Mechanism Analysis Of N-glycosylation In Regulating Stomatal Development

Stomata are micropores on leaf surface bounded by two guard cells and act as the major route for water and gas exchange between the exterior atmosphere.The stomata development is strictly controlled by multiple internal and external factors,in which plant hormones are one of the most efficient regulators,and the metabolic balance of plant hormones is closely related to ?-glucosidase.N-glycosylation is one of the most important and essential protein modification processes in eukaryotes,affecting folding,transport and function of a wide range of proteins.Some studies have shown that the disturbance of N-glycosylation significantly decrease the plant tolerance to drought and high salinity,accompanied by the increase of transpirational water loss.In order to investigate if N-glycosylation participates in the regulation of stomatal development,the stt3 a mutant,defective in the catalytic subunit of oligosaccharyltranferase(OST),was used to analyze the stomatal phenotype and density on leaves under different growth stages.The possible molecular mechanisms are also analyzed.The major discoveries are listed below:1.The transpirational water loss rate increased significantly in stt3a-2,and the stomatal density on the first true leaf was significantly higher than that of the wild type at the same growth stage,accompanied by some abnormal stomatal clusters. Correspondingly,the expression of transcriptional factors in stomatal development SPCH,MUTE and FAMA were up-regulated in stt3a-2 mutant;The introduction of the wild-type STT3 A gene driven by the native or 35 S promoter complemented the stomatal density,salt tolerance and rate of water loss of the stt3a-2 mutant to a level comparable to wild type.The knockout seedlings generated by CRISPR/Cas9 method showed a similar stomatal dysplasia phenotype to stt3a-2.Besides,the root hair density and lateral root length of STT3A-2 were lower compared with the wild type,and Nglycation deficiency also caused the decrease of photosynthetic capacity.2.The contents of ABA and IAA were low in the stt3a-2 mutant and exogenously application of ABA or IAA reduced the stomatal density and the salt-sensitive phenotype of the stt3a-2 in a certain dose-dependent manner.3.At BG1,a ?-glucosidase,hydrolyzing biologically inactive glucose-conjugated hormones to release active form was confirmed being a glycoprotein and its activity and stability were decreased in the stt3a-2;Overexpression of At BG1 in stt3a-2 seedlings partially rescued the salt sensitivity.4.TMM,the key receptor molecular switch for stomatal development,was also regulated by N-glycosylation.The removal of three N-glycosylation sites on TMM protein failed to restore the abnormal stomatal phenotype of the tmm mutant and led to a rapid degradation and mis-targeting of TMM.In addition,the interaction between TMM and ERf was attenuated in the stt3a-2 mutant in BIFC(bimolecular fluorescence complementation)analysis.According to the results above,it is concluded that the loss of N-glycosylation simultaneously causes the abnormality of ?-glucosidase function and the interaction of TMM-ERf,a key stomatal receptor molecule switch.The superposition of these two factors further leads to the up-regulation of SPCH,MUTE and FAMA.The increased stomatal density and transpirational water loss may be the major reasons in the decline of plant tolerance to drought or salt stress.