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 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.

Nitric oxide on/off in fruit ripening

“…B; Rodríguez‐Ruiz et al . ). Concomitantly, the nitrated and nitrosated protein profile remained unchanged.…”

“…B, treatment of pre‐ripe fruits with NO gas maintained the protein nitrosation and nitration levels, but, most importantly, increased about 40% the ascorbate (vitamin C) content (Rodríguez‐Ruiz et al . ).…”

Assessing Nitric Oxide (NO) in Higher Plants: An Outline

“…Some of those processes include seed and pollen germination [42][43][44][45][46], root development and plant growth [47][48][49], stomata movement [50,51], senescence [52,53], flower development [54], and fruit ripening [55][56][57][58].…”

“…Figure 2a shows the phenotype of California sweet pepper (Capsicum annum L.) fruits at different ripening stages: green, breaking point, and red. This fruit is a non-climacteric one where it has been demonstrated that NO content diminishes during ripening, whereas other elements of the RNS metabolism change following patterns, such as an increase of protein nitration and SNOs content accompanied by a decreased S-nitrosoglutahione reductase activity [56][57][58]. In the case of pea (Pisum sativum) plants, behind the characteristic loss of chlorophyll during leaf senescence (Figure 2b), there is also a lower NO content accompanied by a strong enhancement in the generation of ROS and the decline of the antioxidative systems [52,59].…”

“…Thus, it has been demonstrated that the provision of exogenous NO can mitigate the adverse effects of different environmental stresses such as heavy metal stress, salinity, or UV radiation, regulates seed germination, delays fruit ripening, and increases shelf-life and improved life quality of detached flowers [71]. It is remarkable that, in many cases, the beneficial effects of NO are through the stimulation of the antioxidant systems [57,72,73] which, again, highlights the close interrelationship between RNS and ROS metabolisms.…”