Fig. 6: Effect of S-sulfhydration on BraFLC function.
From: Hydrogen sulfide promotes flowering in heading Chinese cabbage by S-sulfhydration of BraFLCs
A S-sulfhydration modification of BraFLCs mediated by H2S. a S-sulfhydration of BraFLC 1 by H2S; b S-sulfhydration of BraFLC 2 by H2S; c S-sulfhydration of BraFLC 3 by H2S; d S-sulfhydration modification of BraFLC 4 by H2S; M: marker; 1, control, FLC + ddH2O; 2, BraFLC treated with 200 µmol L−1 NaHS; 3, BraFLC treated with 100 µmol L−1 NaHS; 4: negative control, DTT (protein + 1 mM DTT). Experimental procedure: the protein was treated with H2S, followed by MMTS, methyl methanethiosulfonate blocking, biotin-HPDP, N-(6-(biotinamido)hexyl)-3′-(2′-pyridyldithio)-propionamide biotin labeling, SDS-PAGE electrophoresis, and transmembrane treatment. Then, the samples were blocked with BSA, subjected to an anti-biotin primary antibody, an AP goat anti-mouse alkaline phosphatase-labeled sheep secondary antibody, and a NBT/BCIP, nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl Phosphate color reaction assay. B Effects of S-sulfhydration on the binding ability of BraFLCs with BraSOC I promoter probes. a The binding ability of S-sulfhydrated BraFLC 1 and b the binding ability of S-sulfhydrated BraFLC 3; M: marker; SOC I 1-1 + FLC1: assay for SOC I 1-1 probes binding with BraFLC 1 without H2S treatment, and SOC I 1-1 + FLC1 (-SH): assay for SOCI 1-1 probe binding with S-sulfhydrated BraFLC 1; the representative symbol for other BraSOC I probes binding with FLC protein are the same as above; “−” negative control. C Effects of S-sulfhydration on the binding ability of BraFLCs with BraFT promoter probes. a The binding ability of S-sulfhydrated BraFLC 1 and b the binding ability of S-sulfhydrated BraFLC 3; M: marker; pFT 1 + FLC1: assay for pFT 1 probe binding with BraFLC 1 without H2S treatment, and pFT 1 + FLC1 (-SH): assay for pFT 1 probe binding with S-sulfhydrated BraFLC 1. The representative symbol for the other BraFT probes binding with BraFLC protein are the same as above; “−” negative control. D BraSOC I and BraFT homolog expression levels revealed by qRT-PCR analysis after treatment with 100 µmol L−1 NaHS solution or 2 mmol L−1 HA. Values are the mean ± SE of three biological replicates; *p < 0.05, **p < 0.01, Student’s t-test
