*2.6. Expression Profiling of HSF Genes*

The RNA-seq data utilized in the current study was retrieved from the maize MaizeGDB database (https://qteller.maizegdb.org/genes\_by\_name\_B73v4.php, accessed on 1 August 2021). Previously, a comprehensive maize gene expression analysis was performed by Stelpflug et al. [59], used in the current study. We analyzed HSF profiles in 3 different growth stages (seed, vegetative, and reproductive) and across 20 different tissues (embryo, endosperm, whole seed, primary root, tap root, whole root, stem and shoot apical meristem, immature leaves, tip of stage 2 leaf, mature leaf tissue, pooled leaves, topmost leaves, vegetative meristem and surrounding tissues, immature tassel, meiotic tassel, anthers, mature pollen, mature female spikelet, pre-pollination cob, immature cob, and silks) (Table S4). Furthermore, the expression patterns were investigated at different timescales in a particular developmental stage to get an overview of the spatiotemporal expression of HSFs. In addition, the expression of *Zm*HSFs was also analyzed under abiotic (heat, drought, salinity, cold, UV) stress conditions. For the construction of the heatmap, FPKM values were used, which are already available on MaizeGDB. The heatmap was constructed using Tbtools.

#### *2.7. Protein 3D Structure, Network Interaction, and Gene Ontology Analysis*

The three-dimensional (3D) structure of maize HSFs was predicted through AlphaFold (https://www.alphafold.ebi.ac.uk/, accessed on 1 August 2021) [60]. For this, the protein IDs were entered into the search bar and the structures were obtained. The protein interaction network analysis was performed using the STRING database (https://string-db.org/, accessed on 1 August 2021) using default parameters, i.e., sequences showing more than 40% identity in the database were included for interaction networking [61]. The net-

work interaction file was downloaded and visualized using Cytoscape V. 3.8.2 (https: //cytoscape.org/, accessed on 1 August 2021) [62]. Gene ontology annotation analysis was performed by uploading the gene IDs of *Zm*HSFs to the GENE ONTOLOGY RESOURCE (http://geneontology.org/, accessed on 1 August 2021) [63].

#### **3. Results**

*3.1. Identification and Chromosomal Distribution of Maize HSFs*

With the availability of the genomic sequences of the number of plant species, including maize, it is now possible to obtain the protein sequences of all the HSF members. In the present study, a total of 25 HSFs were identified from the maize genome (Figure 1; Table 1). All the HSF proteins were surveyed for the presence of DBD and OD through EMBL-EBI, employing HMM. Furthermore, SMART was used to search the HSF-DBD to check the accuracy of the results. After discarding redundant sequences, 25 *Zm*HSFs were selected for analysis. These HSFs were named based on their chromosomal locations (*ZmHSF-01* to *ZmHSF-25*) (Table S1). The characteristics of maize HSF genes are presented in Table 1. All the HSFs were mapped on the chromosomes of maize (Figure 1). The maize genome was shown to possess HSF genes on all of its chromosomes, though the number of HSFs between different chromosomes varied considerably. Chromosome 1 had a maximum of 6 HSFs genes, whereas a single HSF gene copy was localized on each of chromosomes 4, 6, and 10. On the other hand, chromosomes 2, 3, 7, and 9 harbor two gene copies each, while three gene copies were recognized on each of chromosomes 5 and 8. Except for *ZmHSF01*, *ZmHSF02*, *ZmHSF12*, *ZmHSF16*, and *ZmHSF23*, all the other HSF genes were present on the lower arm of the chromosomes.

**Figure 1.** The chromosomal position of HSF genes in maize. The scale on the left side represents megabases (Mb). The chromosome number is indicated at the middle of each bar.


