A conserved asparagine residue in the inner surface of BRI1 superhelix is essential for protein native conformation.

Asparagine-linked glycosylation (ALG, N-glycosylation) is one of the most prevalent protein modifications in eukaryotes and regulates protein folding, trafficking and function. Recently, we reported that the mutation of N154Q significantly led to the ER retention of brassinosteroids insensitive 1 (BRI1), the receptor of brassinosteroids (BRs). However, the mechanism of how the N154 site affects BRI1 structure is still not completely clear. In current study, we found that the removal of N154-glycan with S156A replacement significantly enhanced the ability of bri1 to complement bri1-301 mutant and plasma membrane localization compared with N154Q. In addition, the various mutations on N154 site resulted in bri1 retention in the ER, except for N154D. The 3D modeling suggested that there existed polar contacts around N154 site and the mutations not only destroyed the addition of N-glycan on the site, but also led to the disorder of hydrogen bonds formation. The sequence analysis showed that the N275 shared more similarity with N154 site and the removal of N275-glycan further enhanced the retention of bri1 carrying S156A mutation in the ER. Our results showed that N154 was special and essential for maintaining BRI1 structure and explored the role of those residues and key N-glycans lying in the LRR inner surface on protein conformation.

Comparative Label-Free Quantitative Proteomics Analysis Reveals the Essential Roles of N-Glycans in Salt Tolerance by Modulating Protein Abundance in Arabidopsis.

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.

N-glycosylation is involved in stomatal development by modulating the release of active abscisic acid and auxin in Arabidopsis.

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, little is known about the roles of N-glycosylation in the development of stomata in plants. In the present study, we provide evidence that the Arabidopsis stt3a-2 mutant, defective in oligosaccharyltransferase catalytic subunit STT3, has 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 low endogenous abscisic acid (ABA) and auxin (IAA) content. Exogenous application of ABA or IAA could partially rescue the mutant's salt-sensitive and abnormal stomatal phenotype. Further analyses revealed that the decrease of IAA or ABA in stt3a-2 seedlings was associated with the underglycosylation of ß-glucosidase (AtBG1), catalysing the conversion of conjugated ABA/IAA to active hormone. Our results provide strong evidence that N-glycosylation is involved in stomatal development and participates in abiotic stress tolerance by modulating the release of active plant hormones.

Multiple N-glycans cooperate in balancing misfolded BRI1 secretion and ER retention.

KEY MESSAGE: N-glycans play a protective or monitoring role according to the folding state of associated protein or the distance from structural defects. Asparagine-linked (Asn/N-) glycosylation is one of the most prevalent and complex protein modifications and the associated N-glycans play crucial roles on protein folding and secretion. The studies have shown that many glycoproteins hold multiple N-glycans, yet little is known about the redundancy of N-glycans on a protein. In this study, we used BRI1 to decipher the roles of N-glycans on protein secretion and function. We found that all 14 potential N-glycosylation sites on BRI1 were occupied with oligosaccharides. The elimination of single N-glycan had no obvious effect on BRI1 secretion or function except N154-glycan, which resulted in the retention of BRI1 in the endoplasmic reticulum (ER), similar to the loss of multiple highly conserved N-glycans. To misfolded bri1, the absence of N-glycans next to local structural defects enhanced the ER retention and the artificial addition of N-glycan could help the misfolded bri1-GFPs exiting from the ER, indicating that the N-glycans might serve as steric hindrance to protect the structure defects from ER recognition. We also found that the retention of misfolded bri1-9 by lectins and chaperones in the ER relied on the presence of multiple N-glycans distal to the local defects. Our findings revealed that the N-glycans might play a protective or monitoring role according to the folding state of associated protein or the distance from structural defects.

Trimming of N-Glycans by the Golgi-Localized α-1,2-Mannosidases, MNS1 and MNS2, Is Crucial for Maintaining RSW2 Protein Abundance during Salt Stress in Arabidopsis.

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-ß-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.

A low degenerate primer pool improved the efficiency of high-efficiency thermal asymmetric interlaced PCR to amplify T-DNA flanking sequences in Arabidopsis thaliana.

We employed hi-TAIL-PCR to identify T-DNA loci in our Arabidopsis 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 A. thaliana.