*2.10. Interaction Network Analysis of HSF Proteins in M. sativa*

We predicted probable interactions between MsHSF proteins using the STRING database in order to better comprehend MsHSF protein interactions (Figure 12). The findings demonstrate that while certain proteins, like MsHSF10 and MsHSF16, exhibit direct connections, others, including MsHSF10, MsHSF16, and MsHSF08, exhibit more complex multigene interactions. where it is projected that the major nodes MsHSF01, MsHSF08, and MsHSF13 radiate a significant number of connections to additional nodes.

**Figure 11.** GO enrichment analysis of the MsHSF proteins relative to the GO database. The horizontal axis indicates the enrichment factor, and the size of the circle indicates the number of genes annotated with a given GO term.

**Figure 12.** Interaction network of HSF proteins in *M. sativa*. Nodes represent proteins; central nodes are indicated in blue, and black lines indicate interactions between nodes.

#### **3. Discussion**

#### *3.1. The Characteristics of HSF Gene Family in M. sativa*

HSF is a particular sort of transcription factor that is crucial for plants' ability to resist diverse stressors [35]. The highly conserved plant HSF DBD is located at the N-terminal end where it is able to precisely locate and recognize the heat stress element (HSE) in the promoter of the target gene [36–38]. All MsHSF proteins comprised DBD with three helices and four folds, according to multiple sequence alignment and secondary structure prediction (Figure 2). It's interesting to note that some MsHSF proteins also have additional conserved structural domains; further experimental confirmation is required to determine whether this is a sign of the gene family's functional diversification. Tertiary structural analysis showed that the portion of transcription factors interacting with nucleic acids is conserved in the subfamily. HSF B and HSF C members may not have transcriptional activation because they lack AHA motifs. which is consistent with the results of previous studies [39–42].

Similar gene architectures and conserved protein motifs among members of the same phylogenetic group typically indicate a tight phylogenetic relationship [43]. Short sequences involved in significant biological processes are typically referred to as motif [44]. The presence of Motifs 1–2 in every MsHSF raises the possibility that they may have significant biological roles, however this has not yet been established. Because motif 9 is specific to the HSF A subgroup, it is possible that the *HSF* genes in this subgroup perform a particular role. Additionally, we discovered that the majority of MsHSF share exon-intron architectures and motif distributions within the same evolutionary tree grouping, indicating that genes within a subfamily frequently have comparable biological activities.

Only 16 HSF genes have been found in *M. sativa*, which is less than other plant species and may reflect the lack of expansion of the MsHSF family. MsHSF genes might be further grouped into three categories using homology matching and multispecies matching: A, B, and C. They are clustered in the same way as the members of the *A. thaliana* HSF gene family, with group A having the most genes and group C having the fewest [26]. Three subgroups, A4, A7, and A9, are missing from *M. sativa*, demonstrating that despite HSF family proteins sharing a common ancestor, they have evolved separately in different species. The majority of the alfalfa HSF proteins grouped with the *A. thaliana* HSF proteins but not with the *O. sativa* HSF proteins, suggesting that MsHSF and AtHSF have a tight evolutionary relationship. The HSF proteins of dicotyledons and monocotyledons have evolved in quite different ways.

Gene duplication events have a big impact on how gene families are formed. By supplying the necessary building blocks for the creation of new genes, gene duplication aids in the development of new, functional genes [45]. The majority of gene duplication occurs as tandem and fragmental duplication [46]. The 16 *MsHSF* genes in the *M. sativa* genome contained four homologous gene pairs, all of which underwent WGD (whole genome duplication) or fragmental duplication events and intense purifying selection pressure. These findings imply that WGD or fragmental replication is essential for *MsHSF* gene amplification.

Cis-acting elements are nucleotide sequences found upstream or downstream of genes that regulate their transcriptional levels [47]. When plants react to numerous developmental processes and stressors, they work by binding to certain transcription factors [48]. According to studies, cis-acting elements are present in plant-inducible promoters in response to adverse stress. There are numerous hormone-responsive core promoter elements and binding sites spread across the 16 *M. sativa* HSF promoter regions. According to this, MsHSF might be involved in the communication between several hormone signaling pathways. Aside from the heat stress element, the majority of MsHSF also contained the drought response element MBS, the anaerobic induction response element ARE, and the low temperature response element LTR. This shows that this gene family may control the effects of a variety of abiotic stimuli. These findings imply that MsHSF may interact with hormone signaling pathways that control growth and development as well as stress responses in *M. sativa*.

According to GO enrichment analysis, 15 of the 16 *MsHSF* genes are involved in two biological processes of GO resistance to abiotic stress and synthesis of abiotic stress factors (Figure 11). Several studies have shown that HSF regulates the expression of stressrelated proteins, such as heat shock protein (HSP), which plays an important role in the plant stress response, especially heat stress [49–51]. Therefore, we speculate that the *MsHSF* gene may play a key role in plant resistance to abiotic stresses.

## *3.2. The Potential Roles of Differentially Expressed MsHSF Genes*

In order to protect plants from heat stress, HSPs can raise the denaturation temperature of their proteins. They can also fix damaged proteins, enabling plants to withstand high temperatures [52]. HSFs play a major role in transcriptionally controlling the expression of HSPs. Additionally, the role of the HSFs signaling pathway encompasses many stresses, including cold, osmosis, drought, and salt, in addition to the response to heat stress [53]. It's interesting to note that osmotic pressures such as drought, salinity disruption, and other stresses result in the buildup of ROS, ABA, and H2O2 as well as alterations to cell walls. Ca2+ and ROS are the key factors causing abiotic stress response processes. Therefore, we examined the *M. sativa* transcriptome under ABA, salt, drought, and low temperature stressors. It was discovered that the expression of a considerable number of HSF genes was elevated under these stressful circumstances. This suggests that these *HSF* genes may be involved in some processes in the response of plants to external stresses. In conclusion, *MsHSF* genes are an important class of regulatory genes that control the plant's growth, development, and response to stress.

HSF plays an important role in the plant's response to abiotic stresses because it can achieve resistance to abiotic stresses by regulating the expression of different genes [54,55]. Among the *HSF* genes in plants, HSFA is a major transcriptional activator because it is essential to awaken HSR [56–58]. Although there were some subtle differences in the responses of *MsHSF* genes to different stresses in this study, the first to play a role in resistance was the *MsHSF09* gene in HSFA, which reinforces the important role of HSFA in resistance to abiotic stresses. Unlike HSFA, a considerable number of HSFB and HSFC have not been reported as transcriptional activators, but interestingly, in *M. sativa*, many genes in HSFB, such as *MsHSF07*, *MsHSF08*, and *MsHSF15*, also play important roles in resistance to abiotic stresses, and based on previous studies and analyses, it is clear that genes in HSFB in alfalfa function as transcriptional co-activators of HSFA. Taken together, several MsHSF genes are differentially expressed under abiotic stresses (including heat, salt, or ABA stress), and these results suggest that they may be involved in plant responses to abiotic stresses.
