*4.2. Construction of Phylogenetic Tree and Sequence Comparison*

Whole genome information for *A.thaliana* and *O.sativa* was downloaded from the NCBI database (https://www.ncbi.nlm.nih.gov/) (accessed 2 August 2022). Using MUSCLE technology, the 16 MsHSF protein sequences, 21 AtHSF proteins, and 25 OsHSF sequences found were compared to multiple sequences. The comparison parameters were in multiple comparison mode (other parameters were in default mode), and the obtained comparison results were used to construct a neighbor-joining (NJ) phylogenetic tree that we created in MEGA 7.0 using 1000 bootstrap replications, and the phylogenetic tree was created as an illustration using iTOL (https://itol.embl.de/) (accessed on 2 August 2022).

Intraspecific classification of *M. sativa* HSF sequences was performed based on interspecific phylogenetic trees, and amino acid sequences of conserved structural domains were compared and modified using Jalview software 2.11.2.4 (Andrew, M.W.; Cambridge, MA, USA) (http://www.jalview.org/) (accessed on 2 August 2022), and conserved thematic WebLogo were produced.

We also submitted the Jalview output to SOPMA (https://npsa-prabi.ibcp.fr/cgi-bin/ npsa\_automat.pl?page=npsa\_sopma.html) (accessed on 3 August 2022) for protein secondary structure prediction by using default parameters. For tertiary structure prediction, we completed the analysis via the online website SWISS-MODEL (https://swissmodel. expasy.org/interactive) (accessed on 3 August 2022).
