S-HsfA2 negatively regulates Arabidopsis extreme heat tolerance.

(A) Schematic diagrams of HsfA2-III and its corresponding product S-HsfA2 (top). Mutations in the LxLxLx motif (underlined) generated the dominant version of S-HsfA2 (S-HsfA2L-A). The predicted AlphaFold 3D structure of S-HsfA2 (AFDB accession: AF-J7G1E7-F1) is shown (bottom). (B) Thermotolerance of the WT control, S-HsfA2-overexpressing (S-HsfA2-OE) lines, and S-HsfA2L-A-overexpressing (S-HsfA2L-A-OE) lines in both medium and soil. (C) RNAi-mediated S-HsfA2 knockdown (S-HsfA2-KD) and thermotolerance analyses of S-HsfA2-KD lines. RNAi (targeting the retained intron sequences; see 1A) reduced the abundance of the S-HsfA2-encoding transcript (HsfA2-III) but did not significantly affect the other two HsfA2 transcripts (HsfA2-II and HsfA2) in Arabidopsis plants (12 DAS) exposed to 42°C for 1 h (top). YELLOW-LEAF-SPECIFIC GENE 8 (YLS8) served as a loading control. The RT‒qPCR heatmap of HsfA2-III expression in three lines of S-HsfA2-KD is shown (bottom). For thermotolerance analyses, the survival rates of the seedlings under heat stress are shown. DAS, days after sowing.

The HRE is recognized by S-HsfA2.

HsfA2 binding to HREs was verified by EMSA (A) and Y1H (B) assays. The mutated HRE (mHRE) was used as a negative control. (C) The production of His6-DBD and His6-tDBD was confirmed by immunoblotting with an anti-His6 antibody (left). The mock proteins (empty vector control) were used as a negative control. Coomassie Brilliant Blue (CBB)-stained proteins are shown as a loading control. The binding of His6-DBD and His6-tDBD to HSE or mutated HSE (mHSE) was verified by EMSA (right). For the EMSAs, the specific binding signal is marked with an arrow, while the asterisk indicates a nonspecific signal. The HRE and HSE sequences are shown, and the corresponding mutations are shown in lowercase.

HRE confers heat induction in Arabidopsis.

(A) Qualitative GUS histochemical staining of GUS reporter transgenic Arabidopsis seedlings. Scale bar, 0.5 mm or 1 mm for HRE-35Sm:GUS (#2). Heat-induced GUS activity analyses of HRE-35Sm:GUS transgenic Arabidopsis at the GUS mRNA (B) and GUS protein levels (C). (D) Heat time course analysis of the GUS mRNA level in HRE-35Sm:GUS transgenic Arabidopsis plants. (E) Western blot analysis verified the successful isolation of INPUT nuclear protein (NP) without cytoplasmic protein (CP) contamination using an anti-histone H3 (nuclear marker) antibody and an anti-glucose pyrophosphorylase (UGPase) (cytoplasmic marker) antibody (top). EMSAs revealed that HRE probes, but not mHRE probes, bound to NPs from heat shock-treated (37°C, 42°C) or untreated (22°C) Arabidopsis plants (bottom). The bound complex is indicated by an arrow. (F) GUS accumulation in crossed GUS reporter plants harboring 35S:S-HsfA2-Flag or 35S:S-HsfA2L-A-Flag effector (+effector) or an uncrossed reporter control (-effector). In (C) and (F), GUS expression was determined by immunoblotting assays using anti-GUS and anti-Tubulin antibodies. The GUS protein level is expressed as the relative band intensity of GUS to tubulin [the control (22°C) or the effector was set as 1]. In (B), (C), and (D), the data are presented as the means ± SDs of at least two independent experiments. Different letters indicate significant differences according to one-way ANOVA, P < 0.05.

S-HsfA4c is similar to S-HsfA2 and represents a new HSF.

(A) Schematic diagrams of HsfA4c splice variants and their RT‒PCR splicing analyses in two-week-old Arabidopsis plants under heat stress. The YLS8 gene served as a loading control. gDNA, genomic DNA control. The asterisk indicates the in-frame stop codon. S-HsfA4c binding to the HRE was verified by Y1H (B) and EMSA with bacterially expressed and purified His6-S-HsfA4c (C). (D) Thermotolerance of the 35S:GFP vector control and 35S:S-HsfA4c-GFP overexpression (S-HsfA4c-OE) lines. (E) Two T-DNA HsfA4c insert-knockdown mutants (hsfa4c-1 and hsfa4c-2) confirmed by RT‒qPCR (Student’s t test, *P <0.05; top) and thermotolerance analyses (bottom). For thermotolerance analyses, the survival rates of the seedlings under heat stress are shown. (F) Representative images of the subcellular localization of S-HsfA4c-GFP in Arabidopsis root cells treated without (normal) or with heat stress (42°C for 1 h). DAPI, 4,6-diamidino-2-phenylindole (DAPI; nuclei staining). Scale bar, 40 μm. DAS, days after sowing.

S-HsfB1 is similar to S-HsfA2 and represents a new HSF.

(A) RT‒PCR splicing analyses of HsfB1 and HsfB2a in 7-d-old Arabidopsis plants under heat stress. The YLS8 gene served as a loading control. The structures of the S-HsfB1 and S-HsfB2a splice variants are shown in the right panel. The amino acid residues encoded by the retained introns are indicated above each variant. (B) Representative images showing the subcellular localization of S-HsfB1-RFP or S-HsfB2a-RFP in N. benthamiana epidermal cells. RFP was used as a negative control. DAPI, 4,6-diamidino-2-phenylindole (DAPI; nuclei staining). Scale bar, 50 μm. (C, D) S-HsfB1 or S-HsfB2a binding to the HRE was verified by Y1H and EMSA with bacterially expressed and purified His6-S-HsfB1 or His6-S-HsfB2a. Mock, empty vector control. (E) The 35S:S-HsfB1-RFP-overexpressing (OE) lines were confirmed by western blot analysis and then subjected to thermotolerance assays. (F) The T-DNA HsfB1-knockout mutant (hsfb1-1) confirmed by RT‒PCR and the antisense (targeting the retained intron sequences)-mediated S-HsfB1-knockdown lines (KD-1 and KD-2) verified by RT‒qPCR were subjected to thermotolerance assays. The data are presented as the means ± SDs of three independent experiments. Different letters indicate significant differences according to one-way ANOVA, P < 0.05. DAS, days after sowing.

HSP17.6B was identified as a direct target gene of S-HsfA2.

(A) Schematic diagrams of the HSP17.6B gene (top) showing HRE-HRE-like and ChIP‒qPCR products. (B) Heat-induced GUS expression assays of Arabidopsis plants harboring HSP17.6Bp or HSP17.6Bp lacking the HRE-HER-like (HSP17.6BpΔHRE)-driven GUS reporter transgene via GUS histochemical staining (top) and heat-induced fold (heat stress/normal) analysis, as determined via Western blotting (bottom). For each GUS reporter transgene, mixed Arabidopsis plants (10 DAS) from three independent transgenic lines were treated without (normal) or with heat stress and then subjected to GUS expression analysis. Bar, 0.5 mm. (C) ChIP‒qPCR experiments were performed with an anti-Flag antibody and mouse IgG (mock control) in 35S:S-HsfA2-Flag (S-HsfA2-OE) Arabidopsis plants. (D) RT‒qPCR heatmaps showing heat (45 ℃ for 3 h)-induced expression of HSP17.6B in S-HsfA2-OE and S-HsfA2-KD plants. In (B), (C), and (D), the data are presented as the means ± SDs of at least two independent experiments. The significance of differences between the experimental values was assessed by Student’s t test (*P <0.05).

HSP17.6B positively regulates Arabidopsis tolerance to heat.

(A) Schematic diagrams of the HSP17.6B gene (top) showing T-DNA insertions. RT‒qPCR heatmaps of heat (37 ℃ for 2 h)-induced expression of HSP17.6B in two T-DNA mutants (hsp17.6b-1 and -2), three lines of 35S:HSP17.6B-Flag/hsp17.6b-1 (HSP17.6B-KI), and two lines of 35S:HSP17.6B-Flag (HSP17.6B-OE). (B) Thermotolerance of the WT control, hsp17.6b mutants, and HSP17.6B-OE lines. (C) Thermotolerance of the WT control and HSP17.6B-KI lines. In (B) and (C), the survival rates of the seedlings under heat stress are shown. DAS, days after sowing.

HSP17.6B overexpression represses Arabidopsis growth.

(A) HSP17.6B-OE plants (15 DAS) exhibited defects in growth, as indicated by decreased chlorophyll content and fresh weight. The data are presented as the means ± SDs of at least two independent experiments. The significance of differences between the experimental values was assessed by Student’s t test (*P <0.05). (B) The dwarf but heat-resistant phenotype of the HSP17.6B-OE plants grown on soil. DAS, days after sowing.

S-HsfA2 interacts with the DBD of HsfA2.

(A) Five HSFs that interact with S-HsfA2 were identified via Y2H analysis of 19 Arabidopsis HSFs. (B) GST pull-down verified that S-HsfA2 interacted with HsfA2 in vitro. According to the Y2H (C) and BiFC (D) assays, S-HsfA2 interacted with the DBD of HsfA2.

S-HsfA2 serves as a negative binding regulator of HsfA2.

(A) Schematic diagram of the streptavidin-agarose bead pull-down assay, in which polydeoxyinosinic-deoxycytidylic acid (dI-dC) was used as the nonspecific DNA competitor (left). GST-S-HsfA2 and His6-HsfA2 were incubated for the indicated times (0, 4, and 12 h) and then subjected to a streptavidin-agarose bead pull-down assay to isolate His6-HsfA2 proteins that bind to the biotin-labelled HSE probes. The resulting His6-HsfA2 was analysed by immunoblotting (IB) with an anti-His6 antibody (right). The ability of His6-HsfA2 to bind to the HSE bait is expressed as the relative band intensity of the control (0 h), which was set as 1. The data are the means of two independent experiments. (B) Dual-luciferase (LUC) assays in tobacco leaves. A representative bioluminescence image of the sHSP17.6Bp:LUC reporter coexpressing the indicated effector groups. The effector-driven reporter expression activity was expressed as the relative ratio of LUC to Renilla luciferase (REN) activity (the GUS effector control was set as 1). The data are presented as the means ± SDs of three independent experiments. Different letters indicate significant differences according to one-way ANOVA, P < 0.05.

A proposed model for S-HsfA2 balancing Arabidopsis heat tolerance and growth.

The overexpression of HSP17.6B not only confers thermotolerance but also retards seedling growth, reflecting the hyperactivation of the HSR mediated by HSP17.6B overexpression. Upon exposure to severe heat, S-HsfA2 is generated from the splice variant HsfA2-III. Then, S-HsfA2 balances the gain (extreme heat tolerance, EHT) and loss (growth retardation, GR) of HsfA2-regulated HSP17.6B overexpression to ensure proper growth in two ways: ① antagonistic repression of HSP17.6B overexpression through a noncanonical HSR and ② a negative binding regulator of HSFs that inactivates the HSP17.6B promoter-binding capacity of HsfA2.