Identification Of Hydroxyproline-containing Proteins And Function Of Hong Xians In Double Fertilization Of Rice

The glycosylation modification,one kind of protein modifications,plays indispensable roles in the function of glycoproteins in plants.The hydroxyproline-containing proteins(HCPs)contain hydroxyproline(O,Hyp)residues,of which some HCPs are O-glycosylated at the Hyp residues.To date,studies on HCPs have focused on hydroxyproline-rich glycoproteins(HRGPs),including highly glycosylated arabinogalactan proteins(AGPs),moderately glycosylated extensins(EXTs),and lightly glycosylated proline-rich proteins(PRPs).Among them,the highly heterogeneous AGPs play important roles in the growth and development of higher plants.A large number of glycoproteins have been identified in different species by bioinformatic methods,but their potential glycosylation modification sites are rarely identified.The ECA1-like proteins have five proteins in rice and the proteins are originally identified as "atypical hydroxyproline-deficient AGPs",three of which played an important role in double fertilization and were named “HONGXIANs(HXs)”.HXs were small peptides that were secreted from cells to the extracellular space and might transmit signal to the target proteins by binding to their receptors.In this study,we used mass spectrometry technique,CRISPR/Cas9 gene editing technology,amino acid sequence alignment,phylogenetic tree analysis,protein three-dimensional structure modeling,whole staining and clearing of ovary,pollen tube staining,quantitative PCR,and bioinformatic analysis,cell biology,genetics and molecular biology research methods,identified a large number of HCPs in rice,and identified hydroxylation sites in the proteins.Moreover,we studied the biological functions of Os HXs and preliminary regulatory mechanism and found that Os HXs were essential for rice double fertilization.The main findings of this study were as follows:1.A large number of proteins were extracted by β-Glc Y reagent in different tissues and organs,and 62 HCPs were identified by mass spectrometry technique.Among the 62 HCPs,114 Hyps were identified,the O-linked glycosylation modification might link to the protein backbones of these HCPs via hydroxylated prolines.The results showed that 57 HCPs were specifically or predominantly expressed in different tissues and organs of rice.The number of new HCPs in pistils was 44,of which 41 HCPs were specific,significantly more than the number of other tissues,which indicated that HCPs were critical for panicle development and seed formation in rice.2.By analyzing the Hyp sites in HCPs,it was found that the characteristic glycomodules of AO,OA,OG,VO,LO,and OE were significantly abundant and the prolines were hydroxylated in these glycomodules.In addition,the glycomodules of AO and OA accounted for high proportion among glycomodules,suggesting that alanine residues were more important for the hydroxylation and glycosylation of prolines.Further analysis revealed that the Hyp sites in AGPs were located in the AGPs-like domains,indicating that AGPs-like domains in AGPs were potential important regions for their glycosylation.3.Through the study of the expression of different AGP genes,it was found that AGP genes were expressed in different stages of rice growth and development.Furthermore,six genes were screened for specific expression in rice anthers.These anther-specific genes may play important roles in the development of anthers and even the growth of pollen tubes,which can be used as marker genes for study.The osfla15,osfla22,osfla25 and osela3 mutants were obtained by CRISPR/Cas9 technology,but phenotypic observations showed no obvious defects in plant growth and development,especially in anther development,suggesting that AGPs might have functional redundancy in rice.It will be necessary to obtain multi-gene mutants for further research.4.The HX proteins belonging to the ECA1-like proteins were found to be conserved in angiosperms by amino acid sequence alignment and phylogenetic tree analysis.In the Amborella trichopoda,which is considered to be the most primitive angiosperm,the HXs homologous proteins were also found.Further analysis revealed that there were a total of 124ECA1-like proteins in Arabidopsis,while only 17 in Medicago sativa.In rice,we found that there were only five ECA1-like proteins,three of which had been identified to be HXs proteins.Furthermore,HX2 and HX3 proteins were more homologous.However,phylogenetic tree analysis found that there were no HXs homologous proteins in lower plants such as algae,fungi and lichen,as well as higher plants such as mosses and ferns,suggesting that HXs were produced for double fertilization in angiosperms.5.We obtained various mutants of Os HXs,including hx1,hx1/2,hx2/3,hx1/2/3 through CRISPR/Cas9 technology.Among these mutants,the plants before flowering,the pollen and embryo sac development were normal.However,partial abortion was observed in hx1,hx1/2,and hx1/2/3 mutants in mature panicles,and the seed setting rates were 63.80%,40.19% and5.21%,respectively.6.Using real-time quantitative PCR and fluorescence confocal microscopic observation,it was found that Os HXs were strongly expressed in egg cells in reproductive tissues,but not in sperm cells.What was more interesting was that unlike the At HXs expressed only in egg cells,the Os HXs were also highly expressed in the 15 DAG shoot and root.It implied that HXs in rice were endowed with new functions during biological evolution.7.Further studies showed that parts of the embryo sac could not be fertilized at 3.5 HAP(hours after pollination),indicating that the HXs caused seed abortion by affecting double fertilization in rice.But,there was no obvious seed abortion in the mutant hx2/3,and the significant sterilization in the mutant hx1/2/3 indicated that HX1 was the major protein affecting rice double fertilization,and the HX2/3 played auxiliary roles.8.Through the staining of the embryo sac by schiff reagent,it was found that the proportions of the normally double-fertilized embryo sacs in the mutant hx1/2 at 8 HAP and32 HAP were 26.56% and 36.19%,respectively.Moreover,they were less than the seed-setting rate of mutant hx1/2(40.19%),which indicated that part of the embryo sacs that were not fertilized at 8 HAP were normally fertilized during later development.Therefore,we further observed embryo sacs through schiff reagent staining and found that the unfertilized embryo sacs at 8 HAP restarted the “remediation” mechanism of attracting the pollen tube,and the other pollen tube re-entered the embryo sac to release the second pair of sperm cells,which was the reason that there were a few normal seeds in the hx1/2/3 mutant.9.Through the observation of the rice mutant hx1/2 embryo sacs,it was found that there was a small part of the single fertilization phenomenon,that is,only the egg cells or only the central cells were fertilized.The proportion of single fertilization was about 5.62%.Using the schiff reagent to stain the embryo sac of the mutant hx1/2,it was found that the two sperm cells entering the embryo sac were in pairs and not separated,which might be the reason of the single fertilization.