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.

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.

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.

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.

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 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.

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.