Correlation of β-hexosaminidases and α-mannosidases with changes in protein conjugated N-glycan profiles during peach ripening

Abstract

Recent studies have demonstrated that N-glycan and its metabolic pathway are crucial in the ripening and softening processes of peaches, as evidenced by the significant upregulation of processing enzymes such as α-mannosidase (α-Man) and β-hexosaminidase (β-Hex). However, little is known about the changes of protein conjugated N-glycans in peaches during storage. In this study, a systematic comparison of the N-glycan profiles of three peach cultivars with distinct flesh types, specifically, the soft-melting “Hu Jing”, the hard-melting “Xia Hui 6″, and the stonyhard “Xia Cui” was provided. The study monitored the structure and content changes of these N-glycans during storage. Fourteen different N-glycan structures were identified using Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. A consistent N-glycan profile with varying relative content was observed among three cultivars. MMXF³, GnGnXF³, and MM were characterized as representative structures associated with ripening, showing statistically significant correlations with firmness. The conversion of GnGnXF³ to MMXF³ were proposed to be catalyzed by β-Hexs, which exhibited significantly increased activities in all three cultivars during storage. The degradation of high-mannose N-glycans to yield MM structure were observed, but no significant increase in α-Man activities was observed. Real-time qPCR results demonstrated that distinct gene expression patterns of β-Hexs and α-Mans existed among three cultivars. The expressions of genes hex0020 and man4915 were highest and exhibited a climacteric pattern in the “Hu Jing” cultivar. In contrast, these same genes displayed a double-peak expression pattern in the “Xia Hui 6” cultivar and steadily decreased in the “Xia Cui” cultivar. Our findings provide detailed insights into the changes of N-glycome associated with peach ripening and suggest potential target genes for preventing excessive softening in peaches. However, further investigation is needed to clarify the role of N-glycosylation and its related metabolic enzyme genes in peach fruit ripening and softening.