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.

Keywords: Ascorbate metabolism; Cloning; Galactono-1,4-lactone dehydrogenase; Nitric oxide; Pepper fruit ripening; Reactive nitrogen species.

Graphical abstract

Fig. 1

Binding and transmembrane domains of the galactono-1,4-lactone dehydrogenase from pepper fruits. A, Binding domains for FAD (FAD_binding_4), for FAD/FMN to dehydrogenases (GlcD), and for some UDP-N-acetylmuramate dehydrogenases (MurB) are depicted. B and C, Transmembrane domains. This analysis was carried out at the www.expasy.org site, following the TMHMM (CBS, Denmark) (B) and DAS (Stockholm University) (C) methods. TMHMM, Transmembrane Helices based on the Hidden Markov Model (TMHMM)). DAS, Method of Dense Alignment Surface (DAS). Amino acids are numbered once the mitochondrial targeting signal (Met1-Pro37) is removed. Black arrows in B and C indicate the identified transmembrane domain, whereas blue arrows depict domains with low score to be transmembranal.

Fig. 2

Immunological characterization of galactono-1,4-lactone dehydrogenase from pepper fruits. A, Western blotting was assayed using an antibody raised against an internal consensed sequence consisting in 15 amino acids. GF, green fruit; RF, red fruit. B, GalLDH activity assayed in crude extracts from red fruits in the presence of different antibody dilutions. Activity was expressed as percentage±SEM of the activity measured in the absence of the antibody (100%) after three independent determinations.

Fig. 3

Galactono-1,4-lactone dehydrogenase activity and gene expression and ascorbate content in different organs of pepper plants. A, GalLDH activity. B, GalLDH gene expression determined by qRT-PCR and quantified considering the reference value (1) to green fruit. C, ascorbate content determined by the α, α’-dipyridyl method. Values represent means of three independent experiments±SEM. Student's t-test was only applied for comparison of means between green and red fruits, and no significant differences were observed. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Effect of treatment with exogenous NO gas on the ascorbate content, and GalLDH enzyme activity and gene expression of pepper fruits. Fruits were subjected to treatment with 5 ppm NO, then maintained at room temperature for 10 d, and crude extracts were prepared for analyses as described in Materials and methods. Values represent means of three independent experiments±SEM. Differences between untreated (Control) and NO-treated (+NO) plants were statistically significant (Student's t-test, P≥0.01; n=3).

Fig. 5

In vitro assays of galactono-1,4-lactone dehydrogenase activity from pepper fruits under nitration and nitrosylation conditions. GalLDH activity was assayed in the presence of different concentrations of A) 3-morpholino-sydnonimine (SIN-1, a peroxynitrite donor), B) S-nitrosoglutahione (GSNO, an NO donor) and C) reduced glutathione (GSH). Values represent means of three independent experiments±SEM. Differences among samples treated either with SIN-1 and GSNO were not statistically significant. Treatment of samples with GSH ≥0.5 mM provoked significant activity decreases (Student's t-test, P≥0.005; n=3).

Fig. 6

Electron fluxes in the mitochondria inner membrane in pepper fruits. Complexes I, II, III and IV from the mitochondrial electron transfer chain are depicted. GalLDH, galactono-1,4-lactone dehydrogenase. GalL, galactono-1,4-lactone. NADH DH, dehydrogenase associated to Complex I. UQ pool, ubiquinone pool. Cyt c, cytochrome c. GalLDH contains a transmembrane domain (Tyr46-Pro65) and is able to provide electrons directly to cytochrome c for reduction of oxygen at the Complex IV site.