| Cotton provides the most improtant renewable fiber resource for the textile industry.Cotton fiber development is a complicated process involving several factors,including phytohormone BRs.It has been proved that ovlues invitro culture and cotton field sparing with BRs can promote fiber elongation,while the mechanism that BR involved in regulating elogation remains elusive.The glycogen synthase kinase 3/shaggy kinase(GSK3)is a serine/threonine kinase with important roles in animals.Previous researches have confirmed that plant GSK genes are involved in diverse processes,including floral development,brassinosteroid signaling,and responses to abiotic stresses.To date,the completed genome sequencing of G.hirsutum,G.arboreum and G.raimondii now allow genome-wide analyses of phyletic evolution and biological functions of cotton GSK3 s via comparative genomics.In the present study,we performed genomic identification and phylogenetic analysis for cotton GSK3 gene family with several molecular biological methods on the basis of the published genomic datasets of cotton,Arabidopsis and rice.Meanwhile,based on fiber phosphoproteomics,we investigated the influence of BR signal pathway on cotton fiber development via GSK3 involved phosphorylation.The main results are as follows: 1)Several BR signal pathway related components were identified from the phoaphoproteomics dataAccording to the components of BR signal pathway of Arabidopsis,several cotton orthologous genes such as Gh_A10G1428(AtCDG1),Gh_D08G0486 and Gh_A08G0395(AtBSU1)and three GSK3s(Gh_A12G1106,Gh_A12G0411 and Gh_A01G1558)were identified.Interestingly,Gh_A12G1106 was only existed in the group 20 P.2)Cotton GSK3 s were potentially involved in BR signal pathway to regulate fiber developmentAs the negative regulator of BR signal transduction pathway,GSK3 executed its regulation on BR responsive genes and cell elongation genes by phosphorylation.Therefore,based on the reported substrate characteristics of GSK3,we selected 703 phosphorylated proteins predicted to be GSK3s’ substrates.According to the GO enrichment results of these proteins,some of them were enriched in the terms of microtubule cytoskeleton,cytoskeleton organization/binding,saccharides synthesis/metabolism and lipid synthesis/metabolism(p<0.05).Furthermore,they were significantly enriched in starch and sucrose metabolism pathway,amino sugar and nucleotide sugar metabolism,and other types of O-glycan biosynthesis process pathways(p<0.05).All of the results suggesting that cotton GSK3 s were likely to be involved in regulating fiber development via the above pathways or processes.3)The genomic identification and characteristics of GSK3 in three cotton speciesIn this study,20,15(including 5 different transcripts)and 10 GSK genes were identified and named as GhSK,GrSK and GaSK,respectively.High similarities in GSK3 protein sequences and conserved motifs were observed.Interestingly,the above mentioned three GSK3 s were GhSK13,GhSK41 and GhSK44.4)GhSKs possibly function during cotton fiber development and abiotic responsesThe gene expression patterns of GhSKs were analyzed both by using the published transcriptomic data of TM-1 and qRT-PCR,and these two results kept high consistent with each other.Moreover,the GO annotation result of genes co-expressed with GhSKs indicated that the top 20 GO were mainly enriched for protein phosphorylation,protein kinase activity,integral component of membrane,Calcium ion binding,transmembrane transport,and enzyme inhibitor activity(FDR<0.05).Further,these were also significantly enriched in pentose and glucoronate interconversion,plant-pathogen interaction,as well as starch and sucrose metabolism pathways(Top20 KEGG,FDR<0.05).The above results suggested that some GhSKs were potentially involved in regulating cotton fiber development.The qRT-PCR results indicated that except for GhSK25 showed non-significant response to almost all abiotic stresses,all other members exhibited diversiform response patterns to different abiotic stresses.5)GhSK13was potentially involved in BR pathway regulated cotton fiber developmentFurther experimental results revealed that GhSK13 was not only involved in BR signal pathway,but also interacted with GhAP2 L,Gh_DNF_YB19 and GhHDZ5 both in vivo and vitro.It’s implied that GhSK13 could be implicated in ethylene pathway,microtuble related functions or polysaccharide synthasis or metabolism to regulate fiber development.In conlusion,although cotton GSK3 genes were highly conversed during the evolution process,GhSKs genes possessed versatile functions as they not only played roles in upland cotton response to abiotic stresses,but also maight function in regulating fiber development.Among them,GhSK13 was likely to be implicated to BR signal pathway regulated fiber development by phosphorylating its downstream substrates. |
