Role of ethylene in the regulation of cell death and leaf loss in ozone-exposed European beech

To test the involvement of ethylene in mediating ozone-induced cell death and leaf loss in European beech ( Fagus sylvatica L.), tree seedlings were exposed to proportionally increased or decreased field ozone levels for up to 6 months. Ozone treatment caused cell death and accelerated leaf loss at higher than ambient levels, but had only minor effects at ambient and no effects at subambient ozone levels. The emission of ethylene, the levels of its precursor, 1-aminocyclopropane-1-carboxylate (ACC), and mRNA levels of specific ACC synthase ( FS-ACS2 ) and ACC oxidase ( FS-ACO1 ) isoforms showed a persistent increase and preceded cell death by approximately 2 weeks. Inhibition of ethylene biosynthesis led to reduced lesion formation whereas application of ACC accelerated ozone-induced cell death and leaf loss. Similar results were obtained when adult beech trees were exposed to 2 × ozone by a whole tree free-air canopy exposure system. The results suggest a role of ethylene in amplifying ozone effects under field conditions in this major European broad-leaved tree species.     

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     

Differential induction of tobacco MAP kinases by the defense signals nitric oxide, salicylic acid, ethylene, and jasmonic acid

In tobacco, two mitogen-activated protein (MAP) kinases, designated salicylic acid (SA)-induced protein kinase (SIPK) and wounding-induced protein kinase (WIPK) are activated in a disease resistance-specific manner following pathogen infection or elicitor treatment. To investigate whether nitric oxide (NO), SA, ethylene, or jasmonic acid (JA) are involved in this phenomenon, the ability of these defense signals to activate these kinases was assessed. Both NO and SA activated SIPK; however, they did not activate WIPK. Additional analyses with transgenic NahG tobacco revealed that SA is required for the NO-mediated induction of SIPK. Neither JA nor ethylene activated SIPK or WIPK. Thus, SIPK may function downstream of SA in the NO signaling pathway for defense responses, while the signals responsible for resistance-associated WIPK activation have yet to be determined.     

Ozone-induced ethylene production is dependent on salicylic acid,and both salicylic acid and ethylene act in concert to regulate ozone-induced cell death

Ethylene is known to influence plant defense responses including cell death in response to both biotic and abiotic stress factors. However, whether ethylene acts alone or in conjunction with other signaling pathways is not clearly understood. Ethylene overproducer mutants, eto1 and eto3, produced high levels of ethylene and developed necrotic lesions in response to an acute O3 exposure that does not induce lesions in O3-tolerant wild-type Col-0 plants. Treatment of plants with ethylene inhibitors completely blocked O3-induced ethylene production and partially attenuated O3-induced cell death. Analyses of the responses of molecular markers of specific signaling pathways indicated a relationship between salicylic acid (SA)- and ethylene-signaling pathways and O3 sensitivity. Both eto1 and eto3 plants constitutively accumulated threefold higher levels of total SA and exhibited a rapid increase in free SA and ethylene levels prior to lesion formation in response to O3 exposure. SA pre-treatments increased O3 sensitivity of Col-0, suggesting that constitutive high SA levels prime leaf tissue to exhibit increased magnitude of O3-induced cell death. NahG and npr1 plants compromised in SA signaling failed to produce ethylene in response to O3 and other stress factors suggesting that SA is required for stress-induced ethylene production. Furthermore, NahG expression in the dominant eto3 mutant attenuated ethylene-dependent PR4 expression and rescued the O3-induced HR (hypersensitive response) cell death phenotype exhibited by eto3 plants. Our results suggest that both SA and ethylene act in concert to influence cell death in O3-sensitive genotypes, and that O3-induced ethylene production is dependent on SA.     

Ozone treatment rapidly activates MAP kinase signalling in plants

Brief exposure to ozone, a potent cross-inducer of plant stress responses, leads within minutes to activation of an ERK-type MAP kinase (approximately 46 kDa) in tobacco. This activation process is calcium-dependent and can be blocked both by free radical quenchers and by a specific inhibitor of MEK-1 (MAPKK). Hydrogen peroxide and superoxide anion radicals can substitute for ozone as the activation stimulus, which does not appear to require salicylate as an intermediary. The properties of the ozone-induced MAPK suggest that it may be SIPK (salicylate-induced protein kinase), a tobacco MAPK that is activated by a variety of stress treatments. The ability of ozone to activate SIPK indicates that this protein kinase acts as a very early transducer of redox stress signals in plant cells.     

Involvement of protein kinases and calcium in the * NO-signalling cascade for defence-gene induction in ozonated tobacco plants

This study analyses the signalling pathways triggered by nitric oxide (NO) in response to ozone (O(3)) fumigation of tobacco plants, with particular attention to protein kinase cascades and free cytosolic Ca(2+) in defence-gene activation. NO was visualized with the NO probe DAF-FM. Using a pharmacological approach, the effects of different inhibitors on the expression profiles of NO-dependent defence genes were monitored using RT-PCR. The assay of the kinase activity of the immunoprecipitates complexes shows that O(3) stimulates a 48 kDa salicylic acid (SA)-induced protein kinase (SIPK) in an NO-dependent manner. The O(3)-induced alternative oxidase 1a (AOX1a) and phenylalanine ammonia lyase a (PALa) genes are modulated by phosphorylation by protein kinases, and SIPK might have a role in this up-regulation. By contrast, protein dephosphorylation mediates pathogenesis-related protein 1a (PR1a) expression in O(3)-treated tobacco plants. Ca(2+) is essential, but not sufficient, to promote NO accumulation in ozonated tobacco plants. Intracellular Ca(2+) transients are also essential for PALa up-regulation and cGMP-induced PR1a expression. Partial dependence on intracellular Ca(2+) suggests two different pathways of SA accumulation and PR1a induction. A model summarizing the signalling networks involving NO, SA, and the cellular messengers in this O(3)-induced defence gene activation is proposed.     

Double jeopardy: both overexpression and suppression of a redox-activated plant mitogen-activated protein kinase render tobacco plants ozone sensitive

In plants, the role of mitogen-activated protein kinase (MAPK) in reactive oxygen species (ROS)-based signal transduction processes is elusive. Despite the fact that ROS can induce MAPK activation, no direct genetic evidence has linked ROS-induced MAPK activation with the hypersensitive response, a form of programmed cell death. In tobacco, the major ROS-induced MAPK is salicylate-induced protein kinase (SIPK). We found through gain-of-function and loss-of-function approaches that both overexpression and RNA interference-based suppression of SIPK render the plant sensitive to ROS stress. Transgenic lines overexpressing a nonphosphorylatable version of SIPK were not ROS sensitive. Analysis of the MAPK activation profiles in ROS-stressed transgenic and wild-type plants revealed a striking interplay between SIPK and another MAPK (wound-induced protein kinase [WIPK]) in the different kinotypes. During continuous ozone exposure, abnormally prolonged activation of SIPK was seen in the SIPK-overexpression genotype, without WIPK activation, whereas strong and stable activation of WIPK was observed in the SIPK-suppressed lines. Thus, one role of activated SIPK in tobacco cells upon ROS stimulation appears to be control of the inactivation of WIPK.     

Atmospheric Ozone Interacts with Stress Ethylene Formation by Plants to Cause Visible Plant Injury

Ozone toxicity was studied in peas, beans, and tobacco (BelB and Bel W3). These experiments showed that ozone toxicitywas related to the rates of ethylene biosynthesis. Sensitivityto ozone was reduced if ethylene biosynthesis was inhibitedafter treatment with aminoethoxyvinylglycine (AVG). Similarly,plants that were able to reduce or prevent stress ethylene formationwere less sensitive after both short- and long-term exposureto ozone. Plants conditioned by longer exposures to ozone havelow rates of ethylene formation and this may be why brief ozoneexposures may be more phytotoxic than prolonged fumigations. Key words: Nicotiana tabacum, Phaseolus vulgaris, Pisum sativum, lipid peroxidation, peas, Pinto beans, tobacco     

Salicylic acid modulates ozone-induced hypersensitive cell death in tobacco plants

Ozone-tolerant Bel B and ozone-sensitive Bel W3 tobacco cultivars were subjected to acute ozone fumigation (200 p.p.b. for 3 h) and the subcellular localization of H₂O₂ was then studied. H₂O₂ accumulated on the cell walls and plasma membrane of both cultivars but the accumulation pattern differed greatly. H₂O₂ production was high in both cultivars immediately after fumigation, but, in the tolerant Bel B cultivar, after 7 h was only detected in some spongy cells adjacent to epidermal cells. Instead, in the sensitive Bel W3 cultivar, accumulation was still abundant in the cell walls of palisade, spongy and epidermal cells at this time. Significant changes in apoplastic ascorbate pool were noted in both cultivars in the first hours after fumigation. As the reduced ascorbate content remained unchanged, the marked increase in total ascorbate must have originated from the striking increase in dehydroascorbate, particularly in the ozone-sensitive Bel W3. Exposure of plants to ozone resulted in a marked transient increase in both free and conjugated salicylic acid (SA) as well as an increase in the activity of benzoic acid 2-hydroxylase which catalyses SA biosynthesis. SA induction differed greatly in the two cultivars, in that: (1) SA accumulation was far greater in the ozone-sensitive Bel W3 cv. and (2) the maximum SA peak was delayed in Bel W3 and observed only 7 h after fumigation ended. These results suggest that a high SA content, as documented in the ozone-sensitive Bel W3 cultivar, could trigger the production of ROS with subsequent SA-mediated cell-death.     

Involvement of abscisic acid in ozone-induced puerarin production of Pueraria thomsnii Benth. suspension cell cultures

Exposure to ozone induced a rapid increase in the levels of the sesquiterpene phytohormone abscisic acid (ABA) and the isoflavone puerarin in suspension cell cultures of Pueraria thomsnii Benth. The observed increases in ABA and puerarin were dependent on the concentration of ozone applied to P. thomsnii cell cultures. In order to examine the role of ABA in ozone-induced puerarin production, cell suspensions were pretreated with the ABA biosynthetic inhibitor fluridone. Following ozone exposure, fluridone treatment suppressed ABA accumulation suggesting ABA was normally synthesized de novo through the carotenoid pathway. Fluridone also blocked ozone-induced puerarin production, which could be reversed through application of exogenous ABA. However, in the absence of ozone, ABA itself had no effect on puerarin accumulation in the suspension cells. Taken together, the data indicate that ozone is an efficient elicitor of puerarin production and may be particularly applicable for improving puerarin production in plant cell cultures. Furthermore, we demonstrate that ABA is one factor associated with ozone-induced puerarin production in P. thomsnii cell cultures.     

Plant defence systems induced by ozone

Recent advances in the understanding of the molecular basis of plant response to ozone attack are reviewed. Plants grown in elevated atmospheric ozone are known to undergo several biochemical changes before any actual damage can be detected. These reactions include increases in the activities of enzymes associated with general plant defence mechanisms. Ozone exposure often causes a surge in the production of the plant hormone ethylene, as well as changes in polyamine metabolism and increases in the activities of several phenylpropanoid and flavonoid pathway enzymes. The activities of superoxide dismutase and peroxidases that protect cells from the oxidative damage caused by hydroxyl radicals, H₂O₂ and superoxides also increase. However, ozone-induced changes in plant cells at the gene level are almost unknown. The limited data available suggest close similarities between ozone-induced and pathogen-induced defence responses in plants. Several general defence genes that have been cloned in other studies will soon be applied to studies of gene expression in ozone-exposed plants. The use of molecular biological tools in ozone research should enable the development of highly specific and sensitive molecular markers for biomonitoring ozone-induced injuries in plants.     

Dissection of the ozone-induced calcium signature

The cellular responses of plants to numerous extracellular stimuli are mediated by transient elevations in the concentration of cytosolic free calcium ([Ca2+]cyt). We have addressed the question of how cells can use this apparently ubiquitous system to initiate so many specific and appropriate end responses. We show that the pollutant gas ozone elicits a biphasic Ca2+ response in intact Arabidopsis plants and a subsequent increase in expression of the gene encoding the antioxidant defence enzyme glutathione-S-transferase (GST). The second of the two [Ca2+]cyt peaks, but not the first, could be eliminated either by pre-treatment of plants with lanthanum chloride, or by reducing the duration of ozone fumigation. Under these conditions, ozone-induced GST expression was abolished. These data provide a functional dissection of the ozone Ca2+ signalling pathway and indicate that the second ozone-induced [Ca2+]cyt peak provides the necessary information to direct expression of GST.     

Oxidative burst and cell death in ozone-exposed plants

The phytotoxic air pollutant ozone spontaneously generates reactive oxygen species (ROS) in the leaf apoplast, provokes hypersensitive response-like lesions and induces defence reactions that significantly overlap with pathogen and other oxidative stress responses. Consequently, ozone has been used as a tool to unravel in planta ROS-induced plant defence and cell death mechanisms. Ozone exposure stimulates an oxidative burst in leaves of sensitive plants, resulting in the generation and accumulation of hydrogen peroxide or superoxide anions in distinct species. Accumulation of these ROS precedes the induction of cell death, and both responses co-occur spatially in the periveinal regions of the leaves. The review summarizes some of the recent results that have been obtained concerning the molecular basis of apoplastic ROS production in monocot and dicot species. Signal molecules, in particular ethylene and salicylic acid, control and potentiate the oxidative burst and subsequent cell death in its initiation and propagation phases while jasmonate leads to lesion containment. Amplification mechanisms that result in the production of excess ROS and hypersensitive cell death are discussed as major factors in ozone sensitivity of plant species and cultivars.     

MAP kinases function downstream of HSP90 and upstream of mitochondria in TMV resistance gene N-mediated hypersensitive cell death

Although the involvement of heat shock protein 90 (HSP90), mitogen-activated protein kinase (MAPK) cascades and organelle dysfunction in plant hypersensitive cell death has been suggested, the mutual relationship among them has not been elucidated. Here, we show the molecular network of HSP90, the wound-induced protein kinase (WIPK)/salicylic acid-induced protein kinase (SIPK)-mediated MAPK cascade and mitochondrial dysfunction in tobacco mosaic virus (TMV) resistance gene N-dependent cell death. p50, the Avr component for N, NtMEK2(DD), a constitutively active form of a MAPK kinase of WIPK/SIPK, and a mammalian pro-apoptotic factor Bax were used for cell death induction. Suppression of HSP90 and treatment with geldanamycin, a specific inhibitor of HSP90, compromised p50- but not NtMEK2(DD)- or Bax-mediated cell death accompanying the reduction of NtMEK2, WIPK and SIPK activation. In WIPK/SIPK-double knockdown plants, p50- and NtMEK2(DD)- but not Bax-mediated cell death was suppressed. All three types of cell death induced mitochondrial dysfunction, but they were similarly suppressed by Bcl-xL, which is a mammalian anti-apoptotic factor, and prevents mitochondrial dysfunction in plants as it does in animals in the cell death signal pathway. Taken together with the expression profile of hypersensitive reaction marker genes, it was indicated that the MAPK cascade functions downstream of HSP90 and transduces the cell death signal to mitochondria for N gene-dependent cell death. Furthermore, we found that WIPK and SIPK are functionally redundant in cell death signaling using WIPK/SIPK single or double knockdown plants.     

Phosphorylation of 1-aminocyclopropane-1-carboxylic acid synthase by MPK6, a stress-responsive mitogen-activated protein kinase, induces ethylene biosynthesis in Arabidopsis

Mitogen-activated protein kinases (MAPKs) are implicated in regulating plant growth, development, and response to the environment. However, the underlying mechanisms are unknown because of the lack of information about their substrates. Using a conditional gain-of-function transgenic system, we demonstrated that the activation of SIPK, a tobacco (Nicotiana tabacum) stress-responsive MAPK, induces the biosynthesis of ethylene. Here, we report that MPK6, the Arabidopsis thaliana ortholog of tobacco SIPK, is required for ethylene induction in this transgenic system. Furthermore, we found that selected isoforms of 1-aminocyclopropane-1-carboxylic acid synthase (ACS), the rate-limiting enzyme of ethylene biosynthesis, are substrates of MPK6. Phosphorylation of ACS2 and ACS6 by MPK6 leads to the accumulation of ACS protein and, thus, elevated levels of cellular ACS activity and ethylene production. Expression of ACS6(DDD), a gain-of-function ACS6 mutant that mimics the phosphorylated form of ACS6, confers constitutive ethylene production and ethylene-induced phenotypes. Increasing numbers of stress stimuli have been shown to activate Arabidopsis MPK6 or its orthologs in other plant species. The identification of the first plant MAPK substrate in this report reveals one mechanism by which MPK6/SIPK regulates plant stress responses. Equally important, this study uncovers a signaling pathway that modulates the biosynthesis of ethylene, an important plant hormone, in plants under stress.