Cyanide-resistant Respiratory Pathway Research Articles

The 2′,4′-Dichloro-chalcone Inhibits the In Vitro Growth and Pathogenicity of Fusarium tricinctum and Trichothecium roseum by Activating Cyanide-Resistant Respiration

Chalcones are a class of flavonoids possessing antimicrobial properties and have potential for use as coatings of plant products for the control of postharvest diseases. The effects of 2′,4′-dichloro-chalcone on the in vitro growth and in vivo pathogenicity of Fusarium tricinctum and Trichothecium roseum were investigated. First, 1 µM of 2′,4′-dichloro-chalcone strongly inhibited the mycelial growth and conidial production of F. tricinctum (32.3%) and T. roseum (65.2%) in vitro. Meanwhile, the cell membrane permeability was increased by 25% and 22.5% and the accumulation of reactive oxygen species was increased by 41.7 and 65.4%, respectively, of F. tricinctum and T. roseum. This treatment also significantly inhibited the total respiration rate and activated the cyanide-resistant respiratory pathway in both pathogens. The expression level of AOX was enhanced in F. tricinctum and T. roseum by 52.76 and 39.13%, respectively. This treatment also significantly inhibited the expansion of potato dry rot from F. tricinctum (48.6%) and apple rot spot from T. roseum (36.2%). Therefore, 2′,4′-dichloro-chalcone has potential use as an alternative safety method in the control of postharvest diseases by F. tricinctum and T. roseum in agricultural practices.

Enhanced cyanide-resistant respiration as the predominant respiratory metabolic pathway in abnormal chilling injury behavior of postharvest papaya

An increasing number of studies have demonstrated that reactive oxygen species (ROS) play a crucial role in the chilling injury (CI) of postharvest fruit and vegetables. This is not only associated with the free radical scavenging system but is also intimately connected to the electron transport chain pathway. However, it remains unclear whether ROS is associated with the electron transport chain in the abnormal chilling behavior observed in postharvest papaya. As such, this study investigated the electron transport chain of papaya fruit stored at 1 ℃ and 6 ℃ and its connection to ROS. In comparison to storage at 6 ℃, storage at 1 ℃ suppressed the expression of CpCCO, and induced the upregulation of CpAOX expression in papaya fruit. Throughout the middle and late stages of storage, there was an increase in pyruvate content and ubiquinone reduction level. The increase had two effects: it inhibited cytochrome oxidase (CCO) activity and promoted alternative oxidase (AOX) activity, leading to a blocked cytochrome pathway and an increased proportion of cyanide-resistant respiratory pathways. Consequently, the cyanide-resistant respiratory pathway supplanted the cytochrome pathway as the primary electron transport pathway. This transition effectively curbed the accumulation of ROS, thereby diminishing the damage degree of fruit cell membrane, boosting chilling tolerance, and ultimately inhibiting the CI of papaya fruit, which led to the emergence of abnormal chilling injury.

Postharvest nitric oxide treatment induced the alternative oxidase pathway to enhance antioxidant capacity and chilling tolerance in peach fruit

Nitric oxide (NO) is an important regulator of plant response to cold stress. In this study, NO treatment delayed the development of chilling injury (CI), inhibited the increase in H2O2 content, O2− production rate and decrease in firmness of postharvest peach fruit. Meanwhile, through RNA-seq analysis, NO treatment up-regulated gene expression of PpG-6-PDH, Pp6-PGDH and PpAOX while it down-regulated the expression of PpGPI and PpHK, suggesting that the pentose phosphate respiratory pathway and cyanide-resistant respiratory pathway were promoted and the glycolysis pathway was inhibited. Furthermore, the PpAOX expression was consistent with the trend of PpPOD1/2 expression and H2O2 content, indicating that AOX may play a role in reducing oxidative damage of peach fruit by scavenging H2O2. Thus, it was concluded that NO treatment could induce the cyanide-resistant respiration pathway to enhance antioxidant ability and chilling tolerance in post-harvest peach fruit.

The role of cyanide-resistant respiration in Solanum tuberosum L. against high light stress.

Cyanide-resistant respiration in potato mitochondria is an important pathway for energy dissipation. It can be activated by high light; however, it is unclear what roles cyanide-resistant respiration plays in the response to high light stress in potato. We designed a CRISPR vector for the functional gene StAOX of the potato cyanide-resistant respiratory pathway. Agrobacterium tumefaciens GV3101 was transformed into potato. Hydrogen peroxide level, MDA content, antioxidant activity and cyanide-resistant respiratory capacity of potato leaves under high light stress were determined. Photosynthetic efficiency and chlorophyll content were determined. In addition, the operation of the malate-oxaloacetate shuttle route and transcription level of photorespiration-related enzymes were also examined. The results showed that two base substitutions occurred at the sequencing target site on leaves of the transformed potato. Accumulation of ROS and increased membrane lipid peroxidation were detected in the transformed potato leaves and lower photosynthetic efficiency was observed. The transcription level of the malate-oxaloacetate shuttle route and photorespiration-related enzymes also significantly increased. These results indicate that the cyanide-resistant respiration is an important physiological pathway in potato in response to high light stress. It also suggests that plant cyanide-resistant respiration is closely related to photosynthesis. This implies the unexplored importance of plant cyanide-resistant respiration in plant photosynthesis, energy conversion and carbon skeleton formation.

Cyanide-resistant respiratory pathway is involved in the high-light systemic acquired acclimation of kidney bean (Phaseolus vulgaris)

After exposing one half of a low light-adapted kidney bean (Phaseolus vulgaris) leaf to high light, parameters of chlorophyll (Chl) a fluorescence, such as PSII operating efficiency, PSII maximum efficiency under light, and photochemical quenching, decreased in the opposite half of the same leaf, whereas the capacity of the cyanide-resistant respiratory pathway significantly increased. When one half of the low light-adapted leaf was exposed to low light, the opposite half pretreated with 1 mM salicylhydroxamic acid (SHAM, an inhibitor of the cyanide-resistant respiratory pathway) did not exhibit significant changes in the Chl fluorescence values compared with the without SHAM pretreatment. However, after exposing one half of the low light-adapted leaf to high light, the opposite half pretreated with 1 mM SHAM showed lower Chl fluorescence values than that without SHAM pretreatment. Our results indicate that partial exposure of the low light-adapted leaf to high light can impose a systemic stress on the PSII photochemistry. The enhanced capacity of the cyanide-resistant respiratory pathway may be involved in the maintenance of the photosynthetic performance in the leaf tissues experiencing high light-induced systemic stress.

A GmAOX2b antisense gene compromises vegetative growth and seed production in soybean

The alternative oxidase mediates the cyanide-resistant respiratory pathway in plant mitochondria. In non-thermogenic plants, the role of alternative oxidase in plant growth and development is not well understood. Soybean (Glycine max) lines carrying a GmAOX2b antisense gene had compromised vegetative growth and reproductive performance under typical glasshouse growth conditions. The reduction in vegetative growth was demonstrated by reduction in shoot height, the number of leaves per plant and the green leaf area. Antisense plants also had decreased pod formation and seed to pod ratios, which together led to a reduction in the number and total mass of seed produced. The negative effects of the antisense gene on pod set, seed set, ovule availability and total seed mass were primarily confined to the branches, rather than the main stem. The preferential effect of alternative oxidase suppression in the branches is discussed in relation to the reproductive potential of soybean under stress. Taken together, these results demonstrate that alternative oxidase provides the benefit of sustaining plant vegetative growth and reproductive capacity in soybean.

Alternative oxidase, a determinant of plant gametophyte fitness and fecundity

The alternative oxidase (AOX) is the terminal oxidase that comprises the cyanide-resistant respiratory pathway in plant mitochondria. While the role of AOX in plant thermogenesis is well established, its role in the reproductive development of non-thermogenic species is not well understood. AOX genes can be separated into two groups based on sequence homology, AOX1 and AOX2. Reverse genetic experiments carried out primarily in Arabidopsis and tobacco have largely focussed on examining the role of AOX1-type genes in stress responses. We recently reported a systematic characterisation of the reproductive phenotypes of three AOX2 antisense lines of soybean. This addendum summarises the key evidence in our recent paper that points to a role for AOX in the development and function of both male and female gametophytes. Furthermore, we discuss the relative importance of AOX in the reproductive biology of plant species examined to date and highlight practical implications of our findings to crop improvement research.

D-Erythroascorbic acid activates cyanide-resistant respiration in Candida albicans

Higher plants, protists and fungi possess cyanide-resistant respiratory pathway, which is mediated by alternative oxidase (AOX). The activity of AOX has been found to be dependent on several regulatory mechanisms including gene expression and posttranslational regulation. In the present study, we report that the presence of cyanide in culture medium remarkably retarded the growth of alo1/alo1 mutant of Candida albicans, which lacks d-arabinono-1,4-lactone oxidase (ALO) that catalyzes the final step of d-erythroascorbic acid (EASC) biosynthesis. Measurement of respiratory activity and Western blot analysis revealed that increase in the intracellular EASC level induces the expression of AOX in C. albicans. AOX could still be induced by antimycin A, a respiratory inhibitor, in the absence of EASC, suggesting that several factors may act in parallel pathways to induce the expression of AOX. Taken together, our results suggest that EASC plays important roles in activation of cyanide-resistant respiration in C. albicans.

Characterization of the gene family encoding alternative oxidase from Candida albicans

Candida albicans possesses a cyanide-resistant respiratory pathway mediated by alternative oxidase (AOX), which seems to be encoded by a gene family with two members. Cloning and expression of AOX1a, one of the genes encoding alternative oxidase from C. albicans, has previously been reported [Huh and Kang (1999) J. Bacteriol. 181, 4098-4102]. In the present study we report the isolation of another gene coding for alternative oxidase, designated AOX1b. AOX1b contains a continuous open reading frame that encodes a polypeptide consisting of 365 amino acids. Interestingly, AOX1a and AOX1b were found to be located in tandem on one of the chromosomes of C. albicans. The presence of cyanide in the culture medium remarkably retarded the growth of the aox1a/aox1a mutants. The growth of the aox1b/aox1b mutants and the aox1a/aox1a aox1b/aox1b double mutants was almost completely inhibited in the same medium. beta-Galactosidase reporter assays indicated that, whereas AOX1a was expressed constitutively, the expression of AOX1b was dependent on growth phase and was induced by treatment with cyanide, antimycin A, H(2)O(2), menadione and paraquat. Growth of the cells in media with non-fermentable carbon sources also enhanced the expression of AOX1b. CaSLN1, which encodes a histidine kinase, seems to be involved in the regulation of AOX expression in C. albicans on the basis of the observation that the activity of cyanide-resistant respiration and the expression level of AOX in the casln1/casln1 mutants were found to be significantly low under normal conditions and slightly increased in the presence of respiratory inhibitors compared with the wild-type strain. Like AOX1a, AOX1b could also be functionally expressed in AOX-deficient Saccharomyces cerevisiae and confer cyanide-resistant respiration on the organism.

Characterization of the gene family encoding alternative oxidase from Candida albicans

Candida albicans possesses a cyanide-resistant respiratory pathway mediated by alternative oxidase (AOX), which seems to be encoded by a gene family with two members. Cloning and expression of AOX1a, one of the genes encoding alternative oxidase from C. albicans, has previously been reported [Huh and Kang (1999) J. Bacteriol. 181, 4098–4102]. In the present study we report the isolation of another gene coding for alternative oxidase, designated AOX1b. AOX1b contains a continuous open reading frame that encodes a polypeptide consisting of 365 amino acids. Interestingly, AOX1a and AOX1b were found to be located in tandem on one of the chromosomes of C. albicans. The presence of cyanide in the culture medium remarkably retarded the growth of the aox1a/aox1a mutants. The growth of the aox1b/aox1b mutants and the aox1a/aox1a aox1b/aox1b double mutants was almost completely inhibited in the same medium. β-Galactosidase reporter assays indicated that, whereas AOX1a was expressed constitutively, the expression of AOX1b was dependent on growth phase and was induced by treatment with cyanide, antimycin A, H2O2, menadione and paraquat. Growth of the cells in media with non-fermentable carbon sources also enhanced the expression of AOX1b. CaSLN1, which encodes a histidine kinase, seems to be involved in the regulation of AOX expression in C. albicans on the basis of the observation that the activity of cyanide-resistant respiration and the expression level of AOX in the casln1/casln1 mutants were found to be significantly low under normal conditions and slightly increased in the presence of respiratory inhibitors compared with the wild-type strain. Like AOX1a, AOX1b could also be functionally expressed in AOX-deficient Saccharomyces cerevisiae and confer cyanide-resistant respiration on the organism.

Cyanide-resistant alternative respiration is strictly correlated to intracellular peroxide levels in Acremonium Chrysogenum

A strict correlation between the intensity of the cyanide-resistant alternative respiratory pathway and the intracellular peroxide levels in the cephalosporin C producer filamentous fungus Acremonium chrysogenum was demonstrated. Intracellular peroxide levels increased in a dose-dependent manner after addition of H2O2 to the culture media. A similar phenomenon was observed due to the specific inhibition of catalase by salicylic acid. In both cases, cyanide-resistant respiration was markedly stimulated. On the other hand, both cyanide-resistant respiration and intracellular peroxide levels were effectively suppressed by the lipid peroxyl radical scavenger DL-α-tocopherol, which breaks lipid peroxidation chains effectively. Our findings firmly supported the assumption that there is a connection between the intracellular peroxide levels and the intensity of the alternative respiratory pathway in fungi.

Activities of the cyanide-resistant respiratory pathway in leaves of sun and shade species

Higher plant mitochondria have a cyanide-resistant alternative respiratory pathway of electron transport (AP) that is not coupled to proton translocation. To characterise the ecophysiology of this apparently ‘wasteful’ pathway, we constructed a system consisting of a gas-phase oxygen electrode and an air sampling line for measurement of stable oxygen isotope ratios. With this system, we were able to measure respiratory rates of a small amount of leaf segments of ca 0.6 g fresh weight, and collect about 100 L of the air from the oxygen electrode chamber several times. The 18 O/16 O ratio in the air samples was measured by mass spectrometry. The activity of AP was estimated based on the isotopic discrimination of 18 O. We used the leaves of Alocasia odora (Lodd.) Spach., a shade species, and Spinacia oleracea L. and Phaseolus vulgaris L., sun species. These plants were grown at two levels of photosynthetically active photon flux density (PPFD). Three main findings were: (1) in the leaves of A. odora, the contribution of AP was less than 10% of the total respiratory rate, irrespective of growth PPFD; (2) for the sun species grown at high PPFD, the contribution of AP in the leaves was about 40% early in the night, but decreased dramatically late in the night; and (3) when S. oleracea was grown at low PPFD, the contribution of AP in the leaves declined. The low activity of AP in the leaves of A. odora suggests that the efficiency of adenosine triphosphate (ATP) production (ATP/O 2 ) of this species is high. This may be especially important in shaded environments where input of light energy is low. We also suggest that, in the leaves of sun species, ATP/O 2 changes depending on the conditions.

Effects of light on respiration and oxygen isotope fractionation in soybean cotyledons

ABSTRACTLight effects on electron flow through the cyanide‐resistant respiratory pathway, oxygen isotope fractionation and total respiration were studied in soybean (Glycine max L.) cotyledons. During the first 12 h of illumination there was an increase in both electron partitioning through the alternative pathway and oxygen isotope fractionation by the alternative oxidase. The latter probably indicates a change in the properties of the alternative oxidase. There was no engagement of the alternative oxidase in darkness and its fractionation was 27‰. In green cotyledons 60% of the respiration flux was through the alternative pathway and the alternative oxidase fractionation was 32‰. Exposing previously illuminated tissue to continuous darkness induced a decrease in the electron partitioning through the alternative pathway. However, this decrease was not directly linked with the low cellular sugar concentration resulting from the lack of light because 5 min of light every 12 h was sufficient to keep the alternative pathway engaged to the same extent as plants grown under control conditions.

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.

Defense Mechanism of Rice Plant to Respiratory Inhibition by a Promising Candidate Blasticide, SSF126

A new candidate systemic rice blasticide, SSF126, dose-dependently inhibited NADH oxidation by submitochondrial particles from rice roots. However, oxidation by the root submitochondrial particles was much less susceptible to SSF126 compared to that by submitochondrial particles from mycelial cells ofPyricularia grisea, a pathogen causing rice blast. Interestingly, SSF126 did not completely suppress the respiration by intact rice roots, and the respiratory activity of the roots recovered from inhibition time-dependently even in the presence of SSF126 at concentrations sufficient to fully block oxidation by the submitochondrial particles. This recovery was not due to selective extrusion of SSF126 from the roots, but to switching from the cytochrome pathway to the alternative cyanide-resistant respiratory pathway. In immunoblots of the alternative oxidase, high molecular mass species were detected in the mitochondria from rice roots in addition to low molecular mass species. Quantification of high and low molecular mass species revealed an increase in the amount of a protein corresponding to a 36-kDa species equivalent to a decrease in the amount of a protein corresponding to a 72-kDa species following a 5-h incubation with SSF126. This conversion of the alternative oxidase to the low molecular mass species in the mitochondria was correlated with the respiratory recovery found in intact rice roots, suggesting that the low molecular mass species is the active form of the alternative oxidase and the high molecular mass species is the inactive form. These results suggest that rice plants can block the severe injury caused by limiting the cytochrome pathway by SSF126 through utilization of the alternative pathway promoted by the interconversion of the alternative oxidase protein.

Mutants of the Phytopathogenic FungusMagnaporthe griseaDeficient in Alternative, Cyanide-Resistant, Respiration

The phytopathogenic fungusMagnaporthe griseahas a cyanide-resistant respiratory pathway. The fungicide SSF-126 ((E)-2-methoxyimino-N-methyl-2-(2-phenoxyphenyl) acetamide) blocks the cytochrome electron transport ofM. griseaand induces the alternative respiratory pathway. Twelve mutants ofM. griseamore susceptible to SSF-126 than wild type were identified afterN-methyl-N′-nitro-N-nitrosoguanidine mutagenesis. Five mutants retained a reduced alternative respiration activity, and seven mutants lacked alternative pathway activity. A monoclonal antibody against the maize alternative oxidase cross-reacted against a 40-kDa mitochondrial protein ofM. grisea,indicating that the 40-kDa protein is an alternative oxidase. Immunoblot analysis indicated that the seven completely deficient mutants grouped into two classes: four mutants produced the 40-kDa proteins while the other three mutants failed to produce the functional protein.

The reaction of the plant mitochondrial cyanide-resistant alternative oxidase with oxygen

The dependence of electron flux through the cyanide-resistant respiratory pathway on the redox poise of the ubiquinone pool and oxygen concentration was studied in purified mitochondria isolated from green and etiolated soybean ( Glycine max L. Merr. cv. Ransom) cotyledons at different ages (4 and 10 days after planting), soybean roots and mung bean ( Vigna radiata L.R. Vilcz) hypocotyls. In soybean, the K m of the alternative oxidase with respect to oxygen was found to vary between values of 10 and 20 μM. These are generally higher than values of the K m for oxygen of the alternative oxidase reported previously (0.5 to 2.0 μM). In addition, the value of the K m for oxygen varied with the redox poise of the ubiquinone pool, measured voltametrically; the more reduced the quinone pool, the larger the observed K m. These results are at variance with the behavior expected of the kinetic model developed by Siedow and Moore (1993; Biochim. Biophys. Acta 1142, 165–174) which predicts that the K m for oxygen should decrease as the quinone pool becomes more reduced. A modified kinetic model is developed that incorporates an additional reaction step involving activation of the four-electron reduced oxidase into the earlier kinetic model. The modified model successfully simulates the dependence of the alternative oxidase activity on both ubiquinone pool redox poise and oxygen concentration.

Characterization of the alternative oxidase protein in the yeast Hansenula anomala

The cyanide-resistant respiratory pathway is induced by respiratory inhibitors in the yeast Hansenula anomala. A monoclonal antibody against the alternative oxidase in the higher plant Sauromatum guttatum cross-reacted with a 36-kDa mitochondrial protein induced by antimycin A in H. anomala and with a protein encoded by a cDNA which was previously cloned for an antimycin A-inducible mRNA in the yeast. There was a similarity in the amino acid sequence between the cDNA-encoded protein and the plant alternative oxidase protein. We propose that the 36-kDa mitochondrial protein encoded by the cDNA is a component of alternative oxidase in H. anomala.

Possible involvement of superoxide anion in the induction of cyanide-resistant respiration in Hansenula anomala

A chemiluminescence study showed that Q i site inhibitors such as antimycin A induce O 2 − generation in respiring cyanide-sensitive mitochondria from the yeast, Hansenula anomala. The O 2 − generation was suppressed by radical scavengers such as flavone, butylated hydroxyanisole, and Co 0. Induction of cyanide-resistant respiration in H. anomala cells by Q i site inhibitors was also inhibited by these radical scavengers. Furthermore, antimycin A-induced synthesis of the mitochondrial 36-kDa protein, which is thought to be the alternative oxidase functional in the cyanide-resistant respiratory pathway, was abolished by the addition of flavone. These observations suggest that O 2 − is somehow involved in the induction of cyanide-resistant respiration.

Molecular cloning of cDNA for antimycin A-inducible mRNA and its role in cyanide-resistant respiration in Hansenula anomala

A cDNA for mRNA induced by antimycin A in Hansenula anomala was cloned. The mRNA for the cDNA was expressed in the yeast under the conditions expressing the cyanide-resistant respiration activity. The nucleotide sequence revealed a long open reading frame of 342 codons encoding a protein with a molecular weight of 40,282 in the cDNA. An antibody recognizing the protein encoded by the open reading frame was produced. Immunoblotting of H. anomala proteins with this antibody showed that a 36 kDa protein localized in mitochondria was a mature form of the protein encoded by the cDNA. It is suggested that the cloned cDNA encodes a protein involved in the cyanide-resistant respiratory pathway.