JXB Advance Access originally published online on June 27, 2005
Journal of Experimental Botany 2005 56(418):2195-2201; doi:10.1093/jxb/eri219
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RESEARCH PAPER |
Overexpression of SIPK in tobacco enhances ozone-induced ethylene formation and blocks ozone-induced SA accumulation
1Michael Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, BC, Canada V6T 1Z4
2Department of Wood Science, University of British Columbia, Canada
* Present address and to whom correspondence should be sent: Department of Botany, University of Toronto, Canada. Fax: +1 416 978 5878. E-mail: smarcus{at}interchange.ubc.ca
Received 8 December 2004; Accepted 5 May 2005
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Abstract |
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Ozone induces rapid activation of SIPK, a mitogen-activated protein kinase (MAPK) in tobacco. Through transgenic manipulation it has previously been shown that overexpression of SIPK leads to enhanced ozone-induced lesion formation with concomitant accumulation of ROS. In spite of this hypersensitive phenotype, the effect of this altered SIPK expression on the levels of various hormones that regulate ozone-induced cell death has remained unexplored. The response of both salicylate and ethylene, the major phytohormones that modulate ozone-induced cell death, have now been analysed in SIPK-OX tobacco plants. Ozone treatment strongly induced ethylene formation in the sensitive SIPK-OX plants at ozone concentrations that failed to elicit stress ethylene release in WT plants. By contrast, SIPK-overexpressing plants displayed no ozone-induced SA accumulation, whereas WT plants accumulated SA upon ozone exposure. Epistatic analysis of SIPK-OX function suggests that the ozone-induced cell death observed in SIPK-OX plants is either independent, or upstream, of SA accumulation.
Key words: Cell death, ethylene, ozone, ROS, SA, SIPK
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Tropospheric ozone damages crop plants and forests by entering leaf mesophyll tissue through the stomata and rapidly generating reactive oxygen species (ROS) such as ·O2, HO·, and H2O2 at the cell perimeter (Pasqualini et al., 2002
; Pellinen et al., 1999). These oxidizing agents chemically modify membranes, proteins, and nucleic acids, and trigger oxidant-sensitive signalling processes. In sensitive plant species, exposure to elevated levels of ozone eventually induces localized cell death (lesion formation). The severity of this cell death response is modulated by a number of signalling agents, including protein kinases, salicylic acid, and ethylene (Rao and Davis, 2001; Nakajima et al., 2002; Samuel and Ellis, 2002; Overmyer et al., 2003). For example, plant tissues challenged with acutely toxic levels of ozone rapidly produce a burst of ethylene (‘stress ethylene’) (Mehlhorn and Wellburn, 1987). This ethylene burst appears to promote ozone-induced lesion formation, since blocking it either by application of an ethylene synthesis inhibitor, or by genetic suppression of ACC synthase, can reduce ozone-induced tissue damage (Nakajima et al., 2002; Overmyer et al., 2003).
Salicylic acid also plays a prominent role in controlling ozone-induced cell death in plants. Suppression of SA accumulation in tobacco (cv. Xanthi) through ectopic overexpression of bacterial salicylate hydroxylase (NahG) markedly reduced the plant's sensitivity to ozone (Orvar et al., 1997). Consistent with this positive correlation between SA levels and ozone sensitivity, ozone exposure induces SA accumulation in both the ozone-tolerant (Bel B) and ozone-sensitive (Bel W3) tobacco cultivars, but the accumulation is higher and more prolonged in the sensitive genotype (Pasqualini et al., 2002). However, despite the evidence that salicylic acid and ethylene are both involved in regulating ozone-induced cell death, the signalling matrix connecting these hormones remains largely uncharacterized.
One early signalling component in the ozone response pathway is activation of specific MAPKs (Samuel et al., 2000). Ozone induces rapid MAPK signalling in both tobacco and arabidopsis (Samuel et al., 2000; Ahlfors et al., 2004). In tobacco, the major ozone-activated MAPK is ‘salicylic acid-induced protein kinase’ (SIPK), transgenic manipulation of which leads to increased sensitivity to ozone (Samuel and Ellis, 2002). Oxidant signalling through SIPK thus appears to be directly or indirectly involved in controlling the fate of the challenged cells. Plants that ectopically overexpress SIPK (SIPK-OX) display loss of membrane integrity and rapid lesion formation when challenged with ozone concentrations that leave wild-type plants undamaged (Samuel and Ellis, 2002). This lesion response in SIPK-OX plants is accompanied by an increased accumulation of ROS in the ozone-challenged tissue (Samuel and Ellis, 2002), suggesting that overexpression of SIPK in tobacco interferes with the ability of the cells to maintain cellular homeostasis in the face of an increased oxidative burden.
Since ozone is a strong and rapid activator of SIPK, and activation of SIPK in a gain-of-function system has recently been reported to lead to increased production of ethylene (Kim et al., 2003), which acts as a positive modulator of ozone-induced cell death, it is speculated that SIPK-OX plants might be affected in their ability to regulate ethylene synthesis or response pathways. Alternatively, since suppression of salicylate accumulation has been shown to reduce ozone sensitivity, it seemed possible that the enhanced sensitivity of SIPK-OX plants could reflect perturbation of salicylate signalling.
In order to help position SIPK within the ozone–hormone–cell death response matrix, the response of both salicylate and ethylene signalling during ozone-induced lesion formation in SIPK-OX tobacco plants was examined. It was found that ozone-treatment led to strong induction of ethylene release in the ozone-sensitive SIPK-OX plants while the insensitive WT plants did not release any stress ethylene. Unexpectedly, SIPK overexpression also strongly down-regulated ozone-induced SA accumulation. Thus, while SIPK appeared to modulate the behaviour of two key effectors of programmed cell death in tobacco, the associated suppression of SA formation failed to block oxidant-induced lesion formation.
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Materials and methods |
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Plant material and treatment
Plants of the Nicotiana tabacum genotypes Xanthi, NahG (courtesy of Dr John Ryals, Agricultural Biotechnology Research Unit, CIBA-GEIGY Corp.), and SIPK-OX and NGOX were grown in controlled environment growth chambers with 25/20 °C day/night under a 16 h photoperiod (120–150 µE m–2 s–1, 06.00 h–22.00 h) and RH 60±5%. Six-week-old plants were used for the treatments. Ozone was generated from air with a DELZONE ZO-300 Ozone Generating Sterilizer (DEL Industries) and monitored with a Dasibi 1003-AH ozone analyser (Dasibi Environmental Corp). Ozone exposure regimes were either 500 ppb for 8 h for 1 d (06.00 h–22.00 h), or samples were harvested at the stipulated time points after initiation of ozone exposure. Exposure levels varied no more than ±10% over the course of the treatment. Tissues harvested for molecular and biochemical analysis were frozen in liquid nitrogen and stored at –80 °C until further analysis.
Generation of NGOX lines
NGOX lines were created through fertilization of SIPK-OX pistils with nahG pollen, followed by PCR screening of the kanamycin-resistant F1 lines for the presence of both insertions, using a CaMV 35S forward primer in combination with either a SIPK-specific reverse primer (Samuel and Ellis, 2002) or a nahG-specific reverse primer (5'-GTC GCG CAA CTC GTA TAA CTC-3') (Gaffney et al., 1993).
Protein extraction and western blotting and in-gel kinase assays
Total protein extracts (40–80 µg) were prepared and used for western blotting as described earlier (Samuel et al., 2000; Samuel and Ellis, 2002). The ozone activated MAPKs in tobacco have been previously identified as SIPK and WIPK through immunoprecipitation with SIPK- and WIPK-specific antibodies (Samuel et al., 2002). The anti-pERK antibody employed in this study was previously shown to detect phosphorylated forms of SIPK and WIPK in ozone-exposed WT tobacco tissue, as well as pSIPK in SIPK-OX tissue (Samuel et al., 2000; Samuel and Ellis, 2002).
Northern blotting and RT-PCR analysis
Total RNA was extracted, blotted and probed as described previously (Orvar et al., 1997). Hybridization signals were scanned and analysed using a Storm 860 Phosphor Imager (Amersham Pharmacia Biotech). The PAL (Xanthi nc.) probe was a gift from Monica McQuoid (UBC), while the PR-1a probe was provided by Dr Daniel Klessig, Rutgers, USA.
For RT-PCR, cDNA synthesized from total RNA using a first-strand cDNA synthesis kit (Invitrogen) was used as the amplification template. PCR was performed as described earlier using gene-specific primers designed to target NtACS1, NtACO1, ERF1, and EF1
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- NtACS1 (forward): 5'-GAGAATGAGAAGAACAGCTCA-3';
- NtACS1 (reverse): 5'-TTCTAGCACAATTAACGACGG-3';
- NtACO1 (forward): 5'-CTTCTTTGAGTTGGTGAACC-3';
- NtACO1 (reverse): 5'-GCAGTTGCAATTGGATCCATC-3';
- EF1
(forward): 5'-TCACATCAACATTGTGGTCATTGGC-3';
- EF1
(reverse): 5'-TTGATCTGGTCAAGAGCCTCAAG-3';
- NtERF1 (forward): 5'-ATGAATCAACCAATTTATACAGAG-3';
- NtERF1 (reverse): 5'-TTAACTGACTAATAATTGATGTCG-3'.
- NtACS1 (reverse): 5'-TTCTAGCACAATTAACGACGG-3';
Ethylene measurements
Six-week-old tobacco plants were exposed to ozone (500 ppb) for different times. Leaf discs were excised at the stipulated time-point and incubated under light overnight in a sealed vial (20 ml). A headspace sample (1 ml) was drawn from each vial and ethylene levels were analysed using a gas chromatograph (Hewlett 5890 Packard Series II) equipped with a flame ionization detector (Nakajima et al., 2002).
SA measurements
Six-week-old tobacco plants from WT and SIPK-OX plants were continuously treated with ozone for up to 8 h followed by incubation in clean air for up to 24 h. Leaf blade samples (0.5–1 g tissue) harvested at various intervals were used for measuring free and total SA as described previously ( Li et al., 1999).
Unless otherwise indicated, all experiments were repeated at least once, with consistent results.
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Results |
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Ozone induces an ethylene burst in SIPK-OX leaf tissue
When SIPK-OX plants are continuously exposed to ozone (500 ppb), local lesions appear on the leaves as early as 4–6 h after the initiation of ozone treatment, and continue to develop up to 24–36 h, whereas WT plants are completely lesion-free when treated in the same fashion (Samuel and Ellis, 2002
; Fig. 1a). This response in SIPK-OX plants has previously been shown to be accompanied by abnormally elevated ROS accumulation in the ozone-challenged tissue (Samuel and Ellis, 2002).
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Ozone is known to cause a transient rise in ethylene in sensitive tobacco cultivars, with peak production reported 4 h after the initiation of fumigation (Nakajima et al., 2002
). In order to determine whether the lesion response observed in ozone-treated SIPK-OX plants reflected changes in ethylene formation, ethylene production levels were compared at various intervals in WT and SIPK-OX plants exposed to ozone continuously at concentrations (500 ppb) that fail to elicit lesions in WT tissues. There was no significant difference in basal levels of ethylene production in WT and SIPK-OX plants in the absence of an ozone challenge (Fig. 1b). Exposure of WT plants to 500 ppb ozone also failed to induce any increase in ethylene over the period tested, consistent with the absence of visible tissue damage. However, in the SIPK-OX genotype, ozone exposure (500 ppb) induced rapid ethylene production, which peaked about 2 h after the initiation of fumigation. Since visible lesion formation occurs in SIPK-OX plants 4–6 h after the initiation of ozone exposure, this induced ethylene burst evidently precedes the commitment of leaf tissue to a local cell death programme. The ethylene burst is, in turn, temporally preceded by the even more rapid activation of the oxidant-responsive MAPKs, SIPK and WIPK, in ozone-challenged tobacco tissue (Fig. 2a).
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Ozone induces strong activation of ethylene biosynthetic and responsive genes in SIPK-OX plants
Two enzymatic steps control ethylene biosynthesis. The first is conversion of S-adenosyl-L-methionine to 1-aminocyclopropane-1-carboxylic acid (ACC), catalysed by ACC synthase (ACS). This is generally considered to be the rate-limiting step in the pathway (Kende, 1993
). The subsequent oxidative cleavage of ACC to form ethylene is catalysed by ACC oxidase (Kende, 1993). Both ACS and ACC oxidase are encoded by gene families, and the transcriptional profiles of the various isoforms differ, depending on the applied stress and its timing (Bleecker and Kende, 2000; Wang et al., 2002). When four tomato ACS genes were tested for ozone-responsiveness, only expression of LE-ACS2 was induced (Tuomainen et al., 1997). Tomato ACO (LE-ACO) transcription was also strongly induced by ozone (Tuomainen et al., 1997). Interestingly, the putative orthologues of these genes in tobacco (NtACS1 and NtACO1) were also found to be strongly induced following in vivo activation of SIPK in a transgenic gain-of-function system (Kim et al., 2003). Since such a regulatory link might provide a potential explanation for the heightened ethylene response in the SIPK-OX plants, the transcriptional response of these two biosynthetic genes was examined, as well as the ethylene responsive gene, ERF-1, in ozone-challenged SIPK-OX plants. The basal levels of NtACS1 and NtACO1 expression were similar in both WT and SIPK-OX plants, and that expression was enhanced following ozone exposure (Fig. 2b). However, induction of both genes was stronger in SIPK-OX plants than in WT after 2 h of ozone exposure, a pattern that is consistent with the increased ethylene levels observed in the SIPK-OX plants (Fig. 1b). Ozone also rapidly induced the accumulation of ethylene response factor, ERF-1, in both genotypes as early as 30 min following exposure (Fig. 2c).
Overexpression of SIPK suppresses ozone-induced SA accumulation and PR-1a gene induction
Although both SIPK activity and SA exert a strong influence on the outcome of ozone challenge in plant cells, a direct link between SIPK activation and SA accumulation or SA-induced responses has yet to be defined. However, given the importance of SA in modulating ozone-induced cell death (Rao and Davis, 2001) and the fact that SA acts downstream of ozone-induced MAPK activation (Samuel et al., 2000), it seemed possible that the increased cell death observed in ozone-challenged SIPK-OX tissue might result either from altered SA levels or SA-sensitivity in that genetic background. Therefore, NahG and SIPK-OX tobacco plants were crossed and two F1 progeny were selected that carried both the SIPK-OX and nahG transgenes (NGOX genotype).
When NGOX plants were treated with ozone (500 ppb), and compared with similarly treated SIPK-OX, WT and nahG lines, lesion formation was found to be induced in both the SIPK-OX plants and NGOX lines, while the WT and nahG plants were completely resistant (Fig. 3). The rapid ozone-induced lesion formation in SIPK-OX plants thus appears to be independent of SA accumulation.
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To test whether the endogenous accumulation of SA is altered in SIPK-OX lines, SA levels were measured at 8 h and 24 h after initiation of ozone exposure (500 ppb for 8 h). These time-points were chosen based on previous studies in tobacco that reported 66-fold greater accumulation of SA 24 h after initiation of ozone exposure (Yalpani et al., 1994
). Ozone induced marked increases in SA accumulation in the WT plants by 24 h (16 h post-treatment), while SIPK-OX plants displayed no increase in SA accumulation over this same period (Fig. 4a). Consistent with the failure of SA to respond to the applied stress, PR-1a gene induction by ozone was also completely blocked in the ozone-treated SIPK-OX plants (Fig. 4c).
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Since one of the biosynthetic pathways to SA utilizes phenylalanine as a distal precursor, the question was asked whether over-expression of SIPK might be affecting ozone induction of phenylalanine ammonia lyase (PAL), the enzyme that controls the entry of phenylalanine into the phenylpropanoid pathway. However, northern blot analysis revealed that PAL induction was similar in both WT and SIPK-OX plants (Fig. 4b), indicating that SIPK regulation must be acting elsewhere during SA biogenesis.
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Discussion |
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In this study, it was shown that ectopically expressed SIPK functions simultaneously as a positive regulator of ethylene induction by ozone and a negative regulator of ozone-induced SA accumulation. Ethylene influences a broad spectrum of physiological processes, both during development and in response to stress (Suzuki et al., 1998
). Stress ethylene is rapidly induced in ozone-sensitive tobacco cultivars (Langebartels et al., 1991) and suppression of this response has been correlated with the plant's sensitivity to ozone (Moeder et al., 2002; Nakajima et al., 2002). It was observed that WT plants treated with non-lesion-inducing levels of ozone failed to produce stress ethylene, despite the induction of genes encoding key enzymes of ethylene biosynthesis (Fig. 1), suggesting that the primary control of ethylene formation may operate post-transcriptionally. Both direct and indirect evidence points to a central role for protein kinase activity in this putative post-transcriptional regulatory process. Induction of the activity of ACS by ozone in tomato plants was blocked by K252A, a protein kinase inhibitor, while ACS activity could be induced in the absence of ozone by treatment with calyculin A, a protein phosphatase inhibitor (Tuomainen et al., 1997; Wang et al., 2002). A similar phenomenon was observed in tomato suspension cultures treated with fungal elicitors (Spanu et al., 1994). Recently, the ozone-inducible Le-ACS2 isoform in tomato was shown to be phosphorylated at its C-terminus by a calcium-dependent protein kinase (Tatsuki and Mori, 2001), and the C-terminus of Arabidopsis ACS5 has been shown to affect ACS activity by helping to stabilize the ACS protein (Chae et al., 2003). In Arabidopsis, phosphorylation of ACS2 and ACS6 by ATMPK6 has recently been shown to be responsible for the increased stability of these proteins, which is correlated with increased ethylene production (Liu and Zhang, 2004). Since SIPK is the tobacco orthologue of ATMPK6, this finding is congruent with our hypothesis that, in tobacco, SIPK exerts its effect on the ethylene biosynthetic pathway post-transcriptionally.
SIPK is also strongly activated following calyculin A treatment (Samuel et al., 2000; Liu et al., 2003), and its activation is inhibited by staurosporine and K252A (Zhang et al., 2000). More specifically, recent gain-of-function studies using an upstream activator of SIPK (NtMEK2) demonstrated that SIPK activation was sufficient to induce increased ACS activity and rapid accumulation of ethylene, even though NtACS gene transcription was only induced later (Kim et al., 2003). It was observed that activation of SIPK by ozone treatment occurs within a few min (Samuel et al., 2000), well before the transcriptional activation of the ethylene biosynthesis genes, ACS and ACO, can be detected (Fig. 2a, b). Taken together, these data imply that SIPK is likely to exert its effect on ethylene formation in tobacco by contributing directly or indirectly to rapid post-translational phosphorylation of the ACS protein. The resulting alteration of either ACS activity or stability, or both (Spanu et al., 1994; Tatsuki and Mori, 2001) would be predicted to lead to increased ethylene production.
SIPK activation alone is not sufficient, however, since ozone treatment also induces rapid transient SIPK activation in WT tobacco plants, followed by increased expression of the ethylene biosynthetic genes. However, in WT plants these events do not lead to a stress ethylene burst or to lesion formation. Since the duration of ozone-induced SIPK activation is markedly enhanced in SIPK-OX tissues, such a prolonged SIPK activation may be essential for up-regulating ethylene formation and directing the oxidant-challenged cells into a cell death programme. There is precedent for this model in mammalian MAPK signalling, where prolonged activation of ERK1/2 has been shown to induce apoptosis in neuronal cells (Stanciu et al., 2000), whereas transient activation is known to support cell proliferation (Roux and Blenis, 2004).
While an ethylene burst is triggered in SIPK-OX plants by ozone, the salicylic acid accumulation normally induced by ozone is completely blocked in these plants. Contrary to previous studies in which suppression of salicylic acid accumulation was associated with a reduced ozone sensitivity phenotype (Orvar et al., 1997; Rao et al., 2002), and interfered with the local cell death associated with the pathogen-induced hypersensitive response (Alvarez, 2000), it was found that SIPK-OX plants display rapid lesion formation in response to ozone despite loss of salicylic acid accumulation and SA-dependent responses (Figs 3, 4). There are several possible interpretations of this pattern. It may be that ozone-induced lesions, despite their superficial similarity to HR lesions, result from a different, salicylic acid-independent, cell death process, or that the heightened production of ethylene in the SIPK-OX genotype is sufficient to override the usual requirement of SA for activation of lesion formation. A more trivial explanation would be that the basal levels of SA still present are sufficient to support the canonical cell death pathway.
Where SIPK acts in the salicylic acid biosynthetic pathway to prevent SA accumulation is still not known, except that this negative regulation appears to occur downstream of PAL gene induction (Fig. 4b). It is of interest that PAL, which catalyses the first committed step in the phenylpropanoid pathway needed for salicylic acid formation in tobacco (Yalpani et al., 1993), may itself be directly regulated by phosphorylation. In elicited Phaseolus cells, PAL has been shown to be phosphorylated by a calcium-dependent protein kinase, a modification that apparently increased the lability of the PAL protein (Allwood et al., 1999, 2002).
How the dramatic alterations in stress hormone responses and cell death control observed in the SIPK-OX plants are functionally related to prolonged activation of SIPK at the molecular level requires further exploration. While phosphorylation and stabilization of ACS probably plays an important role, elucidation of additional specific cellular targets for activated SIPK will be necessary in order to understand how this central player in plant stress signal transduction exerts such a profound influence over the fate of challenged cells.
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Acknowledgements |
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We thank D Klessig for providing the PR1-a probe, M McQuoid for providing the tobacco PAL probe, and J Ryals for providing us with the nahG transgenic tobacco seeds. We are also grateful to K Cham for technical assistance with SA measurements and to X Li (Department of Botany, UBC) for providing access to her HPLC. Funding for this research was provided by the Natural Sciences and Engineering Research Council of Canada.
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