Genome ‐ Wide Identification and Expression Analysis of TALE Gene Family in Pomegranate ( Punica granatum L.)

: The three ‐ amino ‐ acid ‐ loop ‐ extension (TALE) gene family is a pivotal transcription factor that regulates the development of flower organs, flower meristem formation, organ morphogenesis and fruit development. A total of 17 genes of pomegranate TALE family were identified and analyzed in pomegranate via bioinformatics methods, which provided a theoretical basis for the functional research and utilization of pomegranate TALE family genes. The results showed that the PgTALE family genes were divided into eight subfamilies (KNOX ‐  Ⅰ , KNOX ‐ Ⅱ , KNOX ‐ Ⅲ , BELL ‐ Ⅰ , BELL ‐ Ⅱ , BELL ‐ Ⅲ , BELL ‐ Ⅳ , and BELL ‐ Ⅴ ). All PgTALEs had a KNOX domain or a BELL domain, and their structures were conservative. The 1500 bp promoter sequence had multiple cis ‐ elements in response to hormones (auxin, gibberellin) and abiotic stress, indicating that most of PgTALE were involved in the growth and development of pomegranates and stress. Function prediction and protein ‐ protein network analysis showed that PgTALE may participate in regulating the development of apical meristems, flowers, carpels, and ovules. Analysis of gene expression patterns showed that the pomegranate TALE gene family had a particular tissue expression specificity. In conclusion, the knowledge of the TALE gene gained in pomegranate may be applied to other fruit as well.


Construction of Phylogenetic tree of PgTALE Gene Family
Multiple sequence alignments of candidate proteins with A. thaliana, E. grandis, P. trichocarpa and V. vinifera TALE gene family proteins were performed using MAFFT [47]. The phylogenetic tree was constructed by using RA × ML -NG [48] with Bootstrap 1000 repeats and the best model of JTT + F + I + G4 selected by ModelFinder [49]. Then, the phylogenetic tree was beautified by using the online software tool EvolView (http://www.evolgenius.info/) [50].

Analysis of PgTALE Conserved Motifs and Gene Structure
The motif type and sequence of the PgTALE family were analyzed by MEME (http://memesuite.org/tools/meme) [51], and the motif characteristics of PgTALE were obtained. According to the protein sequence and gene sequence of the PgTALE gene, the gene structure information of pomegranate TALE was obtained by Perl script (Perl file S1), including intron, exon and upstream and downstream sequence. In addition, a combined figure of phylogenetic tree, conserved motifs and gene structure was drawn by TBtools [52].

Analysis of PgTALE Protein Structure
Protein sequence similarity of more than 35% as a template, the tertiary structure and homologous modelling of PgTALE proteins were analyzed using the SWISS-MODEL (https://swissmodel.expasy.org/) [53], and Ramachandran Plots were used to display protein properties.

Analysis of Cis-elements and Protein-protein Interaction Network of PgTALE Gene Family
To analyze the cis-elements of the promoter region, the 1500 bp sequence upstream of the start codon was obtained from the pomegranate genome sequence by Perl script (Perl file S2), and the sequence was searched by PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [54]. The protein-protein interaction network of the TALE family was analyzed by String (https://string-db.org/) [55].

Expression Analysis of PgTALE Gene Family
RNA-Seq data of tissues and organs closely related to pomegranate were downloaded from the NCBI database (Table 1). Subsequently, Kallisto version 0.44.0 software (California, USA) [56] was used to index the sequence with the 'Taishanhong' transcriptome file to calculate further and analyze gene expression. The corresponding expression levels (TPM values) of the TALE family members were obtained, and the obtained TPM values were converted by Log2 (TPM + 1). Finally, a heat map of the TALE gene was drawn by using the R package heatmap.

Identification and Sequence Analysis of PgTALE Gene Family Members
In this study, 74 homebox gene family members were identified by using the hmmsearch method. The homebox family consists of five families (HD-ZIP, TALE, WOX, HB-PHD, and HBother), and they share a PF number (PF00046). As TALE encodes an atypical structure forming two helices and three additional amino acid residues, 16 candidate members of the TALE gene family were identified, and all candidate proteins were identified to belong to the TALE protein family. 23 candidate members of the TALE gene family were identified by BLASTP. While Pg001623. 1 (Table S2). PgTALE gene family was renamed, the results as shown in Table 2.
The physical and chemical properties of the PgTALEs was analyzed using the ExPASy online tool. The results showed that the length of the 17 PgTALE gene coding regions ranged from 465 bp (PgTALE17) to 2400 bp (PgTALE16). The amino acid length of the TALE protein ranged from 154 aa (PgTALE17) to 799 aa (PgTALE16), and the protein molecular weight ranged from 17605.66 Da (PgTALE17) to 87381.25 Da (PgTALE16). The pI ranged from 5.13 (PgTALE7) to 8.78 (PgTALE16). Among them, the pI of three PgTALE proteins were higher than 7, suggesting that proteins were slightly alkaline; the other 14 PgTALEs were acidic proteins. The grand average of hydropathicity (GRAVY) was between −0.969 to −0.449, suggesting that PgTALEs are all hydrophilic proteins. The number of exons of the PgTALEs was 3-6. Besides, the signal peptide prediction showed that there were no signal peptides in all PgTALE proteins, which belonged to non-secreted proteins. Subcellular localization prediction suggested that all PgTALE proteins were distributed on the nucleus.

Phylogenetic Tree Analysis of PgTALE Gene Family
To clarify the evolutionary relationship and possible biological functions of members of the PgTALE gene family, the phylogenetic tree of the TALE gene was constructed based on the amino acid sequences of the pomegranate, A. thaliana, E. grandis, P. trichocarpa and V. vinifera (Figure 1). Based on the classification of A. thaliana TALE gene family (BELL and KNOX family), the pomegranate BELL proteins were classified into five subfamilies: BELL-Ⅰ (one member), BELL-Ⅱ (two), BELL-Ⅲ (one), BELL-Ⅳ (two) and BELL-Ⅴ (three), and KNOX proteins were classified into 3 subfamilies: KNOX-Ⅰ (five), KNOX-Ⅱ (two), KNOX-Ⅲ (five). In each clade, there are branches from the same species, which may be caused by gene duplications [59].

Analysis of Conserved Motifs and Gene Structures of PgTALE Gene Family
The conserved motifs of PgTALE were identified. 10 conserved motifs (Figure 2

Protein Structure Analysis and Protein Interaction Networks of Pomegranate TALE Gene Family
The spatial structure of proteins plays a role in the biological function of proteins. The tertiary structure of the protein was analyzed, which found that the structure of the PgTALE family members was similar (Figure 3), except for PgTALE17 without a template (protein sequence similarity of less than 35%) that we cannot predict protein tertiary structure. The protein is a multi-chain folded protein, mainly α-helix. The calculation test showed that the Ramachandran Favoured value of the PgTALE family was above 90%, and PgTALE2 and PgTALE15 reached 100%, except PgTALE9 was only 87.27%. The results showed that the PgTALE protein had a stable spatial structure. Protein function prediction suggested that PgTALE2, PgTALE7 and PgTALE15 played roles in meristem function (Figure 4), contributing to the shoot apical meristem (SAM) maintenance and organ separation. They may also be involved in maintaining cells in a meristematic state. In addition, PgTALE14 might involve in the regular pattern of organ initiation. PgTALE11 may be required for SAM formation in embryogenesis. PgTALE12 may be involved in secondary cell wall biosynthesis. PgTALE13 might be required for the SAM to respond appropriately to floral inductive signals.
The protein-protein interaction of PgTALE was analyzed for predicting its potential function, signal transduction and metabolic pathways. It was predicted that there were interactions between PgTALE14 and AG, SEP3, KNAT1, INO and other proteins to regulate ovule development. In addition, BEL1 can form heterodimers with KNAT1, it predicted that PgTALE14 (BELL family) may interact with PgTALE5 (KNOX family) to form heterodimers. PgTALE8 might interact with STM and KNAT6 and enhance the apical meristem of these genes.
From the figure of gene co-expression (Figure 4), we can see the level of co-expression of KNAT1/KNAT3/KNAT6/STM/BEL1/BLH6/ATH1 and other genes. Among them, KNAT1 and KNAT6, KNAT1 and STM were higher than that of other genes. They may participate in or respond to a biological or abiotic stress process, and it may be inferred that PgTALE2/PgTALE5/PgTALE11 may also have similar functions.

Analysis of Cis-elements of PgTALE Gene Family
In this study, the upstream 1500 bp sequence of PgTALE gene was extracted, the possible ciselements in the promoter region were found (Table S3). 13 cis-elements related to abiotic stress were found, which were ABRE, ARE, AuxRR-core, CAAT-box, CGTCA-motif, GARE-motif, LTR, MBS, Pbox, TATC-box, TCA-element, TGA-element and TGACG-motif ( Figure 5; Table S4). AuxRR-core and TGA-element are auxin-responsive elements. CGTCA-motif and TGACG-motif are MeJAresponsiveness elements, while GARE-motif, P-box and TATC-box are gibberellin response elements. The PgTALE genes contain the enhancer response element CAAT-box. 64.7% of the PgTALE genes contain ABA response element ABRE and the cold stress response element LTR. 70.6% of the PgTALE genes contain the antioxidant response element ARE. 41.2% of the PgTALE gene contains MeJAresponsiveness response elements CGTCA-motif and TGACG-motif, the salicylic acid response element TCA-element, 29.4%, 23.5%, 35.3% of the PgTALE genes contain gibberellin response elements GARE-motif, P-box, TATC-box, 29.4% of the PgTALE genes contain the drought stress response element MBS. Besides, only the PgTALE8 gene contains the auxin response element AuxRRcore, and PgTALE12 and PgTALE13 contain the auxin response element TGA-element.

Expression Analysis of PgTALE Gene Family
To further analyze the characteristics and function of the PgTALE genes, the tissue-specific expression of the TALE gene was analyzed ( Figure 6; Table S5). The results showed that the vast majority of PgTALE genes were expressed in different tissues, but PgBLH8 was expressed in trace or no expression in all tissues.
PgTALE5, PgTALE12, and PgTALE15 are expressed during functional male flower development, indicating that these genes may be involved in the female and male organ differentiation; PgTALE1 and PgTALE9 are higher expressed in leaves, bisexual and functional male flower, indicating that they may be related to the differentiation of male and female organs of pomegranate flowers and regulating leaf development. There are also differences in the expression of different PgTALE genes in different tissue, such as PgTALE2 is not expressed in the inner seed coat, outer seed coat and pericarp. The expression of PgTALE9 is the highest in the functional male flower (5.1 m-13.0 mm), and the expression of PgTALE10 is the highest in the pericarp. However, there are some differences in the expression of different PgTALE genes in different pomegranate varieties, such as PgTALE7 and PgTALE14 in the varieties of 'Dabenzi', 'Tunisia' and 'Baiyushizi'. In the same pomegranate variety 'Dabenzi', there are also significant differences in tissue expression between leaves and outer seed coat. For example, the expression of PgTALE16 is higher in leaves, but the lowest in the outer seed coat. ); S10: Female sterility (13.1-25.0 mm); S11: Female sterility (5.1-13.0 mm); S12: Female sterility (3.0-5.0 mm); S13: Inner seed coat of 'Tunisia' (50 days after pollination); S14: Inner seed coat of 'Baiyushizi' (50 days after pollination); S15: Pericarp of 'Wonderful'; S16: Mix of leaves, flowers, fruit and roots of 'nana'; S17: Mix of leaves, flowers, fruit and roots of 'Black127' (cultivars S1-S6 are 'Dabenzi', cultivars S7-S13 are 'Tunisia').

Discussion
The TALE gene family is found in plant meristems and is related to the differentiation and signal transduction of meristems, for example, it can inhibit the expression of the critical enzyme gene ga20ox1 in the GA pathway [60]. In other important fruits belonging to the Rosaceae family, TALE are involved in the rootstock responding to apple cold stress [61], the cherry anthesis [62]. In addition, it regulated tomato fruit development [28]. Currently, the TALE gene family has been found in many plants: 33 AtTALE genes in A. thaliana, 40 LjTALE genes in Lotus japonicas K. [63], 46 GaTALE genes in Gossypium arboretum L., 47 GrTALE genes in G. raimondii L., 88 GbTALE genes in G. barbadense L., 94 GhTALE genes G. hirsutum L. [8], and 35 PtTALE genes in poplar [64], 7 VsTALE genes in Vandenboschia speciose G. [16]. Therefore, the copy number of the TALE gene family in different species is different. At present, the genomic data of three pomegranate varieties have been released in China, but there are no reports on the identification and analysis of pomegranate TALE family genes. In this study, for the first time, 17 TALE genes were identified in the pomegranate. Through the analysis of the physicochemical properties of the protein (Table 1), it was found that pomegranate TALE proteins are all hydrophilic proteins that are consistent with studies in Popular and L. japonicas [63,64]. Domain differences may represent the regulatory effects of promoting or inhibiting. In addition, the PgTALE genes are divided into eight subfamilies (Figure 1), which is consistent with the A. thaliana and cotton TALE gene subfamily classification [8].
The cis-elements exist at the gene promoter site and specifically binds transcription factors to regulate gene transcription. This study found that the PgTALE promoter sequence contained multiple cis-elements related to hormonal response and abiotic stress, which are rich in methyl jasmonate response element, abscisic acid response element and gibberellin response element, which is similar to antecedent studies [8]. It indicated that the promoter of the TALE gene has a certain conservative. Previous studies have found that ABRE is associated with plant drought, ABA induction, and high salt stress in plants [31,65]. In addition, there are a series of elements related to stress, such as ARE, MBS and LTR. The results indicate that PgTALE plays a role in pomegranate abiotic stress. Gene function prediction and protein-protein network analysis also show that the PgTALE family plays a significant role in regulating ovule and inflorescence development. Gene functional prediction and protein-protein network analysis also showed that there are some interactions between PgTALE14, AG and KNAT1 in floral organs; the results were consistent with the previous study [7,25].
The tissue expression analysis of the PgTALE found that most of them were expressed in diverse tissues and varieties, but diverse PgTALE genes were expressed in different tissue varieties and showed specific differences. It was similar to the results of TALE genes in A. thaliana [66]. It can be speculated that the TALE family of pomegranate has similar functions to this family in other plants.
According to the function of BEL1 in A. thaliana, we speculated that its homologous gene PgTALE14 has important regulatory significance in the development of pomegranate ovules [67,68]; PgTALE8, as the homologous gene of ATH1, controls inflorescence development [69,70]. The specificity of tissue and variety expression is speculated to be closely related to its gene function. For example, PgTALE1, PgTALE6, PgTALE9, PgTALE10, PgTALE12 and PgTALE14 had high expression levels in the functional male flowers, bisexual flowers, and fruit tissues. It is predicted that PgTALE may have roles in maintaining flower organ and fruit development. However, due to the inconsistency of some sequencing platforms (Illumina and 454) in RNA-seq data, to a certain extent, it may lead to the uneven sequencing depth among tissue samples and the gap in reading length, which has a certain impact on the analysis results, while the difference in pomegranate varieties also has a certain error on the expression analysis results. After the normalization of RNA-seq data, the error may be reduced.

Conclusions
In this study, 17 PgTALE members were identified in pomegranate and explored their phylogenetic relationships. The PgTALE gene structure of all members of the subfamily is very similar. PgTALE may participate in the apical meristems, flower organ and fruit development, and the subfamily genes may have the same expression pattern. These conclusions are the foundation for the function research of the PgTALE gene and provide a reference for exploring its evolutionary process.

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

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