Study On The Roles Of Three R2R3-MYB Transcription Factors During Cotton(Gossypium Hirsutum)Fiber Secondary Cell Wall Thickening Stage

Cotton is an important economic crop,and cotton fiber is the most important natural textile material in the textile industry.The cotton fiber development includes four consecutive stages,in which fiber cell initiation determines the yield,primary cell wall(PCW)synthesis determines fiber length,and secondary cell wall(SCW)thickening affects fiber strength.Cotton fiber deposits a large amount of cellulose in the SCW thickening period,which makes mature cotton fiber with more than 90%cellulose,and makes fiber become a unique SCW model.How this unique SCW composition forms and what’s its regulation mechanism remain an open question.Previous studies have indicated that R2R3-MYB transcription factors(TFs)are involved in the regulation of SCW biosynthesis.When I was a master student,I identified two cotton R2R3-MYB TFs(GhMYB7 and GhMYB6)from the NCBI database.With the completion of the tetraploid cotton genome sequences,we further screened 36 R2R3-MYB TFs at the genome level.all genes were predominantly expressed or highly expressed in 20 DPA cotton fibers.A functional analysis of GhMYB46_D13 was performed.In this paper,our data shows that GhMYB7 can directly activate the SCW cellulose synthase gene promoters and function as a master regulator of SCW biosynthesis.GhMYB6 acts as a repressor to inhibite the biosynthesis ofproanthocyanidins(PAs)to ensure the white fibers.In addition,GhMYB 46 D13 also can activate the promoters of the SCW component biosynthetic genes,and the GhMYB46_D13 promoter can be activated by GhMYB7.The main results of this paper are as follows:1.GhMYB7 regulates cotton fiber SCW biosynthesisOverexpression and RNA interference of GhMYB7 transgenic cottons were obtained by Agrobacterium-mediated transformation.It was found that GhMYB7 overexpression inhibited the fiber length,through precocious deposition of cellulose by activating the expression of the SCW cellulose synthase genes in advance.Compared to the wild type,the overexpressing lines caused an increase in cell wall thickness.On the other hand,GhMYB7-RNAi transgenic cotton showed thinner fibers compared to wild type.Transactivation analysis indicated that GhMYB7 can activate the promoters of the SCW cellulose synthase genes.The above results were verified by chromatin immunoprecipitation(ChIP)assay.The promoter deletion assay and EMSA revealed that GhMYB7 binds to GhCesA4-2 promoter via the cis-acting element GTTTGGTA.In addition,GhMYB7 overexpression also promotes the biosynthesis of the hemicellulose and lignin.Based on the above results,GhMYB7 is a main regulator that activates the biosynthesis of the three major components of fiber SCW.2.GhMYB6 negatively regulates anthocyanin and PA synthesisGhMYB6 encodes a repressor-type R2R3-transcription factor with a typical EAR repression motif and a TLLLFR repression motif.It was highly expressed during cotton fiber secondary wall thickening stage,.Heterologous expression of GhMYB6 in Arabidopsis can inhibit both the biosynthesis of proanthocyanidins in the seed coats and the deposition of anthocyanins in the seedlings.The accumulation of anthocyanins in the petals of GhMYB6 overexpression transgenic cotton was significantly lower than that in the wild type.GhMYB6 can repress activity of a major PA activator GhTT2-3A and suppress the PA synthesis specific gene GhLAR activity,which prevents PA accumulation in cotton fibers.Overall,GhMYB6 acts as a transcriptional repressor,by inhibiting the accumulation proanthocyanidins to ensures the white fibers.3.Genome-wide identification of R2R3-MYB transcription factors regulating secondary cell wall thickening in cotton fiber developmentWe identified 419 R2R3-MYB genes by systematically examining the cotton genome.A combination of phylogenetic,RNA-seq and co-expression analyses identified 36 R2R3-MYBs either preferentially or highly expressed in 20 DPA fibers as putative SCW regulators.We highlighted on the roles of two MYBs named GhMYB46_D13 and GhMYB46_D9.Heterologous expression of GhMYB46_D13 and GhMYB46_D9 individually in Arabidopsis resulted in ectopic SCW deposition in transgenic plants.Furthermore,both GhMYB46_D13 and GhMYB46_D9 were able to activate the cotton fiber SCW cellulose synthase gene promoters.We further analyzed the function of GhMYB46_D13 during cotton fiber development.GUS histochemistry of GhMYB46_D13P::GUS transgenic cotton showed that the promoter of GhMYB46_D13 was expressed almost exclusively in the cotton fiber,especially in the SCW thickening stage.The fiber length and cell wall thickness of mature cotton fiber in GhMYB46_D13-RNAi transgenic cotton decreased.On the other hand,GhMYB46_D13 overexpressing transgenic cotton plants driven by CaMV 35S are dwarf andsterile,.The mature fiber length of GhMYB46_D13 overexpression transgenic cotton driven by its own promoter also decreased.Transactivation analysis showed that GhMYB46_D13 can activate the SCW cellulose synthase gene promoter,the ChIP assay validated this result in cotton fibers.Meanwhile,GhMYB7 can activate the activity of GhMYB46_D13 promoter,indicating that there is a transcriptional cascade regulatory relationship between GhMYB7 and GhMYB46_D13...