Genome Wide Identification Of CLE Gene Family In Gossypium And The Functional Analysis Of GhCLE5

The CLE gene family encodes important short peptide hormones in plant genomes,but the function of these short peptide hormones in cotton remains largely unknown.Cotton（Gossypium L.）is an important source of natural textile materials.Currently,the main cotton variety is AD heterotetraploid species,which is formed through the process of hybridization and polyploid evolution of A and D diploid donor ancestors.The changes and functional evolution of the CLE gene family during genome duplication and polyploidization in Gossypium are unclear.In the presented study,we characterized the CLE gene family from the putative diploid ancestor species Gossypium herbaceum（A₁）,Gossypium arboreum（A₂）,Gossypium raimondii（D₅）,allotetraploid cotton Gossypium hirsutum（AD₁）,Gossypium barbadense（AD₂）,Gossypium tomentosum（AD₃）,Gossypium mustelinum（AD₄）,Gossypium darwinii（AD₅）.The study found that most members of the CLE gene family are single exon genes with signal peptides.The number of CLE gene family members is relatively stable in the evolution of cotton from diploid to tetraploid,and the number of tetraploid genome was the sum of the number of the diploid genome A and D,and 80%of the CLE gene sequences were conserved during the hybrid polyploidy.In terms of expression,the CLE gene showed a significant tissue expression specificity.The homologous CLE genes in the five tetraploid cotton species has a certain degree of organization consistency,which reflects the conservation of their potential functions.The number of promoter cis-elements related to stress and growth and development on the CLE gene of the tetraploid A subgenome is different from that of the A diploid species.These transcriptional characteristics indicate that the CLE members of cotton may be actively involved in regulating the growth and development of cotton and the formation of adaptive traits.Previous research in our laboratory found that the transgenic Arabidopsis thaliana of the G.hirsutum CLE gene GhCLE5 showed phenotypes such as cauliflower-like inflorescence and short club-shape silique.Therefore,an in-depth functional study of GhCLE5 was carried out.The gene sequence is highly conserved in Gossypium,and there is an amino acid difference between diploid and tetraploid cotton.Five hormones such as cytokinin,abscisic acid,auxin,gibberellin,and salicylic acid,as well as three abiotic stresses of high temperature,salt,and drought,can inhibit the expression of GhCLE5 to varying degrees.The results of subcellular localization showed that GhCLE5 before proteolysis into short peptides was distributed in the cell membrane and nucleus.Ectopic expression of GhCLE5 in Arabidopsis will result in multiple abnormal developmental phenotypes of flat fusion stems and shoot apical meristem（SAM）similar to clv3 mutants.Using yeast two-hybrid experiments and split luciferase activity experiments,it was found that GhCLE5 may bind to the endogenous At CLV3receptors At CLV1,At CLV2 and At CRN in Arabidopsis.It indicates that GhCLE5 may compete with Arabidopsis At CLV3 for binding to the receptor under ectopic expression,hindering the accurate transmission of At CLV3 signal,resulting in a phenotype similar to clv3 mutants.The virus induced gene silencing（VIGS）experiment of cotton confirmed that silencing GhCLE5 can cause differences in cotton plant height.The synthetic GhCLE5 motif polypeptide was used to cultivate Arabidopsis and cotton seedlings with external application.Experiments confirmed that GhCLE5 may not directly affect the root development of Arabidopsis and cotton.At the same time,the external application of GhCLE5 motif polypeptide will down-regulate the expression of endogenous GhCLE5 in cotton.It shows that GhCLE5 motif may also be a kind of short peptide regulatory protein in cotton.From gene family analysis to the exploration of functional genes,this study systematically explored the evolution of the CLE gene family in the evolution of cotton genome from diploid to allotetraploid,and revealed the possibility of CLE genes regulating growth and development in cotton.The molecular mechanism of this lays a foundation for exploring the functional mechanism of cotton CLE gene family members,and provides genetic resources for cotton improvement and molecular breeding.