A defect in cystathionine β-lyase activity causes the severe phenotype of a Nicotiana plumbaginifolia methionine auxotroph

In plants and bacteria, methionine (Met) is synthesised through three consecutive reactions starting at the convergence point of one branch of the aspartate pathway and the sulphur reduction pathway. The substrates O-phosphohomoserine and cysteine converge to cystathionine. which is cleaved to homocysteine. Finally, homocysteine is methylated to Met. The second enzymatic step of Met synthesis, the cleavage of cystathionine to homocysteine, pyruvate and ammonia, is catalysed by cystathionine beta-lyase (CbL). Here, we report the functional complementation and phenotypical reversion of a Nicotiana plumbaginifolia mutant previously assumed to be defective in CbL activity using a heterologous bacterial protein targeted to the chloroplast. Molecular analysis revealed the stable integration and high expression rate of the chimeric gene in the complemented mutant. Up to 500-fold more CbL activity when compared to wild type was measured in partially purified extracts from the complemented mutant. Despite the high rate of overexpression and the strongly increased enzyme activity the content of Met was restored only to wild type levels. Furthermore, no change in free amino acid composition could be determined. These results are discussed with respect to regulation of the fluxes involved in Met biosynthesis. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.