Molecular analysis of pea manganese superoxide dismutase

Superoxide dismutases represent the first element of the multienzymatic cellular defensive system against oxidative stress. The general reaction catalyzed by ail superoxide dismutases Is the reduction of the superoxide anion, a very toxic oxygen derived radical, into hydrogen peroxide. Several Isoenzymes are present in most plants, each of them with a specific subcellular localization. The manganese containing isoform (Mn SOD) is a nuclear encoded, mitochondrial localized enzyme.

Screening a ë gt 10 cDNA library from pea [Pisum sativum) I have isolated and characterized two full length cDNAs coding for Mn SOD. Using these inserts as probes I have isolated, from a X EMBL-3 pea genomic library, a clone comprising the whole MnSOD gene, which has been fully sequenced. Genomic Southern and Genomic Reconstruction analyses show the presence of one copy of the MnSOD gene in the pea genome. Primer extension analysis allowed the identification of a putative transcription initiation site, located at a correct distance from a likely TATA box. Messenger RNA analyses of this gene done on mature and immature organs, from light and dark grown plants, show that this gene is expressed ubiquitously, but at much stronger levels in seedlings and flowers. Developmental and light regulated message heterogeneity is also apparent. Double Mn SOD transcripts appear in young tissues undergoing maturation. Treatment of mature pea leaves with Ethephon showed that this gene is Induced by the plant hormone ethylene. Modification of the cDNA sequences through polymerase chain reaction procedures generated constructs aimed to express Mn SOD into different subcellular organelles. Upon transformation of tobacco tissues and generation of transgenic plants, in situ enzymatic activity gel assays showed that recombinant Mn SODs are expressed in high levels and correctly targeted into chloroplasts and mitochondria. Electrolyte leakage assays demonstrated that the chloroplastic Mn SOD expressing plants have acquired improved resistance against the toxic effects of the herbicide methyl viologen, a strong generator of superoxide anions.