The Plant Cell, Vol. 28: 2, January 2016, www.plantcell.org ã 2016 American Society of Plant Biologists. All rights reserved.
IN BRIEF
Keep Your Cool: A Regulatory Region to Inactivate Heat Stress Transcription Factors under Normal Conditions Heat stress is a problem facing virtually all living organisms. Accordingly, it is perhaps not surprising that some of the cellular components for responding to high temperature are conserved across kingdoms of life. Among these conserved components are heat shock transcription factors (HSFs), which activate the expression of downstream genes in the heat shock response. HSF proteins are divided into three classes, with plant HSF gene families much expanded compared with those in other eukaryotes (Scharf et al., 2012). In plants, class A group 1 HSFs are known as master regulators of the heat shock response, which involves both activation of acclimation mechanisms and negative feedback to ensure that the response is transitory. New work from Ohama et al. (2016) identifies a plant-specific region of HsfA1 that serves to suppress the heat stress response in Arabidopsis thaliana under normal conditions. Sequence alignment of HsfA1s from plants revealed a conserved domain in the central region between known functional domains characteristic of all HSFs. Using deletion analysis, Ohama et al. showed that this region functions as a negative regulator of HsfA1 DNA binding and transactivation activity. One portion of the domain, termed region 1, was particularly important in the heat responsiveness of the transactivation activity. The most highly conserved part of region 1 was designated as the temperature-dependent repression (TDR) domain. The TDR domain contains a QIVKYQP motif conserved in plant and Chlamydomonas reinhardtii HsfA1 proteins. To test whether phosphorylation of the Y in this motif might regulate HsfA1 function, the authors replaced it with D (as a phosphomimic). The Y271D mutation had similar effects on transactivation and DNA binding activity as removal of the entirety of region 1, implying that phosphorylation might allevi-
www.plantcell.org/cgi/doi/10.1105/tpc.15.01064
Figure 1. Plants overexpressing HsfA1d lacking region 1 are thermotolerant. Lines lacking region 1 (dD1-j and dD1-f) tolerate high-temperature treatment better than lines with other versions of HsfA1d or the vector-only control (VC). (Reprinted from Ohama et al. [2016], Figure 6E.)
ate the negative regulation imposed by that region. However, the relevance of this putative phosphorylation remains to be explored, as the authors could not obtain direct evidence of phosphorylation in vivo. Ohama et al. did show that the negative regulatory domain functions as an independent unit. As part of a chimeric fusion protein, the HsfA1d negative regulatory domain repressed a heterologous activation domain. Importantly, this repression occurred in a heat stress-dependent manner and was abolished by mutation of Y271, again pointing to a role for this residue in the regulation of HsfA1 activity. Ohama et al. found that region 1 was important for interaction between HsfA1d and Hsp70 and Hsp90 proteins, both of which are negative regulators of HsfA1 activity. In addition, the authors provide longawaited experimental evidence to support the model that HsfA1 proteins are activated under heat stress by dissociation from Hsp70, which suppresses their activity under normal conditions. Plants overexpressing a version of the HsfA1d-lacking region 1 were thermotolerant (see figure), with some aspects of the heat stress response constitutively activated. However, not all facets of the response were activated and even some direct targets of HsfA1 were not induced.
Thus, region 1 is necessary for regulation of some HsfA1 downstream genes, but not for all of them. Finally, although these plants could survive high temperature, they were stunted, which points to the high cost of the heat stress response and likely helps explain its strict and multifaceted regulation in plants.
Nancy R. Hofmann Science Editor [email protected] ORCID ID: 0000-0001-9504-1152
REFERENCES Ohama, N., Kusakabe, K., Mizoi, J., Zhao, H., Kidokoro, S., Koizumi, S., Takahashi, F., Ishida, T., Yanagisawa, S., Shinozaki, K., and Yamaguchi-Shinozaki, K. (2015). The transcriptional cascade in the heat stress response of Arabidopsis is strictly regulated at the level of transcription factor expression. Plant Cell 28: 181–201. Scharf, K.D., Berberich, T., Ebersberger, I., and Nover, L. (2012). The plant heat stress transcription factor (Hsf) family: structure, function and evolution. Biochim. Biophys. Acta 1819: 104–119.
IN BRIEF
Keep Your Cool: A Regulatory Region to Inactivate Heat Stress Transcription Factors under Normal Conditions Heat stress is a problem facing virtually all living organisms. Accordingly, it is perhaps not surprising that some of the cellular components for responding to high temperature are conserved across kingdoms of life. Among these conserved components are heat shock transcription factors (HSFs), which activate the expression of downstream genes in the heat shock response. HSF proteins are divided into three classes, with plant HSF gene families much expanded compared with those in other eukaryotes (Scharf et al., 2012). In plants, class A group 1 HSFs are known as master regulators of the heat shock response, which involves both activation of acclimation mechanisms and negative feedback to ensure that the response is transitory. New work from Ohama et al. (2016) identifies a plant-specific region of HsfA1 that serves to suppress the heat stress response in Arabidopsis thaliana under normal conditions. Sequence alignment of HsfA1s from plants revealed a conserved domain in the central region between known functional domains characteristic of all HSFs. Using deletion analysis, Ohama et al. showed that this region functions as a negative regulator of HsfA1 DNA binding and transactivation activity. One portion of the domain, termed region 1, was particularly important in the heat responsiveness of the transactivation activity. The most highly conserved part of region 1 was designated as the temperature-dependent repression (TDR) domain. The TDR domain contains a QIVKYQP motif conserved in plant and Chlamydomonas reinhardtii HsfA1 proteins. To test whether phosphorylation of the Y in this motif might regulate HsfA1 function, the authors replaced it with D (as a phosphomimic). The Y271D mutation had similar effects on transactivation and DNA binding activity as removal of the entirety of region 1, implying that phosphorylation might allevi-
www.plantcell.org/cgi/doi/10.1105/tpc.15.01064
Figure 1. Plants overexpressing HsfA1d lacking region 1 are thermotolerant. Lines lacking region 1 (dD1-j and dD1-f) tolerate high-temperature treatment better than lines with other versions of HsfA1d or the vector-only control (VC). (Reprinted from Ohama et al. [2016], Figure 6E.)
ate the negative regulation imposed by that region. However, the relevance of this putative phosphorylation remains to be explored, as the authors could not obtain direct evidence of phosphorylation in vivo. Ohama et al. did show that the negative regulatory domain functions as an independent unit. As part of a chimeric fusion protein, the HsfA1d negative regulatory domain repressed a heterologous activation domain. Importantly, this repression occurred in a heat stress-dependent manner and was abolished by mutation of Y271, again pointing to a role for this residue in the regulation of HsfA1 activity. Ohama et al. found that region 1 was important for interaction between HsfA1d and Hsp70 and Hsp90 proteins, both of which are negative regulators of HsfA1 activity. In addition, the authors provide longawaited experimental evidence to support the model that HsfA1 proteins are activated under heat stress by dissociation from Hsp70, which suppresses their activity under normal conditions. Plants overexpressing a version of the HsfA1d-lacking region 1 were thermotolerant (see figure), with some aspects of the heat stress response constitutively activated. However, not all facets of the response were activated and even some direct targets of HsfA1 were not induced.
Thus, region 1 is necessary for regulation of some HsfA1 downstream genes, but not for all of them. Finally, although these plants could survive high temperature, they were stunted, which points to the high cost of the heat stress response and likely helps explain its strict and multifaceted regulation in plants.
Nancy R. Hofmann Science Editor [email protected] ORCID ID: 0000-0001-9504-1152
REFERENCES Ohama, N., Kusakabe, K., Mizoi, J., Zhao, H., Kidokoro, S., Koizumi, S., Takahashi, F., Ishida, T., Yanagisawa, S., Shinozaki, K., and Yamaguchi-Shinozaki, K. (2015). The transcriptional cascade in the heat stress response of Arabidopsis is strictly regulated at the level of transcription factor expression. Plant Cell 28: 181–201. Scharf, K.D., Berberich, T., Ebersberger, I., and Nover, L. (2012). The plant heat stress transcription factor (Hsf) family: structure, function and evolution. Biochim. Biophys. Acta 1819: 104–119.
