Chemoproteomic profiling of O‐GlcNAcylated proteins and identification of O‐GlcNAc transferases in rice

O‐linked β‐N‐acetylglucosaminylation (O‐GlcNAcylation) is a ubiquitous post‐translation modification occurring in both animals and plants. Thousands of proteins along with their O‐GlcNAcylation sites have been identified in various animal systems, yet the O‐GlcNAcylated proteomes in plants remain poorly understood. Here, we report a large‐scale profiling of protein O‐GlcNAcylation in a site‐specific manner in rice. We first established the metabolic glycan labelling (MGL) strategy with N‐azidoacetylgalactosamine (GalNAz) in rice seedlings, which enabled incorporation of azides as a bioorthogonal handle into O‐GlcNAc. By conjugation of the azide‐incorporated O‐GlcNAc with alkyne‐biotin containing a cleavable linker via click chemistry, O‐GlcNAcylated proteins were selectively enriched for mass spectrometry (MS) analysis. A total of 1591 unambiguous O‐GlcNAcylation sites distributed on 709 O‐GlcNAcylated proteins were identified. Additionally, 102 O‐GlcNAcylated proteins were identified with their O‐GlcNAcylation sites located within serine/threonine‐enriched peptides, causing ambiguous site assignment. The identified O‐GlcNAcylated proteins are involved in multiple biological processes, such as transcription, translation and plant hormone signalling. Furthermore, we discovered two O‐GlcNAc transferases (OsOGTs) in rice. By expressing OsOGTs in Escherichia coli and Nicotiana benthamiana leaves, we confirmed their OGT enzymatic activities and used them to validate the identified rice O‐GlcNAcylated proteins. Our dataset provides a valuable resource for studying O‐GlcNAc biology in rice, and the MGL method should facilitate the identification of O‐GlcNAcylated proteins in various plants.