Biological Function Of Complex N-glycan Formation In Plant

Asparagine-linked glycosylation(Asn/N-glycosylation)is an important protein co-and post-translational modification in eukaryotic cells.In the ER,a pre-assembled lipid-linked tetradecylsaccharide is transferred by oligosaccharyltransferase to Asn residue within the canonical motif N-X-S/T(x can be any amino acid but proline;S/T,serine and threonine)on nascent peptides.N-linked glycans(N-glycans)are crucial signals for ER quality control(ERQC)and ER-associated protein degradation(ERAD).In both plants and animals,with the secretion of correctly folded proteins to Golgi,the associated N-glycans are further modified to form complex N-glycan.Although defects in complex N-glycan formation lead to CDG(congenital disorder of glycosylation)diseases in human and salt-sensitive root phenotypes in plants,the biological significance of complex N-glycan biosynthesis remains largely unknown.Through the phenotypic comparison of two Arabidopsis mutant defective in complex N-glycan against the high salinity stress,it was found that loss function of Golgi N-glycan trimming led to a more sensitive response of plant to the stress.Detailed analyses using genetic,biochemical and histological methods suggested that the trimming of N-glycan in Golgi plays an important role for generating the signal for directing the substrate proteins maintaining the homeostasis or secretion.The main discoveries and results are listed below.I.Phenotypic analyses between the mnsl mns2 and cgll mutants demonstrated NMNS1/2-mediated N-glycan trimming was required for abiotic stress responses in Arabidopsis.Although primary root growth was significantly inhibited both in mnslmns2 and cgll under salt stress,the mnsl mns2 double mutant showed more sensitive phenotype.Moreover,the mnsl mns2 mutant was responded to mild osmotic stress but not cgll.Failure of complex N-glycan formations in both mutants was confirmed by immunoblot assay.Moreover,wild-type plants were reminiscent of mnsl mns2-like salt-sensitive root morphology in the presence of class I a-mannosidase(MNSI)inhibitor.In addition,complementary expression of MNS1 in mnsl mns2 rescued root growth deficiency under salt condition and excluded the potentially allele-dependent salt hypersensitive phenotype.Above results confirmed that abolishment of two functionally redundant mannosidases MNS1 and MNS2 responsible for N-glycan trimming in Golgi led to salt-oversensitivity in plants.II.The mechanism of MNS1/2 mediated ?1,2-mannose cleavage regulating plant responses to abiotic stress was investigated.The N-glycan structures under distinct mutant backgrounds including in the mnsl mns2 and cgll were analyzed.The alg3,ebs3 and ebs4 which lack of arm C on N-glycan displayed weaker root growth arrest upon the medium in the presence of MNSI inhibitor and high dose of salt,indicating MNS1/2-mediated N-glycan trimming may generate a regulatory code regulating client proteins in response to salt stress.?.MNS1/2 mediated N-glycan trimming modulated abundance of RSW2,a glycoprotein which is crucial for cellulose biosynthesis.The aberrant root phenotypes of the mnsl mns2 corresponded to severe cell wall deficiency,which was reminiscent of the rsw2-1.RSW2 was a salt-sensitive gene and the protein abundance decreased significantly in mnsl mns2,which was in agreement with the detection of RSW2-GUS expression.Surprisingly,the triple mutant mnsl mns2 rsw2-1 showed a much severe phenotype even seedling lethal and the western blot analysis showed only the trace amount of rsw2-1 was detected.The genetic cross between ebs3 or ebs4 and rsw2-1 stablized rsw2-1 protein level to some extent in the presence of MNS inhibitor,supporting the pre-mentioned discovery.That is,N-glycan C arm contains regulatory signal for rsw2-1 abundance.IV.The underlying molecular mechanism of N-glycan signal regulating rsw2-1 protein level in Golgi.Genetic cross and phenotypic analyzes showed that mns4 mns5 mutations involved in ERAD had no effect on rsw2-1 abundance,indicating the abundance of rsw2-1 protein may be regulated in an ERAD-independent pathway.V.Comparative proteomic analysis between mnsl mns2 and cgll by label-free mass spectrum.Protein profiling by qualitative and quantitative methods supplied direct evidence that salt stress responsive proteins involving in different pathways were influenced in mnsl mns2 and cgll.Taken together,I drew conclusions that the N-glycan trimming and maturation in Golgi were essential for abiotic stress responses by regulating the levels of client proteins and the N-glycans are crucial regulatory codes in finely controlling protein degradation and secretion in eukaryotic cells.