CONSTITUTIVE NONINDUCIBLE EXPRESSION OF THE ARABIDOPSIS-THALIANA NIA-2 GENE IN 2 NITRATE REDUCTASE MUTANTS OF NICOTIANA-PLUMBAGINIFOLIA

We have isolated a haploid cell line of N. plumbaginifolia, hNP 588, t hat is constitutive and not inducible for nitrate reductase. Nitrate r eductase mutants were isolated from hNP 588 protoplasts upon UV irradi ation. Two of these nitrate reductase-deficient cell lines, nia 3 and nia 25, neither of which contained any detectable nitrate reductase ac tivity, were selected for complementation studies. A cloned Ambidopsis thaliana nitrate reductase gene Nia 2 was introduced into each of the two mutants resulting in 56 independent kanamycin-resistant cell line s. Thirty of the 56 kanamycin-resistant cell lines were able to grow o n nitrate as the sole nitrogen source. Eight of these were further ana lyzed for nitrate reductase enzyme activity and nitrate reductase mRNA production. All eight lines had detectable nitrate reductase activity ranging from 7% to 150% of wild-type hNP 588 callus. The enzyme activ ity levels were not influenced by the nitrogen source in the medium. T he eight lines examined expressed a constitutive, non-inducible 3.2 kb mRNA species that was not present in untransformed controls.