Characterization of a new type of molybdenum cofactor-mutant in cell cultures of Nicotiana tabacum

Abstract

Four allelic putative cnx (molybdenum-cofactor defective) cell lines (O42, P12, P31 and P47) of Nicotiana tabacum var. Xanthi, biochemically and genetically distinct from N. tabacum var. Gatersleben cnxA mutants, were examined further. Their molybdenum-cofactor could efficiently reconstitute NADPH-nitrate reductase activity from Neurospora crassa mutant nit-1 extract only in the presence of exogenous molybdenum unlike that of the wild-type cofactor which could reconstitute NADPH-nitrate reductase activity in either the absence or presence of exogenous molybdenum. Loss of cofactor activity in vivo was not due to a defect in molybdenum uptake into the cells. In vitro nitrate reductase complementation between extracts of each of these four lines and a nia mutant showed that they possessed a functional nitrate reductase haemoflavoprotein subunit. Both constitutive molybdenum cofactor and NADH cytochrome c reductase activity were derepressed in the four cell lines. These results show that the four cell lines are indeed altered at a cnx locus, called cnxB, that the defect is probably in molybdenum processing and that there is a link between synthesis of functional molybdenum cofactor and nitrate reductase aporprotein.