Nitric Oxide (NO) Differentially Modulates the Ascorbate Peroxidase (APX) Isozymes of Sweet Pepper (Capsicum annuum L.) Fruits

Abstract

Nitric oxide (NO) is a free radical which modulates protein function and gene expression throughout all stages of plant development. Fruit ripening involves a complex scenario where drastic phenotypical and metabolic changes take place. Pepper fruits are one of the most consumed horticultural products worldwide which, at ripening, undergo crucial phenotypical and biochemical events, with NO and antioxidants being implicated. Based on previous transcriptomic (RNA-Seq), proteomics (iTRAQ), and enzymatic data, this study aimed to identify the ascorbate peroxidase (APX) gene and protein profiles in sweet peppers and to evaluate their potential modulation by NO during fruit ripening. The data show the existence of six CaAPX genes (CaAPX1–CaAPX6) that encode corresponding APX isozymes distributed in cytosol, plastids, mitochondria, and peroxisomes. The time course expression analysis of these genes showed heterogeneous expression patterns throughout the different ripening stages, and also as a consequence of treatment with NO gas. Additionally, six APX isozymes activities (APX I–APX VI) were identified by non-denaturing PAGE, and they were also differentially modulated during maturation and NO treatment. In vitro analyses of fruit samples in the presence of NO donors, peroxynitrite, and glutathione, showed that CaAPX activity was inhibited, thus suggesting that different posttranslational modifications (PTMs), including S-nitrosation, Tyr-nitration, and glutathionylation, respectively, may occur in APX isozymes. In silico analysis of the protein tertiary structure showed that residues Cys32 and Tyr235 were conserved in the six CaAPXs, and are thus likely potential targets for S-nitrosation and nitration, respectively. These data highlight the complex mechanisms of the regulation of APX isozymes during the ripening process of sweet pepper fruits and how NO can exert fine control. This information could be useful for postharvest technology; NO regulates H2O2 levels through the different APX isozymes and, consequently, could modulate the shelf life and nutritional quality of pepper fruits.

Peroxisomal Proteome Mining of Sweet Pepper (Capsicum annuum L.) Fruit Ripening Through Whole Isobaric Tags for Relative and Absolute Quantitation Analysis

“…Therefore, a considerable number of the identified proteins in pepper peroxisomes belongs to different antioxidant systems devoted to maintaining the ROS homeostasis previously described in peroxisomes from different plant species, and it includes catalase ( Palma et al, 2020a ), CuZn-SOD ( Bueno et al, 1995 ; Corpas et al, 1998b ; Palma et al, 2018 ), and all the enzymatic components of ascorbate-glutathione cycle. The enzymatic members of this cycle are located either in the peroxisomal matrix, such as glutathione reductase (GR) and monodehydroascorbate peroxidase (MDAR), and bound to the membrane, as it has been reported for ascorbate peroxidase (APX) and dehydroascorbate reductase (DAR) ( Corpas et al, 1994 ; Bunkelmann and Trelease, 1996 ; Jiménez et al, 1997 ; Nito et al, 2001 ; Leterrier et al, 2005 ; Narendra et al, 2006 ; Romero-Puertas et al, 2006 ; González-Gordo et al, 2022a ). Furthermore, the presence of some components of the pentose phosphate pathway involved in the generation of NADPH reported here is also in good agreement with previous reports in pea and Arabidopsis peroxisomes ( Corpas et al, 1998a ; Corpas et al, 1999 ; Fernández-Fernández and Corpas, 2016 ; Hölscher et al, 2016 ; Lansing et al, 2020 ).…”

“…During ripening, pepper fruits undergo drastic biochemical and phenotypical changes, being the color shift from green to red the most evident since chloroplasts are transformed into chromoplasts as a consequence of the chlorophyll degradation and the biosynthesis of carotenoids, xanthophylls, and anthocyanins. In previous studies, it has been analyzed the sweet pepper fruit ripening at biochemical and molecular levels including the transcriptome ( González-Gordo et al, 2019 ), as well as the metabolism of NADPH-generating enzymes ( Mateos et al, 2009 ; Muñoz-Vargas et al, 2018 , 2020 ), ROS metabolism ( Rodríguez-Ruiz et al, 2017a , 2019 ; Chu-Puga et al, 2019 ; González-Gordo et al, 2020 , 2022a ; Palma et al, 2020a ), reactive nitrogen species ( Chaki et al, 2015 ; Rodríguez-Ruiz et al, 2017b ), the metabolome ( Guevara et al, 2021 ), and the plastidial and mitochondrial proteomes ( Rödiger et al, 2021 ; González-Gordo et al, 2022b ). However, little is known about the proteome from peroxisomes and how it can be modulated during ripening.…”

Lipoxygenase (LOX) in Sweet and Hot Pepper (Capsicum annuum L.) Fruits during Ripening and under an Enriched Nitric Oxide (NO) Gas Atmosphere

“…The present study aims to analyze the potential relevance of LOXs at the biochemical and gene expression levels using different pepper plant organs and varieties with special attention to sweet pepper fruit during ripening and under an enriched nitric oxide (NO) atmosphere. Very recently, it has been shown that NO delays the ripening process of sweet pepper fruit and modulates its antioxidant system [ 21 , 22 , 23 ], and parallel results have been reported in tomatoes [ 25 ]. The data indicate that the LOX isozyme pattern is differentially modulated among pepper organs, fruit variety, capsaicin content, and ripening stage.…”

“…Fruits without any external damages were selected at three developmental stages: green immature (G), breaking point (BP1), and red ripe (R). For the application of NO, two additional groups were set: fruits treated with 5 ppm NO for 1 h (BP2 + NO) and another group that was not treated with NO (BP2 − NO), which was used as control of BP2 + NO [ 21 , 23 , 59 ]. After 3 days, all fruits were chopped into small cubes (5 mm/edge), frozen under liquid nitrogen, and stored at −80 °C until use.…”

“…In previous studies, the metabolism of reactive oxygen species (ROS) systems using a sweet pepper variety (Melchor) from California-type fruits [ 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 ], as well as three autochthonous Spanish varieties including Padrón, Piquillo, and Alegría riojana, which have dissimilar pungency levels, has been investigated [ 24 ]. Additionally, a preliminary approach reported the fatty acid profile and LOX activity from pepper fruits at several ripening conditions [ 19 ].…”