Introduction
Barley (Hordeum vulgare L.) is an important cereal crop used for malting, brewing industry and for animal feed worldwide. Barley belongs to Gramineae family and ranks fourth after wheat, rice and maize in worldwide economic importance. Being sessile organisms, plants cannot escape the deleterious effects of heat stress that affect plant growth, physiology and development (Lobel et al, 2007). Heat stress causes changes in various physiological and metabolic processes, such as the production of reactive oxygen species (ROS) leading to oxidative damage of DNA, lipids and degradation of proteins (Wahid et al, 2007). Heat stress is one of the main cause of decrease of agriculture production and yield globally by more than 50% (Wang et al, 2004). Being a temperate cereal, both yield and quality of produce of barley is decreased by heat stress (Lobel et al, 2007; Kalra et al, 2008).
Plants signal transduction pathway leading to thermotolerance is driven by heat shock transcription factors (HSFs) and heat shock proteins (HSPs). When the ambient temperature increases, an increase in fluidity of plasma membrane takes place allowing the influx of Ca2+, which thereby binds to their downstream binding proteins, and leads to the activation of several kinases such as calcium dependent protein kinases, phosphatases, and cytoskeleton reorganization further fosters the upregulation of mitogen activated protein kinases etc. (Hirayama and Shinozaki, 2010, Kudla et al, 2010, Wahid et al, 2007). The activated kinases or phosphatases can phosphorylate or dephosphorylate particular transcription factors (TFs), due to which the expression levels of stress-responsive genes have been regulated including HSFs and HSPs (Reddy et al, 2011, Kudla et al, 2010).
Transcription factors play an important role in regulation of gene expression with response to abiotic and biotic stresses. Heat stress transcription factors (HSFs) are the central components of responses to heat stress in plants (Nover and Scharf, 1997; Kotak et al, 2007). Hsfs constitute an important gene family involved in responses to abiotic and biotic stresses as well as in plant growth and development (von Koskull-Doring et al., 2007; Liu et al., 2011; Chauhan et al., 2011).Arabidopsis has 21 HSF genes (Scharf et al., 2012), tomato has 24 (Scharf et al., 2012; Fragkostefanakis et al., 2015), pepper and rice have 25 (Chauhan et al., 2009; Guo et al., 2015), soybean has 52 (Scharf et al., 2012) and wheat counts 56 HSF genes (Xue et al., 2014).
On the basis of structure, a typical Hsf consists of five conserved motifs, including a DNA- binding domain (DBD), which is connected to an oligomerization domain (OD) consisting of hydrophobic heptad repeats (HR-A and HR- B). Beside DBD and OD, a nuclear localization signal (NLS), a nuclear export signal (NES) and an activator peptide motif (AHA) (Mittal et al., 2009; Chauhan et al., 2011; Scharf et al., 2012) are also present. Based on the structural characteristics of their HR-A/B domain plant Hsf genes are divided into 3 classes: “A”, “B”, and “C” (Nover et al., 2001; Baniwal et al., 2004). The Class “A” and “C” Hsfs contain an extended HR-A/B with 21 and 7 amino acid residues between the HR-A and HR-B region, respectively, whereas, class B Hsf lack any such insertion in the HR-A/B region (Nover et al., 2001; Baniwal et al., 2004). In addition to this, class “A” Hsfs contain AHA activation domains (rich in (Aromatic, Hydrophobic, Acidic amino acids) that are absent in class “B” and “C” Hsfs (Doring et al., 2000). Thus, class A type Hsfs are involved in transcriptional activation (Shim et al., 2009), whereas Hsfs in class B serve either as repressors of gene expression (Ikeda et al., 2011) or transcriptional coactivators with class A Hsfs (Wang et al., 2014).
In plants, HSFA1 is constitutively expressed and has a unique function as “master regulator” of heat shock response (Mishra et al., 2002). During normal conditions, HSFA1 is distributed in the cytoplasm (Scharf et al., 1998), and upon activation by heat stress, nuclear localization of HsfA1 starts which then leads to the expression ofHSFA2 and HSFB1 and the formation of hetero oligomer (termed superactivator complexes) between HSFA1 and HSFA2 (Scharf et al., 1998; Heerklotz et al., 2001). The latter drives HSR by enhanced activation of heat stress response genes expression (Chan-Schaminet et al., 2009). HSFA2 becomes the most prominent HSF under heat stress conditions and recruited by HSFA1 upon heat shock (HS) exposures. Class B HSF members cannot activate themselves and function as either repressors of HS gene expression (Czarnecka-Verner et al., 2004; Ikeda and Ohme-Takagi, 2009; Kumar et al., 2009) or as a co-regulator enhancing the activity of class A HSFs and other housekeeping transcription factors in the context of the histone acetyl transferase–like protein1 (HAC1) (Bharti et al., 2004). HSFA2 is also considered as an important linker between heat and oxidative stress responses (Chen et al, 2005) as AtHSFA2 knockout mutants showed reduced response for basal and acquired thermotolerance as well as oxidative stress. On the other hand, overexpression leads to increased tolerance for both heat and oxidative stress. Additionally, overexpression of HSFA2 in Arabidopsis, also resulted in enhanced tolerance to anoxia and submergence stress as well (Banti et al, 2010). Transgenic Arabidopsis overexpressing OsHSFA2e showed thermotolerance in various tissues such as cotyledons, rosette leaves, inflorescence stems and seeds (Yokotani et al, 2008). Charng et al, (2007) reported that HSFA2 is not only essential for HSR upon heat stress but also in maintaining expression of HSP genes in continuous heat stress and recovery period.
In Arabidopsis, other HSFAs, such as HSFA4a and HSFA8, have been suggested to act as reactive oxygen species (ROS) sensors (Davletova et al., 2005), and HSFA4c is involved in root circumnutation, gravitropism and hormonal control of differentiation (Fortunati et al., 2008). On the other hand, HSFA5 is a negative regulator which is specific repressor of HSFA4 isoforms forming a HSFA4-HSFA5 complex (Baniwal et al., 2007).HSFA7a and HSFA7b are HSR factors (Liu et al., 2011) andHSFA9 acts as a master regulator of the expression of HSPsduring seed development (Kotak et al., 2007b). Consequently, it has been suggested that HSFs mediate a cross talk between HS and other abiotic stress-signaling cascades (Kotak et al., 2007a). HSFA6a andHSFA6b transcript levels are particularly induced in response to salt, osmotic, and cold stress (von Koskull-Döring et al., 2007; Hwang et al., 2014). Further, Hwang et al (2016) found that expression ofHSFA6b extensively increased with salinity, osmotic, and cold stresses, and also showed that it plays an important role in the response to ABA and in thermotolerance.
Previously, Chauhan et al (2013) showed that overexpression of a seed preferential HSF from wheat TaHSFA2d (later renamed asTaHSFA6b after classification of Scharff et al, 2012) in Arabidopsis provided increased heat and other abiotic stress tolerance. In the present study, we overexpressed TaHSFA6b in transgenic barley for enhancing thermotolerance observed a decrease in ROS under heat stress conditions, an improved stress tolerance and no negative phenotypical changes.