The N-glycan processing enzymes alpha-mannosidase and beta-D-N-acetylhexosaminidase are involved in ripening-associated softening in the non-climacteric fruits of capsicum.

ABSTRACT

Excessive softening of fruits during the ripening process leads to deterioration. This is of significant global importance as softening-mediated deterioration leads to huge postharvest losses. N-glycan processing enzymes are reported to play an important role during climacteric fruit softening however, to date these enzymes have not been characterized in non-climacteric fruit. Two ripening-specific N-glycan processing enzymes, α-mannosidase (α-Man) and ß-D-N-acetylhexosaminidase (ß-Hex), have been identified and targeted to enhance the shelf life in non-climacteric fruits such as capsicum (Capsicum annuum). The purification, cloning, and functional characterization of α-Man and ß-Hex from capsicum, which belong to glycosyl hydrolase (GH) families 38 and 20, respectively, are described here. α-Man and ß-Hex are cell wall glycoproteins that are able to cleave terminal α-mannose and ß-D-N-acetylglucosamine residues of N-glycans, respectively. α-Man and ß-Hex transcripts as well as enzyme activity increase with the ripening and/or softening of capsicum. The function of α-Man and ß-Hex in capsicum softening is investigated through RNA interference (RNAi) in fruits. α-Man and ß-Hex RNAi fruits were approximately two times firmer compared with the control and fruit deterioration was delayed by approximately 7 d. It is shown that silencing of α-Man and ß-Hex enhances fruit shelf life due to the reduced degradation of N-glycoproteins which resulted in delayed softening. Altogether, the results provide evidence for the involvement of N-glycan processing in non-climacteric fruit softening. In conclusion, genetic engineering of N-glycan processing can be a common strategy in both climacteric and non-climacteric species to reduce the post-harvest crop losses.