Research ArticleResearch Article
Open Access

The Mitogen-Activated Protein Kinase Cascade MKK3–MPK6 Is an Important Part of the Jasmonate Signal Transduction Pathway in Arabidopsis

Fuminori Takahashi, Riichiro Yoshida, Kazuya Ichimura, Tsuyoshi Mizoguchi, Shigemi Seo, Masahiro Yonezawa, Kyonoshin Maruyama, Kazuko Yamaguchi-Shinozaki and Kazuo Shinozaki
The Plant Cell March 2007, 19 (3) 805-818; DOI: https://doi.org/10.1105/tpc.106.046581

Abstract

The plant hormone jasmonic acid (JA) plays a key role in the environmental stress responses and developmental processes of plants. Although ATMYC2/JASMONATE-INSENSITIVE1 (JIN1) is a major positive regulator of JA-inducible gene expression and essential for JA-dependent developmental processes in Arabidopsis thaliana, molecular mechanisms underlying the control of ATMYC2/JIN1 expression remain largely unknown. Here, we identify a mitogen-activated protein kinase (MAPK) cascade, MAPK KINASE 3 (MKK3)–MAPK 6 (MPK6), which is activated by JA in Arabidopsis. We also show that JA negatively controls ATMYC2/JIN1 expression, based on quantitative RT-PCR and genetic analyses using gain-of-function and loss-of-function mutants of the MKK3–MPK6 cascade. These results indicate that this kinase unit plays a key role in JA-dependent negative regulation of ATMYC2/JIN1 expression. Both positive and negative regulation by JA may be used to fine-tune ATMYC2/JIN1 expression to control JA signaling. Moreover, JA-regulated root growth inhibition is affected by mutations in the MKK3–MPK6 cascade, which indicates important roles in JA signaling. We provide a model explaining how MPK6 can convert three distinct signals—JA, pathogen, and cold/salt stress—into three different sets of responses in Arabidopsis.

INTRODUCTION

Plants have the ability to cope with unfavorable environmental factors such as biotic and/or abiotic stress. When plants perceive such stresses, they immediately activate signaling machinery, including gene expression, to change their physiological status as a defense mechanism. One of the universal signaling module functions in response to such external stimuli is the mitogen-activated protein kinase (MAPK) cascade. The MAPK cascade is evolutionarily conserved among the eukaryotes and typically consists of three types of protein kinases, MAPK, MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK) (Chen et al., 2001). By responding to external stimuli, the MAPK cascade plays a critical role in gene expression, metabolism, cell death, proliferation, and differentiation in animals and yeast (Widmann et al., 1999; Chen et al., 2001).

In plants, the MAPK cascade is also involved in various biotic and abiotic stress responses, hormone responses, cell division, and developmental processes (Nakagami et al., 2005). Complete sequencing of the Arabidopsis thaliana genome has revealed that the MAPK family consists of >60 MAPKKK, 10 MAPKK, and 20 MAPK genes (Ichimura et al., 2002). Despite the large number of MAPKs, only three (MPK3, MPK4, and MPK6) have been studied in detail. A biochemical analysis revealed that numerous biotic and abiotic stresses activate these MAPKs (Nakagami et al., 2005). Recently, reverse genetic approaches have been used to elucidate the in planta function of these MAPKs. MPK4 is a negative regulator of systemic acquired resistance and a positive regulator of PDF1.2 expression through EDS1 and PAD4, essential components of salicylic acid–mediated defense responses (Petersen et al., 2000; Brodersen et al., 2006). The substrate protein of MPK4 is MKS1 (Andreasson et al., 2005). Lu et al. (2002) proposed that MPK3 plays a role in abscisic acid–induced postgermination growth arrest. Silencing of MPK6 compromised both gene-for-gene and basal resistance in Arabidopsis (Menke et al., 2004). MPK6 and MPK3 appear to be required for ozone tolerance and reciprocally inhibit activity and duration (Miles et al., 2005).

Several studies using the constitutive active form of Arabidopsis MAPKK have identified downstream MAPKs. For example, MKK4 and MKK5 can activate both MPK3 and MPK6 (Asai et al., 2002; Ren et al., 2002). The 1-aminocyclopropane-1-carboxylic acid (ACC) synthases ACS2 and ACS6 are substrates for MPK6, regulated by MPK6 phosphorylation toward ethylene (ET) induction (Liu and Zhang, 2004). This MKK4/MKK5–MPK3/MPK6 cascade also participates in the proposed microbe-associated molecular pattern signaling pathway containing MEKK1 as a MAPKKK (Asai et al., 2002; He et al., 2006). Other approaches, such as yeast two-hybrid (Y2H) analysis and yeast complementation, suggest that MEKK1 functions upstream of the MKK1/MKK2–MPK4 cascade (Ichimura et al., 1998; Mizoguchi et al., 1998). This cascade plays a role in the MEKK1–MKK2–MPK4/MPK6 cascade during cold and salt stress in Arabidopsis (Teige et al., 2004). Several studies strongly suggest that MKK1 is also the upstream factor of MPK4 and mediates bacterial elicitor, wounding, and H2O2 signals (Huang et al., 2000; Matsuoka et al., 2002; Teige et al., 2004). Although these MAPKs are simultaneously activated by many types of stress, the presence of different phenotypes suggests that MAPK cascade regulation in plants could be much more complicated than it appears.

Jasmonic acid (JA) is an important regulator in response to environmental stresses such as ozone exposure, wounding, water deficit, and pathogen and pest attack (Berger, 2002; Devoto and Turner, 2003). In Arabidopsis, mechanical and biotic wounding activates independent signaling pathways regulating different sets of target genes either at the injury or the systemic site (Leon et al., 2001). JA induces systemic gene expression of VSP, JR1, and Thi2.1 in response to wounding, but it is negatively regulated by the local synthesis of ET (Rojo et al., 1999). However, JA and ET cooperate synergistically to activate basic pathogenesis-related (PR) proteins such as β-CHI, PR3, and PDF1.2 (Xu et al., 1994; Penninckx et al., 1998). Studies using protein kinase/phosphatase inhibitors have indicated that protein kinase activity may negatively regulate JA-induced systemic gene expression and positively regulate JA/ET-induced local gene expression (Rojo et al., 1998). Transgenic tobacco (Nicotiana tabacum) that overexpresses WIPK, a tobacco MPK3 homolog, shows an increased JA level and constitutive expression of JA-inducible Proteinase inhibitor II (Seo et al., 1999). In tobacco, however, JA activated neither SIPK, a tobacco MPK6 homolog, nor WIPK (Kumar and Klessig, 2000). Thus, whether JA directly activates MAPK, leading to the induction of local and/or systemic response genes, remains unknown.

To date, six MAPKKs (MKK1, -2, -4, -5, -6, and -7) have been identified and analyzed in Arabidopsis (Nakagami et al., 2005; Dai et al., 2006). MKK3 is one of the least characterized MAPKKs and has not yet been analyzed phenotypically or biochemically. Here, we describe the functional characterization of Arabidopsis MKK3, a group B MAPKK (Ichimura et al., 2002) and a homolog of tobacco NPK2 (Shibata et al., 1995). MKK3 has a unique domain composition with a C-terminal domain similar to that of nuclear transport factor 2 (Ichimura et al., 2002). Biochemical analyses in planta and in vitro showed that MPK6 is a downstream target of MKK3. Microarray analysis revealed that MKK3 plays a role in JA signaling. Indeed, MPK6 was activated by JA, and this activation depended largely on the MKK3 manner. Loss-of-function and gain-of-function analyses revealed that the MKK3–MPK6 cascade negatively regulates ATMYC2 in both JA-dependent gene expression and inhibition of root growth. Our data indicate that this MKK3–MPK6 cascade is part of an important JA signal transduction pathway in Arabidopsis. Possible roles of the MKK3–MPK6 cascade in JA signaling are discussed.

RESULTS

MKK3 Activates MPK6 Both in Vitro and in Vivo

For their activation, MAPKKs require the phosphorylation of conserved Ser (S) and/or Thr (T) residues in the kinase activation loop (Marshall, 1994). Constitutive activation of the MAPKKs often results from substitution of these S/T residues with Asp (D) or Glu (E), and this method has been used to activate target MAPKs both in vitro and in vivo (Cowley et al., 1994; Mansour et al., 1994). We produced a constitutively active form of MKK3 by substituting S/T with D/D (MKK3DD) and tested whether MKK3 activated the Arabidopsis MAPKs in vitro. We prepared glutathione S-transferase (GST)-tagged wild-type (WT) and DD forms of MKK3, and various GST–MPK proteins, from Escherichia coli. An in vitro phosphorylation assay using kinase-inactive MAPKs as a substrate indicated that MKK3 directly phosphorylates MPK6 (see Supplemental Figure 1A online). Consistently, MKK3WT specifically activated MPK6 (Figure 1A ; see Supplemental Figure 1B online). In some cases, MKK3WT also appeared to activate MPK1 and MPK2 slightly (see Supplemental Figure 1B online). MKK3DD-activated MPK6 showed more kinase activity than MKK3WT-activated MPK6, suggesting that MPK6 may be a downstream MAPK of MKK3 in Arabidopsis.

Figure 1.
Figure 1.

MKK3 Specifically Activates MPK6.

(A) An in vitro activation assay of MKK3. Affinity-purified GST–MPKs and GST–MKK3 were expressed in E. coli. Either MKK3WT (WT) (1 μg) or constitutively active MKK3DD (DD) (1 μg) was incubated with (+) or without (–) each MPK (1 μg) in the kinase reaction mixture with MBP as a substrate, and aliquots of the samples were separated by SDS-PAGE and subjected to autoradiography. Coomassie blue (CBB) staining of the GST–MPKs (white arrowheads) and GST–MKKs (black arrowheads) is shown in the bottom panel.

(B) Enhanced expression of MKK3DD by DEX activates endogenous MPK6 in planta. Steroid-inducible transgenic plants carrying the wild-type or constitutively active MKKs were treated with DEX at the indicated times. The kinase activities of the crude extracts (20 μg) and immune complexes by Ab6NT1 were subsequently analyzed by an in-gel kinase assay. MPK6 protein levels were determined by immunoblot analysis using Ab6NT1 (bottom panels).

(C) MBP kinase activity of MPK1, MPK3, MPK4, and MPK6 in MKK3DD and MKK4DD plants. The MKK3DD- and MKK4DD-induced activation of MPK1, MPK3, MPK4, and MPK6 was determined using an IP-kinase assay. MPK1, MPK3, MPK4, and MPK6 were immunoprecipitated with specific antibodies, and their activity was measured using MBP as a substrate.

(D) Phenotype observed in MKK3DD and MKK4DD plants. The 7-d-old seedlings grown without DEX were transferred to agar plates containing 5 μM DEX. These photographs were taken 5 d after the seedling was transferred. The two independent lines of each transgenic plant had similar phenotypes (data not shown).

(E) H2O2 generation in MKK3DD and MKK4DD plants. The 7-d-old seedlings grown without DEX were transferred to agar plates containing 5 μM DEX for 5 d. H2O2 production was detected by the polymerization of diaminobenzidine.

(F) ET production in steroid-inducible MKK3DD and MKK4DD plants. The 14-d-old seedlings grown without DEX were transferred to 10 μM DEX solution for the indicated times. ET accumulation was measured at the indicated times. Values are means ± sd of three measurements (each with triplicate samples). F.W., fresh weight.

Nt MEK2DD, an active form of tobacco MKK4/MKK5 homolog, fused to a steroid-inducible promoter activates endogenous MPK6/MPK3 in Arabidopsis as well as SIPK/WIPK in tobacco (Yang et al., 2001; Ren et al., 2002). We used this system to analyze MPK6 activation by MKK3 in planta. Four hours after dexamethasone (DEX) treatment, the 46-kD protein kinase was clearly activated in MKK3DD plants (Figure 1B, top panels). By contrast, MKK3WT only slightly activated the 46-kD kinase. An immunocomplex kinase (IP-kinase) assay using the MPK6-specific antibody Ab6NT1 indicated that this 46-kD protein kinase was MPK6 (Figure 1B, middle panels). The protein levels of MPK6 were almost equal during this experiment (Figure 1B, bottom panels). MKK4DD also activated MPK6, as reported previously (Figure 1B, top panels) (Asai et al., 2002). The activation of MPK6 was similarly observed in the two independent lines of each transgene (data not shown).

To further confirm the identity of the activated kinases, we used specific antibodies raised against MPK1, MPK3, MPK4, and MPK6 for IP-kinase assays, using myelin basic protein (MBP) as a substrate. In MKK3DD plants, MPK6 was activated after 4 h of DEX treatment, but the MBP-kinase activity of MPK1, MPK3, and MPK4 was not affected (Figure 1C). These results suggest that the activation of MPK6 by MKK3 is specific. In MKK4DD plants, MPK3 and MPK6 were activated, as reported previously (Asai et al., 2002). These results indicate that the overexpression of constitutively active MKK3DD activates MPK6 in planta.

We compared the morphological and physiological phenotypes of the MKK3DD and MKK4DD plants. As reported previously, MKK4DD plants exhibited strong hypersensitive response–like (HR-like) cell death only when treated with DEX (Figure 1D) (Ren et al., 2002; Kim et al., 2003). However, MKK3DD, MKK3WT, and control plants did not show HR-like cell death with or without DEX treatment (Figure 1D; data not shown). Because reactive oxygen species production often accompanies HR-like cell death, we used diaminobenzidine staining to determine whether H2O2 production was involved in this HR-like cell death phenotype. MKK4DD plants exhibited the reddish brown precipitates of oxidized diaminobenzidine that indicate H2O2 production (Figure 1E), whereas the stain was not detected in either MKK3DD or control plants. MKK4-activated MPK6 phosphorylates ACS2 and ACS6, ACC synthases, and a rate-limiting enzyme for ET biosynthesis and enhances ET production (Liu and Zhang, 2004). Therefore, we measured the ET levels in both MKK4DD and MKK3DD plants. DEX-treated MKK4DD plants showed higher levels of ET accumulation than control plants and MKK3DD plants (Figure 1F). We also tested the ET-dependent phenotype in these plants and found a triple response in dark-grown, DEX-treated MKK4DD plants but not in MKK3DD plants (see Supplemental Figure 2 online). These results suggest that MKK3 and MKK4 have distinct roles in Arabidopsis, although both MAPKKs activate MPK6.

MKK3DD Affects mRNA Levels of JA- and ET-Regulated Genes

To identify genes acting downstream of MKK3, we used a microarray analysis to compare the transcript levels of MKK3DD and MKK4DD plants. The steroid-inducible overexpression system shown in Figure 1B was applied to rule out any possibility of artificial effects, which are sometimes associated with constitutive overexpression systems. Because MPK6 activation becomes significant at 2 to 4 h after DEX treatment (Figure 1B), we prepared total RNA from seedlings treated with DEX for 4 h. The transcriptome profiles revealed that 56 and 100 genes increased in expression more than fivefold in MKK3DD and MKK4DD plants, respectively (Figure 2A ; see Supplemental Tables 1 and 2 online). Among these genes, only 17 were coregulated by both MKK3DD and MKK4DD (see Supplemental Table 3 online), suggesting that MKK3 and MKK4 have different roles in the regulation of gene expression in Arabidopsis.

Figure 2.
Figure 2.

Gene Profiling Analysis of MKK3DD and MKK4DD Transgenic Plants.

(A) Venn diagrams showing a fivefold or greater expression difference in control plants. Total RNA extracted from seedlings after 4 h of DEX treatment was subjected to microarray analysis.

(B) and (C) Relative expression of pathogen- and wounding-inducible genes in MKK3DD and MKK4DD plants. Total RNA was isolated from DEX-treated transgenic plants and subjected to QRT-PCR analysis. Transgenic plants carrying empty vectors were used as a control. The transcript levels of LOX2, PDF1.2, ACS6, and FRK1 were normalized to the expression of β-actin measured in the same RNA samples. Black bars represent MKK3DD plants, and white bars indicate MKK4DD plants. Data are means ± sd of three independent experiments.

MKK3DD and MKK4DD had distinct effects on the expression of JA- and/or ET-regulated genes. Among them, we chose four genes, LOX2, PDF1.2, ACS6, and FLG22-INDUCED RECEPTOR-LIKE KINASE1 (FRK1), as markers for a quantitative real-time PCR (QRT-PCR) analysis to further compare the possible roles of MKK3 and MKK4 in the JA and ET signaling pathways. LOX2 encodes a chloroplast-targeted lipoxygenase that may be involved in the biosynthesis of JA (Bell et al., 1995). PDF1.2 defensin gene expression is induced by pathogen infection (Alternaria brassicicola) and by treatment with JA and ET (Penninckx et al., 1998). ACS6 gene expression is induced by stress (Wang et al., 2002), and the ACS6 protein is phosphorylated by MPK6 (Liu and Zhang, 2004). FRK1 encodes a Leu-rich repeat receptor kinase and is induced by pathogens. Overexpression of either MKK4DD or MKK5DD increases FRK1 expression (Asai et al., 2002). The LOX2 and PDF1.2 genes were upregulated as the activation of the MKK3 cascade was initiated (Figure 2B), and the expression levels of these genes increased in MKK4DD plants after 12 h of DEX treatment. Expression levels of the ACS6 and FRK1 genes increased in MKK4DD plants but not in MKK3DD plants (Figure 2C). Considering the clear difference between MKK3DD and MKK4DD plants in terms of gene expression upon DEX treatment, we postulated that MKK3DD induction may modulate JA signaling in Arabidopsis.

MPK6 Is Activated by JA

We first tested whether JA activated MPK6 in Arabidopsis. We obtained two T-DNA insertion lines of MPK6 (mpk6-4 and mpk6-5) (Figure 3A ), both of which were confirmed as null mutants by immunoblotting (Figure 3B). The mpk6-4 mutant and the plants constitutively overexpressing MPK6 (35S-MPK6-Myc) were then treated with JA and analyzed to determine whether JA activates MPK6 using an in-gel kinase assay (Figure 3C, top panel). Wild-type plants showed JA-dependent activation of the 46-kD protein kinase (MPK6), whereas these activations in the mpk6-4 mutant were specifically diminished. Interestingly, the activity of the 44-kD protein kinase was higher in mpk6-4 than in wild-type plants. The predicted molecular mass of the MPK6 protein with the Myc tags was ∼65 kD in 35S-MPK6-Myc#15 plants, and this protein was recognized by Ab6NT1 (Figure 3C, middle panel). JA activated not only endogenous MPK6 but also the fusion protein MPK6-Myc. We also tested the effect of the JA-insensitive coi1 mutation on MPK6 activation. The coi1 mutation specifically reduced JA-dependent MPK6 activity (Figures 3D and 3E). These data show that JA activates MPK6 and that full MPK6 activation by JA requires COI1.

Figure 3.
Figure 3.

MPK6 Is Activated by JA.

(A) T-DNA knockout mutants of MPK6. Exons are indicated as white boxes. T-DNA is inserted in the second intron (mpk6-4) and the fifth exon (mpk6-5).

(B) Immunoblot analysis of MPK6 in wild-type (Col. [for Columbia]) and mpk6 null plants. Equal protein loading was confirmed by staining the large subunit of ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco LSU) with Coomassie blue.

(C) JA-dependent activation of MPK6. Wild-type, mpk6-4, and 35S-MPK6-Myc#15 plants were treated with 50 μM JA for 10 min, and their protein extracts were subjected to in-gel kinase assay (top panel). Immunoblot analyses of MPK6 (middle panel) and Rubisco LSU stained by Coomassie blue (bottom panel) are shown as additional controls for equal protein amounts.

(D) JA-dependent activity in the coi1 mutant. Wild-type and coi1 plants were treated with JA for the indicated times, and their protein extracts were subjected to in-gel kinase assay.

(E) Relative 46-kD kinase activities in wild-type and coi1 plants in response to 50 μM JA.

(F) Effects of JA on the activation of MPK4 and MPK6 in wild-type plants. MBP kinase activities of MPK4 and MPK6 were determined by IP-kinase assay. MPK4 and MPK6 were immunoprecipitated with specific antibodies, and their activities were measured in tube using MBP as a substrate.

Arabidopsis MPK4 is important for both systemic acquired resistance repression and ET/JA defense signaling (Petersen et al., 2000; Brodersen et al., 2006). Therefore, we examined whether JA actually activates MPK4 in planta using IP-kinase assay, with MBP as a substrate. JA did not activate MPK4 but did activate MPK6, as shown (Figure 3C), with peaking at 5 to 10 min in wild-type plants (Figure 3F). No MPK4 activation by JA is consistent with the results reported by Brodersen et al. (2006).

MKK3 Plays a Key Role in the Activation of MPK6 by JA

To test whether MKK3 was involved in the activation of MPK6 by JA, we analyzed MPK6 activation in wild-type, mkk2-2, mkk3-1, and constitutively overexpressing MKK3 plants (35S-MKK3). The mkk3-1 and mkk2-2 mutants were T-DNA insertion lines of MKK3 and MKK2, respectively. RT-PCR analysis revealed that mkk3-1 and mkk2-2 were null mutants (Figures 4A and 4B ). An in-gel kinase assay showed that the 46-kD activity of MPK6 in wild-type plants was activated by JA (Figures 4C and 4D). As a MAPKK control, we performed the same experiment using the mkk2-2 mutant. No significant difference in MPK6 activation by JA was observed between the mkk2-2 mutant and wild-type plants. Interestingly, MPK6 was only slightly activated in mkk3-1 at 10 min, and the activation was much less than that in wild-type plants. By contrast, in the 35S-MKK3 plants, JA-dependent MPK6 activation was significantly higher than that in wild-type plants. Because JA contains a carboxyl group, we tested whether weak acid treatment (0.012% ethanol solution, pH 4.8) affected MPK6 activity in wild-type plants. Although we detected a slight increase in the 46-kD kinase (MPK6) activity, the activation by this treatment was much weaker than that by JA. This finding shows that the MAPK activation by JA treatment did not occur in response to the low pH of the treatment.

Figure 4.
Figure 4.

MKK3 Mediates the JA-Dependent Activation of MPK6.

(A) T-DNA knockout mutants of MKK3 and MKK2. Exons are indicated as white boxes. T-DNA is inserted in the seventh exon (mkk3-1) and the second intron (mkk2-2).

(B) RT-PCR analysis of MKK3 and MKK2 in wild-type, mkk3-1, and mkk2-2 plants.

(C) Effects of MKK3 on the JA-dependent activation of MPK6. Wild-type, mkk2-2, mkk3-1, and 35S-MKK3#13 plants were treated with JA. As controls, wild-type plants were treated with 0.012% ethanol adjusted to pH 4.8 or unadjusted (Cont.).

(D) Relative 46-kD kinase activities in wild-type, mkk2-2, mkk3-1, 35S-MKK3#13, pH 4.8, and control plants.

(E) Effects of JA on the activation of MPK6 and immunoblot analysis of MPK3, MPK4, and MPK6 proteins in wild-type, mkk2-2, mkk3-1, 35S-MKK3#13, and mpk6-4 plants. Samples were taken at 0 and 10 min after JA treatment and used for IP-kinase assay and immunoblot analysis. Equal protein loading was confirmed by staining the Rubisco LSU with Coomassie blue.

To observe MPK6 activity more directly in the different genotypes used in Figure 4C, we performed an IP-kinase assay of MPK6. This assay clearly demonstrated again that MPK6 is strongly activated by JA in wild-type and mkk2-2 plants, whereas the 35S-MKK3 plant and the mkk3-1 mutant had enhanced and decreased JA-dependent MPK6 activity, respectively (Figure 4E). These findings confirm the results shown in Figure 4C and also show that MKK3 is upstream of MPK6 in JA signaling. Because protein kinase activities of MPK6 were changed in mkk3 and 35S-MKK3, we compared the protein levels of MPK6, MPK3, and MPK4 in wild-type, mkk2-2, mkk3-1, 35S-MKK3#13, and mpk6-4 plants. Immunoblot analysis with specific MAPK antibodies showed that protein levels remained constant with or without JA treatment in these mutants (Figure 4E). These results indicate that the changes in MPK6 activity in different mutant backgrounds were not caused by variation in the protein levels but rather by posttranslational modification of MPK6. These results also suggest that the loss of function and gain of function of MKK3 mainly affected the activity of MPK6.

The MKK3–MPK6 Cascade Affects JA-Regulated Root Growth Inhibition

Exogenously applied JA inhibits root growth in Arabidopsis (Staswick et al., 1992; Feys et al., 1994; Berger et al., 1996). Therefore, we investigated whether JA-dependent control of root growth is affected by loss-of-function mutants and transgenic overexpressors of MKK3 and MPK6 using the JA signaling–related mutants coi1 and atmyc2-3 as controls. The root growth of mkk3-1 and transgenic plants was similar to that of wild-type plants without JA (Figures 5A and 5B ). The mpk6 mutant plants had accelerated root growth compared with wild-type plants. Increasing the concentration of JA caused severe root growth inhibition in two independent alleles of mpk6 mutants. JA-mediated root growth inhibition was also observed in mkk3-1 compared with wild-type plants. This phenotype was rescued by 35S-MKK3, indicating that the JA-sensitive phenotype of the mkk3-1 plants is caused by the loss of function of MKK3 (see Supplemental Figure 3 online). On the other hand, the mkk2-2 mutant was inhibited to a similar level as the wild type. By contrast, two independent lines each of 35S-MKK3 and 35S-MPK6-Myc plants showed less sensitivity to JA, at a level comparable to that of atmyc2-3. The coi1 mutant showed a strong insensitive phenotype to JA, as reported previously (Feys et al., 1994). The mkk2 alleles were already described to have less sensitivity to salt stress (Teige et al., 2004). Compared with wild-type plants, the mkk2-2 mutant also showed decreased sensitivity to 100 mM NaCl, but mkk3-1 did not (Figures 5C and 5D). These results support our hypothesis that the MKK3–MPK6 cascade specifically regulates JA signaling but not the salinity stress response.

Figure 5.
Figure 5.

MKK3 and MPK6 Affect JA-Dependent Root Growth Sensitivity.

(A) Effects of JA on the inhibition of root growth in wild-type (lane 1), mkk2-2 (lane 2), mkk3-1 (lane 3), mpk6-4 (lane 4), mpk6-5 (lane 5), 35S-MKK3#13 (lane 6), 35S-MKK3#14 (lane 7), 35S-MPK6-Myc#15 (lane 8), 35S-MPK6-Myc#16 (lane 9), coi1 (lane 10), and atmyc2-3 (lane 11) plants. Seedlings were grown on Murashige and Skoog (MS) plates with or without 50 μM JA, and the photographs were taken after a 10-d growth period.

(B) Degree of JA-dependent root growth inhibition in each genotype. Root length of these plants was measured after the 10-d growth period. Root growth, in terms of length, was calculated from the results of three independent experiments (n = 8 each). Error bars indicate sd.

(C) Effects of salt on the germination of MKK null mutants. Seedlings of the wild type, mkk2-2, and mkk3-1 were grown on MS plates with (right panels) or without (left panels) 100 mM NaCl, and the photographs were taken after a 10-d growth period.

(D) Germination rates in each genotype. White and black bars represent 0 and 100 mM NaCl, respectively.

The MKK3–MPK6 Cascade Affects Gene Expression Controlled by JA

The PDF1.2 and VSP2 genes are commonly used markers for JA signaling. PDF1.2 is regulated by the JA/ET pathway (Penninckx et al., 1998), and VSP2 is regulated by the JA pathway (Berger et al., 1996), but crosstalk occurs between these two pathways, as revealed by their antagonistic regulation (Boter et al., 2004; Lorenzo et al., 2004). To elucidate the role of the MKK3–MPK6 cascade in the crosstalk between the JA and JA/ET pathways, we investigated mRNA levels of PDF1.2 and VSP2 genes in both loss-of-function mutants and transgenic overexpressors of MKK3 and MPK6 (Figure 6A ). In the absence of JA, the basal expression of PDF1.2 in the 35S-MPK6-Myc plant was higher than that in wild-type plants. JA significantly increased the level of the PDF1.2 transcript in the 35S-MKK3 and 35S-MPK6-Myc plants. The expression level of PDF1.2 in atmyc2-3 was also higher than that in wild-type plants, as reported previously (Boter et al., 2004; Lorenzo et al., 2004). By contrast, the mkk3-1, mpk6-4, and mpk6-5 mutants all showed significantly lower JA-dependent PDF1.2 expression and increased JA-induced VSP2 expression. Consistently, VSP2 expression levels in both 35S-MKK3 and 35S-MPK6-Myc plants were suppressed to an extent similar to that of atmyc2-3. The coi1 mutant largely suppressed JA-dependent expression of the PDF1.2 and VSP2 genes, as reported previously (Devoto and Turner, 2003). The mkk2-2 mutant did not show a significant change in the level of PDF1.2 and VSP2 relative to wild-type plants. These results suggest that the MKK3–MPK6 cascade positively and negatively regulates the expression of PDF1.2 and VSP2, respectively.

Figure 6.
Figure 6.

The MKK3–MPK6 Cascade Negatively Regulates the JA Pathway, Including ATMYC2.

(A) Relative transcription levels of PDF1.2 and VSP2 in wild-type, mkk2-2, mkk3-1, mpk6-4, mpk6-5, 35S-MKK3 (lines 13 and 14), 35S-MPK6-Myc (lines 15 and 16), coi1, and atmyc2-3 plants. Total RNA was isolated from nontreated seedlings (white bars) or seedlings treated with 50 μM JA for 12 h (black bars) and subjected to QRT-PCR analysis. The PDF1.2 and VSP2 transcript levels were normalized to the expression of β-actin measured in the same RNA samples. Data are means ± sd of three independent experiments.

(B) ET production in wild-type, mkk3-1, mpk6-4, 35S-MKK3#13, and 35S-MPK6-Myc#15 plants. The 14-d-old seedlings were treated with (gray bars) or without (white bars) 50 μM JA for 12 h. ET accumulation was measured at the indicated times. Values shown are means ± sd of three measurements (each with triplicate samples). The asterisk represents a significant difference between nontreated wild-type and 35S-MPK6-Myc#15 plants (unpaired t test; P < 0.01). F.W., fresh weight.

(C) Relative transcription levels of PDF1.2 in JA-treated wild-type, 35S-MKK3#13, ein2-5, 35S-MKK3 ein2-5, ein3-1, and 35S-MKK3 ein3-1 plants. Total RNA was isolated from untreated seedlings (white bars) or seedlings treated for 12 h with 50 μM JA (black bars) or 50 μM ACC (gray bars) and subjected to QRT-PCR analysis. PDF1.2 and VSP2 transcript levels were normalized to the expression of β-actin measured in the same samples. Data are means ± sd of three independent experiments.

(D) JA-dependent 46-kD protein kinase activity in wild-type, ein2-5, and ein3-1 plants. These plants were treated with 50 μM JA for the indicated times, and their protein extracts were subjected to in-gel kinase assay.

(E) Relative transcription levels of ATMYC2 in wild-type, mkk2-2, mkk3-1, mpk6-4, mpk6-5, 35S-MKK3 (lines 13 and 14), and 35S-MPK6-Myc (lines 15 and 16) plants. Total RNA was isolated from seedlings treated with 50 μM JA for the indicated times and subjected to QRT-PCR analysis. ATMYC2 transcript levels were normalized to the expression of β-actin measured in the same samples. Data are means ± sd of three independent experiments.

(F) Relative transcription levels of PDF1.2 and VSP2 in wild-type, mkk3-1, atmyc2-3, and mkk3-1 atmyc2-3 plants. Total RNA was isolated from untreated seedlings (white bars) or seedlings treated with JA for 12 h (black bars) and subjected to QRT-PCR analysis. The PDF1.2 and VSP2 transcript levels were normalized to the expression of β-actin measured in the same RNA samples. Data are means ± sd of three independent experiments.

(G) Effects of JA on the inhibition of root growth in wild-type, mkk3-1, atmyc2-3, and mkk3-1 atmyc2-3 plants. Each seedling was grown on MS plates with 50 μM JA. The photographs were taken after a 10-d growth period. Root growth, in terms of length, was calculated from the results of three independent experiments (n = 8 each). Error bars indicate sd.

The MKK3–MPK6 Cascade Functions in Parallel to or Independent of the ET Pathway

To analyze whether the increased PDF1.2 expression by the MKK3–MPK6 cascade is mediated through the ET pathway, we measured ET production in both loss-of-function mutants and transgenic overexpressors of MKK3 and MPK6 treated with or without JA (Figure 6B). The accumulation of ET production in mkk3-1, mpk6-4, and 35S-MKK3#13 plants without JA was similar to that in wild-type plants, and these differences were not statistically significant (mkk3-1, P = 0.397479; mpk6-4, P = 0.329222; 35S-MKK3#13, P = 0.4300025). Only 35S-MPK6-Myc#15 had a greater amount of ET than in wild-type plants, and this difference was statistically significant (35S-MPK6-Myc#15, P = 0.04186 [<0.05]). Basal ET levels increased in 35S-MPK6-Myc#15, possibly as a result of the slight upregulation of ACS, which is a substrate of MPK6 (Liu and Zhang, 2004). Similarly, JA did not significantly affect the ET levels in wild-type, mkk3-1, mpk6-4, 35S-MKK3#13, and 35S-MPK6-Myc#15 plants (wild type, P = 0.871529; mkk3-1, P = 0.875416; mpk6-4, P = 0.617229; 35S-MKK3#13, P = 0.571416; 35S-MPK-Myc#15, P = 0.461129).

We also tested how ET-insensitive ein2 and ein3 mutations affected JA-dependent expression of PDF1.2 by 35S-MKK3. PDF1.2 expression requires concomitant triggering of the JA and ET pathways; if either pathway is not activated, expression cannot occur (Penninckx et al., 1998). In wild-type plants, PDF1.2 gene expression was induced by JA and ACC, as reported previously (Penninckx et al., 1998) (Figure 6C). PDF1.2 expression in the 35S-MKK3 plants treated with JA was higher than that in wild-type plants treated with JA. To test whether ET signaling was required for the JA-dependent induction of PDF1.2 expression, two ET-insensitive mutants, ein2 and ein3, were used to generate the double mutants 35S-MKK3 ein2-5 and 35S-MKK3 ein3-1. The ein2 mutation completely suppressed the JA-dependent induction of PDF1.2 expression in the 35S-MKK3 genetic background. The ein3 mutation also reduced PDF1.2 expression in 35S-MKK3, but suppression by ein3 was weaker than that by ein2. Similar effects by ein2 and ein3 on the JA-dependent induction of PDF1.2 expression were observed in wild-type plants. This may have occurred because EIN3 and its close homologs, EIL1 to EIL3, have redundant functions in ET signaling in Arabidopsis and the ET-insensitive phenotype of ein3 is much weaker than that of ein2 (Stepanova and Alonso, 2005). These results indicate that ET signaling was required for the JA-dependent induction of PDF1.2 expression.

To test whether the ein mutations affect the JA-dependent activation of MPK6, we examined MPK6 activity in ein2-5, ein3-1, and wild-type plants. The MPK6 activity in ein2-5 and ein3-1 mutants was almost the same as that in wild-type plants (Figure 6D). Therefore, the increased levels of PDF1.2 observed in the MKK3–MPK6 cascade may be attributable mainly to JA signaling and be concomitantly upregulated.

The MKK3-MPK6 Cascade Plays a Key Role in the JA-Dependent Negative Regulation of ATMYC2

ATMYC2 encodes a basic helix-loop-helix transcription factor and plays a predominant role in the JA pathway (Boter et al., 2004; Lorenzo et al., 2004). To investigate whether ATMYC2 could function as a downstream factor of the MKK3–MPK6 cascade in JA signaling, the JA-dependent expression of ATMYC2 was tested (Figure 6E). ATMYC2 expression was markedly reduced in the independent lines of 35S-MKK3 and 35S-MPK6-Myc plants, was increased in the JA-treated mkk3-1, mpk6-4, and mpk6-5 mutants, and was similar to that of wild-type plants in the mkk2-2 mutants. This result was unexpected, because JA has been thought to regulate ATMYC2 expression positively. Our results suggest that JA activates the MKK3–MPK6 cascade and are a demonstration of a possible negative regulation of ATMYC2 expression by JA. In addition, these results are consistent with the data on JA-dependent root growth inhibition (Figure 5A).

Next, we analyzed the expression levels of PDF1.2 and VSP2 using a mkk3-1 atmyc2-3 double mutant (Figure 6F). PDF1.2 expression was repressed in the mkk3-1 mutant (Figure 6A). Interestingly, the suppressed PDF1.2 expression in mkk3-1 was reversed to a level similar to that of the atmyc2-3 single mutant by introducing the atmyc2-3 mutation to mkk3-1 (Figure 6F, left panel). The induction of VSP2 expression was consistently repressed in the mkk3-1 atmyc2-3 mutant as well as in the atmyc2-3 mutant (Figure 6F, right panel). These results strongly suggest that ATMYC2 is genetically downstream of MKK3. Along this line, we further examined whether the JA-dependent root growth sensitivity of mkk3-1 was affected by atmyc2-3 (Figure 6G). Without exogenous application of JA, the root growth of these mutants was similar to that of wild-type plants (data not shown). By contrast, mkk3-1 atmyc2-3 showed less sensitivity to JA, with a level comparable to that of atmyc2-3 (Figure 6G). These results indicate that (1) JA regulates ATMYC2 expression both positively and negatively in two independent pathways, (2) ATMYC2 has a role downstream of the MKK3–MPK6 cascade, and (3) the MKK3–MPK6 cascade plays a key role in the JA-dependent negative regulation of ATMYC2.

DISCUSSION

Loss-of-function mutations of mkk3 and mpk6, and transgenic plants overexpressing MKK3 and MPK6, were used to study the roles of the MAPK cascade, MKK3–MPK6, in Arabidopsis. Analyzing these mutants in combination with coi1 and atmyc2-3 revealed that the MKK3–MPK6 cascade plays a key role in JA signaling of Arabidopsis. This is discussed in detail below.

An in Vitro and in Planta Activation of MAPKs by MKK3

We took two approaches to identify the interactions among MEKKs, MAPKKs, and MAPKs in Arabidopsis: Y2H analysis on pairwise protein–protein interactions, and a functional complementation of yeast mutants by cotransformation of MEKK/MAPKK or MAPKK/MAPK. The first potential MAPK cascade in plants, MEKK1–MKK1/MKK2–MPK4 (Ichimura et al., 1998; Mizoguchi et al., 1998), was identified using these approaches. Since then, several other MAPK cascades have been identified using Y2H analysis, functional complementation of yeast mutants by coexpression of the kinases, and transient coexpression assay of those kinases using Arabidopsis mesophyll cell protoplasts. These include MKK4/MKK5–MPK3/MPK6 and MKK2–MPK6 (Asai et al., 2002; Ren et al., 2002; Teige et al., 2004). Here, we used an in vitro activation assay to identify novel functional interactions between MAPKKs and MAPKs. We confirmed MPK6 and MPK3 as target proteins of MKK4 in this system (data not shown), as reported previously, and identified MPK6 as a possible downstream target of one of the least characterized MAPKKs, MKK3. In addition, we note that MPK1 and MPK2 are slightly phosphorylated by MKK3 in vitro (see Supplemental Figure 1B online). Considering that MKK3 interacts with MPK1 in the Y2H analysis (Ichimura et al., 1998) and that MPK1 and MPK2 are closely related group C MAPKs (Ichimura et al., 2002), a functional link may exist between MKK3 and MPK1/MPK2 in Arabidopsis. Alternatively, MPK1 may not be an in vivo target of MKK3, because we did not detect the activation of MPK1 by MKK3DD in our experiment using an IP-kinase assay (Figure 1C). Although MPK1 activity was not affected by MKK3DD under our conditions during the time course we tested, we cannot rule out the possibility that MPK1 may be activated transiently or in specific organs or tissues by MKK3. MPK6 did not interact directly with MKK3 in a Y2H analysis (T. Mizoguchi and K. Shinozaki, unpublished data). It would be worthwhile to test whether MPK6 interacts with MKK3 in vivo.

To determine the specific roles of the MAPKKs in Arabidopsis plants, we used a conditional overexpression system to identify immediate molecular responses in planta. Previous works have reported that overexpression of the constitutively active form of MAPKKs driven by a DEX-inducible promoter can mimic the activation of its MAPK cascade in both tobacco and Arabidopsis (Yang et al., 2001; Ren et al., 2002). Using this system, we confirmed that the novel MKK3–MPK6 cascade identified by the in vitro activation assay in fact functions in Arabidopsis (Figure 1B). Moreover, we found that constitutively active MKK3DD activates MPK6 more strongly in plant cells than that observed in the in vitro assay. This difference may derive from its structural effects, because MKK3 has a longer extra-kinase region on its C terminus than other MAPKKs. The MKK3 protein produced in E. coli cells might not function properly and may require some modification of the C-terminal region to achieve full activation. Domain analysis of the C-terminal region should reveal how it affects MKK3 activation.

Identification of the Protein Kinase Cascade Regulated by the JA Pathway

Pharmacological studies using protein kinase or protein phosphatase inhibitors have revealed the important roles of protein phosphorylation and dephosphorylation in JA signaling (Leon et al., 2001). Treatment of Arabidopsis seedlings with the protein kinase inhibitor staurosporin stimulated the expression of the wound-regulated marker genes VSP and JR1 in the absence of JA. By contrast, the expression of these JA-dependent genes was repressed by the protein phosphatase inhibitor okadaic acid. These results indicate that the JA pathway is negatively regulated by protein kinase cascades. In addition, several reports have suggested that MAPKs are involved in JA signaling (Seo et al., 1999; Petersen et al., 2000; Gomi et al., 2005). However, whether JA in fact activates MAPKs in plants was previously unknown. Here, we have demonstrated JA-dependent MPK6 activity in Arabidopsis plants (Figures 3C and 3F). This study provides evidence that MPK6 is activated by JA in addition to various kinds of abiotic and biotic stresses (Nakagami et al., 2005). Arabidopsis has 10 MAPKKs classified into four groups (A to D) (Ichimura et al., 2002). MKK3 belongs to group B, and its biological function and substrate MAPK remain to be elucidated. This study clearly showed that JA-induced MPK6 activation is dependent mainly on MKK3, because MPK6 activation by JA is strongly repressed in mkk3-1 but enhanced in 35S-MKK3 plants (Figures 4C to 4E). Possibly, other MAPKKs may slightly activate MPK6 in mkk3-1 mutants, because the mkk3 mutation did not completely diminish MPK6 activity. In addition, the coi1 mutation also partially repressed JA-dependent MPK6 activation (Figures 3D and 3E), strongly suggesting a crucial function of the MKK3–MPK6 cascade in JA signaling. However, an apparent inconsistency exists between the degree of JA insensitivity to root growth and the partial suppression of JA-dependent MPK6 activation in the coi1 mutant. Mechanical stress during the experiment could result in MPK6 activation and may explain this inconsistency.

Previous reports indicate that Arabidopsis MPK4 regulates PDF1.2 expression through EDS1/PAD4 downstream or independently of ethylene response factor1 (ERF1) (Petersen et al., 2000; Andreasson et al., 2005). Therefore, we tested whether MPK4 is responsive to JA; however, we did not observe any MPK4 activation (Figure 3F). Considering that MKK3 did not activate MPK4 in planta as well as in vitro (Figures 1A and 1C), we think that MPK4 is not an immediate target of MKK3. In mpk4, ET-induced PDF1.2 induction was blocked (Brodersen et al., 2006). By contrast, 35S-MKK3 plants showed almost wild-type levels of ACC-dependent PDF1.2 induction, not like the JA-dependent PDF1.2 induction (Figure 6C). Because the MKK3-MPK6 cascade should be upstream of ATMYC2, but probably not upstream of ERF1, we think that the MKK3-MPK6 pathway regulates PDF1.2 through ATMYC2 function but the MPK4-EDS1/PAD4 pathway does so downstream or independently of ERF1.

The Function of the MKK3–MPK6 Cascade in the Crosstalk between the JA and JA/ET Pathways in Arabidopsis

We have demonstrated that the novel MKK3–MPK6 cascade plays an important role in JA-controlled root growth and JA- and JA/ET-dependent gene expression (Figures 5 and 6A). Recent studies revealed that the transcription of ET- and JA-induced genes is regulated mainly by two transcription factors, ERF1 and ATMYC2, respectively. ERF1 activates JA/ET-dependent gene expression but represses JA-dependent gene expression (Lorenzo et al., 2003). By contrast, ATMYC2 promotes JA-dependent gene expression and represses JA/ET-dependent genes (Boter et al., 2004; Lorenzo et al., 2004). In Figure 7 , we summarize a model of crosstalk in the JA and ET pathways and MAPK cascades. In this study, we have demonstrated that the MKK3–MPK6 cascade positively regulates the expression of PDF1.2 (Figure 6A) without affecting ERF1 expression (data not shown). Moreover, the JA-activated MKK3–MPK6 cascade did not accumulate ET (Figure 6B), indicating that the MKK3-activated MPK6 does not enhance ET biosynthesis, whereas the MKK4–MPK6 cascade does. Based on these results, we propose a model in which the MKK3–MPK6 cascade may independently mediate the expression of PDF1.2 without affecting ET synthesis. However, as shown previously (Penninckx et al., 1998), 35S-MKK3 ein2-5 and 35S-MKK3 ein3-1 plants further demonstrated that the expression of PDF1.2 regulated by the JA-activated MKK3–MPK6 cascade requires endogenous ET signaling (Figures 6C and 6D). An alternative but still possible explanation for this is that the MKK3–MPK6 cascade might affect ERF activity at the posttranslational level. A rice (Oryza sativa) kinase, BWMK1, belongs to group D of the MAPK family and has been shown to be activated by various stresses and hormones, including JA (Cheong et al., 2003). BWMK1 interacts with a rice homolog of ERFs.

Figure 7.
Figure 7.

Possible Roles of MPK6 Signaling in Arabidopsis.

At least three cascades, MKK2–MPK6, MKK4/MKK5–MPK6, and MKK3–MPK6, function in Arabidopsis. Each plays a specific role in response to different stimuli, and the cascades transduce their signals to adapt to various environmental changes by crosstalking with each other. The MKK2–MPK6 cascade regulates cold and salt stress-responsive genes, the MKK4/MKK5–MPK6 cascade regulates pathogen-responsive genes by activating ET biosynthesis, and the novel MKK3–MPK6 cascade regulates JA signaling. The JA-activated MKK3–MPK6 signal negatively regulates the JA pathway and affects JA-dependent gene expression and root growth sensitivity through ATMYC2.

We also found that the MKK3–MPK6 pathway negatively regulated JA-dependent ATMYC2 expression. Because of the key roles of ATMYC2 in both root growth inhibition and gene expression by JA (Boter et al., 2004; Lorenzo et al., 2004), phenotypes of mkk3 and mpk6 mutants as well as 35S-MKK3 and 35S-MPK6 can be explained by the expression level of ATMYC2. We note that ATMYC2 expression by JA is still increasing in both the 35S-MKK3 and 35S-MPK6 backgrounds. In addition, simple overexpression of JAMYC2 and JAMYC10, potato (Solanum tuberosum) ATMYC2 homologs, is not sufficient to induce JA-inducible genes (Boter et al., 2004; Lorenzo et al., 2004). These findings suggest the presence of unidentified JA-induced positive regulation for ATMYC2, possibly competing with the MKK3–MPK6 pathway. Thus, we propose that ATMYC2 expression is controlled by a finely turned balance of positive and negative pathways (Figure 7). Genetic analysis by producing the mkk3-1 atmyc2-3 double mutant showed that the MKK3–MPK6 cascade is epistatic to ATMYC2 (Figures 6F and 6G). In summary, we propose that the MKK3–MPK6 cascade negatively regulates the JA-induced gene expression of ATMYC2 upstream of the JA pathway (Figure 7). This study demonstrates the negative regulation of ATMYC2 expression by JA.

Specificity of the MAPK Cascade in Arabidopsis

We found that the MKK3–MPK6 and MKK4–MPK6 cascades regulated different functions, as revealed by both morphological and physiological phenotypes and gene expression profiles using steroid-inducible constitutively active MAPKK transgenic plants. In addition, MKK2–MPK6 also did not affect JA signaling, on the basis of JA-dependent MPK6 activation, gene expression, and the root growth assay.

In summary, this study identified the MAPK cascade MKK3–MPK6 in Arabidopsis. Four MAPKKs—MKK2, MKK4, MKK5, and MKK3—activate MPK6 in Arabidopsis (Figure 7). However, the biological functions of the three cascades seem to be different. Presumably, MKK2, MKK4, MKK5, and MKK3 interact with MPK6 to constitute different MAPK complexes in planta to transduce different signals and crosstalk with each other. The MKK3–MPK6 cascade may be involved mainly in JA signaling. This cascade may negatively regulate ATMYC2 function and JA-dependent root growth sensitivity. By contrast, the MKK4/MKK5–MPK6 and MKK2–MPK6 cascades are involved in ET signaling and cold/salt stress signaling, respectively (Kim et al., 2003; Liu and Zhang, 2004; Teige et al., 2004).

In Saccharomyces cerevisiae, high extracellular osmolarity treatment induces the Sln1–Ypd1–Ssk1 two-component osmosensor to activate a MAPK cascade composed of Ssk2 and Ssk22 (MAPKKKs), Pbs2 (MAPKK), and Hog1 (MAPK). A second osmosensor, Sho1, also activates Pbs2 and Hog1 but does so through Ste11 (MAPKKK). The MAPKK Pbs2, thought to serve as a scaffold protein, binds to the Sho1 osmosensor, the MAPKKK Ste11, and the MAPK Hog1. However, under mating signaling, Ste11, Ste7 (MAPKK), and Fus3/Kss1 (MAPKs) constitute the pheromone-responsive MAPK cascade coordinated by the scaffold protein Ste5 (Posas and Saito, 1997). Thus, Ste11 is activated by two distinct signals, the high extracellular osmolarity and the mating pheromone, but regulates different signaling pathways through Pbs2–Hog1 and Ste7–Fus3/Kss1, respectively. Scaffold proteins such as Pbs2 and Ste5 appear to play important roles in selecting upstream and downstream factors of Ste11. In Arabidopsis, four distinct MAPKKs activate MPK6 and regulate different downstream responses. Therefore, distinct scaffold proteins may play a key role in each signaling pathway. Protein complexes that include the MKK3–MPK6, MKK4/MKK5–MPK6, and MKK2–MPK6 cascades need to be identified to understand both the upstream and downstream factors of the MAPK cascades.

METHODS

Preparation of GST Fusion Proteins

Preparation of GST fusion proteins was performed according to Ichimura et al. (2000).

Preparation of Constitutively Active MAPKK Constructs

The constitutively active mutant MAPKKs were generated by QuickChange site-directed mutagenesis (Stratagene) and confirmed by sequencing. The Ser or Thr residues in the activation loop domain [S/T] xxxxx [S/T] were replaced by the acidic Glu amino acids MKK3DD (S235D T241D) and MKK4DD (T224D S230D).

Preparation of Kinase-Inactive MAPK Constructs

The kinase-inactive mutant MAPKs were generated by QuickChange site-directed mutagenesis (Stratagene) and confirmed by sequencing. The conserved Lys residue in the ATP binding domains were replaced by the Met and Arg amino acids MPK1-KN (K61M K62R), MPK3-KN (K67M K68R), MPK4-KN (K72M K73R), and MPK6-KN (K92M K93R).

In Vitro Activation Assay

Each GST–MPK (1 μg) was incubated in 10 μL of kinase reaction buffer (50 mM Tris–HCl, pH 7.5, 1 mM DTT, 10 mM MgCl2, 10 mM MnCl2, 50 μM ATP, and 0.037 MBq of [γ-32P]ATP [60 Ci/mmol]) either with or without GST–MKKs or constitutively active GST–MKKs at 30°C for 30 min. After adding 10 μL of kinase reaction containing 5 μg of MBP, the mixture was incubated again at 30°C. Kinase reactions were stopped after 30 min by adding 4 μL of 6× SDS sample buffer and heating for 4 min at 95°C. Reaction products were analyzed using SDS-PAGE, autoradiography, and Coomassie Brilliant Blue R 250 staining.

Plant Materials and Treatments

The Columbia ecotype of Arabidopsis thaliana was used. The seeds of wild-type, mkk2-2, mkk3-1, mpk6-4, mpk6-5, coi1, atmyc2-3, and transgenic plants were sterilized with 70% ethanol and 0.5% antiformin and sown on MS agar plates containing 1% sucrose. They were grown under continuous light at 22°C. Two-week-old seedlings of each transgenic plant were then transferred to 3 mL of water for 24 h, at which point the water was replaced with 10 μM DEX or 50 μM JA solution. Samples were incubated for the indicated times. The MPK6 T-DNA null lines were obtained from the Kazusa DNA Research Institute; the MKK3 (SALK_051970) and ATMYC2 (SALK_017005) T-DNA null lines were from the ABRC; and the MKK2 T-DNA null line (FLAG_629G03) was obtained from the Institute National de la Recherche Agronomique at the Jean-Pierre Bourgin Institute. The mkk2-2 mutant was originally in ecotype Wassilewskija, and the F3 populations were introgressed twice into Columbia. Lines that were putatively homozygous for T-DNA insertion were subjected to RT-PCR or protein gel blot analysis. The following gene-specific primers were used for RT-PCR analysis: for MKK2, forward (5′-ATGAAGAAAGGTGGATTCAG-3′) and reverse (5′-CACGGAGAACGTACCAGACAG-3′); for MKK3, forward (5′-CAGTTATTCTTATCCAGCTG-3′) and reverse (5′-AAGCCAAGATCTTTGAAACTCGG-3′).

Microarray Analysis

Total RNA was isolated with Trizol reagent (Invitrogen) and used for the preparation of Cy5- and Cy3-labeled cDNA probes. Microarray experiments were performed using the Arabidopsis 2 (Arabidopsis 22K) Oligo Microarray (Agilent Technologies). For each biological replicate, material from five plants was pooled to make a single sample for RNA purification. Two independent transgenic lines of MKK3DD and MKK4DD (MKK3DD#5 and MKK4DD#7 for experiment 1; MKK3DD#3 and MKK4DD#2 for experiment 2) were used for each experiment. All microarray experiments, including the data analysis, were performed according to the manufacturer's manual (http://www.chem.agilent.com/scripts/generic.asp?lpage=11617andindcol=Nandprodcol=Y). The reproducibility of microarray analysis was assayed by a dye swap in each experiment. On the basis of our empirical findings, expression of genes showing average signal intensity values of <500 to ∼1000 in either the Cy3 or the Cy5 channel of the control plants was not always detected reproducibly by RNA gel blot analysis. Thus, under our experimental conditions, genes showing a signal value of <1000 in both Cy3 and Cy5 channels of the control plants were not considered for analysis. Feature extraction and image analysis software (version 7.3; Agilent Technologies) was used to locate and delineate every spot in the array and to integrate each spot's intensity, filtering, and normalization by the Lowess method (P value cutoff of 0.01). The statistical significance of gene expression was tested using a one-way analysis of variance test combined with a Benjamini and Hochberg false-discovery rate multiple correction algorithm (Genespring 7.3) with a corrected P < 0.05 set as cutoff.

QRT-PCR Analysis

Total RNA was treated with RNase-free DNase (Invitrogen). First-strand cDNA produced by the reverse transcription reaction was used as the subsequent template, amplified with real-time PCR master mix, and analyzed using the Gene Amp 7700 sequence detection system (Applied Biosystems). The relative expression levels in each transcript were obtained by normalization to the β-actin gene. The following gene-specific primers for real-time PCR analysis were used: for β-actin2, forward (5′-AGTGGTCGTACAACCGGTATTGT-3′) and reverse (5′-GATGGCATGAGGAAGAGAGAAAC-3′); for ACS6, forward (5′-CCGATGAAGAGTTTGTAGACGAGTT-3′) and reverse (5′-ATCTCAGCGTGCCTTGCAG-3′); for FRK1, forward (5′-GATTTCAACAGTTGTCGCTGGAT-3′) and reverse (5′-ACATCACTCTTTTCGTTCATTTGG-3′); for LOX2, forward (5′-TTGCATCCTCATTTCCGCTAC-3′) and reverse (5′-CCTCCGTTGACAAGACTTTGG-3′); for PDF1.2, forward (5′-TTTGCTGCTTTCGACGCAC-3′) and reverse (5′-CGCAAACCCCTGACCATG-3′); for VSP2, forward (5′-TCAGTGACCGTTGGAAGTTGTG-3′) and reverse (5′-GTTCGAACCATTAGGCTTCAATATG-3′); and for ATMYC2, forward (5′-TCATACGACGGTTGCCAGAA-3′) and reverse (5′-AGCAACGTTTACAAGCTTTGATTG-3′).

Preparation of Protein Extracts and Immunoprecipitation

Preparation of protein extracts and immunoprecipitation were performed as described by Ichimura et al. (2000). The crude extract of whole plants was used for immunoprecipitation.

In-Gel Kinase Assay and Immunocomplex Kinase Assay

The in-gel kinase assay was performed as described previously (Zhang and Klessig, 1997). After immunoprecipitates were washed with 1 mL of reaction buffer without ATP (25 mM Tris-HCl, pH 7.5, 2 mM EGTA, 12 mM MgCl2, 1 mM DTT, and 0.1 mM Na3VO4) (Zhang et al., 1998), kinase assays were performed in 20 μL of the same buffer containing 25 μM ATP, 1 μCi of [γ-32P]ATP, and MBP as a substrate at 30°C for 30 min. The reaction was stopped by the addition of sample buffer. After electrophoresis on a 12% SDS gel, the phosphorylated MBP was visualized by autoradiography.

ET Production

ET was measured according to the methods of Seo et al. (2003).

H2O2 Detection by the 3,3′-Diaminobenzidine Uptake Method

The diaminobenzidine uptake method was performed as described previously (Thordal-Christensen et al., 1997).

Accession Numbers

The microarray data were submitted in MIAME-compliant (for minimum information about a microarray experiment) format to the ArrayExpress database (http://www.ebi.ac.uk/arrayexpress/) and have been assigned the accession number E-MEXP-827. Sequence data from this article can be found in the GenBank/EMBL data libraries under the following accession numbers: ACS6, At4g11280; LOX2, At3g45140; ATMYC2, At1g32640; FRK1, At2g19190; MPK1, At1g10210; MPK2, At1g59580; MPK3, At3g45640; MPK4, At4g01370; MPK5, At4g11330; MPK6, At2g43790; MPK7, At2g18170; MKK2, At4g29810; MKK3, At5g40440; MKK4, At1g51660; PDF1.2, At5g44420; VSP2, At5g24770; and β-actin, At3g18780.

Supplemental Data

The following materials are available in the online version of this article.

  • Supplemental Figure 1. In Vitro Activation Assay of MKKs.

  • Supplemental Figure 2. Phenotype Observed in MKK3DD and MKK4DD Plants.

  • Supplemental Figure 3. Complementation of the JA-Sensitive Phenotype of mkk3-1 by 35S-MKK3.

  • Supplemental Table 1. Prominent Upregulated Genes in Steroid-Inducible MKK3DD Plants.

  • Supplemental Table 2. Prominent Upregulated Genes in Steroid-Inducible MKK4DD Plants.

  • Supplemental Table 3. Prominent Upregulated Genes in Both Steroid-Inducible MKK3DD and MKK4DD Plants.

Acknowledgments

We thank Scott C. Peck for providing the MPK3 antibodies; H. Kobayashi, S. Mizukado, and K. Yoshiwara for technical assistance; the Rice Genome Resource Center at the National Institute of Agrobiological Sciences for the use of the Arabidopsis microarray analysis system; and Y. Nagamura and R. Motoyama for technical support. This research was supported in part by the Bio-oriented Technology Research Advancement Institution.

Footnotes

  • The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Kazuo Shinozaki (sinozaki{at}rtc.riken.jp).

  • www.plantcell.org/cgi/doi/10.1105/tpc.106.046581

  • [OA] Open Access articles can be viewed online without a subscription.

  • [W] Online version contains Web-only data.

  • Received August 14, 2006.
  • Revised November 20, 2006.
  • Accepted February 13, 2007.
  • Published March 16, 2007.

References

  1. Andreasson, E., et al. (2005). The MAP kinase substrate MKS1 is a regulator of plant defense responses. EMBO J. 24: 2579–2589.
  2. Asai, T., Tena, G., Plotnikova, J., Willmann, M.R., Chiu, W.L., Gomez-Gomez, L., Boller, T., Ausubel, F.M., and Sheen, J. (2002). MAP kinase signalling cascade in Arabidopsis innate immunity. Nature 415: 977–983.
  3. Bell, E., Creelman, R.A., and Mullet, J.E. (1995). A chloroplast lipoxygenase is required for wound-induced jasmonic acid accumulation in Arabidopsis. Proc. Natl. Acad. Sci. USA 92: 8675–8679.
  4. Berger, S. (2002). Jasmonate-related mutants of Arabidopsis as tools for studying stress signaling. Planta 214: 497–504.
  5. Berger, S., Bell, E., and Mullet, J.E. (1996). Two methyl jasmonate-insensitive mutants show altered expression of AtVsp in response to methyl jasmonate and wounding. Plant Physiol. 111: 525–531.
  6. Boter, M., Ruiz-Rivero, O., Abdeen, A., and Prat, S. (2004). Conserved MYC transcription factors play a key role in jasmonate signaling both in tomato and Arabidopsis. Genes Dev. 18: 1577–1591.
  7. Brodersen, P., Petersen, M., Nielsen, H.B., Zhu, S., Newman, M.A., Shokat, K.M., Rietz, S., Parker, J., and Mundy, J. (2006). Arabidopsis MAP kinase 4 regulates salicylic acid- and jasmonic acid/ethylene-dependent responses via EDS1 and PAD4. Plant J. 47: 532–546.
  8. Chen, Z., Gibson, T.B., Robinson, F., Silvestro, L., Pearson, G., Xu, B., Wright, A., Vanderbilt, C., and Cobb, M.H. (2001). MAP kinases. Chem. Rev. 101: 2449–2476.
  9. Cheong, Y.H., et al. (2003). BWMK1, a rice mitogen-activated protein kinase, locates in the nucleus and mediates pathogenesis-related gene expression by activation of a transcription factor. Plant Physiol. 132: 1961–1972.
  10. Cowley, S., Paterson, H., Kemp, P., and Marshall, C.J. (1994). Activation of MAP kinase kinase is necessary and sufficient for PC12 differentiation and for transformation of NIH 3T3 cells. Cell 77: 841–852.
  11. Dai, Y., Wang, H., Li, B., Huang, J., Liu, X., Zhou, Y., Mou, Z., and Li, J. (2006). Increased expression of MAP KINASE KINASE7 causes deficiency in polar auxin transport and leads to plant architectural abnormality in Arabidopsis. Plant Cell 18: 308–320.
  12. Devoto, A., and Turner, J.G. (2003). Regulation of jasmonate-mediated plant responses in Arabidopsis. Ann. Bot. (Lond.) 92: 329–337.
  13. Feys, B., Benedetti, C.E., Penfold, C.N., and Turner, J.G. (1994). Arabidopsis mutants selected for resistance to the phytotoxin coronatine are male sterile, insensitive to methyl jasmonate, and resistant to a bacterial pathogen. Plant Cell 6: 751–759.
  14. Gomi, K., Ogawa, D., Katou, S., Kamada, H., Nakajima, N., Saji, H., Soyano, T., Sasabe, M., Machida, Y., Mitsuhara, I., Ohashi, Y., and Seo, S. (2005). A mitogen-activated protein kinase NtMPK4 activated by SIPKK is required for jasmonic acid signaling and involved in ozone tolerance via stomatal movement in tobacco. Plant Cell Physiol. 46: 1902–1914.
  15. He, P., Shan, L., Lin, N.C., Martin, G.B., Kemmerling, B., Nurnberger, T., and Sheen, J. (2006). Specific bacterial suppressors of MAMP signaling upstream of MAPKKK in Arabidopsis innate immunity. Cell 125: 563–575.
  16. Huang, Y., Li, H., Gupta, R., Morris, P.C., Luan, S., and Kieber, J.J. (2000). ATMPK4, an Arabidopsis homolog of mitogen-activated protein kinase, is activated in vitro by AtMEK1 through threonine phosphorylation. Plant Physiol. 122: 1301–1310.
  17. Ichimura, K., Mizoguchi, T., Irie, K., Morris, P., Giraudat, J., Matsumoto, K., and Shinozaki, K. (1998). Isolation of ATMEKK1 (a MAP kinase kinase kinase)-interacting proteins and analysis of a MAP kinase cascade in Arabidopsis. Biochem. Biophys. Res. Commun. 253: 532–543.
  18. Ichimura, K., Mizoguchi, T., Yoshida, R., Yuasa, T., and Shinozaki, K. (2000). Various abiotic stresses rapidly activate Arabidopsis MAP kinases ATMPK4 and ATMPK6. Plant J. 24: 655–665.
  19. Ichimura, K., et al. (2002). Mitogen-activated protein kinase cascades in plants: A new nomenclature. Trends Plant Sci. 7: 301–308.
  20. Kim, C.Y., Liu, Y., Thorne, E.T., Yang, H., Fukushige, H., Gassmann, W., Hildebrand, D., Sharp, R.E., and Zhang, S. (2003). Activation of a stress-responsive mitogen-activated protein kinase cascade induces the biosynthesis of ethylene in plants. Plant Cell 15: 2707–2718.
  21. Kumar, D., and Klessig, D.F. (2000). Differential induction of tobacco MAP kinases by the defense signals nitric oxide, salicylic acid, ethylene, and jasmonic acid. Mol. Plant Microbe Interact. 13: 347–351.
  22. Leon, J., Rojo, E., and Sanchez-Serrano, J.J. (2001). Wound signalling in plants. J. Exp. Bot. 52: 1–9.
  23. Liu, Y., and Zhang, S. (2004). Phosphorylation of 1-aminocyclopropane-1-carboxylic acid synthase by MPK6, a stress-responsive mitogen-activated protein kinase, induces ethylene biosynthesis in Arabidopsis. Plant Cell 16: 3386–3399.
  24. Lorenzo, O., Chico, J.M., Sanchez-Serrano, J.J., and Solano, R. (2004). JASMONATE-INSENSITIVE1 encodes a MYC transcription factor essential to discriminate between different jasmonate-regulated defense responses in Arabidopsis. Plant Cell 16: 1938–1950.
  25. Lorenzo, O., Piqueras, R., Sanchez-Serrano, J.J., and Solano, R. (2003). ETHYLENE RESPONSE FACTOR1 integrates signals from ethylene and jasmonate pathways in plant defense. Plant Cell 15: 165–178.
  26. Lu, C., Han, M.H., Guevara-Garcia, A., and Fedoroff, N.V. (2002). Mitogen-activated protein kinase signaling in post germination arrest of development by abscisic acid. Proc. Natl. Acad. Sci. USA 99: 15812–15817.
  27. Mansour, S.J., Matten, W.T., Hermann, A.S., Candia, J.M., Rong, S., Fukasawa, K., Vande Woude, G.F., and Ahn, N.G. (1994). Transformation of mammalian cells by constitutively active MAP kinase kinase. Science 265: 966–970.
  28. Marshall, C.J. (1994). MAP kinase kinase kinase, MAP kinase kinase and MAP kinase. Curr. Opin. Genet. Dev. 4: 82–89.
  29. Matsuoka, D., Nanmori, T., Sato, K., Fukami, Y., Kikkawa, U., and Yasuda, T. (2002). Activation of AtMEK1, an Arabidopsis mitogen-activated protein kinase kinase, in vitro and in vivo: Analysis of active mutants expressed in E. coli and generation of the active form in stress response in seedlings. Plant J. 29: 637–647.
  30. Menke, F.L., van Pelt, J.A., Pieterse, C.M., and Klessig, D.F. (2004). Silencing of the mitogen-activated protein kinase MPK6 compromises disease resistance in Arabidopsis. Plant Cell 16: 897–907.
  31. Miles, G.P., Samuel, M.A., Zhang, Y., and Ellis, B.E. (2005). RNA interference-based (RNAi) suppression of AtMPK6, an Arabidopsis mitogen-activated protein kinase, results in hypersensitivity to ozone and misregulation of AtMPK3. Environ. Pollut. 138: 230–237.
  32. Mizoguchi, T., Ichimura, K., Irie, K., Morris, P., Giraudat, J., Matsumoto, K., and Shinozaki, K. (1998). Identification of a possible MAP kinase cascade in Arabidopsis thaliana based on pairwise yeast two-hybrid analysis and functional complementation tests of yeast mutants. FEBS Lett. 437: 56–60.
  33. Nakagami, H., Pitzschke, A., and Hirt, H. (2005). Emerging MAP kinase pathways in plant stress signalling. Trends Plant Sci. 10: 339–346.
  34. Penninckx, I.A., Thomma, B.P., Buchala, A., Metraux, J.P., and Broekaert, W.F. (1998). Concomitant activation of jasmonate and ethylene response pathways is required for induction of a plant defensin gene in Arabidopsis. Plant Cell 10: 2103–2113.
  35. Petersen, M., et al. (2000). Arabidopsis MAP kinase 4 negatively regulates systemic acquired resistance. Cell 103: 1111–1120.
  36. Posas, F., and Saito, H. (1997). Osmotic activation of the HOG MAPK pathway via Ste11p MAPKKK: Scaffold role of Pbs2p MAPKK. Science 276: 1702–1705.
  37. Ren, D., Yang, H., and Zhang, S. (2002). Cell death mediated by MAPK is associated with hydrogen peroxide production in Arabidopsis. J. Biol. Chem. 277: 559–565.
  38. Rojo, E., Leon, J., and Sanchez-Serrano, J.J. (1999). Cross-talk between wound signalling pathways determines local versus systemic gene expression in Arabidopsis thaliana. Plant J. 20: 135–142.
  39. Rojo, E., Titarenko, E., Leon, J., Berger, S., Vancanneyt, G., and Sanchez-Serrano, J.J. (1998). Reversible protein phosphorylation regulates jasmonic acid-dependent and -independent wound signal transduction pathways in Arabidopsis thaliana. Plant J. 13: 153–165.
  40. Seo, S., Sano, H., and Ohashi, Y. (1999). Jasmonate-based wound signal transduction requires activation of WIPK, a tobacco mitogen-activated protein kinase. Plant Cell 11: 289–298.
  41. Seo, S., Seto, H., Koshino, H., Yoshida, S., and Ohashi, Y. (2003). A diterpene as an endogenous signal for the activation of defense responses to infection with tobacco mosaic virus and wounding in tobacco. Plant Cell 15: 863–873.
  42. Shibata, W., Banno, H., Ito, Y., Hirano, K., Irie, K., Usami, S., Machida, C., and Machida, Y. (1995). A tobacco protein kinase, NPK2, has a domain homologous to a domain found in activators of mitogen-activated protein kinases (MAPKKs). Mol. Gen. Genet. 246: 401–410.
  43. Staswick, P.E., Su, W., and Howell, S.H. (1992). Methyl jasmonate inhibition of root growth and induction of a leaf protein are decreased in an Arabidopsis thaliana mutant. Proc. Natl. Acad. Sci. USA 89: 6837–6840.
  44. Stepanova, A.N., and Alonso, J.M. (2005). Ethylene signaling pathway. Sci. STKE 2005: cm3.
  45. Teige, M., Scheikl, E., Eulgem, T., Doczi, R., Ichimura, K., Shinozaki, K., Dangl, J.L., and Hirt, H. (2004). The MKK2 pathway mediates cold and salt stress signaling in Arabidopsis. Mol. Cell 15: 141–152.
  46. Thordal-Christensen, H., Zhang, Z., Wei, Y., and Colling, D.B. (1997). Subcellular localization of H2O2 in plants. H2O2 accumulation in papillae and hypersensitive response during the barley–powdery mildew interaction. Plant J. 11: 1187–1194.
  47. Wang, K.L., Li, H., and Ecker, J.R. (2002). Ethylene biosynthesis and signaling networks. Plant Cell 14 (suppl.): S131–S151.
  48. Widmann, C., Gibson, S., Jarpe, M.B., and Johnson, G.L. (1999). Mitogen-activated protein kinase: Conservation of a three-kinase module from yeast to human. Physiol. Rev. 79: 143–180.
  49. Xu, Y., Chang, P., Liu, D., Narasimhan, M.L., Raghothama, K.G., Hasegawa, P.M., and Bressan, R.A. (1994). Plant defense genes are synergistically induced by ethylene and methyl jasmonate. Plant Cell 6: 1077–1085.
  50. Yang, K.Y., Liu, Y., and Zhang, S. (2001). Activation of a mitogen-activated protein kinase pathway is involved in disease resistance in tobacco. Proc. Natl. Acad. Sci. USA 98: 741–746.
  51. Zhang, S., Du, H., and Klessig, D.F. (1998). Activation of the tobacco SIP kinase by both a cell wall-derived carbohydrate elicitor and purified proteinaceous elicitins from Phytophthora spp. Plant Cell 10: 435–450.
  52. Zhang, S., and Klessig, D.F. (1997). Salicylic acid activates a 48-kD MAP kinase in tobacco. Plant Cell 9: 809–824.