Alternative Respiratory Pathway Research Articles

Involvement of a Glycerol-3-Phosphate Dehydrogenase in Modulating the NADH/NAD+ Ratio Provides Evidence of a Mitochondrial Glycerol-3-Phosphate Shuttle in Arabidopsis

A mitochondrial glycerol-3-phosphate (G-3-P) shuttle that channels cytosolic reducing equivalent to mitochondria for respiration through oxidoreduction of G-3-P has been extensively studied in yeast and animal systems. Here, we report evidence for the operation of such a shuttle in Arabidopsis thaliana. We studied Arabidopsis mutants defective in a cytosolic G-3-P dehydrogenase, GPDHc1, which, based on models described for other systems, functions as the cytosolic component of a G-3-P shuttle. We found that the gpdhc1 T-DNA insertional mutants exhibited increased NADH/NAD+ ratios compared with wild-type plants under standard growth conditions, as well as impaired adjustment of NADH/NAD+ ratios under stress simulated by abscisic acid treatment. The altered redox state of the NAD(H) pool was correlated with shifts in the profiles of metabolites concerning intracellular redox exchange. The impairment in maintaining cellular redox homeostasis was manifest by a higher steady state level of reactive oxygen species under standard growth conditions and by a significantly augmented hydrogen peroxide production under stress. Loss of GPDHc1 affected mitochondrial respiration, particularly through a diminished capacity of the alternative oxidase respiration pathway. We propose a model that outlines potential involvements of a mitochondrial G-3-P shuttle in plant cells for redox homeostasis.

Exogenous Putrescine Reduces Sodium and Chloride Accumulation in NaCl-Treated Calli of the Salt-Sensitive Rice Cultivar I Kong Pao

In order to gain information on the putative involvement of polyamines (PAs) in the response of rice cells to salinity, mature embryo-derived calli issued from the salt-sensitive cultivar I Kong Pao were exposed for 3 months to the simultaneous presence of NaCl (0, 150 and 300 mM) and exogenous polyamines (putrescine (Put): 1 and 10 mM; spermidine (Spd): 1 and 10 mM; spermine (Spm): 1 mM). Callus growth, endogenous PAs, Na+, K+ and Cl− concentrations were quantified and analysed in relation to cell viability based on 2,3,5-triphenytetrazolium chloride (TTC) reduction. All exogenous PAs were efficiently absorbed from the external medium. Exogenous Put 1 mM clearly stimulated growth of salt-stressed calli in relation to a decrease in both Na+ and Cl− accumulation. In contrast, Spd 10 mM and Spm 1 mM exacerbated the deleterious impact of NaCl on callus growth and induced a decrease in K+ concentration. While Put helped in the maintenance of cell viability, Spd 10 mM and Spm 1 mM decreased cell viability, mainly in relation to an inhibition of the alternative respiratory pathway. It is proposed that Put may assume positive functions in salt stress resistance in rice.

The concomitant expression and availability of conventional and alternative, cyanide-insensitive, respiratory pathways in Candida albicans

The opportunistic oral pathogen Candida albicans expresses a cyanide-insensitive alternative oxidase (AOX) upon exposure to respiratory inhibitors that act downstream from coenzyme Q, and upon ageing of cells. To investigate whether the conventional pathway is retained when the alternative pathway is induced, cells were grown in the presence of sodium cyanide, a reversible inhibitor of cytochrome oxidase. AOX expression was monitored by Western blotting and the presence of cytochromes associated with complexes III and IV of the conventional pathway was monitored by recording spectra between 500 and 650 nm at 77 K. The activities of complexes III and IV were determined in polarographic and enzyme-kinetic experiments using specific respiratory substrates and inhibitors. Results indicated that complexes III and IV are constitutively expressed and are functional in cells expressing AOX. Furthermore, the enzymatic activities of complexes III and IV were similar in mitochondrial preparations from cells grown with or without cyanide. We next investigated whether both pathways are simultaneously available for electron transfer from the Q pool to molecular oxygen. Respiration was virtually completely inhibited by the combination of cyanide and salicyl hydroxamic acid (SHAM) or antimycin A and SHAM, but only partly inhibited by either of these inhibitors alone. This indicates that electrons can in principle flow either through the conventional or the alternative respiratory pathway. The availability of two electron pathways in C. albicans and the potential use of either pathway endows this pleomorphic fungus with another level at which it can rapidly adjust to altered environmental conditions.

The alternative oxidase from Euglena gracilis

The alternative oxidase (AOX) is an ubiquinol oxidase of the mitochondrial respiratory chain. AOX is an additional terminal oxidase; the electron flux to AOX branches from the classical electron‐transport chain at the level of the ubiquinone pool leading to the direct reduction of oxygen to water in a single four‐electron transfer step. O2 reduction catalyzed by AOX is not coupled to proton pumping and hence not ATP‐producing. The energy is released as heat. AOX is specifically inhibited by salicylhydroxamic acid (SHAM), but is cyanide resistant. AOX was discovered in plant thermogenic tissues and all plant species tested to date can express the alternative pathway. It was long thought that AOX is specific to higher plants, but it is now turning up in many non‐photosynthetic eukaryotes. For example the bloodstream form of the protist Trypanosoma brucei brucei that lacks functional cytochromes, limits most of its energy metabolism to glycolysis while passing the reducing equivalents produced to water via a plant‐like alternative oxidase. It has also been reported that oxidative phosphorylation in the protist Euglena gracilis is supported by an alternative respiratory pathway in which an AOX is involved. We have cloned the mitochondrial AOX from Euglena and have investigated the evolutionary history of this enzyme in prokaryotes and eukaryotes.

High-level expression of alternative oxidase protein sequences enhances the spread of viral vectors in resistant and susceptible plants

The alternative oxidase (AOX) is the terminal oxidase of the cyanide-resistant alternative respiratory pathway in plants and has been implicated in resistance to viruses. When tobacco mosaic virus (TMV) vectors were used to drive very high levels of expression of either AOX or AOX mutated in its active site (AOX-E), virus spread was enhanced. This was visualized as the induction of larger hypersensitive-response lesions after inoculation onto NN-genotype tobacco than those produced by vectors bearing sequences of comparable length [the green fluorescent protein (gfp) gene sequence or antisense aox] or the 'empty' viral vector. Also, in the highly susceptible host Nicotiana benthamiana, systemic movement of TMV vectors expressing AOX or AOX-E was faster than that of TMV constructs bearing gfp or antisense aox sequences. Notably, in N. benthamiana, TMV.AOX and TMV.AOX-E induced symptoms that were severe and ultimately included cell death, whereas the empty vector, TMV.GFP and the TMV vector expressing antisense aox sequences never induced necrosis. The results show that, if expressed at sufficiently high levels, active and inactive AOX proteins can affect virus spread and symptomology in plants.

Respiratory chain supercomplexes in plant mitochondria

Supercomplexes are defined associations of protein complexes, which are important for several cellular functions. This “quintenary” organization level of protein structure recently was also described for the respiratory chain of plant mitochondria. Except succinate dehydrogenase (complex II), all complexes of the oxidative phosphorylation (OXPOS) system (complexes I, III, IV and V) were found to form part of supercomplexes. Compositions of these supramolecular structures were systematically investigated using digitonin solubilizations of mitochondrial fractions and two-dimensional Blue-native (BN) polyacrylamide gel electrophoresis. The most abundant supercomplex of plant mitochondria includes complexes I and III at a 1:2 ratio (I 1 + III 2 supercomplex). Furthermore, some supercomplexes of lower abundance could be described, which have I 2 + III 4, V 2, III 2 + IV 1–2, and I 1 + III 2 + IV 1–4 compositions. Supercomplexes consisting of complexes I plus III plus IV were proposed to be called “respirasome”, because they autonomously can carry out respiration in the presence of ubiquinone and cytochrome c. Plant specific alternative oxidoreductases of the respiratory chain were not associated with supercomplexes under all experimental conditions tested. However, formation of supercomplexes possibly indirectly regulates alternative respiratory pathways in plant mitochondria on the basis of electron channeling. In this review, procedures to characterize the supermolecular organization of the plant respiratory chain and results concerning supercomplex structure and function are summarized and discussed.

Nitrate, NO and haemoglobin in plant adaptation to hypoxia: an alternative to classic fermentation pathways.

The role of nitrate reduction to produce nitric oxide (NO) and its subsequent oxidation by oxyhaemoglobin as a mechanism to maintain plant cell energetics during hypoxia is examined. Nitrate reduction in hypoxic conditions can be considered as an alternative respiratory pathway, with nitrate as an intermediate electron acceptor, contributing to the oxidation of NADH. NO, produced in the reaction, does not accumulate due to the induction of hypoxia-induced (class 1) haemoglobins. These haemoglobins remain in the oxyhaemoglobin form, even at oxygen tensions two orders of magnitude lower than necessary to saturate cytochrome c oxidase. They act, probably in conjunction with a flavoprotein, as NO dioxygenases converting NO back to nitrate, consuming NAD(P)H in the process. The overall system oxidizes 2.5 moles of NADH per one mole of nitrate recycled during the reaction, leading to the maintenance of redox and energy status during hypoxia and resulting in the reduced production of ethanol and lactic acid.

Light regulation of the Arabidopsis respiratory chain. Multiple discrete photoreceptor responses contribute to induction of type II NAD(P)H dehydrogenase genes.

Controlled oxidation reactions catalyzed by the large, proton-pumping complexes of the respiratory chain generate an electrochemical gradient across the mitochondrial inner membrane that is harnessed for ATP production. However, several alternative respiratory pathways in plants allow the maintenance of substrate oxidation while minimizing the production of ATP. We have investigated the role of light in the regulation of these energy-dissipating pathways by transcriptional profiling of the alternative oxidase, uncoupling protein, and type II NAD(P)H dehydrogenase gene families in etiolated Arabidopsis seedlings. Expression of the nda1 and ndc1 NAD(P)H dehydrogenase genes was rapidly up-regulated by a broad range of light intensities and qualities. For both genes, light induction appears to be a direct transcriptional effect that is independent of carbon status. Mutant analyses demonstrated the involvement of two separate photoreceptor families in nda1 and ndc1 light regulation: the phytochromes (phyA and phyB) and an undetermined blue light photoreceptor. In the case of the nda1 gene, the different photoreceptor systems generate distinct kinetic induction profiles that are integrated in white light response. Primary transcriptional control of light response was localized to a 99-bp region of the nda1 promoter, which contains an I-box flanked by two GT-1 elements, an arrangement prevalent in the promoters of photosynthesis-associated genes. Light induction was specific to nda1 and ndc1. The only other substantial light effect observed was a decrease in aox2 expression. Overall, these results suggest that light directly influences the respiratory electron transport chain via photoreceptor-mediated transcriptional control, likely for supporting photosynthetic metabolism.

Mitochondria/nuclear signaling of alternative oxidase gene expression occurs through distinct pathways involving organic acids and reactive oxygen species.

Cultured cells of tobacco (Nicotiana tabacum L. cv Petit Havana) were used to investigate signals regulating the expression of the "model" nuclear gene encoding the alternative oxidase (AOX) (AOX1), the terminal oxidase of the mitochondrial alternative respiratory pathway. Several conditions shown to induce AOX1 mRNA accumulation also result in an increase in cellular citrate concentrations, suggesting that citrate and/or other tricarboxylic acid (TCA) cycle intermediates may be important signal metabolites. In addition, mitochondrial reactive oxygen species (ROS) production has recently been shown to be a factor mediating mitochondria-to-nucleus signaling for the expression of AOX1. We found that the exogenously supplied TCA cycle organic acids citrate, malate and 2-oxoglutarate caused rapid and dramatic increases in the steady-state level of AOX1 mRNA at low, near physiological concentrations (0.1 mM). Furthermore, an increase in AOX1 induced by the addition of organic acids occurs independently of mitochondrial ROS formation. Our results demonstrate that two separate pathways for mitochondria-to-nucleus signaling of AOX1 may exist, one involving ROS and the other organic acids.

Salinity and water stress have contrasting effects on the relationship between growth and cell viability during and after stress exposure in durum wheat callus

Mature embryo-derived calli obtained from three cultivars (cvs.) (Omrabi: drought-resistant; Belikh: salt-resistant; Cando: drought- and salt-sensitive) were exposed for one month to various iso-osmotic concentrations of NaCl, mannitol or polyethylene glycol (PEG) 6000. The lowest relative growth rate (RGR) for all cultivars were observed in the presence of PEG. Correlation between RGR and cell viability estimated by reduction of 2,3,5-triphenytetrazolium chloride (TTC) was significant in the case of salt stress only. NaCl increased the relative importance of the non-phosphorylating alternative respiratory pathway while osmotic stress in contrast increased the relative importance of the CN-sensitive pathway. Neither mineral concentration, nor proline, nor soluble sugar accumulation fully accounted for lowering the internal osmotic potential in PEG-treated calli. Calli exposed to PEG were able to recover while those treated with mannitol were not. After NaCl treatment, only calli obtained from the salt-resistant cultivar Belikh recovered. It is concluded that (i) salinity and water stress have contrasting effects on the relationships between growth and cell viability, (ii) these relationships are not the same during and after the stress treatment, and (iii) maintenance of growth potential after the stress relief is linked to the maintenance of ATP-producing respiratory pathway rather than to a stress-induced stimulation of the alternative pathway.

Evidence for mitochondrial-derived alternative oxidase in the apicomplexan parasite Cryptosporidium parvum: a potential anti-microbial agent target

The observation that Plasmodium falciparum possesses cyanide insensitive respiration that can be inhibited by salicylhydroxamic acid (SHAM) and propyl gallate is consistent with the presence of an alternative oxidase (AOX). However, the completion and annotation of the P. falciparum genome project did not identify any protein with convincing similarity to the previously described AOXs from plants, fungi or protozoa. We undertook a survey of the available apicomplexan genome projects in an attempt to address this anomaly. Putative AOX sequences were identified and sequenced from both type 1 and 2 strains of Cryptosporidium parvum. The gene encodes a polypeptide of 336 amino acids and has a predicted N-terminal transit sequence similar to that found in proteins targeted to the mitochondria of other species. The potential of AOX as a target for new anti-microbial agents for C. parvum is evident by the ability of SHAM and 8-hydroxyquinoline to inhibit in vitro growth of C. parvum. In spite of the lack of a good candidate for AOX in either the P. falciparum or Toxoplasma gondii genome projects, SHAM and 8-hydroxyquinoline were found to inhibit the growth of these parasites. Phylogenetic analysis suggests that AOX and the related protein immutans are derived from gene transfers from the mitochondrial endosymbiont and the chloroplast endosymbiont, respectively. These data are consistent with the functional localisation studies conducted thus far, which demonstrate mitochondrial localisation for some AOX and chloroplastidic localization for immutans. The presence of a mitochondrial compartment is further supported by the prediction of a mitochondrial targeting sequence at the N-terminus of the protein and MitoTracker staining of a subcellular compartment in trophozoite and meront stages. These results give insight into the evolution of AOX and demonstrate the potential of targeting the alternative pathway of respiration in apicomplexans.

MeSA and MeJA increase steady-state transcript levels of alternative oxidase and resistance against chilling injury in sweet peppers ( Capsicum annuum L.)

Methyl salicylate (MeSA) and methyl jasmonate (MeJA) vapors increased resistance against chilling injury in freshly harvested green bell pepper ( Capsicum annuum L. cv Century). The period within 2 days of cold storage was considered the most critical because chilling injury symptom (surface pitting) was not apparent. The expression patterns of alternative oxidase (AOX) and seven other genes involved in defense against oxidative stress before and during the early chilling period suggested that pre-treatment of pepper fruit with MeSA or MeJA vapors increased preferentially the transcript levels of AOX. Overnight treatment with MeSA or MeJA vapors increased transcript levels of AOX (1.5 kb) even at room temperature of 25 °C, whereas no change was observed with untreated control. In addition to the expected 1.5 kb AOX transcript, RNA gel blot analysis revealed an extra 3.5 kb transcript that was induced only at 0 °C. At 0 °C, both AOX transcripts (1.5 and 3.5 kb) reached maximal levels firstly in MeSA treated fruit, secondly in MeJA treated fruit and lastly in controls. Compared with freshly harvested peppers, overnight treatment of wholesaler peppers with MeJA does not increase AOX transcript level and no differences in chilling injury symptom were observed between treated and control fruit. AOX transcript level in peppers from farm and wholesaler were maintained at a high level as long as the fruits were kept at 0 °C. Transcript levels of AOX (1.5 kb) were increased by storage at low temperature but the steady-state mRNA accumulation rate was faster at 0 than at 5 °C. Alternative respiratory pathway was proposed to mediate chilling injury. Here, we show that the increase in AOX transcript levels by MeJA or MeSA before cold treatment was correlated with reduced incidence of chilling injury.

Effects of oxidative stress on production of heterologous and native protein, and culture morphology in batch and chemostat cultures of Aspergillus niger (B1-D)

The effects of addition of menadione (MD) to induce elevated intracellular superoxide levels (superoxide stress) and of elevated oxygen availability (oxygen stress) was investigated in batch and chemostat cultures of a recombinant Aspergillus niger (B1-D). Culture responses to the two stressors were clearly distinct in terms of the changes in recombinant protein production, overall protein synthesis, and morphology. MD addition resulted in a significant increase in intracellular O 2 − levels, but gassing with O 2-enriched air (∼25%) led to decreased intracellular O 2 − levels. Under elevated O 2 availability, the activity of the alternative respiratory pathway was enhanced, reducing endogenous O 2 − generation, but the consequent decreased energy availability in such cultures is proposed to be the main reason leading to the decreased intracellular protein content and foreign protein secretion in batch cultures and chemostat cultures at low dilution rates ( D<0.1 h −1). The negative effects of O 2 enrichment on protein synthesis were less pronounced at high D due to the relative dominance of the main respiratory pathway at high growth rates. Under stressed cultivation conditions, the mean number of tips per clump correlated well with the secretion of recombinant protein. Morphological change with O 2 enrichment was not directly caused by the increased O 2 − generation as proposed in some previous studies, but possibly by the significantly altered culture metabolism.

Genetic modification of alternative respiration has differential effects on antimycin A-induced versus salicylic acid-induced resistance to Tobacco mosaic virus.

Salicylic acid (SA), a natural defensive signal chemical, and antimycin A, a cytochrome pathway inhibitor, induce resistance to Tobacco mosaic virus (TMV). Pharmacological evidence suggested signaling during resistance induction by both chemicals involved alternative oxidase (AOX), sole component of the alternative respiratory pathway (AP). Roles of the AP include regulation of intramitochondrial reactive oxygen species and maintenance of metabolic homeostasis. Transgenic tobacco (Nicotiana tabacum) with modified AP capacities (2- to 3-fold increased or decreased) showed no alteration in phenotype with respect to basal susceptibility to TMV or the ability to display SA-induced resistance to systemic viral disease. However, in directly inoculated tissue, antimycin A-induced TMV resistance was inhibited in plants with increased AP capacities, whereas SA and antimycin A-induced resistance was transiently enhanced in plant lines with decreased AP capacities. We conclude that SA-induced TMV resistance results from activation of multiple mechanisms, a subset of which are inducible by antimycin A and influenced by AOX. Other antiviral factors, potentially including the SA-inducible RNA-dependent RNA polymerase, are regulated by AOX-independent mechanisms.

The effect of modified atmospheres on the rate of quality change in ‘Hass’ avocado

Gas exchange rates and quality changes of avocados ( Persea americana , Mill., cv. ‘Hass’) stored at 7 °C in 32 different modified atmosphere (MA) conditions generated using a flow-through system, were monitored to characterise their functional relationship. An integrated modelling approach was used to link rates of quality loss to the rate of gas exchange. This revealed a close quantitative relationship between the gas exchange of ‘Hass’ avocado fruit on one hand and weight loss, colour change and softening on the other, indicating a direct metabolical link between these processes. High CO 2 lowered the respiratory quotient of avocado, an effect which, based on literature, could be explained by a shift from oxidative phosphorylation into the alternative respiratory pathway. In the model approach, the rates of quality degrading processes were linked to oxidative phosphorylation only, assuming this is where ATP was being produced. At high CO 2 levels at aerobic conditions, unaccounted weight loss data indicated a considerable volatile production which, however, was not qualitatively characterised. Both weight loss and colour change indicated the involvement of anaerobic processes at low O 2 levels. At 2 kPa O 2 and 0 kPa CO 2 no CO 2 injury occurred, while the change in hue was minimal, weight loss was minimised, and softening was almost completely inhibited. At such low O 2 levels, no additional benefit was found from raising the levels of CO 2 .

Microbial respiration and diffusive transport of O2, 16O2, and 18O15O in unsaturated soils and geologic sediments.

Molecular oxygen (O2) in unsaturated geologic sediments plays an important role in soil respiration, biodegradation of organic contaminants, metal oxidation, and global oxygen and carbon cycling, yet little is known about oxygen isotope fractionation during the consumption and transport of O2 in unsaturated zones. We used a laboratory kinetic cell technique to quantify isotope fractionation due to respiration and a numerical model to quantify both consumptive and diffusive fractionation of O2 isotopes at a field site comprised of unsaturated lacustrine sandy materials. The combined use of laboratory-based kinetic cell experiments and field-based isotope transport modeling provided an effective tool to characterize microbial respiration in unsaturated media. Based on results from the closed-system kinetic cells, O2 consumption and isotope fractionation were attributed to the alternative cyanide-resistant respiration pathway. At the field site, the modeled depth profiles for O2 and delta18O matched the measured in situ data and confirmed that the consumption of O2 was via the alternative respiration pathway. If the cyanide-resistant respiration pathway is indeed widespread in soils, its high oxygen isotope enrichment factor could help to explain the discrepancy between the predicted present-day Dole effect (+20.8/1000) and the observed Dole effect (+23.5/1000). Thus, further soil O2 isotope studies are needed to better characterize and model the fractionation of oxygen isotopes during subsurface respiration and the potential impact on the isotopic content of atmospheric O2.

Stimulation of the cyanide-resistant alternative respiratory pathway by oxygen in Acremonium chrysogenum correlates with the size of the intracellular peroxide pool.

The relationship between oxygen input and activity of the cyanide-resistant alternative respiration of submerged cultures of Acremonium crysogenum was investigated. The volumetric oxygen transfer coefficient of the respective cultures correlated positively within almost two ranges of magnitude with the size of the intracellular peroxide pool, which in turn, correlated with the activity of the cyanide-resistant alternative respiratory pathway. Increased aeration also stimulated the glucose uptake rate but had no effect on the total respiration rate or the growth rate. Addition of the lipid peroxyl radical scavenger DL-alpha-tocopherol to A. chrysogenum cultures decreased the rate of intracellular peroxide production as well as glucose uptake. An increase in the cyanide-resistant fraction of total respiration was observed, while growth and the total respiratory activity remained unchanged. We conclude that intracellular peroxides may stimulate the alternative respiration in A. chrysogenum.

Factors Influencing the Efficacy of Myclobutanil and Azoxystrobin for Control of Grape Black Rot.

We studied several factors influencing the efficacy of the demethylation inhibitor (DMI) fungicide myclobutanil and the strobilurin fungicide azoxystrobin for control of grape black rot, caused by the pathogen Guignardia bidwellii (anamorph Phyllosticta ampelicida). The distribution of sensitivities to myclobutanil among G. bidwellii isolates from an "organic" vineyard (no previous exposure to synthetic fungicides, n = 50) and from a commercial vineyard with a history of DMI applications (n = 60) was determined in vitro. There was little difference between the two populations, and the range of sensitivities was narrow; for the composite population of 110 isolates, the value of the mean effective dose for 50% inhibition (ED50) was 0.04 mg/liter, and the most- and least-sensitive isolates were separated by a factor of 16. When applied from 2 to 6 days after inoculating grape seedlings with a suspension containing either 2 × 104 or 1 × 106 conidia per ml, myclobutanil (60 mg/liter) provided complete control of lesion development. When applied beyond 6 days after inoculation but prior to lesion appearance (9 to 11 days after inoculation, depending on temperature), it provided complete control of pycnidium production in those lesions that developed subsequently. In contrast, when applied 2 to 10 days after inoculation with 2 × 104 conidia per ml, azoxystrobin (128 mg/liter) provided only 78 to 63% control of lesion formation and erratic control of pycnidium formation, although conidium production was reduced by 85 to 68% across this range of treatments. Relatively little control was provided by azoxystrobin treatments following inoculation with 1 × 106 conidia per ml. On leaf disks treated with azoxystrobin at 20 mg/liter prior to inoculation, 8 to 43% of conidia from five G. bidwellii isolates germinated, and 4 to 19% formed appressoria. However, these processes were completely to near-completely inhibited when salicylhydroxamic acid (SHAM), which inhibits an alternative respiration pathway utilized to circumvent the activity of strobilurin fungicides, was added to the inoculum at 100 mg/liter. Thus, alternative respiration apparently allowed the conidia to germinate and form appressoria on azoxystrobin-treated leaves. When grape seedlings were sprayed with commercially formulated azoxystrobin at 200 mg/liter and inoculated the next day with G. bidwellii conidia, little or no disease was evident 4 weeks later. However, G. bidwellii pycnidia formed on up to 50% of the leaves from such plants when they were killed with paraquat 1 to 7 days after inoculation. These results suggest that latent infections became established on azoxystrobin-treated leaves and became active after the plants were killed with paraquat.

Alternative Respiration Pathways and Secondary Metabolism in Plants with Different Adaptive Strategies under Mineral Deficiency

The costs of adaptation respiration, the contribution of different biochemical pathways of respiration and alternative oxidases to the adaptation to mineral deficiency were compared in plant species representing different types of adaptive strategies: ruderal (Ru) (Amaranthus retroflexus L. and Leonurus quinquelobatus Gilib.) and predominantly stress-tolerant type (St) (Dactylis glomerata L. and Medicago sativa L.). The adaptation component of the total dark respiration was calculated on the basis of the relative growth rate and the ratio of dark respiration to gross photosynthesis. Adaptation costs were greater in less tolerant species. The inhibitory analysis showed that the contribution of glycolytic pathway decreased and the proportion of oxidative pentose phosphate pathway and residual respiration increased in the intolerant Ru-species under the conditions of abiotic stress. Conversely, in the tolerant St-species, the contribution of glycolysis increased. In all species studied, the contribution of the cytochrome pathway of substrate oxidation was reduced and the contribution of the alternative cyanide-insensitive and residual respiration increased under conditions of mineral deficiency; this phenomenon was particularly pronounced in a less tolerant species L. quinquelobatus. In the species characterized by Ru-strategy, stress increased the content of phenolic compounds and shikimic acid. We suppose that the adaptation respiration costs are associated with alternative biochemical pathways and alternative cyanide-resistant oxidase. The role of these pathways in the plants with St-strategy consists in maintaining the active state of oxidative pathways, whereas in the plants with Ru-strategy, they serve to burn excess metabolites and enhance the synthesis of secondary metabolites, which perform protective functions.

Characterization of the mitochondrial respiratory pathways in Candida albicans

Candida albicans is an opportunistic oral pathogen. The flexibility of this microorganism in response to environmental changes includes the expression of a cyanide-resistant alternative respiratory pathway. In the present study, we characterized both conventional and alternative respiratory pathways and determined their ADP/O ratios, inhibitor sensitivity profiles and the impact of the utilization of either pathway on susceptibility to commonly used antimycotics. Oxygen consumption by isolated mitochondria using NADH or malate/pyruvate as respiratory substrates indicated that C. albicans cells express both cytoplasmic and matrix NADH–ubiquinone oxidoreductase activities. The ADP/O ratio was higher for malate/pyruvate (2.2±0.1), which generate NADH in the matrix, than for externally added NADH (1.4±0.2). In addition, malate/pyruvate respiration was rotenone-sensitive, and an enzyme activity assay further confirmed that C. albicans cells express Complex I activity. Cells grown in the presence of antimycin A expressed the cyanide-insensitive respiratory pathway. Determination of the respiratory control ratio (RCR) and ADP/O ratios of mitochondria from these cells indicated that electron transport from ubiquinone to oxygen via the alternative respiratory pathway was not coupled to ATP production; however, an ADP/O ratio of 0.8 was found for substrates that donate electrons at Complex I. Comparison of antifungal susceptibility of C. albicans cells respiring via the conventional or alternative respiratory pathways showed that respiration via the alternative pathway does not reduce the susceptibility of cells to a series of clinically employed antimycotics (using Fungitest®), or to the naturally occurring human salivary antifungal peptide, histatin 5.