Cotton (Gossypium Hirsutum) MYB7 Regulates The Secondary Cell Wall Biosynthesis

Cotton is one of the most important commercial crops in the world.Cotton fiber is derived from seed coat epidermis and consists of four distinct and overlapping developmental stages:initiation,primary wall formation,secondary wall thickening and maturation.All these features make cotton fiber a model system to study cell wall biogenesis especially cellulose biosynthesis.Mature cotton fiber contains over 90%cellulose with little xylan and lignin.However,by far little is known about the regulation of the biosynthesis of secondary wall of cotton fiber.In this work,we identified a R2R3-MYB transcription factor,previously designated as GhMYB7,functional characterization of GhMYB7 revealed that it was involved in regulation of secondary wall biosynthesis,the results are as follows:1.GhMYB7 is predominantly expressed in developing fibersQuantitative RT-PCR analysis demonstrated that GhMYB7 was predominantly transcribed in developing fibers,its mRNA level was greatest in 0 dpa fiber,the second most abundant in 20 dpa fiber suggesting it may function during the secondary wall thickening stage.2.GhMYB7 is a typical transcription factorAfter transformation of enhanced green fluorescent protein(eGFP)-tagged GhMYB7 gene fusion expression vector to Arabidopsis thaliana,the eGFP fluorescence of 5-day-old roots from transgenic seedlings was observed using a laser confocal microscope,it was found that GhMYB7 protein was localized in the nucleus.In yeast two hybrid systerm self-transcription activation experiment,GhMYB7 can activite expression of His,Ade,LacZ,showing GhMYB7 was a transcriptional activator in yeast.The above results showed GhMYB7 was a typical transcription factor3.Expression of GhMYB7 in Arabidopsis causes ectopic deposition of secondary walls in various tissuesHeterologous expression GhMYB7 in Arabidopsis thaliana led to the up-curly and dark green leaves,shorter and thinner inflorescence stems.Cross sections of basal stems revealed cell wall thickness of vessels and interfascicular fibers was increased in overexpression lines compared with the wild type.Different dyes which stain cellulose and lignin were used to image the cellulose and lignin distribution in plant cell walls.The staining results showed that ectopic deposition of cellulose and lignin was detected in the stem epidermas as well as root cortical cells.In addition,the amount of crystalline cellulose in the cell wall increased in the transgenic lines compared with the wild type.4.Expression of GhMYB7 in Arabidopsis activates a number of secondary wall biosynthetic genes and secondary wall-related transcription factor genesRT-PCR analysis of expression of secondary wall biosynthetic genes using RNAs from seedlings of 4-week-old wild type and GhMYB7 overexpressors demonstrated a substantial increase of transcript level of genes involved in cellulose(CesA4,CesA7 and CesA8),and lignin biosynthesis(4CL1,CCoAOMT1 and PAL1)in transgenic lines,compared with the wild type indicating that the ectopic secondary wall thickening started before cessation of cell elongation,which hindered plant growth and led to dwarfs.In the stems of eight,week old plants,we also observed that overexpression of GhMYB7 activated genes involved in secondary wall cellulose synthesis and lignin biosynthesis.Though we did not stain xylan,we also found that expression of xylan synthesis genes are also upregulated significantly.The results suggest that GhMYB7 was a transcriptional activator capable of regulating the whole secondary wall biosynthesis.Moreover,expression of secondary wall related TFs including AtNSTl,AtNST2,AtSND1,AtMYB20,AtMYB46 and AtMYB58 were all greatly induced.5.GhMYB7 binds to the promoters of AtSND1 and AtCesA7 genesA yeast one-hybrid assay was performed to test whether GhMYB7 could bind to these promoters.It was found that GhMYB7 showed binding to AtCesA4 and AtSND1 promoter.The results showed that GhMYB7 activated expression of downstream target genes such as AtSND1 and AtCesA4.6.Expression of GhMYB7 in Arabidopsis enhances the tolerance to two cellulose-specific dyesFlourescent Brightenter and Pontamin fast scarlet 4b can bind to cellulose.To see if the two dyes can affect the cellulose deposition,a certain concentration of dye was added to the medium,the root elongation of wild type and transgenic lines overexpressing GhMYB7 was measured.It was found that both dyes inhibited the root elongation of wild type more severely than that of the transgenic lines.7.Expression GhMYB7 in E.Coli and preparation of GhMYB7 antibodyGhMYB7-HisTaq fusion protein expression vector was expressed in E.Coli.After IPTG induction and SDS-PAGE,the expressed GhMYB7 protein was detected,the Ni-column purified GhMYB7 protein was used to inject the rabbit and antibody was obtained.8.Generate GhMYB7 overexpression and RNAi-silenced transgenic cotton plantspBI121-GhMYB7 and pBI121-GhMYB7RNAi vectors were transferred into cotton by Agrobacterium-mcdiated genetic transformation.20 lines of GhMYB7 overexpression transgenic cotton were obtained,31 lines of GhMYB7RNAi silenced transgenic plants were acquired,further functional analyses needs to be done.