Senescence-dependent changes in superoxide anion production by illuminated chloroplasts from bean leaves

The formation of superoxide ion radicals ([Formula: see text]) by chloroplasts from senescing leaves ofPhaseolus vulgaris L. was determined by electron-spin-resonance measurements of Tiron (1,2-dihydroxybenzene-3,5-disulfonic acid) semiquinone, a radical species formed when Tiron reacts with[Formula: see text]. The Tiron radical signal obtained from chloroplasts is sensitive to superoxide dismutase (EC 1.15.1.1) confirming that it is derived from[Formula: see text], oxygen-dependent and unaffected by 3-(3,4-dichlorophenyl)-1,1-dimethyl urea and hydroxylamine. Further confirmation of the identity of the radical was obtained by using the diagnostic spin trap 5,5'-dimethyl-1-pyrroline-1-oxide. The production of[Formula: see text] by illuminated chloroplasts increases by about fourfold during the early stages of leaf senescence; it declines again as senescence intensifies. A similar pattern of[Formula: see text] production was noted during aging of isolated chloroplasts in buffer. In addition, heat denaturation of freshly isolated chloroplasts greatly increases their ability to form[Formula: see text] upon illumination, indicating that the radical is formed through a photochemical reaction involving chlorophyll, rather than enzymatically. Accordingly, the rise in[Formula: see text] production during senescence may reflect deteriorative molecular rearrangements in the thylakoid membranes, which expose chlorophyll molecules normally inaccessible to oxygen. The propensity of chloroplasts to produce increased levels of[Formula: see text] with advancing senescence is not counterbalanced by an augmented enzymatic radical-scavenging capability. Moreover, the peak in[Formula: see text] production during leaf senescence coincides temporally with the initiation of lipid peroxidation and the formation of gel-phase lipid in chloroplast membranes, phenomena that are known to be induced by this reactive species of oxygen.