Promoter specificity and interactions between early and late Arabidopsis heat shock factors

Abstract

The class A heat shock factors HsfA1a and HsfA1b are highly conserved, interacting regulators, responsible for the immediate-early transcription of a subset of heat shock genes in Arabidopsis. In order to determine functional cooperation between them, we used a reporter assay based on transient over-expression in Arabidopsis protoplasts. Reporter plasmids containing promoters of Hsf target genes fused with the GFP coding region were co-transformed with Hsf effector plasmids. The GFP reporter gene activity was quantified using flow cytometry. Three of the tested target gene promoters (Hsp25.3, Hsp18.1-CI, Hsp26.5) resulted in a strong reporter gene activity, with HsfA1a or HsfA1b alone, and significantly enhanced GFP fluorescence when both effectors were co-transformed. A second set of heat shock promoters (HsfA2, Hsp17.6CII, Hsp17.6C-CI) was activated to much lower levels. These data suggest that HsfA1a/1b cooperate synergistically at a number of target gene promoters. These targets are also regulated via the late HsfA2, which is the most strongly heat-induced class A-Hsf in Arabidopsis. HsfA2 has also the capacity to interact with HsfA1a and HsfA1b as determined by bimolecular fluorescence complementation (BiFC) in Arabidopsis protoplasts and yeast-two-hybrid assay. However, there was no synergistic effect on Hsp18.1-CI promoter-GFP reporter gene expression when HsfA2 was co-expressed with either HsfA1a or HsfA1b. These data provide evidence that interaction between early and late HSF is possible, but only interaction between the early Hsfs results in a synergistic enhancement of expression of certain target genes. The interaction of HsfA1a/A1b with the major-late HsfA2 may possibly support recruitment of HsfA2 and replacement of HsfA1a/A1b at the same target gene promoters.

Fig. 1

Quantification of interactions between Late HsfA2 and early HsfA1a and HsfA1b. a BiFC Interactions between full-length Hsf-YFP constructs in Arabidopsis protoplasts. No interaction was detected between full length HsfA2 and HsfA1a-ΔOD fusion protein. b Interactions between the oligomerization domains (OD) of HsfA2, HsfA1a and HsfA1b YFP fusion proteins in protoplasts. No interaction was detected between any OD-YFP construct and HsfA1a-ΔOD-YFP constructs. The BiFC was quantified by flow cytometric analysis (see “Methods”). c Schematic diagrams of types of Hsf-YFP constructs, drawings are not to scale. BD DNA binding domain, OD oligomerization domain, NLS nuclear localization sequence, AHA activation domain motif, c-myc and HA tags provided by BIFC vectors, 35-S and nos-ter constitutive CaMV promoter and respectively termination Sequence of the BIFC vector. d BIFC of transfected protoplast suspensions representing examples of HSF interaction (HsfA2-YFP^N152/HsfA2-YFP^c) and respectively no interaction (HsfA1aΔOD-YFP^N152/HsfA2-YFP^c). Dot plots depict the fluorescence of cells, the marked gate delimits the area of BIFC signals, the other dots result from auto-fluorescence of non-transformed protoplasts

Fig. 2

Yeast-two-hybrid interactions between early (HsfA2) and late (HsfA1a and HsfA1b) class A-Hsfs. The interaction between GAL4 bait (BD binding domain) and prey (AD activation domain) HSF fusion proteins in yeast was assayed by spotting serial dilutions (1:10, 1:100, 1:1000) of yeast on selective dropout media: -L (leucine), -W (tryptophan) -H (histidine)

Fig. 3

Promoter specificity of HsfA1a/A1b and expression of target genes. Homomeric (HsfA1a, HsfA1b) or heteromeric (HsfA1a/1b) effectors coexpressed in Arabidopsis protoplasts together with HsfA1a/1b-independent [top panels: Hsp17.6C-CI, -17.6CII, HsfA2] and HsfA1a/1b–dependent [bottom panels: Hsp26.5-P(r), -18.1-CI, -25.3-P] promoter-driven GFP reporter genes. GFP fluorescence was quantified by flow cytometry. Three ug of each promoter::reporter construct were cotransformed with 25 μg effector DNA (for double HSF transformations 12.5 μg of each construct). Data were normalized for transformation efficiency using co-transformation of 5 μg luciferase (LUC) expression plasmid. The bZIP effector expression was used as a negative control for determining endogenous Hsf-background activities in protoplasts. The expression of the effector proteins was determined by Western Blots (see supplement Fig. 1). HS (left half panels): heat stress treatment (3 h, 37°C); RT (right half panels): room temperature (3 h 25°C). Significance indicated by an asterisk, P ≤ 0.05. (Hsp17.6C-CI: HsfA1a vs. HsfA1a/1b t-test P = 0.002, HsfA1b vs. HsfA1a/1b t-test P = 0.001; Hsp25.3-P: HsfA1a vs. HsfA1a/1b t-test P = 0.009, HsfA1b vs. HsfA1a/1b t-test P = 0.05; Hsp18.1-CI: HsfA1a vs. HsfA1a/1b t-test P = 0.04, HsfA1b vs. HsfA1a/1b t-test P = 0.02; Hsp26.5: HsfA1a vs. HsfA1a/1b t-test P = 0.03, HsfA1b vs. HsfA1a/1b t-test P = 0.002)

Fig. 4

Hsp18.1 promoter activation by early and late class A Hsfs. Homomeric (HsfA1a, HsfA2) or heteromeric (HsfA1a/HsfA2, HsfA1b/HsfA2) effectors coexpressed in Arabidopsis protoplasts together the Hsp18.1-CI promoter-driven GFP reporter gene. GFP fluorescence was quantified by flow cytometry. The expression of the effector proteins was determined by Western Blots (see supplement Fig. 1). Three ug of the promoter-reporter construct were cotransformed with 25 μg effector DNA (for double HSF transformations 12.5 μg of each construct). Data were normalized for transformation efficiency using co-transformation of 5 μg luciferase (LUC) expression plasmid. The bZIP effector expression was used as a negative control for determining endogenous HSF-activities of protoplasts (see supplement Fig. 3). HS heat stress treatment (3 h, 37°C), RT room temperature (3 h 25°C)