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

Abstract

The formation of superoxide ion radicals ( $$O_2^{\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle\cdot}$}}{ - } } $$ ) 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 $$O_2^{\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle\cdot}$}}{ - } } $$ . The Tiron radical signal obtained from chloroplasts is sensitive to superoxide dismutase (EC 1.15.1.1) confirming that it is derived from $$O_2^{\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle\cdot}$}}{ - } } $$ , 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 $$O_2^{\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle\cdot}$}}{ - } } $$ by illuminated chloroplasts increases by about fourfold during the early stages of leaf senescence; it declines again as senescence intensifies. A similar pattern of $$O_2^{\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle\cdot}$}}{ - } } $$ production was noted during aging of isolated chloroplasts in buffer. In addition, heat denaturation of freshly isolated chloroplasts greatly increases their ability to form $$O_2^{\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle\cdot}$}}{ - } } $$ upon illumination, indicating that the radical is formed through a photochemical reaction involving chlorophyll, rather than enzymatically. Accordingly, the rise in $$O_2^{\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle\cdot}$}}{ - } } $$ 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 $$O_2^{\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle\cdot}$}}{ - } } $$ with advancing senescence is not counterbalanced by an augmented enzymatic radical-scavenging capability. Moreover, the peak in $$O_2^{\underset{\raise0.3em\hbox{$\smash{\scriptscriptstyle\cdot}$}}{ - } } $$ 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.