Ozone-induced oxidation of Rubisco: from an ELISA quantification of carbonyls to putative pathways leading to oxidizing mechanisms

In an attempt to detect a possible relationship between protein oxidation and ozone (O-3) atmospheric concentration, we used a sensitive enzyme-linked immunosorbent assay (ELISA) method for measuring carbonyl formation in amino acid residues that constitute Rubisco (EC 4.1.1.39) small subunit (Rubisco-SSU). Using open-top chamber technology, bean plants (Phaseolus vulgaris L. cv. Bergamo) were exposed for 21 d (from emergence) to four different atmospheres characterized by average daylight O-3 concentrations of 12, 70, 89 and 109 nL L-1. Rubisco-SSU extracted from primary leaves was fixed specifically on wells coated with anti-SSU antibodies. Aldehydes and ketones, previously derivatized with 2,4-dinitrophenylhydrazine (DNP), were quantified with anti-DNP antibodies conjugated with alkaline phosphatase. A significant positive O-3 effect on carbonyl formation was detected, and the number of carbonyls was found to be linearly increased (r(2)=0.82) when plotted against increasing external O-3 dose expressed as accumulated exposure over a threshold of 40 nL L-1 h (AOT40). Furthermore, these O-3-induced oxidative modifications were connected with a significant reduction in the amount of native SSU that linearly decreased (r(2) =0.95) as AOT40 increased from 0 to about 8295 nL L-1 h. A possible pathway leading to oxidation of Rubisco is proposed, with special reference to O-3 reactivity.