Fig. 6: ILR3 protein accumulation depends on RH24.
From: An RNA helicase coordinates with iron signal regulators to alleviate chilling stress in Arabidopsis
a RH24 interacts with BTS in BiFC assays. The proteins were transiently expressed in N. benthamiana leaves. The plants stably expressing Histone3-mcherry served as a nucleus marker. BTS-nYFP and cYFP-EV or nYFP-EV and RH24-cYFP were used as a negative control. YFP fluorescence was assigned with pseudocolor green. Bar = 25 μm. BF: bright field. b RH24 did not affect the protein levels of BTS. 35S:5’UTR-BTS-T7-3’UTR and 35S:RH24-FLAG or 35S:RH24DAAD-FLAG were expressed in Arabidopsis protoplasts. The protein levels were subjected to immunoblotting assays at 16 h after transformation. c RH24 interacts with ILR3 by BiFC assays. Bar = 25 μm. d ILR3 accumulates in the presence of RH24. 35S:5’UTR-ILR3-T7-3’UTR and 35S:RH24-FLAG or 35S:RH24DAAD-FLAG were expressed in Arabidopsis protoplasts. 50 μM MG132 was added to the samples at 12 h after transfection. After an additional 6 h incubation, the protoplasts were subjected to immunoblot assays. The band abundance was quantified by Image J. The relative protein level of ILR3 was quantitatively analyzed for three repeats (right panel). Data presented as means ± SD (n = 3 biological replicates) by two-factor ANOVA with Tukey’s HSD test (P ≤ 0.05). e RIP-qPCR assays for RH24 and ILR3 mRNA. RIP-qPCR assays were performed using 35S:RH24-FLAG plants. The plants were grown on ½ MS for 14 days. RT-qPCR was used to evaluate the precipitated mRNA. Values are the mean of the percentage of input in three independent experiments. Data presented as means ± SD (n = 3 biological replicates) by two-factor ANOVA with Tukey’s HSD test (P ≤ 0.0001). f Accumulation of ILR3 protein under chilling stress is dependent on RH24. The transgenic plants ILR3-ox-3 and ILR3-ox-3/rh24-1 were grown on ½ MS at 22 °C for 12 days and then treated with 2.5 μM MG132 at 4, 22, or 26 °C for 2 days, respectively. The expression levels of ILR3 were examined by RT-qPCR. The relative protein level of ILR3 was quantitatively analyzed for three repeats (right panel). Data presented as means ± SD (n = 3 biological replicates) by two-factor ANOVA with Tukey’s HSD test (P ≤ 0.05). Source data are provided as a Source Data file.
