Journal of Experimental Botany, Vol. 54, No. 384, pp. 923-933,
March 1, 2003
© 2003 Oxford University Press
Mitochondrial and peroxisomal manganese superoxide dismutase: differential expression during leaf senescence
Received 2 July 2002; Accepted 29 October 2002
1 Department of Plant Science, Rutgers University, New Brunswick, NJ, USA
2 Departamento de Bioquímica, Biología Celular y Molecular de Plantas, Estación Experimental del Zaidín, CSIC, Apdo. 419, E-18080 Granada, Spain
3 To whom correspondence should be addressed. Fax: +34 958 129600. E-mail: luisalfonso.delrio{at}eez.csic.es
4 Present address: Fox Chase Cancer Center, Human Genetics Program, Philadelphia, PA 19111, USA.
Abbreviations: MBP, maltose-binding protein; SOD, superoxide dismutase; Mn-SOD, manganese-containing superoxide dismutase; CuZn-SOD, copper,zinc-containing superoxide dismutase; NBT, nitro blue tetrazolium; IEM, immunoelectron microscopy; PTS, peroxisome targeting signal; BCIP, 5-bromo-4-chloro-3-indoyl phosphate.
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 in creased 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.
Key words: Differential expression, immunocytochemical localization, manganese superoxide dismutase, mitochondria, peroxisomes, Pisum sativum L., senescence, superoxide dismutase.
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