Volume 41, Issue 6, Pages 649-660 (March 2011) Calmodulin-Dependent Activation of MAP Kinase for ROS Homeostasis in Arabidopsis  Fuminori Takahashi, Tsuyoshi Mizoguchi, Riichiro Yoshida, Kazuya Ichimura, Kazuo Shinozaki  Molecular Cell  Volume 41, Issue 6, Pages 649-660 (March 2011) DOI: 10.1016/j.molcel.2011.02.029 Copyright © 2011 Elsevier Inc. Terms and Conditions

Molecular Cell 2011 41, 649-660DOI: (10.1016/j.molcel.2011.02.029) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 1 Effect of Loss and Gain of Function of Arabidopsis MPK8 on ROS Accumulation (A) The RT-PCR analysis of MPK8 gene expression in roots, rosette leaves, cauline leaves, stems, and flowers. The Arabidopsis β-tubulin gene was used as an internal control. (B) Activation of MPK8-GFP in Arabidopsis after various stresses. The MBP kinase activity of MPK8-GFP measured with the IP kinase assay (top) and immunoblot analysis of MPK8-GFP detected with anti-GFP antibody are shown (second from top). MPK6 was activated by these stress conditions, as previously reported, and shown as a control (second from bottom). MPK6 protein levels were determined by an immunoblot analysis using anti-MPK6 antibody (bottom). (C) H2O2 generation in wild-type, mpk8, and MPK8-GFP plants. H2O2 production was detected by polymerization of DAB. Black triangles indicate the cut edge of the wound site. (D) Paraquat resistance in wild-type, mpk8, and MPK8-GFP plants. These experiments were repeated three times with similar results. (E) Chlorophyll content in each genotype under paraquat stress conditions. Chlorophyll a/b was calculated from the results of three independent experiments (n = 15). Error bars indicate the SD. Molecular Cell 2011 41, 649-660DOI: (10.1016/j.molcel.2011.02.029) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 2 In Planta Activation of MPK8 by MKK3 (A) Activation of MPK8-GFP by MKK3, but not by MKK4, in Arabidopsis. MPK8-GFP activity was measured using MBP as substrate (top). Immunoblot analysis of MPK8-GFP was performed using an anti-GFP antibody (middle). Rubisco large subunit was stained with Coomassie brilliant blue (CBB) and is shown as control for equal protein amounts (bottom). The error bars represent the SEM. (B) Effects of loss of function of MKK3 on the activation of MPK8. MPK8-GFP #4 and MPK8-GFP mkk3-1 #33 plants were wounded, and the MPK8-GFP and 66 kDa protein kinase activity was analyzed by in-gel kinase assay. Similar results were observed in two independent experiments. Molecular Cell 2011 41, 649-660DOI: (10.1016/j.molcel.2011.02.029) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 3 Effect of Calcium Signaling for MPK8 Activation under Wounding Ca2+-dependent activation of MPK8 in mkk3-1 mutant. Total proteins from plants expressing the GFP-tagged MPK8 were immunoprecipitated with anti-GFP antibody in the presence of 1 mM CaCl2 or 5 mM EGTA. Endogenous MPK6 proteins were immunoprecipitated with anti-MPK6 antibody. The kinase activity of immunoprecipitated MPK8 or MPK6 was determined with MBP as substrate using the IP kinase assay (top and third from top). Expression of MPK8-GFP, MPK6, or CaM was detected by immunoblot analysis with anti-GFP (second from top), anti-MPK6 (second from bottom), or anti-CaM antibody (bottom). These experiments were repeated twice, with similar results. Molecular Cell 2011 41, 649-660DOI: (10.1016/j.molcel.2011.02.029) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 4 Interaction and Activation between MPK8 and CaMs In Vivo (A) Deletion mapping of the MPK8-interacting domain of CaM4. CaM4 interacted with MPK8 through its C-terminal domain. (B) Intracellular localization of MPK8/CaM4, MPK8/CaM3, and MPK8/CaM7 in a split YFP-based BiFC assay in Arabidopsis protoplasts. 35S-YFPN (YFPN) and 35S-YFPC (YFPC) vector expression split YFP domains alone were used as controls. (C) CoIP of MPK8 and CaM from protoplast extracts. Protein extracts from plants expressing the GFP-tagged MPK8, MPK6, or GFP that were transiently transformed with CaM4 were immunoprecipitated with an anti-GFP antibody. Immunoblot analysis showed the presence of MPK8-GFP, MPK6-GFP, GFP, or CaM4 in the crude protein extracts (Input) or immunoprecipitates (IP) with either anti-GFP antibody or anti-CaM antibody. (D) Ca2+-dependent interaction between MPK8 and CaM. Total proteins from plants expressing the GFP-tagged MPK8 or GFP plants transiently transformed with CaM4 were immunoprecipitated with an anti-GFP antibody, and 1 mM Ca2+ or 5 mM EGTA. The coIP experiments were repeated three times, with similar results. (E) Activation of MPK8 by CaM in vivo. CaM was transiently transformed into Arabidopsis protoplasts from MPK8-GFP or GFP-overexpressing plants. The kinase activity of immunoprecipitated MPK8 was investigated with MBP as substrate using an IP kinase assay (top) or in-gel kinase assay (third from top) in the presence of Ca2+. The autophosphorylation activity of MPK8 is indicated in the second from top panel. CaM, GFP, or MPK8-GFP proteins were prepared by immunoprecipitation (IP) or crude extracts (Input) with anti-CaM or anti-GFP antibodies. (F) Phosphorylation-independent activation of MPK8 by CaM in vivo. Four types of mutated MPK8 and wild-type MPK8 were transiently transformed into Arabidopsis protoplasts. The kinase activity of immunoprecipitated MPK8 was investigated using an IP kinase assay (top). CaM or MPK8-GFP proteins were prepared by immunoprecipitation (IP) or crude extracts (Input) with anti-CaM or anti-GFP antibodies. These experiments were repeated three times, with similar results. Molecular Cell 2011 41, 649-660DOI: (10.1016/j.molcel.2011.02.029) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 5 Wounding Activation of MPK8 with Dual Activators, MKK3 and CaM, In Planta (A) Both MKK3- and CaM-dependent activation of MPK8. Wound-inducible MPK8 activation was partially or largely abolished by CaM inhibitors, W7 or ST-A, in wild-type or the mkk3-1 mutant, respectively. The detached plants were pretreated with 50 μM W5, W7, or ST-A for 2 hr, and then subjected to wounding stress for the indicated times. Immunoblot analysis of MPK8-GFP, MPK6, or CaM was performed using anti-GFP (second from top), anti-MPK6 (second from bottom), or anti-CaM antibodies (bottom). These experiments were repeated three times, with similar results. (B) Phosphorylation-dependent or -independent activation of MPK8 by either MKK3 or CaM in vitro. Immunoblot analysis using 4G10 (phosphotyrosine antibody, top), in-gel kinase assay (second from top), and CBB staining (bottom) are shown. The numeric characters indicate relative level of protein amount or kinase activity. (C) Independent activation of MPK8 by both CaMs and MKK3. The kinase activity of GST-MPK8 was measured using an in-tube kinase assay. The kinase activity of MPK8 without MKK3 and CaMs was defined as 1, and relative fold increases are shown. Similar results were observed in two independent experiments. The numeric characters indicate relative level of kinase activity. Molecular Cell 2011 41, 649-660DOI: (10.1016/j.molcel.2011.02.029) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 6 Analysis of ROS-Mediated Gene Expression in the MPK8 Pathway (A) Relative transcription levels of RbohD, Zat12, and OXI1 in wild-type, mpk8, and MPK8-GFP plants after wounding. Total RNA was isolated from seedlings wounded for the indicated times, and subjected to qRT-PCR analysis. Each transcript level was normalized to the expression of actin2 measured in the same samples. Data are means ± SD (n = 6) of three independent experiments. (B) Effects of CaM inhibitors on the expression of MPK8 downstream genes. Relative transcription levels of RbohD and OXI1 in wild-type, mpk8-1, or mkk3-1 plants after wounding. The detached plants were pretreated with 50 μM W7 or W5 for 2 hr and then wounded for indicated times. Total RNA was isolated from these seedlings and subjected to qRT-PCR analysis. Each transcript level was normalized to the expression of actin2 measured in the same sample. Data are the means ± SD (n = 6) of three independent experiments. Molecular Cell 2011 41, 649-660DOI: (10.1016/j.molcel.2011.02.029) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 7 Suppression of the H2O2 Accumulation and Transient Induction of Zat12 and OXI1 Genes in mpk8 using rbohD (A) H2O2 generation in wild-type, mpk8-1, rbohD-1, and mpk8-1 rbohD-1 plants. H2O2 production was detected by polymerization of DAB. Black triangles indicate the cut edge of the wound site. (B) Relative transcription levels of Zat12 and OXI1 in wild-type, mpk8-1, rbohD-1, and mpk8-1 rbohD-1 plants after wounding. Total RNA was isolated from seedlings after wounding for the indicated times and subjected to qRT-PCR analysis. Each transcript level was normalized to the expression of actin2 measured in the same sample. Data are the means ± SD (n = 6) of three independent experiments. (C) Possible roles of the CaMs–MKK3–MPK8 pathway under mechanical wounding. For details, see text. Molecular Cell 2011 41, 649-660DOI: (10.1016/j.molcel.2011.02.029) Copyright © 2011 Elsevier Inc. Terms and Conditions