**4. Materials and Methods**

#### *4.1. Identification and Sequence Analysis of HSF Genes in M. sativa*

HSF protein sequences from the *A. thaliana* TAIR database (https://www.arabidopsis. org/) (accessed on 7 June 2022) were used as a reference sequence, and members of the putative *MsHSF* gene were sought using *M. sativa* genomic data from the National Genomics Data Center (https://ngdc.cncb.ac.cn/) (accessed on 7 June 2022) and a BLASTP search. We then used the native HMMER 3.0 software (Robert, D.F.; Ashburn, VA, USA) [59], using Hidden Markov (HMM) mapping of the HSF protein (PF0047), downloaded from the pfam database (http://pfam.xfam.org) (accessed on 9 July 2022). The potential gene members of *M. sativa* HSF identified were pooled using these two search techniques. WebCD-search (https://www.ncbi.nlm.nih.gov/cdd) and SMART (http://smart.embl.de/) were used to identify conserved HSF structural domains in all potential *MsHSF* genes (accessed on 9 July 2022). We finally identified 16 *MsHSF* genes and renamed them according to their position on the *M. sativa* chromosome.

We predicted and examined the physicochemical characteristics of all MsHSF potential proteins, including amino acid numbers, molecular weights, and theoretical sites, through the website ExPasy (https://www.expasy.org/) (accessed on 11 August 2022). Cell-PLoc2.0 was used to create subcellular localizations (http://www.csbio.sjtu.edu.cn/bioinf/plantmulti/#) (accessed on 11 August 2022).
