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. 2022 Jul 22;9:uhac163. doi: 10.1093/hr/uhac163

Figure 5.

Figure 5

HsfA1a regulates expression and protein accumulation of HSPs in tomato anthers under heat stress. (A) EMSA analysis showing binding of HsfA1a to HSE sequences of the promoters of HSP17.6A, HSP70-2, HSP90-2, and HSP101. Recombinant HsfA1a was applied for DNA binding assays to HSP17.6A, HSP70-2, HSP90-2, and HSP101 and their HSE motif sequences as the probes. His is displayed as the negative control. Asterisks (*) represent mutated probes. All probes are listed in Supplementary Data Table S4. (B) ChIP–qPCR was used to validate HsfA1a binding to the promoters of HSP17.6A, HSP70-2, HSP90-2, and HSP101 in 35S-HsfA1a-HA-overexpressing (HsfA1a-OE) plants. WT and HsfA1a-OE plants were subjected to heat stress at flowering stage, anther samples were taken and input chromatin was isolated from them. Anti-HA antibody immunoprecipitation was used to epitope-tag the HsfA1a–chromatin complex. Mouse IgG treatment was used as a control reaction. qRT–PCR was used to quantify input and ChIP DNA samples with specific primers for the promoters of HSP17.6A, HSP70-2, HSP90-2, and HSP101 genes. The percentages of input DNA are displayed as ChIP results. (C) Relative expression of HSP17.6A, HSP70-2, HSP90-2, and HSP101 genes in anthers of hsfA1a, WT, and HsfA1a-OE plants under control or heat stress. (D) Accumulation of HSP17.6, HSP70, HSP90, and HSP101 proteins in anthers of hsfA1a, WT, and HsfA1a-OE plants under control or heat stress. Actin was displayed as a loading control in western blotting. Means with the same letter in Tukey’s test indicate a non-significant difference at P < .05. Similar results were obtained in three independent experiments. hsfA1a-3, one line of hsfA1a mutants; HsfA1a-OE5, one line of HsfA1a-OE plants.