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.