Discussion:
A number of studies have been carried out to functionally characterize
the HSF genes for their potential role in thermotolerance and its
signaling (Chauhan et al., 2013, Wang et al., 2017, Zhu et al., 2018).TaA6b is mainly nucleo-cytosolic and heat stress does not affect
its nucleocytoplasmic distribution (Figure 1). Huang et al. (2016)
reported HSFA6b from Arabidopsis to be localized to both the
nucleus and cytosol, with partial but not complete translocation to the
nucleus which is in consistence with our results. Hu et al (2015) have
also found that fourteen Hsfs in Fragaria vesca localized in
nucleus, out of which 6 were also localized in cytosol. Baniwal et al.,
(2007) studied two Hsfs from tomato and found that HsfA4b was
localized in the nucleus, whereas HsfA5 was predominantly
detected in the cytoplasm.
It has been demonstrated that transgenic plants with overexpression of
HSF genes show improved thermotolerance, however, most of the studies
were based on model plants like Arabidopsis and tobacco (Busch et
al. 2005, Li et al., 2005, Zhu et al., 2009, Wu et al., 2018). Heat
induced up-regulation of class A6 HSFs has been observed in wheat andTaHSFA6b was one of the predominantly expressed genes in the
leaves during early heat stress conditions (Xue et al., 2013), which
suggests that the constitutive overexpression of TaHSFA6b may
enhance tolerance to high temperature conditions. Previously, it has
been shown that TaHSFA6b provides tolerance against high
temperature stress in transgenic Arabidopsis (Chauhan et al.,
2013).
In the present study, the role of TaHSFA6b gene in enhanced
thermotolerance of transgenic barley has been investigated. Transgenic
lines of barley constitutively overexpressing the TaHSFA6b gene
under the control of ZmUbi promoter showed notable
thermotolerance (Figure 2). Enhanced thermotolerance of these plants can
be explained by increased expression of abiotic stress responsive genes
including antioxidative enzymes (catalase, glutathione S-transferase and
peroxidase), HSPs, LEA protein and proteins involved in
Ca2+ signaling pathway. Similarly, Xue et al., 2015
reported that TaHSFA6f regulates the transcription of heat
responsive genes involved in heat stress signaling cascade including
Hsps and Golgi anti-apoptotic protein (GAAP).
Excessive generation and accumulation of reactive oxygen species (ROS)
severely affect the plant cell by increased levels of oxidative injuries
leading to destruction of cellular structure and function (Schutzendubel
and Polle, 2002). Barley transgenic lines showed significantly low
levels of superoxide accumulation in comparison to wild type plants
under high temperature conditions (Figure 3A). This difference in
superoxide accumulation can be attributed by the quenching of excessive
ROS levels by upregulated antioxidative defense system. Chloroplasts and
mitochondria are the major sites for ROS production under heat stress
(Suzuki and Mittler, 2006). Ascorbate peroxidases (APX) are the main
enzymes which regulate the release of ROS from their generation site
(Koussevitzky et al. , 2008); upregulated APXs ameliorate the
oxidizing environment created by the high ROS accumulation (Badawi et
al., 2004, De Pinto et al., 2015).
Deficient expression of catalase (CAT) results into increased levels of
peroxide (H2O2), which gives rise to
imbalance in ROS homeostasis; upregulation of CAT removes the excess
H2O2 and maintains the ROS balance
(Vandenabeele et al., 2004).
Transcriptome analysis revealed that TaHSFA6b regulated genes are
mostly the genes which are heat, draught and oxidative stress
responsive. In the present study it was observed that the transcription
of HSPs was positively affected and higher expression of HSPs
demonstrated in RNA-seq data and confirmed by qRT-PCR analysis (HSP70-2,
HSP70-5, HSP90-2, and HSP18.1). Protein folding, intracellular
localization and degradation are considered as the primary functions of
Hsps (Qu et al., 2013), however, the important role of HSPs during heat
and other abiotic stresses has been well studied in different plant
systems (Hu et al., 2009, Jacob et al., 2017). In a heat tolerant
cultivar of rice; N22, a significant upregulation of HSPs was observed
demonstrating the important role of HSPs in plants (Jagadish et al.,
2010).
The constitutive upregulation of chaperonins (Cpn60-1, Cpn60-2, and
Cpn60-10) was also observed in TaHvHSFA6b overexpressing
transgenic barley lines. Chaperonins are the high molecular weight
complex proteins helping the chaperons in protein folding (Evstigneeva
et al., 2001, Reddy et al., 2016). Chaperonins have been reported to
function in folding the newly translated proteins as well as assisting
the chaperons in refolding denatured proteins under stress conditions
(Hill et al., 2001, Levy-Rimler et al., 2002, Wang et al., 2004).
Increased expression of DNAJ proteins was also observed; DNAJ proteins
work as co-chaperones with HSP70 and regulate the protein homeostasis
(Pulido et al., 2017). Wang et al., 2019 reported that DNJ protein ofSolanum lycopersicum (SlDnaJ20) showed heat inducibility and
DNJ20 overexpressing transgenic tomato plants performed better under
heat stress conditions in terms of fresh biomass, total chlorophyll,
chlorophyll fluorescence and less accumulation of ROS.
Heat stress creates oxidizing environment inside the cell by over
production of ROS, which leads to peroxidation of membrane lipids
(Mansoor et al., 2013). The aldehyde dehydrogenase (ALDH) is known for
removing the lipid peroxidation generated reactive aldehydes (Brocker et
al., 2012). The constitutive upregulation of ALDH gene inTaHSFA6b overexpression lines suggests their superiority over
wild type plants in terms of less availability of reactive aldehyde
species, as these are the most cytotoxic substances produced downstream
of the ROS (Mano et al., 2019).
Extreme environmental conditions like high temperature, desiccation,
salinity, and freezing; result into decreased cellular water content in
plants (Fahad et al., 2017). Under high temperature conditions, a steep
decrease in cellular water content was observed in tomato plants
(Morales et al., 2003). Expression of LEA proteins is upregulated to
protect the cell water content and to prevent the dehydration caused
protein denaturation (Kovacs et al., 2008). Goyal et al., (2005)
reported that LEA protein may also act as chaperons and showed that
these proteins can prevent the heat induced inactivation and aggregation
of different enzymes such as citrate synthase. In the present study, LEA
proteins expression levels were remarkably high in transgenic lines in
comparison to wild type plants, which contributes to explaining the
better performance of transgenics over wild type plants.
Global climate change and increasing temperatures are major issues of
international concern. High temperature severely affects the growth,
production and final yield capacity of food crops, therefore, it is
important to understand the plant responses towards heat stress at
physiological, biochemical and molecular levels. HSF and HSP genes have
been well studied and suggested as key players in plant heat stress
response, however there is no sufficient knowledge about the roles and
stress mitigation potentials of individual genes in cereal plants.
Conclusively, the present study investigated and characterized a heat
responsive HSF gene of wheat; TaHSFA6b in barley. TaHSFA6bplays regulatory role in heat shock response pathways. Transgenic lines
of barley showed notable superiority over wild type under high
temperature conditions. The positive alteration in thermotolerance may
be attributed by the coordinated upregulation of defense mechanisms
related genes in transgenics. Transcriptomic analysis of overexpression
lines suggests that TaHSFA6b works as an activator of HSPs as
well as other stress responsive genes. Therefore, we suggest that theTaHSFA6b gene may be used for molecular breeding to generate
heat-tolerant cultivars of temperate cereal crops which are highly
susceptible to heat stress.