Genome-wide identification and expression analysis of auxin response factor (ARF) gene family in Punica granatum

Abstract: 【Objective】Auxin response factor (ARF) is an essential plant-specific transcription factor (TF) which can identify and combine the auxin response elements.It participates in auxin signal transduction and regulates the auxin responses.Generally, the majority of ARF proteins consist of three typical conserved domains:a DNA binding domain (DBD) in the N terminal region, a middle region (MD) and a C-terminal dimerization domain (CTD) .Pomegranate (Punica granatum) is widely cultivated as a functional and emerging fruit tree.Although there is an increasing trend for pomegranate market and cultivated area, there is still a gap in the understanding of its growth and development regulation.ARF transcription factor genes are the central element mediating plant development.Hence, we performed the genome-wide analysis of ARF transcription factor gene family in pomegranate to provide sequence resource for further functional verifications.【Methods】In order to identify ARF gene family genomewide, the pomegranate genomic and transcriptomic data were downloaded from NCBI.HMMER v3.1 software was used to blast the putative ARF candidate genes in pomegranate genome with the HMM profile of the Auxin＿resp domain (PF06507) as a query.After identification of the conserved domains, the invalid and redundant sequences were deleted.The ARF protein sequences of Arabidopsis thaliana and Eucalyptus grandis were obtained from TAIR database and Plant Transcription Factor Database (PlantTFDB) , respectively.This estimation of the pomegranate ARF protein sequence was analyzed using the Protparam software.The molecular weight, theoretical pI, instability index and grand average of hydropathicity were estimated.The positions and numbers of introns and exons of the PgARF genes were detected using the Gene Structure Display Server (GSDS v2.0) .Phylogenetic analysis was performed by MEGA v7.0 program with the neighbor-joining (NJ) method and 1 000 bootstrap replicates.The conserved motifs and domains of the PgARF protein sequences were predicted by the Multiple Em for Motif Elicitation (MEME) and Prosite software, respectively.DNAMAN v8.0 was used for multiple sequence alignments in pomegranate ARF proteins.The protein secondary structure and tertiary structure of the pomegranate ARF proteins were analyzed by SSPro v4.0 and SWISS-MODEL.To elucidate the tissue-specific expression pattern of PgARF genes, the Kallisto tool was employed to quantify the ARF gene expression levels with the pomegranate RNA-Seq data.【Results】A total of 19 putative ARF candidate genes were identified in pomegranate genome.This estimation of the pomegranate ARF proteins indicated that their molecular weight ranged from 61.59 to 124.23 ku with a pIs ranging from 5.35 to 8.86, and all of them were labile and hydrophobic proteins.A NJ phylogenetic tree was constructed using the ARF proteins of pomegranate, A. thaliana and E. grandis, and clustered ARFs into four classes (Class Ⅰ, Ⅱ, Ⅲ and Ⅳ) .The structure analysis of the pomegranate ARF genes indicated that the intron number varied from 1 to 13 and the exon number varied from 2 to 14.Integrated phylogenetic and gene structure analyses deciphered that the exons have been split due to the recent intron insertion.Phylogenetic analyses showed an ARF gene number ratio (1:1:1) for A.thaliana versus E.grandis versus pomegranate except for the A. thaliana-lineage-specific clade in Class Ⅲ.It was inferred to be related with the paleohexaploidy event.Similarly, a ratio (1:2:2) for A.thaliana versus E.grandis versus pomegranate was found in several groups of Clades Ⅲ and Ⅳ.It was inferred to be related with the paleotetraploidy event.Hence, the paleohexaploidy and paleotetraploidy events in pomegranate genome contributed to the expansion of the PgARF gene.The phylogenetic and genomic location analyses indicated a tandem duplication of PgARF16 a and PgARF16 b.Motif and sequence alignment analyses suggested that the B3, Auxin＿resp and Aux/IAA domain of the PgARF proteins were highly conserved.The motif4 and motif 5 belonged to the B3 domain;motif 7 and motif 8 belonged to the Auxin＿resp domain;motif 11 and motif 12 belonged to Aux/IAA domain.The secondary and tertiary structure analysis showed that the helixes were the major part of the ARF protein.The roles of PgARF in the development of pomegranate leaves and flower tissues were inferred from the homologous genes AtARF in A.thaliana.The RNA-Seq expression profile suggested that most of the PgARF genes were highly expressed in root, leaf, flower, floral bud, fruit, inner seed coat, outer seed coat and peel.PgARF4 b was lowly expressed in the inner seed coat, and PgARF5 highly expressed in the outer seed coat.These results suggested tissue-specific expression patterns of ARF family genes.Compared with the other ARF genes, PgARF3 a had a very weak expression.This might be related to the pseudogenization of PgARF3 a resulting in the gene inactivation.Conversely, distinct results were found in PgARF24 which were highly expressed in all tissues.It inferred to be related with the neofunctionalization.【Conclusion】The integration of the gene structure, phylogentic, evolutionary and expression analyses provided a genome-wide trait and expression pattern of ARF in pomegranate.The results provide a reference for further function verifications and auxin signal transduction pathway regulation of PgARF genes.