N‐glycan remodeling by α‐D‐mannosidase and β‐D‐N‐acetylhexosaminidase regulates fruit softening, redox balance, and post‐harvest pathogen resistance

Post-harvest loss of fruits and vegetables poses significant challenges to food security and economic sustainability, primarily due to ripening-associated excessive softening that shortens shelf life and increases susceptibility to pathogens. N-glycans, N-glycoproteins, and their processing enzymes are integral to various plant processes, including fruit ripening. Among these, α-D-mannosidase (α-Man) and β-D-N-acetylhexosaminidase (β-Hex) are key ripening-specific enzymes that modulate fruit softening. Previously, we have shown that RNAi-mediated suppression of α-Man or β-Hex improves fruit shelf life and firmness in both climacteric and non-climacteric fruits. However, the underlying molecular and biochemical basis of fruit softening regulation by α-Man and β-Hex was not well understood. In this study, we developed transgenic tomato (Solanum lycopersicum) plants by silencing α-Man and β-Hex simultaneously using RNAi. Suppression of these enzymes reduces N-glycoprotein degradation, downregulates pectin dissolution, and inhibits ripening-related gene expression. RNAi fruits exhibited enhanced shelf life, greater firmness, reduced reactive oxygen species (ROS) accumulation and increased resistance against post-harvest pathogens without affecting plant growth, fruit development, yield, or nutritional quality. To further explore the molecular mechanism of α-Man and β-Hex function, we purified and quantified N-glycans in RNAi fruits and other ripening-impaired mutants, identifying key N-glycan species. We also carried out iTRAQ-based quantitative proteome profiling to investigate the abundance of proteins in ripened fruit affected by silencing of α-Man and β-Hex. Molecular insights revealed that N-glycan processing and degradation are key events during ripening, influencing cell wall softening, fruit redox state, and post-harvest quality attributes. This study highlights the potential of co-silencing α-Man and β-Hex as a novel approach to extending the shelf life of fruits, regardless of their climacteric behavior, without compromising quality or yield.