Alternative Respiratory Pathway Research Articles

Nitrogen deprivation induces cross-tolerance of Poa annua callus to salt stress

Alternative respiration pathway (AP) is an important pathway which can be induced by environment stresses in plants. In the present study, we show a new mechanism involving the AP in nitrogen deprivation-induced tolerance of Poa annua callus to salt stress. The AP capacity markedly increased under a 600 mM NaCl treatment or nitrogen deprivation pretreatment and reached a maximum under the nitrogen deprivation pretreatment combined with the NaCl treatment (–N+NaCl). Malondialdehyde (MDA) and H2O2 content and Na+/K+ ratio significantly increased under the 600 mM NaCl treatment but less under the–N+NaCl treatment. Moreover, both the nitrogen deprivation and the NaCl stress stimulated the plasma membrane (PM) H+-ATPase activity and increased pyruvate content. The maximal stimulating effect was found under the–N+NaCl treatment. When the AP capacity was reduced by salicylhydroxamic acid (SHAM, an inhibitor of AP), content of MDA and H2O2 and Na+/K+ ratio dramatically increased, whereas PM H+-ATPase activity decreased. Moreover, exogenous application of pyruvate produced a similar effect as the nitrogen deprivation pretreatment. The effects of SHAM on the Poa annua callus were counteracted by catalase (a H2O2 scavenger) and diphenylene iodonium (a plasma membrane NADPH oxidase inhibitor). Taken together, our results suggest that the nitrogen deprivation enhanced the capacity of AP by increasing pyruvate content, which in turn prevented the Poa annua callus from salt-induced oxidative damages and Na+ over-uptake.

The influence of alternative pathways of respiration that utilize branched-chain amino acids following water shortage in Arabidopsis.

During dark-induced senescence isovaleryl-CoA dehydrogenase (IVDH) and D-2-hydroxyglutarate dehydrogenase (D-2HGDH) act as alternate electron donors to the ubiquinol pool via the electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF/ETFQO) pathway. However, the role of this pathway in response to other stresses still remains unclear. Here, we demonstrated that this alternative pathway is associated with tolerance to drought in Arabidopsis. In comparison with wild type (WT) and lines overexpressing D-2GHDH, loss-of-function etfqo-1, d2hgdh-2 and ivdh-1 mutants displayed compromised respiration rates and were more sensitive to drought. Our results demonstrated that an operational ETF/ETFQO pathway is associated with plants' ability to withstand drought and to recover growth once water becomes replete. Drought-induced metabolic reprogramming resulted in an increase in tricarboxylic acid (TCA) cycle intermediates and total amino acid levels, as well as decreases in protein, starch and nitrate contents. The enhanced levels of the branched-chain amino acids in loss-of-function mutants appear to be related to their increased utilization as substrates for the TCA cycle under water stress. Our results thus show that mitochondrial metabolism is highly active during drought stress responses and provide support for a role of alternative respiratory pathways within this response.

Massive gene loss in mistletoe (Viscum, Viscaceae) mitochondria.

Parasitism is a successful survival strategy across all kingdoms and has evolved repeatedly in angiosperms. Parasitic plants obtain nutrients from other plants and some are agricultural pests. Obligate parasites, which cannot complete their lifecycle without a host, may lack functional photosystems (holoparasites), or have retained photosynthesis (hemiparasites). Plastid genomes are often reduced in parasites, but complete mitochondrial genomes have not been sequenced and their mitochondrial respiratory capacities are largely unknown. The hemiparasitic European mistletoe (Viscum album), known from folklore and postulated therapeutic properties, is a pest in plantations and forestry. We compare the mitochondrial genomes of three Viscum species based on the complete mitochondrial genome of V. album, the first from a parasitic plant. We show that mitochondrial genes encoding proteins of all respiratory complexes are lacking or pseudogenized raising several questions relevant to all parasitic plants: Are any mitochondrial gene functions essential? Do any genes need to be located in the mitochondrial genome or can they all be transferred to the nucleus? Can parasitic plants survive without oxidative phosphorylation by using alternative respiratory pathways? More generally, our study is a step towards understanding how host- and self-perception, host integration and nucleic acid transfer has modified ancestral mitochondrial genomes.

Allelic variation on DcAOX1 gene in carrot (Daucus carota L.): An interesting simple sequence repeat in a highly variable intron

The alternative oxidase (AOX) is a key enzyme of the alternative respiration pathway and is involved in plant response to a variety of environmental stresses. The genes encoding for this enzyme are highly polymorphic and thus likely good candidates for molecular marker development to assist plant breeding on stress tolerance. In carrot, DcAOX1 gene has shown to be the most responsive to environmental changes. Therefore, we have studied its allelic variation in different carrot materials, including commercial cultivars, inbred lines, carrot subspecies and carrot crop wild relatives. This work is thus a first step to develop molecular markers based on AOX1 gene. Sequence comparison revealed the existence of high number of single nucleotide polymorphisms (SNPs), as well as several insertion/deletion events (InDels), especially in exon 1 and intron 1. Intron 1 showed to be the most polymorphic region and harbored an insertion event of 400bp, which had highly divergent sequences depending on the carrot genotype. The insertion was located in a region of single tandem repeats (SSR) that was also polymorphic between genotypes. This intronic insertion, together with its associated SSRs, arises as a good source of candidate functional markers (FMs) that, once validated, could greatly contribute to marker assisted selection of new carrot genotypes.

Ethylene is Involved in Brassinosteroids Induced Alternative Respiratory Pathway in Cucumber (Cucumis sativus L.) Seedlings Response to Abiotic Stress.

Effects of brassinosteroids (BRs) on cucumber (Cucumis sativus L.) abiotic stresses resistance to salt, polyethylene glycol (PEG), cold and the potential mechanisms were investigated in this work. Previous reports have indicated that BRs can induce ethylene production and enhance alternative oxidase (AOX) pathway. The mechanisms whether ethylene is involved as a signal molecule which connected BR with AOX in regulating stress tolerance are still unknown. Here, we found that pretreatment with 1 μM brassinolide (BL, the most active BRs) relieved stress-caused oxidative damage in cucumber seedlings and clearly enhanced the capacity of AOX and the ethylene biosynthesis. Furthermore, transcription level of ethylene signaling biosynthesis genes including ripening-related ACC synthase1 (CSACS1), ripening-related ACC synthase2 (CSACS2), ripening-related ACC synthase3 (CSACS3), 1-aminocyclopropane-1-carboxylate oxidase1 (CSACO1), 1-aminocyclopropane-1-carboxylate oxidase2 (CSACO2), and CSAOX were increased after BL treatment. Importantly, the application of the salicylhydroxamic acid (SHAM, AOX inhibitor) and ethylene biosynthesis inhibitor aminooxyacetic acid (AOA) decreased plant resistance to environmental stress by blocking BRs-induced alternative respiration. Taken together, our results demonstrated that ethylene was involved in BRs-induced AOX activity which played important roles in abiotic stresses tolerance in cucumber seedlings.

Nitric oxide is involved in brassinosteroid-induced alternative respiratory pathway in Nicotiana benthamiana seedlings' response to salt stress.

Recent studies reported that brassinosteroids (BRs) can induce plant tolerance to different environmental stresses via the nitric oxide (NO) signaling pathway. Previous reports have indicated that alternative oxidase (AOX) plays an important role in plants under various stresses. The mechanisms governing how NO is involved as a signal molecule which connects BR with AOX in regulating stress tolerance are still unknown. Recently, we found that Nicotiana benthamiana seedlings which were pretreated with BR have more tolerance to salt stress, accompanied with an increase of CN-resistant respiration. Our results suggested that pretreatment with 0.1 μM brassinolide (BL, the most active brassinosteroid) alleviated salt-induced oxidative damage and increased the NbAOX1 transcript level. Application of 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-1-oxyl-3-oxide (cPTIO, an NO scavenger) or virus-induced gene silencing of nitrate reductase (NR) and nitric oxide synthase (NOS)-like enzyme compromised the BRs-induced alternative respiratory pathway. Furthermore, pretreatment with specific chemical inhibitors of NR and NOS or gene silencing experiments decreased plant resistance to salt stress which also compromised BRs-induced salt stress tolerance. In conclusion, NO is involved in BRs-induced AOX capability which plays essential roles in salt tolerance in N. benthamiana seedlings.

Effect of 24-epibrassinolide on Arabidopsis thaliana alternative respiratory pathway under salt stress

Using wild-type (WT) Arabidopsis thaliana plants and two transgenic lines—overexpressing alternative oxidase AOX1a (AOX1a-OE) and antisense AOX1a (AOX1a-AS)—it was observed that brassinosteroids (BRs) are involved in regulation of respiration in A. thaliana leaf, particularly alternative respiratory pathway, under salt stress conditions. This study demonstrates that BRs stimulate total respiration rate, alternative respiratory pathway, and shift capacity of alternative respiratory pathway comparing to cyanide-sensitive pathway. Lowering the level of endogenous BRs with the specific inhibitor of BRs biosynthesis—brassinazole—caused the decrease of alternative respiratory pathway activity in WT plants and transgenic AOX1a-OE and AOX1a-AS lines.

Fermentation and alternative respiration compensate for NADH dehydrogenase deficiency in a prokaryotic model of DJ-1-associated Parkinsonism.

YajL is the closest prokaryotic homologue of Parkinson's disease-associated DJ-1, a protein of undefined function involved in the oxidative stress response. We reported recently that YajL and DJ-1 protect cells against oxidative stress-induced protein aggregation by acting as covalent chaperones for the thiol proteome, including the NuoG subunit of NADH dehydrogenase 1, and that NADH dehydrogenase 1 activity is negligible in the yajL mutant. We report here that this mutant compensates for low NADH dehydrogenase activity by utilizing NADH-independent alternative dehydrogenases, including pyruvate oxidase PoxB and d-amino acid dehydrogenase DadA, and mixed acid aerobic fermentations characterized by acetate, lactate, succinate and ethanol excretion. The yajL mutant has a low adenylate energy charge favouring glycolytic flux, and a high NADH/NAD ratio favouring fermentations over pyruvate dehydrogenase and the Krebs cycle. DNA array analysis showed upregulation of genes involved in glycolytic and pentose phosphate pathways and alternative respiratory pathways. Moreover, the yajL mutant preferentially catabolized pyruvate-forming amino acids over Krebs cycle-related amino acids, and thus the yajL mutant utilizes pyruvate-centred respiro-fermentative metabolism to compensate for the NADH dehydrogenase 1 defect and constitutes an interesting model for studying eukaryotic respiratory complex I deficiencies, especially those associated with Alzheimer's and Parkinson's diseases.

Inhibition of the alternative respiratory pathway at high temperatures leads to higher reactive oxygen species production and downregulation of the antioxidant defense system in squash leaves

In this work, the role of the alternative respiratory pathway in the heat tolerance of squashes was determined. Heat-tolerant C. moschata, heat-susceptible C. maxima and intermediate heat-tolerant ‘Maxchata’ plants were sprayed with an inhibitor of the alternative respiratory pathway, 2 mM salicylhydroxamic acid (SHAM), or water and were subjected to three different temperatures, i.e., 30 °C (control), 37 °C daytime temperature (moderate heat stress) or 42 °C daytime temperature (severe heat stress), for 7 days. The results indicated that total, cytochrome and alternative respirations were high in C. moschata and ‘Maxchata’ at both 37 and 42 °C, but decreased in C. maxima at 42 °C. Moreover, the increase in reactive oxygen species production upon heat exposure was also more apparent in SHAM-treated squashes compared with water-treated ones. The transcript levels of APX1, APX2,CAT1, CAT2, CAT3, Cu/ZnSOD, FeSOD and MnSOD were determined in both the SHAM- and water-treated squash leaves that were exposed to heat stress. It was observed that CAT1 and FeSOD were downregulated in SHAM-treated C. moschata plants under heat stress conditions. In addition, the expressions of CAT2 and MnSOD were lower in SHAM-treated C. moschata and ‘Maxchata’ plants. These results indicate some type of interrelationship between AOX and antioxidant systems. However, the complexity of the regulatory mechanisms and their contribution to the stress tolerance of squashes requires further research.

The alternative respiratory pathway is involved in brassinosteroid-induced environmental stress tolerance in Nicotiana benthamiana.

Brassinosteroids (BRs), plant steroid hormones, play essential roles in modulating cell elongation, vascular differentiation, senescence, and stress responses. However, the mechanisms by which BRs regulate plant mitochondria and resistance to abiotic stress remain largely unclear. Mitochondrial alternative oxidase (AOX) is involved in the plant response to a variety of environmental stresses. In this report, the role of AOX in BR-induced tolerance against cold, polyethylene glycol (PEG), and high-light stresses was investigated. Exogenous applied brassinolide (BL, the most active BR) induced, while brassinazole (BRZ, a BR biosynthesis inhibitor) reduced alternative respiration and AOX1 expression in Nicotiana benthamiana. Chemical scavenging of H2O2 and virus-induced gene silencing (VIGS) of NbRBOHB compromised the BR-induced alternative respiratory pathway, and this result was further confirmed by NbAOX1 promoter analysis. Furthermore, inhibition of AOX activity by chemical treatment or a VIGS-based approach decreased plant resistance to environmental stresses and compromised BR-induced stress tolerance. Taken together, our results indicate that BR-induced AOX capability might contribute to the avoidance of superfluous reactive oxygen species accumulation and the protection of photosystems under stress conditions in N. benthamiana.

An Arabidopsis mitochondria-localized RRL protein mediates abscisic acid signal transduction through mitochondrial retrograde regulation involving ABI4.

The molecular mechanisms of abscisic acid (ABA) signalling have been studied for many years; however, how mitochondria-localized proteins play roles in ABA signalling remains unclear. Here an Arabidopsis mitochondria-localized protein RRL (RETARDED ROOT GROWTH-LIKE) was shown to function in ABA signalling. A previous study had revealed that the Arabidopsis mitochondria-localized protein RRG (RETARDED ROOT GROWTH) is required for cell division in the root meristem. RRL shares 54% and 57% identity at the nucleotide and amino acid sequences, respectively, with RRG; nevertheless, RRL shows a different function in Arabidopsis. In this study, disruption of RRL decreased ABA sensitivity whereas overexpression of RRL increased ABA sensitivity during seed germination and seedling growth. High expression levels of RRL were found in germinating seeds and developing seedlings, as revealed by β-glucuronidase (GUS) staining of ProRRL-GUS transgenic lines. The analyses of the structure and function of mitochondria in the knockout rrl mutant showed that the disruption of RRL causes extensively internally vacuolated mitochondria and reduced ABA-stimulated reactive oxygen species (ROS) production. Previous studies have revealed that the expression of alternative oxidase (AOX) in the alternative respiratory pathway is increased by mitochondrial retrograde regulation to regain ROS levels when the mitochondrial electron transport chain is impaired. The APETALA2 (AP2)-type transcription factor ABI4 is a regulator of ALTERNATIVE OXIDASE1a (AOX1a) in mitochondrial retrograde signalling. This study showed that ABA-induced AOX1a and ABI4 expression was inhibited in the rrl mutant, suggesting that RRL is probably involved in ABI4-mediated mitochondrial retrograde signalling. Furthermore, the results revealed that ABI4 is a downstream regulatory factor in RRL-mediated ABA signalling in seed germination and seedling growth.

Overexpression of Mitochondrial Phosphate Transporter 3 Severely Hampers Plant Development through Regulating Mitochondrial Function in Arabidopsis.

Mitochondria are abundant and important organelles present in nearly all eukaryotic cells, which maintain metabolic communication with the cytosol through mitochondrial carriers. The mitochondrial membrane localized phosphate transporter (MPT) plays vital roles in diverse development and signaling processes, especially the ATP biosynthesis. Among the three MPT genes in Arabidopsis genome, AtMPT3 was proven to be a major member, and its overexpression gave rise to multiple developmental defects including curly leaves with deep color, dwarfed stature, and reduced fertility. Transcript profiles revealed that genes involved in plant metabolism, cellular redox homeostasis, alternative respiration pathway, and leaf and flower development were obviously altered in AtMPT3 overexpression (OEMPT3) plants. Moreover, OEMPT3 plants also accumulated higher ATP content, faster respiration rate and more reactive oxygen species (ROS) than wild type plants. Overall, our studies showed that AtMPT3 was indispensable for Arabidopsis normal growth and development, and provided new sights to investigate its possible regulation mechanisms.

Unraveling the functions of type II-prohibitins in Arabidopsis mitochondria.

In yeast and mammals, prohibitins (PHBs) are considered as structural proteins that form a scaffold-like structure for interacting with a set of proteins involved in various processes occurring in the mitochondria. The role of PHB in plant mitochondria is poorly understood. In the study, the model organism Arabidopsis thaliana was used to identify the possible roles of type-II PHBs (homologs of yeast Phb2p) in plant mitochondria. The obtained results suggest that the plant PHB complex participates in the assembly of multisubunit complexes; namely, respiratory complex I and enzymatic complexes carrying lipoic acid as a cofactor (pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase and glycine decarboxylase). PHBs physically interact with subunits of these complexes. Knockout of two Arabidopsis type-II prohibitins (AtPHB2 and AtPHB6) results in a decreased abundance of these complexes along with a reduction in mitochondrial acyl carrier proteins. Also, the absence of AtPHB2 and AtPHB6 influences the expression of the mitochondrial genome and leads to the activation of alternative respiratory pathways, namely alternative oxidase and external NADH-dependent alternative dehydrogenases.

Whole-plant respiration and its temperature sensitivity during progressive carbon starvation.

Plant respiration plays a critical role in the C balance of plants. Respiration is highly temperature sensitive and small temperature-induced increases in whole-plant respiration could change the C balance of plants that operate close to their light-compensation points from positive to negative. Nonstructural carbohydrates are thought to play an important role in controlling respiration and its temperature sensitivity, but this role has not been studied at the whole-plant level. We measured respiration of whole Ardisia crenata Sims. seedlings and tested the hypothesis that darkness-induced C starvation would decrease the temperature sensitivity of whole-plant respiration. Compared with control plants, sugar and starch concentrations in darkened plants declined over time in all organs. Similarly, whole-plant respiration decreased. However, the temperature sensitivity of whole-plant respiration, expressed as the proportional increase in respiration per 10°C warming (Q10), increased with progressive C starvation. We hypothesise that growth respiration was suppressed in darkened plants and that whole-plant respiration represented maintenance respiration almost exclusively, which is more temperature sensitive. Alternatively, changes in the respiratory substrate during C starvation or increased involvement of alternative oxidase pathway respiration may explain the increase in Q10. Carbohydrates are important for respiration but it appears that even in C-starved A. crenata plants, carbohydrate availability does not limit respiration during short-term warming.

Influence of brassinosteroids on plant cell alternative respiration pathway and antioxidant systems activity under abiotic stress conditions

Influence of brassinosteroids on plant cell alternative respiration pathway and antioxidant systems activity under abiotic stress conditions

Modulation of alternative oxidase to enhance tolerance against cold stress of chickpea by chemical treatments

The alternative oxidase (AOX) is the enzyme responsible for the alternative respiratory pathway. This experiment was conducted to examine the influence on cold tolerance ability of chickpea (Cicer aurentium cv. Müfitbey) seedlings of AOX activator (pyruvate), AOX inhibitor (salicylhydroxamic acid (SHAM)) and an inhibitor of the cytochrome pathway of respiration (antimycin A) treatments. 5mM pyruvate, 2μM antimycin A and 4mM SHAM solutions were exogenously applied to thirteen-day-old chickpea leaves and then the seedlings were transferred to a different plant growth chamber arranged to 10/5°C (day/night) for 48h. Cold stress markedly increased the activities of antioxidant enzymes compared to controls. Pyruvate and antimycin A significantly increased the cold-induced increase in antioxidant activity but SHAM decreased it. Cold-induced increases in superoxide anion, hydrogen peroxide, and lipid peroxidation levels were significantly reduced by pyruvate and antimycin A, but increased by SHAM treatment. Pyruvate and antimycin A application increased both the activity and protein expression of AOX in comparison to cold stress alone. However, SHAM significantly decreased activity of AOX but did not affect its expression. Total cellular respiration values (TCRV) supported the changes in activity and expression of AOX. While TCRV were increased by cold and pyruvate, they were significantly reduced by SHAM and especially antimycin A. These results indicate that pyruvate and antimycin A applications were effective in reducing oxidative stress by activating the alternative respiratory pathway as well as antioxidant activity. Furthermore, direct activation of AOX, rather than inhibition of the cytochrome pathway, was the most effective way to mitigate cold stress.

Decreasing electron flux through the cytochrome and/or alternative respiratory pathways triggers common and distinct cellular responses dependent on growth conditions.

Diverse signaling pathways are activated by perturbation of mitochondrial function under different growth conditions.Mitochondria have emerged as an important organelle for sensing and coping with stress in addition to being the sites of important metabolic pathways. Here, responses to moderate light and drought stress were examined in different Arabidopsis (Arabidopsis thaliana) mutant plants lacking a functional alternative oxidase (alternative oxidase1a [aox1a]), those with reduced cytochrome electron transport chain capacity (T3/T7 bacteriophage-type RNA polymerase, mitochondrial, and plastidial [rpoTmp]), and double mutants impaired in both pathways (aox1a:rpoTmp). Under conditions considered optimal for growth, transcriptomes of aox1a and rpoTmp were distinct. Under adverse growth conditions, however, transcriptome changes in aox1a and rpoTmp displayed a highly significant overlap and were indicative of a common mitochondrial stress response and down-regulation of photosynthesis. This suggests that the role of mitochondria to support photosynthesis is provided through either the alternative pathway or the cytochrome pathway, and when either pathway is inhibited, such as under environmental stress, a common, dramatic, and succinct mitochondrial signal is activated to alter energy metabolism in both organelles. aox1a:rpoTmp double mutants grown under optimal conditions showed dramatic reductions in biomass production compared with aox1a and rpoTmp and a transcriptome that was distinct from aox1a or rpoTmp. Transcript data indicating activation of mitochondrial biogenesis in aox1a:rpoTmp were supported by a proteomic analysis of over 200 proteins. Under optimal conditions, aox1a:rpoTmp plants seemed to switch on many of the typical mitochondrial stress regulators. Under adverse conditions, aox1a:rpoTmp turned off these responses and displayed a biotic stress response. Taken together, these results highlight the diverse signaling pathways activated by the perturbation of mitochondrial function under different growth conditions.

Light regulation of mitochondrial alternative oxidase pathway during greening of etiolated wheat seedlings

This study deals with effects of de-etiolation (48h) of spring wheat (Triticum aestivum L., var. Irgina) seedlings on differential expression of AOX1 genes, levels of AOX protein and the alternative respiratory pathway (AP) capacity. As a result of exposure to continuous irradiation of dark-grown wheat seedlings, the respiratory activity and AP capacity in leaves significantly increased during the first 6h of studies. Expression of AOX1a was up-regulated by light and proved consistent with changes in the AP capacity. Effects on expression of AOX1c were less pronounced. Immunoblot analysis showed three distinct bands of AOX with molecular weights of 34, 36 and 38kDa, with no significant changes in the relative levels during de-etiolation. The lack of a clear correlation between AOX protein amount, AOX1a expression, and AP capacity suggests post-translational control of the enzyme activation. The AOX1a suppression and a decrease in the AP capacity correlated with the sugar pool depletion after 24h of the de-etiolation, which may mean a possible substrate dependence of the AOX activity in the green cells. More efficient malate oxidation by mitochondria as well as the higher AOX capacity during the first 6h of de-etiolation was detected, whereas respiration and AOX capacity with exogenous NADH and glycine increased after 6 and 24h, respectively. We conclude that AOX plays an important role during development of an actively photosynthesizing cell, and can rapidly adapt to changes in metabolism and photosynthesis.

Respiratory supercomplexes of plant mitochondria: Structure and possible functions

Over the last few years a vast amount of information has accumulated on the structural organization of mitochondrial electron-transport chain. The respiratory complexes were shown to be organized into sophisticated dynamic structures, so-called supercomplexes and megacomplexes that ensure sustained and effective operation of the respiratory electron-transport chain. This review considers structural and functional features of plant supercomplexes, as well as their possible role in energy-related adaptation of plants to stresses. It is proposed that supercomplexes provide the first-line defense mechanism that protects the electron-transport chain against reactive oxygen species in stressed plants. Subsequent protective responses involve the alternative respiratory pathways, uncoupling mechanisms, and other antioxidant systems.

Effects of modifying alternative respiration on nitric oxide-induced virus resistance and PR1 protein accumulation.

Nitric oxide (NO) is an important defensive signal in plants but its effects on virus infection are not well understood. Administration of NO-releasing compounds immediately before inoculation of tobacco leaves with potato virus X and tobacco mosaic virus decreased the accumulation of virus, indicating that NO can induce resistance rapidly. Resistance induction was inhibited by co-administration with an NO-scavenging compound or when experiments were done in transgenic tobacco plants expressing increased alternative respiratory pathway capacity due to constitutive expression of the plant mitochondrial enzyme, alternative oxidase (AOX). These results indicate that NO, which inhibits electron transport chain activity, is triggering defensive signalling by inducing changes in mitochondrial reactive oxygen species levels that are in turn regulated by AOX. Experiments using nahG-transgenic plants, which cannot accumulate the defensive plant hormone salicylic acid (SA) showed that NO rapidly induces resistance to virus infection independently of SA. However, this initial state of resistance may be transient. Subsequently, by 5 days post-treatment, NO had caused an increase in pathogenesis-related protein 1 (PR1) expression (a proxy for increased SA biosynthesis), which correlated with a longer-term state of resistance to virus infection. The induction by NO of PR1 accumulation was modified in AOX-transgenic plants. This indicates that the influence of NO on defensive gene expression is in part mediated through its effects on mitochondria.