In vivo 31P and 13C NMR investigations of Rhodococcus rhodochrous metabolism and behaviour during biotransformation processes.

AIMS: The strain Rhodococcus rhodochrous OBT18 was isolated from a water treatment plant used to decontaminate industrial effluents containing benzothiazole derivatives. Aims of the work are to study the central metabolism of this strain and more specifically its behaviour during biodegradation of 2-aminobenzothiazole. METHODS AND RESULTS: In vivo(13)C and (31)P NMR experiments showed that this strain contains storage compounds such as polyphosphates, glycogen and trehalose and produces biosurfactants containing trehalose as sugar unit. Trehalose can be synthesized after reversion of the glycolytic pathway. In vivo(31)P NMR experiments showed that energy metabolism markers such as the intracellular pH and the ATP concentration did not change during biotransformation processes when R. rhodochrous was exposed to potentially toxic compounds including iron complexes and (* )OH radicals. Also R. rhodochrous recovers the normal values of ATP and pH after anoxia/reoxygenation cycle very quickly. CONCLUSIONS: Rhodococcus rhodochrous carbon and energy metabolism is well adapted to different stresses and consequently to live in the environment where conditions are constantly changing. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study can be used to understand the behaviour of this bacterium in natural environments but also in water treatment plants where iron and UV light are present.

Ultrastructural and biochemical characterization of autophagy in higher plant cells subjected to carbon deprivation: control by the supply of mitochondria with respiratory substrates.

Autophagy triggered by carbohydrate starvation was characterized at both biochemical and structural levels, with the aim to identify reliable and easily detectable marker(s) and to investigate the factors controlling this process. Incubation of suspension cells in sucrose-free culture medium triggered a marked degradation of the membrane polar lipids, including phospholipids and galactolipids. In contrast, the total amounts of sterols, which are mainly associated with plasmalemma and tonoplast membranes, remained constant. In particular, phosphatidylcholine decreased, whereas phosphodiesters including glycerylphosphorylcholine transiently increased, and phosphorylcholine (P-Cho) steadily accumulated. P-Cho exhibits a remarkable metabolic inertness and therefore can be used as a reliable biochemical marker reflecting the extent of plant cell autophagy. Indeed, whenever P-Cho accumulated, a massive regression of cytoplasm was noticed using EM. Double membrane-bounded vacuoles were formed in the peripheral cytoplasm during sucrose starvation and were eventually expelled into the central vacuole, which increased in volume and squeezed the thin layer of cytoplasm spared by autophagy. The biochemical marker P-Cho was used to investigate the factors controlling autophagy. P-Cho did not accumulate when sucrose was replaced by glycerol or by pyruvate as carbon sources. Both compounds entered the cells and sustained normal rates of respiration. No recycling back to the hexose phosphates was observed, and cells were rapidly depleted in sugars and hexose phosphates, without any sign of autophagy. On the contrary, when pyruvate (or glycerol) was removed from the culture medium, P-Cho accumulated without a lag phase, in correlation with the formation of autophagic vacuoles. These results strongly suggest that the supply of mitochondria with respiratory substrates, and not the decrease of sucrose and hexose phosphates, controls the induction of autophagy in plant cells starved in carbohydrates.

Early Events Induced by the Elicitor Cryptogein in Tobacco Cells: Involvement of a Plasma Membrane NADPH Oxidase and Activation of Glycolysis and the Pentose Phosphate Pathway.

Application of the elicitor cryptogein to tobacco (cv Xanthi) is known to evoke external medium alkalinization, active oxygen species production, and phytoalexin synthesis. These are all dependent on an influx of calcium. We show here that cryptogein also induces calcium-dependent plasma membrane depolarization, chloride efflux, cytoplasm acidification, and NADPH oxidation without changes in NAD+ and ATP levels, indicating that the elicitor-activated redox system, responsible for active oxygen species production, uses NADPH in vivo. NADPH oxidation activates the functioning of the pentose phosphate pathway, leading to a decrease in glucose 6-phosphate and to the accumulation of glyceraldehyde 3-phosphate, 3- and 2-phosphoglyceric acid, and phosphoenolpyruvate. By inhibiting the pentose phosphate pathway, we demonstrate that the activation of the plasma membrane NADPH oxidase is responsible for active oxygen species production, external alkalinization, and acidification of the cytoplasm. A model is proposed for the organization of the cryptogein responses measured to date.

NMR and plant metabolism.

Recent advances in NMR methodology offer a way to acquire a comprehensive profile of a wide range of metabolites from various plant tissues or cells. NMR is a powerful approach for plant metabolite profiling and provides a capacity for the dynamic exploration of plant metabolism that is virtually unmatched by any other analytical technique.

A precise localization of cardiolipin in plant cells.

Cardiolipin and cytochrome aa3 contents of isolated plant cells (sycamore cells) and their purified mitochondria were measured. Since the cardiolipin/cytochrome aa3 ratio was the same in the intact cells and in the isolated mitochondria it was strongly suggested that cardiolipin is present only in the mitochondria. Furthermore, outer and inner mitochondria membranes of purified sycamore cells and mung bean hypocotyl mitochondria were separated and it was shown that cardiolipin is localized in the inner mitochondrial membrane.

Calcium-dependent lipolytic acyl-hydrolase activity in purified plant mitochondria.

Ageing of isolated potato mitochondria induced by CaCl2 resulted in rapid enzymatic hydrolysis of the membrane phospholipids with the liberation of free fatty acids. The enzyme responsible for this effect was identified as a membrane bound lipolytic acyl-hydrolase which was unmasked by CaCl2. The presence of this lipolytic acyl-hydrolase induced severe functional impairments in the mitochondrial oxidative and phosphorylative properties.

[Oxygen and temperature effects on the fatty acid composition in sycamore cells (Acer pseudoplatanus L.) (author's transl)]. / Role de l'oxygene et de la temperature sur la composition en acides gras des cellules isolees d'erable (Acer pseudoplatanus L.)

Temperature and oxygen effects on the degree of unsaturation of membrane fatty acids have been investigated with sycamore cells in suspension culture. Sycamore cells were incubated with [14C]acetate at temperature varying from 15 to 25 degrees C and at O2 concentration from 12.5 to 305 muM. It was found that: (i) no significant difference was observed in the distribution of radioactivity between oleate and linoleate with different temperatures; (ii) in marked contrast, the aeration conditions during growth of plant cell cultures affected the fatty acid pattern of the total lipids: by maintaining the oxygen concentration below 60 muM, the molar proportion of oleate increased dramatically whereas that of the linoleate decreased. Under these conditions, the aeration of the culture medium (250 muM) induced a rapid transformation of oleate to linoleate. These results cast further doubt on the importance of the temperature on the degree of unsaturation of the fatty acids in sycamore cells, but confirmed evidence that the formation of unsurated fatty acids by plant cells was indeed controlled by the oxygen concentration in solution.

Cooperation and Competition between Adenylate Kinase, Nucleoside Diphosphokinase, Electron Transport, and ATP Synthase in Plant Mitochondria Studied by 31P-Nuclear Magnetic Resonance.

Nucleotide metabolism in potato (Solanum tuberosum) mitochondria was studied using 31P-nuclear magnetic resonance spectroscopy and the O2 electrode. Immediately following the addition of ADP, ATP synthesis exceeded the rate of oxidative phosphorylation, fueled by succinate oxidation, due to mitochondrial adenylate kinase (AK) activity two to four times the maximum activity of ATP synthase. Only when the AK reaction approached equilibrium was oxidative phosphorylation the primary mechanism for net ATP synthesis. A pool of sequestered ATP in mitochondria enabled AK and ATP synthase to convert AMP to ATP in the presence of exogenous inorganic phosphate. During this conversion, AK activity can indirectly influence rates of oxidation of both succinate and NADH via changes in mitochondrial ATP. Mitochondrial nucleoside diphosphokinase, in cooperation with ATP synthase, was found to facilitate phosphorylation of nucleoside diphosphates other than ADP at rates similar to the maximum rate of oxidative phosphorylation. These results demonstrate that plant mitochondria contain all of the machinery necessary to rapidly regenerate nucleoside triphosphates from AMP and nucleoside diphosphates made during cellular biosynthesis and that AK activity can affect both the amount of ADP available to ATP synthase and the level of ATP regulating electron transport.

Transport, Compartmentation, and Metabolism of Homoserine in Higher Plant Cells. Carbon-13- and phosphorus-31-nuclear magnetic resonance studies Carbon-13- and Phosphorus-31-Nuclear Magnetic Resonance Studies

The transport, compartmentation, and metabolism of homoserine was characterized in two strains of meristematic higher plant cells, the dicotyledonous sycamore (Acer pseudoplatanus) and the monocotyledonous weed Echinochloa colonum. Homoserine is an intermediate in the synthesis of the aspartate-derived amino acids methionine, threonine (Thr), and isoleucine. Using 13C-nuclear magnetic resonance, we showed that homoserine actively entered the cells via a high-affinity proton-symport carrier (Km approximately 50-60 mum) at the maximum rate of 8 +/- 0.5 mumol h-1 g-1 cell wet weight, and in competition with serine or Thr. We could visualize the compartmentation of homoserine, and observed that it accumulated at a concentration 4 to 5 times higher in the cytoplasm than in the large vacuolar compartment. 31P-nuclear magnetic resonance permitted us to analyze the phosphorylation of homoserine. When sycamore cells were incubated with 100 mum homoserine, phosphohomoserine steadily accumulated in the cytoplasmic compartment over 24 h at the constant rate of 0.7 mumol h-1 g-1 cell wet weight, indicating that homoserine kinase was not inhibited in vivo by its product, phosphohomoserine. The rate of metabolism of phosphohomoserine was much lower (0.06 mumol h-1 g-1 cell wet weight) and essentially sustained Thr accumulation. Similarly, homoserine was actively incorporated by E. colonum cells. However, in contrast to what was seen in sycamore cells, large accumulations of Thr were observed, whereas the intracellular concentration of homoserine remained low, and phosphohomoserine did not accumulate. These differences with sycamore cells were attributed to the presence of a higher Thr synthase activity in this strain of monocot cells.

Induction of beta-methylcrotonyl-coenzyme A carboxylase in higher plant cells during carbohydrate starvation: evidence for a role of MCCase in leucine catabolism.

Induction of beta-methylcrotonyl-coenzyme A carboxylase (MCCase) activity was observed during carbohydrate starvation in sycamore cells. In mitochondria isolated from starved cells, we noticed a marked accumulation of the biotinylated subunit of MCCase, of which the apparent molecular weight of 74000 was similar to that of the polypeptide from mitochondria of potato tubers. Our results provide evidence for a role of MCCase in the catabolic pathway of leucine, a branched-chain amino acid which transiently accumulates in carbon-starved cells in relation to a massive breakdown of proteins. Furthermore, when control sycamore cells were incubated in the presence of exogenous leucine, this amino acid accumulated in the cells and no induction or accumulation of MCCase was observed, indicating that leucine is not responsible for the induction of its catabolic machinery. Finally, MCCase is proposed as a new biochemical marker of the autophagic process triggered by carbohydrate starvation.

Arrest of mitochondrial biogenesis in copper-treated sycamore cells.

Sycamore suspension cells (Acer pseudoplatanus L.) were grown in the presence of sublethal concentrations of copper (50 microM). During the first 5-6 days of treatment, growth was not affected, but cell respiration (coupled and uncoupled) declined to approximately 60% of its normal value. This decline of respiration was attributed to a progressive diminution of the number of mitochondria in copper-treated cells, based on the demonstration of the concomitant decline of (1) cardiolipin (diphosphatidylglycerol) and cytochrome aa3 (cytochrome oxidase), two specific markers of mitochondrial inner membrane, and (2) fumarase activity, a specific marker of mitochondrial matrix space. In addition, the mitochondria extracted from copper-treated cells presented the same properties as those from control cells, concerning substrate oxidation, cardiolipin and cytochrome aa3 contents, and fumarase activity. These results strongly suggest that copper triggered an arrest of mitochondrial biogenesis, which preceded cell division arrest.

Effect of glyphosate on plant cell metabolism. 31P and 13C NMR studies.

The effect of glyphosate (N-phosphonomethyl glycine; the active ingredient of Roundup herbicide) on plant cells metabolism was analysed by 31P and 13C NMR using suspension-cultured sycamore (Acer pseudoplatanus L) cells. Cells were compressed in the NMR tube and perfused with an original arrangement enabling a tight control of the circulating nutrient medium. Addition of 1 mM glyphosate to the nutrient medium triggered the accumulation of shikimate (20-30 mumol g-1 cell wet weight within 50 h) and shikimate 3-phosphate (1-1.5 mumol g-1 cell wet weight within 50 h). From in vivo spectra it was demonstrated that these two compounds were accumulated in the cytoplasm where their concentrations reached potentially lethal levels. On the other hand, glyphosate present in the cytoplasmic compartment was extensively metabolized to yield aminomethylphosphonic acid which also accumulated in the cytoplasm. Finally, the results presented in this paper indicate that although the cell growth was stopped by glyphosate the cell respiration rates and the level of energy metabolism intermediates remained unchanged.

Protection against photoinhibition in the alpine plant Geum montanum.

Geum montanum L. is an alpine plant usually found at altitudes between 1700 and 2600 m. Its wintergreen leaves can be subjected to very low temperatures and at the same time receive high photon flux densities at the beginning of the growth season when the snow melts. We report results of a study, performed with classical methods of biophysics, showing that leaves of G. montanum were remarkably tolerant to sunlight even at low temperatures. This tolerance results from the interplay of photorespiration and CO2 photosassimilation. When temperatures approach 0°C, responses include stomatal opening and CO2 uptake even under desiccation stress. This permits linear electron transport that is sufficient to avoid the excessive reduction of the electron transport chain which is known to lead to photodamage. In addition, excitation energy was shifted from photosystem (PS)II to PSI which is a very efficient energy quencher. Sensitivity of P700 in PSI to oxidation by far-red light was decreased and rates of dark reduction of photooxidized P700 were increased by actinic illumination, suggesting activation of cyclic electron transport. Consistent with this, far-red light was able to decrease the quantum yield of PSII (measured by the F v/F m ratio of chlorophyll fluorescence). We suggest that cyclic electron transport decreases the lumenal pH under strong light. In the presence of zeaxanthin, this increases energy dissipation at the PSII level. At low temperatures, P700 remained strongly oxidized under high irradiation while the primary electron acceptor of PSII, QA, was largely reduced. This shows efficient control of electron transport presumably at the level of the cytochrome b/f complex and suggests formation of a protective transthylakoid proton gradient even when linear electron transport is much reduced in the cold. Thus, several mechanisms cooperate to effectively protect the photosynthetic apparatus of G. montanum from photodamage. We see no indication of destructive "photostress" in this species during the growth season under alpine low-temperature and drought conditions.

Growth of Suspension-cultured Acer pseudoplatanus L. Cells in Automatic Culture Units of Large Volume.

A phytostat to mass culture higher plant cells in liquid medium is described. This apparatus allowed the culture in batch, turbidostat and chemostat of 20 liters of cells. Automatic control of cell suspension growth was based on culture turbidity. Changes with time of certain cell characteristics, particularly cell respiration and phospholipid content, indicated that the test time to harvest large amounts of sycamore cells (Acer pseudoplatanus L.) in good physiological state was about 2 days before the end of the exponential phase of growth, when the cell density reached one million cells per milliliter of culture.

Excretion of laccase by sycamore (Acer pseudoplatanus L.) cells. Purification and properties of the enzyme.

A laccase-type polyphenol oxidase is excreted by sycamore cells (Acer pseudoplatanus L.) cells. The enzyme has been purified by classical purification techniques. It is a blue copper protein of Mr 97 000, containing 45% carbohydrate and 0.24% copper. This protein consists of one single unit and the copper content corresponds to four copper atoms per protein molecule. The specific activity of the purified extracellular sycamore-cell laccase measured at pH 6.6 (optimum pH) and in the presence of 20mM-4-methhylcatechol (optimum substrate conditions) corresponded to an oxygen uptake of 32 000 nmol of O2/min per mg of protein. Under these conditions, the catalytic-centre activity of the enzyme reached 100 s-1. The excretion of laccase by sycamore cells is significant, being about 2% of the total protein synthesized by the cells during the exponential phase of growth, and is independent of cell growth. The physiological significance and the problems raised by the passage of this protein across the cytoplasmic membrane are discussed.

Mitochondria of Isolated Plant Cells (Acer pseudoplatanus L.): II. Copper Deficiency Effects on Cytochrome C Oxidase and Oxygen Uptake.

The effects of copper deficiency on cell culture growth, cell respiration, mitochondrial oxidative properties, and electron transport chain have been studied with suspension-cultured sycamore cells (Acer pseudoplatanus L.). Within the range of the copper concentration studied (0.1-25 mug/1 of culture medium), the mean rate of cell division is independent of copper concentration. An initial copper concentration lower than 2 mug/1 limited the maximum density of population reached at the stationary phase of growth.On a protein basis, the uncoupled O(2) uptake rates were about the same for normal and copper-deficient cells. In contrast, the half-maximal inhibition of O(2) uptake rate was obtained at greater KCN concentration in the normal cells (20 muM) compared to copper-deficient cells (2 muM). Similar results were obtained with the normal and copper-deficient sycamore cell mitochondria.In the copper-deficient mitochondria, the concentration of the cytochrome aa(3) was less than 0.02 nmol/mg mitochondrial protein or 1/20 of the normal rate. The b- and c-type cytochrome content was invariant with copper depletion. It appeared that cytochrome aa(3) is present in large excess in normal cells. This work also indicated that cytochrome c is a very mobile molecule.

Excretion of laccase by sycamore (Acer pseudoplatanus L.) cells. Effects of a copper deficiency.

Copper-deprived sycamore (Acer pseudoplatanus) cells do not excrete molecules of active laccase in their culture medium. In the range of 2-100 micrograms of copper initially present per litre of nutrient solution, the total laccase activity measured in the cell suspensions at the end of the exponential phase of growth was closely proportional to the amount of added copper. However, copper-deprived cells excreted the laccase apoprotein (laccase without copper) at the same rate as copper-supplied cells excreted the active, copper-containing, laccase. When the culture medium was initially supplied with limiting amounts of copper, the active laccase was excreted until all copper molecules were metabolized. Thereafter, the laccase apoprotein was excreted. Consequently, at the end of the exponential phase of growth, the cell supernatants contained a mixture of apoprotein and copper-containing laccase. After purification and concentration, this mixture of copper-containing laccase (blue) and laccase apoprotein (slightly yellow) showed a yellow-green colour. Under copper-limiting culture conditions an equivalent decrease of Type 1, Type 2 and Type 3 Cu2+ was observed. Addition of copper to copper-deficient enzyme solutions does not result in a recovery of the enzyme activity. However, when added to copper-deficient sycamore-cell suspensions, copper induced a recovery of the excretion of active enzyme, at a normal rate, within about 10 h. The first molecules of active laccase were excreted after 3-4 h.

O2-triggered changes of membrane fatty acid composition have no effect on Arrhenius discontinuities of respiration in sycamore (Acer pseudoplatanus L.) cells.

Sycamore cells (Acer pseudoplatanus L.) in suspension culture were grown at 25 degrees C in culture medium containing two oxygen concentrations: 250 microM O2 (standard conditions) and 10 microM O2 (O2-limiting conditions). The decrease of O2 concentration in the culture medium did not modify significantly the relative proportion of each phospholipid. In contrast, the molar proportion of fatty acids was dramatically changed in all lipid classes of the cell membranes; the average percentage of oleate increased from 3 to 45% whereas that of linoleate decreased from 49 to 22%. When normal culture conditions were restored (250 microM O2), oleate underwent a rapid desaturation process; the loss of oleic acid was associated with a stoichiometric appearance of linoleic acid at a rate of about 4 nmol of oleate desaturated/h/10(6) cells. Under these conditions, no change in the Arrhenius-type plots of the rate of sycamore cell respiration was observed; the values of the transition temperature and of the Arrhenius activation energy (Ea) associated with the cell respiration as well as with the respiration-associated enzymes remained unchanged. Thus it was concluded that the fact that a strong decrease in the fraction of unsaturated fatty acid residues present in the mitochondria had no effect on electron transport rates and Arrhenius plot discontinuities casts doubt on the significance of such changes in terms of chilling injury. Finally it is suggested that some of the Arrhenius discontinuities observed at the level of membrane enzyme could be the consequence of intrinsic thermotropic changes in protein arrangement independent of lipid fluidity.

Regulation of intracellular pH values in higher plant cells. Carbon-13 and phosphorus-31 nuclear magnetic resonance studies.

The regulation of the cytoplasmic and vacuolar pH values (pHc and pHv) in sycamore (Acer pseudoplatanus L.) cells was analyzed using 31P and 13C nuclear magnetic resonance spectroscopy. Suspension-cultured cells were compressed in the NMR tube and perfused with the help of an original arrangement enabling a tight control of the pH (external pH, pHe) of the carefully oxygenated circulating nutrient medium. Intracellular pH values were measured from the chemical shifts of: CH2-linked carboxyl groups of citric acid below pH 5.7; orthophosphate between pH 5.7 and 8.0; 13C-enriched bicarbonate over pH 8.0. pHc and pHv were independent of pHe over the range 4.5-7.5. In contrast intracellular pH values decreased rapidly below pHe 4.5 and increased progressively at pHe over 7.5. There was an acceleration in the rate of O2 consumption accompanied with a decrease in cytoplasmic ATP concentration as pHe decreased. When the rate of O2 consumption was approaching the uncoupled O2 uptake rate, a loss of pHc control was observed. It is concluded that as pHe decreased, the plasma membrane ATPase consumed more and more ATP to reject the invading H+ ions in order to maintain pHc at a constant value. Below pHe 4.5 the efficiency of the H+ pump to react to back leakage of H+ ions became insufficient, leading to an acidification of pHc and to an alkalinization of pHe. On the other hand, over pHe 7.5 a passive influx of OH- ions was observed, and pHc increased proportionally to the increase of pHe. Simultaneously appreciable amounts of organic acids (malate and citrate) were synthesized by cells during the course of the alkalinization of the cytoplasmic compartment. The synthesis of organic acids which partially counteract the alkalinization of the cytoplasmic compartment may result from a marked activation of the cytoplasmic phosphoenolpyruvate carboxylase induced by an increase in cytoplasmic bicarbonate concentration. The fluctuations of pHv followed a similar course to that of pHc. It is concluded that the vacuole, which represents a potentially large H+ ions reservoir, can counteract H+ (or OH-) ion invasion observed at acidic (or alkaline) pHe contributing to the homeostasis of pHc.

Is the cytosolic pi concentration a limiting factor for plant cell respiration?

The substrate-dependent O(2) uptake by sycamore (Acer pseudoplatanus L.) cell mitochondria in the presence of ADP and limiting Pi concentrations has been measured. The Pi concentration for half-maximum O(2) uptake rate was found to be in the range 20 to 50 micromolar for all the substrates tested. (31)P NMR of intact sycamore cells indicated that the Pi concentration in the cytoplasm was in the range 5 to 6 millimolar, approximately 100-fold higher than the Pi concentration required for maximum O(2) uptake rates by isolated mitochondria. When sycamore cells were transferred to a culture medium devoid of Pi, the cytoplasmic Pi concentration decreased from 6 to less than 3 millimolar, but the intact cell respiration remained practically constant for at least 4 days. These results strongly suggest that, in vivo, the respiration rate of sycamore cells is not limited by the quantity of Pi supplied to the mitochondria.