Differential abilities of nitrogen dioxide and nitrite to nitrate proteins in thylakoid membranes isolated from Arabidopsis leaves

Abstract

Exposure of Arabidopsis leaves to nitrogen dioxide (NO₂) results in nitration of specific chloroplast proteins. To determine whether NO₂ itself and/or nitrite derived from NO₂ can nitrate proteins, Arabidopsis thylakoid membranes were isolated and treated with NO₂-bubbled or potassium nitrite (KNO₂) buffer, followed by protein extraction, electrophoresis, and immunoblotting using an anti-3-nitrotyrosine (NT) antibody. NO₂ concentrations in the NO₂-bubbled buffer were calculated by numerically solving NO₂ dissociation kinetic equations. The two buffers were adjusted to have identical nitrite concentrations. Both treatments yielded an NT-immunopositive band that LC/MS identified as PSBO1. The difference in the band intensity between the 2 treatments was designated nitration by NO₂. Both NO₂ and nitrite mediated nitration of proteins, and the nitration ability per unit NO₂ concentration was ∼100-fold greater than that of nitrite.

Keywords: Arabidopsis; PsbO; nitrite; nitrogen dioxide; protein tyrosine nitration; thylakoid membranes.

Figure 1.

Protein nitration in isolated thylakoid membranes due to treatment with NO₂-bubbled buffer or KNO₂ buffer. Thylakoid membranes were incubated with NO₂-bubbled buffer containing 0 to 36.8 μM NO₂ and 0 to 4.35 mM NO₂⁻ or with KNO₂ buffer containing 0 to 4.35 mM NO₂⁻ for 60 min at 25°C in light. (A) Immunoblot with an antibody against 3-NT (lanes 1, 2, and 5) and SYPRO Ruby stain (lanes 3, 4, and 6) of proteins extracted from isolated thylakoid membranes before (lanes 1 and 3) and after treatment with NO₂-bubbled buffer containing 36.8 µM NO₂ and 4.35 mM NO₂⁻ (lanes 2 and 4) or KNO₂ buffer containing 4.35 mM NO₂⁻ (lanes 5 and 6). (B) Immunoblot with an antibody against 3-NT of proteins from isolated thylakoid membranes following incubation with NO₂-bubbled or KNO₂ buffer. (C) FC_PSBO1 by NO₂-bubbled buffer or FC_PSBO1 by KNO₂ buffer plotted against of NO₂ and NO₂⁻ concentrations. The concentration of NO₂ corresponds to that in NO₂-bubbled buffer, and the concentration of NO₂⁻ corresponds to that in NO₂-bubbled and KNO₂ buffer. FC_PSBO1 by NO₂-bubbled buffer = (intensity of the NT-immunopositive band of PSBPO1 after incubation with NO₂-bubbled buffer) / (intensity of the same band before incubation with NO₂-bubbled buffer). FC_PSBO1 by KNO₂ buffer = (intensity of the NT-immunopositive band of PSBPO1 after incubation with KNO₂ buffer) / (intensity of the same band before incubation with KNO₂ buffer). Data represent means of 3 independent experiments ± SD. Asterisk indicates that the FC_PSBO1 value of NO₂-bubbled buffer was significantly higher (*, p < 0.05; **, p < 0.01) than that of FC_PSBO1 by KNO₂ buffer, as assessed by Student's t-test. Statistical analyses were performed using GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA, USA).