Molecular Mechanism Of Secondary Cell Wall Formation And Flavonoid Accumulation In Woodland Strawberry Fruit Regulated By FvVND4c

Strawberry is one of the most popular fruit crops thanks to the unique flavor and aroma of its berries.Strawberry fruits easily soften and rot after ripening,resulting in a short shelf life and high storage and transportation costs,which greatly limits the development of strawberry industry.The softening of strawberry fruit is mainly due to the change of the structure and composition of the cell wall.Therefore,the research on the cell wall of strawberry fruit can provide new genetic resources and theoretical basis for the improvement of the quality of strawberry fruit.The diploid strawberry(Fragaria vesca)is a model plant for strawberry research because of its small genome,short growth cycle and facile of genetic transformation.Our previous studies have found that the NAC family protein Fv NST1 b regulates the formation of secondary cell wall in strawberry fruits,providing molecular basis into cell wall modification in strawberry.However,the function of its close homologous genes FvVND4s(FvVND4 a,FvVND4 b,FvVND4c)remains uncertain.In this study,we examined the function of FvVND4 s by the in vitro cultural system that induces the formation of secondary cell wall from strawberry mesophyll cells.Transient overexpression and transcriptomic analysis were used for the phenotype observation and function identification of FvVND4 s in the fruit of strawberry.Combined with SELEX and luciferase reporting experiment,we illustrated that FvVND4 c regulates the secondary cell wall formation and flavonoids accumulation in strawberry fruit by activating the downstream gene Fv MYB46.The main results are as follows:(1)Establishment of an in vitro cultural system to induce formation of secondary cell wall from mesophylls in Fragaria vesca.Mesophylls began to differentiate into xylem cells on the 7th day of induction culture.During the secondary cell wall formation,expressions of FvVND4 a and FvVND4 c significantly increased,indicating that they may be involved in the formation of secondary cell wall.(2)Transient overexpression of FvVND4 a,FvVND4 b and FvVND4 c in strawberry fruits showed that FvVND4 b and FvVND4 c induced thickening of secondary cell wall and accumulation of flavonoids.Transcriptional analysis showed that FvVND4 c is involved in the biosynthetic regulation of lignin and flavonoid.(3)qRT-PCR analysis showed that overexpression of FvVND4 c induced the expression of Fv MYB46 and Fv MYB83.Combined with SELEX and luciferase reporting results,Fv MYB46 and Fv MYB83 are the downstream genes of FvVND4 c.(4)Transient overexpression of Fv MYB46 and Fv MYB83 in strawberry fruits showed that only Fv MYB46 could induce secondary cell wall formation and flavonoid accumulation,as well as the expression of related functional genes.(5)Transcriptomic comparison for strawberry fruits overexpressed with FvVND4 c and Fv MYB46 showed that both FvVND4 c and Fv MYB46 were involved in the regulation of lignin and flavonoid biosynthesis.In summary,our results indicate that FvVND4 c regulates secondary cell wall formation and flavonoids accumulation by activating the expression of downstream gene Fv MYB46,which provides valuable gene resources and theoretical basis for the improvement of the quality of strawberry fruit.