The Study Of Transcriptional Regulation Mechanism Of Cotton Fibre Early Development

Cotton is an important economic crop and the regulatory mechanism underlyingcotton fibre development is always the focus of cotton research. Previous studies haveshown that MYB transcription factors play important roles in fibre development.R2R3-MYB transcription factor GhMYB25is a key positive regulator of fibreinitiation and elongation. However, whether it defines a new genetic pathway of fibredevelopment remains unknown. In contrast to the positive role of R2R3-MYBtranscription factors, R3-MYB transcription factors negatively regulate trichomedifferentiation. However, cotton R3-MYB transcription factors remain to be clonedand functionally analysed. BR is an important hormone that regulates fibredifferentiation and elongation, but the molecular mechanism of this regulation needsto be analysed.To understand the transcriptional regulation mechanism during cotton fibre earlydevelopment, we chose the high-quality fibre bearing cultivar, Gossypium barbadense,as the main resource. By combination of the effects of gene cloning, evolution study,expression analysis, protein-DNA binding, protein-protein interaction and functionalstudy by transgenic plants, we achieved the results as follows.1. An L1box binding protein, GbML1interacts with GbMYB25to regulateearly fibre development. A new HD-Zip IV family member, designated as GbML1, was cloned from earlystage ovules by RACE technique. GbML1could form homodimers by its ZLZdomain and this dimer formation is critical for its binding to DNA. GbML1couldspecifically bind to L1box by its HD domain. Moreover, GbML1bound to its ownpromoter and a fibre-specific promoter. The expression of GbML1and GbMYB25wassimilar during cotton ovule development and both proteins could be localized intonucleus. Furthermore, GbML1could interact with the C terminal part of GbMYB25by its START-SAD domain and the three dimentional structure of START domainwas important for the binding. Though GbML1itself was a weak activator, the C2domain of GbMYB25had strong activation activity. Thus the complex formed byGbML1and GbMYB25both had the DNA specific binding activity andtranscriptional activation activity. This complex could bind to the promoters of fibrespecific genes and then activated their expression. Overexpression of GbML1inArabidopsis increased the trichome number in the leaf and stem epidermis, whichsupported that GbML1was a key regulator in cotton fibre differentiation anddevelopment.2. SANT/MYB transcription factors are involved in regulating fibre developmentWe also cloned two SANT/MYB transcription factors from the early ovulelibrary by RACE techinique.The sequence of GbRL1was similar to RAD, and they fell into the same group inthe phylogenetic tree analysis. Furthermore, GbRL1had strong expression in petalsand ectopic overexpression of GbRL1in Arabidopsis had similar developmentalalterations as overexpression of RAD. These results supported that GbRL1was thehomolog of RAD in petal development. Unlike the restricted expression of RAD,GbRL1had strong expression in the ovules at initiation stage. The expression ofGbRL1could also be detected in the fibres during fibre elongation. Thus, GbRL1might also play a role in ovule and fibre development. The cloning and analysis of theGbRL1promoter revealed that GbRL1might be controlled by the MADS box geneswhich controlled the flower organ identity and flowering-time. GbRL1had an unusual big intron which contained the binding site for WUS, thus WUS might also controlGbRL1expression.On the contrary, the expression of GbRL2was undetectable on-3DPA andvery weak on0DPA, but increased during fibre elongation. The expression of GbRL2in ovules was stronger than fibres on+8DPA. GhRL2had different expressionpattern in fibreless mutant fl compared with its wild type Xu-142. Before anthesis (-3DPA), GhRL2was expressed strongly in the ovules of the fl mutant while in signal inthe WT was still not detectable. While the GhRL2signal was weak in the fl mutantovules at+5DPA, the signal in WT ovules was still stong. This expression pattern aswell as the fact that GbRL2belonged to the R3-MYB like family suggested thatGbRL2might be a negative regulator of cotton fibre differentiation. Besides theregulatory element for MADS box transcription factors, there was an L1box in theGbRL2promoter, suggesting that GbRL2might be cotrolled by L1bindingtranscription factors, such as GbML1.3. SRN1is involved in multiple organs’ developmentSRN1was considered as a reporter gene for BR treatment. Our results showedthat SRN1encoded a nucleus-localized transcription activator and the transcriptionactivation activity was contributed by its C terminal. SRN1was preferentiallyexpressed in the vascular tissues of different organs, and the expression of SRN1wasalso detected in the epidermis such as guard cells and the seed coat. The repression ofSRN1expression let to the abnormal lateral shoot and reduced male fertiliy caused byboth few spores and thick secondary wall. Overexpression of SRN1resulted in smalland light seeds with low geminating rate. Overexpression of SRN1also increased thepigmentation accumulation in cotyledons and leaves, increased the width ofhypocotyls, repressed the hypocotyl elongation and leaf expansion, changed theepidermal morphology of cotyledons and leaves, reduced the apical dominance, andled to fusions of various organs. SRN1might control SAM development by activatingSTM-CUC pathway. Moreover, SRN1protein could bind to L1box and negativelyregulate the expression HD-Zip IV family members, such as PDF2in Arabidopsis. SRN1was also shown to be a positive regulator of cellulose synthase genes, such asCesA4, CesA7and CesA8. Overexpression of SRN1increased the auxin content incotyledons. The SRN1protein might be regulated by phosphorylation anddegredation.