br Material and methods br Results and discussion Unlike

Material and methods
Results and discussion Unlike many fruits such as bananas and tomatoes, known as climacteric fruits, in which ripening is regulated by a burst of dexamethasone acetate biosynthesis and an increase in respiration, pepper fruits, which do not follow this pattern, are called non-climacteric [32]. However, pepper fruit ripening is a highly regulated physiological process which is externally characterized by a drastic shift from green to red/yellow/purple/orange color depending on the variety. At the biochemical level, NO metabolism has been reported to be involved in this process through the regulation of some enzymatic activities by NO-mediated post-translational modifications, including, in particular, the antioxidant catalase [3], [7]. However, information on endogenous NO metabolism in pepper fruit remains very scarce. Interestingly, our knowledge of the applications of exogenous NO in the field of postharvest horticulture has begun to increase given the possible beneficial effects of different fruits when exposed to an enriched and controlled NO environment [33]. For example, the exogenous application of NO to some climateric fruits such as banana reduces ethylene production; this is associated with a reduction in the activity of 1-aminocyclopropane-1-carboxylate oxidase (ACO), an enzyme involved in the final step of ethylene production, which consequently causes a delay in fruit ripening [34]. To gain a deeper insight into the endogenous NO metabolism of pepper fruit, we focused on the potential involvement of GSNO reductase during the ripening stage of pepper fruits as well as its correlation with the protein-nitrosothiol profile in this physiological process. The enzyme GSNO reductase (GSNOR) catalyzes the NADH-dependent reduction of GSNO to GSSG and NH3. GSNO functions as a mobile reservoir of NO and can therefore affect the trans-nitrosation equilibrium between GSNO and S-nitrosylated proteins [35], [36]. There is evidence to show that GSNOR activity, and consequently SNO content, are involved in the mechanism of response to different biotic [27], [37], [38], [39], [40] and abiotic stresses including heavy metal [24], mechanical wounding [25], [41], [42] as well as low and high temperatures [25], [37], [39], [40]. Additionally, the important role played by GSNOR at different stages of plant development has also been reported [28], [43], [44], [45], [46]. However, to our knowledge, no information exists on the role played by GSNOR and SNOs in fruit ripening. Fig. 1A shows that GSNO reductase activity is down-regulated by 43% in red pepper as compared to the corresponding activity in green pepper. The analysis of pepper samples by native polyacrylamide gel electrophoresis and staining for GSNOR activity showed a single band in green pepper but was virtually undetectable in red pepper (Fig. 1B), which is closely in line with data on activity detected by spectrophotometric assays. To study the protein content of this enzyme, an antibody against GSNOR, previously characterized in sunflower and pea samples [27], [47], was used. Fig. 1C shows the immunoblot analysis of pepper fruits, in which an immuno-reactive band of approximately 45 kDa was detected, whose pattern showed lower protein expression at the red stage. This resembles the pattern described in different plant species [27], [43], [47], [48]. Additionally, the mRNA expression of pepper GSNOR was analyzed by semi-quantitative RT-PCR (Fig. 1D). The mRNA showed no significant differences between green and red pepper fruits. Similar behavior was observed in a set of gene expression patterns (including catalase and superoxide dismutase) during pepper ripening [2], suggesting that regulation in this plant species may occur at the post-translational level. In this regard, recent data also reveal that GSNOR can undergo a process of S-nitrosylation which provoked its inhibition and consequently a rise in the content of total SNOs [49]. Given their direct correlation with GSNOR activity, the level of total SNOs was analyzed during the pepper fruit ripening stage. In this context, a novel approach to plant samples based on DAF gels, previously described in relation to animal systems, was used to evaluate endogenous protein SNOs [30]. Briefly, the samples were separated by non-reducing SDS–PAGE, and NO bound to S-nitrosylated proteins was photolytically released by UV light. The NO released was then detected using a specific fluorescence probe for NO. To our knowledge, as no information exists on this technique as applied to plant samples, we carried out systematic controls to corroborate that DAF gels can reliably detect endogenous S-nitrosylated proteins in our system. Fig. 2A shows that the fluorescence signal, corresponding to the detection of S-nitrosylated proteins, was either absent or significantly reduced compared to control in the presence of chemicals capable of either decomposing SNOs such as ascorbate and CuCl; N-ethylmaleimide (NEM) which block free thiols [31], [50], [51], [52], [53], [54], [55]; or different reducing agents (DTT, GSH or ME). Fig. 2B shows that the content levels of endogenous S-nitrosylated proteins are higher in red pepper as compared to green pepper, which closely correlates with the lower GSNOR activity detected previously. More recently, similar behavior has been reported in Arabidopsis seedlings exposed to paraquat-induced oxidative stress, in which GSNOR activity was inhibited, with a concomitant increase in cellular SNOs [49]. In vitro analysis of recombinant GSNO activity under oxidative conditions appears to indicate that the inhibition of this activity correlates with Zn2+ release of GSNOR protein. Moreover, another set of data has shown that GSNOR S-nitrosation inhibits GSNOR activity, thus facilitating GSNO accumulation and consequently enhancing total SNOs [49].