Compartmentation and export of 14CO2 fixation products in mesophyll protoplasts from the C4-plant Digitaria sanguinalis

Isolated mesophyll protoplasts, and protoplast extracts containing intact chloroplasts, from the C₄ species Digitaria sanguinalis have been used to study Compartmentation and export of C₄ acids, using different C₃ precursors as substrate for ¹⁴CO₂ fixation. Mg²⁺ was necessary for maximum ¹⁴CO₂ fixation rates with both protoplasts and protoplast extracts, whereas Mg²⁺ was inhibitory for oxaloacetate and phosphoglycerate reduction. This inhibition could be overcome by preincubating the materials in the light with excess of EDTA before addition of Mg²⁺. Under these conditions pyruvate as substrate for ¹⁴CO₂ fixation induced mainly malate formation, whereas phosphoglycerate as substrate induced oxaloacetate formation, indicating competition for available NADPH between oxaloacetate and phosphoglycerate reduction. Oxaloacetate could be exported from the protoplasts at rates comparable to the rates of ¹⁴CO₂ fixation in intact leaves (200 μmol/mg Chl × h). This product probably passed the plasma membrane by simple diffusion, whereas the export of malate and aspartate seemed to be regulated, with the size of the intraprotoplast pool being relatively independent of the export rate. It is concluded that transport via the plasma membrane-cell wall path may play a role in metabolite flow during photosynthesis in C₄ plants.     

Relationship between leaf development and primary photosynthetic products in the C4 plant Portulaca oleracea L.

Summary The photosynthetic products of Portulaca oleracea differ greatly depending on leaf age and length of exposure to ¹⁴CO₂. Mature leaves of P. oleracea fix ¹⁴CO₂ primarily into organic and amino acids during a 10-s exposure period. Less than 2% of the ¹⁴CO₂ fixed appears in phosphorylated compounds. In contrast, incorporation into amino acids can account for over 60% of the total ¹⁴CO₂ fixed by young leaves in an equal time period, and incorporation into alanine alone can account for up to one half of this amount. Senescent leaves display a quantitative shift of primary products toward phosphorylated compounds with a concomitant reduction of the label residing in malate and asparate. About 8 times more phosphoglyceric acid is produced in senescent leaves than in mature leaves. The aspartate/ malate ratio is not constant and depends on the length of time the leaves are exposed to ¹⁴CO₂ and the age of the leaves under study. It appears as if the stage of leaf development is one of the most important factors determining the operation of a particular enzyme system in C₄ plants.     

Glutamic Acid metabolism and the photorespiratory nitrogen cycle in wheat leaves: metabolic consequences of elevated ammonia concentrations and of blocking ammonia assimilation

The effects of methionine sulfoximine and ammonium chloride on [¹⁴C] glutamate metabolism in excised leaves of Triticum aestivum were investigated. Glutamine was the principal product derived from [U¹⁴C]glutamate in the light and in the absence of inhibitor or NH₄Cl. Other amino acids, organic acids, sugars, sugar phosphates, and CO₂ became slightly radioactive. Ammonium chloride (10 mm) increased formation of [¹⁴C] glutamine, aspartate, citrate, and malate but decreased incorporation into 2-oxoglutarate, alanine, and ¹⁴CO₂. Methionine sulfoximine (1 mm) suppressed glutamine synthesis, caused NH₃ to accumulate, increased metabolism of the added radioactive glutamate, decreased tissue levels of glutamate, and decreased incorporation of radioactivity into other amino acids. Methionine sulfoximine also caused most of the ¹⁴C from [U-¹⁴C]glutamate to be incorporated into malate and succinate, whereas most of the ¹⁴C from [1-¹⁴C]glutamate was metabolized to CO₂ and sugar phosphates. Thus, formation of radioactive organic acids in the presence of methionine sulfoximine does not take place indirectly through “dark” fixation of CO₂ released by degradation of glutamate when ammonia assimilation is blocked. When illuminated leaves supplied with [U-¹⁴C] glutamate without inhibitor or NH₄Cl were transferred to darkness, there was increased metabolism of the glutamate to glutamine, aspartate, succinate, malate, and ¹⁴CO₂. Darkening had little effect on the labeling pattern in leaves treated with methionine sulfoximine.     

The Influence of Leaf Age on C(4) Photosynthesis and the Accumulation of Inorganic Carbon in Flaveria trinervia, a C(4) Dicot

Characteristics of C₄ photosynthesis were examined in young, mid-age, and mature leaves of Flaveria trinervia (an NADP-malic enzyme-type C₄ dicot). The turnover of [4-¹⁴C] (malate plus aspartate) following a pulse with ¹⁴CO₂ was similar in leaves of different ages (apparent half-time of 18-25 seconds). However, the rate of ¹⁴CO₂ incorporation in mid-age leaves was about 1.5-fold higher than in young leaves, and about 2.5-fold higher than in mature leaves. The rate of ¹⁴CO₂ fixation was proportional to the total active pool of malate plus aspartate but was not correlated with the total photosynthetically derived inorganic carbon pool. The leaf's ability to concentrate inorganic carbon photosynthetically declined during leaf expansion, from 29 down to 7 nanomoles per milligram chlorophyll. Similarly, the active aspartate pool also declined during leaf expansion, from about 123 down to 20 nanomoles per milligram chlorophyll. Enhanced metabolism of aspartate to CO₂ and pyruvate in young leaves is suggested to facilitate the maintenance of high CO₂ levels in bundle sheath cells which are thought to have a higher conductance to CO₂.     

Regulation of oxaloacetate, aspartate, and malate formation in mesophyll protoplast extracts of three types of c(4) plants

The use of mesophyll protoplast extracts from various C₄ species has provided an effective method for studying light-and substrate-dependent formation of oxaloacetate, malate, and asparate at rates equivalent to whole leaf C₄ photosynthesis. Conditions regulating the formation of the C₄ acids were studied with protoplast extracts from Digitaria sanguinalis, an NADP-malic enzyme C₄ species, Eleusineindica, an NAD-malic enzyme C₄ species, and Urochloa panicoides, a phosphoenolpyruvate (PEP) carboxykinase C₄ species. Light-dependent induction of CO₂ fixation by the mesophyll extracts of all three species was relatively low without addition of exogenous substrates. Pyruvate, alanine and α-ketoglutarate, or 3-phosphoglycerate induced high rates of CO₂ fixation in the mesophyll extracts with oxaloacetate, malate, and aspartate being the primary products. In all three species, it appears that pyruvate, alanine, or 3-phosphoglycerate may serve as effective precursors to the formation of PEP for carboxylation through PEP-carboxylase in C₄ mesophyll cells. Induction by pyruvate or alanine and α-ketoglutarate was light-dependent, whereas 3-phosphoglycerate-induced CO₂ fixation was not.     

Metabolism of alpha-Ketoglutarate by Roots of Woody Plants

The uptake and metabolism of α-ketoglutarate-5-¹⁴C by peach, apple, and privet root tissues were studied over various time intervals. As much as 80% of the absorbed ¹⁴C appeared as ¹⁴CO₂ in 320 minutes in peach roots. Apple and privet roots were less effective in this conversion with the bulk of the ¹⁴C found in the organic acid fraction. This indicates differences in organic acid metabolism among species of woody plants.

The ¹⁴C accumulated in malate earlier and in larger quantities than in citrate. Both glutamate and aspartate were labeled in 10 minutes and glutamate was labeled as early as 3 minutes. The labeling pattern does not clearly distinguish between the synthesis of glutamate by glutamic dehydrogenase or by transamination with oxaloacetate.

The rapid metabolism of α-ketoglutarate to glutamate by the 3 species studied indicates the presence of enzyme systems important in amino acid synthesis in the roots of woody plants.

     

Variations in short term products of inorganic carbon fixation in exponential and stationary phase cultures of Aphanocapsa 6308

Aphanocapsa 6308 metabolizes both NaHCO₃ and Na₂CO₃. The short term incorporation (5-s) metabolic pattern and the patterns of incorporation of bicarbonate for exponential versus stationary phase cultures differ, however. Cells were equilibrated for 10 min in air and distilled water prior to injection of either NaH¹⁴CO₃ at pH 8.0, or Na₂ ¹⁴CO₃ at pH 11.0. Hot ethanol extracts were analyzed via paper chromatography and autoradiography for products of CO₂ fixation. At 5 s, malate (51.5%) predominates slightly as a primary bicarbonate fixation product over 3-phosphoglycerate (40.3%); 3-phosphoglycerate is the primary product of carbonate fixation. At 60 s, the carbonate and bicarbonate labelling patterns are similar. Cells in stationary phase fix in 5 s a greater proportion of bicarbonate into malate (36% vs. 14% for 3-phosphoglycerate) than do cells in exponential growth. Likewise, 60 s incorporations show a large amount of bicarbonate fixed into aspartate (30.9%) in stationary phase cells over that of exponential phase (11.6%). These data suggest an operative C₄ pathway for purposes not related to carbohydrate synthesis but rather as compensation for the incomplete tricarboxylic acid cycle in cyanobacteria. The enhancement of both aspartate fixation and CO₂ fixation into citrulline in stationary phase correlates with an increase in cyanophycin granule production which requires both aspartate and arginine.Nonstandard Abbreviations 3-PGA 3-phosphoglyceric acid - TCA tricarboxylic acid     

Variation with age in the photosynthetic carbon fixation pattern by leaves of Amaranthus paniculatus and Oryza sativa: Change in the primary carboxylation but no shift from C4 or C3 pathway

Abstract The pattern of photosynthetic carbon fixation by leaves of Amaranthus paniculatus L. (a C₄ plant) and Oryza sativa L. (a C₃ plant) varied with age. Younger leaves of A. paniculatus incorporated ¹⁴CO₂ into malate and aspartate while senescent leaves fixed predominantly into phosphoglycerate (PGA) and sugar phosphates. Only developing leaves of O. sativa formed malate/aspartate whereas mature and senescent leaves produced PGA/sugar phosphates as the initial labelled products. Correspondingly the ratio of phosphoenolpyruvate/ribulose bisphosphate (RuBP) carboxylase activities was higher in younger leaves of A. paniculatus and developing leaves of O. sativa than in older leaves. However, pulse chase experiments revealed that the main donors of carbon to end products, irrespective of leaf stage, were C₄ acids and PGA in A. paniculatus and O. sativa respectively. The results suggest that although an apparent change from initial β-carboxylation to RuBP carboxylation occurs during leaf ontogeny in both the plants, the overall leaf photosynthesis remains C₄ or C₃. The high rate of ¹⁴CO₂ incorporation into PGA/sugar phosphates by senescent leaves of A. paniculatus is suggested to be partly due to the increased intercellular spaces in their mesophyll, allowing greater access of CO₂ directly to RuBP carboxylase in the bundle sheath.     

Equilibration of Label in Malate during Dark Fixation of CO(2) in Kalanchoë fedtschenkoi

In vitro studies of dark ¹⁴CO₂ fixation with isolated cell aggregates of Kalanchoë fedtschenkoi showed that malate synthesized after 20 sec is predominantly (85 to 92%) labeled at carbon 4, while after 20 min only 65 to 69% of the radioactivity was located in this position. The intramolecular labeling pattern of malate could not be changed by supplementing the cells with carboxylation reaction substrates such as ribulose diphosphate or phosphoenolpyruvate. The kinetic decline of label at carbon 4 of malate occurs independently of CO₂ fixation, since 4-¹⁴C-labeled aspartate fed to the cells gave rise to malate labeled 62% at carbon 4 after 20 min. Furthermore, the cells were capable of converting fed malate to fumarate. It is concluded that synthesis of malate during dark CO₂ fixation is accomplished by a single carboxylation step via phosphoenolpyruvate carboxylase and labeling patterns observed in malate are a consequence of the action of fumarase.     

The C-4 pathway in Pennisetum purpureum

Summary The anatomical structure of leaf tissue of P. purpureum, and the short term labelling pattern following exposure to ¹⁴CO₂ in the lighht, have been investigated. Both the arrangement of photosynthetic tissue in two layers around the vascular tissue, and the early labelling of malate and aspartate, characteristic of C-4 plants were observed. The structure of the epidermis and the arrangement of stomata is such that CO₂ must pass through non-chloroplast-containing tissue before reaching the chloroplasts. At 0.05% CO₂ in air the rate of photosynthetic CO₂ assimilation was about 70 moles/mg chl·h. This increased to over 700 moles/mg chl·h at saturating concentrations of CO₂. At 0.05% CO₂ negative slopes were obtained from percentage plots for malate, which was the major product. As the CO₂ concentration was increased, sugar phosphates became the major product. At saturating concentrations of CO₂, both malate and aspartate had positive initial slopes and a negative slope was observed for phosphoglyceric acid. These results are discussed in relation to the contribution of C-4 metabolism to photosynthesis in P. purpureum.     

Malate synthesis by dark carbon dioxide fixation in leaves

The rates of dark CO₂ fixation and the label distribution in malate following dark ¹⁴CO₂ fixation in a C-4 plant (maize), a C-3 plant (sunflower), and two Crassulacean acid metabolism plants (Bryophyllum calycinum and Kalanchoë diagremontianum leaves and plantlets) are compared. Within the first 30 minutes of dark ¹⁴CO₂ fixation, leaves of maize, B. calycinum, and sunflower, and K. diagremontianum plantlets fix CO₂ at rates of 1.4, 3.4, 0.23, and 1.0 μmoles of CO₂/mg of chlorophyll· hour, respectively. Net CO₂ fixation stops within 3 hours in maize and sunflower, but Crassulaceans continue fixing CO₂ for the duration of the 23-hour experiment.

A bacterial procedure using Lactobacillus plantarum ATCC No. 8014 and one using malic enzyme to remove the β-carboxyl (C₄) from malate are compared. It is reported that highly purified malic enzyme and the bacterial method provide equivalent results. Less purified malic enzyme may overestimate the label in C₄ as much as 15 to 20%.

The contribution of carbon atom 1 of malate is between 18 and 21% of the total carboxyl label after 1 minute of dark CO₂ fixation. Isotopic labeling in the two carboxyls approached unity with time. The rate of increase is greatest in sunflower leaves and Kalanchoë plantlets. In addition, Kalanchoë leaves fix ¹⁴CO₂ more rapidly than Kalanchoë plantlets and the equilibration of the malate carboxyls occurs more slowly. The rates of fixation and the randomization are tissue-specific. The rate of fixation does not correlate with the rate of randomization of isotope in the malate carboxyls.

     

CO(2) Metabolism in Corn Roots. I. Kinetics of Carboxylation and Decarboxylation

The kinetics of ¹⁴CO₂ carboxylation and decarboxylation in corn root tips were determined to ascertain the sequence of product formation and subsequent utilization, and to obtain further evidence to predict the enzymes mediating the carboxylation and decarboxylations. The carboxylation data indicated that the first product was oxaloacetate followed by malate and aspartate. Malate was the first stable product which could be detected. Decarboxylation data indicated that a large fraction of the ¹⁴CO₂ release and turnover of ¹⁴C was accountable for by a decrease in malate: however, essentially all labeled amino acids turned over rapidly and at a greater rate than organic acids. The data generally support the hypothesis that CO₂ fixation in corn root tips is via P-enolpyruvate carboxylase and malic dehydrogenase and that subsequent malate metabolism is for the most part by direct decarboxylation, possibly by the malic enzyme.     

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     

Photosynthetic activities of isolated bundle sheath cells in relation to differing mechanisms of C-4 pathway photosynthesis.

Photosynthetic activities of bundle sheath cell strands isolated from several C₄ pathway species were examined. These included species that decarboxylate C₄ acids via either NADP-malic enzyme (Zea mays, NADP-malic enzyme-type), NAD-malic enzyme (Atriplex spongiosa and Panicum miliaceum, NAD-malic enzyme-type) or phosphoenolpyruvate carboxykinase (Chloris gayana and Panicum maximum, phosphoenolpyruvate carboxykinase-type). Preparations from each of these species fixed ¹⁴CO₂ at rates ranging between 1.2 and 3.5 μmol min^?1 mg^?1 of chlorophyll, with more than 90% of the ¹⁴C being assimilated into Calvin cycle intermediates. With added HCO₃^? the rate of light-dependent O₂ evolution ranged between 2 and 4 μmol min^?1 mg^?1 of chlorophyll for cells from NAD-malic enzyme-type and phosphoenolpyruvate carboxykinase-type species but with Z. mays cells there was no O₂ evolution detectable. Most of the ¹⁴CO₂ fixed by Z. mays cells provided with H¹⁴CO₃^? plus ribose 5-phosphate accumulated in the C-1 of 3-phosphoglycerate. However, 3-phosphoglycerate reduction was increased several fold when malate was also provided. Cells from all species rapidly decarboxylated C₄ acids under appropriate conditions, and the CO₂ released from the C-4 carboxyl was reassimilated via the Calvin cycle. Malate decarboxylation by Z. mays cells was dependent upon light and an endogenous or exogenous source of 3-phosphoglycerate. Bundle sheath cells of NAD-malic enzyme-type species rapidly decarboxylated [¹⁴C]malate when aspartate and 2-oxoglutarate were also provided, and [¹⁴C]aspartate was decarboxylated at similar rates when 2-oxoglutarate was added. Cells from phosphoenolpyruvate carboxykinase-type species decarboxylated [¹⁴C]aspartate when 2-oxoglutarate was added and they also catalyzed a slower decarboxylation of malate. Cells from NAD-malic enzyme-type and phosphoenolpyruvate carboxykinase-type species evolved O₂ in the light when C₄ acids were added. These results are discussed in relation to proposed mechanisms for photosynthetic metabolism in the bundle sheath cells of species utilizing C₄ pathway photosynthesis.     

Acetate and carbon dioxide assimilation by Desulfovibrio vulgaris (Marburg), growing on hydrogen and sulfate as sole energy source

Desulfovibrio vulgaris (Marburg) was grown on hydrogen plus sulfate as sole energy source and acetate plus CO₂ as the sole carbon sources. The incorporation of U-¹⁴C acetate into alanine, aspartate, glutamate, and ribose was studied. The labelling data show that alanine is synthesized from one acetate (C-2 + C-3) and one CO₂ (C-1), aspartate from one acetate (C-2 + C-3) and two CO₂ (C-1 + C-4), glutamate from two acetate (C-1–C-4) and one CO₂ (C-5), and ribose from 1.8 acetate and 1.4 CO₂. These findings indicate that in Desulfovibrio vulgaris (Marburg) pyruvate is formed via reductive carboxylation of acetyl-CoA, oxaloacetate via carboxylation of pyruvate or phosphoenol pyruvate, and -ketoglutarate from oxaloacetate plus acetyl-CoA via citrate and isocitrate. Since C-5 of glutamate is derived from CO₂, citrate must have been formed via a (R)-citrate synthase rather than a(S)-citrate synthase. The synthesis of ribose from 1.8 mol of acetate and 1.4 mol of CO₂ excludes the operation of the Calvin cycle in this chemolithotrophically growing bacterium.     

Photosynthetic Characteristics of C(3)-C(4) Intermediate Flaveria Species : III. Reduction of Photorespiration by a Limited C(4) Pathway of Photosynthesis in Flaveria ramosissima

The initial products of photosynthesis by the C₃ species Flaveria cronquistii, the C₄ species F. trinervia, and the C₃-C₄ intermediate species F. ramosissima were determined using a pulse-chase technique with ¹⁴CO₂-¹²CO₂. The intermediate species F. ramosissima incorporated at least 42% of the total soluble ¹⁴C fixed into malate and aspartate after 10 seconds of photosynthesis in ¹⁴CO₂, as compared with 90% for the C₄ species F. trinervia and 5% for the C₃ species F. cronquistii. In both F. ramosissima and F. trinervia, turnover of labeled malate and aspartate occurred during a chase period in ¹²CO₂, although the rate of turnover was slower in the intermediate species. Relative to F. cronquistii, F. ramosissima showed a reduced incorporation of radioactivity into serine and glycine during the pulse period. These results indicate that a functional C₄ pathway of photosynthesis is operating in F. ramosissima which can account for its reduced level of photorespiration, and that this species is a true biochemical intermediate between C₃ and C₄ plants.     

Extremely high activities of phosphoenolpyruvate carboxylase in thermogenic tissues of Araceae

Phosphoenolpyruvate carboxylase activity in extracts of a wide range of thermogenic tissues of the Araceae was shown to be in the range 10–100 mol g^-1 fresh weight min^-1 (0.5–3.7 mol mg^-1 protein min^-1). Such high activities were not found in non-thermogenic tissues of the Araceae or in thermogenic tissues of Aristolochia brasiliensis Mart. and Zucc., Victoria amazonica Schomb. and Encephalartos barteri Carruth. During development and thermogenesis in the club of Arum maculatum L. the high activities of the carboxylase did not lead to any marked accumulation of citrate, isocitrate, 2-oxoglutarate, fumarate, malate and oxaloacetate. Clubs of Arum maculatum and of Arum italicum Miller readily fixed ¹⁴CO₂ in the dark, mostly into aspartate, malate, alanine and glutamate. Pulse and chase experiments showed that most of the fixed carbon was very rapidly metabolized to CO₂. The detailed distribution suggest that this occurred largely by decarboxylation of C-4 acids. It is suggested that thermogenic tissues of the Araceae are characterized by very high activities of phosphoenolpyruvate carboxylase, and that in vivo this leads to synthesis of C-4 acids which are promptly decarboxylated.     

Studies on the mechanism of glycerate 3-phosphate synthesis in tomato and maize leaves

The net carbon incorporation in maize (Zea mays) and tomato (Lycopersicum esculentum) leaves was mainly the result of the carboxylation of ribulose 1,5-diphosphate. In both of these organisms synthesis of glycerate 3-phosphate was studied during short chase experiments (2 or 3 seconds in ¹⁴CO₂ then 8 to 27 seconds in unlabeled CO₂). Changes in the radioactivity in the individual carbon atoms of glycerate 3-phosphate, malate, and aspartate are consistent with the formation, in both leaves, of 2 molecules of glycerate 3-phosphate for each CO₂ molecule incorporated. The CO₂, before reacting with ribulose 1,5-diphosphate, is first incorporated in an intracellular CO₂ pool which has a different composition according to the species. This pool is constituted in tomato by volatile compounds (50 nanomoles per gram of fresh weight) more or less in equilibrium with atmospheric CO₂. In maize the pool consists of carbon atoms 4 of malate and aspartate (for at least 80% of the pool) and volatile compounds which correspond, in all, to 540 nanomoles per gram of fresh weight where atmospheric CO₂ enters through an irreversible reaction.     

Photosynthetic Induction in a C(4) Dicot, Flaveria trinervia: II. Metabolism of Products of CO(2) Fixation after Different Illumination Times

The metabolism of fixed ¹⁴CO₂ and the utilization of the C-4 carboxyl of malate and aspartate were examined during photosynthetic induction in Flaveria trinervia, a C₄ dicot of the NADP-malic enzyme subgroup. Pulse/chase experiments indicated that both malate and aspartate appeared to function directly in the C₄ cycle at all times during the induction period (examined after 30 seconds, 5 minutes and 20 minutes illumination). However, the rate of loss of ¹⁴C-label from the C-4 position of malate plus aspartate was relatively slow after 30 seconds of illumination, compared to treatments after 5 or 20 minutes of illumination. Similarly, the appearance of label in other photosynthetic products (e.g. 3-phosphoglycerate, sugar phosphates, alanine) during the chase periods was generally slower after only 30 seconds of leaf illumination, compared to that after 5 of 20 minutes illumination. This may be due to the lower rate of photosynthesis after 30 seconds illumination. The appearance of label in carbons 1→3 of each C₄ acid during the chase periods was relatively slow after either 30 seconds or 5 minutes illumination, while there was a relatively rapid accumulation of label in carbons 1→3 of both C₄ acids after 20 minutes illumination. Thus, while the turnover rate of the ¹⁴C-4 label in both C₄ acids increased only during the first 5 minutes of the induction period, only later during induction is there an increased rate of appearance of label in other carbon atoms of the C₄ acids. The implied source of ¹⁴C for labeling of the 1→3 positions of the C₄ acids is an apparent carbon flux from 3-phosphoglycerate of the reductive pentose phosphate pathway to phosphoenolpyruvate of the C₄ cycle.     

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