The role of phytohormone signaling in ozone-induced cell death in plants

Ozone is the main photochemical oxidant that causes leaf damage in many plant species, and can thereby significantly decrease the productivity of crops and forests. When ozone is incorporated into plants, it produces reactive oxygen species (ROS), such as superoxide radicals and hydrogen peroxide. These ROS induce the synthesis of several plant hormones, such as ethylene, salicylic acid, and jasmonic acid. These phytohormones are required for plant growth, development, and defense responses, and regulate the extent of leaf injury in ozone-fumigated plants. Recently, responses to ozone have been studied using genetically modified plants and mutants with altered hormone levels or signaling pathways. These researches have clarified the roles of phytohormones and the complexity of their signaling pathways. The present paper reviews the biosynthesis of the phytohormones ethylene, salicylic acid, and jasmonic acid, their roles in plant responses to ozone, and multiple interactions between these phytohormones in ozone-exposed plants.Key words: cross-talk, ethylene, jasmonic acid, ozone, phytohormones, programmed cell death, salicylic acid, signaling pathways     

Analysis of cytosolic isocitrate dehydrogenase and glutathione reductase 1 in photoperiod‐influenced responses to ozone using Arabidopsis knockout mutants

Oxidative stress caused by ozone (O₃) affects plant development, but the roles of specific redox‐homeostatic enzymes in O₃ responses are still unclear. While growth day length may affect oxidative stress outcomes, the potential influence of day length context on equal‐time exposures to O₃ is not known. In Arabidopsis Col‐0, day length affected the outcome of O₃ exposure. In short‐days (SD), few lesions were elicited by treatments that caused extensive lesions in long days (LD). Lesion formation was not associated with significant perturbation of glutathione, ascorbate, NADP(H) or NAD(H). To investigate roles of two genes potentially underpinning this redox stability, O₃ responses of mutants for cytosolic NADP‐isocitrate dehydrogenase (icdh) and glutathione reductase 1 (gr1) were analysed. Loss of ICDH function did not affect O₃‐induced lesions, but slightly increased glutathione oxidation, induction of other cytosolic NADPH‐producing enzymes and pathogenesis‐related gene 1 (PR1). In gr1, O₃‐triggered lesions, salicylic acid accumulation, and induction of PR1 were all decreased relative to Col‐0 despite enhanced accumulation of glutathione. Thus, even at identical irradiance and equal‐time exposures, day length strongly influences phenotypes triggered by oxidants of atmospheric origin, while in addition to its antioxidant function, the GR‐glutathione system seems to play novel signalling roles during O₃ exposure.     

Leaf stage-associated resistance is correlated with phytohormones in a pathosystem-dependen manner

It has been reported in several pathosystems that disease resistance can vary in leaves at different stages. However, how general this leaf stage-associated resistance is, and the molecular mechanism(s) underlying it, remain largely unknown. Here, we investigated the effect of leaf stage on basal resistance, effectortriggered immunity(ETI) and nonhost resistance, using eight pathosystems involving the hosts Arabidopsis thaliana, Nicotiana tabacum, and N. benthamiana and the pathogens Sclerotinia sclerotiorum, Pseudomonas syringae pv. tabaci, P. syringae pv. tomato DC3000, and Xanthomonas oryzae pv. oryzae(Xoo). We show evidence that leaf stage-associated resistance exists ubiquitously in plants, but with varying intensity at different stages in diverse pathosystems. Microarray expression profiling assays demonstrated that hundreds of genes involved in defense responses, phytohormone biosynthesis and signaling, and calcium signaling, were differentially expressed between leaves at different stages. The Arabidopsis mutants sid1, sid2-3, ein2, jar1-1, aba1 and aao3 lost leaf stage-associated resistance to S. sclerotiorum, and the mutants aba1 and sid2-3 were affected in leaf stage-associated RPS2/Avr Rpt2t-conferred ETI, whereas only the mutant sid2-3 influenced leaf stage-associated nonhost resistance to Xoo. Our results reveal that the phytohormones salicylic acid,ethylene, jasmonic acid and abscisic acid likely play an essential, but pathosystem-dependent, role in leaf stageassociated resistance.     

Artificial elevation of glutathione contents in salicylic acid‐deficient tobacco (Nicotiana tabacum cv. Xanthi NahG) reduces susceptibility to the powdery mildew pathogen Euoidium longipes

• The effects of elevated glutathione levels on defence responses to powdery mildew (Euoidium longipes) were investigated in a salicylic acid‐deficient tobacco (Nicotiana tabacum cv. Xanthi NahG) and wild‐type cv. Xanthi plants, where salicylic acid (SA) contents are normal.
• Aqueous solutions of reduced glutathione (GSH) and its synthetic precursor R‐2‐oxothiazolidine‐4‐carboxylic acid (OTC) were injected into leaves of tobacco plants 3 h before powdery mildew inoculation.
• SA‐deficient NahG tobacco was hyper‐susceptible to E. longipes, as judged by significantly more severe powdery mildew symptoms and enhanced pathogen accumulation. Strikingly, elevation of GSH levels in SA‐deficient NahG tobacco restored susceptibility to E. longipes to the extent seen in wild‐type plants (i.e. enhanced basal resistance). However, expression of the SA‐mediated pathogenesis‐related gene (NtPR‐1a) did not increase significantly in GSH or OTC‐pretreated and powdery mildew‐inoculated NahG tobacco, suggesting that the induction of this PR gene may not be directly involved in the defence responses induced by GSH.
• Our results demonstrate that artificial elevation of glutathione content can significantly reduce susceptibility to powdery mildew in SA‐deficient tobacco.

     

Increased intracellular H2O2 availability preferentially drives glutathione accumulation in vacuoles and chloroplasts

One biochemical response to increased H₂O₂ availability is the accumulation of glutathione disulphide (GSSG), the disulphide form of the key redox buffer glutathione. It remains unclear how this potentially important oxidative stress response impacts on the different sub‐cellular glutathione pools. We addressed this question by using two independent in situ glutathione labelling techniques in Arabidopsis wild type (Col‐0) and the GSSG‐accumulating cat2 mutant. A comparison of in situ labelling with monochlorobimane (MCB) and in vitro labelling with monobromobimane (MBB) revealed that, whereas in situ labelling of Col‐0 leaf glutathione was complete within 2 h incubation, about 50% of leaf glutathione remained inaccessible to MCB in cat2. High‐performance liquid chromatography (HPLC) and enzymatic assays showed that this correlated tightly with the glutathione redox state, pointing to significant in vivo pools of GSSG in cat2 that were unavailable for MCB labelling. Immunogold labelling of leaf sections to estimate sub‐cellular glutathione distribution showed that the accumulated GSSG in cat2 was associated with only a minor increase in cytosolic glutathione but with a 3‐ and 10‐fold increase in plastid and vacuolar pools, respectively. The data are used to estimate compartment‐specific glutathione concentrations under optimal and oxidative stress conditions, and the implications for redox homeostasis and signalling are discussed.     

Cytosolic NADP‐dependent isocitrate dehydrogenase contributes to redox homeostasis and the regulation of pathogen responses in Arabidopsis leaves

Cytosolic NADP‐dependent isocitrate dehydrogenase (cICDH) produces 2‐oxoglutarate (2‐OG) and NADPH, and is encoded by a single gene in Arabidopsis thaliana. Three allelic lines carrying T‐DNA insertions in this gene showed less than 10% extractable leaf ICDH activity, but only relatively small decreases in growth compared to wild‐type Col0. Metabolite profiling by gas chromatography–time of flight–mass spectrometry (GC–TOF–MS) and high‐performance liquid chromatography (HPLC) revealed that loss of cICDH function produced only small effects on leaf compounds involved in carbon and nitrogen assimilation. To analyse whether cICDH contributes to NADPH production under conditions of oxidative stress, the icdh mutation was introduced into the cat2 background, in which increased availability of H₂O₂ causes perturbed redox homeostasis and induction of stress‐related genes. Accumulation of oxidized glutathione and pathogen‐related responses were enhanced in double cat2 icdh mutants compared to cat2. Single icdh mutants presented constitutive induction of PR genes, and enhanced resistance to bacteria in icdh, cat2 and cat2 icdh was quantitatively correlated with PR gene expression. However, the effect of icdh in both Col0 and cat2 backgrounds was not associated with enhanced accumulation of salicylic acid (SA). The results suggest that cICDH, previously considered mainly as an enzyme involved in amino acid synthesis, plays a role in redox signalling linked to pathogen responses.     

Enhanced oxidative stress in the ethylene-insensitive (ein3-1) mutant of Arabidopsis thaliana exposed to salt stress

To better understand the role of ethylene signaling in plant stress tolerance, salt-induced changes in gene expression levels of ethylene biosynthesis, perception and signaling genes were measured in Arabidopsis thaliana plants exposed to 15 days of salinity. Among the genes analyzed, EIN3 showed the highest expression level increase under salt stress, suggesting a key role for this ethylene-signaling component in response to salt stress. Therefore, we analyzed the salt stress response over 15 days (by adding 100 mM NaCl to the nutrient solution) in the ein3-1 mutant compared to the wild-type (Col-0) in terms of growth, oxidative stress markers (lipid peroxidation, foliar pigments and low-molecular-weight antioxidants) and levels of growth- and stress-related phytohormones (including cytokinins, auxins, gibberellins, abscisic acid, jasmonic acid and salicylic acid). The ein3-1 mutant grew similarly to wild-type plants both under control and salt stress conditions, which was associated with a differential time course evolution in the levels of the cytokinins zeatin and zeatin riboside, and the auxin indole-3-acetic acid between the ein3-1 mutant and the wild-type. Despite showing no signs of physiological deterioration under salt stress (in terms of rosette biomass, leaf water and pigment contents, and PSII efficiency) the ein3-1 mutant showed enhanced lipid peroxidation under salt stress, as indicated by 2.4-fold increase in both malondialdehyde and jasmonic acid contents compared to the wild-type. We conclude that, at moderate doses of salinity, partial insensitivity to ethylene might be compensated by changes in endogenous levels of other phytohormones and lipid peroxidation-derived signals in the ein3-1 mutant exposed to salt stress, but at the same time, this mutant shows higher oxidative stress under salinity than the wild-type.     

Role of ethylene signalling in growth and systemic resistance induction by the plant growth‐promoting fungus Penicillium viridicatum in Arabidopsis

The plant growth‐promoting fungi (PGPF) have long been known to improve plant growth and suppress plant diseases. The PGPF Penicillium viridicatum GP15‐1 elicited plant growth and induced systemic resistance (ISR) in Arabidopsis thaliana against Pseudomonas syringae pv. tomato DC3000 (Pst), leading to a restriction of pathogen growth and disease development. Examination of local and systemic genes indicated that GP15‐1 did not modulate the expression of any of the tested defence‐related marker genes involved in salicylic acid (SA), jasmonic acid (JA) and ethylene signalling pathways. Subsequent challenge of GP15‐1‐colonized plants with Pst bacterium primed Arabidopsis plants for enhanced activation of the JA‐inducible Atvsp (vegetative storage protein) gene at a later stage of infection. To assess the contribution of different signalling pathways in GP15‐1‐elicited plant growth and ISR, Arabidopsis genotypes implicated in SA signalling expressing the nahG transgene (NahG) or carrying disruption in NPR1 (npr1), JA signalling (jar1) and ethylene signalling (ein2) were tested. The GP15‐1‐induced plant growth and ISR were fully compromised in an ein2 mutation. Root colonization assay revealed that the inability of the ein2 mutant to express GP15‐1‐induced plant growth and ISR was not associated with reduced root colonization by GP15‐1. In conclusion, our results demonstrate the ethylene signalling pathway is involved in plant growth promotion and ISR elicitation by the PGPF P. viridicatum GP15‐1 in Arabidopsis. These results provide evidence that ethylene signalling has a substantial role in plant growth and disease resistance.     

Involvement of ethylene in reversal of salt‐inhibited photosynthesis by sulfur in mustard

Sulfur (S) assimilation results in the synthesis of cysteine (Cys), a common metabolite for the formation of both reduced glutathione (GSH) and ethylene. Thus, ethylene may have regulatory interaction with GSH in the alleviation of salt stress. The involvement of ethylene in the alleviation of salt stress by S application was studied in mustard (Brassica juncea cv. Pusa Jai Kisan). First, the effects of 0, 0.5, 1.0 and 2.0 mM SO₄^2? were studied on photosynthetic and growth parameters to ascertain the S requirement as sufficient‐S and excess‐S for the plant. In further experiments, the effects of sufficient‐S (1 mM SO₄^2?) and excess‐S (2 mM SO₄^2?) were studied on the alleviation of salt stress‐induced by 100 mM NaCl, and ethylene involvement in the alleviation of salt stress by S. Under non‐saline condition, excess‐S increased ethylene with less content of Cys and GSH and adversely affected photosynthesis and growth. In contrast, excess‐S maximally alleviated salt stress due to high demand for S and optimal ethylene formation, which maximally increased GSH and promoted photosynthesis and growth. The involvement of ethylene in S‐mediated alleviation of salt stress was further substantiated by the reversal of the effects of excess‐S on photosynthesis by aminoethoxyvinylglycine (AVG), ethylene biosynthesis inhibitor. The studies suggest that plants respond differentially to the S availability under non‐saline and salt stress and excess‐S was more potential in the alleviation of salt stress. Further, ethylene regulates plants' response and excess S‐induced alleviation of salt stress and promotion of photosynthesis.     

Azospirillum brasilense ameliorates the response of Arabidopsis thaliana to drought mainly via enhancement of ABA levels

Production of phytohormones is one of the main mechanisms to explain the beneficial effects of plant growth‐promoting rhizobacteria (PGPR) such as Azospirillum sp. The PGPRs induce plant growth and development, and reduce stress susceptibility. However, little is known regarding the stress‐related phytohormone abscisic acid (ABA) produced by bacteria. We investigated the effects of Azospirillum brasilense Sp 245 strain on Arabidopsis thaliana Col‐0 and aba2‐1 mutant plants, evaluating the morphophysiological and biochemical responses when watered and in drought. We used an in vitro‐grown system to study changes in the root volume and architecture after inoculation with Azospirillum in Arabidopsis wild‐type Col‐0 and on the mutant aba2‐1, during early growth. To examine Arabidopsis development and reproductive success as affected by the bacteria, ABA and drought, a pot experiment using Arabidopsis Col‐0 plants was also carried out. Azospirillum brasilense augmented plant biomass, altered root architecture by increasing lateral roots number, stimulated photosynthetic and photoprotective pigments and retarded water loss in correlation with incremented ABA levels. As well, inoculation improved plants seed yield, plants survival, proline levels and relative leaf water content; it also decreased stomatal conductance, malondialdehyde and relative soil water content in plants submitted to drought. Arabidopsis inoculation with A. brasilense improved plants performance, especially in drought.     

Regulation of epinasty induced by 2,4‐dichlorophenoxyacetic acid in pea and Arabidopsis plants

The herbicide 2,4‐dichlorophenoxyacetic acid (2,4‐D) causes uncontrolled cell division and malformed growth in plants, giving rise to leaf epinasty and stem curvature. In this study, mechanisms involved in the regulation of leaf epinasty induced by 2,4‐D were studied using different chemicals involved in reactive oxygen species (ROS) accumulation (diphenyleniodonium, butylated hydroxyanisole, EDTA, allopurinol), calcium channels (LaCl₃), protein phosphorylation (cantharidin, wortmannin) and ethylene emission/perception (aminoethoxyvinyl glycine, AgNO₃). The effect of these compounds on the epinasty induced by 2,4‐D was analysed in shoots and leaf strips from pea plants. For further insight into the effect of 2,4‐D, studies were also made in Arabidopsis mutants deficient in ROS production (rbohD, rbohF, xdh), ethylene (ein 3‐1, ctr 1‐1, etr 1‐1), abscisic acid (aba 3.1), and jasmonic acid (coi 1.1, jar 1.1, opr 3) pathways. The results suggest that ROS production, mainly ·OH, is essential in the development of epinasty triggered by 2,4‐D. Epinasty was also found to be regulated by Ca²⁺, protein phosphorylation and ethylene, although all these factors act downstream of ROS production. The use of Arabidopsis mutants appears to indicate that abscisic and jasmonic acid are not involved in regulating epinasty, although they could be involved in other symptoms induced by 2,4‐D.     

Role of ethylene in alleviation of cadmium-induced photosynthetic capacity inhibition by sulphur in mustard

Sulphur (S) assimilation leads to the formation of glutathione (GSH) and alleviation of cadmium (Cd) stress. GSH is synthesized from its immediate metabolite cysteine, which also serves as a metabolite for ethylene formation through S‐adenosyl methionine. To assess the role of ethylene in S‐induced alleviation of Cd stress on photosynthesis, the effects of S or ethephon (ethylene source) on GSH and ethylene were examined in mustard (Brassica juncea L. cv. Varuna). Sufficient‐S at 100 mg S kg^?1 soil alleviated Cd‐induced photosynthetic inhibition more than excess‐S (200 mg S kg^?1 soil) via ethylene by increased GSH. Under Cd stress, plants were less sensitive to ethylene, despite high ethylene evolution, and showed photosynthetic inhibition. Ethylene sensitivity of plants increased with ethephon or sufficient‐S, triggering the induction of an antioxidant system, and leading to increased photosynthesis even under Cd stress. The effects of ethephon and S under Cd stress were similar. The effects of S were reversed by ethylene biosynthesis inhibitor, aminoethoxyvinylglycine (AVG), suggesting that ethylene plays an important role in S‐induced alleviation of Cd stress on photosynthesis.     

Parasitism by Cuscuta pentagona sequentially induces JA and SA defence pathways in tomato

While plant responses to herbivores and pathogens are well characterized, responses to attack by other plants remain largely unexplored. We measured phytohormones and C₁₈ fatty acids in tomato attacked by the parasitic plant Cuscuta pentagona, and used transgenic and mutant plants to explore the roles of the defence‐related phytohormones salicylic acid (SA) and jasmonic acid (JA). Parasite attachment to 10‐day‐old tomato plants elicited few biochemical changes, but a second attachment 10 d later elicited a 60‐fold increase in JA, a 30‐fold increase in SA and a hypersensitive‐like response (HLR). Host age also influenced the response: neither Cuscuta seedlings nor established vines elicited a HLR in 10‐day‐old hosts, but both did in 20‐day‐old hosts. Parasites grew larger on hosts deficient in SA (NahG) or insensitive to JA [jasmonic acid‐insensitive1 (jai1) ], suggesting that both phytohormones mediate effective defences. Moreover, amounts of JA peaked 12 h before SA, indicating that defences may be coordinated via sequential induction of these hormones. Parasitism also induced increases in free linolenic and linoleic acids and abscisic acid. These findings provide the first documentation of plant hormonal signalling induced by a parasitic plant and show that tomato responses to C. pentagona display characteristics similar to both herbivore‐ and pathogen‐induced responses.     

Differential ozone sensitivity among <Emphasis Type="Italic">Arabidopsis</Emphasis> accessions and its relevance to ethylene synthesis

We compared the physiological and molecular responses of two Arabidopsis accessions, Col-0 and Ws-2, to ozone (O(3)) exposure. Observation of visible injury as well as ion-leakage analysis demonstrated clear differences between the O(3)-tolerant accession Col and the O(3)-sensitive accession Ws. RNA-blot analysis showed that O(3)-induced increases in mRNA levels of several ethylene-inducible genes and a salicylic acid-inducible gene were substantially higher in Ws than in Col. The time-course of induction of various mRNA levels shows that the expression of ethylene-inducible genes was rapidly, and more strongly, induced by O(3) in Ws than in Col, suggesting that Ws exhibits higher ethylene-signaling. Both the level of mRNA for an O(3)-inducible 1-aminocyclopropane-1-carboxylate synthase and the level of ethylene generation after 3 h of O(3)-exposure were higher in Ws than in Col. O(3)-induced leaf damage was attenuated by pretreatment with ethylene biosynthesis- and signaling-inhibitors, indicating that ethylene signaling is required for O(3)-induced leaf injury in Ws. On the other hand, an ethylene-overproducing mutant of Col, eto1-1, displayed significantly increased O(3)-induced leaf injury compared to wild type plants. These results indicate that the difference in O(3) sensitivity is dependent on the difference in ethylene production rate between these two accessions. Finally, we investigated the relationship between the degree of leaf damage and the level of ethylene evolution in 20 different Arabidopsis accessions. Based on the result, the accessions were classified into four types. However, most of them showed significant correlation between the ethylene production level and the degree of leaf injury, suggesting that ethylene signaling is an important factor in the natural variety of O(3) sensitivity among Arabidopsis accessions.