Quantitative proteomics analysis identified new interacting proteins of JAL30 in Arabidopsis.

Jacalin-related lectins (JALs) are a unique group of plant lectins derived from the jacalin protein family, which play important roles in plant defense responses. JAL30/PBP1 (PYK10 binding protein 1) interacts with inactive PYK10, exerting negative regulatory control over the size of the PYK10 complex, which is formed and activated upon insect or pathogen invasion. However, the precise interplay between JAL30 and other components remains elusive. In this study, we found JAL30 as a nucleocytoplasmic protein, but no obvious phenotype was observed in jal30-1 single mutant. Through immunoprecipitation (IP) enrichment combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS), dozens of new JAL30 interacting proteins were found in addition to several reported ones. Gene Ontology (GO) analysis revealed that these interacting proteins were highly related to the wounding and bacterial stimuli, suggesting their potential involvement in the jasmonate (JA) response. Importantly, the expression of JAL30 was induced by MeJA treatment, further highlighting its relevance in plant defense mechanisms. A novel JAL30 interacting protein, ESM1, was identified and its interaction with JAL30 was confirmed by Co-Immunoprecipitation. Moreover, ESM1 was found as an O-GlcNAcylated protein, suggesting that JAL30 may possess glycosylated protein binding ability, particularly in O-GlcNAcylated protein and peptide recognition. Overall, our study provides valuable insights into the interacting protein network and biological function of JAL30, demonstrates the interaction between JAL30 and ESM1, and uncovers the potential significance of JAL30 in plant defense system, potentially through its association with PYK10 complex or JA response. SIGNIFICANCE: The biological functions of lectin proteins, including defense responses, immunity responses, signal transduction, have been well studied. Lectin proteins were also utilized to enrich glycosylated proteins for their specific carbohydrates binding capability. Jacalin-related lectins (JALs) were found to involve in plant defense mechanism. However, it is not yet clear whether JALs could use for enrichment of glycosylated proteins. In this study, we used label-free quantification method to identify interacting proteins of JAL30. A novel interacting protein, ESM1, as an O-GlcNAcylated protein was found. ESM1 has been reported to take part in defense against insect herbivory. Therefore, our findings provided experimental evidence to confirm that JALs have potential to be developed as the bio-tools to enrich glycosylated proteins. Finally, our data not only illustrated the vital biological role of JALs in plants, but also verified unique function of JAL30 in recognizing O-GlcNAcylated proteins.