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JXB Advance Access originally published online on August 28, 2009
Journal of Experimental Botany 2009 60(15):4221-4234; doi:10.1093/jxb/erp263
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© 2009 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

RESEARCH PAPER

Protein targets of tyrosine nitration in sunflower (Helianthus annuus L.) hypocotyls

Mounira Chaki1, Raquel Valderrama1, Ana M. Fernández-Ocaña1, Alfonso Carreras1, Javier López-Jaramillo3, Francisco Luque1, José M. Palma2, José R. Pedrajas1, Juan C. Begara-Morales1, Beatriz Sánchez-Calvo1, María V. Gómez-Rodríguez1, Francisco J. Corpas2,* and Juan B. Barroso1

1Grupo de Señalización Molecular y Sistemas Antioxidantes en Plantas, Unidad Asociada al CSIC (EEZ), Departamento de Bioquímica y Biología Molecular, Universidad de Jaén, Spain
2Departamento de Bioquímica, Biología Celular y Molecular de Plantas, Estación Experimental del Zaidín, CSIC, Apartado 419, E-18080 Granada, Spain
3Instituto de Biotecnología, Universidad de Granada, Spain

* To whom correspondence should be addressed: javier.corpas{at}eez.csic.es

Tyrosine nitration is recognized as an important post-translational protein modification in animal cells that can be used as an indicator of a nitrosative process. However, in plant systems, there is scant information on proteins that undergo this process. In sunflower hypocotyls, the content of tyrosine nitration (NO2-Tyr) and the identification of nitrated proteins were studied by high-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) and proteomic approaches, respectively. In addition, the cell localization of nitrotyrosine proteins and peroxynitrite were analysed by confocal laser-scanning microscopy (CLSM) using antibodies against 3-nitrotyrosine and 3'-(p-aminophenyl) fluorescein (APF) as the fluorescent probe, in that order. The concentration of Tyr and NO2-Tyr in hypocotyls was 0.56 µmol mg–1 protein and 0.19 pmol mg–1 protein, respectively. By proteomic analysis, a total of 21 nitrotyrosine-immunopositive proteins were identified. These targets include proteins involved in photosynthesis, and in antioxidant, ATP, carbohydrate, and nitrogen metabolism. Among the proteins identified, S-adenosyl homocysteine hydrolase (SAHH) was selected as a model to evaluate the effect of nitration on SAHH activity using SIN-1 (a peroxynitrite donor) as the nitrating agent. When the hypocotyl extracts were exposed to 0.5 mM, 1 mM, and 5 mM SIN-1, the SAHH activity was inhibited by some 49%, 89%, and 94%, respectively. In silico analysis of the barley SAHH sequence, characterized Tyr448 as the most likely potential target for nitration. In summary, the present data are the first in plants concerning the content of nitrotyrosine and the identification of candidates of protein nitration. Taken together, the results suggest that Tyr nitration occurs in plant tissues under physiological conditions that could constitute an important process of protein regulation in such a way that, when it is overproduced in adverse circumstances, it can be used as a marker of nitrosative stress.

Key words: Nitric oxide, nitroproteomics, nitrotyrosine, peroxynitrite, protein tyrosine nitration, reactive nitrogen species

Received 28 May 2009; Revised 31 July 2009 Accepted 4 August 2009


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