Dual selective nitration in Arabidopsis: Almost exclusive nitration of PsbO and PsbP, and highly susceptible nitration of four non-PSII proteins, including peroxiredoxin II E.

Protein tyrosine nitration is a selective process, as revealed in studies of animals. However, evidence for selective protein nitration in plants is scarce. In this study, Arabidopsis plants were exposed to air with or without nitrogen dioxide at 40 ppm for 8 h in light. Proteins extracted from whole leaves or isolated chloroplasts were subjected to 2D PAGE followed by SYPRO Ruby staining and immunoblotting using an anti-3-nitrotyrosine antibody. We determined the relative intensity of a spot on an immunoblot (designated RISI), and relative intensity of the corresponding spot on SYPRO Ruby gel (designated RISS). Proteins that exhibited a high RISI value and/or a high RISI/RISS ratio were considered selectively nitrated. In whole leaf proteins from exposed plants, all immunopositive spots were identified as PsbO1, PsbO2 or PsbP1 by PMF. Thus, nitration was exclusive to PsbO and PsbP, extrinsic proteins of photosystem II (PSII). Their RISI/RISS ratio was ≤1.5. Non-exposed plants showed very faint nitration. In purified chloroplast proteins, PsbO and PsbP accounted for >80% of the total RISI values, while four non-PSII proteins, including peroxiredoxin II E, exhibited high RISI/RISS ratios (2.5∼6.6). Tyr(9) of PsbO1 was identified as a nitration site. Thus, nitration is selective for two PSII and four non-PSII proteins in Arabidopsis.