High-throughput Proteomic And Phosphoproteomic Analyses Of Mature Maize Pollen

Corn is the largest food crop in China.In flowing plants,diploid sporophyte is developed into haploid gametophytes,which undergoes mitosis and meiosis.The mature male pollen consists of three haploid cells,which are all derived from a single haploid meiotic product of a pollen mother cell.And two of three cells are sperm cells,one of them is a vegetative cell.The pollen grain plays an essential role in sexual reproduction of higher plants,with its primary function of delivering sperm cells to the female gametophyte via the formation of a pollen tube.This process depends on cell-cell communication and rapid cellular responses.Mature pollen from most plant species is metabolically quiescent;include maize,however,after pollination,it germinates quickly and gives rise to a pollen tube to transport sperms into the embryo sac.Various studies have emphasized that a mature,dehydrated pollen grain contains all the transcripts and proteins required for germination and initial pollen tube growth without de novo transcription.Therefore,it is important to explore the role of post-translational modifications(here phosphorylation),through which many processes induced by pollination are probably regulated.In this study we target maize mature pollen as experimental material,In order to investigate posttranslational modification state of mature pollen,the protein phosphorylation map of maize pollen is constructed by mass spectrometry technology.Using HPLC-MS,100990 peptides from 6750 unique proteins were counted in maize pollen totally.To investigate the phosphorylation state in maize pollen,we enriched phosphoproteins and phosphopeptides using Strong cation exchange chromatography-Immobilized Metal Affinity Chromatography,a broad coverage of pollen phosphoproteins with 5291 phosphorylation sites corresponding to 2257 phosphoproteins was obtained.Among these sites,there were 4314 phosphoserine(pS)sites,764 phosphothreonine(pT)sites,and 213 phosphotyrosine(pY)sites.The sites of pS,pT,and pY separately occupy 81.53%,14.44%,and 4.03%of all phosphorylation location.By using Motif-X,twenty-three phosphoserine motifs,four phosphothreonine motifs were identified.In summary,this study enhances our knowledge on the role of phosphorylation in plant reproduction,provides a framework to understand the dynamics of protein phosphorylation during pollination,and suggests processes that may be regulated by phosphorylation during pollen development.