Figure 7.

MPK3 and MPK6 interact with PTP1 and MKP1, which negatively regulate H₂O₂ production in Arabidopsis. A, Interactions of PTP1 and MKP1 with MPK3 and MPK6 in the yeast two-hybrid system. The experiments were performed three times with similar results. B, Co-IP of MPK3 and MPK6 interactions with PTP1 and MKP1. Total proteins were extracted from 15-d-old wild-type Columbia-0 (Col-0) and transgenic 35S:PTP1 and 35S:MKP1 lines. Input and immunoprecipitated proteins were analyzed by independently immunoblotting with anti-MYC, anti-MPK3, and anti-MPK6 antibodies. C, The kinase activities of MPK3 and MPK6 were detected by immunoblotting using anti-phospho-p44/42 MAPK antibodies (p-MPK6 and p-MPK3). Seedlings (7 d old) of the wild type, the ptp1 and mkp1 mutants, and the 35S:PTP1 and 35S:MKP1 lines were treated with 200 μg mL⁻¹ Vd-toxins, and the total proteins were then extracted at various times for immunoblot analysis. β-Actin was used as the loading control. D, Quantification of the kinase activity levels of MPK3 and MPK6 in C using ImageJ software. Results are presented three independent biological replicates. Error bars indicate sd; n = 3. E, H₂O₂ was detected in the leaves of the wild type, the ptp1 and mkp1 mutants, and the 35S:PTP1 and 35S:MKP1 lines using a fluorescence assay with H₂DCF-DA, as described in “Materials and Methods.” Bar = 20 μm. F, Quantification of the H₂DCF-DA fluorescence intensities in E. Error bars indicate sd; n = 8. G, Cell death induced by Vd-toxins in cotyledons of the wild type, the ptp1 and mkp1 mutants, and the 35S:PTP1 and 35S:MKP1 lines. Cotyledons of 14-d-old seedlings were treated 14 h with 150 μg mL⁻¹ Vd-toxins and stained with Trypan Blue. H, Quantification of the cell death rates induced by Vd-toxins in G using ImageJ software. Error bars indicate sd; n = 9. Different letters represent significant differences at P < 0.05 by one-way ANOVA with Tukey’s honestly significant difference posthoc tests. All experiments were repeated three times.