*3.5. Domain Analysis and Physio-Chemical Properties*

The modular structure and the functional domains of HSFs have been studied and described extensively [22]. The HSF-type DBD was highly conserved and consisted of approximately 100 amino acids (Figure 6). The locations of DBD and OD were predicted using SMART and MARCOIL (Table 5). The DBD of most maize HSFs was located at the beginning of the N-terminal. Few exceptions were *Zm*HSF-03, *Zm*HSF-10, *Zm*HSF-14, and *Zm*HSF-15. As expected, the linker length between DBD and OD of HSFBs was larger than HSFAs and HSFCs.

The physio-chemical properties of HSFs such as amino acid length, Mw, and pI were investigated using Expasy (Table 5). In addition, the amino acid composition of each group was analyzed using the online tool CoPId (Table S3). The amino acid length of class A HSF ranged from 350 (*Zm*HSF-23) to 528 (*Zm*HSF-14), for class B, 298 (*Zm*HSF-08) to 414 (*Zm*HSF-03), and in class C the amino acid length ranged from 257 (*Zm*HSF-13) to 348 (*Zm*HSF-21). The pI of class A HSFs ranged from 4.70 (*Zm*HSF-17) to 8.87 (*Zm*HSF-10). For class B it varied from 5.00 (*Zm*HSF-19) to 9.53 (*Zm*HSF-18), and for class C, pI ranged from 5.85 (*Zm*HSF-13) to 8.09 (*Zm*HSF-21). For class A, the Mw varied from 38.154 (*Zm*HSF-23) to 58.138 (*Zm*HSF-14), for class B it ranged from 33.258 (*Zm*HSF-18) to 44.381 (*Zm*HSF-03). While for class C HSFs, Mw ranged from 27.836 (*Zm*HSF-13) to 37.409 (*Zm*HSF-21). In general, the pI of class A HSFs was in acidic ranges except for *Zm*HSF-10, which was shown to be in a slightly basic range. The pI of class B was in slightly acidic and basic ranges. Finally, for class C the pI was in similar ranges as class B HSFs. The average amino acid composition of *Zm*HSFs ranged from 1.1 (cysteine) to 10.0 (alanine) (Figure 7A). The average amino acid composition of class A HSFs ranged from 0.8 (cysteine) to 8.7 (alanine) (Figure 7B). In contrast, the average amino acid composition of class B ranged from 1.3 (tryptophan) to 12.6 (alanine) (Figure 7C). Finally, the class C amino acid composition ranged from 1.1 (tryptophan) to 11.2 (alanine) (Figure 7D).

**Figure 6.** Multiple sequence alignment of maize HSFs. The highly conserved DBD consists of four antiparallel β-stranded sheets (β1, β2, β3, β4) and a bundle of three α-helices (α1, α2, α3) which form the secondary structure. Rectangular boxes represent α-helices while square boxes represent β-stranded sheets.



**Figure 7.** (**A**) The overall amino acid composition of maize HSFs, (**B**) class A HSFs, (**C**) class B HSFs, and (**D**) class C HSFs.
