| Cotton is one of the most important economic crops in the world.It supplies the necessary raw materials for the textile industry.WRKY transcription factors as important regulatory proteins play a vital role in plant growth and development as well as stress response.There are few reports about the functions of WRKY transcription factors in cotton,nor the reports about the function of WRKY transcription factors in cotton fibers.In our previous study,more than 20 cotton WRKY transcription factors were identified.In this paper,two WRKY transcription factors were identified,and the molecular mechanism of GhWRKY16 promoting cotton fiber elongation was studied in depth.In addition,the mechanism of a LIM protein(GhXLIM6)regulating cotton fiber development was also studied.The main findings are as follows:1.Analysis of subcellular localization and regulatory elements of cotton WRKY transcription factorSubcellular localization analysis indicated that the two cotton WRKY transcription factors,GhWRKY16 and GhWRKY34,were localized in the nucleus.Yeast one-hybrid results showed that both WRKY transcription factors were able to bind to the "W-box" elements of the promoter region of the downstream target genes.2.Functional study of GhWRKY16 and GhWRKY34 in transgenic ArabidopsisOverexpression of GhWRKY34 in Arabidopsis makes the transgenic plants show stronger salt tolerance.Under NaCl treatment,the contents of Na+ and K-in the leaves and roots of the plants were determined.GhWRKY34 was able to maintain the lower Na+/K-ratio in the cells,and the expression levels of SOS1 and SOS2 were significantly up-regulated in the transgenic plants,suggesting GhWRKY34 could maintain Na+/K-homeostasis by regulating the expression of SOS1 and SOS2 in the SOS pathway,thereby increasing the salt tolerance of transgenic Arabidopsis lines.Overexpression of GhWRKY16 in Arabidopsis thaliana promoted the initiation and development of trichomes in transgenic plants.Statistical analysis showed that the number of trichomes and the proportion of 4 branches trichomes in transgenic Arabidopsis leaves increased.Given the homology of Arabidopsis trichomes cells to cotton fiber cells(seed epidermal hairs),the above results suggest that GhWRKY16 may play an important role during cotton fiber development.3.Functional study of GhWRKY16 in the initiation and elongation of cotton fiber cellsGh WRKY16 is predominantly expressed in the initiation and elongation of cotton fibers,and its expression level reach to the highest in 9 days post-anthesis(DPA)fibers.GhWRKY16 RNAi transgenic cottons were obtained by Agrobacterium-mediated cotton transformation.The results of freehand section and scanning electron microscopy showed that the number of fiber protrusions on the ovule surface of GhWRKY16 RNAi transgenic cotton(T0 generation)and its progeny lines(T1-T3 generation)was significantly less than wild type,and the fibers growth rate was slower than wild type.The length of mature cotton fiber was measured.The results showed that the length of mature cotton fiber of transgenic cotton was significantly shorter than wild type;meanwhile the fuzz was less or absent.In vitro ovule culture experiments indicated that down-regulation of GhWRKY16 expression affects the elongation of cotton fibers.The above results indicate that GhWRKY16 may play a positive regulatory role during the initiation and elongation of cotton fibers development.4.Molecular mechanism of GhWRKY16 regulating cotton fiber elongationReal-time RT-PCR results showed that the expression levels of some important regulatory genes(GhHOX3 and GhMYB109)associated with cotton fiber elongation were significantly down-regulated in the elongation of GhWRKY16 RNAi transgenic cotton,and the expression levels of cellulose synthase genes associated with primary wall synthesis were also significantly down-regulated.Bioinformatics analysis revealed that the "W-box" cis-acting elements existed in the GhHOX3 and GhMYB109 promoters.EMSA and CHIP-qPCR experiments confirmed that GhWRKY16 can directly bind to the promoters to regulate their expression.The dual luciferase reporter assay showed that GhWRKY16 is capable of activating the transcriptional activity of downstream target genes.These results indicate that GhWRKY16 promotes cotton fiber elongation by positively regulating the expression of GhHOX3 and GhMYB109.In previous reports,WRKY transcription factors were frequently modified by phosphorylation of MAPKs.Through screening analysis,we found the expression pattern of a cotton phosphokinase(GhMPK9)in cotton fiber development was similar to that of GhWRKY16,and they showed co-expression.Yeast two-hybrid,bimolecular fluorescence complementation and pull-down experiments have shown that GhWRKY16 could interact with GhMPK9 protein.Moreover,there are potential phosphorylation sites of MPK kinases in the GhWRKY16 protein sequence,suggesting that GhWRKY 16 may be modified by GhMPK9 phosphorylation.The experimental analysis of phosphorylation modification of GhWRKY16 protein and the influence of the phosphorylation on the function of GhWRKY 16 in cotton fiber development remain to be further studied.5、Study on the function and regulation mechanism of GhXLIM6 during cotton fiber developmentLIM domain proteins are cysteine-rich proteins and are often considered as actin bundlers or transcription factors in plants.However,the roles of XLIM proteins in plants(especially in cotton)have been unexplored in detail so far.In this study,we identified a cotton XLIM protein(GhXLIM6)that is preferentially expressed in cotton fiber during whole elongation stage and early secondary cell wall(SCW)synthesis stage.The GhXLIM6-silenced transgenic cotton produces shorter fibers,compared with wild type.Meanwhile,the cellulose content in fibers of GhXLIM6 RNAi cotton is lower than that in wild type.GhXLIM6 acts as a transcription factor to suppress GhKNL1 expression through binding the PAL-box element of GhKNL1 promoter,and subsequently regulate expression of CesA genes related to cellulose biosynthesis and deposition in SC Ws of cotton fibers. |
