Short heat shock factor A2 confers heat sensitivity in Arabidopsis: Insights into heat resistance and growth balance
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eLife assessment
This paper reports a novel mechanism of regulation of the heat shock response in plants that acts as a brake to prevent hyperactivation of the stress response. The findings are valuable to understand and potentially manipulate the plant's response to heat stress and the presented evidence is overall solid. However, in some cases, the data are either poorly presented or insufficient to support the primary claims.
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Abstract
Cells prevent heat damage through a highly conserved canonical heat stress response (HSR) in which heat shock factors (HSFs) bind heat shock elements (HSEs) to activate heat shock proteins (HSPs). Plants generate short HSFs that originate from HSF splicing variants, but little is known about S-HSFs. Although an enhanced canonical HSR confers thermotolerance, its hyperactivation inhibits plant growth. How this process is prevented to ensure proper plant growth has not been determined. Here, we report that Arabidopsis S-HsfA2, S-HsfA4c, and S-HsfB1 confer extreme heat (45°C) sensitivity and represent new kinds of HSF with a unique truncated DNA-binding domain (tDBD) that binds a new heat-regulated element (HRE). The HRE conferred a minimal promoter response to heat and exhibited heat stress sensing and transmission patterns. We used S-HsfA2 to investigate whether and how S-HSFs prevent hyperactivation of the canonical HSR. HSP17.6B, a direct target gene of HsfA2, conferred thermotolerance, but its overexpression caused HSR hyperactivation. We revealed that S-HsfA2 alleviated this hyperactivation in two different ways. 1) S-HsfA2 negatively regulates HSP17.6B via the HRE-HRE-like element, thus constructing a noncanonical HSR (S-HsfA2-HRE- HSP17.6B ) to antagonistically repress HsfA2-activated HSP17.6B expression. 2) S-HsfA2 binds to the DBD of HsfA2 to prevent HsfA2 from binding to HSEs, eventually attenuating HsfA2-activated HSP17.6B promoter activity. Overall, our findings underscore the biological importance of S-HSFs, namely, preventing plant heat tolerance hyperactivation to maintain proper growth.
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eLife assessment
This paper reports a novel mechanism of regulation of the heat shock response in plants that acts as a brake to prevent hyperactivation of the stress response. The findings are valuable to understand and potentially manipulate the plant's response to heat stress and the presented evidence is overall solid. However, in some cases, the data are either poorly presented or insufficient to support the primary claims.
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Reviewer #1 (Public review):
In the present work, Chen et al. investigate the role of short heat shock factors (S-HSF), generated through alternative splicing, in the regulation of the heat shock response (HSR). The authors focus on S-HsfA2, an HSFA2 splice variant containing a truncated DNA-binding domain (tDBD) and a known transcriptional-repressor leucin-rich domain (LRD). The authors found a two-fold effect of S-HsfA2 on gene expression. On the one hand, the specific binding of S-HsfA2 to the heat-regulated element (HRE), a novel type of heat shock element (HSE), represses gene expression. This mechanism was also shown for other S-HSFs, including HsfA4c and HsfB1. On the other hand, S-HsfA2 is shown to interact with the canonical HsfA2, as well as with a handful of other HSFs, and this interaction prevents HsfA2 from activating gene …
Reviewer #1 (Public review):
In the present work, Chen et al. investigate the role of short heat shock factors (S-HSF), generated through alternative splicing, in the regulation of the heat shock response (HSR). The authors focus on S-HsfA2, an HSFA2 splice variant containing a truncated DNA-binding domain (tDBD) and a known transcriptional-repressor leucin-rich domain (LRD). The authors found a two-fold effect of S-HsfA2 on gene expression. On the one hand, the specific binding of S-HsfA2 to the heat-regulated element (HRE), a novel type of heat shock element (HSE), represses gene expression. This mechanism was also shown for other S-HSFs, including HsfA4c and HsfB1. On the other hand, S-HsfA2 is shown to interact with the canonical HsfA2, as well as with a handful of other HSFs, and this interaction prevents HsfA2 from activating gene expression. The authors also identified potential S-HsfA2 targets and selected one, HSP17.6B, to investigate the role of the truncated HSF in the HSR. They conclude that S-HsfA2-mediated transcriptional repression of HSP17.6B helps avoid hyperactivation of the HSR by counteracting the action of the canonical HsfA2.
The manuscript is well written and the reported findings are, overall, solid. The described results are likely to open new avenues in the plant stress research field, as several new molecular players are identified. Chen et al. use a combination of appropriate approaches to address the scientific questions posed. However, in some cases, the data are inadequately presented or insufficient to fully support the claims made. As such, the manuscript would highly benefit from tackling the following issues:
(1) While the authors report the survival phenotypes of several independent lines, thereby strengthening the conclusions drawn, they do not specify whether the presented percentages are averages of multiple replicates or if they correspond to a single repetition. The number of times the experiment was repeated should be reported. In addition, Figure 7c lacks the quantification of the hsp17.6b-1 mutant phenotype, which is the background of the knock-in lines. This is an essential control for this experiment.
(2) In Figure 1c, the transcript levels of HsfA2 splice variants are not evident, as the authors only show the quantification of the truncated variant. Moreover, similar to the phenotypes discussed above, it is unclear whether the reported values are averages and, if so, what is the error associated with the measurements. This information could explain the differences observed in the rosette phenotypes of the S-HsfA2-KD lines. Similarly, the gene expression quantification presented in Figures 4 and 5, as well as the GUS protein quantification of Figure 3F, also lacks this crucial information.
(3) The quality of the main figures is low, which in some cases prevents proper visualization of the data presented. This is particularly critical for the quantification of the phenotypes shown in Figure 1b and for the fluorescence images in Figures 4f and 5b. Also, Figure 9b lacks essential information describing the components of the performed experiments.
(4) Mutants with low levels of S-HsfA2 yield smaller plants than the corresponding wild type. This appears contradictory, given that the proposed role of this truncated HSF is to counteract the growth repression induced by the canonical HSF. What would be a plausible explanation for this observation? Was this phenomenon observed with any of the other tested S-HSFs?
(5) In some cases, the authors make statements that are not supported by the results:
(i) the claim that only the truncated variant expression is changed in the knock-down lines is not supported by Figure 1c;
(ii) the increase in GUS signal in Figure 3a could also result from local protein production;
(iii) in Figure 6b, the deletion of the HRE abolishes heat responsiveness, rather than merely altering the level of response; and
(iv) the phenotypes in Figure 8b are not clear enough to conclude that HSP17.6B overexpressors exhibit a dwarf but heat-tolerant phenotype. -
Reviewer #2 (Public review):
Summary:
The authors report that Arabidopsis short HSFs S-HsfA2, S-HsfA4c, and S-HsfB1 confer extreme heat. They have truncated DNA binding domains that bind to a new heat-regulated element. Considering Short HSFA2, the authors have highlighted the molecular mechanism by which S-HSFs prevent HSR hyperactivation via negative regulation of HSP17.6B. The S-HsfA2 protein binds to the DNA binding domain of HsfA2, thus preventing its binding to HSEs, eventually attenuating HsfA2-activated HSP17.6B promoter activity. This report adds insights to our understanding of heat tolerance and plant growth.
Strengths:
(1) The manuscript represents ample experiments to support the claim.
(2) The manuscript covers a robust number of experiments and provides specific figures and graphs in support of their claim.
(3) The …Reviewer #2 (Public review):
Summary:
The authors report that Arabidopsis short HSFs S-HsfA2, S-HsfA4c, and S-HsfB1 confer extreme heat. They have truncated DNA binding domains that bind to a new heat-regulated element. Considering Short HSFA2, the authors have highlighted the molecular mechanism by which S-HSFs prevent HSR hyperactivation via negative regulation of HSP17.6B. The S-HsfA2 protein binds to the DNA binding domain of HsfA2, thus preventing its binding to HSEs, eventually attenuating HsfA2-activated HSP17.6B promoter activity. This report adds insights to our understanding of heat tolerance and plant growth.
Strengths:
(1) The manuscript represents ample experiments to support the claim.
(2) The manuscript covers a robust number of experiments and provides specific figures and graphs in support of their claim.
(3) The authors have chosen a topic to focus on stress tolerance in a changing environment.Weaknesses:
(1) One s-HsfA2 represents all the other s-Hsfs; S-HsfA4c, and S-HsfB1. s-Hsfs can be functionally different. Regulation may be positive or negative. Maybe the other s-hsfs may positively regulate for height and be suppressed by the activity of other s-hsfs.
(2) Previous reports on gene regulations by hsfs can highlight the mechanism.
(3) The Materials and Methods section could be rearranged so that it is based on the correct flow of the procedure performed by the authors.
(4) Graphical representation could explain the days after sowing data, to provide information regarding plant growth.
(5) Clear images concerning GFP and RFP data could be used.
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