Molecular Mechanism Of G65V Substituted Actin Inhibiting Cotton Cell Elongation

Cotton fiber is the largest source of natural fiber used for the textile industry,which has important economic value.Ligon lintless-1(Li1)is an upland cotton(G.hirsutum)mutant controlled by an incomplete dominant gene,displaying dwarf plants,distorted stems,wrinkled leaves and petals,and in particular the extremely shortened fibers.It is an ideal material in studying of the mechanisms underling the fiber cell development regulatory network.In this study,we performed map-based cloning and functional analysis of the Li1 gene,and the key results are as follows:Map-based cloning of the Li1 gene.Using of the Li1 mutant and a G.barbadense cv.Hai7124 as parents,two F2 segregation populations were developed.The Li1 gene was delimited into a 630-kb region in chromosome D04 of the G.hirsutum genome,which contains eleven annotated genes.Gh D04G0865 has a base mutation in the Li1 mutant,and the SNP marker designed by this base mutation was found to co-segregated with the mutant phenotype.Gh D04G0865 was expressed in all tissues except for the 0 DPA ovules,which was highly consistent with the phenotypic characteristics of the Li1 mutant.Thus Gh_D04G0865 was considered as the candidate gene.CDS of Gh_D04G0865 is 1,134 bp in length,and the predicted actin protein sequence containing 377 amino acid residues.The 194th base in the CDS of Gh_D04G0865 changed from guanine(G)to thymine(T)in the Li1 mutant,which resulted in the G65V amino acid substitution in its encoding protein.Functional verification of the Li1 gene.To investigate whether the mutated Gh_D04G0865 is responsible for the Li1 mutant,we performed gene silencing experiments in cotton and gene transformation experiments in Arabidopsis..Silencing of Gh_D04G0865 has little effect on WT plants,probably due to the functional redundancy of other cotton actin genes;while reduced expression of mutated Gh_D04G0865 in the Li1 plants produced slighter degree of mutant phenotype,which showed more flattened leaves and straightened stems.To investigate the effect of G65V,ACT2 and it’s mutant type gene ACT2G65V under the control of ACT2’s native promoter A2P was constructed into expression vector,and used for Arabidopsis transformation experiments.A2P::ACT2 transformed Arabidopsis had little difference with the wild type,while A2P::ACT2G65V transformed Arabidopsis displayed a phenotype similar to that of the Li1 mutant,such as dwarf plants and twisted organs.These results are in agreement with the dominant genetic characteristics of the Li1 mutant and indicate that the mutated Gh_D04G0865 is responsible for the Li1 mutant.Given the dominant inherited feature of the Li1 mutant,Gh_D04G0865 was named as Ghli1 and GhLi1 in wild and mutant type,respectively.Physiological mechanism of Lii mutation.The Li1 fiber was no longer elongate after 10 DP A,and the Li1 fibers density was significantly higher than that of WT fibers.Transmission electron microscope(TEM)analysis revealed that the secondary cell wall(SCW)of Li1 fiber was significantly thickened.Cellulose content in Li1 fiber was significantly higher than that of WT.Further observation showed that the F-actin arrangement shifted from longitudinal into horizontally or obliquely orientations,and the amount of F-actin in Li1 cells was significantly reduced than the WT.Vesicles in WT fiber cells were evenly distributed,whereas they were aggregated into patches in the Li1 fiber cells.As a result,a large number of cellulose synthase complexes(CSC)were inserted into the plasma membrane,and produced shortened and thickened fiber cell.These results suggested that F-actin regulated fiber elongation and secondary wall biosynthesis by mediating the intracellular transportation.Molecular mechanism of GhLi1 functional variation.Multiple alignment of the actin protein sequences showed the 65th glycine is highly conserved,indicate that it may be very important for actin’s basic function.Molecular model of the actin and simulation of the G65V mutation in F-actin showed that the G65V may affect the physical interaction among G-actin molecules.Biochemical experiments confirmed that Ghli1 could polymerize into F-actin,while GhLi1 was defective in polymerization ability.Moreover,GhLi1 affects actin polymerization by inhibiting the nucleation and elongation processes.Compared with WT,the F:G-actin ratio in Li1 tissues with GhLi1 expression were significantly decreased,indicated that the GhLi1 disturbed the actin dynamics in Li1 cells.Our results suggested that G65V resulted in GhLi1 functional variation by affecting of molecular interaction among actins.