**1. Introduction**

Normal plant growth is often affected by a variety of adverse environmental factors such as drought, high salinity, temperature extremes and other abiotic stresses [1]. Abiotic stress inhibits normal plant growth, development, and function by speeding up chlorophyll degradation, disrupting chloroplast membrane activities, and decreasing photosynthetic efficiency, as shown by a number of studies [2–4]. In response to external stimuli, plants' bodies produce signals that trigger the phosphorylation of downstream proteins, which in turn trigger a number of transcription factors [5]. Ultimately, plant-associated resistance genes and associated defense systems are induced, thus altering the ability of the plant to adapt to its environment [6–8].

Plants have developed multiple defense mechanisms and strategies to cope with adverse conditions and respond accordingly [9–11]. Under abiotic stress, induction of numerous proteins, including transcription factors (TF), can regulate the expression of specific

**Citation:** Ma, J.; Zhang, G.; Ye, Y.; Shang, L.; Hong, S.; Ma, Q.; Zhao, Y.; Gu, C. Genome-Wide Identification and Expression Analysis of HSF Transcription Factors in Alfalfa (*Medicago sativa*) under Abiotic Stress. *Plants* **2022**, *11*, 2763. https:// doi.org/10.3390/plants11202763

Academic Editor: Małgorzata Nykiel

Received: 23 September 2022 Accepted: 17 October 2022 Published: 19 October 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

functional genes and enhance plant resistance through signal transduction pathways [12]. Reactive oxygen species (ROS) scavenger enzymes and HSP are important functional proteins induced by HS, and their corresponding genes are targets of several HS-responsive TFs [13]. Previous studies have shown that HSFA6b is essential in *Arabidopsis thaliana* as a downstream regulator of the ABA-mediated heat stress response (HSR) [14,15].

HSF is a class of transcription factors that are widely present in eukaryotes [16]. Since the first *HSF* gene was isolated from *Solanum lycopersicum* in 1990, HSF has been reported in *A. thaliana*, *Oryza sativa*, *Glycine max* and other plants with the continuous improvement of genome sequencing technology [17–21]. The number of HSF gene family members varies widely among plants, with the highest number in *Triticum aestivum* containing 56 [22]. *G. max* [23], *Zea mays* [24], and *A. thaliana* [25] contained 52, 30, and 21, respectively. HSF plays a crucial role in the transmission and receipt of signals, the detection of heat shock components, the regulation of downstream genes, and the induction of plant stress responses [26]. The important role of HSF in plant responses to abiotic stresses can be well established.

Alfalfa (*Medicago sativa*) is one of the most economically valuable crops in the world and the most widely farmed legume fodder grass [27]. However, as it matures and flourishes, *M. sativa* is regularly damaged by a number of abiotic factors, such as salinity, cold and drought, which have a detrimental effect on *M. sativa* quality and output [28–31]. Therefore, it is particularly important to breed *M. sativa* germplasm resources with high resistance to stress. HSF is a class of transcription factors that are widely found in eukaryotes. They are crucial in the transmission and receipt of signals, the identification of heat shock components and the control of downstream genes, and the induction of plant responses to abiotic stresses [26,32–34]. Studies on the HSF gene family have been reported in many species but have not yet undergone a thorough analysis in *M. sativa*. Therefore, in this study, we identified the MsHSF family gene in *M. sativa* by integrating conserved motifs, gene structure, chromosome mapping, promoter cis-elements, and their phylogenetic relationships, and we analyzed the expression of HSF genes in *M. sativa* under four abiotic stresses. These results will deepen our current understanding of the evolutionary relationships and functional differentiation of *M. sativa HSF* genes and provide valuable information for further studies on the role of HSF genes in *M. sativa* for resistance under abiotic stresses.
