Dataset Information

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, ?-mannosidase (?-Man) and ?-D-N-acetylhexosaminidase (?-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 ?-Man and ?-Hex from capsicum, which belong to glycosyl hydrolase (GH) families 38 and 20, respectively, are described here. ?-Man and ?-Hex are cell wall glycoproteins that are able to cleave terminal ?-mannose and ?-D-N-acetylglucosamine residues of N-glycans, respectively. ?-Man and ?-Hex transcripts as well as enzyme activity increase with the ripening and/or softening of capsicum. The function of ?-Man and ?-Hex in capsicum softening is investigated through RNA interference (RNAi) in fruits. ?-Man and ?-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 ?-Man and ?-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.

SUBMITTER: Ghosh S

PROVIDER: S-EPMC3003805 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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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.

Ghosh Sumit S Meli Vijaykumar S VS Kumar Anil A Thakur Archana A Chakraborty Niranjan N Chakraborty Subhra S Datta Asis A

Journal of experimental botany 20101028 2

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 β-D-N-acetylhexosaminidase (β-Hex), have b ...[more]

PMID: 21030387

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Project description:BACKGROUND: Climacteric fruit exhibit high ethylene and respiration levels during ripening but these levels are limited in non-climacteric fruit. Even though capsicum is in the same family as the well-characterised climacteric tomato (Solanaceae), it is non-climacteric and does not ripen normally in response to ethylene or if harvested when mature green. However, ripening progresses normally in capsicum fruit when they are harvested during or after what is called the 'Breaker stage'. Whether ethylene, and components of the ethylene pathway such as 1-aminocyclopropane 1-carboxylate (ACC) oxidase (ACO), ACC synthase (ACS) and the ethylene receptor (ETR), contribute to non-climacteric ripening in capsicum has not been studied in detail. To elucidate the behaviour of ethylene pathway components in capsicum during ripening, further analysis is therefore needed. The effects of ethylene or inhibitors of ethylene perception, such as 1-methylcyclopropene, on capsicum fruit ripening and the ethylene pathway components may also shed some light on the role of ethylene in non-climacteric ripening. RESULTS: The expression of several isoforms of ACO, ACS and ETR were limited during capsicum ripening except one ACO isoform (CaACO4). ACS activity and ACC content were also low in capsicum despite the increase in ACO activity during the onset of ripening. Ethylene did not stimulate capsicum ripening but 1-methylcyclopropene treatment delayed the ripening of Breaker-harvested fruit. Some of the ACO, ACS and ETR isoforms were also differentially expressed upon treatment with ethylene or 1-methylcyclopropene. CONCLUSIONS: ACS activity may be the rate limiting step in the ethylene pathway of capsicum which restricts ACC content. The differential expression of several ethylene pathway components during ripening and upon ethylene or 1-methylclopropene treatment suggests that the ethylene pathway may be regulated differently in non-climacteric capsicum compared to the climacteric tomato. Ethylene independent pathways may also exist in non-climacteric ripening as evidenced by the up-regulation of CaACO4 during ripening onset despite being negatively regulated by ethylene exposure. However, some level of ethylene perception may still be needed to induce ripening especially during the Breaker stage. A model of capsicum ripening is also presented to illustrate the probable role of ethylene in this non-climacteric fruit.

Project description:Superoxide radical (O2 •-) is involved in numerous physiological and stress processes in higher plants. Fruit ripening encompasses degradative and biosynthetic pathways including reactive oxygen and nitrogen species. With the use of sweet pepper (Capsicum annuum L.) fruits at different ripening stages and under a nitric oxide (NO)-enriched environment, the metabolism of O2 •- was evaluated at biochemical and molecular levels considering the O2 •- generation by a NADPH oxidase system and its dismutation by superoxide dismutase (SOD). At the biochemical level, seven O2 •--generating NADPH-dependent oxidase isozymes [also called respiratory burst oxidase homologs (RBOHs) I-VII], with different electrophoretic mobility and abundance, were detected considering all ripening stages from green to red fruits and NO environment. Globally, this system was gradually increased from green to red stage with a maximum of approximately 2.4-fold increase in red fruit compared with green fruit. Significantly, breaking-point (BP) fruits with and without NO treatment both showed intermediate values between those observed in green and red peppers, although the value in NO-treated fruits was lower than in BP untreated fruits. The O2 •--generating NADPH oxidase isozymes I and VI were the most affected. On the other hand, four SOD isozymes were identified by non-denaturing electrophoresis: one Mn-SOD, one Fe-SOD, and two CuZn-SODs. However, none of these SOD isozymes showed any significant change during the ripening from green to red fruits or under NO treatment. In contrast, at the molecular level, both RNA-sequencing and real-time quantitative PCR analyses revealed different patterns with downregulation of four genes RBOH A, C, D, and E during pepper fruit ripening. On the contrary, it was found out the upregulation of a Mn-SOD gene in the ripening transition from immature green to red ripe stages, whereas a Fe-SOD gene was downregulated. In summary, the data reveal a contradictory behavior between activity and gene expression of the enzymes involved in the metabolism of O2 •- during the ripening of pepper fruit. However, it could be concluded that the prevalence and regulation of the O2 •- generation system (NADPH oxidase-like) seem to be essential for an appropriate control of the pepper fruit ripening, which, additionally, is modulated in the presence of a NO-enriched environment.

Dataset Information

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.

Publications

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.

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