Cytochrome Pathway Activity Research Articles

Exogenous Proline Alleviated Low Temperature Stress in Maize Embryos by Optimizing Seed Germination, Inner Proline Metabolism, Respiratory Metabolism and a Hormone Regulation Mechanism

Proline (Pro) is not only an important osmotic adjustment substance, but it also plays an important role in regulating plant abiotic stress resistance. The maize varieties, Xinxin 2 (low temperature insensitive) and Damin 3307 (low temperature sensitive), were chosen as materials, setting a normal temperature for germination (22 °C/10 °C, 9d), low temperature germination (4 °C/4 °C, 5d) and normal temperature recovery (22 °C/10 °C, 4d), combined with a proline (15 mmol·L−1) soaking treatment, to reveal the seed germination and regulation mechanism in maize embryos. The results showed that proline significantly improved the germination potential, germination rate, germination index and vigor index of seeds under low temperature stress, increasing the length of the coleoptile and radicle, increasing the dry and fresh weight of young buds (coleoptile + radicle), and increasing the activity of α-amylase. Proline enhanced the activities of Δ1-pyrroline-5-carboxylic acid synthetase (P5CS) and ornithine aminotransferase (OAT) in maize embryos under low temperature stress, enhanced the proline synthesis pathways, and further enhanced proline accumulation. Proline induced the activity of proline dehydrogenase (ProDH) in the early stage of low temperature stress and stress relief. Under low temperature stress, the activities of hexokinase (HXK), phosphofructokinase (PFK), pyruvate kinase (PK), isocitrate dehydrogenase (IDH), succinate dehydrogenase (SDH), malate dehydrogenase (MDH) and glucose-6-phosphate dehydrogenase (G-6-PDH) and glucose-6-phosphate dehydrogenase (G-6-PDH and 6-P-GDH) in maize embryos were decreased, resulting in a decrease in the glycolysis (EMP) pathway, tricarboxylic acid (TCA) cycle and pentose phosphate pathway (PPP). Proline alleviated the inhibition of key enzyme activities of the EMP pathway, TCA cycle and PPP of maize embryos under low temperature stress, increased the activities of cytochrome oxidase (COX) and alternative oxidase (AOX), increased the ATP content, alleviated the inhibition of low temperature stress on main cytochrome pathway activity (ρVcyt), while further increasing the total respiratory activity (Vt) and the actual operational activity of the alternative pathway (ρValt) during seed germination at the initial stage of low temperature stress, as well as improving the inhibition of the Vt and ρValt in the middle and late stages of low temperature stress. Under low temperature stress, the content of abscisic acid (ABA) increased significantly, while gibberellin (GA), auxin (IAA) and zeatin nucleoside (ZR) decreased significantly. Proline alleviated the decrease in IAA, ZR and GA contents in maize embryos under low temperature stress, reduced the increase in the ABA content, and reduced the inhibition of low temperature on seed germination.

Lung mitochondria adaptation to endurance training in rats

We elucidated the impact of eight weeks of endurance training on the oxidative metabolism of rat lungs. Adult 3.5-month-old male rats were randomly allocated to a treadmill training group or a sedentary group as control. In the lungs, endurance training raised the expression level of the oxygen sensors hypoxia inducible factor 1α (HIF1α) and lysine-specific demethylase 6A (KDM6A) as well as stimulated mitochondrial oxidative capacity and mitochondrial biogenesis, while lactate dehydrogenase activity was reduced. Endurance training enhanced antioxidant systems (the coenzyme Q content and superoxide dismutase) in lung tissue but decreased them (and uncoupling protein 2) in lung mitochondria. In the lung mitochondria of trained rats, the decreased Q content and Complex I (CI) activity and the enhanced cytochrome pathway activity (CIII + CIV) may account for the diminished Q reduction level, resulting in a general decrease in H2O2 formation by mitochondria. Endurance training enhanced oxidation of glutamate and fatty acids and caused opposite effects in functional mitochondrial properties during malate and succinate oxidation, which were related to reduced activity of CI and increased activity of CII, respectively. In addition, endurance training downregulated CI in supercomplexes and upregulated CIII in the CIII2+CIV supercomplex in the oxidative phosphorylation system. We concluded that the adaptive lung responses observed could be due to hypoxia and oxidative stress induced by strenuous endurance training.

Targeted Proteomics Approach Toward Understanding the Role of the Mitochondrial Protease FTSH4 in the Biogenesis of OXPHOS During Arabidopsis Seed Germination

Seed germination provides an excellent model to study the process of mitochondrial biogenesis. It is a complex and strictly regulated process which requires a proper biogenesis of fully active organelles from existing promitochondrial structures. We have previously reported that the lack of the inner mitochondrial membrane protease FTSH4 delayed Arabidopsis seed germination. Here, we implemented a targeted mass spectrometry-based approach, Multiple Reaction Monitoring (MRM), with stable-isotope-labeled standard peptides for increased sensitivity, to quantify mitochondrial proteins in dry and germinating wild-type and ftsh4 mutant seeds, lacking the FTSH4 protease. Using total seed protein extracts we measured the abundance of the peptide targets belonging to the OXPHOS complexes, AOX1A, transport, and inner membrane scaffold as well as mitochondrial proteins that are highly specific to dry and germinating seeds. The MRM assay showed that the abundance of these proteins in ftsh4 did not differ substantially from that observed in wild-type at the level of dry seed and after stratification, but we observed a reduction in protein abundance in most of the examined OXPHOS subunits in the later stages of germination. These changes in OXPHOS protein levels in ftsh4 mutants were accompanied by a lower cytochrome pathway activity as well as an increased AOX1A amount at the transcript and protein level and alternative pathway activity. The analyses of the steady-state transcript levels of mitochondrial and nuclear genes encoding OXPHOS subunits did not show significant difference in their amount, indicating that the observed changes in the OXPHOS occurred at the post-transcriptional level. At the time when ftsh4 seeds were fully germinated, the abundance of the OXPHOS proteins in the mutant was either slightly lowered or comparable to these amounts in wild-type seeds at the similar developmental stage. By the implementation of an integrative approach combining targeted proteomics, quantitative transcriptomics, and physiological studies we have shown that the FTSH4 protease has an important role in the biogenesis of OXPHOS and thus biogenesis of mitochondria during germination of Arabidopsis seeds.

Bud dormancy breaking affects respiration and energy balance of bilberry shoots in the initial stage of growth

Respiration and heat production in the shoots of bilberry (Vaccinium myrtillus L.) were studied at the beginning of growth after breaking bud dormancy by means of transfer of the shoots to indoor conditions (November–April) and upon natural sprouting in spring (May). The buds released from dormancy at the beginning of winter sprouted slower and showed lower respiratory activity than the buds that started growing in May. In May, cytochrome respiratory pathway in sprouting buds was 1.3 times more active than energetically ineffective alternative pathway, whereas activity of cytochrome pathway in December was 1.4 times lower as compared with the alternative. In November–December, the rate of heat evolution by the buds was 3–5 times lower than in April–May. In case of early breaking of bud dormancy, the share of respiration energy dissipated as heat was 30% on average. In the buds whose growth was induced later, the value of this parameter was twice as much. The ratio between heat evolution and respiration depended on temperature. High temperature more intensely activated heat evolution than respiration, which caused a decrease in the level of metabolic energy available for growth. In the temperature range of 5–15°C characteristic of the beginning of vegetation, the share of respiration energy dissipated as heat was 2–3 times lower than at 20–30°C, which reflects a great adaptability of V. myrtillus to climatic conditions of the region. Our data suggest that progression through a full cycle of winter dormancy is physiologically important for shoot growth. Early dormancy release brought about changes in respiration and energy balance of the shoots in the initial stage of extra-bud growth.

Mitochondrial Alternative Pathway-Associated Photoprotection of Photosystem II is Related to the Photorespiratory Pathway.

Respiratory electron transport has two ubiquinol-oxidizing pathways, the cytochrome pathway (CP) and the alternative pathway (AP). The AP, which is catalyzed by the alternative oxidase (AOX), is energetically wasteful but may alleviate PSII photoinhibition under light conditions excessive for photosynthesis. However, its mechanism remains unknown. We used Arabidopsis aox1a mutants lacking AOX activity and studied the mutation's effects on photoinhibition by measuring the decrease in the maximum quantum yield of PSII (Fv/Fm) after high light exposure. Since the CP compensates for the lack of AOX, we monitored the extent of photoinhibition under conditions where CP activity is partially inhibited by antimycin A. When leaves were exposed to high light at 350 µmol m-2 s-1, the decline in Fv/Fm was significantly faster in the aox1a mutants than in the wild type. However, under conditions where photorespiration was suppressed by high CO2 or low O2 levels, the decline in Fv/Fm was suppressed in the aox1a mutants, but not in the wild type, making the difference between the wild type and mutants small. Our results demonstrate that the lack of the AP causes an acceleration of PSII photoinhibition in relation to the photorespiratory pathway, suggesting that the AP can support the activity of the photorespiratory pathway under high light conditions.

Respiratory flexibility and efficiency are affected by simulated global change in Arctic plants

Laboratory studies indicate that, in response to environmental conditions, plants modulate respiratory electron partitioning between the 'energy-wasteful' alternative pathway (AP) and the 'energy-conserving' cytochrome pathway (CP). Field data, however, are scarce. Here we investigate how 20-yr field manipulations simulating global change affected electron partitioning in Alaskan Arctic tundra species. We sampled leaves from three dominant tundra species - Betula nana, Eriophorum vaginatum and Rubus chamaemorus - that had been strongly affected by manipulations of soil nutrients, light availability, and warming. We measured foliar dark respiration, in-vivo electron partitioning and alternative oxidase/cytochrome c oxidase concentrations in addition to leaf traits and mitochondrial ultrastructure. Changes in leaf traits and ultrastructure were similar across species. Respiration at 20°C (R(20)) was reduced 15% in all three species grown at elevated temperature, suggesting thermal acclimation of respiration. In Betula, the species with the largest growth response to added nutrients, CP activity increased from 9.4 ± 0.8 to 16.6 ± 1.6 nmol O(2) g(-1) DM s(-1) whereas AP activity was unchanged. The ability of Betula to selectively increase CP activity in response to the environment may contribute to its overall ecological success by increasing respiratory energy efficiency, and thus retaining more carbon for growth.

Impact of oxidative stress on Acanthamoeba castellanii mitochondrial bioenergetics depends on cell growth stage

Addition of a moderate (1.4mM) concentration of H(2)O(2) to protozoon Acanthamoeba castellanii cell cultures at different growth phases caused a different response to oxidative stress. H(2)O(2) treatment of exponentially growing cells significantly delayed their growth; however, in mitochondria isolated from these cells, no damage to their bioenergetic function was observed. In contrast, addition of H(2)O(2) to A. castellanii cells approaching the stationary phase did not influence their growth and viability while seriously affecting mitochondrial bioenergetic function. Although mitochondrial integrity was maintained, oxidative damage was revealed in the reduction of cytochrome pathway activity, uncoupling protein activity, and the efficiency of oxidative phosphorylation as well as the membrane potential and the endogenous ubiquinone reduction level of the resting state. An increase in the alternative oxidase protein level and activity as well as an increase in the membranous ubiquinone content were observed in mitochondria isolated from late H(2)O(2)-treated cells. For the first time, the regulation of ubiquinone content in the inner mitochondrial membrane is shown to play a role in the response to oxidative stress. A physiological role for the higher activity of the alternative oxidase in response to oxidative stress in unicellular organisms, such as amoeba A. castellanii, is discussed.

Response of mitochondrial thioredoxin PsTrxo1, antioxidant enzymes, and respiration to salinity in pea (Pisum sativum L.) leaves.

Mitochondria play an essential role in reactive oxygen species (ROS) signal transduction in plants. Redox regulation is an essential feature of mitochondrial function, with thioredoxin (Trx), involved in disulphide/dithiol interchange, playing a prominent role. To explore the participation of mitochondrial PsTrxo1, Mn-superoxide dismutase (Mn-SOD), peroxiredoxin (PsPrxII F), and alternative oxidase (AOX) under salt stress, their transcriptional and protein levels were analysed in pea plants growing under 150 mM NaCl for a short and a long period. The activities of mitochondrial Mn-SOD and Trx together with the in vivo activities of the alternative pathway (AP) and the cytochrome pathway (CP) were also determined, combined with the characterization of the plant physiological status as well as the mitochondrial oxidative indicators. The analysis of protein and mRNA levels and activities revealed the importance of the post-transcriptional and post-translational regulation of these proteins in the response to salt stress. Increases in AOX protein amount correlated with increases in AP capacity, whereas in vivo AP activity was maintained under salt stress. Similarly, Mn-SOD activity was also maintained. Under all the stress treatments, photosynthesis, stomatal conductance, and CP activity were decreased although the oxidative stress in leaves was only moderate. However, an increase in lipid peroxidation and protein oxidation was found in mitochondria isolated from leaves under the short-term salinity conditions. In addition, an increase in mitochondrial Trx activity was produced in response to the long-term NaCl treatment. The results support a role for PsTrxo1 as a component of the defence system induced by NaCl in pea mitochondria, providing the cell with a mechanism by which it can respond to changing environment protecting mitochondria from oxidative stress together with Mn-SOD, AOX, and PrxII F.

Basic energetic parameters of Acanthamoeba castellanii mitochondria and their resistance to oxidative stress.

The purpose of this study was establishing the basic energetic parameters of amoeba Acanthamoeba castellanii mitochondria respiring with malate and their response to oxidative stress caused by hydrogen peroxide in the presence of Fe(2+) ions. It appeared that, contrary to a previous report (Trocha LK, Stobienia O (2007) Acta Biochim Polon 54: 797), H(2)O(2)-treated mitochondria of A. castellanii did not display any substantial impairment. No marked changes in cytochrome pathway activity were found, as in the presence of an inhibitor of alternative oxidase no effects were observed on the rates of uncoupled and phosphorylating respiration and on coupling parameters. Only in the absence of the alternative oxidase inhibitor, non-phosphorylating respiration progressively decreased with increasing concentration of H(2)O(2), while the coupling parameters (respiratory control ratio and ADP/O ratio) slightly improved, which may indicate some inactivation of the alternative oxidase. Moreover, our results show no change in membrane potential, Ca(2+) uptake and accumulation ability, mitochondrial outer membrane integrity and cytochrome c release for 0.5-25 mM H(2)O(2)-treated versus control (H(2)O(2)-untreated) mitochondria. These results indicate that short (5 min) incubation of A. castellanii mitochondria with H(2)O(2) in the presence of Fe(2+) does not damage their basic energetics.

Changes in respiratory mitochondrial machinery and cytochrome and alternative pathway activities in response to energy demand underlie the acclimation of respiration to elevated CO2 in the invasive Opuntia ficus-indica.

Studies on long-term effects of plants grown at elevated CO(2) are scarce and mechanisms of such responses are largely unknown. To gain mechanistic understanding on respiratory acclimation to elevated CO(2), the Crassulacean acid metabolism Mediterranean invasive Opuntia ficus-indica Miller was grown at various CO(2) concentrations. Respiration rates, maximum activity of cytochrome c oxidase, and active mitochondrial number consistently decreased in plants grown at elevated CO(2) during the 9 months of the study when compared to ambient plants. Plant growth at elevated CO(2) also reduced cytochrome pathway activity, but increased the activity of the alternative pathway. Despite all these effects seen in plants grown at high CO(2), the specific oxygen uptake rate per unit of active mitochondria was the same for plants grown at ambient and elevated CO(2). Although decreases in photorespiration activity have been pointed out as a factor contributing to the long-term acclimation of plant respiration to growth at elevated CO(2), the homeostatic maintenance of specific respiratory rate per unit of mitochondria in response to high CO(2) suggests that photorespiratory activity may play a small role on the long-term acclimation of respiration to elevated CO(2). However, despite growth enhancement and as a result of the inhibition in cytochrome pathway activity by elevated CO(2), total mitochondrial ATP production was decreased by plant growth at elevated CO(2) when compared to ambient-grown plants. Because plant growth at elevated CO(2) increased biomass but reduced respiratory machinery, activity, and ATP yields while maintaining O(2) consumption rates per unit of mitochondria, we suggest that acclimation to elevated CO(2) results from physiological adjustment of respiration to tissue ATP demand, which may not be entirely driven by nitrogen metabolism as previously suggested.

Cytochrome and alternative pathway activity in roots of thermal and non‐thermal Agrostis species in response to high soil temperature

Partitioning of respiration between the cytochrome pathway (CP) and the alternative pathway (AP) may play an important role in plant adaptation to extreme environments. We examined changes in partitioning between CP and AP, and viability of roots associated with plant exposure to high soil temperature for two Agrostis species: Agrostis scabra Willd., a species adapted to high‐temperature soils in geothermal areas in Yellowstone National Park, and Agrostis stolonifera L. (cv. Penncross) a heat‐sensitive grass widely used in cool‐climate regions. Roots of A. scabra and A. stolonifera were exposed to soil temperature of 37 or 20°C, while shoots were exposed to 20°C for 28 days. Root viability decreased, and total root respiration increased for both species at 37°C. The decline in root viability and the increase in respiration rates were less pronounced for A. scabra than for A. stolonifera. A larger proportion of total root respiration was attributed to the AP in A. scabra compared with that in A. stolonifera when both species were exposed to 37°C. At 7 and 14 days at 37°C, the relative proportion of respiration passing through AP increased by 12 and 10%, respectively, in A. scabra, whereas in A. stolonifera, AP increased by 4 and 1%, respectively. Our results suggest that maintaining a higher proportion of AP at a high soil temperature may contribute to root thermo‐tolerance in A. scabra in comparison with A. stolonifera, and alternative respiration may play an important role in plant adaptation to high soil temperature.

The effect of growth at low temperature on the activity and expression of the uncoupling protein in Acanthamoeba castellanii mitochondria

Mitochondria of amoeba Acanthamoeba castellanii, a non-photosynthetic soil amoeboid protozoon, possess an uncoupling protein (AcUCP) that mediates free fatty acid-activated proton re-uptake dissipating the proton electrochemical gradient built up by respiration. The present study provides the first evidence that UCP could be a cold response protein in unicellulars. In mitochondria isolated from an amoeba batch culture grown temporarily at low temperature (6 °C), the content of AcUCP was increased and correlated with an increase in the linoleic acid (LA)-stimulated UCP-mediated carboxyatractyloside-resistant state 4 respiration, as compared to a control culture (routinely grown at 28 °C). Moreover, the cytochrome pathway activity was found to be insensitive to the cold exposure of amoeba cells, as indicated by respiration and membrane potential measurements as well as by an absence of change in the adenine nucleotide translocator and cytochrome oxidase expression levels. Furthermore, in mitochondria from the low-temperature-grown cells, at fixed LA concentration, the increased contribution of AcUCP activity to total mitochondrial phosphorylating respiration accompanied by lower coupling parameters was found, as was confirmed by calculation of this contribution using ADP/O measurements.

Changes in mitochondrial electron partitioning in response to herbicides inhibiting branched-chain amino acid biosynthesis in soybean.

The adaptation of the respiratory metabolism in roots of soybean (Glycine max L. Merr. cv Ransom) treated with herbicides that inhibit the enzyme acetolactate synthase (ALS) was analyzed. A new gas phase dual-inlet mass spectrometry system for simultaneous measurement of 34O2 to 32O2 and O2 to N2 ratios has been developed. This system is more accurate than previously described systems, allows measurements of much smaller oxygen gradients, and, as a consequence, works with tissues that have lower respiration rates. ALS inhibition caused an increase of the alternative oxidase (AOX) protein and an accumulation of pyruvate. The combination of these two effects is likely to induce the activation of the alternative pathway and its participation in the total respiration. Moreover, the start of the alternative pathway activation and the increase of AOX protein were before the decline in the activity of cytochrome pathway. The possible role of AOX under ALS inhibition is discussed.

Involvement of the alternative oxidase in respiration of Yarrowia lipolytica mitochondria is controlled by the activity of the cytochrome pathway.

The degree of involvement of cyanide-resistant alternative oxidase in the respiration of Yarrowia lipolytica mitochondria was evaluated by comparing the rate of oxygen consumption in the presence of cyanide, which shows the activity of the cyanide-resistant alternative oxidase, and the oxidation rate of cytochrome c by ferricyanide, which shows the activity of the main cytochrome pathway. The oxidation of succinate by mitochondria in the presence of ferricyanide and cyanide was associated with oxygen consumption due to the functioning of the alternative oxidase. The subsequent addition of ADP or FCCP (an uncoupler of oxidative phosphorylation) completely inhibited oxygen consumption by the mitochondria. Under these conditions, the inhibition of the alternative oxidase by benzohydroxamic acid (BHA) failed to affect the reduction of ferricyanide at the level of cytochrome c. BHA did not influence the rate of ferricyanide reduction by the cytochrome pathway occurring in controlled state 4, nor could it change the phosphorylation quotient ATP/O upon the oxidation of various substrates. These data indicate that the alternative system is unable to compete with the cytochrome respiratory chain for electrons. The alternative oxidase only transfers the electrons that are superfluous for the cytochrome respiratory chain.

The energy-conserving and energy-dissipating processes in mitochondria isolated from wild type and nonripening tomato fruits during development on the plant.

Bioenergetics of tomato (Lycopersicon esculentum) development on the plant was followed from the early growing stage to senescence in wild type (climacteric) and nonripening mutant (nor, non-climacteric) fruits. Fruit development was expressed in terms of evolution of chlorophyll a content allowing the assessment of a continuous time-course in both cultivars. Measured parameters: the cytochrome pathway-dependent respiration, i.e., the ATP synthesis-sustained respiration (energy-conserving), the uncoupling protein (UCP) activity-sustained respiration (energy-dissipating), the alternative oxidase(AOX)-mediated respiration (energy-dissipating), as well as the protein expression of UCP and AOX, and free fatty acid content exhibited different evolution patterns in the wild type and nor mutant that can be attributed to their climacteric/nonclimacteric properties, respectively. In the wild type, the climacteric respiratory burst observed in vitro depended totally on an increse in the cytochrome pathway activity sustained by ATP synthesis, while the second respiratory rise during the ripening stage was linked to a strong increase in AOX activity accompanied by an overexpression of AOX protein. In wild type mitochondria, the 10-microM linoleic acid-stimulated UCP-activity-dependent respiration remained constant during the whole fruit development except in senescence where general respiratory decay was observed.

Metabolic regulation of leaf respiration and alternative pathway activity in response to phosphate supply

In this study the question whether the alternative respiratory pathway acts as an electron bypass for the cytochrome pathway under conditions of growth on limited phosphorus in leaves of bean (Phaseolus vulgaris L.), tobacco (Nicotiana tabacum L.) and Gliricidia sepium Walp was investigated. The oxygen isotope fractionation technique was used to assess the in vivo activities of the cytochrome and alternative respiratory pathways in the absence of added inhibitors. The response of respiration to low phosphorus supply varied among species. Growth at low phosphorus reduced cytochrome pathway activity in bean and tobacco. Alternative pathway activity increased only in bean leaves in response to low phosphorus and not in tobacco. In the case of G. sepium, cytochrome pathway activity remained unchanged whereas the alternative pathway activity increased with low nutritional phosphorus. At low phosphorus, alternative oxidase protein levels increased in the leaves of bean and G. sepium but not in tobacco, suggesting a dependence of alternative pathway activity on protein level. Alternative pathway activity was also not correlated with soluble carbohydrate concentration in bean or tobacco at any phosphorus level. These results show that the alternative pathway does not always act as an electron bypass in response to the downstream restriction of the cytochrome pathway imposed by low phosphorus supply. These results suggest that factors in addition to cellular carbohydrate level and adenylate control can act to regulate alternative pathway activity.

The alternative oxidase in roots of Poa annua after transfer from high‐light to low‐light conditions

The activity of the alternative pathway can be affected by a number of factors, including the amount and reduction state of the alternative oxidase protein, and the reduction state of the ubiquinone pool. To investigate the importance of these factors in vivo, we manipulated the rate of root respiration by transferring the annual grass Poa annua L. from high-light to low-light conditions, and at the same time from long-day to short-day conditions for four days. As a result of the low-light treatment, the total respiration rate of the roots decreased by 45%, in vitro cytochrome c oxidase capacity decreased by 49%, sugar concentration decreased by 90% and the ubiquinone concentration increased by 31%, relative to control values. The absolute rate of oxygen uptake via the alternative pathway, as determined using the 18O-isotope fractionation technique, did not change. Conversely, the cytochrome pathway activity decreased during the low-light treatment; its activity increased upon addition of exogenous sugars to the roots. Interestingly, no change was observed in the concentration of the alternative oxidase protein or in the reduction state of the protein. Also, there was no change in the reduction state of the ubiquinone pool. In conclusion, the concentration and activity of the alternative oxidase were not changed, even under severe light deprivation.

Growth and respiration of Petunia hybrida cells in chemostat cultures: A comparison of glucose-limited and nitrate-limited cultures

Nitrate-limited and glucose-limited chemostat cultures of Petunia hybrida cells were compared at a specific biomass (+extracellular product) formation rate of 0.0042 C.mol/C.mol h. The composition of the biomass differed considerably in both culture types. The N/C (mol/mol) ratio in the biomass was almost four times lower in the nitrate-limited than in the glucose-limited cultures. On a dry weight basis (g/g DW) the biomass in the nitrate-limited cultures contained about 2.5 times less ions and protein N and about 2.5 times more carbohydrates than the biomass in the glucose-limited cultures. On a fresh weight basis (mmol/g FW) the biomass in nitrate-limited and glucose-limited cultures differed mainly in carbohydrate content. The yields of biomass on glucose and oxygen were generally higher in the nitrate-limited than in the glucose-limited cultures. Average values for these parameters were 0.27 C . mol biomass/C . mol glucose and 0.42 C . mol biomass/mol O(2) in the glucose-limited cultures and 0.34 C . mol biomass/C . mol glucose and 0.55 C . mol biomass/mol O(2) in the nitrate-limited cultures. On a C . mol basis the total respiration was about 25% and the maximally attainable cytochrome pathway activity (measured in the presence of hydroxamate) about 30% higher in the glucose-limited than in the nitrate-limited cultures. The maximally attainable activity of the alternative pathway (measured in the presence of KCN) was significantly lower in the glucose-limited cultures. On an organic N ( approximately protein) basis all respiratory parameters were significantly higher in the nitrate-limited cultures. In the presence of the respiratory uncoupler carbonyl cyanide p-trifluoromethoxy phenylhydrazone (FCCP) and excess glucose, cellular respiratory activity shows its maximal activity; under these conditions the total respiration increased more than 150% in the glucose-limited and only 30% in the nitrate-limited cultures. It is suggested that glucose-limited cultures are able to react more flexibly to changes in the environmental conditions than nitrate-limited cultures. (c) 1996 John Wiley & Sons, Inc.

Effects of water stress on development, operation and gene expression of cyanide-resistant respiratory pathway in wheat

Osmotic dehydration of wheat seedlings in -0.5 MPa polyethylene glycol (PEG) solutions for 24, 48 and 72 h resulted in mild, moderate and severe water stress respectively in leaves, but only caused mild water stress in roots as reflected by the changes in relative water content (RWC). In response to the above water stress conditions, leaf total respiratory rate (V(t)) decreased progressively, and the alternative pathway (AP) capacity (V(alt)) and its actual operation activity (rhoV(alt)) decreased more severely. Water stress also led to continuous reduction in cytochrome pathway (CP) activity ((rho' V (cyt)) and different changes in the contribution of rhoV(alt) and rho'V(cyt) to V(t), in leaves, with rhoV(alt)/ V(t), decreasing and rho'V(cyt)/V(t) increasing. The change pattern of root V(t) was similar to that of its RWC, while root V(alt) and rhoV(alt) were found to decrease during the first 24 h of stress and thereafter recover to a level close to that of the control (0 h). These data indicate that the alternative pathway is sensitive to water stress and can adapt mild water stress. The results of northern hybridization using an alternative oxidase gene (Aox) as probe revealed that there was a good correlation between V(alt) (also rhoV(alt)) and Aox mRNA levels in both leaves and roots, suggesting that water stress affects the development and operation of AP respiration through affecting the expression of the alternative oxidase gene.

Induction of alternative oxidase synthesis by herbicides inhibiting branched‐chain amino acid synthesis

Sycamore suspension cells (Acer pseudoplatanus L.) were incubated in the presence of sulfonylurea and imidazolinone herbicides. These inhibitors of acetolactate synthase (ALS), a key enzyme of branched‐chain amino acid synthesis, triggered a dramatic induction of the alternative oxidase (AOX). AOX activity increased in treated cells, eventually exceeding cytochrome (cyt) pathway activity. This induction of AOX activity was correlated with the accumulation of a 35 kDa AOX protein in isolated mitochondria, detected by Western blotting with a monoclonal antibody against Sauromatum guttatum AOX. It was preceded by the accumulation of putative 1.6 kb AOX mRNA, detected using an Aox cDNA probe from soybean. The metabolic perturbations induced by the herbicides rather than the herbicide molecules themselves were responsible for this induction of AOX. However, α‐oxobutyrate (one of the substrates of ALS) and its transamination product, α‐aminobutyrate, which accumulated after herbicide treatment, were not involved. The inhibition of branched‐chain amino acid synthesis was probably somehow responsible for the AOX induction since: (i) a mixture of those amino acids (leucine, isoleucine, valine) prevented AOX induction by ALS inhibitors; (ii) the herbicide Hoe 704, a potent inhibitor of acetolactate reducto‐isomerase (the enzyme following ALS in the branched‐chain amino acid pathway), also triggered AOX induction.