*3.8. Expression Pattern of Zea mays HSFs under Abiotic Stresses*

Maize growth, development, and yield is adversely affected by several abiotic stresses [70]. Therefore, to examine maize HSFs expression under different abiotic stress events, RNAseq data was analyzed and a heat map was constructed (Figure 10; Table S5). In response to HS, members of class A and B HSFs showed the highest expression. Interestingly, under the drought stress, only the transcript of *ZmHSF-05* was moderately overexpressed. Under cold stress, three members of class A HSFs (*ZmHSF-05*, *ZmHSF-06*, and *ZmHSF-16*) showed relatively higher expression. Moderate expression of *ZmHSF-05*, *ZmHSF-06*, *ZmHSF-04*, and *ZmHSF-19* was observed under UV stress. Under salinity stress, *ZmHSF-05* and *ZmHSF-14* showed the highest expression. Under abiotic stress conditions, *ZmHSF-01*, *ZmHSF-02*, *ZmHSF-05*, *ZmHSF-06*, *ZmHSF-07*, *ZmHSF-09*, *ZmHSF-12*, *ZmHSF-13*, *ZmHSF-14*, *ZmHSF-15*, *ZmHSF-19*, *ZmHSF-23*, and *ZmHSF-25* are upregulated (Figure 10). However, some HSFs were only induced by a particular stress. For example, higher transcripts of *ZmHSF-24* are only detected after HS treatment. Similarly, ZmHSF-16 is slightly overexpressed after cold treatment. While, some HSFs showed almost no expression under any stress condition. These include *ZmHSF-03*, *ZmHSF-08*, *ZmHSF-10*, *ZmHSF-11*, *ZmHSF-17*, *ZmHSF-18*, *ZmHSF-20*, *ZmHSF-21*, and *ZmHSF-22* (Figure 10).

**Figure 10.** Expression profiles of maize HSFs under different abiotic stress conditions. Color bar at the top right represents the log-transformed FPKM values. Green represents higher, red lower, and yellow, medium transcript values.

#### *3.9. Functional Annotation of Maize HSFs*

HSFs have been reported to play a major role not only under stressful conditions but also in plant growth and development [16,69]. Therefore, the regulatory functions of maize HSFs were predicted through GO annotation investigation based on the biological process (BP), molecular function (MF), and cellular component (CC) classes (Figure 11; Table S6). The BP annotation analysis indicated that maize HSFs are mainly involved in cellular response to heat (GO:0034605), response to heat (GO:0009408), response to temperature stimulus (GO:0009266), regulation of transcription by RNA polymerase II (GO:0006357), response to abiotic stimulus (GO:0009628), cellular response to stress (GO:0033554), regulation of RNA biosynthetic process (GO:2001141), etc. (Figure 11). With regard to MF annotation analysis, it was revealed that maize HSFs are mostly involved in RNA polymerase II cis-regulatory region sequence-specific DNA binding (GO:0000978), cis-regulatory region

sequence-specific DNA binding (GO:0000987), RNA polymerase II transcription regulatory region sequence-specific DNA binding (GO:0000977), transcription regulatory region nucleic acid binding (GO:0001067), transcription cis-regulatory region binding (GO:0000976), sequence-specific double-stranded DNA binding (GO:1990837), double-stranded DNA binding (GO:0003690), etc. (Figure 11). The CC annotation study showed that ZmHSFs are majorly involved in the nucleus (GO:0005634), intracellular membrane-bounded organelle (GO:0043231), membrane-bounded organelle (GO:0043227), intracellular organelle (GO:0043229), organelle (GO:0043226), etc. (Figure 11). To conclude, the GO annotation study confirms the role of maize HSFs in regulating abiotic stresses and plant metabolism.
