*3.4. Gene Structure and Protein Motif Analysis*

To investigate the structural relationship among the HSF genes, the intron–exon organization of all the targeted HSFs was analyzed using GSDS software. The intron–exon structure and number play a key role in the evolution of gene families [67,68]. The gene structure analysis was in line with the phylogenetic relationship among maize HSFs (Figure 4). In general, the intron and exon numbers were shown to be highly consistent. Particularly, 92% (23/25) HSFs contain only one intron except for HSF-02 and HSF-24 (Figure 4). Similarly, HSF-02 and HSF-24 were shown to contain three and five exons. In contrast, the rest of the HSF genes contained two exons. Further, 17 HSFs contained 5 UTR and 3 UTR. The HSF genes belonging to the same class and sub-class showed a similar intron–exon pattern in terms of intron number, exon length, intron phase, and overall gene length (Figure 4).

**Figure 4.** The intron–exon structure of maize HSF genes. A black line represents the introns. Exons are represented by a yellow rectangle and 5 UTR and 3 UTR by reddish-pink wedges. The gene structure of *Zea mays* HSFs was in alignment with the phylogenetic relationship.

MEME was used to identify the conserved motifs/regions responsible for DNAbinding, oligomerization, nuclear localization, nuclear export, and biological activation of HSFs (Figure 5). In total, 20 motifs designated as motifs 1–20 were identified among maize HSF proteins (Table 4). The highly conserved DBD is represented by motifs 1, 2, and 4. Motif 3 corresponds to OD of class A and C HSFs. The HR-A core region of OD is represented by motif 5 and is present in all HSFs. OD of class B HSFs is depicted by motif 7. Motif 6 constitutes the NLS of class A HSFs and is present in eight members. The AHA motif is shown by motif 8 and is present in 11 class A HSFs. The NES of class A HSFs is represented by motif 16. Motif 15 constitutes the NLS of Class B2 HSFs. Certain HSFs also possess class-specific conserved motifs i.e., *Zm*HSF-02 and *Zm*HSF-24 contain motifs 10 and 13, HSFs of subclass B1 harbor motif 14, sub-class C1 HSFs motif 19, the sub-class B2 HSFs motif 11, and sub-class A4 members (*Zm*HSF-16, *Zm*HSF-20) contain motif 18. Motifs 9 and 12 are present in the same members of class A HSFs. Four members of class A

HSFs contain motif 17. Motif 18 is found in two members of class A HSFs. Finally, motif 20 is found in members of sub-class A2 and A6 HSFs.

**Figure 5.** Conserved motifs in maize HSFs as identified by MEME. The motifs were identified by using full-length protein sequences of *Zm*HSFs. Name of HSFs and p-values are indicated on the left side. Each motif is represented by a unique color, as indicated at the bottom.


