Alternative fluorimetric-based method to detect and compare total S-nitrosothiols in plants

Author(s)

Mioto, Paulo Tamaso ; Rodriguez-Ruiz, Marta ; Mot, Augustin Catalin ; Zuccarelli, Rafael ; Corpas, Francisco J. ; Freschi, Luciano ; Mercier, Helenice

• Document type: Journal article
• Source: NITRIC OXIDE-BIOLOGY AND CHEMISTRY; v. 68, p. 7-13, AUG 1 2017.
• Web of Science Citations: 1

Abstract

Nitric oxide (NO) is an important signaling molecule occurring in virtually all organisms, whose mechanism of action relies mainly on its interaction with proteins or peptides by nitrosylation, forming compounds such as S-nitrosothiols (SNO). The Saville reaction and the ozone-based chemiluminescence method are the main techniques used for nitrosylated protein quantification. While the Saville assay is not very sensitive, the ozone-based chemiluminescence is expensive and time-consuming. Here we propose a method in which the protein-bound NO groups are exposed to UV light, cleaving the bond and allowing the quantification of the derived NO molecules by diamino-rhodamine (DAR) dyes. The DAR based method was shown to be specific in plant tissues by pre-treatment of the samples with reducing agents and parallel EPR analysis. Spike-and-recovery assays revealed 72% recovery after a GSNO spike. Moreover, the method was significantly more sensitive than the Saville reaction, and this increase in sensitivity was crucial for detecting the reduced levels of nitrosylated proteins in plant species other than Arabidopsis. The method presented here is a suitable alternative to compare plant samples, allowing simple and fast detection of nitrosylated proteins. (C) 2017 Elsevier Inc. All rights reserved. (AU)

• FAPESP's process: 13/18056-2 - Interaction between light, hormonal and nitric oxide signaling during plastidial biogenesis and differentiation and nutraceutical compounds accumulation in tomato fruits
• Grantee: Luciano Freschi
• Support Opportunities: Regular Research Grants
• FAPESP's process: 13/15108-1 - Biosynthesis, transport and mechanism of action of nitric oxide durin crassulacean acid metabolism (CAM) induction in Guzmania monostachia
• Grantee: Paulo Tamaso Mioto
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 11/50637-0 - Competence for Crassulacean Acid Metabolism (CAM) expression in epiphytic bromeliad: signaling, modulation of expression, transcriptional profile and interaction with the nitrogen metabolism
• Grantee: Helenice Mercier
• Support Opportunities: Research Projects - Thematic Grants