Quantification and Localization of S-Nitrosothiols (SNOs) in Higher Plants.
S-nitrosothiols (SNOs) are a family of molecules produced by the reaction of nitric oxide (NO) with -SH thiol groups present in the cysteine residues of proteins and peptides caused by a posttranslational modification (PTM) known as S-nitrosylation (strictly speaking S-nitrosation) that can affect the cellular function of proteins. These molecules are a relatively more stable form of NO and consequently can act as a major intracellular NO reservoir and, in some cases, as a long-distance NO signal. Additionally, SNOs can be transferred between small peptides and protein thiol groups through S-transnitrosylation mechanisms. Thus, detection and cellular localization of SNOs in plant cells can be useful tools to determine how these molecules are modulated under physiological and adverse conditions and to determine their importance as a mechanism for regulating different biochemical pathways. Using a highly sensitive chemiluminescence ozone technique and a specific fluorescence probe (Alexa Fluor 488 Hg-link phenylmercury), the methods described in this chapter enable us to determine SNOs in an nM range as well as their cellular distribution in the tissues of different plant species.
Group of Biochemistry and Cell Signaling in Nitric Oxide, Department of Biochemistry and Molecular Biology, University of Jaén, Campus "Las Lagunillas", 23071, Jaén, Spain. jbarroso@ujaen.es.
Juan B
JB
Barroso
Group of Biochemistry and Cell Signaling in Nitric Oxide, Department of Biochemistry and Molecular Biology, University of Jaén, Campus "Las Lagunillas", 23071, Jaén, Spain.
Raquel
R
Valderrama
Group of Biochemistry and Cell Signaling in Nitric Oxide, Department of Biochemistry and Molecular Biology, University of Jaén, Campus "Las Lagunillas", 23071, Jaén, Spain.
Alfonso
A
Carreras
Group of Biochemistry and Cell Signaling in Nitric Oxide, Department of Biochemistry and Molecular Biology, University of Jaén, Campus "Las Lagunillas", 23071, Jaén, Spain.
Mounira
M
Chaki
Group of Biochemistry and Cell Signaling in Nitric Oxide, Department of Biochemistry and Molecular Biology, University of Jaén, Campus "Las Lagunillas", 23071, Jaén, Spain.
Juan C
JC
Begara-Morales
Group of Biochemistry and Cell Signaling in Nitric Oxide, Department of Biochemistry and Molecular Biology, University of Jaén, Campus "Las Lagunillas", 23071, Jaén, Spain.
Beatriz
B
Sánchez-Calvo
Group of Antioxidants, Free Radicals and Nitric Oxide in Biotechnology, Food and Agriculture, Department of Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del Zaidín, CSIC, Apartado 419, 18080, Granada, Spain. javier.corpas@eez.csic.es.
Francisco J
FJ
Corpas
D049449:Luminescence; D010944:Plants / Q000378:metabolism*; D026403:S-Nitrosothiols / Q000378:metabolism*
D026403:S-Nitrosothiols
Chemiluminescence; Confocal laser scanning microscope; Fluorescent probes; Nitric oxide; Reactive nitrogen species; S-nitrosothiol