Genome-Wide Identification and Comprehensive Analysis of the AP2/ERF Gene Family in Pomegranate Fruit Development and Postharvest Preservation

Pomegranate (Punica granatum L.) is a kind of fruit with significant economic, ecological and health values. AP2/ERF transcription factors belong to a large group of factors mainly found in plants and play key roles in plant growth and development. However, AP2/ERF genes in pomegranate and their implication in development and postharvest preservation have been little described. In this study, 116 PgAP2/ERF genes in pomegranate were identified and renamed based on their chromosomal distributions. Phylogenetic relationship with genes from other species, structures, duplications, annotations, cis-elements in promoter sequences, and protein-protein interaction networks among PgAP2/ERF proteins were comprehensively explored. Expression analysis revealed several PgAP2/ERFs associated with the phenotypes of pomegranate seed hardness, including PgAP2/ERF5, PgAP2/ERF36, PgAP2/ERF58, and PgAP2/ERF86. Subsequent analysis indicated that many differentially expressed PgAP2/ERF genes are potentially important regulators of pomegranate fruit development. Furthermore, expression of more than one-half of PgAP2/ERFs was repressed in ‘Tunisian soft seed’ pomegranate fruit under low-temperature cold storage. The results showed that 1-MCP implicated in promoting postharvest preservation of ‘Tunisian soft seed’ pomegranate upregulated the PgAP2/ERF4, PgAP2/ERF15, PgAP2/ERF26, PgAP2/ERF30, PgAP2/ERF35 and PgAP2/ERF45 genes compared to those under low-temperature cold storage. This indicates that these genes are important candidate genes involved in pomegranate postharvest preservation. In summary, the findings of the present study provide an important basis for characterizing the PgAP2/ERF family genes and provide information on the candidate genes involved in pomegranate fruit development and postharvest preservation.


Introduction
Pomegranates (P. granatum) are fruit that originated from Central Asia and are currently widely grown worldwide. Iran, India, and China contribute 80% of the global production of pomegranates [1]. Pomegranates have significant economic and ecological values and have attracted immense attention. They are widely consumed as fresh products and widely applied in industries for making dyestuffs, cosmetics, and medicines. Moreover, pomegranates have antibacterial, anticancer, heart protection, and anti-inflammatory activities [2][3][4].
concentration of 1-MCP was 1.0 µL/L. Fruit peels were collected at 0, 15,30,45, and 60 days post treatment and photos of the pomegranate fruit were taken. The samples were then immediately frozen in liquid nitrogen and stored at −80 • C for subsequent gene expression analysis.
Ethylene production during the postharvest preservation experiment was evaluated by gas chromatography (GC 2010PLUS, Shimadzu, Kyoto, Japan) [30]. Two fruit were placed in a 2 L box sealed with an air-tight lid equipped with a rubber stopper at room temperature for three hours to collect the ethylene gas released by the fruit. Three boxes were used for the three technical replicates for each biological replicate. Subsequently, 1 mL of gas was obtained from each box and subjected to GC analysis with three replicates using helium as a carrier gas (35 mL min −1 ), hydrogen (40 mL min −1 ), and air (350 mL min −1 ). Injector and detector temperatures were set at 110 and 200 • C, and a 10 µL L −1 ethylene standard was used for equipment calibration.

Identification and Annotation of AP2/ERF Genes in the Pomegranate Genome
The 'Tunisian soft-seed' pomegranate genome was retrieved from the NCBI Sequence Read Archive database (https://www.ncbi.nlm.nih.gov/assebly/GCF_007655135.1 accessed on 16 March 2021). Protein sequences of the 147 AtAP2/ERFs were obtained from TAIR webserver (http://www.arabidopsis.org/ accessed on 16 March 2021). HMMER and BLASTP tools were used to screen the candidate PgAP2/ERF genes according to the AP2 domain model retrieved from Pfam database and AtAP2/ERF protein sequences. NCBI CDD (Conserved Domain Database; https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/ bwrpsb.cgi accessed on 18 March 2021) webserver and family assignment algorithms were used to identify the PgAP2/ERF genes. The identified PgAP2/ERF genes were renamed according to their positions on the pomegranate chromosomes. Isoelectric points (pI) and molecular weights (MW) of the PgAP2/ERF proteins were predicted using the ExPASy proteomic webserver (https://web.expasy.org/compute_pi/ accessed on 18 March 2021).

Analysis of Phylogenetic Relationships, Conserved Motifs, Gene Structures and Duplications of PgAP2/ERF Genes
Phylogenetic trees were built based on full-length protein sequences of PgAP2/ERFs by the maximum likelihood (ML) method with 1000 bootstrap values using MEGA software (version 7.0, Mega Limited, Auckland, New Zealand). Conserved motifs in PgAP2/ERF genes were analyzed using the MEME webserver (https://meme-suite.org/meme/tools/ meme accessed on 15 May 2021). Exon-intron structures and chromosomal locations of PgAP2/ERF genes were visualized using TBtools (SCAU) [31]. Gene duplications of PgAP2/ERFs were analyzed using the Multiple Collinearity Scan tool kit in TBtools with the default parameters [31]. Duplicate segments of the PgAP2/ERFs of pomegranate and other plants were then selected using the Dual Systeny Plotter function in TBtools [31]. Ratios of nonsynonymous substitution (Ka) to synonymous substitution (Ks) of PgAP2/ERF orthologous gene pairs were calculated using TBtools [31].
A total of 116 members of the PgAP2/ERF gene family were retrieved from the 'Tunisian soft-seed' pomegranate genome and were renamed from PgAP2/ERF1 to PgAP2/ERF116 according to their positions on the chromosomes (Table S3). The length of the proteins encoded by these genes ranged from 130 to 612 amino acids, and the molecular weights of the proteins ranged from 14,692.26 to 84,711.64 Da (Table S3). The theoretical isoelectric points of these proteins ranged from 4.57 to 11.65, with PgAP2/ERF79 having the lowest isoelectric point of 4.57 whereas PgAP2/ERF102 had the highest isoelectric point (11.65). The proteins comprised 79 acidic proteins and 38 basic proteins (Table S3). Prediction of subcellular location showed that 77 out the 116 PgAP2/ERF proteins were localized in the nucleus whereas the other proteins were localized in the nucleus and cytoplasm, except for the PgAP2/ERF64 protein that was localized in the cytoplasm (Table S3). These results provide a theoretical basis for further studies on the purification, activity, and function of PgAP2/ERF proteins.

Phylogenetic Analysis and Classification of PgAP2/ERF Genes
Phylogenetic analysis showed that the PgAP2/ERF gene family comprised four subfamilies, including 102 PgERF genes, 10 PgAP2 genes, 3 PgRAV genes, and one PgSoloist gene ( Figure 1). The number of PgERF subfamily genes accounted for 88% of the entire gene family and there were seven genes encoding C-repeat binding factors (CBFs) (Figure 1; Table S3), which has been reported previously [36]. The ERF subfamily was further divided into 12 ERF groups according to the phylogenetic results ( Figure 1). In fact, 11 ERF groups were identified because ERF XI had no pomegranate analog ( Figure 1). The ERF Group V had a relatively distant genetic relationship from the other subfamilies, forming an independent branch (Figure 1). The ERF Group VIII comprised twenty PgERFs, the highest number in all the ERF groups. The ERF Group XII comprised only two gene members, which was the lowest number among all the ERF groups ( Figure 1). Table S3), which has been reported previously [36]. The ERF subfamily was further divided into 12 ERF groups according to the phylogenetic results ( Figure 1). In fact, 11 ERF groups were identified because ERF Ⅺ had no pomegranate analog ( Figure 1). The ERF Group Ⅴ had a relatively distant genetic relationship from the other subfamilies, forming an independent branch ( Figure 1). The ERF Group Ⅷ comprised twenty PgERFs, the highest number in all the ERF groups. The ERF Group Ⅻ comprised only two gene members, which was the lowest number among all the ERF groups ( Figure 1).

Conserved Motifs and Gene Structures of PgAP2/ERF Genes
Conserved motifs and gene structures of the PgAP2/ERF family genes were explored using the MEME webserver ( Figure 2; Figure S1). The results showed that genes clustered

Conserved Motifs and Gene Structures of PgAP2/ERF Genes
Conserved motifs and gene structures of the PgAP2/ERF family genes were explored using the MEME webserver ( Figure 2; Figure S1). The results showed that genes clustered on adjacent branches on the phylogenetic tree of the same subfamily exhibited highly conserved motifs (Figure 2a). Each PgAP2/ERF gene showed motif 1 and 3, except the Soloist subfamily gene PgAP2/ERF114 only having motif 3, and motifs 4-10 were differentially distributed in different PgAP2/ERF genes, indicating the variability in this gene family. Moreover, the AP2 domain was conserved in all PgAP2/ERF genes (Figure 2a). In addition, 80 PgERFs and 3 PgRAVs showed one intron, and the other 17 PgERFs had two introns, whereas 10 PgAP2 and 1 PgSoloist had at least six introns and the PgAP2/ERF47 gene had the greatest number of introns (9) (Figure 2b). These findings showed a relatively high degree of conservation in the PgAP2/ERF gene family.
subfamily gene PgAP2/ERF114 only having motif 3, and motifs 4-10 were differentially distributed in different PgAP2/ERF genes, indicating the variability in this gene family. Moreover, the AP2 domain was conserved in all PgAP2/ERF genes (Figure 2a). In addition, 80 PgERFs and 3 PgRAVs showed one intron, and the other 17 PgERFs had two introns, whereas 10 PgAP2 and 1 PgSoloist had at least six introns and the PgAP2/ERF47 gene had the greatest number of introns (9) (Figure 2b). These findings showed a relatively high degree of conservation in the PgAP2/ERF gene family. Figure 2. Phylogenetic relationships, gene structure, and conserved protein motifs of the PgAP2/ERF genes. The phylogenetic tree was constructed based on the full-length sequences of the PgAP2/ERF proteins using the MEGA 7 tool. (a) Motif profile of the PgAP2/ERF proteins. Motifs numbered 1-10 are displayed in differently colored boxes. Sequence information for each motif is presented in Figure S1. (b) Exon-intron structures of the PgAP2/ERF genes. Protein lengths can be estimated using the scales at the bottom.
indicate a lower selection pressure [38]. The six homologous gene pairs in the prese study displayed Ka/Ks ratios above 1.0, and 37 homologous gene pairs exhibited ratio below 1.0 (Table S5), indicating that these genes underwent the purification selection pre sure.
Syntenic analysis of the PgAP2/ERF genes and AP2/ERFs from dicotyledonou plants, Arabidopsis and grape, as well as from monocotyledonous plants, rice and maiz were further conducted ( Figure 4). The results revealed that 127, 127, 53, and 52 synten gene pairs were present between the AP2/ERF genes in pomegranate and those in Ar bidopsis, grape, maize, and rice, respectively. Among them, 21 common syntenic gen pairs were observed ( Figure 4; Table S6), and it indicated that these genes may have e isted before the differentiation of monocotyledons and dicotyledons.   Gene collinearity analysis of the PgAP2/ERF gene family showed three tandem duplication events and 43 segmental duplication events (indicated by homologous gene pairs), involving 68 PgAP2/ERFs, accounting for 59% members of the whole gene family (Figure 3b; Table S4). The Ka/Ks ratio of the homologous gene pair can be used as an indicator of selection pressure in the process of gene evolution. Ka/Ks ratios less than 1.0 indicate a lower selection pressure [38]. The six homologous gene pairs in the present study displayed Ka/Ks ratios above 1.0, and 37 homologous gene pairs exhibited ratios below 1.0 (Table S5), indicating that these genes underwent the purification selection pressure.
Syntenic analysis of the PgAP2/ERF genes and AP2/ERFs from dicotyledonous plants, Arabidopsis and grape, as well as from monocotyledonous plants, rice and maize, were further conducted ( Figure 4). The results revealed that 127, 127, 53, and 52 syntenic gene pairs were present between the AP2/ERF genes in pomegranate and those in Arabidopsis, grape, maize, and rice, respectively. Among them, 21 common syntenic gene pairs were observed ( Figure 4; Table S6), and it indicated that these genes may have existed before the differentiation of monocotyledons and dicotyledons.

Cis-Elements in the Promoters of PgAP2/ERF Genes
Cis-elements of all the promoter sequences, 2000 bp upstream of the PgAP2/ERF coding sequences, were predicted using the PlantCARE webserver ( Figure 5a). The cis-elements of the PgAP2/ERF promoters can be generally assigned to four categories: light-responsive elements, hormone-responsive elements, stress-responsive elements as well as growthand development-responsive elements ( Figure 5b). Promoters of all the PgAP2/ERFs were characterized by light-responsive elements, such as G-box and Box4, and were also characterized by hormone-responsive elements ( Figure 5). A total of 106 PgAP2/ERF promoters exhibited the abscisic acid (ABA) response element ABRE, 98 PgAP2/ERF promoters were characterized by two methyl jasmonate (MEJA)-response elements, 43 genes exhibited the gibberellin (GA)-response element P-box, 39 PgAP2/ERF promoters showed the presence of the ethylene (ETH)-response element ERE, and 36 PgAP2/ERF promoters had the auxin (AUX)-response element TGA ( Figure 5). Moreover, all PgAP2/ERF promoters exhibited stress-responsive elements, including the anaerobic ARE, the low-temperature responsive LTR, the drought-inductive MBS, as well as the defense-and stress-responsive TC-rich repeats and WUN ( Figure 5). Growth and development elements were observed in the promoters of 63 PgAP2/ERFs. RY-elements involved in seed-specific regulation were observed in 14 PgAP2/ERF promoters and HD-Zip1 elements, involved in the differentiation of palisade mesophyll cells, were present in 7 PgAP2/ERF promoters ( Figure 5).

Tissue-Specific Expression of PgAP2/ERF Genes
Transcriptomic data of different tissues (leaf, root, flower, peel, inner, and outer seed coats) of 'Dabenzi', a hard-seeded pomegranate cultivar, and 'Tunisian soft-seed', a softseeded pomegranate cultivar, were retrieved from the NCBI SRA webserver for tissuespecific expression analysis of PgAP2/ERF genes (Figure 8). A high variance in expression levels of the PgAP2/ERF genes was observed across the investigated tissues ( Figure 8). PgAP2/ERF20 and PgAP2/ERF42 genes were specifically expressed in pomegranate leaves; PgAP2/ERF18, PgAP2/ERF112, and PgAP2/ERF64 were specifically expressed in pomegranate roots; PgAP2/ERF45 and PgAP2/ERF59 were specifically expressed in pomegranate flowers; and the PgAP2/ERF8 gene was mainly expressed in pomegranate peels ( Figure 8). In addition, PgAP2/ERF23 and PgAP2/ERF107 were specifically expressed in pomegranate inner seed coats; and PgAP2/ERF75 and PgAP2/ERF89 were specifically expressed in pomegranate outer seed coats (Figure 8). The number of PgAP2/ERF genes highly expressed in pomegranate roots was higher relative to that in the other investigated pomegranate tissues. The number of PgAP2/ERF genes expressed in inner and outer seed coats of 'Tunisian soft-seed' was higher compared with that expressed in inner and outer seed coats of 'Dabenzi'. Differential expression of PgAP2/ERF genes was observed between the seed coats of 'Dabenzi' and 'Tunisian soft-seed' pomegranates. For example, PgAP2/ERF5, PgAP2/ERF36, PgAP2/ERF88, and PgAP2/ERF97 were specifically expressed in 'Dabenzi' seed coats, whereas PgAP2/ERF99, PgAP2/ERF58, PgAP2/ERF86, and PgAP2/ERF87 were only expressed in 'Tunisian soft-seed' seed coats (Figure 8).

Tissue-Specific Expression of PgAP2/ERF Genes
Transcriptomic data of different tissues (leaf, root, flower, peel, inner, and outer seed coats) of 'Dabenzi', a hard-seeded pomegranate cultivar, and 'Tunisian soft-seed', a soft-seeded pomegranate cultivar, were retrieved from the NCBI SRA webserver for tissuespecific expression analysis of PgAP2/ERF genes ( Figure 8). A high variance in expression levels of the PgAP2/ERF genes was observed across the investigated tissues ( Figure 8). PgAP2/ERF20 and PgAP2/ERF42 genes were specifically expressed in pomegranate leaves; PgAP2/ERF18, PgAP2/ERF112, and PgAP2/ERF64 were specifically expressed in pomegranate roots; PgAP2/ERF45 and PgAP2/ERF59 were specifically expressed in pomegranate flowers; and the PgAP2/ERF8 gene was mainly expressed in pomegranate peels (Figure 8). In addition, PgAP2/ERF23 and PgAP2/ERF107 were specifically expressed in pomegranate inner seed coats; and PgAP2/ERF75 and PgAP2/ERF89 were specifically expressed in pomegranate outer seed coats (Figure 8). The number of PgAP2/ERF genes highly expressed in pomegranate roots was higher relative to that in the other investigated pomegranate tissues. The number of PgAP2/ERF genes expressed in inner and outer seed coats of 'Tunisian soft-seed' was higher compared with that expressed in inner and outer seed coats of 'Dabenzi'. Differential expression of PgAP2/ERF genes was observed between the seed coats of 'Dabenzi' and 'Tunisian soft-seed' pomegranates. For example, PgAP2/ERF5, PgAP2/ERF36, PgAP2/ERF88, and PgAP2/ERF97 were specifically expressed in 'Dabenzi' seed coats, whereas PgAP2/ERF99, PgAP2/ERF58, PgAP2/ERF86, and PgAP2/ERF87 were only expressed in 'Tunisian soft-seed' seed coats (Figure 8). Genes 2022, 13, x FOR PEER REVIEW 13 of 24

Expression Profile of PgAP2/ERF Genes during Pomegranate Fruit Development
Transcriptomic data of the inner and outer seed coats of 'Tunisian soft-seed' pomegranate at 50, 95, and 140 days after flowering (DAF) and transcriptomic data of the peels of 'Taishanhong' pomegranate at 60, 90, and 150 DAF ( Figure 9) were retrieved from the NCBI SRA database to explore the characteristics of PgAP2/ERFs during pomegranate fruit development. Notably, the whole seeds at 50 DAF were used towing to the challenge in visually distinguishing the inner and outer seed coats [39].
PgAP2/ERFs were assigned to six groups according to their expression profiles in inner and outer seed coats of pomegranate (Figure 9a). Most of the genes from Groups Ⅰ and Ⅱ were upregulated during pomegranate fruit development, and higher expression levels were observed in the inner seed coats than in the outer seed coats, especially at 140 DAF (Figure 9a). On the contrary, most genes from Group Ⅲ exhibited higher expression levels in the outer seed coats than in the inner seed coats mainly at 140 DAF (Figure 9a). Genes from Groups Ⅳ, Ⅴ, and Ⅵ showed relatively higher expression levels in the outer seed coats at 95 DAF, in the inner seed coats at 95 DAF stage, and the whole seeds at 40 DAF, respectively. PgAP2/ERF1 and PgAP2/ERF12 genes were not expressed in all seed coat samples (Figure 9a).

Expression Profile of PgAP2/ERF Genes during Pomegranate Fruit Development
Transcriptomic data of the inner and outer seed coats of 'Tunisian soft-seed' pomegranate at 50, 95, and 140 days after flowering (DAF) and transcriptomic data of the peels of 'Taishanhong' pomegranate at 60, 90, and 150 DAF ( Figure 9) were retrieved from the NCBI SRA database to explore the characteristics of PgAP2/ERFs during pomegranate fruit development. Notably, the whole seeds at 50 DAF were used towing to the challenge in visually distinguishing the inner and outer seed coats [39].
PgAP2/ERFs were assigned to six groups according to their expression profiles in inner and outer seed coats of pomegranate (Figure 9a). Most of the genes from Groups I and II were upregulated during pomegranate fruit development, and higher expression levels were observed in the inner seed coats than in the outer seed coats, especially at 140 DAF (Figure 9a). On the contrary, most genes from Group III exhibited higher expression levels in the outer seed coats than in the inner seed coats mainly at 140 DAF (Figure 9a). Genes from Groups IV, V, and VI showed relatively higher expression levels in the outer seed coats at 95 DAF, in the inner seed coats at 95 DAF stage, and the whole seeds at 40 DAF, respectively. PgAP2/ERF1 and PgAP2/ERF12 genes were not expressed in all seed coat samples (Figure 9a). Ⅰ were downregulated during peel development, whereas expressions of all genes from Group Ⅱ were upregulated from 60 DAF to 90 DAF and downregulated from 90 DAF to 150 DAF (Figure 9b). Group Ⅲ comprised 15 genes mainly upregulated in peels at 150 DAF and 12 genes that were not expressed during peel development. Genes in Group Ⅳ showed relatively higher expression levels at 60 DAF relative to the expression levels at the other stages (Figure 9b).  PgAP2/ERFs were assigned to four groups according to their expression patterns in peels during pomegranate development (Figure 9b). Expressions of all genes from group I were downregulated during peel development, whereas expressions of all genes from Group II were upregulated from 60 DAF to 90 DAF and downregulated from 90 DAF to 150 DAF (Figure 9b). Group III comprised 15 genes mainly upregulated in peels at 150 DAF and 12 genes that were not expressed during peel development. Genes in Group IV showed relatively higher expression levels at 60 DAF relative to the expression levels at the other stages (Figure 9b).

Expression Profiles of PgAP2/ERF Genes during Pomegranate Postharvest Preservation
RNA-Seq data of the 'Tunisian soft-seed' pomegranate fruit during LTC were obtained (unpublished) to explore the possible roles of the PgAP2/ERF genes in postharvest preservation of pomegranate fruit. The results revealed six different expression clusters and important roles of PgAP2/ERFs during postharvest preservation ( Figure 10). Group I included 22 genes such as PgAP2/ERF3, PgAP2/ERF109, PgAP2/ERF15, PgAP2/ERF25 and PgAP2/ERF35, which were upregulated at 15 DAT (days after the treatment) and downregulated at 45 DAT. Group VI comprised 9 genes that were downregulated at 15 DAT and relatively upregulated at 45 DAT, whereas PgAP2/ERF88, PgAP2/ERF110, PgAP2/ERF22, and PgAP2/ERF32 in group II were relatively upregulated after LTC (Figure 10). Group III comprised 42 PgAP2/ERFs, which were directly repressed at 15 DAT, including PgAP2/ERF4, PgAP2/ERF113, PgAP2/ERF8, PgAP2/ERF105, PgAP2/ERF26, PgAP2/ERF30, and PgAP2/ERF58, etc. (Figure 10). In addition, 14 genes in Group IV were downregulated during the LTC period and 15 genes in Group V were not upregulated until 45 DAT ( Figure 10).  Postharvest application of 1-MCP is an efficient strategy used for the prevention of fruit damage under low-temperature storage [19]. Therefore, the preservation effect of 1-MCP under LTC (1-MCP + LTC) on the postharvest 'Tunisian soft-seed' pomegranate fruit was explored in the present study ( Figure 11). Time series of fruit were sampled to further evaluate the relationship between PgAP2/ERFs and postharvest preservation (Figures 11 and 12). The LTC fruit showed extensive browning appearance with significantly higher browning indexes compared with the 1-MCP + LTC fruit from 30 DAT (Figure 11a,b), indicating a positive effect of 1-MCP on improving the exterior quality of the postharvest pomegranate fruit. The ethylene production level of the 1-MCP + LTC fruit was also significantly lower relative to the LTC fruit at 30 and 60 DAT (Figure 11c).  Further, we screened the 12 genes shown in Figure 12 from Group III as well as PgAP2/ERF34 from Group IV, in which the genes mainly were downregulated after LTC. Compared to fruit at 0 DAT, the expression of PgAP2/ERF4, PgAP2/ERF8, PgAP2/ERF15, PgAP2/ERF26, PgAP2/ERF34, and PgAP2/ERF35 was directly downregulated at 15 DAT and then slightly upregulated from 30 DAT under LTC. However, PgAP2/ERF4, PgAP2/ERF15, PgAP2/ERF26, and PgAP2/ERF35 were significantly upregulated by the application of 1-MCP under LTC, especially from 15 to 30 DAT, despite their expression levels being lower than those at 0 DAT. Then, these three genes were downregulated after 45 DAT but almost with no significance between LTC and 1-MCP + LTC. On the contrary, PgAP2/ERF8 and PgAP2/ERF34 were more downregulated by the application of 1-MCP under LTC, especially from 15 to 30 DAT, compared to those under LTC. Afterwards, the two genes expressed with no significance between LTC and 1-MCP + LTC (Figure 12). There were also PgAP2/ERF30 and PgAP2/ERF 58 that were significantly upregulated by the application of 1-MCP under LTC, especially from 15 to 30 DAT, but the two genes expressed significantly lower from 45 DAT than those under LTC. The expression levels of PgAP2/ERF45 were significantly higher under 1-MCP + LTC than those under LTC. PgAP2/ERF25, PgAP2/ERF105, and PgAP2/ERF113 were not upregulated by the application of 1-MCP under LTC except for those at 15 DAT, and they mostly expressed significantly more highly under LTC than 1-MCP + LTC ( Figure 12).

Discussion
AP2/ERF gene family is a major plant transcriptional factor family and is implicated in various developmental processes in plants [22]. The AP2/ERF gene family has been reported in several fruit trees, such as grapes, Chinese jujube, sugarcane, Chinese lotus, and pineapple [26,27,[40][41][42]. However, studies have not explored the presence and function of the AP2/ERF gene family in pomegranate. Pomegranate fruit exhibits excellent nutritional qualities and health-promoting phytochemicals. Therefore, the present study was conducted to explore detailed gene information of pomegranate and evaluate the role of genes in the AP2/ERF gene family in pomegranate fruit development and postharvest preservation.
In total, 116 members of the PgAP2/ERF gene family were identified in the present study (Figure 1), based on the genomic information of 'Tunisian softseed' pomegranate available in public databases [37]. The number of AP2/ERF gene

Discussion
AP2/ERF gene family is a major plant transcriptional factor family and is implicated in various developmental processes in plants [22]. The AP2/ERF gene family has been reported in several fruit trees, such as grapes, Chinese jujube, sugarcane, Chinese lotus, and pineapple [26,27,[40][41][42]. However, studies have not explored the presence and function of the AP2/ERF gene family in pomegranate. Pomegranate fruit exhibits excellent nutritional qualities and health-promoting phytochemicals. Therefore, the present study was conducted to explore detailed gene information of pomegranate and evaluate the role of genes in the AP2/ERF gene family in pomegranate fruit development and postharvest preservation.
In total, 116 members of the PgAP2/ERF gene family were identified in the present study (Figure 1), based on the genomic information of 'Tunisian soft-seed' pomegranate available in public databases [37]. The number of AP2/ERF gene family members depends on the number of ERF subfamily members [43]. In the current study, the numbers of PgAP2/ERF gene family and ERF subfamily members (116, 102, P. granatum) were not significantly different relative to those of Arabidopsis (147, 122) and rice (164, 139 O. sativa) [21], but they were both less compared with the number of genes in cotton (269, 222 Gossypium raimondii) and celery (206, 172 Apium graveolens) [44,45]. Previous studies have reported that most genes in the same cluster present similar conserved motifs and exon-intron patterns [26]. The findings of the present study indicated that PgAP2/ERFs in the same PgAP2/ERF subfamily exhibited highly conserved motifs and gene structures. Notably, most PgAP2/ERFs similarly processed motif1, motif3, and AP2 domain (Figure 2), further indicating high conservation in the same subfamily as well as in the whole gene family.
Gene duplication events occurring during genome evolution are the main factors that lead to gene expansion and evolution, and they play important roles in improving plant adaptability to various environmental stresses [46][47][48]. In the present study, tandem repeat events and segmental duplication events were identified in pomegranate the PgAP2/ERF gene family, and syntenic pairs observed in AP2/ERF genes of pomegranate compared with those in Arabidopsis, grape, maize, and rice (Figures 3 and 4). These results showed that gene duplication events were also key factors in the evolution and expansion of the PgAP2/ERF gene family.
Promoter cis-elements play key roles in regulation of gene expression [49]. The presence of different cis-elements in promoters of PgAP2/ERF genes indicates that they are involved in different regulatory networks related to ETH-, ABA-, and MEJA-associated pathways and responses to stresses ( Figure 5). Similar results have been reported for AP2/ERF genes in other plant species [26,41,50]. Seventy PgAP2/ERFs containing LTR elements were identified, which included 7 PgCBFs ( Figure 5, Table S3), implying their potential roles in the response of pomegranates to cold. CBF genes belong to the AP2/ERF gene family and bind to a cis-element (DRE/CRT/LTRE) with a conserved CCGA core sequence, implicated in inducing expression of genes associated with increased cold tolerance [51,52]. Moreover, some transcription factors in the AP2/ERF family are modulated by other AP2/ERF-type factors to regulate the physiological processes of plants. For example, CBF2 negatively regulates the expression of CBF1 and CBF3, thus decreasing plant tolerance to multiple stresses [22]. PPI analysis in this study also showed some possible complex regulation interactions among the different PgAP2/ERFs but these PgAP2/ERFs include no PgCBF (Figure 7).
Transcriptomic analysis is usually used to study the mRNA expression levels of specific tissue or cells transcribed during a certain period, and to explore the related genes and phenotypes [53]. Transcriptomic analysis in the present study indicated PgAP2/ERF5, PgAP2/ERF36, PgAP2/ERF88, and PgAP2/ERF97 exhibited high expression levels in seed coats of hard-seeded 'Dabenzi' pomegranate, whereas PgAP2/ERF99, PgAP2/ERF58, PgAP2/ERF86, and PgAP2/ERF87 showed high expression levels in seed coats of soft-seeded 'Tunisian soft seed' pomegranate ( Figure 8). In fact, AP2-like genes have been reportedly implicated in modulating seed hardness as these genes exhibit genetic divergences in soft-and hardseeded pomegranate cultivars [37]. The differential expression of these genes above were likely related to the different phenotypes of soft-seeded and hard-seeded pomegranates, which is an important trait of pomegranate fruit that determines consumer preference.
Differentially expressed AP2/ERF genes are implicated in fruit development during the ripening process of several kinds of fruit and vegetable plants, such as grape (V. Vinifera), banana (M. acuminata), peach (P. persica), and tomato (Solanum lycopersicum) [11,[54][55][56]. Many mechanisms of ERF regulating fruit development have been well exposed. MaERF11 is a potential repressor, whereas MaERF9 is an activator of the MaACO1 gene family during banana fruit development by regulating cell expansion [9] and PpERF3 regulates PpNCED2/3 expression to promote ABA biosynthesis during fruit ripening in peach [57].
The results in the current study showed that PgAP2/ERFs with different expression levels are importantly involved in pomegranate fruit development (Figure 9), which deserved investigation of the possible roles of PgAP2/ERFs in pomegranate fruit development.
Cold storage under low temperature is widely used to maintain postharvest fruit quality, including pomegranate fruit [18]. Previous studies reported several differentially expressed PgAP2/ERFs by comparing the transcriptomic responses of the 'Wonderful' pomegranate fruit under three pairwise postharvest preservation treatments [15][16][17][18]. AP2/ERFs have proven to be important regulators of the preservation of different fruit, such as grapes, apples, loquats, and bananas, under LTC [11][12][13]56]. In 'Tunisian soft-seed' pomegranate fruit peels, lots of PgAP2/ERFs were found differentially expressed during LTC ( Figure 10). It has been known that 1-MCP can keep postharvest storage performances of non-climacteric pomegranate fruit by modulating decaying scald incidence, peel browning, ethylene production, and other physical processes [6,19,20]. The present study also proved that 1-MCP can help the postharvest preservation of 'Tunisian soft-seed' pomegranate fruit under LTC through decreasing peel browning and ethylene production ( Figure 11). Here, PgAP2/ERFs were also found importantly involved in the postharvest preservation of 'Tunisian soft-seed' pomegranate fruit by the application of 1-MCP under LTC. For example, PgAP2/ERF4, PgAP2/ERF15, PgAP2/ERF26, PgAP2/ERF30, PgAP2/ERF35, and PgAP2/ERF58 were significantly upregulated by the application of 1-MCP under LTC, which were downregulated after LTC (Figure 12), which indicated their possible roles in pomegranate postharvest preservation.
PgAP2/ERF4 was implicated in the 'negative regulation of ethylene-activated signaling pathway' GO term ( Figure 6; Table S7) and is a homologue of MdERF4 (Figure 1). MdERF4 was proven to be involved in negatively regulating ethylene-mediated salt tolerance [58]. Therefore, we suggested that PgAP2/ERF4 might act as an important candidate regulator implicated in negatively regulating the ethylene-associated pathway in the effects of 1-MCP on the postharvest preservation of 'Tunisian soft-seed' pomegranate fruit. PgAP2/ERF15 was identified as a CBF gene and was homologous to AtCBF1 (Figure 1; Table S3), which plays important roles in plant cold tolerance [59,60]. Recent studies have explored the roles of CBFs in fruit postharvest preservation during cold storage. For example, MiCBF1 is induced in mango fruit under hot water treatment; CmCBF1 and CmCBF3 are induced in Hami melon fruit under LTC; and the AcCBF gene is induced in kiwifruit fruit under nitric oxide treatment. They all contribute to enhancing the cold tolerance of fruit to improve postharvest preservation [61][62][63]. Moreover, PgAP2/ERF30 is homologous to MaERF11, and MaERF11 is a crucial transcriptional repressor of ethylene biosynthesis in decaying banana fruit and postharvest ripening [9,64]. These findings indicate that PgAP2/ERF15 and PgAP2/ERF30 are also important candidate genes in pomegranate postharvest preservation.

Conclusions
A comprehensive and systematic genome-wide identification and analysis of AP2/ERF genes in the pomegranate genome was performed using bioinformatics tools and algorithms. A total of 116 PgAP2/ERF genes were identified and classified into four subfamilies with detailed information of the chromosomal distribution, phylogenetic analysis, gene structures, duplications, and their possible biological functions. This study found PgAP2/ERF genes were possibly, and importantly, involved in pomegranate fruit development through transcriptomic analysis. Moreover, the PgAP2/ERF4, 15,26,30,35, and 58 genes were screened as crucial candidate genes that might play important roles in pomegranate postharvest preservation based on the PgAP2/ERF expression profiles of 'Tunisian soft-seed' pomegranate fruit under LTC and 1-MCP + LTC. The detailed regulation mechanisms of pomegranate fruit development and postharvest preservation are to be further investigated.

Supplementary Materials:
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/genes13050895/s1, Figure S1: The sequence information for each motif (Motif 1-Motif 10); Table S1: Accession numbers of RNA-seq data from NCBI; Table S2: Primers used for qRT-PCR; Table S3: Gene information of the AP2/ERF gene family in the pomegranate genome; Table S4: Gene duplication pairs of the PgAP2/ERF genes within the pomegranate genome; Table S5: Ka/Ks ratios of the duplicated PgAP2/ERF genes in pomegranate; Table S6: Syntenic gene pairs between AP2/ERF genes in pomegranate and those in Arabidopsis thaliana; Table S7: GO annotations of the PgAP2/ERF genes.   Table S1) from six different pomegranate tissues and organs were retrieved from NCBI Sequence Read Archive data-base (http://www.ncbi.nlm.nih.gov/sra accessed on 15 May 2021).