Molecular Characterization And Functional Analysis Of The Yucca Flavin Monooxygenase Genes From Strawberry (Fragaria L.)

Auxin is involved either directly or indirectly in every aspect of plant development, virtually playing the integrative role in coordinating plant growth and development. Notably, auxin plays a key role in fruit initiation, seed development, and fruit ripening. Auxin has been regarded as the main signal molecule coordinating the growth and initiation of ripening in strawberry fruit. While the molecular mechanism regulating auxin biosynthesis in strawberry remains unknown.Using a homologous cloning strategy, combining with RACE technique, we isolated two FaYUC genes from cultivated strawberry (Fragaria × ananassa Duch.). Significant expression of FaYUC1 and FaYUC2 was found in vegetative meristems and reproductive organs, with overlapping but distinct patterns. During fruit development, both transcripts of FaYUC1 and FaYUC2 in achenes reach a peak around large green fruit stage. But the sudden rise in FaYUC2 transcripts level is much steeper and begins earlier than that in FaYUC1. FaYUC2 is also obviously expressed in the receptacles from green fruits, hinting another auxin source for receptacle development, other than achenes.Two over-expression vectors were generated:pCAMBIA35S:FvYUC1 and pCAMBIA35S:FvYUC2 and introduced into Arabidopsis. FvYUC1 and FvYUC2 over-expression Arabidopsis plants exhibit typical auxin over-accumulation phenotype in many aspects, such as the narrow and downward curled leaves, strong apical dominance, short and hairy root. FvYUC1 and FvYUC2 over-expression in Arabidopsis reduced plant fecundity, due to this ectopic expression disturbed floral meristems initiation and floral organs development. Arabidopsis mutants yuc2 and yuc4 ectopically expressing FvYUC2 also exhibit phenotypes typical of auxin over-accumulation, and their fecundity significantly reduces. The plant height of yuc164 mutant was completely restored after FvYUC2 ectopic expression, but the mutants were still sterile even with partially restored floral organs. Like their homologs in other plants, FvYUC1 and FvYUC2 over-expression in Arabidopsis lead to typical auxin over-accumulation phenotype, which indicates that they play roles in auxin biosynthesis vie YUCCA pathway.Transgenic strawberry plants over-expressing FvYUC1 and FvYUC2 were generated. They exhibit abnormal leaves morphology, such as longer petiole, rolled downwardly leaf margin. Quantitative real-time PCR analysis show elevated transcripts levels of FvYUC1 and FvYUC2. Significantly increased transcripts levels can be associated with their leaves phenotype. We proposeed that over-expressing FvYUC1 and FvYUC2 resulted in auxin over production, which lead to change in leaf form. dsRNAi transgenic strawberry lines were generated. dsRNAi suppression of FvYUCl resulted in significant changes:Slight round-shaped leaf, closer and partly overlaping trifoliate leaves, reduced number of vein and serration, and shield-shaped serrations were observed; dwarfish and the crown diameter smaller; rarely produce runner, while wild type strawberry can produce runner. These gain- and loss-of-function assays in strawberry functionally demonstrated the roles of FvYUC1 and FvYUC2 in strawberry leaf and runner development.After woodland strawberry genome sequence information was released, we predicted eight potential FvYUC genes in woodland strawberry genome. Eight true FvYUC genes, encoding FMO proteins with the conserved binding motifs for FAD and NADPH, were isolated from woodland strawberry vie cloning experiment validation.A tissue- and stage-specific expression pattern for each member of eight FvYUC genes was revealed, distinct but partially overlapping. All members of FvYUC family are involved in fruit development, though preferentially or specifically expressed at different stages. Of the FvYUC family, FvYUC6 and FvYUC8 are highly valuated for their interesting expression profiles, which are specifically confined to fruits. Notably, expression of FvYUC8 is limited to mature flowers, while FvYUC6 transcripts level is dramatically high in green fruits.Our research identified a YUCCA genes family consisted of eight members. FvYUC1 and FvYUC2 over-expression in Arabidopsis lead to typical auxin over-accumulation phenotype. In transgenic strawberry plants with gain- and loss-of-function, the auxin balance between tissues alter, which affected leaf and runner development. In conclusion, auxin synthesized by YUCCA pathway is essential for strawberry growth and development.