Malate Pool Research Articles

Abstract 2867: Ceramide-dependent mitophagy leads to metabolic reprogramming in aging T-cells associated with reduced anti-tumor T-cell functions

Abstract Changes in mitochondrial bioenergetics in aging T-cells play a key role in the decrease in T-cell function and tumor resistance with aging. The bioactive sphingolipid ceramide, induced by aging stress, is a major player in these changes, as its accumulation at the mitochondrial membranes of both mouse and human aging T-cells induces ceramide-dependent mitophagy, which decreases T-cell viability, cytokine secretion, and anti-tumor activity. Using knock-out mouse models and pharmacological inhibition, we show that C14-ceramide accumulation in the mitochondria and mitophagy in the T-cells of aging mice are dependent on Ceramide Synthase 6 (CerS6), an enzyme responsible for C14/C16-ceramide synthesis. Interestingly, sphingosine kinase 2 (SphK2), another enzyme involved in ceramide metabolism inhibits HDAC1/2 function in aging T-cells, epigenetically inducing CerS6 expression and the resultant ceramide-dependent mitophagy, which ultimately decreases T-cell anti-tumor function. Both CerS6-/- and SphK2-/- aging T-cells were protected from the reduction in T-cell function, and their anti-tumor function was maintained in adoptive-cell transfer experiments to mice with melanoma. Besides lipid signaling, other metabolic changes associated with this aging-induced process have not been explored, and the utility of correcting these metabolic changes to restore T-cell function has not been evaluated. We studied the total hydrophilic metabolome of aging T-cells undergoing ceramide-dependent mitophagy to assess the metabolic changes occurring in these cells. Our findings show a general depletion of tricarboxylic acid cycle (TCA) metabolites in aging T-cells, with pools of fumarate, malate, and argininosuccinate significantly decreasing in both mouse and human aging T-cells. Interestingly, these changes were mirrored by similar changes in human cancer cells undergoing ceramide-dependent mitophagy, suggesting that these changes might constitute a general metabolic signature for this specific mitophagy process with deleterious effects on anti-tumor functions of aging T-cells. Both in vitro and in vivo supplementation of fumarate to mouse T-cells seemed to correct some of the functional defects observed in aging, with a protection of T-cell viability, cytokine production, and tumor killing capacity in co-cultures with tumor cells. The fumarate supplementation also led to a decrease in ceramide-dependent mitophagy in the aging T-cells, indicating an interplay between ceramide-dependent mitophagy and fumarate metabolism. Overall, these studies help explain the mechanisms behind aging-related dysregulation of T-cell anti-tumor activity, which can be restored by inhibiting ceramide-dependent mitophagy by pharmacological and genetic tools or by reprogramming fumarate metabolism in aging T cells. Citation Format: Mohamed Faisal Kassir, Han Gyul Lee, Natalia Oleinik, Paramita Chakraborty, John J. Lemasters, Zdzislaw Szulc, Xue-Zhong Yu, Shikhar Mehrotra, Besim Ogretmen. Ceramide-dependent mitophagy leads to metabolic reprogramming in aging T-cells associated with reduced anti-tumor T-cell functions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2867.

Soybean NADP-Malic Enzyme Functions in Malate and Citrate Metabolism and Contributes to Their Efflux under Al Stress.

Malate accumulation has been suggested to balance Al-induced citrate synthesis and efflux in soybean roots. To test this hypothesis, characteristics of Al-induced accumulation and efflux of citrate and malate were compared between two soybean genotypes combining a functional analysis of GmME1 putatively encode a cytosolic NADP-malic enzyme. Similar amounts of citrate were released, and root elongation was equally inhibited before 8 h of Al treatment of Jiyu 70 and Jiyu 62 cultivars. Jiyu 70 began to secrete more citrate and exhibited higher Al resistance than did Jiyu 62 at 12 h. A sustained increase in internal malate and citrate concentrations was observed in Jiyu 70 at 24 h of Al treatment. However, Jiyu 62 decreased its malate concentration at 12 h and its citrate concentration at 24 h of Al treatment. GmME1 localized to the cytoplast and clustered closely with cytosolic malic enzymes AtME2 and SgME1 and was constitutively expressed in the roots. Al treatment induced higher NADP-malic enzyme activities and GmME1 expression levels in Jiyu 70 than in Jiyu 62 within 24 h. Compared with wild-type hairy roots, over-expressing GmME1 in hairy roots (GmME1-OE) produced higher expression levels of GmME1 but did not change the expression patterns of either of the putative citrate transporter genes GmAACT1 and GmFRDL or the malate transporter gene GmALMT1, with or without Al treatment. GmME1-OE showed a higher internal concentration and external efflux of both citrate and malate at 4 h of Al stress. Lighter hematoxylin staining and lower Al contents in root apices of GmME1-OE hairy roots indicated greater Al resistance. Comprehensive experimental results suggest that sustaining Al-induced citrate efflux depends on the malate pool in soybean root apices. GmME1 encodes a cytosolic malic enzyme that contributes to increased internal malate and citrate concentrations and their external efflux to confer higher Al resistance.

Short‐term dynamics of isotopic composition of leaf‐respired CO2 upon darkening: measurements and implications

Recent advances in understanding the metabolic origin and the temporal dynamics in delta(13)C of dark-respired CO(2) (delta(13)C(res)) have led to an increasing awareness of the importance of plant isotopic fractionation in respiratory processes. Pronounced dynamics in delta(13)C(res) have been observed in a number of species and three main hypotheses have been proposed: first, diurnal changes in delta(13)C of respiratory substrates; second, post-photosynthetic discrimination in respiratory pathways; and third, dynamic decarboxylation of enriched carbon pools during the post-illumination respiration period. Since different functional groups exhibit distinct diurnal patterns in delta(13)C(res) (ranging from 0 to 10 per thousand diurnal increase), we explored these hypotheses for different ecotypes and environmental (i.e. growth light) conditions. Mass balance calculations revealed that the effect of respiratory substrates on diurnal changes in delta(13)C(res) was negligible in all investigated species. Further, rapid post-illumination changes in delta(13)C(res) (30 min), which increased from 2.6 per thousand to 5 per thousand over the course of the day, were examined by positional (13)C-labelling to quantify changes in pyruvate dehydrogenase (PDH) and Krebs cycle (KC) activity. We investigated the origin of these dynamics with Rayleigh mass balance calculations based on theoretical assumptions on fractionation processes. Neither the estimated changes of PDH and KC, nor decarboxylation of a malate pool entirely explained the observed pattern in delta(13)C(res). However, a Rayleigh fractionation of (12)C-discriminating enzymes and/or a rapid decline in the decarboxylation rate of an enriched substrate pool may explain the post-illumination peak in delta(13)C(res). These results are highly relevant since delta(13)C(res) is used in large-scale carbon cycle studies.

Operation of the xanthophyll cycle and degradation of D1 protein in the inducible CAM plant, Talinum triangulare, under water deficit.

Changes in photochemical activity induced by water deficit were investigated in Talinum triangulare, an inducible CAM plant. The aim was to analyse the interactions between C3 photosynthesis, induction and activity of CAM, photosynthetic energy regulation and the mechanisms responsible for photoprotection and photoinhibition under water stress. Gas exchange, chlorophyll a fluorescence, titratable acidity, carotenoid composition and relative contents of the PSII reaction centre protein (D1) were measured. A decrease in xylem tension (psi) from -0.14 to -0.2 MPa substantially decreased daytime net CO2 assimilation and daily carbon gain, and induced CAM, as shown by CO2 assimilation during the night and changes in titratable acidity; a further decrease in psi decreased nocturnal acid accumulation by 60%. Non-photochemical quenching of chlorophyll a fluorescence (NPQ) increased with water deficit, but decreased with a more severe drought (psi below -0.2 MPa), when CAM activity was low. NPQ was lower at 0900 h (during maximum decarboxylation rates) than at 1400 h, when malate pools were depleted. Down-regulation of PSII activity related to the rise in NPQ was indicated by a smaller quantum yield of PSII photochemistry (phiPSII) in droughted compared with watered plants. However, phiPSII was larger at 0900 h than at 1400 h. The de-epoxidation state of the xanthophyll cycle increased with drought and was linearly related to NPQ. Intrinsic quantum yield of PSII (FV/FM) measured at dusk was also lower in severely stressed plants than in controls. Under maximum photosynthetic photon flux and high decarboxylation rates of organic acids, the D1 content in leaves of droughted plants showing maximal CAM activity was identical to the controls; increased drought decreased D1 content by more than 30%. Predawn samples had D1 contents similar to leaves sampled at peak irradiance, with no signs of recovery after 12 h of darkness. It is concluded that under mild water stress, early induction of CAM, together with an increased energy dissipation by the xanthophyll cycle, prevents net degradation of D1 protein; when water deficit is more severe, CAM and xanthophyll cycle capacities for energy dissipation decline, and net degradation of D1 proceeds.

The Effect of Irradiance on Carboxylating/Decarboxylating Enzymes and Fumarase Activities in Mesembryanthemum crystallinum L. Exposed to Salinity Stress

Abstract: In Mesembryanthemum crystallinum plants, treated for 9 days with 0.4 M NaCl at low light intensities (80 ‐ 90 or 95 ‐ 100 μE m‐2 s‐1; λ = 400 ‐ 700 nm), no day/night malate level differences (Δmalate) were detected. At high light (385 ‐ 400 μE m‐2 s‐1) strong stimulation of PEPC activity, accompanied by a Δmalate of 11.3 mM, demonstrated the presence of CAM metabolism. This indicates that, to evolve day/night differences in malate concentration, high light is required. Salt treatment at low light induces and increases the activity of NAD‐ and NADP‐malic enzymes by as much as 3.7‐ and 3.9‐fold, while at high light these values reach 6.4‐ and 17.7‐fold, respectively. The induction of activity of both malic enzymes and PEPC (phospoenolpyruvate carboxylase) take place before Δmalate is detectable. An increase in SOD (superoxide dismutase) was observed in plants cultivated at high light in both control and salt‐treated plants. However, in salt‐treated plants this effect was more pronounced. Carboxylating and decarboxylating enzymes seem to be induced by a combination of different signals, i.e., salt and light intensity. Plants performing CAM, after the decrease of activity of both the decarboxylating enzymes at the beginning of the light period, showed an increase in these enzymes in darkness when the malate pool reaches higher levels. In CAM plants the activity of fumarase (Krebs cycle) is much lower than that in C3 plants. The role of mitochondria in CAM plants is discussed.

Environmental hypoxia affects osmotic and ionic regulation in freshwater midge-larvae

The effect of anaerobic metabolism on the osmotic and ionic regulation of the extracellular fluid was examined. Larvae of three species, characterized by different hypoxia tolerance, were studied: Chaoborus crystallinus, Culex pipiens and Chironomus gr. plumosus. The use of the capillary electrophoresis technique made it possible to determine approximately 15 different ions from individual hemolymph samples. The hemolymph concentration of both inorganic and organic anions and cations as well as the osmolality were measured. A correlation between the hypoxia tolerance and the capability to avoid net changes in the ion concentration or in the osmolality of the three species studied here is proposed: Culex larvae, which have the lowest hypoxia tolerance, show a very large and very rapid lactate accumulation in their hemolymph under experimental hypoxia. This lactate accumulation is not compensated for by a change in the concentration of any other ion. Chaoborus larvae, with a medium hypoxia tolerance, utilize their very large hemolymph malate pool as a source of anaerobic energy. It is converted into succinate, thus inducing little net changes in the sum of the anions. There is a marked increase of the hemolymph osmolality, though. Chironomus larvae have the highest hypoxia tolerance and there are remarkably little changes in their hemolymph under hypoxia. Although these larvae are described as relying mainly on ethanol fermentation under environmental anaerobiosis, we demonstrated a marked lactate fermentation in severe hypoxia. The lactate accumulation observed in our study was compensated by a concomittant decrease of the hemolymph chloride concentration.

Changes in primary metabolism in connection with alkaloid biosynthesis in solanaceous cell suspensions: a 13C-NMR study

Carbon-13 nuclear magnetic resonance (NMR) spectroscopy was used in the direct comparison of acetate metabolism in suspension cultures of Datura stramonium, an alkaloid producer in vitro, and Nicotiana phumbaginifolia, a non-producer. Growth of cells in the presence of 4 mM [2- 13C] acetate, resulted in a considerably slower uptake of the substrate from medium in D. stramonium than in N. plumbaginifolia. Initial labeling, primarily of the intracellular glutamine and malate pools was observed in N. plumbaginifolia, and putrescine and GABA pools in D. stramonium. The high activity of glutamine synthetase observed in N. plumbaginifolia, which can lead to the non-synthesis of intracellular putrescine by metabolic deviation of glutamate, would be a first explanation for non-biosynthesis of alkaloids for these cell suspensions. On the contrary, the observation of putrescine labeling is in agreement with alkaloid biosynthesis through D. stramonium cell cultures.

Models of Metabolism in Rhizopus oryzae

A flux analysis of glucose metabolism in Rhizopus oryzaewas achieved using [ 14C-labeled glucose and acetate. The rates of glucose utilization and end product production were estimated, and metabolite pool sizes and specific radioactivities were determined. These data were analysed using a specific radioactivity curve-matching program called TFLUX. The analysis is consistent with the existence of separate mitochondrial and cytosolic pools of pyruvate, malate and fumarate.

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 μmol · 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 μmol 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 μmol · 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.

13C nuclear magnetic resonance studies of malate and citrate synthesis and compartmentation in higher plant cells.

The synthesis of malate and citrate by sycamore cells (Acer pseudoplatanus L.) perfused with KH13CO3 was analyzed using 13C NMR. To perform in vivo experiments, cells were compressed in a 25-mm tube and perfused with an arrangement enabling tight control of the circulating nutrient medium. An original method using paramagnetic Mn2+ that induced a complete loss of the vacuolar malate and citrate signals was developed to discriminate between cytoplasmic and vacuolar pools of malate and citrate. Our results indicated the following. (a) The accumulation of appreciable amounts of malate in sycamore cells required rather high (1 mM) concentrations of bicarbonate at all the pH values tested. (b) Malate was equally labeled at C-1 and C-4, suggesting that malate labeled at C-1 was produced by randomization of C-1 and C-4 by mitochondrial fumarase. Indeed, the separation of the intact organelles from the lysed protoplasts indicated that fumarase activity was essentially limited to the mitochondria. Similarly, citrate was equally enriched at C-1 and C-5 + C-6 carboxyls. (c) Malate appeared first in the cytoplasmic compartment; and when a threshold of cytoplasmic malate concentration was attained, malate molecules were expelled into the vacuole, where they accumulated. On the other hand, citrate accumulated steadily in the vacuole. Pulse-chase experiments demonstrated the central role played by the tonoplast in governing the vacuolar influx of citrate and the permanent exchange of malate between the cytoplasm and the vacuole.

Compartmentation of soluble carbohydrates, of starch and of malate in motor organs (pulvini) and other parts of Phaseolus coccineus L. leaves

Quantitative histochemistry was used to investigate the tissue-specific compartmentation of soluble carbohydrates (sucrose, glucose, fructose), starch and malate in the laminar pulvinus, leaf blade and petiole of Phaselous coccineus L. at day and night positions of diurnal leaf movement. Total carbohydrate levels measured in a series of cross sections along individual pulvini of 24-d-old plants showed only small differences between the day and night positions of the respective leaf. In contrast, the level of malate changed during diurnal leaf movement, especially in the central part of a pulvinus. The levels of glucose and fructose in the pulvinus increased towards the transition zones between the pulvinus and lamina, and pulvinus and petiole, and this trend was even more pronounced for starch. By contrast, sucrose levels were highest in the pulvinus proper. The transverse compartmentation of metabolites was studied in distinct, approx. 0.5-mm-thick tissue slices from the central part of a pulvinus. These were dissected further into up to 14 distinct subsamples (bundle, bundle sheath, motor tissues, flanks). Irrespective of the position of the leaf (day or night), the central vascular core and the surrounding bundle sheath had high levels of sucrose (up to 500 mmol-(kg DW)(-1)) and low levels of glucose and fructose (below 100 mmol-(kg DW)(-1)), while in the cortex the situation was reversed. In the night position the level of sucrose decreased by approx. 30% in the bundle sheath and the central vascular core but not in the other sections. We thus suggest that because of the relatively small diurnal changes in their cortical pools, soluble sugars are not involved in the osmotic processes resulting in leaf movement. In contrast, pulvini from 14-d-old plants showed an interesting diurnal change in starch and malate pools in the outermost layer of the extensor. Here starch increased at night while the malate pool was lowered nearly stoichiometrically. Inverse pool sizes were found in the day position of the respective leaves. Although less significant, the opposite diurnal variation occurred in samples taken from the flexor region. We thus were able to locate areas of different carbohydrate activities in the laminar pulvinus of P. coccineus. The central vascular core, including the bundle sheath, is involved in temporary storage of photoassimilates, and the cortical regions are responsible for osmotically driven leaf movement. The results are discussed with respect to guard-cell physiology.

Effects of Abrupt or Progressive Drought on the Photosynthetic Mode of Crassula sieberiana Cultivated under Different Daylengths

Well-watered plants of Crassula sieberiana were shown to perform C3-type of photosynthesis independently of photoperiodic regimes. Abrupt drought conditions shifted the plants to CAM or CAM-like situations according to CO2-exchange patterns, levels and diel oscillation of malate pools and phosphoenolpyruvate carboxylase capacity (EC 4.1.1.31 PEPC). Progressively increased levels of drought under long days resulted, at each step, in reversible increase in PEPC extractable activity, independently of other CAM criteria (CO2 exchange patterns and diel oscillation of malate). Immunotitration experiments showed that the increase in PEPC was due to de novo synthesis of the enzyme. No correlation could be found between PEPC synthesis and water parameters of the leaves. The primary triggering element for the observed changes remains to be discovered.

Channeling of TCA cycle intermediates in cultured Saccharomyces cerevisiae

Oxidation of [3-13C]propionate was studied in cultured yeast cells, and the distribution of label in the 2- and 3-positions of alanine was detected by 13C NMR. [3-13C]Propionate forms [2-13C]succinyl-CoA in the mitochondria which then enters the citric acid cycle and forms malate through two symmetrical intermediates, succinate and fumarate. If these symmetrical intermediates randomly diffuse from one enzyme to the next in mitochondria as is normally assumed, then 13C labeling in malate C2 and C3 must be equal. However, any direct transfer of metabolites from site to site between succinate thiokinase, succinate dehydrogenase, and fumarase would result in an uneven distribution of 13C in malate C2 and C3 and any molecules derived from malate. Since pyruvate may be derived from malate via the malic enzyme and subsequently converted into alanine by transamination, any 13C asymmetry in alanine C2 and C3 must directly reflect the 13C distribution in the malate pool. During oxidation of [3-13C]propionate, we detect a significant quantity of labeled alanine, where 13C enrichment in C3 is significantly higher than that in C2. Inhibition of succinate dehydrogenase with malonate or creating conditions that increase the chances of a back-reaction (from malate to fumarate) result in a significant decrease in the asymmetric labeling of alanine. Ubiquinone-deficient yeast cells (having only 10% of the oxidative capacity of wild-type cells) could slowly oxidize propionate, but in this case the 13C labeling was equal in the C2 and C3 of alanine, showing that isotope randomization had occurred.(ABSTRACT TRUNCATED AT 250 WORDS)

Observation of Cytoplasmic and Vacuolar Malate in Maize Root Tips by 13C-NMR Spectroscopy

The accumulation of malate by maize (Zea mays L.) root tips perfused with KH(13)CO(3) was followed by (13)C nuclear magnetic resonance spectroscopy. In vivo nuclear magnetic resonance spectra contained distinct signals from two pools of malate in maize root tips, one at a pH approximately 5.3 (assigned to the vacuole) and one at a pH > 6.5 (assigned to the cytoplasm). The ratio of cytoplasmic to vacuolar malate was lower in 12 millimeter long root tips than in 2 millimeter root tips. The relatively broad width of the signals from C1- and C4-labeled vacuolar malate indicated heterogeneity in vacuolar pH. During the 3 hour KH(13)CO(3) treatment, (13)C-malate accumulated first primarily in the cytoplasm, increasing to a fairly constant level of approximately 6 millimolar by 1 hour. After a lag, vacuolar malate increased throughout the experiment.

The Influence of Leaf Age on C4 Photosynthesis and the Accumulation of Inorganic Carbon in Flaveria trinervia, a C4 Dicot

Characteristics of C(4) photosynthesis were examined in young, mid-age, and mature leaves of Flaveria trinervia (an NADP-malic enzyme-type C(4) dicot). The turnover of [4-(14)C] (malate plus aspartate) following a pulse with (14)CO(2) was similar in leaves of different ages (apparent half-time of 18-25 seconds). However, the rate of (14)CO(2) 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 (14)CO(2) 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 CO(2) and pyruvate in young leaves is suggested to facilitate the maintenance of high CO(2) levels in bundle sheath cells which are thought to have a higher conductance to CO(2).

Enzymic and Photosynthetic Characteristics of Reciprocal F1 Hybrids of Flaveria pringlei (C3) and Flaveria brownii (C4-Like Species)

The activities of key C(4) enzymes in gel-filtered, whole-leaf extracts and the photosynthetic characteristics for reciprocal F(1) hybrids of Flaveria pringlei (C(3)) and F. brownii (C(4)-like species) were measured to determine whether any inherited C(4)-photosynthetic traits are responsible for their reduced CO(2) compensation concentration values (AS Holaday, S Talkmitt, ME Doohan Plant Sci 41: 31-39). The activities of phosphoenolpyruvate carboxylase, pyruvate, orthophosphate dikinase, and NADP-malic enzyme (ME) for the reciprocal hybrids are only about 7 to 17% of those for F. brownii, but are three- to fivefold greater than the activities for F. pringlei. The low activities of these enzymes in the hybrids appear to be the result of a partial dominance of F. pringlei genes over certain F. brownii genes. However, no such dominance occurs with respect to the expression of genes for NADP-malate dehydrogenase, which is as active in the hybrids as in F. brownii. In contrast to the situation with the enzymes above, cytoplasmic factors appear to determine the inheritance of NAD-ME. The NAD-ME activity in each hybrid is comparable to that in the respective maternal parent. Pulse-chase (14)CO(2) incorporation analyses at ambient CO(2) levels indicate that the hybrids initially assimilate 7 to 9% of the total assimilated CO(2) into C(4) acids as compared to 3.5% for F. pringlei. In the hybrids, the percentage of (14)C in malate decreases from an average of 6.5 to 2.1% after a 60-second chase in (12)CO(2)/air. However, this apparent C(4)-cycle activity is too limited or inefficient to substantially alter CO(2) exchange from that in F. pringlei, since the values of net photosynthesis and O(2) inhibition of photosynthesis are similar for the hybrids and F. pringlei. Also, the ratio of the internal to the external CO(2) concentration and the initial slopes of the plot of CO(2) concentration versus net photosynthesis are essentially the same for the hybrids and F. pringlei. At 45 micromoles CO(2) per mole and 0.21 mole O(2) per mole, the hybrids assimilate nearly fivefold more CO(2) into C(4) acids than does F. pringlei. Some turnover of the malate pool occurs in the hybrids, but the labelling of the photorespiratory metabolites, glycine and serine, is the same in these plants as it is in F. pringlei. Thus, although limited C(4)-acid metabolism may operate in the hybrids, we conclude that it is not effective in altering O(2) inhibition of CO(2) assimilation. The ability of the hybrids to assimilate more CO(2) via phosphoenolpyruvate carboxylase at low levels of CO(2) than does F. pringlei may result in an increased rate of reassimilation of photorespiratory CO(2) and CO(2) compensation concentrations below that of their C(3) parent. If the hybrids do possess a limited C(4) cycle, it must operate intracellularly. They are not likely to have inherited an intercellular compartmentation of C(4) enzymes, since F. brownii has incomplete compartmentation of key C(3) and C(4) enzymes.

Photosynthesis in marine macroalgae: evidence for carbon limitation

During a shipboard expedition to Andros Island (Bahamas), photosynthetic measurements for the macroalgae Cladophoropsis membranacea (Chlorophyta), Dilophus guineensis, Turbinaria turbinata, and Lobophora variegata (Phaeophyta), and Laurencia papillosa (Rhodophyta), taken directly from their marine habitat, showed that only Cladophoropsis was saturated at seawater inorganic carbon levels (2.5 mM). The photosynthetic k0.5 values for inorganic carbon ranged from 1.1 to 3.2 mM. Decreasing the pH at 2.5 mM inorganic carbon, and thus enhancing the CO2 by 30-fold, only slightly increased photosynthesis, suggesting that bicarbonate was the major assimilated form of inorganic carbon. At 2.5 mM inorganic carbon, only Lobophora exhibited a Warburg effect on photosynthesis (49%), but at 0.5 mM, Turbinaria and Laurencia were also inhibited by O2. Ribulosebisphosphate carboxylase–oxygenase appeared to be the predominant carboxylation enzyme, but in Dilophus and Laurencia extracts, its activity was rivaled by phosphoenolpyruvate carboxylase and carboxykinase. Malate pools were detected in Turbinaria and Laurencia, and in the latter they were greater at night than during the day. However, this diel fluctuation was too small to implicate crassulacean acid metabolism. The data indicate that the bicarbonate concentration in seawater is insufficient to overcome O2 inhibition effects on photosynthesis, unless the macroalga has some form of CO2 concentrating system, based on bicarbonate uptake or C4 acid metabolism. In addition, the inorganic carbon in seawater may be a nutrient limiting the photosynthesis and productivity of certain macroalgae.

Characterization of Acetate and Pyruvate Metabolism in Suspension Cultures of Zea mays by 13C NMR Spectroscopy

Carbon-13 nuclear magnetic resonance (NMR) spectroscopy has been applied to the direct observation of acetate and pyruvate metabolism in suspension cultures of Zea mays (var Black Mexican Sweet). Growth of the corn cells in the presence of 2 millimolar [2-(13)C]acetate resulted in a rapid uptake of the substrate from the medium and initial labeling (0-4 hours) of primarily the intracellular glutamate and malate pools. Further metabolism of these intermediates resulted in labeling of glutamine, aspartate, and alanine. With [1-(13)C]acetate as the substrate very little incorporation into intermediary metabolites was observed in the (13)C NMR spectra due to loss of the label as (13)CO(2). Uptake of [3-(13)C]pyruvate by the cells was considerably slower than with [2-(13)C]acetate; however, the labelling patterns were similar with the exception of increased [3-(13)C] alanine generation with pyruvate as the substrate. Growth of the cells for up to 96 hours with 2 millimolar [3-(13)C]pyruvate ultimately resulted in labeling of valine, leucine, isoleucine, threonine, and the polyamine putrescine.

CAM induction by photoperiodism in green callus cultures from a CAM plant

Abstract Green calli obtained from leaves of the CAM‐inducible plant Kalanchoe blossfeldiana cv. Montezuma were grown either under long‐day or short‐day regimes in the Phytotron of Gif‐sur‐Yvette. Callus cells were found to be CAM inducible by the short‐day treatment, according to levels and diurnal oscillations of malate pools and phosphor‐enolpyruvate (PEP) carboxylase (EC.4.1.1.31) capacity. De novo synthesis of PEP carboxylase was shown to occur under the short‐day regime. In spite of continuous net CO2 output, CAM‐like patterns of CO2 exchange by calli were obtained under the short‐day treatment. After 2 months under both photoperiodic conditions, spontaneous organogenesis in callus tissues gave rise to numerous shootlets which were determined as photoperiod dependent for CAM; the same was so for plants originating from these shootlets.

Characterization of carbon metabolism in Opuntia ficus-indica Mill. exhibiting the idling mode of Crassulacean acid metabolism

Upon transfer from well-watered conditions to total drought, long-day-grown cladodes of Opuntia ficus-indica Mill. shift from full Crassulacean acid metabolism (CAM) to CAM-idling. Experiments using (14)C-tracers were conducted in order to characterize the carbon-flow pattern in cladodes under both physiological situations. Tracer was applied by (14)CO2 fumigations and NaH(14)CO3 injections during the day-night cycle. The results showed that behind the closed stomata, mesophyll cells of CAM-idling plants retained their full capacity to metabolize CO2 in light and in darkness. Upon the induction of CAM-idling the level of the capacity of phosphoenolpyruvate carboxylase (EC 4.1.1.31) was maintained. By contrast, malate pools decreased, displaying finally only a small or no day-night oscillation. The capacity of NADP-malic enzyme (EC 1.1.1.40) decreased in parallel with the reduction in malate pools. Differences in the labelling patterns, as influenced by the mode of tracer application, are discussed.