Synergistic Effects Of Brassinosteroid And Gibberellin On Promoting Cotton Fiber Elongation And Their Molecular Mechanism

Cotton is the most important fiber crop.Upland cotton（Gossypium hirsutum）is the most widely grown cotton species in the world.Cotton fibers are derived from single cells of the ovule outer epidermis,the development process of which can be divided into four stages:differentiation and initiation,elongation,secondary wall synthesis and maturation.Among the four mutually distinguishable and partially overlapped stages,elongation is crucial for cotton fiber development,which determines the final length and of cotton fiber.The in-depth study of the molecular mechanism of cotton fiber development not only helps to fully reveal the regulatory mechanisms of plant cell differentiation and growth,but also can provide theoretical basis for improving fiber production and quality.Plant hormones play an important role in the regulation of cotton fiber development.There exist comlicated inter-regulatory relationships between different hormones.Studying the interaction between hormones allows us to further understand the growth and development of plants.Previous studies have shown that brassinosteroid（BR）and gibberellin（GA）can promote the elongation of cotton fiber cells.To explore the possible molecular mechanisms and the interactions between BR and GA,in this paper,I analyzed the relationship of them in promoting fiber elongation using in vitro ovule culture experiments.In addition,I also analyzed the genes that are commonly regulated by BR and GA using RNA-seq,especially those involved in cell elongation.To further clarify whether there is any interaction between BR and GA at the signalling level in cotton fiber,I identified all cotton BZR1 proteins which are key transcription factors in the BR signaling pathway.At the same time,a comprehensive analysis of the interaction between BZR1 and DELLA,a key transcription factor in the GA signaling pathway,were conducted.The main results are shown as followed:1.Exogenous GA and BR promoted the elongation of cotton fiber in vitroTo explore the effects of GA and BR on cotton fiber development,1 DPA Ovules were treated with bioactive GA（GA₃）or BR（24-eBR）,respectively.The results showed the fiber length significantly increased with the concentrations of GA₃ and 24-eBR,indicating that both exogenous GA and BR promoted the elongation of cotton fiber.When2μM 24-eBR and 0.1μM GA₃ were simultaneously added to the medium,the fiber length reached to 12.2 mm,which was much higher compared to BR or GA treatments,and also the superimposed value（8.59 mm）of the two treatments.These results demonstrated that the BR and GA synergistically promoted the elongation of cotton fiber.2.BR and GA involved in regulating the genes related to fiber elongationTo understand the molecular mechanism underlying synergistic effect of BR and GA on promoting fiber elongation,we compared BR-and GA-induced genes in elongating fibers by RNA-seq.A total of 89 genes commonly induced by 24-eBR and GA₃ were identified.Among them,many genes are involved in cell elongation,including arabinogalactan peptides（AGP14,FLA2）,xyloglucan endotransglucosylase/hydrolase（XTH9,XTHB）,aquaporin（TIP1-1）,actin binding protein（Profilin）,etc.We further validated the expression of the above genes by RT-PCR,the results showed that these genes were induced by both BR and GA.3.BR regulated cotton fiber elongation by promoting GA biosynthesisThe results of in vitro ovule culture experiments and electron microscopy scans showed that BR-induced fiber elongation is inhibited by addition of 10μM PAC（GA synthesis inhibitor）.RT-PCR analysis showed that several GA synthase genes,including KAO2,GA20ox-1,and GA20ox-2,were induced by BR,and their transcript levels increased with 24-eBR concentrations,GA2ox-2 involved in GA metabolism was inhibited by BR.These results indicated that BR affected the expression of enzymes involved in GA biosynthesis and metabolism,resulting in higher GA levels,and promoting the development of cotton fiber.4.Interaction between BR-BZR1 signaling pathway and GA-DELLA signaling pathwayTo explore the possible interactions between BR and GA signalling,the cotton homologs of BZR1,a key transcription factor in the BR signaling,were comprehensively identified.It was found that there are seven pairs of BZR1 homologs in upland cotton.Phylogenetic analysis showed that GhBZRs can be divided into two branches,highly homologous to AtBZR1 and AtBEH,respectively.Sequence alignment analysis revealed that all 7 pairs of GhBZRs contained typical domains of BZR proteins.Transcriptomic data and RT-PCR analysis showed that GhBZR1-1A/1D predominantly express in various tissues of G.hirsutum.Interactions between BZR1 and DELLA protein（GhGAI1A/GhGAI1A）,key negative regulators of GA signaling pathway,were detected with Y2H and BiFC metods.The results showed that GhBZR1-1A/1D and GhBZR1-2A/2D interacted with GhGAI1A/1D,suggesting crosstalk of BR and GA signaling in upland cotton might be mediated by the interaction BZR1 and DELLA.