Abstract: 【Objective】Anthocyanins are important substance basis of coloration and intrinsic nutritional quality in Pomegranate (Punica granatum L.). Studies about anthocyanin associated genes in pomegranate are very important. F3’H is an important kind of key enzymes in anthocyanin biosynthesis pathways. However, the role of PgF3’H in aril anthocyanin biosynthesis is little known. This study introduced the PgF3’H gene family information in the whole pomegranate genome, for example, length, location, gene structure, cis- elements in promoters, and analysis their potential roles in anthocyanin biosynthesis and coloration of pomegranate arils. 【Methods】The Tunisian soft seed pomegranate genome (PRJNA355913) download from NCBI was used to identified the PgF3’H gene family. Their chromosomal distributions, sequences and structures, and phylogenetic relationships as well as cis-elements in promoters were analyzed through bioinformatics. Furthermore, transcriptional expression of PgF3’Hsbased on the transcriptomic data from arils of three pomegranate cultivars (Tunisian soft seed with red color arils, Meilisuan with dark red colorand Zimei with purple red color arils) at different developental stages were analyzed to explore the possible roles of PgF3’Hs in aril anthocyanin biosynthesis and coloration. Agrobacterium mediated- heterogenous transformation inArabidopsis was used to determine the PgF3’H3 function. 【Results】ThreePgF3’H genes in pomegranate were identified,which were renamed PgF3’H1,PgF3’H2 andPgF3’H3. Their sequences are 2055-3933 bp, their proteins contain 208-280 amino acids, and the 2D and 3D protein structures were predicted， primarily with α helices and random coils. The three PgF3’Hstogether with44 F3’H genesfrom other plant species were used to construct the phylogenetic tree. The results showed that PgF3’H3/AtF3’Hare orthologs and clustered in Group Ⅱ, while PgF3’H1/PgF3’H2are paralogues and clustered in Group Ⅲ. Moreover, PgF3’H1/PgF3’H2 have a comparatively close distance to FoF3’H (Flos Osmanthi Fragrantis) and OsF3’H2/OsF3’H3 (Oryza sativa). Bioinformatic analysis showed that PgF3’Hshave conservative structures with 1-10 motifs with a similar order, and conserved function domain CYP75B. Furthermore, all of PgF3’H genes contained five kinds of cis- elements on their promoter regions, including growth and development, light, MYB, hormone and stress. The PgF3’H2 and PgF3’H3 promoters have 29 and 24 cis- elements, respectively, which were much more than those of PgF3’H1. This possibly implicated their more important and active roles than PgF3’H1. Remarkably, there are 4 TGAACG-boxes and 4 CGTCA-boxes related to growth and development, and 4 AUX associated cis- elements on the PgF3’H2 promoter, whereas the PgF3’H3 promotercontains 5 G-boxes related to response to light and 6 ABA associated cis- elements. Moreover, all of 3 PgF3’H genes have at least one MYB transcription factor binding sites. Subsequently, the transcriptomic data of different color arils from Tunisian soft seed and Meilisuan at the five developmental stages and Zimei at the three developmental stages was used to investigate PgF3’H expression patterns. The results indicated thateach PgF3’Hshowed similar expression trendsin the three kinds of arils during fruit development. PgF3’H1 displayed an extremely low transcriptional level all the time in each kind of arils, indicating that it might have little implication in anthocyanin biosynthesis and coloration of pomegranate arils. The expression levels of PgF3’H3 was dominantly increased beforefruit expanding period, indicating that PgF3’H3 might be responsible foraril coloration at early stages of fruit development. Noticeably, the PgF3’H2 with quite high expression levels in each kind of arils during developmentshowed two increasing leaps at the turning and ripening stage. Therefore, PgF3’H2 and PgF3’H3 might be much contributed to anthocyanin biosynthesis and coloration of pomegranate arils. Combined the results above, PgF3’H2 and PgF3’H3 possibly regulated by light and hormones, were speculated playing important roles in pomegranate anthocyanin biosynthesis and coloration. Finally, PgF3’H3were cloned andshowed a relative high similarity with VvF3’H (Vitis vinifera L.) and MdF3’H (Malus domestica). PgF3’H3 was then transformed into Arabidopsistodemonstrate its function of regulating anthocyanin biosynthesis and coloration. The younglines of PgF3’H3 over-expressed Arabidopsis had red cotyledons with the average anthocyanin level of 0.308 3 mg/g, however, young WT lines showed a green color with no detection of anthocyanins. Also, the carotenoid and chlorophyll contents were significantly lower in PgF3’H3 over-expression lines than those of WT. 【Conclusion】Three identified PgF3’H genes all had the conserved function domain CYP75B and conserved gene structures. PgF3’H2 and PgF3’H3 might play important roles in anthocyanin biosynthesis and coloration of pomegranate arils, and their expression might be regulated by light and hormone. PgF3’H3 was suggested involved in aril coloration before fruit expansion, whereas, PgF3’H2 was probably implicated in continuous coloration of arils. Furthermore, the function of PgF3’H3 regulating anthocyanin biosynthesis and coloration was primarily determined by heterogenous transformation. This study deepened the understanding of anthocyanin biosynthesis and coloration mechanismin pomegranate arils, and also supplied important basis for pomegranate colorimprovement.
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