Evolutionary Analysis And Functional Characterization Of The GLV Peptide Family

Plant hormones play important role in various aspects of plant growth and development.GLV(GOLVEN)/RGF(Root meristem growth factor)/CLEL(CLE-Like)is a group of 13-18 amino acid secreted peptides involved in different developmental processes,such as root stem cell niche maintenance and root pattern formation.Although the GLV peptides have been studied in Arabidopsis and some other species,Wide-ranging phylogenetic analysis of the family across the plant kingdom has not yet been undertaken.To illuminate the evolutionary process of the GLV gene family within the plant kingdom,a comprehensive survey was undertaken to identify and characterize GLV genes in 24 plant species that represent a variety of plant lineages.203 GLV were identified and each of them containing a typical GLV domain.The GLV gene family was analyzed comprehensively with respect to signal peptide,chemical properties,gene structure,phylogeny,clustering,motif analysis,molecular evolutionary rate,selection pressure,the search of upstream regulation area cis-acting element and expression pattern.No GLV was identified in the algal,suggesting that the GLV family may originated from land plants.Gene structure analysis showed that the introns of GLV family genes were diverse in length and number,suggesting the event of intron loss and insertion that contributed to the diversity of gene structure.Motif analysis indicated that some motifs are unique to species,indicating the evolutionary diversity and specificity of the GLV family.Phylogenetic analysis indicated that the GLV gene family is mainly divided into 9 groups.Gene expansion was found in angiosperm.Segmental duplication and tandem duplication may be important factors in the expansion of angiosperm GLV gene family.Analysis of evolutionary rates and selection properties for orthologous genes and GLVs in different groups indicated that purifying selection is the dominant force for the evolution of the GLV gene family,suggesting the conservation of GLV gene function.Most of the GLV expression levels in the roots are relatively high and members of the same group have similar expression patterns.However,a few duplicated genes have different expression patterns,indicating that neofunctionalization might have occurred during evolution.To further uncover molecular mechanisms of GLV in other plant species,four GLV homologous genes in tomato(dicot),maize(monocot)and moss were selected and were artificially synthesized,and then treated Arabidopsis seedlings by adding the polypeptide in the culture medium.We also cloned and overexpressed these genes in wild type Arabidopsis to study their functions.It was found that all these genes are function in the regulation of root gravitropic responses and lateral root development.In GLV peptide-induced roots,the root apical meristem(RAM)was enlarged and consisted of an increased number of cells.These results indicated that the GLV peptides are biologically active.The Auxin-TPST-GLVs/RGFs/CLELs-RGIs-PLT pathway is known to be responsible for the maintenance of stem cell niche.To further understand the evolution and function of GLVs/RGFs/CLELs-RGIs signaling pathway,117 RGIs were identified in 24 plant genomes including 2 RGI genes in moss.Phylogenetic analysis showed that 117 RGI genes were divided into four major branches,with the gene expansion in angiosperms.Expression pattern analysis indicated that the expression of RGI family genes in roots was generally higher.Two,seven and seven RGIs were identified in moss,tomato and maize,respectively.Some RGI genes were cloned via a Gateway technology and transformed into a quintuple Arabidopsis mutant rgi1,2,3,4,5.We found that most RGI genes from different plant species can partially restore the short root phenotype of rgi 1,2,3,4,5,suggesting that homologues genes of AtRGI in these species may possess similar biological functions.Our research shed light on several key factors in GLV gene family evolution and provided genomic characteristics of GLV gene family,which may provide a valuable resource to future research regarding the roles of GLV genes in plant growth and development.