Characterization of leucine aminopeptidase (LAP) activity in sweet pepper fruits during ripening and its inhibition by nitration and reducing events

Abstract

Key message Pepper fruits contain two leucine aminopeptidase (LAP) genes which are differentially modulated during ripening and by nitric oxide. The LAP activity increases during ripening but is negatively modulated by nitration. Abstract Leucine aminopeptidase (LAP) is an essential metalloenzyme that cleaves N-terminal leucine residues from proteins but also metabolizes dipeptides and tripeptides. LAPs play a fundamental role in cell protein turnover and participate in physiological processes such as defense mechanisms against biotic and abiotic stresses, but little is known about their involvement in fruit physiology. This study aims to identify and characterize genes encoding LAP and evaluate their role during the ripening of pepper (Capsicum annuum L.) fruits and under a nitric oxide (NO)-enriched environment. Using a data-mining approach of the pepper plant genome and fruit transcriptome (RNA-seq), two LAP genes, designated CaLAP1 and CaLAP2, were identified. The time course expression analysis of these genes during different fruit ripening stages showed that whereas CaLAP1 decreased, CaLAP2 was upregulated. However, under an exogenous NO treatment of fruits, both genes were downregulated. On the contrary, it was shown that during fruit ripening LAP activity increased by 81%. An in vitro assay of the LAP activity in the presence of different modulating compounds including peroxynitrite (ONOO−), NO donors (S-nitrosoglutathione and nitrosocyteine), reducing agents such as reduced glutathione (GSH), l-cysteine (l-Cys), and cyanide triggered a differential response. Thus, peroxynitrite and reducing compounds provoked around 50% inhibition of the LAP activity in green immature fruits, whereas cyanide upregulated it 1.5 folds. To our knowledge, this is the first characterization of LAP in pepper fruits as well as of its regulation by diverse modulating compounds. Based on the capacity of LAP to metabolize dipeptides and tripeptides, it could be hypothesized that the LAP might be involved in the GSH recycling during the ripening process.

Persulfidome of Sweet Pepper Fruits during Ripening: The Case Study of Leucine Aminopeptidase That Is Positively Modulated by H2S

“…In a previous study using S -nitrosoglutathione (GSNO) and nitroso-cysteine (NO-Cys), it was found that these NO donors caused the downregulation of the pepper LAP activity. However, this inhibition was not due to the NO released from these compounds because when GSH and Cys were used as internal controls, the same downregulation of LAP activity was also observed, thus indicating that these effects were due to the reductants [ 31 ]. To get deeper insights into the modulation of LAP activity, the pepper fruit samples were incubated with GSNO and NaHS that may compete for the same Cys residue as a target.…”

“…Plant leucine aminopeptidase (LAP) is a metalloenzyme, often requiring metal ions such as zinc or manganese for its catalytic activity [ 101 , 102 , 103 , 104 ]. LAP is typically found in the cytoplasm and plastids but can also be localized in other cellular compartments including mitochondria and peroxisomes, depending on the plant species and specific physiological conditions [ 31 , 43 , 105 , 106 , 107 , 108 , 109 , 110 ]. It plays a significant role in plant development [ 111 , 112 , 113 ] and its response to environmental adverse conditions including abiotic (Gu et al [ 114 ]; Gu et al [ 115 ]) and biotic stresses [ 116 , 117 ].…”

“…Recently, in pepper fruit, we have shown that LAP activity increased during ripening. The enzyme was also a target of NO-derived PTMs (tyrosine nitration and S -nitrosation), and S -cyanylation, with its activity being either negatively or positively regulated, respectively [ 31 ]. This seems to be correlated with the protein degradation associated with the disassembly of some organelles, such as it occurs in chloroplasts that transformed into chromoplasts during pepper ripening.…”

“…In previous studies, we have provided evidence of the relevance of the metabolism of reactive oxygen and nitrogen species (ROS and RNS, respectively) in the ripening of sweet pepper fruits and how the application of exogenous nitric oxide (NO) could modulate these processes at different levels [ 26 ]. Thus, enzymes like peroxidases, superoxide dismutases (SODs), NADPH oxidases, and ascorbate peroxidases (APXs), among others, have been shown to undergo numerous changes at the transcriptomic, proteomic, and biochemical levels [ 27 , 28 , 29 , 30 , 31 ].…”

“…This study provides the first comparative persulfidome of sweet pepper fruit at two stages of ripening including immature green and ripe red. Among the identified target proteins for persulfidation, the leucine aminopeptidase (LAP), which was previously shown to be modulated by NO [ 31 ], was selected to evaluate how H 2 S can affect its activity. Thus, the obtained data indicate that H 2 S exerts a positive effect on LAP activity which was also reversed by a reductant reagent (dithiothreitol), indicating that this process is reversible.…”