The Functions Of Arabidopsis Oligosaccharyltransferase Complex

Asparagine(ASN,N)-linked protein glycosylation because of their glycan branched maintaining structure variability and binding specificity ligands,is becoming one of the most important,generally,and complex protein modification during and after protein translational bioprocess.N-linked protein glycosylation plays a major roles in many eukaryotic proteins,it can promote protein folding,quality assurance,and endoplasmic reticulum-associated degradation as well as serving in protein sorting,regulatory functions,and in signal transduction.In angiosperms,double fertilization is a complex and precise fertilization system.It involves the joining of female gametophyte with male gametophyte and both the communication female and male gametophytes.When pollen tube enters the ovule,pollen tube releases two sperm cells,sperm cells fuse with egg cell and central respectively in the process known as double fertilization.The last stage of male gametophyte development,pollen tube burst(or PT reception),is controlled by the communication between male and female gametophytes and this biological process is the hot spot of present research.We found one mutant named ptbl-1(pollen tube burst 1-1)which is phenocopy fer-1.Homozygotes ptbl mutant plants could not be recovered for mutant pollen grains degenerate before maturation.Aniline blue staining showed that the pollen tube fails to burst and continually grows inside the embryo sac.We then found that in ptb1-1 a T-DNA was inserted in the eighth exon of At5g66680 by TAIL-PCR.The At5g66680 genomic fragments could successfully complement the ptb1-1/PTB1-1 phenotype.Another allele mutant ptbl-2/PTB1-2 showed similar phenotype with ptbl-1.The location of PTB1-GFP fusion protein shows that PTB1 expressed in majority development process of embryo sac of Arabidopsis,especially in synergid cell.It is also highly expressed in pollen grains.Besides,specific expression of PTB1 in the synergid cells could recover the phenotype of abortion ovule,indicating PTB1 played its role effectively in synergid cell.Bioinformatics has shown that PTB is a protein ortholog of human OST48 or yeast Wbp1,one of subunit of the oligosaccharyltransferase(OST)complex.OST complex is responsible for the transfer of the N-linked glycan precursor onto Asn residues of candidate proteins in the endoplasmic reticulum and then complete the process of N-linked protein glycosylation.We preformed a systematic site-diredted mutagenesis study and generated mutant versions of FER laking varing numbers of potential N-glycosylation sites in FER.The mutant versions of FER were transformed into fer-1 respectively to assess their functionality.The results showed that specific N-glycosylation genetic locus was essential for FER function.Biochemical test result also showed that FER was glycosylated in wild type Arabidopsis.On the other hand,the subcellular localization of FER-GFP fusion protein was affected in ptb1.The results suggested that PTB1 could influence the reception of pollen tube by glycosylating FER.In Arabidopsis thaliana,OST complex had not been systematically investigated yet.Through co-IP assay and mass spectrometry analysis,we found some other crucial genes that are homologous with genes of OST complex in yeast,including STT3A/B,OST1A/B,OST2,OST3B,OST6 and then confirm their interactions among them by BiFC.Together,these results confirm OST complex directed FER N-linked glycosylation is critical for the interaction between the female and male gametophytes in pollen tube reception.