RESUMEN
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.
Asunto(s)
Fluorometría/métodos , Óxido Nítrico/metabolismo , Proteínas de Plantas/metabolismo , Plantas/metabolismo , S-Nitrosotioles/análisis , Fluorometría/instrumentación , Mediciones Luminiscentes/métodos , Óxido Nítrico/efectos de la radiación , Nitritos/química , Nitrosación , Plantas/química , Rodaminas/química , Rodaminas/efectos de la radiación , S-Nitrosoglutatión/metabolismo , Rayos Ultravioleta , Flujo de TrabajoRESUMEN
The enzyme S-nitrosoglutathione reductase (GSNOR) has an important role in the metabolism of S-nitrosothiols (SNO) and, consequently, in the modulation of nitric oxide (NO)-mediated processes. Although the mitochondrial electron transport chain is an important target of NO, the role of GSNOR in the functionality of plant mitochondria has not been addressed. Here, we measured SNO content and NO emission in Arabidopsis thaliana cell suspension cultures of wild-type (WT) and GSNOR overexpressing (GSNOR(OE)) or antisense (GSNOR(AS)) transgenic lines, grown under optimal conditions and under nutritional stress. Respiratory activity of isolated mitochondria and expression of genes encoding for mitochondrial proteins were also analyzed. Under optimal growth conditions, GSNOR(OE) had the lowest SNO and NO levels and GSNOR(AS) the highest, as expected by the GSNO-consuming activity of GSNOR. Under stress, this pattern was reversed. Analysis of oxygen uptake by isolated mitochondria showed that complex I and external NADH dehydrogenase activities were inhibited in GSNOR(OE) cells grown under nutritional stress. Moreover, GSNOR(OE) could not increase alternative oxidase (AOX) activity under nutritional stress. GSNOR(AS) showed constitutively high activity of external NADH dehydrogenase, and maintained the activity of the uncoupling protein (UCP) under stress. The alterations observed in mitochondrial protein activities were not strictly correlated to changes in gene expression, but instead seemed to be related with the SNO/NO content, suggesting a post-transcriptional regulation. Overall, our results highlight the importance of GSNOR in modulating SNO and NO homeostasis as well mitochondrial functionality, both under normal and adverse conditions in A. thaliana cells.