ABSTRACT
Several isoforms of superoxide dismutase (SOD) with a high isoelectric point (pI) have been identified by isoelectric focusing chromatography in protein extracts from Scots pine (Pinus sylvestris) needles. One of these isoforms, a CuZn-SOD with a pI of about 10 and thus denoted hipI-SOD, has been isolated and purified to apparent homogeneity. A cDNA encoding the hipI-SOD protein was cloned and sequenced. Northern hybridization of mRNA isolated from different organs and tissues showed that hipI-SOD has a markedly different pattern of expression compared with chloroplastic and cytosolic SOD. Furthermore, the transcript levels of hipI-SOD and cytosolic SOD were found to respond differently to mechanical wounding, treatment with oxidized glutathione, paraquat, and ozone. Immunogold electron microscopy localized the hipI-SOD in the plasma membrane of sieve cells and the Golgi apparatus of albuminous cells. Moreover, high protein density was also detected in extracellular spaces such as secondary cell wall thickenings of the xylem and sclerenchyma and in intercellular spaces of parenchyma cells.
Subject(s)
Cycadopsida/genetics , Superoxide Dismutase/genetics , Adaptation, Physiological , Amino Acid Sequence , Biological Transport , Blotting, Northern , Cell Membrane/metabolism , Cell Wall/metabolism , Cycadopsida/metabolism , Cycadopsida/ultrastructure , DNA, Complementary , DNA, Plant , Gene Expression , Golgi Apparatus/metabolism , Isoenzymes/genetics , Isoenzymes/metabolism , Molecular Sequence Data , Oxidation-Reduction , Superoxide Dismutase/isolation & purification , Superoxide Dismutase/metabolismABSTRACT
Several CuZn-superoxide dismutases (SODs; EC 1.15.1.1) were cloned from hybrid aspen (Populus tremula L. x tremuloides Michx.). Two of the cloned genes encode representatives of a novel type of CuZn-SOD and we named it HipI-SOD because of its high isoelectric point (> or =9). The SODs were cloned by screening a cDNA library with a probe based on a Scots pine (Pinus sylvestris L.) CuZn-SOD that is predominantly located extracellularly. The expression pattern of HipI-SOD was examined using a Northern blot technique and compared with the expression patterns of cytosolic and chloroplastic SODs. Distinct expression patterns were observed for the three types of CuZn-SOD, with HipI-SODs showing strong expression in apical tissues. Southern blots as well as protein analysis suggest that these novel HipI-SODs belong to a small gene family, one member of which might be monomeric.
