Fluorimetric-Based Method to Detect and Quantify Total S-Nitrosothiols (SNOs) in Plant Samples.

Accumulating experimental evidence indicates that S-nitrosylation (technically S-nitrosation) events have a central role in plant biology, presumably accounting for much of the widespread influence of nitric oxide (NO) on developmental, metabolic, and stress-related plant responses. Therefore, the accurate detection and quantification of S-nitrosylated proteins and peptides can be particularly useful to determine the relevance of this class of compounds in the ever-increasing number of NO-dependent signaling events described in plant systems. Up to now, the quantification of S-nitrosothiols (SNOs) in plant samples has mostly relied on the Saville reaction and the ozone-based chemiluminescence method, which lacks sensitivity and are very time-consuming, respectively. Taking advantage of the photolytic properties of S-nitrosylated proteins and peptides, the method described in this chapter allows simple, fast, and high-throughput detection of SNOs in plant samples.

Nitro-oxidative metabolism during fruit ripening.

Pepper (Capsicum annuum L.) and tomato (Solanum lycopersicum L.), which belong to the Solanaceae family, are among the most cultivated and consumed fleshy fruits worldwide and constitute excellent sources of many essential nutrients, such as vitamins A, C, and E, calcium, and carotenoids. While fruit ripening is a highly regulated and complex process, tomato and pepper have been classified as climacteric and non-climacteric fruits, respectively. These fruits differ greatly in shape, color composition, flavor, and several other features which undergo drastic changes during the ripening process. Such ripening-related metabolic and developmental changes require extensive alterations in many cellular and biochemical processes, which ultimately leads to fully ripe fruits with nutritional and organoleptic features that are attractive to both natural dispersers and human consumers. Recent data show that reactive oxygen and nitrogen species (ROS/RNS) are involved in fruit ripening, during which molecules, such as hydrogen peroxide (H2O2), NADPH, nitric oxide (NO), peroxynitrite (ONOO-), and S-nitrosothiols (SNOs), interact to regulate protein functions through post-translational modifications. In light of these recent discoveries, this review provides an update on the nitro-oxidative metabolism during the ripening of two of the most economically important fruits, discusses the signaling roles played by ROS/RNS in controlling this complex physiological process, and highlights the potential biotechnological applications of these substances to promote further improvements in fruit ripening regulation and nutritional quality. In addition, we suggest that the term 'nitro-oxidative eustress' with regard to fruit ripening would be more appropriate than nitro-oxidative stress, which ultimately favors the consolidation of the plant species.

The contribution of weak CAM to the photosynthetic metabolic activities of a bromeliad species under water deficit.

The Crassulacean acid metabolism (CAM) can be a transitory strategy for saving water during unfavourable conditions, like a dry season. In some cases, CAM can also contribute to the maintenance of photosynthetic integrity, even if carbon gain and growth are impaired. CAM occurs in different intensities, being stronger or weaker depending on the degree of nocturnal malic acid accumulation. For example, Guzmania monostachia is an epiphytic tank bromeliad that shows an increase in its nocturnal organic acid accumulation and a variable CAM behaviour when exposed to water deficit. In this context, this study aimed at investigating whether the weak CAM displayed by this species may mitigate the harmful effects of water limitation on its photosynthetic activity. To this, bromeliads were submitted to well-watered and water deficit conditions. Guzmania monostachia plants under water deficiency conditions showed a reduction on atmospheric carbon assimilation without exhibiting changes in PSII integrity and carbohydrate production while showed an increase in nocturnal malic acid accumulation. Additionally, spots with high PSII efficiency in the leaf portion with a greater nocturnal malic acid accumulation were observed in plants exposed to water shortage conditions. These high-efficiency spots might be associated with a greater malate decarboxylation capacity. Also, the malic acid contributed to approximately 50% of the total carbon assimilated under water deficit. These results suggest that weak CAM may participate in photo-protection and it appears to meaningfully contribute to the overall carbon balance, being an important metabolic strategy to maintain plant fitness during water deficit periods.

Detection of Protein S-nitrosothiols (SNOs) in Plant Samples on Diaminofluorescein (DAF) Gels.

In plant cells, the analysis of protein S-nitrosothiols (SNOs) under physiological and adverse stress conditions is essential to understand the mechanisms of Nitric oxide (NO)-based signaling. We adapted a previously reported protocol for detecting protein SNOs in animal systems ( King et al., 2005 ) for plant samples. Briefly, proteins from plant samples are separated via non-reducing SDS-PAGE, then the NO bound by S-nitrosylated proteins is released using UV light and, finally, the NO is detected using the fluorescent probe DAF-FM (Rodriguez-Ruiz et al., 2017). Thus, the approach presented here provides a relatively quick and economical procedure that can be used to compare protein SNOs content in plant samples and provide insight in NO-based signaling in plants.