N-glycosylated proteome reveals zinc sulphate-induced high enrichment of melatonin-related glycosylated proteins in hull-less barley

Melatonin, an indoleamine compound, can enhance the endurance of plant tissues against adverse conditions, such as various environmental stresses. In this study, hull-less barley seeds were used as experimental materials to analyse the N-glycosylation proteomics induced under ZnSO 4 stress. LC-MS/MS-based quantitative proteomics was employed to study N-glycosylation modifications. Under 0.8 mM ZnSO 4 stress conditions, melatonin content reached its highest level at 13.41 ng/g. The melatonin was significantly correlated with the reducing sugar content (P<0.01). A total of 273 glycosylation sites, corresponding to 256 glycosylated peptides and 210 unique glycosylated proteins, were identified in the three sample groups (CK, Zn, and S). The results indicated that the abundance of glycosylated proteins, including peptidases, peroxidases, glycosidases, and several featureless proteins, was significantly altered in high-melatonin barley seeds under ZnSO 4 stress. Glycosylation sites were analysed with their modification sites mainly located in the α-helix structure and irregularly coiled region. The functional enrichment analyses revealed that these significantly altered glycoproteins were mainly involved in phenylpropanoid biosynthesis, carbohydrate metabolism, protein processing in the endoplasmic reticulum, and hydrogen peroxide catabolism. These findings offer a deeper understanding of the molecular mechanisms that trigger the production of melatonin-related glycosylated proteins in barley seeds under ZnSO 4 stress. They also supply valuable data to aid in the cultivation of barley seed varieties with high melatonin content. • melatonin was significantly negatively correlated with the reducing sugar. • First analysis of melatonin enrichment mechanism using N-glycosylated proteomics. • the α-helix structure and irregularly coiled region are main modification sites. • Glycosylation of glycosidases, peptidases, and peroxidases represents potential targets for melatonin enrichment.