Genome-Wide Identification and In Silico Analysis of ZF-HD Transcription Factor Genes in Zea mays L.

Zinc finger-homeodomain proteins are amongst the most prominent transcription factors (TFs) involved in biological processes, such as growth, development, and morphogenesis, and assist plants in alleviating the adverse effects of abiotic and biotic stresses. In the present study, genome-wide identification and expression analyses of the maize ZHD gene family were conducted. A total of 21 ZHD genes with different physicochemical properties were found distributed on nine chromosomes in maize. Through sequence alignment and phylogenetic analysis, we divided ZHD proteins into eight groups that have variations in gene structure, motif distribution, and a conserved ZF domain. Synteny analysis indicated duplication in four pairs of genes and the presence of orthologues of maize in monocots. Ka/Ks ratios suggested that strong pure selection occurred during evolution. Expression profiling revealed that the genes are evenly expressed in different tissues. Most of the genes were found to make a contribution to abiotic stress response, plant growth, and development. Overall, the evolutionary research on exons and introns, motif distributions, and cis-acting regions suggests that these genes play distinct roles in biological processes which may provide a basis for further study of these genes’ functions in other crops.


Introduction
The zinc finger-homeodomain (ZF-HD) TFs, containing a conserved zinc finger (ZF) domain in the N-terminal and a homeodomain (HD) in the C-terminal, are members of a plant-specific TF superfamily [1]. The ZF-HD gene family plays an important role in plant developmental processes and stress responses. ZHD genes are plant-specific, nearly 3.07 percent (https://knoema.com/atlas/World/topics/Agriculture/Crops-Production-Quantity-tonnes/Maize-production) (accessed on 1 September 2020). Abiotic stresses, such as intense water logging, extreme temperature, and drought, affect maize production significantly [31]. Drought, high salinity conditions, and extreme temperatures all cause transcription factors (TFs), such as ZHDTF, to interact with cis-elements or other TF proteins to respond to various stresses in signal transduction pathways of stress response which control plant growth and development by protecting plants [32,33]. Since maize crop yield is highly damaged by abiotic stresses, it is essential to identify the ZF-HD gene family roles in this crop.
To date, no study has been carried out on the ZF-HD/ZHD gene family in maize (Z. mays). So far, functional analysis of ZmZHD9 has been performed to identify the role of the gene in the case of drought stress [34]. We proceeded to adopt a bioinformatics approach for a genome-wide characterization and evolutionary analysis of ZHD genes and their encoded proteins in the maize genome [35]. We intended to analyze chromosomal locations, gene structures, promoter elements, evolutionary relationships, distinct tissue expressions, duplication patterns, and miRNA patterns. Our results lay the groundwork for exploring the mechanisms of ZHD genes in response to various abiotic stresses in maize.

Identification of ZHD Family Genes and Sequence Analysis of Their Proteins
In Z. mays, twenty-one ZF-HD genes were found. The names of the genes identified were chosen from those given by GrassTFDB [36] (Table 1). Out of 10 chromosomes, chromosome number 9 of maize did not possess any one of the 21 ZF-HD domains. The lengths of the protein sequences varied from 89 aa to 655 aa, whereas their molecular weights ranged from 9.8 kDa to 71.6 kDa (Table 1). In both cases, ZmZHD13 and ZmZHD18 showed the minimum and maximum amino acid lengths and molecular weights ( Table 1). The genes ZmZHD10, ZmZHD12, ZmZHD15, ZmZHD16, and ZmZHD17 are associated with theoretical pI values less than 7, while the rest of them showed values higher than 7 (Table 1), which shows the values where the amino acids can be neutral. The GRAVY values of ZmZHDs ranged from −0.985 to −0.138, demonstrating that the proteins are hydrophilic in nature (Table 1). Although all the ZmZHD proteins are in the nucleus, some of them are also located in the cell wall and chloroplasts (Table 1). From all the parameters, we can predict that ZmZHD proteins have diverse physicochemical properties.

Sequence Alignment and Phylogenetic Tree Construction
To conclude the evolutionary analysis, we performed a multiple sequence alignment and built a phylogenetic tree for maize ZmZHD proteins and ZHD proteins of other species using protein sequences of 21 maize ZHDs, 14 rice ZHDs, 15 Arabidopsis ZHDs, 16 foxtail millet ZHDs, 14 sorghum ZHDs, 11 barley ZHDs, 21 purple false brome (Brachypodiumdistachyon) ZHDs, and 13 Heller's rosette grass (Dichantheliumoligosanthes) ZHDs. In two multiple sequence alignments (MSAs), MSA1 contains only ZmZHD proteins ( Figure 1). Motif 1 and Motif 4 are presented, which represent the ZF (zinc finger) domains (Supplementary Materials, Table S1). ZHD proteins are classified into eight groups, I to VIII, according to the popularized tobacco and wheat ZHD family classifications [19,37]. Clade 1 is the smallest class, containing about 7.2% of the total 125 ZHD proteins, and Clade VIII is the largest class, containing 22.4% of the total 125 ZHD proteins. (Figure 2). In the largest group, around 21% of maize and barley proteins, about 14% of rice and foxtail millet proteins are present ( Figure 2). Clade VII is next to the largest one and contains about 17% of the ZHD proteins found in all the mentioned species. Among these, approximately 19% are found in maize, 15% in rice, millet, and sorghum, and 10% in Arabidopsis and barley ( Figure 2). In Clades III and VIII, all plant proteins are present asymmetrically.     Phylogenetic analysis of full-length ZHD protein sequences from Z. mays (Zm, maize), Arabidopsis thaliana (At, Arabidopsis), Oryzasativa (Os, rice), Sorghum bicholor (Sb, sorghum), Brachypodiumdistachyon (Bd, purple false brome), and Dichantheliumoligosanthes (Do, Heller's rosette grass). Red circles, red checkmarks, and red triangle, red rectangular, and red stars indicate Arabidopsis, and rice sequences, maize respectively.

Chromosomal Location, Gene Structure, and Motif Composition Analysis
Twenty-one ZmZHDs were mapped onto 10 Z. mays chromosomes, according to their locations (Supplementary File S1, Figure S1). ZmZHD genes are not symmetrically distributed across all of the 10 chromosomes. None of the 21 genes is located on chromosome number 9 (Chr9). Chr4 contains four genes, while Chr1 and Chr2 contain 3 genes, and Chr3, Chr5, and Chr10 contain 2 genes each (Supplementary File S1, Figure S1). By inputting the entire lengths of the ZmZHD protein sequences, a phylogenetic tree was created to examine the evolutionary connections among the 21 ZmZHD genes. This tree was split into two classes, Class I and Class II, which were further divided into four and three subclasses, respectively ( Figure 3A). We identified at least two motifs and a maximum of seven motifs in ZHD proteins using MEME ( Figure 3C). All contain Motif 1 and Motif 4 in their sequences, and these constituted the most highly conserved parts of the ZF domain. Class I genes contain a greater number of motifs than Class II genes. Interestingly, Motif 2 and Motif 3 are present in all the members of class I, while they are only present in Subclass IIb. Motif 9 was detected in the specific subfamily subclass Ic. Almost all the ZmZHD genes have no introns in their sequences ( Figure 3B). Only about 24% of the genes have introns ranging from 1 to 4, while only two of the genes have UTR regions in their sequences ( Figure 3B). the ZF domain. Class I genes contain a greater number of motifs than Class II genes. Interestingly, Motif 2 and Motif 3 are present in all the members of class I, while they are only present in Subclass IIb. Motif 9 was detected in the specific subfamily subclass Ic. Almost all the ZmZHD genes have no introns in their sequences ( Figure 3B). Only about 24% of the genes have introns ranging from 1 to 4, while only two of the genes have UTR regions in their sequences ( Figure 3B).

Analysis of Cis-Elements in ZmZHD Promoters
Even though cis-acting elements are non-coding DNA sequences, they influence transcriptomic processes in gene promoter regions. Plant CARE software was used to identify around 25 cis-acting regions from the 2000 bp upstream regions of the genomic sequences of the ZmZHD genes ( Figure 4). ZmZHD genes contain plant-growth-and development-related promoters, such as circadian, the O2-site, as-1, the AAGAA-motif,

Analysis of Cis-Elements in ZmZHD Promoters
Even though cis-acting elements are non-coding DNA sequences, they influence transcriptomic processes in gene promoter regions. Plant CARE software was used to identify around 25 cis-acting regions from the 2000 bp upstream regions of the genomic sequences of the ZmZHD genes ( Figure 4). ZmZHD genes contain plant-growth-and developmentrelated promoters, such as circadian, the O2-site, as-1, the AAGAA-motif, the CCAATmotif, the GCN4-motif, and the RY-element ( Figure 4). Abscisic acid-responsive ABRE, the MeJA-responsive CGTCA-motif, the gibberellic acid-responsive GARE motif, salicylic acid-responsive TCA-element, TGA-element, and auxin-responsive AUxRR-core are hormone-responsive cis-elements found in the ZmZHD promoters ( Figure 4). Stress-related components were found in several promoters, including TC-rich repeats implicated in defense and stress response, as well as low-temperature-response (LTR)-related motifs, and MYB and MYB binding sites were found in all ZmZHD promoters, these being implicated in the induction of drought, high-salt, and low-temperature responses ( Figure 4). These results indicated that ZHD genes in maize are mainly responsible for hormonal and biotic and abiotic stress tolerance.
AUxRR-core are hormone-responsive cis-elements found in the ZmZHD promoters ( Figure 4). Stress-related components were found in several promoters, including TC-rich repeats implicated in defense and stress response, as well as low-temperature-response (LTR)-related motifs, and MYB and MYB binding sites were found in all ZmZHD promoters, these being implicated in the induction of drought, high-salt, and low-temperature responses ( Figure 4). These results indicated that ZHD genes in maize are mainly responsible for hormonal and biotic and abiotic stress tolerance.

Synteny and Evolutionary Analysis of ZmZHDGenes
Gene duplication is a common occurrence in all organisms that results in the creation of new functional genes from previously existing ones, which drives evolution. As a result, we used Advanced Circos in TBtools to perform a microsynteny analysis to evaluate duplications among the ZmZHD genes. In four gene pairs, segmental duplications were discovered ( Figure 5). To further investigate the gene duplications in the ZHD gene family, a duel synteny analysis was performed including maize and four other plants: sorghum, foxtail millet, the Oryza indica group, and Arabidopsis ( Figure 6). The results showed that all the monocots, i.e., sorghum, foxtail millet, and the Oryza indica group, have 21 syntenic relations with maize while Arabidopsis has only one ( Figure 6). As a result, there was more genetic overlap found between maize and monocot genomes than between Z. mays and dicot genomes. In addition, all monocot genes had orthologues in maize, implying that maize has undergone additional whole-genome duplication (WGD) events during its evolution. We computed Ka, Ks, and Ka/Ks values for four homologous ZmZHD gene pairs to examine evolutionary limitations and selection pressures on the ZmZHD genes (Supplementary File S2, Table S2). Ks values can be used to retrodict the time of whole-genome duplication (WGD) occurrences, since they indicate the background base substitution rate [38,39]. The Ks values for the ZmZHD gene pairs varied

Synteny and Evolutionary Analysis of ZmZHDGenes
Gene duplication is a common occurrence in all organisms that results in the creation of new functional genes from previously existing ones, which drives evolution. As a result, we used Advanced Circos in TBtools to perform a microsynteny analysis to evaluate duplications among the ZmZHD genes. In four gene pairs, segmental duplications were discovered ( Figure 5). To further investigate the gene duplications in the ZHD gene family, a duel synteny analysis was performed including maize and four other plants: sorghum, foxtail millet, the Oryza indica group, and Arabidopsis ( Figure 6). The results showed that all the monocots, i.e., sorghum, foxtail millet, and the Oryza indica group, have 21 syntenic relations with maize while Arabidopsis has only one ( Figure 6). As a result, there was more genetic overlap found between maize and monocot genomes than between Z. mays and dicot genomes. In addition, all monocot genes had orthologues in maize, implying that maize has undergone additional whole-genome duplication (WGD) events during its evolution. We computed Ka, Ks, and Ka/Ks values for four homologous ZmZHD gene pairs to examine evolutionary limitations and selection pressures on the ZmZHD genes (Supplementary File S2, Table S2). Ks values can be used to retrodict the time of whole-genome duplication (WGD) occurrences, since they indicate the background base substitution rate [38,39]. The Ks values for the ZmZHD gene pairs varied from 0.04 to 91.87, implying that a large-scale ZmZHD gene duplication event occurred between 7066.56 and 2.88 million years ago (MYA) (Supplementary File S2, Table S2). The gene pairs' Ka/Ks ratios were all less than 1.0, suggesting that these genes were subjected to strong purifying selection during evolution.

Construction of a PPI Network and Expression Profiling of ZmZHD Genes in Various Tissues
To predict potential interactions among the proteins, we used the STRING database (https://string-db.org/) accessed on 1 September 2022. Only 8 proteins, ZmZHD2, ZmZHD3, ZmZHD3, ZmZHD3, ZmZHD3, ZmZHD3, ZmZHD3, ZmZHD3, ZmZHD5, ZmZHD6, ZmZHD7, ZmZHD11, ZmZHD16, and ZmZHD20, out of the 21 were correlated at the medium level (0.400) and at the highest level (0.900) of confidence ( Figure 7). Each of the eight proteins is interconnected with the other four. ZmZHD6, for example, is closely linked to ZmZHD2, ZmZHZD5, ZmZHD7, and ZmZHD20. The core nodes of ZmZHD6 and ZmZHD11 only have high confidence levels for their associated proteins (highest confidence level, 0.900) (Figure 7 and Supplementary File S2, Table S3).
Specific gene expression patterns in certain developmental activities in plants can usually be predicted with tissue-specific transcriptome data. Among ZmZHD genes, ZmZHD11 is expressed in almost all of the 16 tissues in maize plants. The expression levels of the Group A proteins, ZmZHD2, ZmZHD4, ZmZHD11, and ZmZHD21, for the sixteen tissues were high in comparison to those of the others (Figure 8 and Supplementary File S2, Table S4). The protein expression levels were even for all the tissues, for example, maize unpollinated silk (US), vegetative meristem (VM), pericarp and aleurone (PA), embryo after pollination (EmAP), endosperm after pollination (EnAP), internode (IN), mature leaf (ML), mature female spikelet (MFS), primary root (PR), secondary root (SR), root differentiation zone (RDZ), root elongation zone (REZ), stomatal divisional zone of the leaf (SDZL), tip of ear primordium (TEP), germinated embryo (GEM), and growth zone of leaf (GZL) tissues. We can predict that the expression profiles of the Group A genes are much higher than those of the others.  Table S2). The gene pairs' Ka/Ks ratios were all less than 1.0, suggesting that these genes were subjected to strong purifying selection during evolution.

MiRNA Target Site Prediction and Validation
miRNAs cleave mRNA or inhibit translation to produce proteins and regulate target gene expressions. About 29 miRNA families were found in the maize genome, with 188 members, and 26 of 29 families exhibited perfect and sometimes nearly perfect tar-get sequences. All the mature miRNA sequences were predicted against the CDSs of ZmZHD genes and 77 miRNAs were shown to be present (Supplementary File S2, Table S5). miRNA167 has ten target sites in ZmZHD5, miRNA4109 has seven target sites in ZmZHD1, and miRN4099 and miRN4186 have four target sites in ZmZHD10 and ZmZHD21, respectively ( Figure 9). One miRNA, miR2275, has target sites in different genes, such as ZmZHD1, ZmZHD15, ZmZHD1, ZmZHD4, ZmZHD15, ZmZHD20, and ZmZHD1, and most of the rest of the miRNAs have one or two target sites (Supplementary File S2, Table S5). These results reveal the correlations of Zma-miRNA167 and miRNA4109 with other miRNA families.
2022, 13, x FOR PEER REVIEW 9 Figure 6. Synteny analysis of the maize genome with one monocot (A) and three dicot (B genomes. The gray lines represent aligned blocks between the paired genomes, and the red indicate syntenic ZHD gene pairs. We performed both dual synteny and specific gene synteny analyses for the maize genome, this being one of the most important fields in compa genomic analysis, as it is the basis of evolutionary studies at both the gene and genome leve used the species-specific gene family protein sequences, but not in the synteny analysis, as m the causes have not been properly studied, for instance, the chromosomal gene positions are enigmatic. Insofar as we could find well-researched sequences online, we retrieved an al-synteny-analyzed them. In addition, a specific gene family from the maize genome sy analysis was studied to comprehend duplication occurrences and internal evolutionary proc

Construction of a PPI Network and Expression Profiling of ZmZHD Genes in Various T
To predict potential interactions among the proteins, we used the STRING dat (https://string-db.org/) accessed on 1 September 2022. Only 8 proteins, ZmZ  ZmZHD3, ZmZHD3, ZmZHD3, ZmZHD3, ZmZHD3, ZmZHD3, ZmZHD3, ZmZ  ZmZHD6, ZmZHD7, ZmZHD11, ZmZHD16, and ZmZHD20, out of the 21 were corr at the medium level (0.400) and at the highest level (0.900) of confidence (Figure 7) Figure 6. Synteny analysis of the maize genome with one monocot (A) and three dicot (B) plant genomes. The gray lines represent aligned blocks between the paired genomes, and the red lines indicate syntenic ZHD gene pairs. We performed both dual synteny and specific gene family synteny analyses for the maize genome, this being one of the most important fields in comparative genomic analysis, as it is the basis of evolutionary studies at both the gene and genome levels. We used the species-specific gene family protein sequences, but not in the synteny analysis, as most of the causes have not been properly studied, for instance, the chromosomal gene positions are quite enigmatic. Insofar as we could find well-researched sequences online, we retrieved and dual-synteny-analyzed them. In addition, a specific gene family from the maize genome synteny analysis was studied to comprehend duplication occurrences and internal evolutionary processes. We performed in silico protein-protein interaction analyses within the family for phylogenetic profiling and to identify structural patterns and homologous pairs, intracellular localizations, and post-translational modifications among the proteins. Furthermore, we considered the interaction and involvement of major signalling or stress pathways, though we avoid discussion of these subjects due to their complexity.
Specific gene expression patterns in certain developmental activities in plants can usually be predicted with tissue-specific transcriptome data. Among ZmZHD genes, ZmZHD11 is expressed in almost all of the 16 tissues in maize plants. The expression levels of the Group A proteins, ZmZHD2, ZmZHD4, ZmZHD11, and ZmZHD21, for the sixteen tissues were high in comparison to those of the others (Figure 8 and Supplementary File S2, Table S4). The protein expression levels were even for all the tissues, for example, maize unpollinated silk (US), vegetative meristem (VM), pericarp and aleurone (PA), embryo after pollination (EmAP), endosperm after pollination (EnAP), internode (IN), mature leaf (ML), mature female spikelet (MFS), primary root (PR), secondary root (SR), root differentiation zone (RDZ), root elongation zone (REZ), stomatal divisional zone of the leaf (SDZL), tip of ear primordium (TEP), germinated embryo (GEM), and growth zone of leaf (GZL) tissues. We can predict that the expression profiles of the Group A genes are much higher than those of the others. We performed in silico proteinprotein interaction analyses within the family for phylogenetic profiling and to identify structural patterns and homologous pairs, intracellular localizations, and post-translational modifications among the proteins. Furthermore, we considered the interaction and involvement of major signalling or stress pathways, though we avoid discussion of these subjects due to their complexity.

MiRNA Target Site Prediction and Validation
miRNAs cleave mRNA or inhibit translation to produce proteins and regulate target gene expressions. About 29 miRNA families were found in the maize genome, with 188 members, and 26 of 29 families exhibited perfect and sometimes nearly perfect target sequences. All the mature miRNA sequences were predicted against the CDSs of ZmZHD genes and 77 miRNAs were shown to be present (Supplementary File S2, Table S5). miRNA167 has ten target sites in ZmZHD5, miRNA4109 has seven target sites in ZmZHD1, and miRN4099 and miRN4186 have four target sites in ZmZHD10 and ZmZHD21, respectively (Figure 9). One miRNA, miR2275, has target sites in different genes, such as ZmZHD1, ZmZHD15, ZmZHD1, ZmZHD4, ZmZHD15, ZmZHD20, and

Discussion
A conserved zinc finger (ZF) domain on the N-terminal and a homeodomain on the C-terminal is present in the zinc finger-homeodomain (ZF-HD). The homeodomain possesses a highly conserved structure containing approximately 60 amino acids. The homeodomain is folded into a recognition helix, which has a characteristic three-helix structure that is attached to the main sulcus of DNA, forming a special link with DNA [40]. ZF-HD transcription factors are only present in plants and play an important role in plant growth and development, as well as biotic and abiotic stress alleviation [41]. About 21 ZmZHD genes were found in maize in TFDB and BLASTp searches.
ZHD genes are exhibited only in terrestrial plants and expanded during angiosperm evolution [1,42]. Various evolutionary and structural analyses have been performed for the structural analysis of ZmZHD domains. These involved various methods, such as sequence alignment, phylogenetic tree, gene structure, motif organization, synteny, and gene duplication analyses. The function of a gene family is determined by the extent and the types of conserved regions basically present in an appropriate sequence alignment. Multiple sequence alignments showed that Motif 1 and Motif 4, which is popularly known as the ZF dimer (Figure 1), are consistent with those of other plant species [1,42], indicating that ZHD proteins have similar structures. ZmZHD genes were categorized into eight groups (I-VIII) in our tree analysis (Figure 2), which is consistent with earlier phylogenetic research on the crops [37,[43][44][45][46][47]. Except for Groups I, IV, and VII, ZF-HD/ZHD proteins from Arabidopsis, rice, and maize were found to be in the majority of the groupings (Figure 2). Maize and rice were found in Groups I and IV, but only Arabidopsis and maize were found in Group VII (Figure 2), indicating a protein divergence from both monocots and dicots. Evolutionary insights can be extracted from the gene structures of the gene families [26]. In our gene structure study, most of the genes in the

Discussion
A conserved zinc finger (ZF) domain on the N-terminal and a homeodomain on the C-terminal is present in the zinc finger-homeodomain (ZF-HD). The homeodomain possesses a highly conserved structure containing approximately 60 amino acids. The homeodomain is folded into a recognition helix, which has a characteristic three-helix structure that is attached to the main sulcus of DNA, forming a special link with DNA [40]. ZF-HD transcription factors are only present in plants and play an important role in plant growth and development, as well as biotic and abiotic stress alleviation [41]. About 21 ZmZHD genes were found in maize in TFDB and BLASTp searches.
ZHD genes are exhibited only in terrestrial plants and expanded during angiosperm evolution [1,42]. Various evolutionary and structural analyses have been performed for the structural analysis of ZmZHD domains. These involved various methods, such as sequence alignment, phylogenetic tree, gene structure, motif organization, synteny, and gene duplication analyses. The function of a gene family is determined by the extent and the types of conserved regions basically present in an appropriate sequence alignment. Multiple sequence alignments showed that Motif 1 and Motif 4, which is popularly known as the ZF dimer (Figure 1), are consistent with those of other plant species [1,42], indicating that ZHD proteins have similar structures. ZmZHD genes were categorized into eight groups (I-VIII) in our tree analysis (Figure 2), which is consistent with earlier phylogenetic research on the crops [37,[43][44][45][46][47]. Except for Groups I, IV, and VII, ZF-HD/ZHD proteins from Arabidopsis, rice, and maize were found to be in the majority of the groupings (Figure 2). Maize and rice were found in Groups I and IV, but only Arabidopsis and maize were found in Group VII (Figure 2), indicating a protein divergence from both monocots and dicots. Evolutionary insights can be extracted from the gene structures of the gene families [26]. In our gene structure study, most of the genes in the ZF-HD/ZHD gene family do not contain introns or UTRs ( Figure 3B), and this phenomenon can be observed in many species [21,42,48]; the loss of introns might lead to an immediate response to abiotic stress.
On the contrary, five genes have one to four introns ( Figure 3B), indicating the structural divergence of the maize ZF-HD/ZHD gene family. Except for tomatoes, our findings imply that ZF-HD/ZHD family genes have been largely conserved in evolution, along with their functions. In previous studies of ZF-HD/ZHD genes in other plants, researchers have concluded that these genes have undergone severe purifying selection, and their functions cannot be differentiated [42,[48][49][50][51][52]. Motif analysis showed various conserved motifs of ZmZHD proteins in Classes I and II, revealing similar motifs present in the same subclass in the phylogenetic tree that are functionally similar ( Figure 3A,C). Gene duplication mechanisms, such as segmental duplication, tandem repeats, and retro-and/or replicate transposition, contributed to biological evolution [53]. Many gene families have been documented to have expanded as a result of segmental duplication [54]. The gene pairs' Ka/Ks ratios indicate that they have undergone purifying selection during genome-wide evolution, and the ZmZHD gene family's duplication times vary from 2.88 to 7066.56 MYA (Supplementary File S2, Table S2).
Abiotic stresses adversely affect the growth and development of maize and ultimately affect economic production. ZF-HD/ZHD TFs play pivotal roles in the biological processes of plants [28]. For instance, in Arabidopsis, overexpressed ZF-HD1 upregulated several stress-inducible genes, eventually leading to significant increases in drought resistance [23]. So far, in maize crops, the expression patterns of ZmZHDs under abiotic stress have not yet been properly investigated. TF mechanisms depend on the cis-elements present in the related genes, which actually regulate the stress signalling and expression of the responsible genes. Numerous cis-elements were identified in a promoter region analysis of plant hormones and abiotic stresses (Figure 4), reflecting the ZmZHD gene expression roles in the external environment. Abscisic acid-responsive ABRE, MeJA-responsive CGTCAmotif, gibberellic acid responsive GARE-motif, salicylic acid-responsive TCA-element, TGA-element, and auxin-responsive AUxRR-core are hormone responsive cis-elements found in the ZmZHD promoters ( Figure 4). Stress-related components were found in several promoters, including TC-rich repeats implicated in defense and stress response, as well as low-temperature response (LTR)-related motifs, and MYB and MYB binding sites were found in all ZmZHD promoters, these being implicated in the induction of drought, high-salt, and low-temperature responses. This shows that ZF-ZHD genes are stimulated by stress and that they are involved in stress-mediated pathways. Several studies have shown ZF-HD/ZHD gene participation in the case of abiotic/biotic stress. For example, the ZF-HD/ZHD gene family in Arabidopsis, tomato, cotton, grape, and Chinese cabbage was found to be involved in fighting various stress conditions, such as salt, drought, heat, and cold, by regulating stress-related hormones, such as ABA [1,27,42]. MYB and ARE help plants endure abiotic stress and are found in all the maize ZHD genes, especially ZmZHD8 and ZmZHD20, in the case of MYB, and ZmZHD9, in the case of ARE ( Figure 5). These findings suggest that these genes are responsible for maize tolerance to abiotic stress. ZF-HD is involved in a variety of biological activities in plants, including growth, development, and stress reduction [55]. Specific biological activities are determined by tissue-specific expression patterns [56]. The majority of Arabidopsis ZF-HD/ZHD genes are located in floral tissues, indicating that they play a role in floral development regulation [42]. ZmZHD2, ZmZHD5, and ZmZHD21 exhibited greater expression patterns in floral tissues in this investigation (Figure 8), indicating that they may be involved in maize pollination [56,57]. The greater expression patterns of Cluster I and II genes in the heatmap for floral (unpollinated silk, endosperm after pollination, female spikelet, tip of ear primordium) and vegetative tissues ( Figure 8) may have implications for their growth and development. ZmZHD6 and ZmZHD11 are the key genes in our study. They interact with other genes (Figure 7) and the ZF-HD protein dimerization region, including proteins, ZF-HD homeobox protein, and zinc finger-homeodomain protein (Supplementary File S2, Table S3).
MicroRNA was found to regulate the cellular responses of plants under stress conditions, such as salinity, cold, and dehydration [57][58][59][60]. Stress-responsive transcription factors (TFs) or functional genes are mainly targeted by several miRNAs [61]. Thus, miRNA may be involved in responses to stress conditions. Ten miR167 genes found in the maize ZHD genes (Supplementary File S2, Table S5) represent auxin response factor protein annotations found in maize [62]. This microRNA involved in maize shoot and leaf development enhances auxin response [62]. Another microRNA that regulates five ZmZHD genes (Supplementary File S2, Table S5) has not had its function elucidated yet. All of these results provide a valuable foundation for further future molecular investigations of ZmZHD genes and pave the way for the development of new varieties of maize.
For the following reasons, we favored the neighbor-joining method above others. The basic objective was to reconstruct phylogenetic trees using evolutionary distance information. The idea behind this approach is to identify the out pairs at each level of clustering, starting with a star-shaped tree, that have the shortest overall branch length. Using this technique, the branch lengths and topology of a parsimonious tree can be easily determined. This method's primary goal is to establish relationships between sequences based on their genetic distances; however, the evolutionary model does not account for this. We checked and rechecked the ZF dimer (PF04770) domain in both the HMMER and Conserved Domains Database (CDD) and the Resources-NCBI database for confirmation. Both are good materials for bioinformatics, with advantages and disadvantages. We used BLASTp and HMMER for different purposes. To discover our chosen gene family sequences and to check the published data, we performed a BLASTp search of our domain sequences against the genome databases or annotation projects for the chosen plant species.

Chromosomal Locations, Motif Compositions, and Exon and Intron Distributions
The chromosomal locations of the 21 genes identified on the 10 maize chromosomes were mapped using the MapGene2Chrom web v2 web tool [71], and information was gleaned from Maize GDB (https://www.maizegdb.org/) accessed on 1 September 2022. The positions of the conserved motifs of these 21 ZHD protein sequences were analyzed by setting a maximum width of 50, a minimum width ≥6, and a motif number of 10; other parameters were set to default in the online MEME tool (http://meme-suite.org/) accessed on 1 September 2022 [72]. For exon and intron distributions, the gene structure display server (GSDS) web tool [43] was used to align genomic sequences with the CDSs of the 21 maize ZHD genes.

Cis-Acting Elements and Functional Prediction
Putative cis-elements in maize ZHD genes of about 5 to 10 bp were retrieved using the Plant CARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) accessed on 1 September 2022 [73] web-based tool. The 2000 bp upstream sequence for each gene from the start codon was extracted from Phytozome 13 (https://phytozome-next.jgi.doe.gov/) accessed on 1 September 2022 for cis-regulatory element extraction, as the upstream region contained cis-elements that bound the transcription factors that regulate target genes [74].

Synteny Analysis of ZHD Proteins and ks/ka Ratios
Protein sequences of Z. mays were compared with protein sequences from the rice indica group, sorghum, and foxtail millet using TBtools software. Synteny relationships and duplication events among the ZHD proteins were predicted using MCScan [75], and the findings about gene duplications with gene pairs were used to identify duplications in the ZmZHD genes of maize along with those of several monocots and dicots, the results being visualized in TBtools [76]. An NCBI BLAST search was run considering 80% sequence similarity against each of the maize ZHD proteins to determine gene duplications [77]. The protein sequences of duplicated gene pairs were first aligned in Clustal Omega [78]. Then, the sequence alignments of proteins and their associated cDNA sequences were used, by means of the PAL2NAL online tool, to determine the relevant codon alignments [79]. Finally, Ks and Ks values were estimated using PAML's CODEML software and the generated codon alignments [36]. The synonymous substitution rate (Ks), nonsynonymous substitution rate (Ka), and Ka/Ks ratio were calculated for homologous gene pairs using Ka/Ks Calculator2.0 [46]. The equation T = Ks/2λ (where λ = 6.5 × 10 −9 ) was used to compute evolutionary divergence periods within the ZHD gene family.

Protein-Protein Interaction and Z. mays L. RNA-Sequencing Data Analysis
Protein-protein interactions were assessed with the aid of the STRING database, with a medium (0.400) confidence level, to determine the interrelationships among the 21 ZmZHD proteins. The Expression Atlas database (https://www.ebi.ac.uk/gxa/home) accessed on 1 September 2022 was used to obtain ZmZHD RNA-sequencing data [80], which were previously collected and analyzed by Walley et al. [81]. We collected data for a total of 16 tissues of Z. mays L.: unpollinated silk (US), vegetative meristem (VM), pericarp and aleurone (PA), embryo after pollination (EAM), endosperm after pollination (EAP), internode (IN), mature leaf (ML), female spikelet (FL), primary root (PR), secondary root (SR), root differentiation zone (RDZ), root elongation zone (REZ), stomatal divisional zone of the leaf (SDZF), tip of ear primordium (TED), germinated embryo (GE), and growth zone of the leaf (GZL) tissues. In the acquisition of expression data, the fragments per kilobase of exon model per million mapped reads (FPKM) unit was utilized. The FPKM values were transferred to log2 (FPKM + 1) form and then a heatmap was built using the pheatmap package in Rstudio [82].

MiRNA Target Site Prediction
To determine the target sites of the 21 genes in the ZmZHD gene family in maize, first, mature miRNA was retrieved from the PmiREM server (https://www.pmiren.com/) accessed on 1 September 2022. Then, the CDSs of the 21 genes were searched against mature miRNAs using the online server tool PsRNA (https://www.zhaolab.org/psRNATarget/) accessed on 1 September 2022, with the default parameters [83]. The linkages between the predicted miRNAs were constructed using Cytoscape software (https://www.omicshare. com/tools/) accessed on 1 September 2022 [84].

Conclusions
In this study, we divided the 21 ZHD genes in maize (Z. mays) into two groups through phylogenetic analysis. Based on evolutionary research, these proteins were classified into seven subgroups. Exon-intron arrangements, motifs, and cis-acting regions were all comparable for the genes grouped. These Z. mays ZHD genes may play a role in biological processes and environmental stress control, according to the promoter and expression profiles of tissue-specific RNA sequencing studies. This study provides a foundation for exploring the roles of these genes in stressful environments and investigating their molecular mechanisms.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/genes13112112/s1: Supplementary File S1, Figure S1: Chromosomal location of the ZmZHD genes in the 10 chromosomes of Z. mays. Figure S2: Synteny analysis of the maize genome with A. thaliana and Oryza sativa. And Figure S3: Synteny analysis of the maize genome with S. bicolor and S. italic; Supplementary File S2, Table S1: List of 10 motifs along with description. Table S2: Ks, Ka and Ks/Ka calculation and divergent time of the duplication maize ZHD pairs. Table S3a: Interaction among proteins with confidence level. Table S3b: List of node annotations. Table S4: Tissue specific transcriptome data in FPKM unit. Table S5: List of miRNA found in ZmZHD genes along with target alignments. Table S6: Sequences of the protein sequences of the selected species.

Conflicts of Interest:
The authors declare no conflict of interest.