The Origin,Evolution And Functional Divergence Of Land Plant GH5₁1 Cellulases

In plants,cellulases are encoded by large glycosyl hydrolase gene families,which can hydrolyze 1,4-?-glucan chain molecules and play important roles in plant growth and many essential physiological processes.Cellulases are known as multi-component enzyme systems,including the endoglucanase,exoglucanase and ?-glucosidase.A combination of these three types of enzymes is necessary for the complete hydrolysis of cellulose.Plant GH5₁1 genes encode a cell-wall degrading enzyme,belonging to a subfamily of Glycoside hydrolase 5,which are widely present in land plants.Although the functions of this family were not clearly detected,many databases annotated the enzymes encoded by GH5₁1 genes as cellulases.In addition,the origin and evolution patterns of plant GH5₁1 genes were not detected.To illustrate the origin and evolution pattern of land plant GH5₁1 genes and to preliminarily reveal their roles in plant growth,a combination of bioinformatics and experimental biology were preformed to analyze the origin,evolution and putative functions of this gene family.In addition,we also examined the expression patterns of GH5₁1 genes in rice and Arabidopsis.The morphological and physiological phenotypes of Arabidopsis mutants were identified to investigate the potential function of land plant GH5₁1 genes.The main results were shown as follows:1.GH5₁1 genes were not present in green algae,and were firstly appeared in Bryophytes.The members of GH5₁1 subfamily are widely spread in land plants.During the evolution of land plants,the GH5₁1gene subfamilies have experienced massive gene duplication and loss events.No GH5₁1 homologs were found in the genomes of fern Selaginella moellendorffii and basal angiosperm Amborella trichopoda,suggesting gene loss had occurred in these two species.In addition,the distribution of the gene numbers were also unbalanced among the genomes of land plants due to lineage and/or species-specific expansion.The ancestors of Graminaceae and Brassicaceae were found to only possess one GH5₁1 gene,while several duplication events contributed to multiple copies of this subfamily in these two families,respectively.2.Blast results revealed that GH5₁1 genes are distributed widely in bacterial and fungal groups.In the phylogenetic trees,all the selected land plants GH5₁1 genes form a single branch with high bootstrap,which fell into the branches of fungi.In addition,we noticed that land plant GH5₁1 proteins have the highest similarity with those in fungi.These results suggested the ancestor of land plants acquired GH5₁1 gene from fungi through an ancient horizontal gene transfer event.3.Adaptive evolution analysis indicated that land plant GH5₁1 genes have undergone positive selection after a short period of origination from HGT,while the predominant constraint was the purifying selection after the split of main land plant species,suggesting GH5₁1 gene families have experienced functional adaptive evolution after the HGT event.4.There were five Arabidopsis and four rice GH5₁1 genes.Different tissue expression patterns were found among different homologs in rice and Arabidopsis,suggesting that functional divergence might have occurred after the gene duplication.In addition,multiple gene expression of rice and Arabidopsis were regulated by abiotic stress,such as drought,heat,salt and ABA etc.,suggesting that GH5₁1 gene might have functions responding to stresses.5.To elucidate the biological function of GH5₁1 genes,three Arabidopsis GH5₁1 gene mutants were selected to detect their morphological and physiological phenotypes.Mutants of atgh5₁1a and atgh5₁1c showed a significant increase in the width and length of rosette leaves.atgh5₁1c also delayed flowering time under short photoperiods.However,atgh5₁1d did not show any changes in morphological phenotypes.The cellulase activities and endoglucanase activities decreased in all selected mutants,suggesting that Arabidopsis GH5₁1 genes might encode cellulase,belonging to endoglucanase.In addition,the contents of cellulose and lignin increased in all detected mutants,and the contents of glucose and sucrose decreased in atgh5₁1a and atgh5₁1c mutant and the contents of fructose increased,while the contents of three type of oligosaccharide did not change in atgh5₁1d mutant.All these results indicated that Arabidopsis GH5₁1 genes might encode a novel cellulase and belong to endoglucanase.Our analyses revealed that the land plant GH5₁1 genes originated through an HGT event from fungi.Further evidence revealed massive gene duplication and loss events contributed to the evolution of land plant GH5₁1 gene families.According to the analyses of the rice and Arabidopsis GH5₁1 genes expression patterns and three Arabidopsis mutants,we revealed that the duplicated genes have undergone functional divergence.We preliminarily proved the GH5₁1 genes encode cellulases that belong to endoglucanase through analysis of Arabidopsis GH5₁1 mutants.Our results will not only provide the theoretical basis on revealing the origin and molecular evolution of land plants,but also provide references for further functional detection of plant GH5₁1 genes.