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

SUMMARY 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.