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

Abstract

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 (NO(2)-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 NO(2)-Tyr in hypocotyls was 0.56 micromol 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.

Peroxisomes Are Required for in Vivo Nitric Oxide Accumulation in the Cytosol following Salinity Stress of Arabidopsis Plants

“…B, of proteins under stress conditions in animal cells (Radi, 2004;Szabó et al, 2007). To evaluate this possible correlation between ONOO 2 and protein nitration in Arabidopsis under salinity conditions, ONOO 2 was analyzed in roots by CLSM using the fluorescence probe 3#-(p-aminophenyl) fluorescein (APF; Chaki et al, 2009aChaki et al, , 2009b, while the presence of Tyr nitration was studied by immunoblot analysis using a well-characterized antibody against 3-nitrotyrosine (Valderrama et al, 2007;Corpas et al, 2008;Chaki et al, 2009aChaki et al, , 2009b. Figure 10A shows the location of ONOO 2 in the control roots of Arabidopsis wild-type seedlings and ONOO 2 significantly increased in roots under salinity stress (Fig.…”

Nitration is exclusive to defense-related PR-1, PR-3 and PR-5 proteins in tobacco leaves

“…[1][2][3][4][5] Plant proteomic studies have identified a variety of nitrated proteins in the leaves and seedlings of Arabidopsis [Arabidopsis thaliana (L.) Heynh. ], [6][7][8][9] the hypocotyls of sunflower (Helianthus annuus L.), 10,11 and the roots of pea (Pisum sativum L.). 12 Each of these organs has shown nitration of a distinctly different set of proteins, and thus nitration is considered specific for organs analyzed.…”

Biosíntesis, señalización y modo de acción del óxido nítrico en Arabidopsis thaliana

“…Sin embargo, recientemente, se han descrito diferentes trabajos enfocados a caracterizar este tipo de modificación en plantas (Valderrama et al, 2007;Romero-Puertas et al, 2007;Corpas et al, 2008;Chaki et al, 2009b;Chaki et al, 2009a;Bechtold et al, 2009;Cecconi et al, 2009;Corpas et al, 2009). Parece que las hemoglobinas de plantas pueden facilitar la nitración in vitro de proteínas tal como ocurre en modelos animales (Sakamoto et al, 2004).…”

“…Por otro lado, el número de proteínas nitradas identificadas en plantas es mucho menor que en modelos animales (Corpas et al, 2007). Basándose en la detección por Western-blot de proteínas separadas en geles bidimensionales mediante un anticuerpo que detecta 3-nitrotirosina y la posterior identificación de los proteínas inmunoreactivas, se han identificado 12 proteínas de Arabidopsis que ven incrementado su patrón de nitración en respuesta a la bacteria fitopatógena Pseudomonas syringae (Cecconi et al, 2009) y 21 proteínas de girasol que sufren el proceso de nitración bajo condiciones fisiológicas en ausencia de estrés (Chaki et al, 2009b). La gran mayoría de las proteínas identificadas regulan procesos del metabolismo como la RuBisCO, la Fructosas bifosfatasa y la Sadenosyl-homocisteina hidrolasa.…”

“…La gran mayoría de las proteínas identificadas regulan procesos del metabolismo como la RuBisCO, la Fructosas bifosfatasa y la Sadenosyl-homocisteina hidrolasa. Proteínas similares a 14-3-3 y calmodulina son las únicas identificadas con una potencial función en señalización (Chaki et al, 2009b). Curiosamente, ninguna de las proteínas nitradas identificadas tras la infección por Pseudomonas syringae se encuentra enmarcada dentro de las proteínas de respuesta a estrés (Cecconi et al, 2009), lo que se podría esperar tras el bien establecido papel del NO y de las especies reactivas de nitrógeno causantes de la nitración en las interacciones planta-patógeno (Delledonne et al, 1998;Saito et al, 2006;Besson-Bard et al, 2008a;Chaki et al, 2009a).…”