Research On The Molecular Mechanism Of Co-regulation Of Anthocyanin Biosynthesis By Light-responsive Transcription Factor RhHY5 And R2R3-MYBs In Rose

Roses(Rosa hybrida)are one of the most important ornamental and economic plants in the world,exhibiting a variety of flower colors and fragrances.During the greenhouse cultivation,insufficient light conditions often result in poorly colored petals of the red roses,thus leading to the decline of ornamental value.Understanding the molecular mechanism of how light affects the rose petal color is significant for the cultivation and variety improvement of roses.Previous researches have found that the red color of the rose petals is primarily due to the accumulation of anthocyanin.However,to date,there is no comprehensive report on the mechanism of light-mediated anthocyanin biosynthesis in roses.In this study,R.hybrida ‘Burgundy Iceberg’ is used as plant material,and we report a regulatory module of anthocyanin biosynthesis comprising RhHY5,RhMYB114 a,and RhMYB3 b.The main results are as follows:(1)Transcriptome analysis to screen transcription factors related to anthocyanin biosynthesis: By performing light/dark treatments on R.hybrida ‘Burgundy Iceberg’and by analyzing the total anthocyanin content and transcriptomes at three different developmental stages: flower bud(S1);flower in early opening stage(S2)and fully open flower(S3),we found that dark treatment significantly inhibited the anthocyanin content and the expression of anthocyanin structural genes at stages 2 and especially stage 3.Two anthocyanin biosynthesis-related R2R3-MYB genes RhMYB114 a and RhMYB3 b,as well as a light-responsive gene RhHY5 are selected as candidate genes.(2)Functional verification of the candidate genes: By stable transformation in the callus of ‘Old Blush’,we found that the overexpressing of RhMYB114 a significantly induces the total anthocyanin content and the expression level of anthocyanin structural genes in the callus,whereas the overexpressing of RhMYB3 b significantly represses the total anthocyanin content and the expression level of most anthocyanin structural genes in the callus,suggesting that RhMYB114 a and RhMYB3 b act as an activator and a repressor,respectively,of anthocyanin biosynthesis in the rose.Besides,transient overexpression of RhHY5 activates the accumulation of anthocyanin and the expression of anthocyanin structural genes in petals and pedicel of R.hybrida ‘Burgundy Iceberg’flower bud,whereas the transient silencing of RhHY5 inhibited the anthocyanin biosynthesis and the expression of anthocyanin structural genes in R.hybrida‘Burgundy Iceberg’ petals,indicating that RhHY5 is the anthocyanin activator,as well.(3)Analysis of the regulatory network of rose anthocyanin biosynthesis under light/dark conditions: By performing the Y1 H,Y2H,Y3 H,BIFC,dual-LUC,firefly luciferase complementation assay,and transient transformation in apple,we found that both RhMYB114 a and RhMYB3 b can form the MBW complexes with Rhb HLH3 and Rh WD40 and can directly bind to the promoters of Rh CHS,Rh F3’H,Rh ANS,and Rh GT1,activating and repressing the promoter activity of these structural genes,respectively.RhMYB3 b also inhibits the formation of MYB114a-b HLH3-WD40 complex and anthocyanin biosynthesis by directly repressing the expression of RhMYB114 a and by competing with RhMYB114 a for binding to Rhb HLH3.Besides,RhHY5 activates RhMYB114 a expression and represses that of RhMYB3 b by directly binding their promoters,thus positively regulating anthocyanin biosynthesis in the rose.RhHY5 directly interacts with the MYB114a-b HLH3-WD40 complex and enhances the RhMYB114a-caused transcriptional activation of the anthocyanin structural genes’ promoter.Overall,this study explores two R2R3-MYB transcription factors and a HY5 transcription factor that regulate anthocyanin biosynthesis in rose petals,and reports a complex light-mediated regulatory network of anthocyanin biosynthesis,providing a theoretical basis for greenhouse cultivation and molecular breeding of rose flower color.