The expression of heat shock proteins (Hsps) induced by nonlethal heat treatment confers acquired thermotolerance (AT) to organisms against subsequent challenges of otherwise lethal temperature. After the stress signal is removed, AT gradually decays, with decreased Hsps during recovery. AT of sufficient duration is critical for sessile organisms such as plants to survive repeated heat stress in their environment, but little is known regarding its regulation. To identify potential regulatory components, we took a reverse genetics approach by screening for Arabidopsis (Arabidopsis thaliana) T-DNA insertion mutants that show decreased thermotolerance after a long recovery (2 d) under nonstress conditions following an acclimation heat treatment. Among the tested mutants corresponding to 48 heat-induced genes, only the heat shock transcription factor HsfA2 knockout mutant showed an obvious phenotype. Following pretreatment at 37°C, the mutant line was more sensitive to severe heat stress than the wild type after long but not short recovery periods, and this could be complemented by the introduction of a wild-type copy of the HsfA2 gene. Quantitative hypocotyl elongation assay also revealed that AT decayed faster in the absence of HsfA2. Significant reduction in the transcript levels of several highly heat-inducible genes was observed in HsfA2 knockout plants after 4 h recovery or 2 h prolonged heat stress. Immunoblot analysis showed that Hsa32 and class I small Hsp were less abundant in the mutant than in the wild type after long recovery. Our results suggest that HsfA2 as a heat-inducible transactivator sustains the expression of Hsp genes and extends the duration of AT in Arabidopsis.

In Arabidopsis, there are four homologs of class A1 heat shock factor (HSFA1) genes, which likely encode the master regulators of heat shock response (HSR). However, previous studies with double knockout (KO) mutants were unable to confirm this point probably due to functional redundancy. Here, we generated a quadruple KO (QK) and four triple KO mutants to dissect their functions. Our data show that members of the HSFA1 group not only play a pivotal role in HSR but also are involved in growth and development. Alterations in morphology and retardation in growth were observed in the quadruple but not in triple KO mutants. The basal and acquired thermotolerance capacity was dramatically decreased in the QK mutant but varied in triple KO mutants at different developmental stages. The transcriptomics profiles suggested that more than 65% of the heat stress (HS)-up-regulated genes were HSFA1 dependent. HSFA1s were also involved in the expression of several HS genes induced by H2O2, salt and mannitol, which is consistent with the increased sensitive phenotype of the QK mutant to the stress factors. In conclusion, the Arabidopsis HSFA1s function as the master regulators of HSR and participate as important components in other abiotic stress responses as well.

Plants and animals share similar mechanisms in the heat shock (HS) response, such as synthesis of the conserved HS proteins (Hsps). However, because plants are confined to a growing environment, in general they require unique features to cope with heat stress. Here, we report on the analysis of the function of a novel Hsp, heat-stress-associated 32-kD protein (Hsa32), which is highly conserved in land plants but absent in most other organisms. The gene responds to HS at the transcriptional level in moss (Physcomitrella patens), Arabidopsis (Arabidopsis thaliana), and rice (Oryza sativa). Like other Hsps, Hsa32 protein accumulates greatly in Arabidopsis seedlings after HS treatment. Disruption of Hsa32 by T-DNA insertion does not affect growth and development under normal conditions. However, the acquired thermotolerance in the knockout line was compromised following a long recovery period (.24 h) after acclimation HS treatment, when a severe HS challenge killed the mutant but not the wild-type plants, but no significant difference was observed if they were challenged within a short recovery period. Quantitative hypocotyl elongation assay also revealed that thermotolerance decayed faster in the absence of Hsa32 after a long recovery. Similar results were obtained in Arabidopsis transgenic plants with Hsa32 expression suppressed by RNA interference. Microarray analysis of the knockout mutant indicates that only the expression of Hsa32 was significantly altered in HS response. Taken together, our results suggest that Hsa32 is required not for induction but rather maintenance of acquired thermotolerance, a feature that could be important to plants.

There are 21 heat shock factor (HSF) homologs in Arabidopsis (Arabidopsis thaliana), of which members of class A1 (HSFA1a/HSFA1b/ HSFA1d/HSFA1e) play the major role in activating the transcription of heat-induced genes, including HSFA2. Once induced, HSFA2 becomes the dominant HSF and is able to form heterooligomeric complexes with HSFA1. However, whether HSFA2 could function independently as a transcription regulator in the absence of the HSFA1s was undetermined. To address this question, we introduced a Cauliflower mosaic virus 35S promoter:HSFA2 construct into hsfa1a/hsfa1b/hsfa1d/hsfa1e quadruple knockout (QK) and wild-type (Wt) backgrounds to yield transgenic lines A2QK and A2Wt, respectively. Constitutive expression of HSFA2 rescued the developmental defects of the QK mutant and promoted callus formation in A2QK, but not in A2Wt, after heat treatment. Transcriptome analysis showed that heat stress response genes are differentially regulated by the HSFA1s and HSFA2; the genes involved in metabolism and redox homeostasis are preferentially regulated by HSFA2, while HSFA1-preferring genes are enriched in transcription function. Ectopic expression of HSFA2 complemented the defects of QK in tolerance to different heat stress regimes, and to hydrogen peroxide, but not to salt and osmotic stresses. Furthermore, we showed that HSFA1a/HSFA1b/HSFA1d are involved in thermotolerance to mild heat stress at temperatures as low as 27°C. We also noticed subfunctionalization of the four Arabidopsis A1-type HSFs in diverse abiotic stress responses. Overall, this study reveals the overlapping and distinct functions of class A1 and A2 HSFs and may enable more precise use of HSFs in engineering stress tolerance in the future.