Structure and function analysis of R2R3-MYB gene family from passion fruit (Passiflora edulis)

Abstract:【Objective】The R2R3-MYB transcription factors (TFs) are widely involved in the regulation of various biological processes in plants, including primary and secondary metabolism, growth and development, and response to environmental factors and plant hormones. In this study, the expression of R2R3-MYB gene family in passion fruit was systematically analyzed and its regulatory mechanism during flower and fruit development was explored.【Methods】In this study, the hidden Markov Model profile (HMM) of the MYB binding domain (PF00249) was obtained from the Pfam database, which wasused as the seed model for the HMMER search of candidate PeMYB genes from the passion fruit genome in TBtools. The redundant sequences were removed manually. The SMART and the NCBI-CDD database were used to cross-check the presence of the MYB domain in candidate sequences. The physicochemical features of the potential MYB protein sequences, including molecular weight, theoretical isoelectric point (pI), and the number of amino acids were further analyzed using ExPASy. The MYB protein sequences of Arabidopsis were downloaded from the Plant Transcription Factor Database. Based on the alignment of the R2R3-MYB protein sequence of passion fruit and Arabidopsis thaliana, a neighbor- joining phylogenetic tree was constructed by MEGA. The conserved motifs, gene structure and chromosome location were analyzed with TBtools. We also predicted the putative miRNA target sites of PeMYB genes by the online psRNATarget Server. Eight PeMYB genes clustered with Arabidopsis R2R3-MYB gene members from the above subgroups of phylogenetic trees were selected for qRTPCR analysis under cold, heat, osmotic, and salt stress. In addition, the candidate PeMYB genes were analyzed by qRT-PCR analysis in the peel of passion fruit at different developmental stages after the pollination.【Results】A total of 99 R2R3- MYB genes were identified from passion fruit genome. The lengths of the protein sequences of PeMYB ranged from 113 (PeMYB78) to 1013 (PeMYB39) amino acids, and molecular weight (MWs) varied from 12.89 ku (PeMYB78) to 113.94 ku (PeMYB39). Moreover, the theoretical pI of PeMYBs was from 4.73 (PeMYB16) to 10.15 (PeMYB76). According to the topological structure of the phylogenetic tree and the classification of AtMYBs, the 99 R2R3-PeMYBs were divided into 36 groups (P1-P36). Most subgroups contained the R2R3-MYB members from both passion fruit and Arabidopsis, suggesting that they might be derived from a common ancestor. The structural analysis, including motifs and gene structure, illustrates that the R2R3-PeMYBs in the same subgroup have relative conserved structural features. The result showed that the number of exons varied from 1 to 13 in PeMYB genes. Most R2R3-PeMYB genes (53.3%) had similar distributions of three exons and two introns and a few R2R3-PeMYBs (PeMYB58, PeMYB60, PeMYB82) contained 12 or more exons. Motif 1, motif 2, and motif 3 were located in the N-terminal of almost all R2R3-PeMYB protein sequences, which contain conserved tryptophan residues and are related to R2 and R3 domains. The R2R3-PeMYB genes were randomly distributed on 9 passion fruit chromosomes. Most genes were located at the distal ends of the chromosomes. Chromosome 1 had the maximum number of R2R3-MYB genes (28 genes, 28.3%), followed by chromosome 6, which had 15 (15.2%) genes. The gene duplication event analysis revealed 37 pairs of segmentally duplicated genes and 7 pairs of tandemly duplicated genes were identified in R2R3-PeMYBs. The six tandem duplication events were on chromosomes 1, 2, 4, 5, and 6 and most segmental duplication events occurred on chromosome 1. Of the 99 genes, 54 (54.5% ) produced 37 segmental duplication gene pairs suggesting that segmental duplication events contributed to the expansion of the R2R3-MYB family of passion fruit. Based on transcriptome data and qRT-PCR analysis, it was found that R2R3-PeMYB genes showed different expression patterns in different floral tissues at different developmental stages. Most of the R2R3-MYB genes (eg: PeMYB79 and PeMYB80) were expressed in the early development stage of floral tissues and showed a decreasing trend in the late development stages. A few genes showed high expression patterns throughout the development of different floral tissues. At all stages of ovule development, some R2R3-PeMYB genes, like PeMYB34 and PeMYB39, were highly expressed. In addition, the high expression of PeMYB62 and PeMYB63 in the peel color transformation period of passion fruit indicated that they may be involved in the anthocyanin synthesis pathway of passion fruit. Moreover, PeMYB62 and PeMYB63 genes were the targeted genes of Ped-miR828 family, and miR828 has been reported to be involved in the anthocyaninsynthesis pathway. This further indicates that the two genes are likely to play an important role in the anthocyanin synthesis pathway. These results indicated that R2R3-PeMYB genes play an important role in the development of different floral tissues and fruit. At the same time, eight R2R3-PeMYB genes also showed different responses to abiotic stress (cold, heat, salt and osmotic stresses), these R2R3-PeMYB genes can be significantly regulated by at least two stress treatments. The result indicates that R2R3- PeMYB genes were also induced by various abiotic stress.【Conclusion】In this study, 99 R2R3-PeMYB gene family members were identified and found to play an important role in flower and fruit development and stress induction of passion fruit, which provided valuable clues for future research on the function and evolution of R2R3-MYB genes in flower and fruit development.