Glycoside-specific metabolomics reveals the novel mechanism of glycinebetaine-induced cold tolerance by regulating apigenin glycosylation in tea plants.

Glycosylation is a key modification that affects secondary metabolites under stress and is influenced by glycinebetaine (GB) to regulate plant stress tolerance. However, the complexity and detection challenges of glycosides hinder our understanding of the regulatory mechanisms of their metabolic interaction with GB during stress. A glycoside-specific metabolomic approach utilizing cone voltage-induced in-source dissociation was developed, achieving precise and high-throughput detection of glycosides in tea plants by narrowing the target ion range by 94.3%. Combined with enzyme activity assays, exogenous spraying, and gene silencing, this approach helps investigate the role of GB-glycosides cascade effect in enhancing cold tolerance of tea plants. Our method demonstrated that silencing betaine aldehyde dehydrogenase (CsBADH1) in tea plants altered 60 glycoside ions while reducing GB content and cold tolerance, indicating that glycosylation affects GB-mediated cold tolerance. By combining glycoside-specific with conventional metabolomics, isorhoifolin, a GB-regulated cold response metabolite was discovered, and its precursor apigenin was found to be a new cold tolerance metabolite that enhanced cold tolerance by scavenging reactive oxygen species. This study reveals a new mechanism by which GB mediated cold tolerance in tea plants through regulating apigenin glycosylation, broadening our understanding of the role of glycosylation in plant cold tolerance.