Gbpdf1(Protodemal Factor1) Is Involved In Cotton Fiber Initiation VIA The Core Cis-Element Hdzip2Atathb2

Fiber is the main product of cotton, which is the most important fiber crop in the world. Cotton fiber cells are highly elongated single-cell trichomes derived from the epidermal layer of the ovules. Because of its special structure, cotton fiber was considered as an excellent model for the research of cell elongation and secondary cell wall biosynthesis. The mechanism of fiber elongation and the thicking of secondary cell wall got improved process. However, the molecular basis of fiber initiation remains largely unknown, because the stage is rapid and it’s difficult to visualize. The initiation period contained two biological processes:the differentiation of pre-fibers from protodermal cells on the surface of cotton ovules (fiber cell fate determination) and the cells balloon out from cotton ovule protodermal cells (formation of morphologically visible fiber initials). The quantity, as well as the quality of fiber is dependent on the timing and numbers of fiber cells from each ovule. The research of fiber initiation was so essential that it could provide an effective strategy to improve the yield and quality of cotton fiber by gene engineering.To unravel the role of PDF1function in cotton fiber development, we used bioinformatics, molecular genetics during fiber initiation and early differentiation. The main results are as follow:1. The structure and sequences comparison of PDF1genes from G barbadense3-79and G hirsutumTM-1Three and two PDF1-homologous genes were obtained from the genomic DNA of G. barbadense and G. hirsutum, respectively. The five sequences were classified into three types according to different number of the introns:Type Ⅰ (without intron), Type Ⅱ (one intron), and Type Ⅲ (two introns). The alignment of the putative amino acids showed that the PDF1proteins in Type Ⅲ had different repeats of8-aa " SYGGGSPP" motifs. In addition, conserved C-terminus was found among the PDF1proteins from different sources of plants. 2. PDF1-suppressed cotton showed retarded fiber differentiationPDF1-silenced cotton plants were generated and the ovules around the day of anthesis were surveyed. Scanning electron micrographs of the RNAi transformed cotton ovules indicated that the fiber development was delayed. The wild-type fibers began to balloon out from the epidermal surface12h before flowering, whereas the PDF1-silenced plants showed no trichome protrusion on the surface of the ovules. Fiber elongation in the RNAi transformants was retarded compared to both wild-type and control plants in the1DPA ovules. Comparison of the mature fiber length between the PDF1-RNAi transgenic plants and control plants revealed that the reduction expression of PDF1in fiber resulted in shorter fibers in the RNAi transformants than in the wild type.3. The potential role of PDF1in cotton fiber developmentThe H2O2content in the GbPDFl knockdowns3-DPA ovules was much higher than the one of wild-type, however, no more ethylene was induced by the high accumulation of H2O2in the knockdowns. The pectin biosynthesis of the PDF1-suppressed cotton fibers and ovules might be disturbed because of the down-regulation of two putative glycosyltransferases together with the reduced carbon source. We concluded that GbPDF1plays a critical role in H2O2homeostasis and steady biosynthesis of ethylene and pectin during fiber development.4. Interacting proteins of GbPDF1The full-length GbPDF1fused with the GAL4-DNA binding domain was used to screen a yeast two-hybrid library, using the cDNAs from cotton ovules and fibers to construct prey vectors. Thirteen candidate GbPDFl-interacting proteins were identified. Three putative proteins (PPIP1, PPIP2and PPIP3) were obtained after being re-transformed into the bait strains and determined by bimolecular fluorescence complementation. The interaction partners of GbPDFl may involve cellular signaling or metabolism:the regulation of protein degradation, the controlling of antioxidation metabolism, intracellular protein or lipid-binding signaling pathways. 5. Overexpression of GbPDFl promotes primary root length in tobaccoThe homozygous tobacco lines carried CaMV35S:GbPDFl were obtained, the primary roots were significantly longer than that of wild-type. These data were confirmed by three independent repeats.6. The expression pattern analyses of GbPDFl promotersTwo GbPDF1gene promoters, differing in the lengths of the5’flanking regions (PGbPDF1-1,1679bp; PGbPDF1-2,1285bp), were cloned from G. barbadense3-79by genome-walking. The two promoters shared a nearly identical390-bp sequence upstream of the translational initiation codon. PGbPDF1-2, as well as PGbPDF1-1preferentially drives GUS expressed in the cotton ovules and fibers. The GUS activity was highest in the0DPA ovules and decreased gradually during fiber development.7. The identification of the essential cis-element for the basal transcription of GbPDFlProgressive deletions of the GbPDF1promoter showed that a236-bp promoter fragment was sufficient for basal GbPDF1transcription in cotton. Mutation of putative regulatory sequences showed that HDZIP2ATATHB2, an element within the fragment, was essential for PGbPDF1-1expression. The binding activity of this cis-element and nuclear extracts from5DPA fiber-bearing cotton ovules was specific.In summary, our findings indicated that GbPDFl is required for the transition of pre-fiber cells from the cotton ovule protodermal cells, possibly by controlling the normal concentration of H2O2and steady biosynthesis of ethylene and pectin. Moreover, the regulatory element HDZIP2ATATHB2located in the promoter of GbPDF1was essential for its expression.