Effects of 1-Methyl-4-Phenylpyridinium on Isolated Rat Brain Mitochondria: Evidence for a Primary Involvement of Energy Depletion

Abstract: The effects of 1-methyl-4-phenylpyridinium (MPP⁺) on the oxygen consumption, ATP production, H₂O₂ production, and mitochondrial NADH-CoQ₁ reductase (complex I) activity of isolated rat brain mitochondria were investigated. Using glutamate and malate as substrates, concentrations of 10–100 µ M MPP⁺ had no effect on state 4 (−ADP) respiration but decreased state 3 (+ADP) respiration and ATP production. Incubating mitochondria with ADP for 30 min after loading with varying concentrations of MPP⁺ produced a concentration-dependent decrease in H₂O₂ production. Incubation of mitochondria with ADP for 60 min after loading with 100 µ M MPP⁺ caused no loss of complex I activity after washing of MPP⁺ from the mitochondrial membranes. These data are consistent with MPP⁺ initially binding specifically to complex I and inhibiting both the flow of reducing equivalents and the production of H₂O₂ by the mitochondrial respiratory chain, without irreversibly damaging complex I. However, mitochondria incubated with H₂O₂ in the presence of Cu²⁺ ions showed decreased complex I activity. This study provides additional evidence that cellular damage initiated by MPP⁺ is due primarily to energy depletion caused by specific binding to complex I, any increased damage due to free radical production by mitochondria being a secondary effect.     

The effects of oligomycin on content of adenylates in mesophyll protoplasts,chloroplasts and mitochondria from Pb<Superscript>2+</Superscript> treated pea and barley leaves

The effects of oligomycin on photosynthesis and respiration in relation to ATP production in chloroplasts and mitochondria were investigated in protoplasts isolated from the detached pea (Pisum sativum L cv. Iłowiecki.) and barley (Hordeum vulgare L. cv. Gunilla) leaves treated 5 mM Pb(NO₃)₂. The oligomycin (OM), an inhibitor of oxidative phosphorylation at 0.1 μM concentration caused the inhibition of photosynthesis rate in the protoplasts from both the control and the Pb-treated pea leaves. The respiration rate and ATP/ADP ratio in the protoplasts and the activity of ATPase in mitochondria, were also diminished in the control protoplasts. These effects were not observed in the protoplasts and mitochondria isolated from the Pb-treated leaves. Oligomycin, an inhibitor of photophosphorylation at 10 μM concentration decreased ATPase activity in chloroplasts from both the control and the Pb- treated leaves. Using the method of rapid fractionation of barley protoplasts it was shown that the ATP/ADP ratio in the mitochondria from Pb-treated leaves was largely suppressed (from 1.8 to 0.4) by OM under nonphotorespiratory conditions (high CO₂), whereas under photorespiratory conditions (low CO₂) this ratio was high (5.3) and under OM decreased less (to 3.1). Our results indicate that oligomycin, in organelle isolated from Pb-treated leaves, had no inhibitory effect on the mitochondrial ATPase, whereas it inhibited chloroplasts ATPase. We suggest that Pb ions affected the catalytic cycle and/or conformational changes of ATPase in pea chloroplasts differently than in mitochondria. The differences in Pb responses may reflect fine mechanisms for the regulation of ATP production in the plant cells under stress conditions.     

Developmental changes of enzymes of malate metabolism in relation to respiration, photosynthesis and nitrate assimilation in peach leaves

The developmental profile of the activities of some enzymes involved in malate metabolism, namely phosphoenolpyruvate carboxylase (PEPC; EC 4. 1. 1. 31), NAD⁺-linked (EC 1. 1. 1. 37) and NADP⁺-linked (EC 1. 1. 1. 82) malate dehydrosenase (MDH), NAD⁺linked (EC 1. 1. 1. 39) and NADP⁺-linked (EC 1. 1. 1. 40) malic enzyme (ME), has been determined in leaves of peach [ Prunus persica (L.) Batsch cv. Maycrest], a woody C₃ species. In order to study the role of these enzymes, their activities were related to developmental changes of photosynthesis, respiration, and capacity for N assimilation. Activities of PEPC, NAD(P)⁺-MDH and NADP⁺-ME were high in young expanding leaves and decreased 2- to 3-fold in mature ones, suggesting that such enzymes play some role during the early stages of leaf expansion. In leaves of peach, such a role did not seem to be linked to C₃ photosynthesis or nitrate assimilation, in that photosynthetic O₂ evolution and activities of nitrate reductase (EC 1. 6. 6. 1) and glutamine synthetase (EC 6. 3. 1. 2) increased during leaf development. In contrast, leaf respiration strongly decreased with increasing leaf age. We suggest that in expanding leaves of this woody species the enzymes associated with malate metabolism have anaplerotic functions, and that PEPC may also contribute to the recapture of respiratory CO₂.     

Importance of oxidative electron transport over oxidative phosphorylation in optimizing photosynthesis in mesophyll protoplasts of pea (Pisum sativum L.)

The role of mitochondrial respiration in optimizing photosynthesis was assessed in mesophyll protoplasts of pea ( Pisum sativum L., cv. Arkel) by using low concentrations of oligomycin (an inhibitor of oxidative phosphorylation), antimycin A (inhibits cytochrome pathway of electron transport) and salicylhydroxamic acid (SHAM, an inhibitor of alternative oxidase). All three compounds decreased the rate of photosynthetic O₂ evolution in mesophyll protoplasts, but did not affect chloroplast photosynthesis. The inhibition of photosynthesis by these mitochondrial inhibitors was stronger at optimal CO₂ (1.0 m M NaHCO₃) than that at limiting CO₂ (0.1 m M NaHCO₃). We conclude that mitochondrial metabolism through both cytochrome and alternative pathways is essential for optimizing photosynthesis at limiting as well as at optimal CO₂. The ratios of ATP to ADP in whole protoplast extracts were hardly affected, despite the marked decrease in their photosynthetic rates by SHAM. Similarly, the decrease in the ATP/ADP ratio by oligomycin or antimycin A was more pronounced at limiting CO₂ than at optimal CO₂. The mitochondrial oxidative electron transport, through both cytochrome and alternative pathways, therefore akppears to be more important than oxidative phosphorylation in optimizing photosynthesis, particularly at limiting CO₂ (when ATP demand is expected to be low). Our results also confirm that the alternative pathway has a significant role in contributing to the cellular ATP, when the cytochrome pathway is limited.     

Mitochondria from Zea mays leaf tissues: Differentiation of carbon assimilation and photorespiratory activity between mesophyll and bundle sheath cells

A procedure was developed to obtain intact and purified mitochondria from mesophyll and bundle sheath tissues of Zea mays L. cv. I.N.R.A. 180, an NADP⁺-malic enzyme type C₄ plant. There was little cross-contamination between the two mitochondrial fractions.
Both types of mitochondria oxidized NADH, succinate and malate with respiratory control. In mesophyll mitochondria malate oxidation was highly sensitive to KCN (85–90% inhibition of first state 3) and showed good respiratory control. In bundle sheath mitochondria malate oxidation was less sensitive to cyanide (75-80% inhibition) and showed poor respiratory control. Malate and NADH appeared to be the best substrates for respiratory activity. Mesophyil mitochondria could not oxidize glycine, whereas bundle sheath mitochondria could.
The results indicate that mesophyll and bundle sheath mitochondria of Zea mays are differentiated, not only with respect to the decarboxylation of malate but also with respect to the decarboxylation phase of photorespiration.     

Multisite Interactions Between Pb2+ and Protein Kinase C and Its Role in Norepinephrine Release from Bovine Adrenal Chromaffin Cells

Abstract: We investigated the interaction between Pb²⁺ and protein kinase C (PKC) in the Pb²⁺-induced release of norepinephrine (NE) from permeabilized adrenal chromaffin cells. Our analysis of endogenous PKC activity in permeabilized cells suggests that Pb²⁺ interacts with the adrenal enzyme at multiple sites. Pb²⁺ activates the enzyme through high-affinity ( K _A(Pb) = 2.4 × 10⁻¹² M ) interactions and inhibits the enzyme by competitive and noncompetitive interactions with nanomolar-( K _i = 7.1 × 10⁻⁹ M ) and micromolar- ( K '_i = 2.8 × 10⁻⁷ M ) affinity sites, respectively. Activation of PKC by 12- O -tetradecanoylphorbol 13-acetate (TPA) in Ca²⁺-deficient, Pb²⁺-containing medium, enhances the Pb²⁺-induced NE release from permeabilized chromaffin cells by lowering the concentration of Pb²⁺ required for half-maximal activation of the secretory response from 7.5 × 10⁻¹⁰ to 5.7 × 10⁻¹¹ M . The PKC inhibitors staurosporine and pseudosubstrate PKC (19–36) abolish the effect of TPA without affecting the Pb²⁺-induced secretion in the absence of TPA. These results indicate that (a) Pb²⁺ is a partial agonist of PKC, capable of both activating and inhibiting the enzyme and (b) synergistic activation of PKC by TPA and Pb²⁺ results in increased sensitivity of exocytosis to Pb²⁺ but is not obligatory for Pb²⁺-triggered secretion.     

Effects of 2-iodobenzanilide on potassium uptake, H+ extrusion and K+-stimulated ATPase of corn roots

The carboxanilide systemic fungicide 2-iodobenzanilide (2-IB) after 2 h pretreatment at 0.25 m M inhibited K⁺ and SO₄^2- uptake by excised corn roots ( Zea mays L., cv. Dekalb 342) up to ca 70 and 40%, respectively. Proton extrusion from corn roots was also reduced by ca 50% after 1 h contact, and the microsomal K⁺-stimulated ATPase activity from corn roots and pea stems ( Pisum sativum L., cv. Alaska) inhibited by 50 and 72%, respectively. In contrast, the Mg²⁺-ATPase activities of microsomes and mitochondria at pH 6.0 and 8.7, respectively, were unaffected. After 2 h of preincubation with 0.25 m M 2-IB, O₂ consumption by corn roots and pea stems was inhibited by 12 and 18%, respectively. ATP content of corn roots was not altered by 2-IB treatment. Therefore, energy availability "in vivo" was unaffected and the primary effect on corn roots is suggested to be at the plasmalemma ATPase which forms the proton gradient.
With isolated pea stem mitochondria, 0.25 m M 2-IB inhibited O₂ consumption by ca 60% when NADH or malate plus pyruvate were added as substrates; when succinate was used O₂ consumption was unaffected. The mode of action on isolated mitochondria was different from that shown for carboxin and also formerly attributed to the whole class of carboxanilide fungicides.     

The localization of serine hydroxymethyltransferase in leaves of C3 and C4 species

The intracellular distribution of serine hydroxymethyltransferase (EC 2.1.2.1) was studied in young wheat ( Triticum aestivum L. cv. Starke II) leaves by fractionation of protoplasts and further purification of peroxisomes and chloroplasts. Essentially all of the activity in wheat leaves was located in the mitochondria. Within the mitochondria the enzyme was mainly in the matrix as shown by centrifugation of sonicated wheat mitochondria. In the C₄ plants, Zea mays (L. cv. Earliking), Panicum miliaceum and Panicum maximum (cv. Australia) belonging to different C₄ types, serine hydroxymethyltransferase was almost exclusively found in bundle sheath cells. The location of this enzyme in leaves is consistent with its role relative to glycine decarboxylation during photorespiration.     

K+ATP channel opening prevents succinate-dependent H2O2 generation by plant mitochondria

The role of a recently identified K⁺_ATP channel in preventing H₂O₂ formation was examined in isolated pea stem mitochondria. The succinate-dependent H₂O₂ formation was progressively inhibited, when mitochondria were resuspended in media containing increasing concentration of KCl (from 0.05 to 0.15  M ). This inhibition was linked to a partial dissipation of the transmembrane electrical potential (ΔΨ) induced by KCl. Conversely, the malate plus glutamate-dependent H₂O₂ formation was not influenced. The succinate-sustained H₂O₂ generation was also unaffected by nigericin (a H⁺/K⁺ exchanger), but completely prevented by valinomycin (a K⁺ ionophore). In addition, cyclosporin A (a K⁺_ATP channel opener) inhibited this H₂O₂ formation, while ATP (an inhibitor of the channel opening) slightly increased it. The inhibitory effect of ATP was strongly stimulated in the presence of atractylate (an inhibitor of the adenine nucleotide translocase), thus suggesting that the receptor for ATP on the K⁺ channel faces the intermembrane space. Finally, the succinate-dependent H₂O₂ formation was partially prevented by phenylarsine oxide (a thiol oxidant).     

Light-enhanced dark respiration in leaves and mesophyll protoplasts of pea in relation to photorespiration,respiration and some metabolites content

The respiration rate of leaves and mesophyll protoplasts of pea (Pisum sativum L.), from plants which were previously kept in darkness for 24 h was doubled following a period of photosynthesis at ambient level of O₂ (21 %), whereas the low level of O₂ (1 % and 4 % for leaves and protoplasts, respectively) reduced this light-enhanced dark respiration (LEDR) to the rate as noted before the illumination. Similarly to respiration rate, the oxygen at used concentrations had no effect on the ATP/ADP ratio in the dark-treated leaves. However, the ATP/ADP ratio in leaves photosynthesizing at 21 % O₂ was higher (up to 40 %, dependence on CO₂ concentration in the range 40–1600 1 dm⁻³) than in those photosynthesizing at 1 % O₂ or darkened at air (21 % O₂). Also, at 1 % O₂ the accumulation of malate was suppressed (by about 40 %), to a value noted for leaves darkened at 21 % O₂. The dark-treatment of leaves reduced the ability of isolated mitochondria to oxidize glycine (by about twofold) and succinate, but not malate. Mitochondria from both the light- and dark-treated leaves did not differ in qualitative composition of free amino acids, however, there were significant quantitative differences especially with respect to aspartate, alanine, glutamate and major intermediates of the photorespiratory pathway (glycine, serine). Our results suggest that accumulation of photorespiratory and respiratory metabolites in pea leaves during photosynthesis at 1 % O₂ is reduced, hence the suppression of postillumination respiration rate.     

Seasonal differences in photosynthesis between the C3 and C4 subspecies of Alloteropsis semialata are offset by frost and drought

The regional abundance of C₄ grasses is strongly controlled by temperature, however, the role of precipitation is less clear. Progress in elucidating the direct effects of photosynthetic pathway on these climate relationships is hindered by the significant genetic divergence between major C₃ and C₄ grass lineages. We addressed this problem by examining seasonal climate responses of photosynthesis in Alloteropsis semialata , a unique grass species with both C₃ and C₄ subspecies. Experimental manipulation of rainfall in a common garden in South Africa tested the hypotheses that: (1) photosynthesis is greater in the C₄ than C₃ subspecies under high summer temperatures, but this pattern is reversed at low winter temperatures; and (2) the photosynthetic advantage of C₄ plants is enhanced during drought events. Measurements of leaf gas exchange over 2 years showed a significant photosynthetic advantage for the C₄ subspecies under irrigated conditions from spring through autumn. However, the C₄ leaves were killed by winter frost, while photosynthesis continued in the C₃ plants. Unexpectedly, the C₄ subspecies also lost its photosynthetic advantage during natural drought events, despite greater water-use efficiency under irrigated conditions. This study highlights previously unrecognized roles for climatic extremes in determining the ecological success of C₃ and C₄ grasses.     

Kinetics of leaf oxygen uptake represent in planta activities of respiratory electron transport and terminal oxidases

We present, for the first time, the oxygen response kinetics of mitochondrial respiration measured in intact leaves (sunflower and aspen). Low O₂ concentrations in N₂ (9–1500 ppm) were preset in a flow-through gas exchange measurement system, and the decrease in O₂ concentration and the increase in CO₂ concentration as result of leaf respiration were measured by a zirconium cell O₂ analyser and infrared-absorption CO₂ analyser, respectively. The low O₂ concentrations little influenced the rate of CO₂ evolution during the 60-s exposure. The initial slope of the O₂ uptake curve on the dissolved O₂ concentration basis was relatively constant in leaves of a single species, 1.5 mm s⁻¹ in sunflower and 1.8 mm s⁻¹ in aspen. The apparent K _0.5(O₂) values ranged from 0.33 to 0.67 μ M in sunflower and from 0.33 to 1.1 μ M in aspen, mainly because of the variation of the maximum rate, V _max (leaf temperature 22°C). The initial slope of the O₂ response of respiration characterizes the catalytic efficiency of terminal oxidases, an important parameter of the respiratory machinery in leaves. The plateau of the response characterizes the activity of the mitochondrial electron transport chain and is subject to regulations in accordance with the necessity for ATP production. The relatively low oxygen conductivity of terminal oxidases means that in leaves, less than 10% of the photosynthetic oxygen can be reassimilated by mitochondria.     

Direct and indirect effects of Cd2+ on photosynthesis in sugar beet (Beta vulgaris)

Sugar-beet plants ( Beta vulgaris L. cv. Monohill) were cultivated for 4 weeks in a complete nutrient solution. Indirect effects of cadmium were studied by adding 5, 10 or 20 μ M CdCl₂ to the culture medium while direct effects were determined by adding 1, 5, 20, 50 or 2 000 μ M CdCl₂ to the assay media. The photosynthetic properties were characterized by measurement of CO₂ fixation in intact plants, fluorescence emission by intact leaves and isolated chloroplasts, photosystem (PS) I and PSII mediated electron transport of isolated chloroplasts, and CO₂-dependent O₂ evolution by protoplasts. When directly applied to isolated leaves, protoplasts and chloroplasts. Cd²⁺ impeded CO₂ fixation without affecting the rates of electron transport of PSI or PSII or the rate of dark respiration. When Cd²⁺ was applied through the culture medium the capacity for, and the maximal quantum yield of CO₂ assimilation by intact plants both decreased. This was associated with: (1) decreased total as well as effective chlorophyll content (PSII antennae size), (2) decreased coupling of electron transport in isolated chloroplasts, (3) perturbed carbon reduction cycle as indicated by fluorescence measurements. Also, protoplasts isolated from leaves of Cd²⁺-cultivated plants showed an increased rate of dark respiration.     

Yeast mitochondria import ATP through the calcium-dependent ATP-Mg/Pi carrier Sal1p, and are ATP consumers during aerobic growth in glucose

Sal1p, a novel Ca²⁺-dependent ATP-Mg/Pi carrier, is essential in yeast lacking all adenine nucleotide translocases. By targeting luciferase to the mitochondrial matrix to monitor mitochondrial ATP levels, we show in isolated mitochondria that both ATP-Mg and free ADP are taken up by Sal1p with a K _m of 0.20 ± 0.03 mM and 0.28 ± 0.06 mM respectively. Nucleotide transport along Sal1p is strictly Ca²⁺ dependent. Ca²⁺ increases the V _max with a S _0.5 of 15 μM, and no changes in the K _m for ATP-Mg. Glucose sensing in yeast generates Ca²⁺ transients involving Ca²⁺ influx from the external medium. We find that carbon-deprived cells respond to glucose with an immediate increase in mitochondrial ATP levels which is not observed in the presence of EGTA or in Sal1p-deficient cells. Moreover, we now report that during normal aerobic growth on glucose, yeast mitochondria import ATP from the cytosol and hydrolyse it through H⁺-ATP synthase. We identify two pathways for ATP uptake in mitochondria, the ADP/ATP carriers and Sal1p. Thus, during exponential growth on glucose, mitochondria are ATP consumers, as those from cells growing in anaerobic conditions or deprived of mitochondrial DNA which depend on cytosolic ATP and mitochondrial ATPase working in reverse to generate a mitochondrial membrane potential. In conclusion, the results show that growth on glucose requires ATP hydrolysis in mitochondria and recruits Sal1p as a Ca²⁺-dependent mechanism to import ATP-Mg from the cytosol. Whether this mechanism is used under similar settings in higher eukaryotes is an open question.     

Variations in the dorso-ventral organization of leaf structure and Kranz anatomy coordinate the control of photosynthesis and associated signalling at the whole leaf level in monocotyledonous species

Photosynthesis and associated signalling are influenced by the dorso-ventral properties of leaves. The degree of adaxial/abaxial symmetry in stomatal numbers, photosynthetic regulation with respect to light orientation and the total section areas of the bundle sheath (BS) cells and the surrounding mesophyll (M) cells on the adaxial and abaxial sides of the vascular bundles were compared in two C₄[ Zea mays (maize) and Paspalum dilatatum ] and one C₃[ Triticum turgidum (Durum wheat)] monocotyledonous species. The C₃ leaves had a higher degree of dorso-ventral symmetry than the C₄ leaves. Photosynthetic regulation was the same on each side of the wheat leaves, as were stomatal numbers and the section area of the BS relative to that of the M cells (BS/M section area ratio). In contrast, photosynthetic regulation in maize and P. dilatatum leaves showed a marked surface-specific response to light orientation. Compared to the adaxial sides of the C₄ monocotyledonous leaves, the abaxial surfaces had more stomata and the BS/M section area ratio was significantly higher. Differences in dorso-ventral structure, particularly in Kranz anatomy, serve not only to maximize photosynthetic capacity with respect light orientation in C₄ monocotyledonous leaves but also allow adaxial and abaxial-specific signalling from the respective M cells.     

The role of photorespiration in redox and energy balance of photosynthetic plant cells: A study with a barley mutant deficient in glycine decarboxylase

Protoplasts and mitochondria were isolated from leaves of homozygous barley ( Hordeum vulgare L.) mutant deficient in glycine decarboxylase complex (GDC, EC 2.1.2.10) and wild-type plants. The photosynthetic rates of isolated protoplasts from the mutant and wild-type plants under saturating CO₂ were similar, but the respiratory rate of the mutant was two-fold higher. Respiration in the mutant plants was much more strongly inhibited by antimycin A than in wild-type plants and a low level of the alternative oxidase protein was found in mitochondria. The activities of NADP- and NAD-dependent malate dehydrogenases were also increased in mutant plants, suggesting an activation of the malate-oxaloacetate exchange for redox transfer between organelles. Mutant plants had elevated activities of NADH- and NADPH-dependent glyoxylate/hydroxypyruvate reductases, which may be involved in oxidizing excess NAD(P)H and the scavenging of glyoxylate. We estimated distribution of pools of adenylates, NAD(H) and NADP(H) between chloroplasts, cytosol and mitochondria. Under photorespiratory conditions, ATP/ADP and NADPH/NADP ratios in the mutant were higher in chloroplasts as compared to wild-type plants. The cytosolic NADH/NAD ratio was increased, whereas the ratio in mitochondria decreased. It is concluded that photorespiration serves as an effective redox transfer mechanism from the chloroplast. Plants with a lowered GDC content are deficient in this mechanism, which leads to over-reduction and over-energization of the chloroplasts.     

Cytoplasmic free Ca2+ dynamics in single tomato (Lycopersicon esculentum) protoplasts subjected to chilling temperatures

Protoplasts were isolated from leaves of tomato seedlings ( Lycopersicon esculentum Mill., cv. Marmande) at the 2nd to 4th true leaf stage and were loaded with the calcium binding tetra[acetoxymethyl⁺] ester of the fluorescent stilbene chromophore, Fura 2. Although the loading efficiency of the dye in these protoplasts was low, many protoplasts loaded only in the cytosol were always obtained. Changes in the cytosolic calcium concentration ([Ca²⁺]_cyt) were determined in single protoplasts in a temperature-controlled perfusion chamber by use of fluorescence photometry microscopy after excitation at 340 and 380 nm. When the protoplasts were subjected to chilling temperatures (10–15°C) by a circulating solution, the [Ca²⁺]_cyt increased in 64% of the analysed protoplasts. Depending on the initial resting level of [Ca²⁺]_cyt, three main types of kinetics were obtained in these protoplasts: (1) In 21% of the protoplasts, [Ca²⁺]_cyt increased to a maximum within 10–20 s from the start of temperature decrease, followed by a fast decrease; (2) in 11% of the protoplasts, the [Ca²⁺]_cyt both increased and decreased somewhat slower; and (3) in 32% a constant increase of [Ca²⁺]_cyt was obtained 1 min after the start of temperature decrease.     

Elevated pyruvate,orthophosphate dikinase (PPDK) activity alters carbon metabolism in C3 transgenic potatoes with a C4 maize PPDK gene

Changes in carbon metabolism and δ¹³C value of transgenic potato plants with a maize pyruvate,orthophosphate dikinase (PPDK; EC 2.7.9.1) gene are reported. PPDK catalyzes the formation of phospho enol pyruvate (PEP), the initial acceptor of CO₂ in the C₄ photosynthetic pathway. PPDK activities in the leases of transgenic potatoes were up to 5.4‐fold higher than those of control potato plants (wild‐type and treated control plants). In the transgenic potato plants, PPDK activity in leaves was negatively correlated with pyruvate content (r²= 0.81), and was positively correlated with malate content (r²= 0.88). A significant increase in the δ¹³C value was observed in the transgenic potato plants, suggesting a certain contribution of PEP carboxylase as the initial acceptor of atmospheric CO₂. These data suggest that elevated PPDK activity may alter carbon metabolism and lead to a partial operation of C₄‐type carbon metabolism. However, since parameters associated with CO₂ gas exchange were not affected, the altered carbon metabolism had only a small effect on the total photosynthetic characteristics of the transgenic plants.     

Rapid, stimulation-induced reduction of C12-resorufin in motor nerve terminals: linkage to mitochondrial metabolism

The Alamar blue (resazurin) assay of cell viability monitors the irreversible reduction of non-fluorescent resazurin to fluorescent resorufin. This study focused on the reversible reduction of C₁₂-resorufin to non-fluorescent C₁₂-dihydroresorufin in motor nerve terminals innervating lizard intercostal muscles. Resting C₁₂-resorufin fluorescence decreased when the activity of the mitochondrial electron transport chain (ETC) was accelerated with carbonyl cyanide m -chloro phenyl hydrazone, and increased when ETC activity was inhibited with cyanide. Trains of action potentials (50 Hz for 20–50 s), which reversibly decreased NADH fluorescence and partially depolarized the mitochondrial membrane potential, produced a reversible decrease in C₁₂-resorufin fluorescence which had a similar time course. The stimulation-induced decrease in C₁₂-resorufin fluorescence was blocked by inhibitors of ETC complexes I, III, and IV and by carbonyl cyanide m -chloro phenyl hydrazone, but not by inhibiting mitochondrial ATP synthesis with oligomycin. Mitochondrial depolarization and the decreases in C₁₂-resorufin and NADH fluorescence depended on Ca²⁺ influx into the terminal, but not on vesicular transmitter release. These results suggest that the reversible reduction of C₁₂-resorufin in stimulated motor nerve terminals is linked, directly or indirectly, to the reversible oxidation of NADH and to Ca²⁺ influx into mitochondria, and provides an assay for rapid changes in motor terminal metabolism.     

The response of stomata to CO2 relates to its effect on respiration and ATP level

External ATP enhanced stomatal opening of Commelina communis L. differently from EDTA. ATP was more effective in opening stomata than EDTA, when both were applied in amounts yielding equivalent free Ca²⁺ concentration. The stimulation by ATP depended upon its de-phosphorylation and was not due to the P₁ released. Hence an energetical contribution of external ATP appears possible. Increase in CO₂ concentration increased the stimulation of stomatal opening by ATP and diminished the internal ATP level, ATP/(ADP+AMP) ratio and respiration rate.