RESUMEN
An approach to understand vines (Vitis vinifera) defense mechanism against heavy metal stress by isolation and determination of Hg-phytochelatins (PCs) complexes was performed. PCs are important molecules involved in the control of metal concentration in plants. PCs complex toxic metals through -SH groups and stores them inside cells vacuole avoiding any toxic effect of free metals in the cytosol. The Hg-PCs identification was achieved by determination of Hg and S as hetero-tagged atoms. A method involving two-dimensional chromatographic analysis coupled to atomic spectrometry and confirmation by tandem mass spectrometry is proposed. An approach involving size exclusion chromatography coupled to inductively coupled plasma mass spectrometry on roots, stems, and leaves extracts describing Hg distribution according to molecular weight and sulfur associations is proposed for the first time. Medium-low molecular weight Hg-S associations of 29-100 kDa were found, suggesting PCs presence. A second approach employing reversed-phase chromatography coupled to atomic fluorescence spectrometry analysis allowed the determination of Hg-PCs complexes within the mentioned fractions. Chromatograms showed Hg-PC2, Hg-PC3 and Hg-PC4 presence only in roots. Hg-PCs presence in roots was confirmed by ESI-MS/MS analysis.
Asunto(s)
Complejos de Coordinación/metabolismo , Mercurio/metabolismo , Fitoquelatinas/metabolismo , Vitis/metabolismo , Adaptación Fisiológica , Cromatografía Líquida de Alta Presión , Cromatografía de Fase Inversa , Complejos de Coordinación/aislamiento & purificación , Mercurio/aislamiento & purificación , Fitoquelatinas/aislamiento & purificación , Hojas de la Planta/metabolismo , Raíces de Plantas/metabolismo , Tallos de la Planta/metabolismo , Estrés FisiológicoRESUMEN
Rice has the predilection to take up arsenic in the form of methylated arsenic (o-As) and inorganic arsenic species (i-As). Plants defend themselves using i-As efflux systems and the production of phytochelatins (PCs) to complex i-As. Our study focused on the identification and quantification of phytochelatins by HPLC-ICP-MS/ESI-MS, relating them to the several variables linked to As exposure. GSH, 11 PCs, and As-PC complexes from the roots of six rice cultivars (Italica Carolina, Dom Sofid, 9524, Kitrana 508, YRL-1, and Lemont) exposed to low and high levels of i-As were compared with total, i-As, and o-As in roots, shoots, and grains. Only Dom Sofid, Kitrana 508, and 9524 were found to produce higher levels of PCs even when exposed to low levels of As. PCs were only correlated to i-As in the roots (r=0.884, P <0.001). However, significant negative correlations to As transfer factors (TF) roots-grains (r= -0.739, P <0.05) and shoots-grains (r= -0.541, P <0.05), suggested that these peptides help in trapping i-As but not o-As in the roots, reducing grains' i-As. Italica Carolina reduced i-As in grains after high exposure, where some specific PCs had a special role in this reduction. In Lemont, exposure to elevated levels of i-As did not result in higher i-As levels in the grains and there were no significant increases in PCs or thiols. Finally, the high production of PCs in Kitrana 508 and Dom Sofid in response to high As treatment did not relate to a reduction of i-As in grains, suggesting that other mechanisms such as As-PC release and transport seems to be important in determining grain As in these cultivars.