compared with related data for C₃-C₄ intermediate species from other genera they support the hypothesis that, during evolution of C₄ photosynthesis, an efficient capacity for CO₂ reassimilation evolved prior to a CO₂-concentrating mechanism.Abbreviations C₃, C₄ assimilated CO₂ initially found in 3-phosphoglycerate (C₃) or malate and aspartate (C₄) - D reassimilation coefficient - R _n , R _t net, total CO₂ evolution as measured with 0.03 and 3% CO₂, respectively - RuBP ribulose-1,5-bisphosphate - TPS true photosynthesis     

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     

Photosynthetic carbon metabolism of the cool-temperate C4 grass Spartina anglica Hubb.

The aim of this work was to describe the photosynthetic carbon metabolism of the cooltemperate C₄ grass Spartina anglica. With the exception of pyruvate, phosphate dikinase and pyruvate kinase, the maximum catalytic activities in leaves of putative enzymes of the C₄ cycle of a phosphoenolpyruvate-carboxykinase C₄ plant were considerably in excess of the observed, steady-state rate of photosynthesis, and were comparable with the maximum catalytic activities of key enzymes of the reductive pentose-phosphate pathway. Radioactive carbon from ¹⁴CO₂ supplied to attached leaves during steady-state photosynthesis appeared first in malate and aspartate from which it moved to intermediates of the reductive pentose-phosphate pathway, and then to sucrose. These experiments show that photosynthetic carbon metabolism in this cool-temperate C₄ plant is similar to that of C₄ plants of hotter climates.     

CO2 and malate metabolism in starch-containing and starch-lacking guard-cell protoplasts

Isolated, purified mesophyll and guard-cell protoplasts of Vicia faba L. and Allium cepa L. were exposed to ¹⁴CO₂ in the light and in the dark. The guard-cell protoplasts of Vicia and Allium did not show any labeling in phosphorylated products of the Calvin cycle, thus appearing to lack the ability to reduce CO₂ photosynthetically. In Vicia, high amounts of radioactivity (35%) appeared in starch after 60-s pulses of ¹⁴CO₂ both in the light and in the dark. Presumably, the ¹⁴CO₂ is fixed into the malate via PEP carboxylase and then metabolized into starch as the final product of gluconeogenesis. This is supported by the fact that guard-cell protoplasts exposed to malic acid uniformly labeled with ¹⁴CO₂ showed high amounts of labeled starch after the incubation, whereas cells labeled with [4-¹⁴C]malate had minimal amounts of labeled starch (1/120).In contrast, the starch-deficient Allium, guard-cell protoplasts did not show any significant ¹⁴CO₂ fixation. However, adding PEP to an homogenate stimulated ¹⁴CO₂ uptake, thus supporting the interpretation that the presence of starch as a source of PEP is necessary for incorporating CO₂ and delivering malate. With starch-containing Vicia guard-cell protoplasts, the correlation between changes in volume and the interconversion of malate and starch was demonstrated. It was shown that the rapid gluconeogenic conversion of malate into starch prevents an increase of the volume of the protoplasts, whereas the degradation of starch to malate is accompanied by a swelling of the protoplasts.Abbreviations GCPs guard-cell protoplasts - MCPs mesophyll cell protoplasts - PEP phosphoenolpyruvate - DTT dithiothreitol - 3-PGA 3-phosphoglyceric acid - RiBP ribulose 1,5 bisphosphate - MDH malate dehydrogenase - MES 2-(N-morpholino)ethane sulfonic acid - CAM crassulacean acid metabolism     

CO2 exchange characteristics during dark-light transitions in wild-type and mutant Chlamydomonas reinhardii cells

A burst of net CO₂ uptake was observed during the first 3–4 min after the onset of illumination in both wild-type Chlamydomonas reinhardii in which carbonic anhydrase was chemically inhibited with ethoxyzolamide and in a mutant of C. reinhardii (ca-1-12-1C) deficient in carbonic anhydrase activity. The burst was followed by a rapid decrease in the CO₂ uptake rate so that net evolution often occurred. After a 2–3 min period of CO₂ evolution, net CO₂ uptake again increased and ultimately reached a steady-state, positive rate. From [¹⁴CO₂]-tracer studies it was determined that CO₂ fixation proceeded at a nearly linear rate throughout the period of illumination. Thus, prior to reaching a steady state, there was a rapid accumulation of inorganic carbon inside the cells which apparently reached a supercritical concentration and the excess was excreted, causing a subsequent efflux of CO₂. A post illumination burst of net CO₂ efflux was also observed in ethoxyzolamide-inhibited wild type and ca-1 mutant cells, but not in the unihibited wild type. [¹⁴CO₂]-tracer experiments revealed that this burst was the result of a collapse of a large internal inorganic carbon pool at the onset of darkness rather than a photorespiratory post-illumination burst. These results indicate that upon illumination, chemical or genetic inhibition of carbonic anhydrase initially causes an accumulation of excess inroganic carbon in C. reinhardii cells, and that unknown regulatory mechanisms correct for this imbalance by first excreting the excess inorganic carbon and then, after several dampened oscillations, achieving an equilibrium between bicarbonate uptake, bicarbonate dehydration, and CO₂ fixation.     

Kinetic interactions of glycine with substrates and effectors of phosphenolpyruvate carboxylase from maize leaves

Glycine enhanced the sensitivity of maize phosphenolpyruvate carboxylase to the activator glucose 6-phosphate and reduced the sensitivity of the enzyme to the inhibitors malate and aspartate. The effects of glycine on the kinetic constants for these other effectors were greater than its effect on the K_m for substrate, raising the K_i(malate) 11-fold and reducing K_{a(glucose6-P)} 7-fold, while reducing the K_m(PEP) by 3-fold. Kinetically saturating levels of glycine and glucose 6-phosphate acted synergistically to raise K_i(malate) higher than that observed with either activator alone. Glycine and glucose 6-phosphate also synergistically reduced aspartate inhibition. Dual inhibitor analysis indicated that aspartate and malate bind in a mutually exclusive manner, and thus probably compete for the same inhibitor site. In contrast, the synergism between glycine and glucose 6-phosphate indicate that these activators bind at separate sites. Glycine also reduced the K_m(Mg) by 3-fold but had no significant effect on the K_m of bicarbonate.Abbreviation PEP phosphoenolpyruvate