The Cloning And Functional Characterization Of Genes GhGRAM31 And GhTCP15 During Cotton Fiber Development

Cotton fiber provides a perfect material to investigate plant single cell development.Decoding the gene regulatory network involved in dynamic fine-tuned process of cotton fiber development,and uncovering key regulatory node genes will contribute to the improvement of cotton fiber quality.The GRAM domain proteins in plants are involved in growth and stress response processes,but they have not been functionally characterized in cotton fiber development yet.Previous studies showed that TCP transcription factors could regulate fiber development,but the underlying mechanism remains obscure.In the present study,the functions of GhGRAM31 and GhTCP15 in regulating cotton fiber development were explored,the results may provide solid supports for fiber development regulation and quality improvement in cotton,the main results were presented in two parts as follows:Part one: GhGRAM31 regulates cotton fiber elongation.In this study,we performed genome-wide identification of GRAM domain genes in four cotton species,and analyzed the expression patterns of GRAM domain gene members during fiber development in G.hirsutum.Then the biological function and regulatory mechanism of GhGRAM31 in cotton fiber development was investigated,the main results are as follows:1.A total of 164 GRAM domain protein members were identified in four cotton species,G.arboretum.G.raimondii.G.hirsutum and G.barbadense,which were clustered into two subfamilies.The expression patterns of GRAM domain genes in G.hirsutum during fiber development were divided into four clusters.Members from Cluster IV were preferentially expressed in 5-10 DPA fiber at elongation stage,suggesting that they might be involved in the regulation of fiber elongation.GhGRAM31,a homolog of Arabidopsis At GRE5,was predominantly expressed in 10 DPA fibers,and was selected for further research.2.To investigate the biological function of GhGRAM31 in fiber development,the GhGRAM31 RNAi and overexpression vectors were developed and was used to transform G.hirsutum cv.YZ1.The mature fibers quality investigation over three successive years revealed that the fiber elongation of GhGRAM31 RNAi plants was hindered.Downregulation of GhGRAM31 led to strikingly shorter mature fiber with worse fiber quality,as well as lower lint percentage and smaller naked seeds size than control.In contrast,the overexpression plants produced longer fibers.3.To analyze the effect of GhGRAM31 on expression regulatory network,q RT-PCR analysis of key genes was performed,and results showed that the expression of the cell wall related genes CTL2 and Ces A7 were significantly up-regulated in RNAi plants.However,CHIT3,SLR1 and several fiber development related transcription factors were downregulated in RNAi plants,resulting in a disturbance of the fiber development gene regulatory network.This in turn affect fiber development.4.In order to further explore its function mode,yeast two-hybrid(Y2H)experiment was conducted to screen the interacting proteins of GhGRAM31.Another two GRAM domain proteins,GhGRAM5 and GhGRAM35,were identified as GhGRAM31 interacting candidates.The interaction of between GhGRAM31 and GhGRAM5,GhGRAM31 and GhGRAM35 was verified in pull-down,LCI and Bi FC assays.Further experiment revealed that GhGRAM5 could interact with WD40 factor GhTTG1,while GhGRAM35 could interact with HD-Zip factor GhHD1 and GhHOX1.Dual-luciferase reporter assays showed that the co-expression of GhGRAM31 and GhGRAM35 could enhance the transcriptional activation activity of GhHD1 on downstream genes.These results indicated that GhGRAM31 may regulate fiber development by participating in a complex protein interaction network.In summary,this study provided plentiful information of cotton GRAM family members,and provides a vast of candidate gene resources for fiber quality improvement.Phenotype analysis of cotton GhGRAM31 RNAi and overexpression lines found that GhGRAM31 positively regulates fiber elongation in cotton.Further experiments revealed that GhGRAM31 regulates fiber development via a complex protein interaction network.Part two: GhTCP15 regulates cotton fiber elongation and cell wall thickening.Plant-specific TCP transcription factors are involved in evolution and a wide range of biological processes.They may constitute central regulatory nodes in a variety of signaling pathways.Therefore,analyzing the functions and mechanisms of TCP transcription factors will enrich plant cell networks.In this study,we conducted biological function and mechanism analysis of fiber preferential expressed gene GhTCP15 during cotton fiber development,the main results are as follows:1.GhTCP15,a homolog of Arabidopsis At TCP15,encoding a Class I TCP transcription factor.The expression pattern analysis revealed that GhTCP15 was predominantly expressed in fibers at 10 DPA.To investigate the biological function of GhTCP15 in fiber development,GhTCP15 overexpression vector driven by fiberpreferential Gb EXPA2 promoter was developed and transformed into G.hirsutum cv.YZ1.The mature fibers quality investigation was repeated for successive years which revealed that the fiber elongation of GhTCP15 overexpression plants was hindered.Overexpression of GhTCP15 led to significantly shorter mature fiber with worse fiber quality,as well as lower uniformity index and higher short fiber index than control.Examination of the paraffin embedded mature fiber,in cross section,showed that GhTCP15 overexpression cotton fiber had significantly thicker cell wall than control.These results suggested a consequential role of GhTCP15 in regulating fiber cell elongation and second cell wall formation.2.To analyze the underlying regulatory mechanism of GhTCP15,Chromatin immunoprecipitation sequencing(ChIP-seq)were performed using transgenic line overexpressing GhTCP15 fused with GFP tag.The ChIP-seq showed a strong enrichment of cis-element in GhTCP15 binding promoter sequences,GTGGGNCC motif,which was consistent with the core sequence recognized by Class Ⅰ TCP in Arabidopsis.The data indicated the highly conservation of the binding sequence of TCP transcription factors.3.To analyze the effect of GhTCP15 on expression regulatory network,thereby RNAseq analysis was performed using 10 DPA fibers from GhTCP15 fiber-specific overexpression line driven by Gb EXPA2 promoter and wide type donor plants.Compared with wild type fibers,totally 830 differentially expressed genes(DEGs)were identified.DEGs were significantly enriched for biological process terms,especially in cell wall biogenesis and synthesis,and secondary wall biogenesis and synthesis.The promoters of139 DEGs possessed GhTCP15 potential binding cis-elements,which were also enriched in cell wall and secondary wall related processes.4.ChIP-q PCR,Y1 H and dual-luciferase reporter assays showed that GhTCP15 could directly bind GhEXPA1 promoter and activate its expression.5.In order to further explore the interacting proteins of GhTCP15,and insight into the function mode,Y2 H experiment was conducted using GhTCP15 as bait.Several interacting candidates were identified,including Class Ⅰ TCP transcription factor GhTCP8 and ClassⅡ TCP transcription factor GhTCP4.LCI and Bi FC assays confirmed the interactions in vivo.It was speculated that GhTCP15 might interact with multiple TCP transcription factors including GhTCP4 and GhTCP8,and participate in regulating the expression of cell wallrelated genes,thereby affecting fiber development.To sum up,the combined analysis of ChIP-seq and RNA-seq showed that GhTCP15 could regulate cotton fiber elongation and cell wall thickening though directly affecting the expression of cell wall-related genes.In addition,GhTCP15 may interact with factors such as GhTCP4 and GhTCP8 to finely orchestrate the network of cotton fiber development.These data revealed an important role of GhTCP15 in regulation of fiber development.