Functional Study Of PeSCL23 And PeMYB4L In The Formation Of Phalaenopsis Flower Type And Flower Color

Phalaenopsis aphrodite Rchb.f.is a perennial epiphytic herb in the orchid family.With the improvement of people's living standards,people's demand for flowers continues to increase,and Phalaenopsis is more and more popular.On the one hand,Phalaenopsis is beautiful in color,which is of great significance in attracting insects and pollination,and has a high value in horticultural ornamental.On the other hand,lip,the unique flower organ of Phalaenopsis,can provide a landing site for insects,and is benefit for its reproduction and evolution.However,the molecular mechanism of flower color formation and lip development of Phalaenopsis is still unclear.In this study,we investigated the effects of MYB and GRAS family on anthocyanin synthesis and lip development of Phalaenopsis respectively,by methods of ATAC-seq,RNA-seq,and gene silencing technology,with the orchid model flower Phalaenopsis equestris and a variety of commercial Phalaenopsis as test materials.The finding might help improve flower color and flower shape through genetic engineering.The main results are as followed:The lip is a unique flower organ in orchids,which is used to attract pollinators,and has high value in horticultural ornamental.In order to explore the molecular regulation mechanism of orchid lip development,high-throughput sequencing technology for chromatin accessibility by transposase(ATAC-seq)was firstly used to analyze differentially peak-associated genes in triple-lipped TPL and single-lipped SL.A key transcript PeSCL23 was uncovered to be involved in the regulation of lip development.Through the analysis of the organ expression profile of different tissues of Phalaenopsis equestris,it was found that the expression level of PeSCL23 in the flower organ was significantly higher than that in the vegetative organ.In addition,PeSCL23 transcript was significantly induced in developmental flower organ.Virusinduced gene silencing(VIGS)showed that the false lip of the mutant became a true lip after silencing PeSCL23,indicating that PeSCL23 negatively regulates the lip development.Quantitative co-expression analysis of PeSCL23 mutants after silencing showed that the expressions of key genes PeMADS3 and PeMADS9 in lip development were significantly increased in the PeSCL23-VIGS line compared with the wild type,further indicating that PeSCL23 may regulate key genes of lip development in P-codes model.Yeast two-hybrid experiment verified that PeSCL23 did not interact with PeMADS3 and PeMADS9.We further performed transcriptome analysis on different flower organs SL(single-lip lip)and SP(single-lip petal)of Phalaenopsis equestris.Among Differentially expressed genes,eight MADS-box transcription factors were discovered that might be involved in the development process of Phalaenopsis lips.By yeast two-hybrid and bimolecular fluorescence complementation techniques,it was found that PeSCL23 can interact with the MYB family transcription factor PeKAN2;Additionally,PeSCL23 and PeKAN2 could inhibited the expression of each other by western blot assay.VIGS also verified that the false lip petal of PeKAN2-silenced plants changed into real petal,indicating that PeKAN2 positively regulates lip development.Based on all the above results,this study established a new molecular mechanism of PeSCL23-PeKAN2-PeMADS3 in regulating orchid lip development,and revealed the upstream regulatory network of MADS-box genes.Phalaenopsis spp.,one genus of Orchidaceae,have become very popular worldwide for their fascinating flowers with various colors and pigmentation patterns.Several R2R3-MYB transcription factors have been reported to function in anthocyanin accumulation in Phalaenopsis spp.However,its molecular mechanism underlying the detailed regulatory pathway remains poorly understood.In this study,we identified a novel subgroup 2 R2R3-MYB transcription factor PeMYB4 L,the expression profile of which was concomitant with red color formation in Phalaenopsis spp.flowers.Virusinduced gene silencing(VIGS)and transient overexpression assay verified that PeMYB4 L promotes anthocyanin accumulation in flower tissues.In addition,PeMYB4 L could directly regulates the expression of Phalaenopsis spp.chalcone synthase gene(PeCHS)through MYBST1(GGATA)binding site.It's interesting that the basic-helix-loop-helix(b HLH)protein PeMYC4 shows opposite expression pattern from PeMYB4 L in anthocyanin accumulation.Furthermore,PeMYC4 was verified to form MYB-b HLH complex with PeMYB4 L,and attenuated the expression of PeCHS and weakened anthocyanin production,indicating a novel regulatory model of MYBb HLH complex.Our findings uncover the detailed regulatory pathway of MYB-b HLH,and might provide a new insight into the complicated anthocyanin pigmentation in Phalaenopsis spp.In summary,this study focused on the flower type and flower color of Phalaenopsis equestris,with Phalaenopsis equestris and commercial Phalaenopsis as materials.On the one hand,we established a new molecular mechanism of PeSCL23-KAN2-MADS3 in regulating orchid lip development.On the other hand,we revealed that PeMYC4 inhibits the binding of PeMYB4 L to its downstream target gene PeCHS,which further explains the formation mechanism of flower color and indicates that the formation of flower color is the balance of gene expression regulation.This study provides new insights on flower type and flower color,and provides an important theoretical basis for improving the quality of Phalaenopsis.