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First published online August 23, 2002; 10.1105/tpc.002337 American Society of Plant Biologists Double JeopardyBoth Overexpression and Suppression of a Redox-Activated Plant Mitogen-Activated Protein Kinase Render Tobacco Plants Ozone SensitiveBiotechnology Laboratory, University of British Columbia, Vancouver, British Columbia, V6T 1Z3 Canada 1 To whom correspondence should be addressed. E-mail bee{at}interchange.ubc.ca; fax 604-822-2114
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.
Mitogen-activated protein kinase (MAPK) modules form a key part of the eukaryotic signal transduction network that links environmental inputs to a wide range of modifications of cellular functions, ranging from cell division to cell death. In plants, MAPK signaling has been implicated in defense against pathogens and herbivores, in cellular responses to auxin, abscisic acid, and other phytohormones, in cell cycle control, in the induction of programmed cell death, and in responses to abiotic stresses such as UV light and ozone (Zhang and Klessig, 1997  ; Kovtun et al., 1998; Romeis et al., 1999; Heimovaara-Dijkstra et al., 2000; Samuel et al., 2000; Nishihama et al., 2001; Yang et al., 2001; Miles et al., 2002).
A variety of stress responses have been found to involve the rapid activation of a specific subset of plant MAPKs, notably Arabidopsis MPK6 (Ichimura et al., 2000
Exposure to ozone immediately creates an oxidizing environment in plant tissues and triggers an array of cellular responses, including the accumulation of antioxidants, elicitation of pathogenesis-related proteins, deposition of phenols, induction of ethylene synthesis, suppression of primary metabolic activities such as photosynthesis, and eventually cell death (Darrall, 1989
One of the earliest responses elicited by ozone and other ROS generators in plants is the activation of specific MAPKs (Samuel et al., 2000
The rapid activation of these MAPKs suggests that their action on downstream targets could be important for the modulation of the cellular response to increased oxidative damage, but direct evidence for that role is lacking in plants. No intracellular substrates have been identified for either SIPK or WIPK, nor have loss-of-function genotypes been assessed for their ability to control redox stress. Stable overexpression or suppression of SIPK or WIPK in transgenic tobacco apparently did not result in the alteration of its activity (Yang et al., 2001
The previously reported inability to produce SIPK-suppressed lines, and the lack of phenotype or alteration of SIPK activity reported for overexpression lines (Yang et al., 2001
Infiltration of fully grown tobacco leaves with a suspension of Agrobacterium tumefaciens cells carrying a SIPK-FLAG overexpression construct resulted in the accumulation of the epitope-tagged SIPK protein in the infiltrated tissue within 48 h. In unstressed cells, endogenous SIPK was not phosphory-lated at the TXY motif found in the activation loop of the kinase, as indicated by the absence of any signal in the control lane of a protein gel blot (Figure 1C) prepared using an anti-pMAPK antibody that specifically recognized the doubly phosphorylated protein. In the infiltrated tissue, however, at least a portion of the pool of SIPK became activated by 48 h after infiltration, with even greater activation observed by 72 h. In the same period, the infiltrated zones showed signs of tissue collapse, and by 96 h, these zones became completely necrotic (Figure 1A).
When leaves were coinfiltrated with Agrobacterium carrying the SIPK-FLAG overexpression construct plus an RNAi construct that targeted SIPK, both expression and activation of SIPK-FLAG were suppressed completely (Figures 1B and 1C). The cell death induced by the overexpression of SIPK-FLAG in the infiltrated zones also was eliminated (Figure 1A). The cell death associated with the spontaneous activation of SIPK in overexpression (OX) transgenic cells suggested that it might be difficult to recover stably transformed lines using this construct, but cocultivation of tobacco leaf discs with the appropriate Agrobacterium culture and selection on kanamycin yielded a number of transgenic lines that were found to ectopically express a range of levels of SIPK-FLAG (Figure 2A). No spontaneous activation of SIPK was detected in these lines, all of which displayed normal growth and development phenotypes.
Transformation of tobacco leaf discs with the SIPK-RI construct also yielded stable transgenic lines, although with a sharply reduced frequency. In the recovered RI lines, silencing of endogenous SIPK expression was observed to varying degrees, ranging from partial reduction in both SIPK mRNA and protein to elimination of both products (Figures 3B and 3C). The specificity of this silencing was shown by the continued expression in most of the recovered RI lines of the closely related NTF4 MAPK gene, whose cDNA sequence is 89% identical to that of SIPK (Figure 3D). The RI lines again showed largely normal growth and development phenotypes, although the most severely suppressed lines showed some modest tendency to dwarfing (data not shown).
Plants of both the OX and RI lines showed no signs of spontaneous cell death under normal growth conditions. However, exposure of mature OX or RI leaves to levels of ozone that caused no visible injury to wild-type plants (500 parts per billion [ppb]) resulted in the rapid appearance of small necrotic lesions on leaves of both the transgenic genotypes (Figures 2B and 3E). The kinetics of this oxidative stress damage were quite different. Lesions consistently appeared on the leaves of OX plants as early as 4 to 6 h, but visually similar lesions only appeared on RI leaves  24 h later. In plants challenged with lower ozone concentrations (250 ppb), an analogous pattern was observed except that the necrotic responses were delayed until 48 h (OX) and 72 h (RI) (data not shown). When leaf discs prepared from the wild-type, OX, and RI genotypes were assayed for the loss of membrane integrity and associated ion leakage resulting from ozone exposure (500 ppb), differential timing of the damage response also was observed (Figure 4).
To assess in situ the relative levels of hydrogen peroxide accumulation induced by ozone exposure, control and ozone-treated leaf halves were infiltrated with 3,3'-diaminobenzidine solution. The staining patterns revealed no detectable levels of hydrogen peroxide in untreated leaves of any of the genotypes or in leaves of wild-type plants after 8 h of ozone exposure. However, strong 3,3'-diaminobenzidine staining was observed in both the OX and RI lines after ozone treatment (Figure 4B). The observation that overexpression of SIPK-FLAG in infiltrated leaves was accompanied by the spontaneous activation of MAPK and by cell death raised the question of whether activation of the ectopically expressed protein was necessary for the induction of cell death. Therefore, site-directed mutagenesis was used to create a version of SIPK-FLAG in which the TEY motif found in the activation loop of SIPK had been converted to an AEF sequence. This modification yielded a kinase that retained a low level of basal activity when the recombinant protein was assayed in vitro against myelin basic protein (Figure 5A), but it could not be activated further through dual phosphorylation of the activation loop by upstream MAPK kinases. Unlike the SIPK-FLAG construct, when transiently expressed in tobacco leaves, the SIPK(AEF)-FLAG construct failed to cause cell death in the infiltrated zone (data not shown).
Stably transformed tobacco plants expressing high levels of SIPK(AEF)-FLAG also were recovered readily after Agrobacterium cocultivation, and these plants displayed no visibly altered phenotype. Despite accumulating similar levels of the epitope-tagged kinase (Figure 5B), the ozone sensitivity of these SIPK(AEF) transgenic lines did not differ from that of wild-type plants (data not shown). This finding indicates that the heightened ozone sensitivity observed in SIPK-OX transgenic lines requires not only that the ectopically expressed kinase be expressed at high levels within the plant cell but that it have the capacity to become activated. The activation status of both SIPK and WIPK in tobacco tissue extracts can be assessed either on protein gel blots using a phosphospecific antibody or by immunoprecipitation with antibodies that discriminate between SIPK and WIPK, followed by in gel or in vitro kinase activity assays. When the various transgenic and wild-type tobacco lines were monitored during a 30-min period of ozone exposure, striking differences in the pattern of kinase activation were observed among these genotypes (Figure 6).
As reported previously (Samuel et al., 2000 ), ozone treatment led to the rapid activation of SIPK in leaves of wild-type plants. This was accompanied by a much weaker activation of the smaller kinase, WIPK (Figure 6A). In the OX4 genotype, ozone exposure also led to SIPK activation, but the level of activation appeared to be depressed relative to the wild-type response, despite the presence of far greater amounts of ectopically expressed SIPK in the OX cells (Figures 6B and 6C). No activation of WIPK was detected in the OX tissue samples. The SIPK(AEF) genotype presented a kinase activation profile that was very similar to that of the wild type. This indicates that flooding the cell with a nonactivatable version of SIPK (a potential dominant-negative form) does not interfere with the ability of the upstream MAPK cascade elements to transmit oxidant-induced signals to their cognate MAPKs. Exposure of the RI genotype to ozone, on the other hand, yielded a very different MAPK activation profile. Very weak or no SIPK activation was detected, as would be predicted for a genotype in which SIPK expression has been suppressed by post-transcriptional gene silencing (Figures 6A and 6C). Instead, ozone exposure produced strong and specific activation of WIPK. The identity of these highly activated kinases in ozone-treated leaves of each genotype was confirmed through immunoprecipitation of the 30-min ozone-treated protein extracts with either SIPK- or WIPK-specific antibodies, followed by in gel kinase assays (Figures 6D and 6E).
Aside from the unexpected massive activation of WIPK, the stability of that activation also was strikingly different in this genetic background. Normally, when oxidants trigger a rapid activation of SIPK, it is a transient response. The activation is effectively lost within 1 h, even under conditions of continuous oxidant stimulus, as seen in Figure 7A (wild-type lane). However, in the RI genotype, WIPK was not only activated rapidly but the pool of this MAPK remained continuously active for up to 8 h after the initiation of the response (Figure 7A, RI lane). Although normally there is far less WIPK than SIPK present in tobacco leaves (Zhang and Klessig, 1998b
Interestingly, kinase activation by ozone in the OX genotype also was prolonged abnormally, relative to that seen in ozone-treated wild-type plants, but in this case, the active kinase was SIPK rather than WIPK (Figure 7B). In addition, unlike the hyperactivated WIPK pool, the extended activation of SIPK in the OX line was more transient and disappeared within 4 h. This is approximately the time at which visible lesions began appearing on ozone-treated OX leaves. Examination of the temporal response of the two genes (GST [glutathione S-transferase] and cAPX [cytosolic ascorbate peroxidase]) whose expression was induced strongly by ozone treatment revealed that the loss of SIPK signaling in the RI genotype resulted in a delayed response in the expression of both genes. In the OX line, the prolonged activation of SIPK signaling resulted in the suppression of GST induction, whereas APX gene expression was unaffected (Figures 8A and 8B).
Plant cells must deal constantly with ROS from a range of sources, including photooxidation, mitochondrial electron transport, flavin oxidase by-products, and environmental insults such as UV light, ozone, and ionizing radiation. Against this background, ROS pulses ("oxidative bursts") also can occur within cells, usually as very early responses to localized challenges to cellular integrity such as wounding and pathogen assault. These pulses may serve in multiple functions, including activation of redox protection mechanisms, modulation of intracellular signal transduction pathways, and transmission of systemic signals to neighboring cells.
A severe oxidative challenge that overwhelms local protective measures ultimately will lead to cell death. The archetype for this outcome is the HR response induced during incompatible host–pathogen interactions. Similar lesions are induced by exposure to increased levels of ozone or UV light. The exact process by which cellular integrity fails is unclear, but the notion that HR represents a form of genetically programmed cell death is supported by the identification of numerous mutants affected in the process of lesion formation (Richberg et al., 1998 The correlation of ROS pulses with the cell death process has been described extensively. Treatments such as chilling, wounding, pathogen infection, UV irradiation, and ozone exposure rapidly induce ROS accumulation in plant cells, followed later by lesion development. However, despite these correlative observations, a functional link between ROS accumulation and local lesion formation has yet to be defined.
It is striking that so many stresses that elicit ROS accumulation in plant cells consistently appear to activate MAPK modules as one of their earliest effects (Seo et al., 1995
Links between ROS-associated cell death and MAPK signaling have been reported for a number of nonplant systems. Hydrogen peroxide–induced cell death in cultured mammalian oligodendrocyte cells is inhibited by PD98059, a specific inhibitor of MEK, the upstream kinase of the ERK1/2 MAPK (Bhat and Zhang, 1999
There also is evidence that ROS-activated MAPKs may play analogous roles in plant cells. Cell death induced in Arabidopsis cell suspension cultures by treatment with a bacterial elicitor (harpin) is inhibited when the cells are treated with the MEK inhibitor PD98059 (Desikan et al., 1999
Genetic manipulation experiments also have implicated MAPK activation in the cell death process. In Arabidopsis plants overexpressing constitutively active forms of the MAPK kinases AtMEK4 and AtMEK5 under the control of an inducible promoter, HR-like lesions appeared after induction with dexamethasone, and lesion formation was preceded by the activation of endogenous MAPKs and the accumulation of hydrogen peroxide (Ren et al., 2002 We have confirmed that ectopic SIPK overexpression leads to the appearance of high levels of the activated kinase in Agrobacterium-infiltrated tobacco tissue and to rapid cell death (Figure 1). On the other hand, when stably transformed tobacco plants were produced that overexpressed epitope-tagged SIPK (Figure 2), they displayed no visible phenotype. When exposed to ozone, however, the transgenic SIPK-OX plants proved to be much more sensitive than the nontransgenic parental line, indicating that ROS-induced cell death was controlled less effectively in the overexpression genotype. Although this pattern is consistent with the results of NtMEK2 or SIPK-OX transient expression, its physiological relevance remains uncertain, because we know little about the effects of the accumulation of nonphysiological levels of active signal components on cellular function. To unambiguously identify a functional relationship between ROS activation of SIPK and ROS-induced cell death, we turned to the creation of defined loss-of-function mutants.
The modification of SIPK function in transgenic tobacco plants using either conventional gene-silencing methods (cosuppression and antisense-mediated suppression) or overexpression of dominant-negative forms proved ineffective (Yang et al., 2001 Given the sensitivity of SIPK-OX lines to ozone, it might have been predicted that the absence of this kinase would have no effects, or perhaps even positive effects, on the ozone sensitivity of the SIPK-RI lines. Instead, after ozone treatments that induced no visible damage on wild-type plants, the SIPK-RI lines developed numerous lesions on their middle leaves within 24 h. Thus, the inability of the suppressed genotype to generate and activate SIPK compromises the cell's ability to manage ROS stress and to control cell death, although apparently on a different time scale from that observed in SIPK-OX plants. Which facet of ROS-stress management has been compromised in SIPK-OX and SIPK-RI plants is not clear. No constitutive hydrogen peroxide accumulation was detected in any of the genotypes, suggesting that their heightened ozone sensitivity is not the consequence of a preexisting accumulation of ROS. Instead, it appears that alteration of the normal ozone-induced MAPK activation process, through either unregulated overexpression or suppression, creates an inability to cope with increased redox stress. Examination of the transcriptional activity of two genes whose mRNAs accumulate rapidly after ozone exposure showed that the response of both genes was affected differently (Figure 8).
Expression of cAPX, which encodes a major ROS-scavenging enzyme, was induced less effectively by ozone in RI plants, whereas it was unaffected in the OX line. Antisense suppression of cAPX was shown previously to create hypersensitivity to both ozone (Orvar and Ellis, 1997
The delayed response of the antioxidant genes in the RI line could result in increased early accumulation of ROS (Figure 4B), which could lead to a necrotic cell death process. In the OX line, although the cAPX gene response to ozone appeared to be normal, the antioxidant response clearly was unable to contain the increasing ROS levels associated with extended SIPK activation (Figure 4B). MAPK activation has been linked previously to increased ROS accumulation in Arabidopsis (Ren et al., 2002
Another aspect of the link between SIPK activation and cell death is revealed in the pattern of MAPK activation in ROS-stressed plants. The activation of SIPK by ozone occurred within 10 min in SIPK-OX plants but was not reversed for 4 h, by which time cell death already was becoming visible. This outcome is similar to the association of the prolonged activation of mammalian ERK with the induction of programmed cell death in neurons (Stanciu et al., 2000
The absence of SIPK in the SIPK-RI genotype also led to premature cell death under redox stress conditions, but in this case, the hyperactivated species observed was WIPK rather than SIPK. There have been other indications that WIPK plays a central role in plant stress signaling. This gene was identified originally on the basis of its rapid and transient induction upon wounding of tobacco leaves (Seo et al., 1995
WIPK activity, either alone or together with SIPK, has been suggested to be involved in the induction of cell death in cultured tobacco cells by specific fungal elicitor treatments (Zhang et al., 2000
How SIPK elimination leads to the prolonged hyperactivation of WIPK is unknown, but various possibilities suggest themselves. If NtMEK2 is the sole upstream MAPK kinase responsible for the activation of both SIPK and WIPK, these two MAPKs may normally compete for binding to NtMEK2. However, basal levels of SIPK in unstimulated tobacco cells are much higher (10-fold) than those of WIPK (Zhang and Klessig, 1998b
Alternatively, one of the normal roles of activated SIPK may be the direct or indirect regulation of WIPK activity. Both dual-specificity phosphoprotein phosphatases (MKP) and Ser/Thr phosphatases have been implicated in inactivating MAPK pathways in mammalian and plant models (Brondello et al., 1997
In this regard, it is interesting that Arabidopsis plants in which a dual-specificity phosphatase (AtMKP-1) has been mutated by T-DNA insertional mutagenesis display increased activation of an unidentified
Plant Material and Treatment Tobacco (Nicotiana tabacum) plants of all genotypes were grown for 6 weeks in soil under controlled environmental conditions (25/20°C, 16-h-light/8-h-dark cycle) and then exposed to ozone (500 parts per billion) and harvested as described previously (Orvar and Ellis, 1997 ).
Recombinant Protein Production
The SIPK(AEF) gene construct then was cloned into pGEX 4T-3. The recombinant glutathione S-transferase (GST) fusion proteins were expressed in Escherichia coli BL21 cells by induction with 0.1 mM isopropylthio-
Intron-Spliced Hairpin Loop RNA–SIPK Construct
Binary Vector Construction and Plant Transformation
Agrobacterium-mediated transformation of tobacco (cv Xanthi-nc) was performed using a leaf disc cocultivation procedure. Transformants were selected on half-strength Murashige and Skoog (1962)
The confirmed transgenic lines were transferred to soil and grown to maturity, and seeds were collected. The T1 seeds were germinated on half-strength Murashige and Skoog (1962)
Transient Transformation Using Agrobacterium Infiltration
RNA Gel Blot and RT-PCR Analysis The cDNA was synthesized from total RNA extracted from control and ozone-exposed tissues of the different genotypes/treatments using a first-strand cDNA synthesis kit (Invitrogen, Carlsbad, CA). RT-PCR was performed using gene-specific primers designed to target either SIPK (25 cycles) or NTF4 (30 cycles). The number of cycles was adjusted so that the amplification was within the linear range. As an internal control, 18S ribosomal cDNA was amplified using a 1:4 ratio of 18S-specific primers to competitor's DNA fragments provided by Ambion (Austin, TX).
Protein Extraction and Protein Gel Blot Analysis
Immune Complex Kinase Assay
In Vitro Kinase Assays
Ion-Leakage Assay
In Situ Staining for Hydrogen Peroxide Upon request, all novel materials described in this article will be made available in a timely manner for noncommercial research purposes. No restrictions or conditions will be placed on the use of any materials described in this article that would limit their use for noncommercial research purposes.
We thank Y. Ohashi for providing the anti-WIPK and anti-SIPK antibodies and H. Hall for assistance with the 3,3'-diaminobenzidine staining experiments. Funding for this research was provided by the Natural Sciences and Engineering Research Council of Canada.
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.002337. Received February 13, 2002; accepted May 15, 2002.
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Abstract
INTRODUCTION
-galactoside for 4 h at 25°C, followed by purification according to the manufacturer's protocol (Amersham Pharmacia). The different constructs were sequenced to confirm the changes and the absence of mismatches. 
-32P– labeled ATP (>5000 Ci/mmol) (Amersham Pharmacia) in a 20-µL reaction mixture (20 mM Hepes, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 5 mM 
