Figure 5.

Expression of the gephyrin P1 cDNA in the N. plumbaginifolia cnxA Moco mutant. (A) Restoration of molybdoenzyme activities. Protoplasts of in vitro-grown plants of the N. plumbaginifolia mutant line D70 defective in the locus cnxA were transformed with the gephyrin P1 cDNA driven by the Cauliflower Mosaic Virus 35S promoter. Transgenic selected colonies were grown as callus cultures and assayed for nitrate reductase and xanthine dehydrogenase activities. Because of the defect in Moco synthesis, the cnxA mutant has only 12.5 ± 8.9% of the wild-type level of nitrate reductase activity. Colonies transformed with the gephyrin cDNA show nitrate reductase activities ranging from 67.5 ± 31.7% (D70/585, presented here) up to 133.3 ± 38.2% of the wild-type level. Wild-type activity levels of 100% represent a NADH–nitrate reductase activity of 1.23 μmol of nitrite formed per gram of fresh weight per hour. Mean values ± SD of nitrate reductase activity determined in three independent subculturings of the cells are shown with duplicate measurements for each subculture. The same callus colonies as described above were tested for xanthine dehydrogenase activity by using an in situ gel assay (30). Because of the defect in Moco synthesis, the cnxA mutant has only trace amounts of xanthine dehydrogenase activity, whereas the plants (D70/585) transformed with the gephyrin cDNA show strongly increased enzyme activities. (B) Normalization of the mutant phenotype. All plants shown were grown in vitro and transferred on the same day into soil fertilized with potassium nitrate as the nitrogen source. The Moco-defective cnxA mutant line D70 (Left) is unable to grow in soil and dies. (Center) The N. plumbaginifolia wild type is shown. (Right) A representative gephyrin-expressing cnxA mutant plant (line D70/585) is shown. The wild type-like phenotype of this plant is representative of 10 gephyrin-transformed and regenerated cnxA-mutant plants.