Asparagine (Asn/N)-linked glycans are important for protein folding, trafficking, and endoplasmic reticulum-associated degradation in eukaryotes. The maturation of glycoproteins involves the trimming of mannosyl residues by mannosidases and addition of other sugar molecules to three-branched N-glycans in the Golgi. However, the biological importance of Golgi-mediated mannose trimming is not fully understood. Here, we show that abolishment of two functionally redundant mannosidases, MNS1 and MNS2, responsible for α-1,2-mannose trimming on the A and C branches of plant N-glycans lead to severe root growth inhibition under salt stress conditions in Arabidopsis. In contrast, mutants with defects in the biosynthesis of the oligosaccharide precursor displayed enhanced salt tolerance in the absence of mannose trimming. However, mutation in EBS3, which is required for the formation of the branched N-glycan precursor, suppressed the salt-sensitive phenotype of mns1 mns2 double mutant. Interestingly, we observed that cellulose biosynthesis was compromised in mns1 mns2 roots under high salinity. Consistently, abundance of a membrane anchored endo-β-1,4-endoglucanase (RSW2/KOR) that plays a key role in cellulose biosynthesis and its mutant variant rsw2-1 were modulated by α-1,2-mannose trimming under salt stress. Overexpression of RSW2 could partially rescue the salt-sensitive phenotype of mns1 mns2. Taken together, these results suggest that MNS1/2-mediated mannose trimming of N-glycans is crucial in modulating glycoprotein abundance to withstand salt stress in plants.

Abstract Asparagine-linked glycosylation (N-glycosylation) is one of the most important protein modifications in eukaryotes, affecting the folding, transport, and function of a wide range of proteins. However, it is still less known about the roles of N-glycosylation in the development of stomata in plants. In the present study, we provided evidence that the stt3a-2 mutant, defective in oligosaccharyltransferase (OST) catalytic subunit STT3, had a greater transpirational water loss and weaker drought avoidance, accompanied by aberrant stomatal distribution. Through physiological, biochemical, and genetic analyses, we found that the abnormal stomatal density of stt3a-2 was partially attributed to a low endogenous abscisic acid (ABA) and auxin (IAA) content. Exogenous application of ABA or IAA could partially rescue the mutant salt-sensitive and abnormal stomatal phenotype. Further analyses revealed that the decrease of IAA or ABA in stt3a-2 seedlings was associated with the under-glycosylation of β-glucosidase (AtBG1), catalyzing the conversion of conjugated ABA/IAA to active hormones. Our results provide strong evidence that N-glycosylation is involved in stomatal development and participates in abiotic stress tolerance by affecting the release of active plant hormones.

Many pieces of evidence show that the adaptive response of plants to salt stress requires the maturation of N-glycan on associated proteins. However, it is still little known about the salt-responsive glycoproteins that function in this process. In the present study, we identified salt-responsive glycoproteins in wild-type (WT) Arabidopsis and two mutants defective in N-glycan maturation, mns1 mns2 and cgl1. A total of 97 proteins with abundance changes of >1.5‐ or <0.67-fold were identified against salt stress by label-free liquid chromatography coupled mass spectrometry (LC-MS/MS) quantitative analyses. A comparison of differentially abundant glycoproteins (DAGs) indicated the substrate preferences regulated by MNS1/MNS2 and CGL1. In addition, the DAGs in mns1 mns2 hardly form functional regulatory networks in STRING analysis. Comparably, the regulatory network in cgl1 was visible and shared overlapping with that in WT. Such difference may supply the evidence to partially explain the lower salt sensitivity of mutant cgl1 than mns1 mns2. We further confirmed that two N-glycosylation clients, peroxidases PRX32 and PRX34, were involved in the salt stress response since the double mutants showed enhanced salt sensitivity. Together, our study provided proteomic evidence that N-glycans are crucial for modulating stress-responsive protein levels, and several novel glycoproteins responsible for salt stress tolerance in Arabidopsis were listed. Data are available via ProteomeXchange with identifier PXD006893.

We employed hi-TAIL-PCR to identify T-DNA loci in our activation tagging library and only a total of 28 (39%) insertion sites from 72 samples were characterized when the recommended primer pools, C1 and C2 were used. By comparison, we found C1 harboring relatively low degeneracy was more efficient to amplify the flanking sequences of T-DNA insertion than C2. We replaced the degenerate sequences in long arbitrary degenerate (LAD) primers with a piece of 16-bp degenerate sequence originally used in TAIL-PCR, which had the relatively low degeneracy. Our results showed that the new LAD primer pool N increased the valid amplifications and a total of 37 (51%) T-DNA loci were identified, indicating a more effective amplification of T-DNA flanking sequences in.

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