Molecular Mechanism Of Polytubey Block Regulated By Female Gametophyte In Arabidopsis Thaliana

During double fertilization in angiosperm,pollen tube is formed after pollen germination.Under the guidance of pistil tissue,pollen tube carries two sperm cells to embryo sac and releases sperm cells to complete double fertilization.Thus,female tissues evolve a series of strategies to attract pollen tubes into the micropylar end.And among these strategies,synergid cells are the masterpiece of life evolution.Synergid cells form filiform apparatus to secret a series of species specific chemo attractants,such as EA1 in monocot plants Zea mays and LURE1 in dicot plants Torenia fourniere and Arabidopsis thaliana.Once pollen tube arriving at the micropylar end of ovule and accomplishing its mission to transport a pair of sperms,the ovules will refuse excess pollen tube attractions.This phenomenon,namely polytubey barrier,is confirmed to be gamete fusion dependent,that is,when the first pollen tube enters,if the sperm and egg cell fuse fail,the second pollen tube can enter the embryo sac.Only the final cessation of pollen tube attraction depends on successful double fertilization.But how do plants perceive sperm-egg fusion successfully,how do they send signals to prevent the extra pollen tubes,what is the essence of the signals,and how are they transmitted? So far,there is no answer to all these questions.We speculated that egg cell may be the key to answer the above questions,and based on this,we carried out a series of studies.Firstly,we isolated the egg cells,carried out transcriptome sequencing and constructed the egg cells c DNA libraries.We found two proteases,Egg Cell-specific 1(ECS1)and ECS2,which were specifically expressed in egg cell.We found that they are involved in the regulation of polytubey block.The main experimental results are as follows:1.Two aspartic protease ECS1/2 were selected from the egg cell libraries and expressed specifically in the egg cell.ECS1/2 are highly homologous and belong to the A1 subfamily of the aspartic protease family,suggesting that they may have similar biological functions.The expression level of ECS1/2 is rapidly decreasing after fertilization and disappeared at the stage of elongated zygote.2.We found that ECS1/2 rapidly secreted towards to synergid cells area during double fertilization,secretion process was triggered by sperm-egg fusion.We found that ECS1/2 did not secret when pollen tube grew into the micropylar end of ovule and pollen tube burst.However,upon sperm cell enters into egg cell,the secretion process began.3.We demonstrated that ECS1/2 is involved in the regulation of polytubey block.In ecs1ecs2 double mutants,there was a certain proportion of polytubey phenotype.The loss of ECS1/2 function led to the ovule attracting the second pollen tube after fertilization,which indicated that ECS1/2 played an important role in polytubey block.The secretory activity of ECS1/2 plays an important role in their biological functions.4.We found that the substrate of ECS1/2 was pollen tube attractor LURE1.Different experiments confirmed that ECS1/2 could cleave and degrade LURE1 both in vivo and in vitro.It ensured that ovules could clear LURE1 immediately after sperm–egg fusion,thus preventing the attraction of redundant pollen tubes.In summary,we demonstrated that proteases ECS1/2 are exclusively expressed in the egg cell and could be secreted from egg cells to accumulate in the persistent synergid cell soon after fertilization.ECS1/2 secretion was triggered only by sperm–egg fusion,and the signal peptide of ECS1/2 is necessary for its secretion and function.ECS1/2 secreted outside the zygote can directly bind and cleave LURE1,the pollen tube attractant,to reduce its activity,thus preventing the attraction of redundant pollen tubes.This study was the first to reveal that egg cells can sense the successful fertilization process to block polytubey.It could control the number of visiting male partners for maximum offspring of plants.