Contents lists available at ScienceDirect Redox Biology journal homepage: www.elsevier.com/locate/redox Research paper Characterization of the galactono-1,4-lactone dehydrogenase from pepper fruits and its modulation in the ascorbate biosynthesis. Role of nitric oxide ☆ Marta Rodríguez-Ruiz a,1 , Rosa M. Mateos b,1 , Verónica Codesido c , Francisco J. Corpas a , José M. Palma a, ⁎ a Group of Antioxidants, Free Radicals and Nitric Oxide in Biotechnology, Food and Agriculture, Dept. Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del Zaidín, CSIC, C/ Profesor Albareda, 1, 18008 Granada, Spain b University Hospital Puerta del Mar, Avenida Ana de Viya, 21, Cádiz 11009, Spain c Phytoplant Research S.L, Rabanales 21 - The Science and Technology Park of Córdoba, C/ Astrónoma Cecilia Payne, Edificio Centauro, módulo B-1, 14014 Córdoba, Spain ARTICLE INFO Keywords: Ascorbate metabolism Cloning Galactono-1,4-lactone dehydrogenase Nitric oxide Pepper fruit ripening Reactive nitrogen species ABSTRACT Pepper fruit is one of the highest vitamin C sources of plant origin for our diet. In plants, ascorbic acid is mainly synthesized through the L-galactose pathway, being the L-galactono-1,4-lactone dehydrogenase (GalLDH) the last step. Using pepper fruits, the full GalLDH gene was cloned and the protein molecular characterization accomplished. GalLDH protein sequence (586 residues) showed a 37 amino acids signal peptide at the N- terminus, characteristic of mitochondria. The hydrophobic analysis of the mature protein displayed one transmembrane helix comprising 20 amino acids at the N-terminus. By using a polyclonal antibody raised against a GalLDH internal sequence and immunoblotting analysis, a 56 kDa polypeptide cross-reacted with pepper fruit samples. Using leaves, flowers, stems and fruits, the expression of GalLDH by qRT-PCR and the enzyme activity were analyzed, and results indicate that GalLDH is a key player in the physiology of pepper plants, being possibly involved in the processes which undertake the transport of ascorbate among different organs. We also report that an NO (nitric oxide)-enriched atmosphere enhanced ascorbate content in pepper fruits about 40% parallel to increased GalLDH gene expression and enzyme activity. This is the first report on the stimulating effect of NO treatment on the vitamin C concentration in plants. Accordingly, the modulation by NO of GalLDH was addressed. In vitro enzymatic assays of GalLDH were performed in the presence of SIN-1 (peroxynitrite donor) and S-nitrosoglutahione (NO donor). Combined results of in vivo NO treatment and in vitro assays showed that NO provoked the regulation of GalLDH at transcriptional and post-transcriptional levels, but not post-translational modifications through nitration or S-nitrosylation events promoted by reactive nitrogen species (RNS) took place. These results suggest that this modulation point of the ascorbate biosynthesis could be potentially used for biotechnological purposes to increase the vitamin C levels in pepper fruits. 1. Introduction Ascorbic acid (vitamin C) is one of the most powerful antioxidants synthesized in the majority of living beings, excepting primates (including humans), guinea pigs, bats and some birds [32,55,74,79,96,97]. Within plant cells, this molecule is ubiquitous and can be detected in many subcellular loci, although it is also found in the apoplast ([46,47,79,80,88]). As an antioxidant, it can directly interact with hydroxyl radicals (·OH), superoxide radicals (O 2 ·- ), hydrogen peroxide (H 2 O 2 ) and singlet oxygen [21,22,44,89]. Furthermore, ascorbate is an electronic donor for the ascorbate peroxidase (APX; EC. 1.11.1.11) activity to remove the hydrogen peroxide, either as an individual reaction but also as the first stage of the ascorbate-glutathione cycle in plants (Foyer-Halliwell-Asada path- http://dx.doi.org/10.1016/j.redox.2017.02.009 Received 5 December 2016; Received in revised form 15 January 2017; Accepted 12 February 2017 ☆ Note: Sequence data from this article have been deposited in the EMBL/GenBank data libraries under accession number AY547352 and AY572427 for the Capsicum annuum L GalLDH complete cDNA and actin mRNA, partial cDNA, respectively. ⁎ Corresponding author. 1 These authors equally contributed to this work. E-mail addresses: marta.rodriguez@eez.csic.es (M. Rodríguez-Ruiz), mateosrosa@hotmail.com (R.M. Mateos), v.codesido@phytoplant.es (V. Codesido), javier.corpas@eez.csic.es (F.J. Corpas), josemanuel.palma@eez.csic.es (J.M. Palma). Redox Biology 12 (2017) 171–181 Available online 20 February 2017 2213-2317/ © 2017 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). MARK