CRISPR-Cas9 mediated deletions of FvMYB46 reduces fruit set and biosynthesis of flavonoids in Fragaria vesca

Abstract

Secondary metabolites produced by the phenylpropanoid pathway, which is regulated by transcription factors of the MYB family, play crucial roles in this early phase of fruit development. The MYB46 transcription factor is a key regulator of secondary cell wall structure and lignin and flavonoid biosynthesis in many plants, but little is known about its activity in flowers and berries in F. vesca. For functional analysis of FvMYB46, we designed a CRISPR-Cas9 construct with an endogenous F. vesca specific U6-promoter for efficient and specific expression of two gRNAs targeting the first exon of FvMYB46. This generated mutants with an in frame 81-bp deletion of the first conserved MYB-domain or an out of frame 82-bp deletion potentially knocking out the gene function. In both types of mutant plants, pollen germination and the frequency of flowers developing to mature berries was significantly reduced compared to wild type. Transcriptomic analysis of flowers demonstrated that FvMYB46 is positively regulating the expression of genes involved in pollen germination, homeostasis of reactive oxygen species (ROS) and the phenylpropanoid pathway, including secondary cell wall biosynthesis and flavonoid biosynthesis, while has a negative impact on carbohydrate metabolism. In FvMYB46-mutant flowers, the flavonols and flavan-3-olscontent, especially epicatechin, quercetin-glucoside and kaempferol-3-coumaroylhexoside were reduced, and we observed a local reduction of lignin content in anthers. Together these results suggest that MYB46 control fertility and efficient fruit set by regulating cell wall structure, flavonoid biosynthesis, carbohydrate metabolism and ROS- signaling in flowers and early fruit development in F. vesca.