Regulation Of Cotton Lint Fiber Initiation And Development By GhMML4-GhWDR Complex

Cotton is one of the most important economic crop in the world,and the resulting spinning fiber is an important source of raw materials for the textile industry.Cotton fiber is differentiated and developed from the outer epidermal cells of ovule,and the number of epidermal cell differentiation determines the yield and quality of the final fiber.Cotton fiber can be divided into fuzz and lint,epidermal cells coated on th surface of seed that initiate 3 days before flowering will development into lint,epidermal cells coated on th surface of seed that initiate 3-5 days after flowering will development into fuzz,the yield of lint is one of the important factors to determine cotton fiber lint percentage.Therefore,it is of great theoretical value and practical significance to elucidate the mechanism of the initiation and development of cotton fiber from the molecular level to improve the yield of cotton fiber.MYB transcription factor is a kind of large family of transcription factors in plants,which has a wide range of biological functions and is involved in almost every aspect of plant growth,development and metabolism.The 9th and 15th subgroup of R2R3 MYB,respectively,are MIXTA-Like and GL1-Like genes,which are of great significance for the differentiation and branching of epidermal cells,but the direct involvement of GL1-Like gene in cotton fiber development has been reported less.There are 10 MIXTA genes in cotton,collectively referred to as GhMMLs.Three of the them have been reported to be directly involved in the initiation and differentiation of cotton fibers,one is involved in the development of the petal trichome.The expression of such genes is mostly restricted during initiation stage,compared to the wild type,expression is significantly reduced in the fuzzless and lintless mutant,it is suggested that this kind of gene plays an important role in the differentiation and development of cotton fiber.We found one important gene GhMML4＿D12 that controlling lint fiber initiation and development by map-based cloning before,in order to explain how GhMML4＿D12 regulates the development of cotton lint fiber,in this study,we used GhMML4＿D12 as bait protein to screen yeast fiber specific library,a WD40 family transcription factor member which named GhWDR was found,and then the protein interaction and function of the gene were further analyzed.Studies are as follows:Screening for proteins that interact with GhMML4＿D12 We used GhMML4＿D12 that controlling lint fiber as bait protein to screen yeast fiber specific library,the candidate interaction proteins were analyzed,and the WD40 transcription factor was obtained with high frequency,we named GhWDR.This gene does not belong to the same clade as the cotton GhTTG1-4 and AtTTG1 which function in Arabidopsis trichome development,and it is considered that the function of the gene has been differentiated,which is of unique significance to the development of cotton fibers.Since there has no[DE]Lx2[RK]x3Lx6Lx3R core amino acid sequence interact with Basic-helix-loop-helix(bHLH)transcription factor on GhMML4＿D12,so we never found any bHLH transcriptional factors in yeast screen.Therefore,it is proposed that GhMML4-GhWDR is one unique protein complex that regulates the initiation and development of cotton lint fiber,which is different from the GL1-GL3-TTG1 triplet protein complex which regulates the development of Arabidopsis leaf trichome.Interaction between GhMML4＿D12 and GhWDR We used Yeast Two Hybrid(Y2H),Bimolecular florescence complementation(BiFC)and In Vitro pull down to investigate the interaction between GhMML4＿D12 and GhWDR.We found that GhMML4＿D12 which comes from recessive naked seed-2(referred to hereafter as n2 NSM)could interact with GhWDR,and the mutated form of GhMML4＿D12(GhMML4＿D12m)which derived from Gossypium hirsutum cv.Xuzhou 142(referred to hereafter as XZ142 FLM)could not interact with GhWDR,and it is considered that interaction between GhMML4＿D12 and GhWDR resulted the initiation and development of lint fiber in n2 NSM.We firstly have found that MYB protein can interact directly with WD40 protein,it is different with Arabidopsis,which MYB protein interacts directly with bHLH protein and then forms a complex through the interaction of bHLH and WD40 protein,thus,differentiation and development of cotton fibers have evolved a newly and unique protein complex.Further investigation showed that the 250th to 267th amino acids of GhMML4＿D12 determined its interaction with GhWDR;and the WD40 repeat region on GhWDR determined the interaction with GhMML4＿D12.Functional investigation of GhWDR in cotton In order to investigate whether GhWDR have effect on fiber development,8 over-expression lines were obtained by transforming in cotton W0 using CaMV35S to drive Gh WDR then fused with GFP reporter gene(35S::GhWDR::GFP).4 of them were used for further analysis,0 DPA ovule were observed by scanning electron microscopy(SEM),we found that over-expression Gh WDR could significantly enhance the number of epidermal cells on the ovule surface that could development into fiber;over express GhWDR could also significantly improve the lint percentage of cotton;but have little effect on trichome development of the other parts in cotton.Therefore,it is considered that GhWDR is of great significance for the initiation and development of cotton fibers.Functional investigation of Gh WDR in Arabidopsis We used cotton native promoter to drive GhWDR gene then fused with GFP reporter gene to transform into Arabidopsis glabrous mutant ttg1,and found that GhWDR could rescue the leaf trichome,root hair position and anthocyanin synthesis,it is implyed that cotton GhWDR and Arabidopsis AtTTG1 share functional redundancy.GhWDR was further divided into three parts,N-Terminal domain(GhWDR-N),WD40 conservative domain(GhWDR-R)and C-Terminal domain(GhWDR-C),we used CaMV35S promoter to drive GhWDR-N,GhWDR-R and GhWDR-C to transform into ttg1,we found that WD40 conservative domain and C-Terminal domain could rescue ttg1 to wild type,and it is considered that WD40 conservative domain and C-Terminal domain are of great significance for the function of Gh WDR.Transcriptional regulation between GhMML4＿D12 and GhWDR GhMML4＿D12 and GhWDR both are transcription factors,which have transcriptional activation effect.The transcription regulatory relationship between them was investigated by Yeast One Hybrid(Y1H),transcient expression assay and Dual-Luciferase(Dual-Luc)assay,and it was found that GhMML4＿D12 could enhance the transcriptional activation of itself in n2 NSM,but GhWDR could not activate its own transcription or GhMML4＿D12.Both GhMML4＿D12m and GhWDR could not activate transcription of themselves or each other in XZ142 FLM.Therefore,it is considered that GhMML4＿D12 activate the transcriptional activation of itself have great significance for the function in the initiation and development of lint fiber in n2 NSM.