**1. Introduction**

In recent years, there has been an increasing trend to focus on the responses of plants to abiotic stresses due to global climate change [1]. Particularly, research has been focused on plant heat stress (HS) tolerance mechanisms, as higher temperatures have a negative effect on plant growth and production [2,3]. Although plants are susceptible to HS throughout their lifespan, reproductive tissues are specifically characterized by

**Citation:** Haider, S.; Rehman, S.; Ahmad, Y.; Raza, A.; Tabassum, J.; Javed, T.; Osman, H.S.; Mahmood, T. In Silico Characterization and Expression Profiles of Heat Shock Transcription Factors (HSFs) in Maize (*Zea mays* L.). *Agronomy* **2021**, *11*, 2335. https://doi.org/10.3390/ agronomy11112335

Academic Editor: Carlos Iglesias

Received: 6 October 2021 Accepted: 16 November 2021 Published: 18 November 2021

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vulnerability to HS [4,5]. Heatwaves are expected to become more frequent in the future, and atmospheric temperature is on course to rise ~4 ◦C by the end of this century [6,7]. HS negatively affects plant morphology, physiology, and growth in diverse ways [5,8]. HS alters plasma membrane fluidity, creates proteotoxic stress, causes the overproduction of reactive oxygen species (ROS), and dismantles cellular organization by inducing the collapse of the cytoskeleton apparatus [3,8–10]. Nonetheless, plants possess an efficient system that allows them to perceive the environmental stimulus, activate the signaling pathways, and alter their gene expression to ensure survival [9,11,12]. A major step in this process is the activation of stress-inducible genes, the expression of which is controlled by transcription factors (TFs). TFs represent a group of regulatory proteins that control the expression pattern of genes under various developmental and stressful conditions [13,14]. Several TFs families including heat shock transcription factors (HSFs), WRKY (named due to conserved WRKYGQK motif), v-myb avian myeloblastosis viral oncogene homolog (MYB), Petunia NAM, *Arabidopsis* ATAF1/2 and CUC2 (NAC), and dehydration responsiveelement binding transcriptional activator (DREB), etc., have been shown to positively regulate HS-responsive gene expression and improve plant HS tolerance [14,15]. Among them, the most comprehensively analyzed family is HSFs, the members of which are considered the master regulators of plant HS-response (HSR) and are also involved in regulating plant responses to other abiotic and biotic stress conditions [12,16]. Since the first HSF gene was identified in yeast [17], the HSF gene family has been characterized in several plant species [18,19]. The pioneering study by Nover et al. [20] allowed the identification of the HSF gene family in various plant species, including essential crop plants [19]. The Heatster database (http://www.cibiv.at/services/hsf, accessed on 1 August 2021) currently holds 848 HSF sequences from 33 different plant species.

Generally, HSFs are divided into three classes, i.e., A, B, and C, based on phylogenetic analysis and structural properties [21]. All the HSFs contain two highly conserved domains, the DNA-binding domain (DBD), which binds with "heat-shock elements" (5 nGAAnnTTCn-3 ) present in regulatory sequences of target genes, through helix-turn-helix (HTH) motif, and oligomerization domain (OD), which has a bipartile heptad repeat pattern of the hydrophobic-associated region (HR-A/B) and is responsible for the trimerization of HSFs [20,22]. Based on the linker length between the HR-A/B region, HSFs are classified into different classes. The linker length comprises 21 amino acid residues in the case of class A and 7 for class C HSFs. On the other hand, HSFs of class B, lack any insertion. The classification of HSFs is also supported by the length of the linker between DBD and OD, 9–39 amino acids for class A, 50–78 for class B, and 14–49 for class C [20–22]. The HSFs of class A are transcriptional activators, and class B are repressors [20,21]. However, in tomato, the HSFB1 possesses both co-activator and repressor functions [23,24]. The class C HSFs are activators like class A [25]. However, they lack activator peptide motif (AHA), and thus cannot initiate transcription on their own [19,20]. The AHA motif is present towards the C-terminal of HSFs and is specific to class A HSFs [26]. In addition to these domains, HSFs also possess nuclear localization signals (NLS), and some contain nuclear export signals (NES) [27]. The NLS and NES are responsible for the nuclear import and export of HSFs, an essential step in cellular functioning [27].

The function of HSFs as the master regulators of plant HSR has been demonstrated mainly in *Arabidopsis* and tomatoes [28,29]. In tomato, the overexpression of *HSFA1a*, improved plant thermotolerance, while co-suppression mutants were susceptible to HS [28]. The mutant plants and their fruits were characterized by extreme sensitivity to HS. *HSFA1a*, *HSFA2*, and *HSFB1* control the fundamental HSF network in tomato [30]. The role of the master regulator is shared by *HSFA1a*, *HSFA1b*, and *HSFA1d* in *Arabidopsis* [29]. In *hsfa1a/b/d* triple mutants, the expression of TFs and chaperons was severely hampered under HS, while the expression of several genes was downregulated even under normal growth conditions [29]. *HSFA2* is responsible for the extension of acquired thermotolerance (AT) in *Arabidopsis* [31]. In *hsfa2* mutants, the duration of AT was compromised, and the expression of HS-inducible genes was downregulated. Lämke et al. [32] reported that HSFA2 promotes

the sustained activation of several HS-memory genes through methylation of the target genomic region [32]. The transcripts of *HSFA2* are undetectable under normal conditions. However, after HS, the *HSFA2* becomes the most strongly induced HSF in plants [18,33]. Yoshida et al. reported that the overexpression of *HSFA3* improves plant thermotolerance, while the T-DNA mutants showed reduced thermotolerance [34]. Lin et al. reported that *HSFA2*, *HSFA4a*, and *HSFA7a* are essential for HSR and cytoplasmic protein response. HSFs have also been characterized in several crop plants [35]. It was reported that *OsHSFA4a* and its homolog in wheat *TaHSFA4a*, confers cadmium tolerance to plants [36]. The expression of *TaHSFA2-10* is induced in response to HS, oxidative stress, salicylic acid, and its overexpression improves plant thermotolerance [37]. In addition, HSFs are also involved in the regulation of growth and development in *Arabidopsis thaliana* [16]. For example, *HSFA9* is expressed specifically during embryogenesis and maturation in *Arabidopsis* seeds [38]. Albihlal et al. [39] reported that in *Arabidopsis*, at least 85 development-associated genes are controlled by *HSFA1b* [39]. The authors proposed that the *HSFA1b* allows plants to adjust growth and development under continuously varying environments by transducing external stimuli to stress-associated and development-related genes.

The HSF gene family has been characterized in several plant species, including *Arabidopsis thaliana* [20], *Oryza sativa* [40], *Zea mays* [41], *Glycine max* [42], *Populus trichocarpa* [43], *Solanum lycopersicum* [44], *Brachypodium distachyon* [45], and *Triticum aestivum* [46]. However, the role of HSFs in plant growth and development and in responses to stresses other than HS, is not very well understood in maize. Computational biology approaches provide a convenient and reliable platform upon which further wet-lab studies could be carried out. Here, we perform an extensive in silico analysis of maize HSFs to gain better insights into the genomic distribution, phylogeny, gene duplication history, gene structure and protein motif, physio-chemical properties, gene annotation, protein networks, and expression profiling of maize HSFs in growth and development and tolerance to multiple abiotic stresses.
