Molecular imaging of glycans has been actively pursued in animal systems for the past decades. However, visualization of plant glycans remains underdeveloped, despite that glycosylation is essential for the life cycle of plants. Metabolic glycan labeling in Arabidopsis thaliana by using N-azidoacetylglucosamine (GlcNAz) as the chemical reporter is reported. GlcNAz is metabolized through the salvage pathway of N-acetylglucosamine (GlcNAc) and incorporated into N-linked glycans, and possibly intracellular O-GlcNAc. Click-labeling with fluorescent probes enables visualization of newly synthesized N-linked glycans. N-glycosylation in the root tissue was discovered to possess distinct distribution patterns in different developmental zones, suggesting that N-glycosylation is regulated in a developmental stage-dependent manner. This work shows the utility of metabolic glycan labeling in elucidating the function of N-linked glycosylation in plants.

Molecular imaging of glycans has been actively pursued in animal systems for the past decades. However, visualization of plant glycans remains underdeveloped, despite that glycosylation is essential for the life cycle of plants. Metabolic glycan labeling in Arabidopsis thaliana by using Nazidoacetylglucosamine (GlcNAz) as the chemical reporter is reported. GlcNAz is metabolized through the salvage pathway of N-acetylglucosamine (GlcNAc) and incorporated into Nlinked glycans, and possibly intracellular O-GlcNAc. Clicklabeling with fluorescent probes enables visualization of newly synthesized N-linked glycans. N-glycosylation in the root tissue was discovered to possess distinct distribution patterns in different developmental zones, suggesting that N-glycosylation is regulated in a developmental stage-dependent manner. This work shows the utility of metabolic glycan labeling in elucidating the function of N-linked glycosylation in plants.Plants synthesize diverse glycans that are essential for their life cycles. The cell wall, an extracellular matrix surrounding plant cells, is mostly composed of high-molecular-weight polysaccharides, including cellulose, hemicellulose, and pectin. [1,2] Furthermore, glycoproteins modified with N-or/ and O-linked glycans are synthesized in plants and play important functional roles. [3][4][5] Comparing to glycans in animals, plant glycosylation has received relatively less attention for the past several decades.[6] As a result, development of techniques for visualizing glycans has mainly focused on animal glycosylation. [7][8][9] In particular, a chemical reporter strategy based on metabolic labeling of glycans with unnatural monosaccharide analogues containing a bioorthogonal functional group (for example, an azide or alkyne) has emerged as a powerful method for glycan imaging and functional studies in animal systems. [10][11][12] The promise of generating renewable energy from glycan-enriched biomass has lately sparked a growing interest in plant glycobiology. [13] The glycan labeling and imaging techniques developed in animal systems, if can be adapted for plants, will be invaluable for probing biosynthesis and biological function of plant glycans.The glycan biosynthetic pathways in plants possess many distinct features, and a variety of unique plant glycans have been identified.[6] Therefore, evaluation of unnatural sugar reporters in plants is critical for developing metabolic glycan labeling methods for plants. For example, 6-alkynyl fucose (FucAl), a fucose analogue containing an alkyne, was used to metabolically label the fucose-containing pectin in cell walls, and no incorporation of FucAl into fucosylated proteins was observed.[14] Distinctly, FucAl primarily labels fucosylated proteins in mammalian cells. [15] Moreover, an azido analogue of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo), a monosaccharide that does not exist in animals, was recently exploited to metabolically label pectin.[16] Metabolic incorporation of FucAl and azido Kdo into pectins has enabled c...

Protein O-GlcNAcylation is a ubiquitous posttranslational modification occurring both in animals and plants. While thousands of O-GlcNAcylated proteins have been identified in animals, the plant O-GlcNAcylated proteome remains poorly studied. Herein we report the development of a chemoproteomic strategy for profiling of O-GlcNAcylated proteins in Arabidopsis based on the metabolic glycan labeling (MGL) method. We first demonstrated that both Nazidoacetylglucosamine (GlcNAz) and N-azidoacetylgalactosamine (GalNAz) can metabolically label O-GlcNAc with azides in Arabidopsis seedlings. Arabidopsis UDP-galactose 4epimerases were found to interconvert UDP-GalNAz and UDP-GlcNAz, supporting the existence of a GalNAc metabolism pathway. By tagging the azide-incorporated O-GlcNAc with alkyne-biotin via click chemistry, the O-GlcNAcylated proteins were enriched and analyzed by mass spectrometry. We identified 645 candidate O-GlcNAcylated proteins in Arabidopsis seedlings, of which 592 were newly identified. The identified O-GlcNAcylated proteins were enriched in various plant-specific processes such as hormone responses. By coexpression of a selected list of the identified proteins with SECRET AGENT, the Arabidopsis O-GlcNAc transferase, we validated that the MGL-identified proteins were O-GlcNAcmodified. Our work establishes a powerful tool for profiling plant O-GlcNAylation and provides an invaluable resource for investigating the functional role of O-GlcNAc in Arabidopsis.