*2.8. Expression Analysis of HSF Genes in M. sativa in Response to Abiotic Stresses*

To investigate the expression levels of *MsHSF* genes under abiotic stress, we analyzed the expression patterns of *MsHSF* genes under treatment with cold stress, abscisic acid (ABA), drought, and salt using published transcriptome data (Figure 10). The results of the analysis showed that the genes that functioned in *M. sativa* under different stress treatments were slightly different compared to the control. However, the genes that appeared to be the first to function and to be highly expressed in the immediate period of abiotic stress were *MsHSF09* and *MsHSF15*, while *MsHSF07* and *MsHSF08* gradually became more highly expressed as the time of stress increased to counteract the external stimuli. By the later stages of stress, it is *MsHSF04*, *MsHSF05* and *MsHSF13* that play a role. Interestingly, most of the *MsHSF* genes were induced to increase in response to abiotic stress, except for *MsHSF02* and *MsHSF16*, which were suppressed in response to cold stress, which may be related to cellular trauma in *M. sativa* during cold stress.


**Figure 9.** A heat map representation of *MsHSF* expression between different tissues. The values in the rectangle represent the magnitude of the gene expression.

#### *2.9. GO Annotation and Enrichment Analysis of M. sativa HSF Protein*

Plants have evolved complex mechanisms to sense and respond to biotic and abiotic stresses, and HSF is an important component of these defense systems. We carried out GO annotation and enrichment analysis on 16 MsHSF proteins in order to learn more about the biological functions of this protein (Figure 11). MsHSF was enriched for 55 biological processes, 1 cellular component, and 2 molecular functions in comparison to the entire GO database. According to the GO enrichment data, MsHSF transcription factors are primarily involved in biological processes including responding to abiotic stimuli, responding to temperature stimuli, responding to heat, and responding to xenobiotic stimuli. The findings again suggest that HSF genes play an extremely important role in resisting abiotic stresses.
