title: Characterization of the galactono-1,4-lactone dehydrogenase from pepper fruits and its modulation in the ascorbate biosynthesis. Role of nitric oxide.
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 56kDa 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.
authors.affiliation: 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. Electronic address: marta.rodriguez@eez.csic.es.
authors.forename: Marta
authors.initials: M
authors.lastname: Rodríguez-Ruiz
authors.affiliation: University Hospital Puerta del Mar, Avenida Ana de Viya, 21, Cádiz 11009, Spain. Electronic address: mateosrosa@hotmail.com.
authors.forename: Rosa M
authors.initials: RM
authors.lastname: Mateos
authors.affiliation: 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. Electronic address: v.codesido@phytoplant.es.
authors.forename: Verónica
authors.initials: V
authors.lastname: Codesido
authors.affiliation: 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. Electronic address: javier.corpas@eez.csic.es.
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Corpas
authors.affiliation: 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. Electronic address: josemanuel.palma@eez.csic.es.
authors.forename: José M
authors.initials: JM
authors.lastname: Palma
mesh_terms: D001205:Ascorbic Acid / Q000096:biosynthesis*; D002212:Capsicum / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D035264:Flowers / Q000201:enzymology / Q000235:genetics; D005638:Fruit / Q000201:enzymology / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D008928:Mitochondria / Q000378:metabolism; D008958:Models, Molecular; D009569:Nitric Oxide / Q000494:pharmacology; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D021382:Protein Sorting Signals
chemical_list: D010940:Plant Proteins; D021382:Protein Sorting Signals; D009569:Nitric Oxide; D044925:Oxidoreductases Acting on CH-CH Group Donors; C093910:galactonolactone dehydrogenase; D001205:Ascorbic Acid
keywords: Ascorbate metabolism; Cloning; Galactono-1,4-lactone dehydrogenase; Nitric oxide; Pepper fruit ripening; Reactive nitrogen species