Genome-wide Identification Of The Mitogen-activated Protein Kinase(MAPK) Family In Cotton(Gossypium Hirsutum) Reveals The Role Of GhMPK6 In Fiber Elongation

As an important economic crop,cotton provides important raw materials for the textile industry.However,the regulation mechanism during cotton fiber development remains uncertain.Previous studies shown that mitogen-activated protein kinases(MAPKs)are important regulators of plant growth and development as well as abiotic and biotic response.However,there are few reports about the function of MAPK in cotton fiber development.In our study,56 upland cotton MAPK genes were identified and analyzed,and the function of GhMAPK protein family members GhMPK6,which was dominantly expressed during cotton fiber elongation,was further investigated.The specific research results are as follows:1.Identification and analysis of MAPK family genes in upland cotton56 upland cotton MAPK genes were identified by using known Arabidopsis MAPK protein sequences and rice MAPK protein sequences as queries to conduct a homologous blast search against cotton(G.hirsutum)protein databases(http://cottonfgd.org/blast/,Gossypium hirsutum,NAU and Gossypium hirsutum,HAU).Of these,28 were from the A genome and 28 were from the D genome.According to the similarity with the Arabidopsis MAPK protein sequences,the 56 GhMAPK genes identified in upland cotton were named GhMPK1A/D-GhMPK20aA/D,and were divided into four sub-branches A,B,C and D.These MAPK genes were unevenly distributed on 22 chromosomes in the cotton genome,and there were no MAPK genes on At_Chr6,Dt_Chr6,At_Chr13,and Dt_Chr13.The exons and introns of the GhMAPK gene family differ greatly in position,number,and length,but almost all GhMAPK members in the same evolutionary subgroup contain similar gene structure and relatively conserved protein domains.Almost all members of the GhMAPK family have MAPK kinase-conserved Ser/Thr protein phosphatase domains,ATP-binding domains,and kinase activation loop domains,and the kinase activation loops in the three sub-branches A,B,and C are TEY-type,and in D sub-branch is TDY type.Analysis of the identified 56 GhMAPK gene expression patterns in cotton tissues and cotton fibers at various developmental stages revealed that GhMAPKs expression patterns were different in different tissues.GhMAPK genes in the same group have different expression patterns,while GhMAPK genes in different groups have similar expression patterns,indicating that there may be functional differences in GhMAPK genes during cotton development.2.GhMPK6 are involved in regulating cotton fiber developmentIt was found that several GhMAPK genes(including GhMPK6)were predominantly expressed in elongated cotton fibers by expression analyzing of GhMAPK genes in cotton tissues.First,we used anti-pERK phosphorylation antibodies to detect phosphorylation levels of GhMPK6 in cotton fiber at different developmental stages,including 0 DPA(days post anthesis)ovules and 3 to 21 DPA fibers.Results showed that the phosphorylation of GhMPK6 can be detected in 0 DPA ovules,and maintain high levels in 3 to 9 DPA fibers.Then the phosphorylation levels gradually decreased in 12 to 15 DPA fibers,and were hardly detected in 18 to 21 DPA fibers.Results indicated that GhMPK6 plays an important role in the initiation and elongation of cotton fiber.3.GhMPK6 regulate cotton fiber elongation in response to phytohormone signalsCotton fiber development is inseparable from the phytohormone,cotton ovules cultured in vitro were treated with appropriate concentrations of phytohormone.It was found that after exogenous application of phytohormone,there was no significant difference in the transcription and protein levels of GhMPK6 in fibers compared to the control.However,it was found that when exogenous BL(the most extensive BR in plants),ACC(ethylene synthesis precursor)and IAA were added,the phosphorylation levels of GhMPK6 in fibers was activated,and when BRZ(BR synthesis inhibitor)and AVG(ethylene synthesis inhibitor)reduce the endogenous BR and ethylene content,and the phosphorylation levels of GhMPK6 in the fiber reduced.Moreover,exogenous GA does not affect the phosphorylation levels of GhMPK6.Above results indicated that GhMPK6 may regulate cotton fiber development by participating in the transduction of phytohormone signals.4.Analysis of the interaction between GhMPK6 and upstream GhMAKKsGhMAKKs,which are highly expressed during cotton fiber elongation,were identified as candidate interaction proteins for GhMPK6 by expression analysis.It was found that GhMKK2-2/4 can interact with GhMPK6 in vitro and in vivo through yeast two-hybrid,BiFC assay and Pull-down assay.It suggested that GhMKK2-2/4-GhMPK6 may be involved in the development of cotton fiber elongation.