The mitogenactivated protein kinases WIPK and ... - Wiley Online Library

The Plant Journal (2007) 49, 899–909

doi: 10.1111/j.1365-313X.2006.03003.x

The mitogen-activated protein kinases WIPK and SIPK regulate the levels of jasmonic and salicylic acids in wounded tobacco plants Shigemi Seo1,2,*†, Shinpei Katou1,2,†, Hideharu Seto3, Kenji Gomi1,2 and Yuko Ohashi1,2 Plant–Microbe Interactions Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, 305-8602, Japan, 2 Program for Promotion of Basic Research Activities for Innovative Bioscience, Minato-ku, Tokyo, 105-0001, Japan, and 3 RIKEN, Wako-shi, Saitama 351-0198, Japan

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Received 1 September 2006; revised 18 October 2006; accepted 27 October 2006. * For correspondence (fax þ81 29 838 7469; e-mail [email protected]). † These authors contributed equally to this work.

Summary In tobacco (Nicotiana tabacum), wounding causes rapid activation of two mitogen-activated protein kinases (MAPKs), wound-induced protein kinase (WIPK) and salicylic acid (SA)-induced protein kinase (SIPK), and the subsequent accumulation of jasmonic acid (JA). Our previous studies suggested that activation of WIPK is required for the production of wound-induced JA. However, the exact role of WIPK remains unresolved. We generated transgenic tobacco plants in which either WIPK or SIPK were silenced using RNA interference to define the roles of WIPK and SIPK in the wound response. In addition, transgenic tobacco plants were generated in which both WIPK and SIPK were silenced to examine the possibility that they have redundant roles. Wound-induced JA production was reduced compared with non-silenced plants in all of the WIPK-, SIPKand WIPK/SIPK-silenced plants. Transgenic plants over-expressing NtMKP1, a gene encoding tobacco MAPK phosphatase, which inactivates WIPK and SIPK, also exhibited reduced JA production in response to wounding. In both WIPK/SIPK-silenced and NtMKP1-over-expressing plants, wounding resulted in an abnormal accumulation of both SA and transcripts for SA-responsive genes. These results suggest that WIPK and SIPK play an important role in JA production in response to wounding, and that they function cooperatively to control SA biosynthesis. Keywords: mitogen-activated protein kinase, wound signal transduction, jasmonic acid, salicylic acid.

Introduction Wounding caused by mechanical tissue damage or herbivory affects the growth and reproduction of plants, and permits pathogens to penetrate the plant body through wounded tissues (Bostock and Stermer, 1989; Bowles, 1990). Plants cope with wounding by activating the expression of a set of genes encoding defence-related proteins such as the pathogenesis-related (PR) proteins that are mostly involved in the restoration of damaged tissues and protection against insect or herbivore attack (Ryan, 1990; Walling, 2000). Wounding causes a rapid accumulation of jasmonic acid (JA) (Baldwin et al., 1997; Conconi et al., 1996; Creelman et al., 1992; Weber et al., 1997). Exogenous application of JA or its methyl ester (MeJA) induces the expression of many wound-responsive genes including the basic PR and proteª 2006 The Authors Journal compilation ª 2007 Blackwell Publishing Ltd

inase inhibitor II (PI-II) genes (Creelman and Mullet, 1997; Farmer et al., 1998; Pauw and Memelink, 2005). These findings suggest that JA functions as an endogenous signal that mediates the defence responses of plants to wounding. Protein phosphorylation/dephosphorylation have been implicated in the signalling leading to both JA biosynthesis and the expression of JA-responsive genes (Farmer and Ryan, 1992; Menke et al., 1999; Le´on et al., 1998). Mitogenactivated protein kinases (MAPKs) are components of signalling pathways that transduce diverse extracellular stimuli to multiple intracellular responses. In response to these stimuli, MAPKs are activated via phosphorylation by an upstream MAPK kinase (MAPKK) (MAPK Group, 2002; Nakagami et al., 2005; Widmann et al., 1999). The tobacco 899

900 Shigemi Seo et al. (Nicotiana tabacum) MAPKs, salicylic acid (SA)-induced protein kinase (SIPK) (Zhang and Klessig, 1997) and wound-induced protein kinase (WIPK) (Seo et al., 1995), are stress-responsive. Wounding activates SIPK and WIPK in tobacco as well as their corresponding MAPK homologues in other plant species (Bo¨gre et al., 1997; Holley et al., 2003; Ichimura et al., 2000; Menke et al., 2004; Romeis et al., 1999; Xiong and Yang, 2003; Zhang and Klessig, 1998a). Activation of these MAPKs occurs within a few minutes after wounding, preceding the accumulation of JA. We have previously shown that transgenic tobacco plants in which expression of the WIPK gene is co-suppressed by the introduction of a WIPK cDNA clone fail to accumulate JA after wounding, whereas WIPK-over-expressing plants exhibit elevated JA levels (Seo et al., 1995, 1999). Based on these results, we concluded that activation of WIPK is required for the production of wound-induced JA. However, we cannot rule out the possibility that the over-expression of WIPK interferes with the expression of genes other than WIPK, because the introduced cDNA contains a region homologous to other MAPK genes. Subsequently, it was reported that over-production of NtMEK2DD, a constitutively active form of a tobacco MAPKK upstream of WIPK and SIPK, results in activation of both MAPKs, but not in an increase in JA levels (Kim et al., 2003). Furthermore, treatment of tobacco plants with a diterpenoid compound induced activation of WIPK, but not production of JA (Seo et al., 2003). Thus, the exact role of WIPK as an inducer of JA biosynthesis has remained unresolved. To address this deficiency, we generated transgenic tobacco plants in which WIPK was specifically silenced by RNA interference (RNAi). Although SIPK in tobacco and its orthologues in other plant species have been implicated in wound signalling (Holley et al., 2003; Ichimura et al., 2000; Zhang and Klessig, 1998a), no studies on their roles in wound responses using loss-of-function mutants or transgenic plants have been reported. Therefore, transgenic tobacco plants in which SIPK was silenced were generated and used to study its role in the wound response of tobacco plants. Furthermore, to examine the possibility that WIPK and SIPK play redundant roles in the wound response, we used transgenic tobacco plants in which both WIPK and SIPK were silenced by RNAi, or in which NtMKP1, which encodes a tobacco MAPK phosphatase (Yamakawa et al., 2004), was over-expressed. Silencing of both WIPK and SIPK has been accomplished by virus-induced gene silencing (VIGS) in Nicotiana benthamiana (Kanzaki et al., 2003; Katou et al., 2005b). Although the VIGS technique is a powerful strategy for suppressing a specific gene, infiltration of Agrobacterium carrying a construct for VIGS causes injury to the tissue. Moreover, Agrobacterium infection and the subsequent systemic infection of virus could trigger additional defence responses not directly related to wounding. Thus, N. benthamiana plants with a VIGS construct are inadequate for

temporal and spatial analyses of the wound response. Consequently, stable transgenic plants were used in this study. Results Generation of SIPK-, WIPK- and WIPK/SIPK-silenced tobacco plants To silence SIPK, WIPK or both, inverted repeat (IR) constructs (SIPKIR, WIPKIR and WIPK/SIPKIR; Figure 1a) that include portions of the coding and 3¢-untranslated regions from SIPK and WIPK cDNA clones were introduced into tobacco plants using Agrobacterium tumefaciens. Two second-generation transgenic lines for each construct were used for further analysis. There were no observable differences in the phenotypes of transformant lines and wild-type plants. WIPK transcripts accumulate after wounding (Seo et al., 1995; Zhang and Klessig, 1998a). Reverse transcriptasepolymerase chain reaction (RT-PCR) and RNA blot analyses confirmed that accumulation of WIPK transcripts was inhibited in WIPK-silenced plants (lines WIPKIR-2 and WIPKIR-6) and WIPK/SIPK-silenced plants (lines WIPK/SIPKIR-2 and WIPK/SIPKIR-3) (Figure 1b,c), and SIPK transcript accumulation was lower in the SIPKIR plants (lines SIPKIR-3 and SIPKIR-4) and WIPK/SIPKIR plants than in the WIPKIR and control lines. The ability of the IR constructs to specifically silence their target genes was estimated by determining the quantity of transcripts for the known tobacco MAPK genes NtMPK4, Ntf3 and Ntf4. None of the IR constructs appreciably affected expression of the NtMPK4 or Ntf3 genes, but expression of the Ntf4 gene, which has a 94% sequence identity to SIPK, was suppressed in the SIPKIR and WIPK/ SIPKIR lines (Figure 1b). Because we could not obtain SIPKsilenced plants that had no influence on the expression of Ntf4, even with IR constructs made with other regions of the SIPK cDNA, and because it has been reported that Ntf4 has functional redundancy with SIPK (Ren et al., 2006), we used these SIPKIR and WIPK/SIPKIR lines. Immunoblot analysis confirmed that SIPK protein was specifically reduced in the SIPKIR and WIPK/SIPKIR lines not only after infection with tobacco mosaic virus (TMV; Figure 1d) but also after wounding (Figure 1e, second panel). These SIPKIR and WIPK/SIPKIR lines also had reduced SIPK activity (Figure 1e, top panel). Because WIPK protein levels were equal in healthy leaves of all lines in a preliminary experiment (data not shown), we examined accumulation of WIPK protein after infection with TMV, a stress that induces accumulation of a large amount of the WIPK protein (Zhang and Klessig, 1998b). Two major bands corresponding to the predicted molecular mass of WIPK were detected in control plants (Figure 1d). There are several lines of evidence indicating that the higher molecular mass band probably

ª 2006 The Authors Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 49, 899–909

Roles of WIPK and SIPK in wound signalling 901

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Figure 1. Analysis of SIPK-, WIPK- and WIPK/SIPK-silenced plants. (a) Structure of the WIPK and SIPK cDNAs and schematic representation of the constructs used to transform tobacco plants. Boxes below individual cDNAs indicate the regions used for vector construction. UTR, untranslated region; El, 5¢ upstream region of the CaMV 35S promoter; 35S, CaMV 35S promoter; X, 5¢ untranslated region of TMV; GUS, coding region of the b-glucuronidase gene; NOS, nopaline synthase gene terminator. (b) RT-PCR analysis of tobacco MAPK genes in leaves 1 h after wounding. The number of PCR cycles is indicated on the right. (c) RNA blot analysis of the WIPK gene in leaves before (0 h) and after (1 h) wounding. Ethidium bromide staining of rRNAs (rRNA) indicates the gel loading for total RNA. (d) Immunoblot analysis of TMV-inoculated leaves with anti-SIPK (a-SIPK) and anti-WIPK (a-WIPK) antibodies using a 12% gel. Coomassie brilliant blue R250 (CBB) staining indicates the gel loading for total protein. The arrowhead, arrow and open arrow indicate SIPK, WIPK and a protein that reacts non-specifically with the antiWIPK antibodies, respectively. Asterisks indicate the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Size markers are indicated at left in kDa. (e) Myelin basic protein (MBP) kinase activity and protein levels of SIPK and WIPK in leaves before (0 min) and after (10 min) wounding. [32P]MBP indicates the position of phosphorylated MBP. For detection of the WIPK protein, a modified immunoblot analysis was performed. The open arrowhead indicates the position of IgG. For (b)–(e), a transgenic line carrying the vector alone (Cont.) was used as a control line.


902 Shigemi Seo et al. corresponds to the WIPK protein. Firstly, the upper band was almost undetectable in the WIPKIR and WIPK/SIPKIR lines. Secondly, the lower band was almost equal in all lines. Thirdly, the accumulation pattern of the upper band is similar to the expression pattern of WIPK reported by Zhang and Klessig (1998a,b), who showed that WIPK is almost undetectable in healthy leaves but is readily detected in leaves after infection with TMV. Fourthly, anti-WIPK antibodies used in the current work were raised against a peptide corresponding to a region unique to WIPK. Based on these results, the upper band is the most likely to be WIPK, and the lower band is a protein that reacts non-specifically with anti-WIPK antibodies. Although we previously showed, using the same antibody, that the amount of WIPK remains constant before and after wounding (Seo et al., 1999; Yamakawa et al., 2004), it is likely that the protein we described as WIPK is the nonspecific protein. Therefore, we re-investigated the protein levels and enzymatic activity of WIPK after wounding. We failed to detect the band corresponding to the WIPK protein in healthy or wounded leaves using the method used for the immunoblot data shown in Figure 1(d). We modified the method to detect the basal level of the WIPK protein. This modified immunoblot analysis revealed that the band corresponding to the WIPK protein was undetectable in the WIPKIR and WIPK/SIPKIR lines (Figure 1e, bottom panel). In the control and SIPKIR lines, the amount of WIPK remained constant after activation by wounding. Importantly, the lower, non-specific protein shown in Figure 1(d) was not precipitated by anti-WIPK antibodies, confirming that the protein reacts non-specifically with our anti-WIPK antibodies. Consistent with reduced WIPK protein levels, the wound-induced activity of WIPK was reduced in the WIPKIR and WIPK/SIPKIR lines (Figure 1e, third panel). Over-production of the NtMKP1 protein compromises wound-induced activation of both SIPK and WIPK in tobacco plants The activity of MAPKs is strictly regulated via phosphorylation. MAPK phosphatases are dual-specificity phosphatases that dephosphorylate both serine/threonine and tyrosine residues and act as a negative regulator of MAPKs. Previously, we have shown that NtMKP1, a tobacco MAPK phosphatase, dephosphorylates and inactivates SIPK (Katou et al., 2005a), and over-expression of NtMKP1 represses wound-induced activation of both WIPK and SIPK (Yamakawa et al., 2004). Wound responses of NtMKP1-over-expressing plants were tested in this study to confirm the results obtained from analyses of WIPK/SIPK-silenced plants. Three independent transgenic lines over-expressing the NtMKP1 cDNA (Figure 2a; lines NtMKP1OX-8, NtMKP1OX-10 and NtMKP1OX-13) were used for each analysis. The

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Figure 2. Analysis of transgenic tobacco plants transformed with the NtMKP1 gene. (a) Schematic representation of the NtMKP1 expression vector (Yamakawa et al., 2004). (b) Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of the NtMKP1 and actin genes in healthy leaves. The number of PCR cycles is indicated on the right. (c) MBP kinase activity of salicylic acid (SA)-induced protein kinase, woundinduced protein kinase and NtMPK4 and NtMKP1 protein level in leaves before (0 min) and after (10 and 30 min) wounding. Immunoblot analysis with anti-NtMKP1 antibodies (a-MKP1) was performed using a 10% gel. The antiNtMKP1 antibodies reacted non-specifically with the large Rubisco subunit (bottom panel). For (b) and (c), a transgenic line carrying the vector alone was used as the control line.

NtMKP1OX-8 and NtMKP1OX-13 lines contained large amounts of NtMKP1 transcripts (Figure 2b) and NtMKP1 protein (Figure 2c). The NtMKP1OX-10 line and the control line exhibited the same level of NtMKP1 transcript accumulation and contained undetectable levels of NtMKP1 protein. Wound-induced activities of SIPK and WIPK were lower in the two NtMKP1-over-expressing lines than in the NtMKP1OX-10 and control lines (Figure 2c). NtMPK4 activity was not correlated with the amount of NtMKP1 protein. The morphological phenotypes of the NtMKP1-over-expressing lines were apparently normal. Effect of repression of SIPK and WIPK activation on woundinduced accumulation of JA and JA biosynthetic gene transcripts JA accumulation peaks 1–2 h after wounding in tobacco plants (Baldwin et al., 1997). Leaves of the SIPKIR, WIPKIR and WIPK/SIPKIR lines were wounded, and levels of JA were


Roles of WIPK and SIPK in wound signalling 903

Figure 3. Accumulation of jasmonic acid (JA) in SIPK-, WIPK- and WIPK/ SIPK-silenced plants after wounding. The amount of JA is expressed as ng g)1 FW. Values are the means SD from three independent measurements. Inset: JA concentrations before wounding. Data were subjected to Student’s t-test. *P > 0.1 versus the control line. **P < 0.008 versus the control line.

determined 0 and 90 min after wounding. There was no significant difference in JA levels before wounding in any of the lines (Figure 3, 0 min). However, wound-induced JA production was reduced in the SIPKIR, WIPKIR and WIPK/ SIPKIR lines. The percentages of JA relative to controls were 18% in SIPKIR-3, 24% in SIPKIR-4, 48% in WIPKIR-2, 28% in WIPKIR-6, 30% in WIPK/SIPKIR-2 and 11% in WIPK/SIPKIR-3 (Figure 3, 90 min). Similarly, reduced JA production was also observed in NtMKP1OX-over-expressing plants. The percentages of JA relative to control plants were 19% in NtMKP1OX-8 and 20% in NtMKP1OX-13. There was no significant difference in the levels of JA after wounding between the NtMKP1OX10 line and the control line. JA induction by wounding is accompanied by transcriptional activation of enzymes involved in JA biosynthesis such as allene oxide synthase (AOS) and allene oxide cyclase (AOC) (Schaller et al., 2005). Accumulation of AOS and AOC transcripts after wounding was inhibited to a large extent in wounded leaves of the SIPKIR and WIPK/SIPKIR lines (Figure 4). In the WIPKIR lines, wounding resulted in a slight decrease in AOS and AOC transcripts compared with the control line.

Figure 5. Accumulation of ethylene in SIPK-, WIPK-, WIPK/SIPK-silenced and NtMKP1-overexpressing plants after wounding. (a) Ethylene concentrations (nl g)1 FW) in the SIPKIR, WIPKIR, WIPK/SIPKIR and control lines before (0 h) and after (2 h) wounding. Values are the means SD from three independent measurements. Data were subjected to Student’s t-test.*P > 0.06 versus the control line. **P < 0.04 versus the control line. (b) Ethylene concentrations in the NtMKP1OX and control lines before (0 h) and after (4 h) wounding. Values are the means SD from three independent measurements. *P > 0.1 versus the control line. **P < 0.05 versus the control line.

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Figure 4. Expression of the NtAOS and NtAOC genes in wounded SIPK-, WIPK- and WIPK/SIPK-silenced plants. Leaves were wounded, harvested at the times indicated after wounding, and subjected to RNA blot analysis. Similar results were obtained from three independent experiments.

Effect of repression of SIPK and WIPK activation on woundinduced accumulation of ethylene The Arabidopsis SIPK orthologue AtMPK6 induces ethylene production by phosphorylating 1-aminocyclopropane-1carboxylic acid (ACC) synthase, the rate-limiting enzyme of ethylene biosynthesis (Liu and Zhang, 2004). Endogenous ethylene was also measured in the MAPK-silenced lines. Ethylene released from intact seedlings was measured as a pre-wounding baseline because of the difficulty of measuring ethylene released from mature leaves without applying any wound stresses such as detaching, punching or cutting. There was no significant difference in the amounts of ethylene released by any of the lines (Figure 5a, 0 h). Similarly, NtMKP1-overpressing lines released the same amount of ethylene as the control and non-over-expressing lines (Figure 5b, 0 h). However, wound-induced ethylene production was reduced in the SIPKIR and WIPK/SIPKIR lines. The percentages of ethylene relative to control plants were 49% in SIPKIR-3, 80% in SIPKIR-4, 60% in WIPK/SIPKIR-2 and 21% in WIPK/SIPKIR-3 (Figure 5a, 2 h). Similarly, NtMKP1-overexpressing lines exhibited reduced ethylene production in

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904 Shigemi Seo et al. response to wounding (Figure 5b, 4 h). There was no significant difference in the levels of wound-induced ethylene between the two WIPKIR lines and the control line.

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Effect of repression of SIPK and WIPK activation on the accumulation of PR gene transcripts In previous studies using transgenic tobacco plants overexpressing WIPK, we have shown that WIPK is involved in the induction of expression of the basic PR-1 and PI-II genes (Seo et al., 1995, 1999). Wound-induced accumulation of basic PR-1 and PI-II transcripts was assayed in the MAPK-silenced and NtMKP1-over-expressing lines by RNA blot analysis. The expression of the basic PR-1 and PI-II genes in the transformant lines varied among replicate experiments. The data shown in Figure 6 are an example. In this case, the amounts of basic PR-1 and PI-II transcripts in the SIPKIR, WIPKIR and WIPK/SIPKIR lines were only slightly less than in the control line (Figure 6a), whereas there was no marked difference in the amounts of these transcripts among the two NtMKP1 over-expression lines and the non-over-expression and control lines over the period tested (Figure 6b). In another case, wounding resulted in the same level of basic PR-1 and PI-II transcript accumulation in the two WIPK/SIPKIR lines and in the control line. The cause of these experimental variations remains unclear. Accumulation of acidic PR-1 and acidic PR-2 gene transcripts, which are not induced by wounding (Brederode et al., 1991), was observed in the WIPK/SIPKIR and NtMKP1-over-expressing lines. Accumulation of acidic PR-1 and acidic PR-2 transcripts in these lines after wounding was invariably observed. Accumulation of SA and SA b-glucoside (SAG) in WIPK/ SIPK-silenced and NtMKP1-over-expressing plants after wounding SA functions as a signal that induces expression of acidic PR genes (Linthorst et al., 1990; Malamy et al., 1990). To assess whether the wound induction of acidic PR gene expression in the WIPK/SIPKIR and NtMKP1-over-expressing lines is mediated by SA, the endogenous levels of SA and its sugar conjugate, SAG, were measured 24 h after wounding. The levels of SA in the WIPK/SIPKIR-2 and WIPK/SIPKIR-3 lines were three- and sevenfold higher, respectively, than in the control line (Figure 7a). These two lines also accumulated large amounts of SAG. Similarly, in the NtMKP1-overexpressing lines, wounding caused an accumulation of SA and SAG (Figure 7b). In contrast, no such accumulation was observed in the SIPKIR and WIPKIR lines after wounding. These results suggest that the induction of acidic PR gene expression in wounded WIPK/SIPK-silenced and NtMKP1over-expressing plants is attributable to elevated SA and SAG levels.

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Figure 6. RNA blot analysis of defence-related genes in wounded SIPK-, WIPK-, WIPK/SIPK-silenced and NtMKP1-over-expressing plants. (a) Leaves of the SIPKIR, WIPKIR, WIPK/SIPKIR and control (Cont.) lines were wounded, harvested at the times indicated after wounding, and subjected to RNA blot analysis. (b) Leaves of the NtMKP1OX and control (Cont.) lines were wounded, harvested at the times indicated after wounding, and subjected to RNA blot analysis.

Discussion The results obtained from JA quantification in WIPKsilenced plants indicate that WIPK is involved in the production of wound-induced JA, and confirm our previous studies (Seo et al., 1995, 1999). The reduced JA production in SIPK-silenced plants indicates that SIPK also participates in JA production in wounded tobacco plants. Support for this role for WIPK and SIPK can also be found from a recent report indicating that silencing of a tobacco receptor-like protein kinase reduces activation of both WIPK and SIPK and the accumulation of JA after wounding (Takabatake et al., 2006). A gain-of-function analysis showed that overexpression of NtMEK2DD results in activation of WIPK and


Roles of WIPK and SIPK in wound signalling 905 Figure 7. Accumulation of SA and SAG in SIPK-, WIPK-, WIPK/SIPK-silenced and NtMKP1-overexpressing plants after wounding. (a) SA and SAG concentrations (ng g)1, FW) in the SIPKIR, WIPKIR, WIPK/SIPKIR and control lines before (0 h) and after (24 h) wounding. (b) SA and SAG concentrations (ng g)1, FW) in the NtMKP1OX and control lines before (0 h) and after (24 h) wounding. Values are the means SD from three independent measurements.

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SIPK but not an increase in endogenous JA levels (Kim et al., 2003). The simplest explanation for these contradictory results is that activation of both WIPK and SIPK is necessary but not sufficient to induce JA biosynthesis. Wounding induces various physiological responses consisting of a complex network of multiple signal transduction pathways (de Bruxelles and Roberts, 2001; Farmer et al., 1998; Ryan, 2000). It is known that many factors are involved in the regulation of wound-induced JA biosynthesis (Howe, 2004; Huang et al., 2004; O’Donnell et al., 1996). It is thus likely that multiple factors, including WIPK and SIPK, function in the pathways leading to JA biosynthesis in wounded tobacco plants. Because over-expression of NtMEK2DD is thought to activate only the NtMEK2/WIPK/SIPK-mediated signal transduction pathway, it is possible that the activation of both WIPK and SIPK by NtMEK2DD over-expression is insufficient to induce JA biosynthesis. There was no significant difference in the amounts of JA in healthy leaves between the MAPK-silenced and control plants. This suggests that WIPK and SIPK are not involved in constitutive JA production in a healthy, non-wounded state. There are reports suggesting the involvement of MAPKs in the induction of JA biosynthesis in plant species other than tobacco. Treatment of tomato suspension cultured cells or leaves with systemin, a polypeptide that activates JA biosynthesis (Ryan, 2000), caused rapid activation of tomato MAPKs orthologous to SIPK (Holley et al., 2003; Stratmann and Ryan, 1997). Lee et al. (2004) reported that rice (Oryza sativa) plants over-expressing MK1, which encodes the Capsicum orthologue of WIPK, display elevated JA levels. It will be important to determine whether MAPK-mediated JA production occurs in plant species other than tobacco. Because silencing of SIPK affected expression of the Ntf4 gene, we cannot rule out the possibility that repression of Ntf4 activation contributes to the reduction in JA production. The same possibility applies for the NtMKP1-over-expressing plants. In this case, constitutive dephosphorylation of Ntf4 by NtMKP1 would lead to a reduction in Ntf4 activation. Ntf4 is phosphorylated by the same upstream MAPKK, NtMEK2, as

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SIPK and WIPK (Ren et al., 2006; Voronin et al., 2004). Furthermore, Ntf4 has a similar expression pattern as SIPK, and its activation, like that of SIPK, induces hypersensitive cell death (Ren et al., 2006). It is likely then that Ntf4 plays a similar role in wound signalling as SIPK, but this supposition requires further Ntf4 functional studies for confirmation. Although expression of JA biosynthetic genes is transcriptionally induced in response to wounding (Schaller et al., 2005), the physiological function of this induction is not fully understood. It is thought that the rapid production of JA after wounding is regulated by wound-induced substrate generation and/or post-translational modification of pre-existing JA biosynthetic enzymes (Stenzel et al., 2003; Wasternack et al., 2006). It is therefore unclear whether reduced expression of the AOS and AOC genes in MAPKsilenced plants is responsible for reduced JA production in these plants. However, there is a possibility that WIPK and SIPK regulate JA biosynthesis by phosphorylating preexisting enzymes such as AOS and AOC. The mechanism that underlies regulation of JA biosynthesis by WIPK and SIPK remains to be clarified. Expression of the basic PR-1 and PI-II genes was induced by wounding in MAPK-silenced and NtMKP1-over-expressing plants with variable expression patterns. Because JA levels still increased substantially in these plants after wounding, the synthesized JA may be sufficient to induce defence-response genes such as the PR genes. In addition, in MAPK-silenced and NtMKP1-over-expressing plants, wound-induced production of ethylene was not completely abolished. Residual JA and/or ethylene may induce expression of the basic PR-1 and PI-II genes. Because the analysis of basic PR-1 and PI-II gene expression in wounded MAPK-silenced and NtMKP1-over-expressing plants suffered from experimental variation, we could not clarify the role of WIPK and SIPK in the regulation of basic PR-1 and PI-II gene expression. Previously, we have shown that primary transformants over-expressing NtMKP1 exhibit enhanced PI-II gene expression in response to wounding (Yamakawa et al., 2004). This could result from


906 Shigemi Seo et al. varied expression of the PI-II gene. Menke et al. (2004) observed that infection of AtMPK6-silenced Arabidopsis plants with a bacterial pathogen resulted in a reduction in PR-1 gene expression, but the reduction also varied among experiments. These findings indicate that caution should be used when interpreting any correlation between the expression of a given gene and repression of WIPK/AtMPK3 activation and/or SIPK/AtMPK6 activation. Based on the observation that transgenic plants over-expressing WIPK, in which the endogenous WIPK gene is suppressed, exhibit reduced expression of the basic PR-1 and PI-II genes (Seo et al., 1995), we previously concluded that WIPK is involved in expression of the basic PR-1 and PI-II genes. However, as mentioned above, we cannot rule out the possibility that the expression of the basic PR-1 and PI-II genes observed in these WIPK-over-expressing plants is a consequence of a complex set of interactions. Highly controlled studies to determine whether WIPK and SIPK regulate expression of the basic PR-1 and PI-II genes are obviously required. In general, mechanical wounding does not induce SA synthesis (Malamy et al., 1990). However, in WIPK/SIPKsilenced and NtMKP1-over-expressing plants, wounding abnormally induced production of SA and SAG. On the other hand, we found that, despite their reduced JA production, neither WIPK- nor SIPK-silenced plants produce SA or SAG in response to wounding. Although it is known that there is an antagonistic relationship between JA and SA at the biosynthetic and signalling levels (Kariola et al., 2005; Niki et al., 1998; Sano et al., 1996; Spoel et al., 2003), our results suggest that a reduction in JA production does not always result in induction of SA biosynthesis. In fact, it has been reported that JA and SA do not act antagonistically (Mur et al., 2006). One possible explanation for the difference in the induction of SA biosynthesis between these transgenic plants is the presence of a factor (not JA) involved in the inhibition of SA biosynthesis. In this model, the unknown factor and JA would function cooperatively or synergistically to inhibit SA biosynthesis, and its functional expression would require regulation by either WIPK or SIPK. It is therefore possible that, in a situation in which WIPK and SIPK are simultaneously suppressed, the inhibitory effects of the unknown factor and JA on SA biosynthesis induction are impaired, which triggers production of SA and SAG. In the case of a situation in which WIPK or SIPK are suppressed individually, the inhibitory effect of the factor would remain, because the unknown factor is partially activated by the remaining MAPK. Further analysis of the MAPK-silenced and NtMKP1-over-expressing plants is required to test this possibility. We have previously shown that WIPK-overexpressing plants produce SA and SAG in response to wounding (Seo et al., 1995), a phenomenon observed in these WIPK-over-expressing plants that may be explained by the action of the proposed factor. In this case, over-

expression of WIPK may affect expression of the SIPK gene, which would have the result of impairing the inhibitory effects of JA and the unknown factor on SA biosynthesis induction. Our results suggest that WIPK and SIPK function cooperatively in mediating cross-talk between JA and SA at least at the biosynthetic level. SIPK has been reported to regulate SA biosynthesis (Samuel et al., 2005). Other candidates for mediating cross-talk between JA and SA are NtMPK4 (Gomi et al., 2005) and AtMPK4 (Petersen et al., 2000). NtMPK4silenced tobacco plants exhibit inhibited PI-II expression in response to wounding, suggesting that activation of NtMPK4 is required for wound-induced expression of the PI-II gene (Gomi et al., 2005). NtMKP4 is thought to function in signalling pathways between JA and PI-II in the tobacco wound response (Gomi et al., 2005). However, whether or not the wound signal transduction pathways mediated by NtMPK4 and WIPK and SIPK are the same is unknown. The relationship among these three MAPKs in wound signalling remains to be clarified. Experimental procedures Vector construction and tobacco transformation A 472 bp region of the SIPK cDNA (corresponding to positions 1049– 1520; Zhang and Klessig, 1997) was amplified by PCR, and the resulting products were inserted into the BamHI and KpnI sites of a modified (Ohtsubo et al., 1999) binary vector, pBE2113 (Mitsuhara et al., 1996), separated by a 989 bp region of the GUS gene (corresponding to positions 821–1809; Jefferson et al., 1986), to obtain the SIPKIR construct. Likewise, the amplified fragments of a 422 bp region of the WIPK cDNA (corresponding to positions 979–1400; Seo et al., 1995) were inserted into pBE2113 to obtain the WIPKIR construct. The amplified SIPK and WIPK fragments were ligated and inserted into pBE2113 to obtain the WIPK/SIPKIR construct. All constructs were verified by sequencing. The three pBE2113 constructs were used to transform tobacco (Nicotiana tabacum cv. Samsun NN) by the leaf disc co-cultivation method with A. tumefaciens LBA4404 (Horsch et al., 1985). Kanamycin-resistant transformants were screened for a reduction in the wound-induced MBP kinase activity of SIPK, WIPK or both. The generation of transgenic tobacco plants over-expressing the NtMKP1 cDNA has been described previously (Yamakawa et al., 2004).

Plant growth conditions, wound stress treatment and inoculation with TMV Kanamycin-resistant seedlings from T1 seeds of the transgenic lines were transferred to pots containing vermiculite and grown in a chamber maintained at 25C with 16 h of light (120 lmol m)2 sec)1). Unless otherwise stated, the upper and middle fully expanded leaves of 2-month-old plants were used for inoculation with TMV and wound stress treatments. Inoculation of leaves with TMV was performed as described previously (Seo et al., 2003). All of the inoculated leaves were incubated at 30C for 48 h, followed by incubation at 20C for 24 h under continuous light. Leaves to be used for stress treatments were cut into small pieces with a razor blade.


Roles of WIPK and SIPK in wound signalling 907 RNA extraction, RNA blot and RT-PCR analyses Total RNA was extracted using TRIzol reagent (Invitrogen, http:// www.invitrogen.com/) according to the manufacturer’s instructions. RNA blot analysis was performed as described previously (Seo et al., 1999). RT-PCR was performed using an RT-PCR High-Plus kit (Toyobo, http://www.toyobo.co.jp) and the following primers: for WIPK, 5¢CGGTGGAGGTCAATTCCCTG-3¢ and 5¢-CATTTACCAAAAGGTTGCTC-3¢; for SIPK, 5¢-GATGATGTCTGATGCGGGGGCGG-3¢ and 5¢GACAGTGCTCCTCAGATAAA-3¢; for NtMPK4, 5¢-GATCACCTGATGATGCCAGT-3¢ and 5¢-GAGCTGTGAACTCACCTCCA-3¢; for Ntf3, 5¢GGAGCTCTTAGGGAGGAAAC-3¢ and 5¢-GCGCGGAAACCTTATTAGAG-3¢; for Ntf4, 5¢-TGTACACCAGCTACGTCTGC-3¢ and 5¢-GAAAGTCACACAGACATTTCC-3¢; for NtMKP1, 5¢-TTGCCGATACTAGTAAAGAGGAGTTAATGT-3¢ and 5¢-CATGACCATCCTCACACATG-3¢; for actin, 5¢-CAGGGTTTGCTGGAGATGATGCTC-3¢ and 5¢-TGAATGCCTGCAGCTTCCATTCC-3¢.

Protein extraction and immunoblot analysis Protein extraction was performed according to the procedure described by Seo et al. (1999), except that there was no MgCl2 in the extraction buffer. Samples (50 lg of total protein per lane) were separated by electrophoresis on SDS–polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore, http://www.millipore.com). After blocking with 5% w/v skim milk, membranes were incubated with affinity-purified anti-WIPK, antiSIPK or anti-NtMKP1 (Katou et al., 2005a) antibodies in 3% skim milk at a 1:2000 dilution. The membranes were incubated with alkaline phosphatase-conjugated anti-rabbit IgG antibody (Sigma http:// www.sigmaaldrich.com) at a dilution of 1:2000 for 1 h at room temperature. After washing, the antibody–antigen complexes were visualized with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium (KPL, http://www.kpl.com). For a highly sensitive immunoblot analysis of the WIPK protein, 200 lg of total protein was incubated with 16 lg of affinitypurified anti-WIPK antibodies for 1 h on ice. After binding for 2 h at 4C to 20 ll of protein A–Sepharose (Amersham, http:// www.amershambiosciences.com/), the immunoprecipitates were washed four times with immunoprecipitation buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 10 mM b-glycerophosphate, 10 mM NaF and 1 mM Na3VO4). Sepharose beads were then suspended in 20 ll antigen peptide (0.1 mg ml)1) used for the generation of anti-WIPK antibodies (Seo et al., 1999), and incubated for 30 min on ice. After centrifugation at 20 000 g for 5 sec, the supernatant was recovered. Elution by the antigen peptide was repeated twice. Supernatants obtained from three elutions were combined and concentrated under vacuum. The residue was suspended in SDS gel loading buffer and subjected to immunoblot analysis with anti-WIPK antibodies as described above.

Kinase assays MBP kinase activity of SIPK, WIPK and NtMPK4 was measured as described previously (Seo et al., 1999).

Quantification of JA and SA Extraction and quantification of JA were performed essentially as described previously (Baldwin et al., 1997), except that [2H2] ()-JA was used as an internal standard (Nojiri et al., 1992). Quantification

of SA and SAG was performed as described previously (Seo et al., 1995).

Measurement of ethylene For measurement of ethylene released from seedlings, T1 seeds of transgenic lines were plated on half-strength Murashige and Skoog medium containing 50 mg l)1 kanamycin in a polystyrene Petri dish and grown in a chamber maintained at 25C with 16 h of light. Fourteen days after seeding, the dish was sealed with vinyl tape and incubated under continuous light. After 24 h, a sample was withdrawn from the headspace and analysed for ethylene as described previously (Ohtsubo et al., 1999). For measurement of ethylene released from wounded leaves, leaf discs punched out from each line were incubated in a sealed 25 ml glass vial under 100% humidity. After 2 h, a 1 ml sample was withdrawn from the headspace and analysed for ethylene.

Acknowledgements This work is supported by the Program for the Promotion of Basic Research Activities for Innovative Biosciences. This work is supported in part by grant from the Japan Society for the Promotion of Science Research Fellowship for Young Scientists (to S.K.).

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