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.