Genome-wide analysis of the pomegranate F3’H family and their roles in aril anthocyanin biosynthesis

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 of their potential roles in anthocyanin biosynthesis and coloration of pomegranate arils.【Methods】The genome of the Tunisian soft seed pomegranate (PRJNA355913) download from NCBI was used to identify 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, the transcriptional expression of the PgF3’Hs based on the transcriptomic data from the arils of three pomegranate cultivars (Tunisian soft seed with red color arils, Meilisuan with dark red color and Zimei with purple red colorarils) at different developental stages were analyzed to explore the possible roles of the PgF3’Hs in aril anthocyanin biosynthesis and coloration. The Agrobacterium mediated- heterogenous transformation in Arabidopsis was used to determine the PgF3’H3 function.【Results】Three PgF3’H genes in pomegranate were identified, which were renamed PgF3’H1, PgF3’H2 and PgF3’H3. Their sequences were 2055-3933 bp, their proteins contain 208-280 amino acids. The 2D and 3D protein structures were predicted, primarily with α helices and random coils. The three PgF3’Hs together with 44 F3’H genes from other plant species were used to construct the phylogenetic tree. The results showed that the PgF3’ H3/AtF3’H were orthologs and clustered in Group Ⅱ, while the PgF3’H1/PgF3’H2 were paralogues and clustered in Group Ⅲ. Moreover, the PgF3’H1/PgF3’H2 had a comparatively close distance to the FoF3’H (Flos osmanthi Fragrantis) and the OsF3’H2/OsF3’H3 (Oryza sativa). Bioinformatic analysis showed that the PgF3’Hs had conservative structures with 1-10 motifs with a similar order, and conserved function domain CYP75B. Furthermore, all of the 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 had 29 and 24 cis- elements, respectively, which were much more than those of the PgF3’H1. This possibly implied that they had more important and active roles than PgF3’H1. Remarkably, there were 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 promoter contained 5 G-boxes related to response to light and 6 ABA associated cis- elements. Moreover, all of the 3 PgF3’H genes had 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 were used to investigate the PgF3’H expression patterns. The results indicated that each PgF3’H showed similar expression trends in the three kinds of arils during fruit development. The 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 the pomegranate arils. The expression levels of the PgF3’H3 was dominantly increased before fruit expanding period, indicating that the PgF3’H3 might be responsible for aril coloration at early stages of fruit development. Noticeably, the PgF3’H2 with quite high expression levels in each kind of arils during development showed two increasing leaps at the turning and ripening stage. Therefore, the PgF3’H2 and PgF3’H3 might be much contributed to the anthocyanin biosynthesis and coloration of the pomegranate arils. Combined the results above, the PgF3’H2 and PgF3’H3 possibly were regulated by light and hormones, were speculated to play important roles in the anthocyanin biosynthesis and coloration in pomegranate. Finally, the PgF3’H3 was cloned and showed a relative high similarity with the VvF3’H (Vitis vinifera L.) and the MdF3’H (Malus domestica). The PgF3’H3 was then transformed into Arabidopsis todemonstrate its function of regulating anthocyanin biosynthesis and coloration. The young lines of the PgF3’H3 over-expressed Arabidopsis had red cotyledons with the average anthocyanin level of 0.308 3 mg·g-1 , however, the young WT lines showed a green color with no detection of anthocyanins. Also, the carotenoid and chlorophyll contents were significantly lower in the PgF3’H3 over- expression lines than those of the WT.【Conclusion】The three identified PgF3’H genes all had the conserved function domain CYP75B and conserved gene structures. The 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. The PgF3’H3 was suggested to be involved in aril coloration before fruit expansion, whereas, the PgF3’H2 was probably implicated in continuous coloration of arils. Furthermore, the function of the PgF3’H3 in regulating anthocyanin biosyn-thesis and coloration was primarily determined by heterogenous transformation. This study deepened the understanding of anthocyanin biosynthesis and coloration mechanism in pomegranate arils, and also supplied important basis for pomegranate color improvement.