Mitochondrial and peroxisomal manganese superoxide dismutase: Differential expression during leaf senescence

Abstract

In pea (Pisum sativum L.) leaves, manganese superoxide dismutase (Mn-SOD) is mainly localized in mitochondria as well as in peroxisomes. In this work, the effect of leaf senescence on the peroxisomal and mitochondrial Mn-SOD was studied in detached leaves from pea plants which were incubated in the dark at 25°C for 3-11 d. Northern blots hybridized with a cDNA encoding mitochondrial Mn-SOD from pea leaves and a mitochondrial Mn-SOD transit peptide-specific probe showed increased Mn-SOD transcript levels during leaf senescence, due in part to increased mitochondrial Mn-SOD mRNA. Recombinantly-expressed mitochondrial Mn-SOD was used to raise polyclonal antibodies which cross-reacted with Mn-SOD in peroxisomes purified from pea leaves. Western blot assays of crude extracts with the antibodies to pea mitochondrial Mn-SOD showed that the levels of total Mn-SOD protein gradually increased with leaf senescence. By native PAGE, the total Mn-SOD activity of pea leaves increased with senescence. EM immunocytochemistry was used to distinguish mitochondrial and peroxisomal Mn-SOD in senescent leaves. Increased Mn-SOD labelling in mitochondria was intensified, whereas the immunogold labelling of peroxisomes did not change with senescence. Overall, these results show that mitochondrial and peroxisomal Mn-SOD expression is regulated differently. The expression of mitochondrial Mn-SOD is induced during the senescence of pea leaves, whereas peroxisomal Mn-SOD could be post-translationally activated. Previously described results showing decreased mitochondrial Mn-SOD activity and increased peroxisomal Mn-SOD activity may be reflective of post-translational events regulating enzymatic activity during leaf senescence.