Genome-wide Analysis Of PMEI Gene Family In Rosaceae Species And Functional Analysis Of PbrPMEI23/39/41

Cell wall is an important structure of plant cells,which plays important roles in cell morphology maintenance,stress resistence,material transportation and signal transmission.Pectin is the main component of plant cell wall polysaccharide,which is the main part of cell wall.After been secreted into the pollen tube cell wall,highly methylesterified pectin will be de-methylesterified by pectin methylesterase（PME）.The carboxyl group of de-methylesterified pectin will bind with extracellular Ca²⁺to harden the cell wall,which hinders the extension of cell walls.On the other hand,de-methylesterification of pectin will acidify the extracellular environment and thus activates pectin lyase and polygalacturonase,which soften the pollen tube cell wall.Pectin methylesterase inhibitor（PMEI）can regulate PME activity by binding to the active site.PMEI gene family has been identified in several plant species.However,the research of PMEI in pear is deficient and the key PMEI genes of pear that regulate pollen tube growth have not been clarified.In this paper,’Dangshansuli’was used as the experimental material.Identification and evolutionary analysis of PMEI gene family member in seven Rosaceae species were performed by bioinformatic analysis and molecular biological experiments.The functions of the key genes involved in pollen tube growth were studied.The main findings are as follows:1.Through bioinformatics analysis,423 PMEI genes were identified in Arabidopsis thaliana and seven Rosaceae species（black raspberry,woodland strawberry,sweet cherry,apple,peach,Japanese apricot and pear）.Phylogenetic analysis,structural analysis and evolutionary analysis of Arabidopsis thaliana and pear（including three pear cultivars,namely‘Dangshansuli’,‘Bartlett’and‘Zhongai 1’pear）were carried out.According to the analysis results,the PMEI gene family of Arabidopsis thaliana and pear were divided into five subfamilies（A,B,C,D and E）.Gene and protein structural analyses also supported the results of subfamily classification.Whole-genome duplication and tandem duplication were two major duplication events in the PMEI gene family of Arabidopsis thaliana and pear.Purification selection was the main driving force for the evolution of PMEI gene family in Arabidopsis thaliana and pear.2.According to transcriptome data,18 PbrPMEI genes with high expression in pear pollen were selected.The expression pattern of these 18 genes during pollen tube growth were detected.PbrPMEI23/39/41 were selected for the increasing expression during pollen tube growth.Subcellular localization of these three genes showed that they were all localized in the cell wall,cell membrane or cytoplasm.3.Biological functions of PbrPMEI23,PbrPMEI39,and PbrPMEI41 were studied.Phenotypic statistics of transgenic Arabidopsis thaliana showed that heterologous overexpression of PbrPMEI23,PbrPMEI39,and PbrPMEI41 could promote the growth of Arabidopsis pollen tubes.Exogenous application of recombinant proteins PbrPMEI23-His,PbrPMEI39-His,and PbrPMEI41-His in the culture medium of pear pollen tubes could change the distribution of methylesterified pectin at the tip of pear pollen tubes and promote the growth of pear pollen tubes.These results indicated that PbrPMEI23,PbrPMEI39,and PbrPMEI41 could affect the pollen tube growth by regulating the cell wall of pear pollen tubes.In summary,PMEI family members from seven Rosaceae species were identified,the phylogenetic and evolutionary history of PMEI gene in Arabidopsis thaliana and pear were analyzed.According to the transcriptome data and expression pattern analysis,three PbrPMEIs that specifically expressed in pollen were selected because their expression levels were continuously increasing during pear pollen growth.The promoting effects of these three PbrPMEI genes on pollen tube growth and the potential mechanism were preliminarily revealed,which enriched the understanding of the role of PMEI in regulating pollen tube cell wall.