Hsf involvement in temperature and other abiotic stresses

Class A Hsfs have been characterized as main transcriptional activators which are responsible for induction of HS genes including other Hsfs. In tomato, among four HsfA1 genes, HsfA1a was defined as the master regulator of thermotolerance (Mishra et al., 2002). This was shown in experiments using transgenic plants exhibiting a co-suppression of HsfA1a expression (A1CS) which had drastically reduced thermotolerance when exposed to elevated temperatures even though the overall plant growth and development were not impaired under control conditions (Mishra et al., 2002). The reason behind this is that the HS-induced expression of HsfA2, HsfB1 and chaperones was almost completely diminished due to HsfA1a suppression. In contrast to this finding, A. thaliana does not have a single master regulator but all four of the class A Hsfs contribute to thermotolerance and HS-responsive gene expression (Liu et al., 2011; Yoshida et al., 2011). Furthermore, the hsfa1a/b/d/e quadruple knockout (KO) mutant plants were not only showing an impaired HSR, but also defects in growth and development suggesting that beyond the well-established role in HSR, the basal activity of some Hsfs is important for physiological processes. However, the hsfa1a/b/d triple KO mutants did not have developmental defects and

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exhibited a severe thermosensitive phenotype even at a mild HS exposure of 27°C (Yoshida et al., 2011; Liu and Charng, 2013). The reason for this peculiarity in single and shared master regulator function between tomato and A. thaliana might be explained by the fact that the RNAi effect in the tomato co-suppression plants might have targeted all the HsfA1s (Scharf et al., 2012). However, this needs further investigation.

4.4.2.2 HsfA2 is important for acquired thermotolerance

Although HsfA2 is structurally similar to HsfA1a it is only induced in response to HS and belongs to the most prominent Hsfs in tomato, A. thaliana and rice. It accumulates at high levels in plants exposed to prolonged heat and recovery conditions after HS exposure (Scharf et al., 1998;

Charng et al., 2006; Nishizawa et al., 2006; Schramm et al., 2008). Interestingly, ectopic expression of HsfA2 could complement the defects of the hsfa1a/b/d/e quadruple KO mutant regarding tolerance to different HS regimes and to hydrogen peroxide even though HS-genes were showing differential regulation by these factors (Liu and Charng, 2013). Analysis of tomato plants with suppressed HsfA2 levels revealed that HsfA2 is involved in the regulation of Hsp expression in a tissue-specific manner. Young seedlings of HsfA2 knock-down plants were not sensitive to a single heat exposure (BTT), however, HsfA2 was necessary for young seedlings to acquire thermotolerance (Fragkostefanakis et al., 2016). Interestingly, pollen viability and germination rate upon HS exposure were also significantly affected by HsfA2 suppression because HsfA2 is essential for the high expression of Hsps during pollen development (Fragkostefanakis et al., 2016).

Similarly, A. thaliana HsfA2 was important for extension of ATT, as shown by a hypocotyl elongation assay (Charng et al., 2006). This was related to a reduction of transcript levels of highly heat-inducible genes and lower protein levels of Hsa32 and class CI sHsps in the mutant compared to wild-type plants. Interestingly, both A. thaliana and tomato HsfA2 were shown to be important for the regulation of other stress-related genes like APX members, GALACTINOL SYNTHASE 1 (GOLS1), Hsa32 and MBF1c (Charng et al., 2006; Nishizawa et al., 2006; Schramm et al., 2006; Nishizawa-Yokoi et al., 2009; Fragkostefanakis et al., 2015b). These findings suggest involvement of HsfA2 in regulating stress related genes beyond Hsps. Furthermore, A. thaliana HsfA2 KO plants were sensitive to light and oxidative stress and anoxia, whereas HsfA2 overexpression plants had an increased thermotolerance and resistance to other stresses like salinity, oxidative stress and anoxia (Nishizawa et al., 2006; Ogawa et al., 2007; Zhang et al., 2009).

4.4.2.3 HsfA6 and HsfA7 members

According to the evolutionary relationship of Hsfs from nine plant species obtained by alignment of the N-proximal parts containing the DBD and OD region of 250 Hsfs, HsfA2/A6/A7 have the highest amino acid sequence similarity and the closest phylogenetic relationship (Nover et al., 2001; Scharf et al., 2012). Several studies extensively studied the role HsfA2 in transgenic plants; however, limited studies are available which investigate the importance of HsfA6 and HsfA7.

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In tomato, it was shown that transcript abundance of HsfA6b and HsfA7 were enhanced more strongly in heat stressed anthers of HsfA2 knock-down plants compared to wild-type plants (Fragkostefanakis et al., 2016). This indicates the existence of a feedback regulatory mechanism between HsfA2, HsfA6b and HsfA7. A similar increase in expression upon loss of HsfA2 was not observed in leaves, indicating that such mechanisms might be tissue or even cell specific (Fragkostefanakis et al., 2016). According to transcriptome studies HsfA7 was shown to be one of the most significantly upregulated Hsfs upon a HS treatment (Busch et al., 2005; Charng et al., 2006; Cortijo et al., 2017) and it was proposed to play an important role in the cytosolic protein response (CPR) (Sugio et al., 2009). An A. thaliana HsfA7a KO mutant had a decreased viability upon a gradual acclimation temperature treatment and an ATT treatment (Larkindale and Vierling, 2007). This indicates that it is one of the Hsfs which contribute to heat acclimation in A thaliana. However, even though a close phylogenetic relationship exists, the loss of HsfA2 could not be compensated by the presence of HsfA7a/A7b in A. thaliana (Charng et al., 2006).

Furthermore, the HsfA7 KO lines did not show a drastic thermotolerance defect comparable to the loss of HsfA2 (Charng et al., 2006). This points out, that although similarities exist, these Hsfs may not have simply redundant functions (Nover et al., 2001). Overexpression of the rice HsfA7 (OsHsfA7) in A. thaliana plants resulted in increased expression of GolS2 and some Hsps like Hsp101 upon HS exposure. The transgenic plants also had an improved thermotolerance upon a harsh temperature treatment (Liu et al., 2009). Furthermore, when OsHsfA7 was overexpressed in the rice background this resulted in increased drought and salinity stress resistance (Liu et al., 2013a).

A. thaliana HsfA6b was recently shown to act as a downstream regulator of the ABA-mediated stress response and participate in ABA-mediated salt and drought resistance while thermotolerance tests showed that HsfA6b is required for thermotolerance acquisition (Huang et al., 2016). This suggests that ABA-signalling plays an important role in the complexity of the HSR. Overexpression of HsfA6f in wheat resulted in improved thermotolerance by the stronger upregulation of several Hsps, as well as previously unknown Hsf target genes such as Golgi-antiapoptotic protein (GAAP) and the large isoform of Rubisco activase (Xue et al., 2015).

4.4.2.4 Other Hsf members

A. thaliana HsfA3 is induced in response to drought and heat and this is directly transcriptionally regulated by DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN (DREB2A) which is a transcription factor mediating expression of genes mainly involved in drought stress (Sakuma, 2006; Schramm et al., 2008). Overexpression of DREB2A can lead to induction of HsfA3 expression and other HS-inducible genes and an increased thermotolerance, while DREB2A KO mutants had a reduced thermotolerance (Sakuma, 2006).

HsfA4a was shown to play a key role in ROS sensing (Davletova, 2005). A. thaliana plants expressing a dominant negative mutant of HsfA4a had an impaired response to oxidative (H2O2) stress (Davletova, 2005).

HsfB1 and HsfB2b were not found to be directly involved in the regulation of the onset of the HSR (Kumar et al., 2009). However, in hsfB1/hsfB2b double KO plants Pdf genes were identified

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as the major targets of an Hsf-dependent negative regulation. These genes are involved in immunity against infection by necrotrophic microorganisms, which implicates the interplay of Hsfs in the regulation of biotic stress responses (Kumar et al., 2009). On the other hand, Ikeda et al. (2011) showed that HsfB1 and HsfB2b suppress the HSR under non-stress conditions as shown by reduction of HsfA2, HsfA7a and HsfB2b transcript levels under control conditions and in the attenuation period (Ikeda et al., 2011). In addition, HsfB1 and HsfB2b were also important for the establishment of ATT in Arabidopsis thaliana (Ikeda et al., 2011).

Taken together, all these findings highlight the involvement of Hsfs in stress signalling cascades other than the ones activated in response to heat and there is a remarkable functional specificity of the different Hsfs participating in a certain abiotic stress response. It is also important to emphasize that knockouts of Hsfs are required in order to study and evaluate their involvement in regulation of HS-gene expression. Many studies mentioned above have shown that, when analysed in detail, a remarkable functional diversification can be found. However, there are not always obvious phenotypes, most probably due to functional redundancy among Hsfs (Scharf et al., 2012).