Wide-Range Portrayal of AP2/ERF Transcription Factor Family in Maize (Zea mays L.) Development and Stress Responses

The APETALA2/Ethylene-Responsive Transcriptional Factors containing conservative AP2/ERF domains constituted a plant-specific transcription factor (TF) superfamily, called AP2/ERF. The configuration of the AP2/ERF superfamily in maize has remained unresolved. In this study, we identified the 229 AP2/ERF genes in the latest (B73 RefGen_v5) maize reference genome. Phylogenetic classification of the ZmAP2/ERF family members categorized it into five clades, including 27 AP2 (APETALA2), 5 RAV (Related to ABI3/VP), 89 DREB (dehydration responsive element binding), 105 ERF (ethylene responsive factors), and a soloist. The duplication events of the paralogous genes occurred from 1.724–25.855 MYA, a key route to maize evolution. Structural analysis reveals that they have more introns and few exons. The results showed that 32 ZmAP2/ERFs regulate biotic stresses, and 24 ZmAP2/ERFs are involved in responses towards abiotic stresses. Additionally, the expression analysis showed that DREB family members are involved in plant sex determination. The real-time quantitative expression profiling of ZmAP2/ERFs in the leaves of the maize inbred line B73 under ABA, JA, salt, drought, heat, and wounding stress revealed their specific expression patterns. Conclusively, this study unveiled the evolutionary pathway of ZmAP2/ERFs and its essential role in stress and developmental processes. The generated information will be useful for stress resilience maize breeding programs.


Introduction
Dynamic environmental ordeals, including biotic and abiotic stresses, are considered to be vital stimuli affecting the plants' growth, reproduction, and productivity [1,2]. They have adverse effects on important field crops, such as wheat, rice, and maize, etc. [3] causing a more than 50% reduction in major crop yields worldwide [4]. The growth and developmental processes of maize are subsequently affected by biotic and abiotic factors, such as a scarcity of water, saline stress, and low-and high-temperature stresses that can cause a significant loss in productivity [5,6]. To strive against these environmental stresses, plants have evolved stress-responsive mechanisms, including the quantifiable expression of genes to cope with the stresses at the molecular level [7]. A plant's response to stress conditions is regulated by the expression of profuse genes working in some fundamental metabolic pathways, i.e., cell metabolism, stress-related proteins, enzymes, secondary metabolites [8], carbohydrates, amino acids, and lipid metabolisms [9,10]. Transcription factors emerged as key regulators in various signaling networks, playing a significant role by improving the growth and development of plants under stress conditions. Transcription The physical positions of ZmAP2/ERFs on chromosomes were obtained from maize genome annotation (Zea_mays. B73_RefGen_v5) and mapped to maize chromosomes by using Circos v0.52 [40]. The location of the 229 ZmAP2/ERFs on 10 maize chromosomes was visualized by using MapChart 2.32 [40]. To calculate non-synonymous (ka) and synonymous (ks) substitution of each duplicated ZmAP2/ERF, the KaKs_calculator 2.0 [41] was used. To search homologous gene pairs among maize, rice, sorghum, and Arabidopsis, BLASTP was performed. Multiple Collinearity Scan toolkit (MCScanX) was implemented to investigate the duplication events, with the default parameters [42]. The intron and exon organization were analyzed by using the TBtools 0.665 [43]. The motif analysis of ZmAP2/ERFs was conducted by using (MEME: http://meme-suite.org/, accessed on 17 July 2022) [44].

ZmAP2/ERFs Expression Profiling by RNA-seq Data
Expression quantification of all ZmAP2/ERFs in maize plant [45], under abiotic stresses [45], wounding and oral secretions (OS) to wounds [46], O.F insect attack and JA stresses [47] were obtained from the transcriptomic data that were downloaded from NCBI's database (accession number GSE50191, PRJNA335771, PRJNA380272, PRJNA299127). Obtained transcriptomic reads were mapped to the maize genome as reference (B73_RefGen_v5) and it was analyzed by using HISAT2 (v2.0.5) [46]. The number of reads were counted by HTSeq (v0.7.1) [47]. The hierarchical clustering of ZmAP2/ERF genes was created using average linkage with Euclidean distance method by using R software to visualize the expression profile in eight maize plant tissues based on the log10 (FPKM + 1) values of ZmAP2/ERFs, shown by heat map.

Experimental Material and Treatments
Maize inbred line B73 was grown up to the seedling stage in pots (10. 250-300 mmolm −2 s −1 following completely randomized design (CRD). At the V3 stage, plants were subjected to environmental stresses. For salt stress, plants were treated with 200 mM NaCl; after 2h, tissues were collected. For drought stress, the 6-day-old maize seedlings were grown without irrigation until the V3 stage. For heat and cold stresses, seedlings were subjected to 42 • C and 4 • C for 2 h, respectively. Wounding was applied to leaves and samples were collected after 1.5 and 6 h. Samples were collected and instantly stored in liquid nitrogen.

Expression Quantification by qRT-PCR
Total RNA was extracted by using TRIzol reagent (Invitrogen, CA, USA) and cDNA was synthesized by using a PrimeScript 1st strand cDNA Synthesis Kit (TaKaRa, Okinawa, Japan). Gene-specific primers (Supplementary Table S13) were designed by using Quant-Prime3. The relative expression of the genes was calculated by using the Ct method, and the qRT-PCR was carried out using a real-time detection system (Roche Diagnostics, Schlieren, Switzerland). Each reaction contained cDNA, 2X SYBR Premix Ex Taq (TaKaRa, Japan), and primers as per recipe [12].

Phylogenetic Classification of ZmAP2/ERFs Family Members
A methodical approach was carried out to find the ZmAP2/ERF subfamily members by using the publicly available genome datasets. The AP2 and ERF keywords were used as queries to find the newest version of the maize genome (V5) in the MaizeGDB and Gramene. Then, BLAST searches were performed by using all of the AP2/ERF sequences to re-examine the acquired sequences. Initially, 236 presumed Maize AP2/ERF members were identified. Further verification of this family led to the deletion of seven false-positive sequences. Finally, a total of 229 ZmAP2/ERF members were identified and grouped into four subfamilies (Table S1). Each ZmAP2/ERF member was specified by the AP2/ERF family name based on the standards determined by the Gene Nomenclature Committee (AGNC) [23] then labeled following the Maize nomenclature (Table S1). Nine erroneously predicted AP2/ERFs models were manually curated. Different traits, i.e., the length of the coding sequence, the chromosomal location, number of exons, number of introns, ZmAP2/ERF domain, subfamily characterizations, variant transcripts, name, and descriptions, are presented in (Table S1).
ZmAP2/ERFs were categorized into five subfamilies: 89 DREB, 105 ERF, 27 AP2, and five RAV members, along with one soloist (Figure 1). The RAVs and soloist members are shown adjacent to the AP2 transcription factor family. A phylogenetic tree was constructed to explore the evolutionary relationship between the AP2/ERF transcription family members in maize. The phylogenetic tree was constructed in the neighbor-joining method by using the full amino acid sequences of ZmAP2/ERF proteins. Resultantly, the dendrogram demonstrates that the ZmAP2/ERFs were grouped into five distinct families shown in (Figure 1). The phylogenetic tree (un-rooted) breaks up the ZmAP2/ERFs family into groups based on the conservation of group-specific domains among the proteins. Six groups (A1-A6 and B1-B6) have been identified in the DREB and ERF subfamilies, respectively. Zm00001eb241420 is the only member of the AP2/ERF family which is different from the other family members and categorized as a soloist (Figure 1). The phylogenetic analysis of the AP2/ERF superfamily in maize indicated that it has the greatest number of members. The AP2/ERF is the major transcription factor family in plants, with 147 members in Arabidopsis [21], 170 members in rice [38,48], 288 members in sunflower [39,49], and 380 members in the soybean genome [40,50]. members. The AP2/ERF is the major transcription factor family in plants, with 147 members in Arabidopsis [21], 170 members in rice [38,48], 288 members in sunflower [39,49], and 380 members in the soybean genome [40,50].

Conserved Motif Analysis and Intron-Exon Organization in ZmAP2/ERF TFs
To obtain further information, the identified amino acid sequences of the maize AP2/ERF members were advanced to a conserved motif analysis by using Multiple Em for Motif Elicitation (MEME). A total of ten conserved motifs were found in putative ZmAP2/ERF proteins, and they were named motif 1-10 based on the individual's E-value (Figures 2c and S1). On the N-terminal, motif 7 was the most recurrent, which was found in 48 out of the 53 ZmAP2/ERF members, and motif 10 was the second most common motif at this terminal. The results show that, within the same family, the proteins carrying the same conserved motifs were highly similar in function to each other. Some motifs were

Conserved Motif Analysis and Intron-Exon Organization in ZmAP2/ERF TFs
To obtain further information, the identified amino acid sequences of the maize AP2/ERF members were advanced to a conserved motif analysis by using Multiple Em for Motif Elicitation (MEME). A total of ten conserved motifs were found in putative ZmAP2/ERF proteins, and they were named motif 1-10 based on the individual's E-value ( Figure 2c and Figure S1). On the N-terminal, motif 7 was the most recurrent, which was found in 48 out of the 53 ZmAP2/ERF members, and motif 10 was the second most common motif at this terminal. The results show that, within the same family, the proteins carrying the same conserved motifs were highly similar in function to each other. Some motifs were absent among certain families; for example, the ZmAP2 family has motif 5, but the ZmERF family does not have motif 5 ( Figure S1). These results reflect the functional divergence in ZmAP2/ERF members. In addition to this, their LOGOS were obtained by the server MEME ( Figure S1). Hence, these results suggested that most ZmAP2/ERF members carry exceptional features due to differences in their amino acid sequences. These conserved To expand vision into the evolution of ZmAP2/ERF TFs, their coding and genomic sequences were compared to determine the exon-intron organizations (Figure 2b). The ZmAP2/ERF TFs' structure was analyzed via the GSDS online suite to obtain more information regarding their conservation and diversification. The number of exons and introns in ZmAP2/ERF members range from 1-10 and 1-4, respectively. The ZmAP2 family members contain 6, 8, 9, or 10 exons, and the majority of ZmAP2/ERFs significantly share a highly conserved structure within the same family or subfamily. Members of a group generally have alike structures, such as how ZmERFs have two exons and two introns. Collectively, the conserved motif configurations and structural similarity of ZmAP2/ERFs strongly support the consistency of the group classifications.

Chromosomal Arrangement, Paralogous Gene Identification, and Synteny Analysis of the ZmAP2/ERF Transcription Factor Family
The localization of the predicted ZmAP2/ERFs was illustrated using the Map Chart software on their corresponding chromosomes in maize ( Figure S2). The analysis indicates that the 229 ZmAP2/ERFs were randomly located across 1-10 chromosomes, and their distribution is (38,26,16,26,24,21,21,18,20, and 17 genes, respectively). Chromosome 1 possesses thirty-eight ZmAP2/ERFs members and the remaining nine chromosomes carry from sixteen to twenty-six ZmAP2/ERFs. Localization revealed that about 78% of the ZmAP2/ERFs were positioned on chromosomal arms in the maize genome ( Figure S2).
The two important events of transcription factor gene family expansion are segmental and tandem duplications. The evolution and expansion of TFs are usually linked with the duplication of the genome, i.e., segmental or tandem duplications [51]. Segmental duplication results in a discrete occurrence of gene family members on different chromosomes [52]. Tandem amplification results in groups of duplicated genes on the same chromosome [53]. The segmental duplication of maize AP2/ERFs using circos is shown in (Figure 3).
The ZmAP2/ERFs were unequally dispersed on the 10 Maize linkage groups (LG), as showed by chromosomal lineage analysis in (Figure 3) (Table S2). As per the illustrations of Holub [54], the occurrence of two or more genes within 200 kb of a chromosomal region is termed as a tandem duplication event. On maize linkage groups 5, 6, 7, 8, 9, and 10, ZmAP2/ERF genes were grouped into 6 tandem duplication event regions. The analysis presented that several ZmAP2/ERFs were generated through duplicate events that played a key role in their evolution ( Figure 3). The analysis reflects that, during evolution, they are likely to undergo an intense significant selection.
The divergence of maize ZmAP2/ERF genes is calculated using the Ka and Ks rate per site per year. The non-synonymous (Ka) and synonymous (Ks) substitutions and Ka/Ks rates per site per year for maize genes have been calculated (Table S3). The nonsynonymous/synonymous value can estimate the selective pressure on duplicated genes, i.e., it can indicate neutral selection and can find out the selective pressure for replicating genes. The estimated time of duplication was calculated by using the formula T = Ks/2λ. The majority of the segmentally duplicated ZmAP2/ERF gene pairs showed a Ka/Ks ratio >1). It revealed that non-synonymous changes are more common than synonymous changes in ZmAP2/ERFs. A Ka/Ks ratio > 1 indicates positive selection, a Ka/Ks ratio = 1 indicates neutral selection, and a Ka/Ks ratio < 1 shows purifying selection. The analysis showed that the lowest value is among the Zm00001d009103-Zm00001d019744 pair (Ka/Ks value = 0.88). It explained that duplications of the paralogous genes in maize occurred from 1.724 to 25.855 MYA. The duplication of Zm00001d001907 and Zm00001d026563 occurred very recently: their divergence time is 1.724 MYA.
Comparative syntenic map of maize with Arabidopsis, rice, and sorghum was created to advance the insight into the evolutionary mechanism of the ZmAP2/ERFs (Figure 4). Total 35 AtAP2/ERFs presented a syntenic linkage to Arabidopsis, 153 in rice and 187 in sorghum (Table S4). The collinear pairing of ZmAP2/ERFs was greater with sorghum and rice than with Arabidopsis. In maize, chromosome number five shows no synteny relationship with A. thaliana. The analysis depicted that maize, sorghum, and rice (monocots) share a large number of collinear gene pairs as compared to dicots. The Ka/Ks values can be used to calculate the selection pressure for duplicating genes. Comparative syntenic map of maize with Arabidopsis, rice, and sorghum was created to advance the insight into the evolutionary mechanism of the ZmAP2/ERFs ( Figure  4). Total 35 AtAP2/ERFs presented a syntenic linkage to Arabidopsis, 153 in rice and 187 in sorghum (Table S4). The collinear pairing of ZmAP2/ERFs was greater with sorghum and rice than with Arabidopsis. In maize, chromosome number five shows no synteny relationship with A. thaliana. The analysis depicted that maize, sorghum, and rice (monocots) share a large number of collinear gene pairs as compared to dicots. The Ka/Ks values can be used to calculate the selection pressure for duplicating genes.

Promoter Region Analysis of Maize AP2/ERFs
The cis-acting regulatory elements of the promoter region are directed to the expression associated with different stresses. To study the regulatory functions, we examined the cis-acting elements in the promoter region of all ZmAP2/ERFs by using the Plant CARE database. The 1500 kb upstream region of the start codon (ATG) was used to analyze cis-acting regulatory elements of ZmAP2/ERFs. The analysis was carried out to find various types of cis-regulatory elements as shown in ( Figure S3A).
Briefly, the majority of the ZmAP2/ERFs are involved in signal transduction pathways, i.e., the phytohormonal signaling pathway, stress-related pathways, and other regulatory elements. The following analysis illustrated that 28.41% of the ZmAP2/ERFs were responsive to phytohormones, followed by 27.38% of ZmAP2/ERFs being responsive to biotic and abiotic stresses, while other observed key regulatory elements were reactive to light regulations, defense-related actions, and the binding of proteins. The results indi-cated that ZmAP2/ERFs have varied functions and are involved in many biotic-abiotic and phytohormonal signal transductions ( Figure S3B).

Promoter Region Analysis of Maize AP2/ERFs
The cis-acting regulatory elements of the promoter region are directed to the expression associated with different stresses. To study the regulatory functions, we examined the cis-acting elements in the promoter region of all ZmAP2/ERFs by using the Plant CARE database. The 1500 kb upstream region of the start codon (ATG) was used to analyze cisacting regulatory elements of ZmAP2/ERFs. The analysis was carried out to find various types of cis-regulatory elements as shown in ( Figure S3A).
Briefly, the majority of the ZmAP2/ERFs are involved in signal transduction pathways, i.e., the phytohormonal signaling pathway, stress-related pathways, and other regulatory elements. The following analysis illustrated that 28.41% of the ZmAP2/ERFs were responsive to phytohormones, followed by 27.38% of ZmAP2/ERFs being responsive to biotic and abiotic stresses, while other observed key regulatory elements were reactive to light regulations, defense-related actions, and the binding of proteins. The results indicated that ZmAP2/ERFs have varied functions and are involved in many biotic-abiotic and phytohormonal signal transductions ( Figure S3B).
The ( Figure S3A) presents many cis-elements existing in the promoter region of ZmAP2/ERFs (G-box, ARE, GC-motif, I-box, O2-site, TATA-box, LS7, ATC, and GATT). The expression of ZmAP2/ERFs may be triggered by several plant hormones, i.e., SA, ET, JA, ABA, GA, and auxin. In addition, the presence of biotic and abiotic stress expression elements (as GC-motif, LTR, WUN-motif, MBS11, MbS1, TC-Rich repeats) in several ZmAP2/ERFs promoter regions indicated that these might involve different growth and stress regulations, i.e., heat, cold, salt, and drought, governing the development of the endosperm and flavonoid biosynthesis.

Expression Analysis of ZmAP2/ERFs in Maize Tissues
To explore the mechanism of ZmAP2/ERFs in maize, their expression pattern was analyzed in different tissues and developmental gradients of maize, including unpollinated silk, female-spikelet, primary roots, vegetative meristem, internode, germinated embryo, endosperm, leaf, and pollen at different developmental stages. The RNA-Seq Atlas of maize offers high-resolution expression data in nine different tissue samples. Publicly available RNA-seq data on NCBI were used to analyze their expression (Tables S5  and S6). It is reported that, among plants, the AP2/ERF family plays a significant role in the developmental processes [28].

Expression Analysis of ZmAP2/ERFs in Maize Tissues
To explore the mechanism of ZmAP2/ERFs in maize, their expression pattern was analyzed in different tissues and developmental gradients of maize, including unpollinated silk, female-spikelet, primary roots, vegetative meristem, internode, germinated embryo, endosperm, leaf, and pollen at different developmental stages. The RNA-Seq Atlas of maize offers high-resolution expression data in nine different tissue samples. Publicly available RNA-seq data on NCBI were used to analyze their expression (Tables S5 and S6). It is reported that, among plants, the AP2/ERF family plays a significant role in the developmental processes [28].
ZmERF85, ZmERF82, and ZmDREB5 were highly regulated in pericarp tissues 18 days after pollination. After 24 days of pollination, ZmDREB69 was overexpressed in the embryo tissues. ZmERF22 overexpressed in the embryo after 16 days of pollination. Among whole-seed-24DAP, ZmERF44 was upregulated after 24 days of pollination. ZmERF23 was overexpressed among endosperm tissues after 16 days of pollination. ZmERF8 and ZmERF45 were overexpressed in silk R1. In immature cob, at the V18 stage, ZmERF64 was overexpressed. ZmDREB60, ZmDREB63, and ZmDREB64 were overexpressed in an-thers_R1. ZmDREB81 and ZmDREB28 were overexpressed among the internode before pollination. ZmERF10 was overexpressed in the eighth leaf at the V9 stage. ZmERF90 was overexpressed in the thirteenth leaf. Among immature leave_V9, ZmDREB74 and Zm-DREB55 were overexpressed. ZmAP2_4 was overexpressed in pooled_leaves_V1. Among primary root_Z4_7DAS, ZmDREB17, ZmDREB18, and ZmDREB45 were overexpressed. Among primary root_GH_6DAS, ZmDREB9, ZmDREB32, and ZmDREB84 were highly expressed. Among root_CP_3DAS, ZmDREB16 was overexpressed. Among root cortex tissues, the ZmERFs and ZmDREBs were overexpressed. ZmDREB48, ZmDREB6, and ZmDREB58 were highly expressed in root elongation zone tissues at the five-day stage. (Figure S4A,B) The maximum number of ZmAP2 genes was overexpressed in pollen. The ZmAP2 genes were mostly overexpressed in vegetative meristem tissues, such as ZmAP2-22, ZmAP2-21, and ZmAP2-7. ZmAP2-21 and ZmAP2-2 were upregulated among the primary roots. The ZmRAVs were overexpressed in pollen tissues, i.e., ZmRAV2. ZmRAV5 and ZmRAV1 were overexpressed in the vegetative meristem, and ZmRAV3 was highly regulated in germinated embryos. The soloist was overexpressed in the leaf tissues. (Figure S4B).

Expression Profiling of ZmAP2/ERFs in Response to Biotic and Abiotic Stresses
The advanced study has put forward the observation that AP2/ERFs play an important role in plant growth and development. The maize AP2/ERF expression analysis was analyzed for their response to biotic, abiotic, and phytohormonal applications. The ZmAP2/ERFs' expression under salt, drought, heat, cold, and ABA was analyzed using publicly available maize transcriptomic data [55]. Expression quantification of ZmAP2/ERFs was also performed under different conditions, i.e., wounding, OA, OF, and JA treatment, by using publicly available maize transcriptomic data [56,57].
In the O.S (oral secretions) from Mythimna separata insects and wounding treatment, both the ERF and DREB gene families were highly responsive. Wounding and OS treatment samples were taken at 1.5 and 6 h, and expression profiling was analyzed as shown in the heat map ( Figure 5) (Tables S9 and S12). ZmAP2-2, ZmAP2-17, ZmERF58, ZmERF14, ZmDREB21, and ZmERF57 were overexpressed in the control condition but had shown no expression regulation under OS and wounding treatments.

Relative Expression of ZmAP2/ERFs by qRT-PCR
For further confirmation that the ZmAP2/ERFs' expression is influenced by cold, salt, drought, heat, and wound 1.5-and 6-h stresses, we selected the overlap expression of biotic and abiotic stresses in 15 ZmAP2/ERFs genes for qRT-PCR ( Figure 6). The ZmDREB5 was highly responsive to wounding at 1.5 and 6 h. It showed no regulation under drought stress. ZmDREB77 showed its regulation among all stresses but was expressed the least under the W6 treatment. ZmDREB24 was expressed under all the conditions, but it was highly expressed under cold conditions. ZmRAV1 was expressed the least among all the abiotic stresses, but it was highly expressed under W1.5 and W6 stress conditions. ZmDREB30 was expressed under all stresses, and was highly expressed under cold, w1.5, and w6 treatments. ZmRAV3 was significantly overexpressed under cold conditions. The Venn diagram concludes that there are 11 ZmAP2/ERF genes expressed solely under heat stress ( Figure S5). The nine ZmAP2/ERF genes (ERF53, ERF87, ERF98, ERF44, ERF39, ERF23, ERF18, DERB71, and DERB53) were exclusively expressed under cold stress. The eight ZmAP2/ERF genes (ZmDREB19, ZmAP2-2, ZmDREB74, ZmDREB45, ZmDREB60, DREB79, DREB52, and ERF95) were uniquely expressed under salt stress. Five ZmAP2/ERF genes (ZmERF12, ZmERF59, ZmERF19, ZmERF41, ZmERF105, Zmsoloist, ZmDREB88, ZmERF94) were significantly regulated under OS_1.5h and the ZmDREB85 gene was expressed specifically under OS_6h treatment. Six genes were significantly regulated under W_1.5h and only ZmERF51 was uniquely expressed under treatment W_6h. The transcription abundance of ERF genes in maize under wounding, with or without oral secretions (OS) from M. separata is shown in (Figure S5, Table S12).

Relative Expression of ZmAP2/ERFs by qRT-PCR
For further confirmation that the ZmAP2/ERFs' expression is influenced by cold, salt, drought, heat, and wound 1.5-and 6-h stresses, we selected the overlap expression of biotic and abiotic stresses in 15 ZmAP2/ERFs genes for qRT-PCR ( Figure 6). The ZmDREB5 was highly responsive to wounding at 1.5 and 6 h. It showed no regulation under drought stress. ZmDREB77 showed its regulation among all stresses but was expressed the least under the W6 treatment. ZmDREB24 was expressed under all the conditions, but it was highly expressed under cold conditions. ZmRAV1 was expressed the least among all the abiotic stresses, but it was highly expressed under W1.5 and W6 stress conditions. Zm-DREB30 was expressed under all stresses, and was highly expressed under cold, w1.5, and w6 treatments. ZmRAV3 was significantly overexpressed under cold conditions. Zm-DREB34 was expressed under both biotic and abiotic stresses, and highly expressed under w1.5 treatment. ZmERF31, ZmERF33, ZmERF37, and ZmERF35 were highly expressed under cold conditions. ZmERF30, ZmDREB80, and ZmDREB78 were overexpressed under w1.5 treatment. ZmERF50 was highly expressed under abiotic conditions as compared to biotic stress conditions, and significantly expressed under drought conditions. ZmDREB34 was expressed under both biotic and abiotic stresses, and highly expressed under w1.5 treatment. ZmERF31, ZmERF33, ZmERF37, and ZmERF35 were highly expressed under cold conditions. ZmERF30, ZmDREB80, and ZmDREB78 were overexpressed under w1.5 treatment. ZmERF50 was highly expressed under abiotic conditions as compared to biotic stress conditions, and significantly expressed under drought conditions. Figure 6. Expression profiling of fifteen selected ZmAP2/ERFs in response to cold, heat, salt, drought, and wounding stress (W1.5, W6) treatments. Data were normalized to Actin2 and asterisks on bars indicate SD (* p < 0.05, ** p < 0.01).

Discussion
AP2/ERF is a ubiquitous family, composed of a large number of TFs with the ability to form complex stress-responsive networks [58]. It responds to biotic and abiotic stresses with erratic dynamic arrays: a number of them are stimulated rapidly and perpetually, however, some of them are induced by continued stress, which suggests that they may have a reciprocated effect on each other's activity [58]. The AP2/ERF transcription family performs a substantial role in various developmental stages of the plant, i.e., it plays a Figure 6. Expression profiling of fifteen selected ZmAP2/ERFs in response to cold, heat, salt, drought, and wounding stress (W1.5, W6) treatments. Data were normalized to Actin2 and asterisks on bars indicate SD (* p < 0.05, ** p < 0.01).

Discussion
AP2/ERF is a ubiquitous family, composed of a large number of TFs with the ability to form complex stress-responsive networks [58]. It responds to biotic and abiotic stresses with erratic dynamic arrays: a number of them are stimulated rapidly and perpetually, however, some of them are induced by continued stress, which suggests that they may have a reciprocated effect on each other's activity [58]. The AP2/ERF transcription family performs a substantial role in various developmental stages of the plant, i.e., it plays a significant role in the transcriptional regulation involved in complex growth, dynamical environmental stresses, seed germination, and floral development [58][59][60]. In the evolution of the Apetala2/Ethylene family, paralogous genes play a fundamental role [59]. These TFs act as a significant element in several plant mechanisms, as has been extensively studied in several plants, i.e., Arabidopsis [31], sorghum [60], rice [61], wheat [62], soybeans [33], grapes [37], castor beans [63], peaches [64], Hazel [65], Arachis hypogaea [66], and Medicago truncatula [67].
In the current study, the evolutionary processes of ZmAP2/ERFs were considered to find the variations in the members resulting in their novel functions. This family was extensively investigated, however, there is still diminutive knowledge about the maize AP2/ERF TF family. Due to the continuous updating of the maize genome database, a wide-range identification and characterization of the AP2/ERF transcription family remain to be further explicated in the latest version of the maize genome. In the current study, the ZmAP2/ERF family was investigated by using the V5 of the maize genome, resulting in the identification of 229 members with the AP2/ERF domain, varying as compared to the previous studies. Phylogenetic analysis and chromosomal localization were performed, which identified that maize followed a parallel distribution pattern of ZmAP2/ERF similar to that of other plant species [31,61]. Based on former classifications, the ZmAP2/ERFs were categorized into groups, i.e., DREB, AP2, ERF, RAV, and Soloist [68,69]. The ZmAP2/ERF enquiry led to the classification and identification of 229 members, with 105 ERF subfamily members, 27 AP2 subfamily members, 89 DREB subfamily members, 5 RAV subfamily members, and 1 soloist. Additionally, it is divided into the subfamilies DREBI-DREBIV and ERFV-ERFX. Each subfamily has distinct and prominent characteristics. The whole classification and distribution of ZmAP2/ERFs is comparable to that of other field crops [60][61][62].
Structural analysis of the ERF subfamily members revealed that 80% of them have no introns, whereas AP2 subfamily genes have 3-9 introns (Figure 2). The structural analysis of ZmAP2/ERFs revealed their similarity to SbAP2/ERFs [60]. The structural variation provides huge diversity in genome evolution. Generally, the ethylene-responsive factors are characterized by few introns, but among ZmERFs, the total number of introns is higher than in other plants. In total, 20 genes in A. thaliana [31] and 41 genes in O. sativa harbor introns [61]. It was identified that transposable elements (TE) are present in the introns of ZmERFs, which might have played a crucial role during whole-genome duplications and rearrangement events. These events might be involved in upsurging the number of genes and introns in Z. mays.
Among transcription factors, conserved motifs play a significant role in gene functioning. They are often associated with protein-protein interactions and different transcriptional activities. Motif analysis of ZmAP2/ERFs showed that the majority of ZmAP2 confined Motif-1, Motif-2, Motif-3, and Motif-4 ( Figure 2). Among the ZmERF subfamily, Motif-7, Motif-8, and Motif-10 were identified, concluding that they execute a central role in gene regulations. In the following study, the chromosomal location and segmental duplications analysis suggested that some ZmAP2/ERFs might have evolved by duplication and work as a major driving force for evolution. The promoter analysis revealed that ZmAP2/ERFs contained manifold ABRE, signifying that these elements are involved in ABA-dependent responses to salt in addition to water scarcity stresses.
In the above study, expression quantification of 229 ZmAP2/ERFs was detected. Apetala genes regulate crop yield and seed quality by controlling the development of embryonic cells and floral organs [70], whereas the ethylene-responsive factor controls the ET signaling network by binding to the promoter region (GCC box) of pathogenesis-related genes and affecting the fruit ripening [62,71]. Members of the RAV family play a principal role in the plant's growth and developmental processes, i.e., leaf senescence [72]. AtRAVs and AtAP2s play vital roles in developmental processes, i.e., shoot and root apical meristem maintenance, flower initiation, etc. [59,60]. The results of this study showed that ethyleneresponsive factors were significantly upregulated in silk and cob. The DREB factors regulate the root elongation and a significant upregulation of ZmRAVs was identified in pollen and meristem tissues.
AP2/ERFs affect the hormone-mediated stress responses, i.e., ABA and ET [62,63]. The subfamily of ethylene-responsive factors are the foremost downstream controlling elements of the ethylene signaling pathway [37,[73][74][75]. Abscisic acid protects the plant against stresses by inducing stomatal closure, modifying root architecture, and synthesizing osmolytes [76,77]. ZmDREB39 and ZmDREB89 are upregulated in response to the application of both ABA and JA.
Jasmonic acid is a crucial signaling molecule for a plant's growth and defense. It has a synergistic interaction with ethylene that initiates the defense-related genes in response to insect attacks and infection by different types of pathogens [78,79]. The factor ERF1 (At3g23240) acts as an integrator of jasmonic acid and ethylene signaling pathways in A. thaliana [80].
In this study, ZmAP2-17, ZmRAV2, ZmERF56, ZmERF27, ZmERF58, ZmDREB42, and ZmDREB85 were upregulated while ZmERF18 was downregulated in response to the Jasmonic acid treatment. Inclusively, the above findings provide an insight into the potential functional roles of the ZmAP2/ERF family and the candidate factors that will be used for the genetic improvement of maize.  Table S13

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