CLCuMuB βC1 represses JA responses though interfering with the integrity of SCF^COI1.

(A) Total root length of HA-βC1 transgenic (#2 HA-βC1 and #3 HA-βC1) and wild-type (#2 WT and #3 WT) N. benthamiana seedlings was measured every 24 h beginning at the 6th day after sowing (n ≥11). Bars represent SEM. #2 HA-βC1 and #2 WT were presented on same plates, while #3 HA-βC1 and #3 WT were presented on same plates. These experiments were repeated 3 times. (B) Jasmonate sensitivity was measured as root growth inhibition. Six-day-old seedlings (n ≥10) were grown on MS contained with 50 μM MeJA for additional 4 days. Bars represent SEM. The raw data were analysed by Mann-Whitney rank sum test to show the significance level at 0.05 (*). (C) Relative expression level of marker genes of jasmonate responses in mock- or MeJA-treated HA-βC1 transgenic and wild-type N. benthamiana seedlings determined by quantitative real-time PCR. #2 HA-βC1 and #2 WT were presented on same plates, while #3 HA-βC1 and #3 WT were presented on same plates. HA-βC1-expressing lines are compared with their corresponding control in each condition. Actin was used as the internal control. Bars represent SEM. The raw data were analysed by two-sample t-test to show the significance level at 0.05 (*), 0.01 (**) and 0.001 (***). These experiments were repeated at least twice. (D) CLCuMB βC1 enhanced degradation of COI1 in vitro. The purified Myc-COI1 protein was added to total protein extracts from N.benthamiana which expressed HA-nLUC or HA-βC1, incubated at 25°C for the indicated time periods, and subjected to immunoblot analysis with the anti-Myc antibody. Intensity was detected through Total Lab TL120. The PVDF membrane was stained with Ponceaux to visualize the large subunit of ribulose-1,5-bisphosphate as the loading control. (E) Quantitative analysis of the relative abundance of COI1 in the presence of HA-nLUC or HA-βC1 for the time periods indicated. The abundance of COI1 at the start point (0-h) was set to 100% as a reference for calculating its relative abundance after different incubation periods. Error bars represent SD. The experiment was repeated three times.