Constitutive non-inducible expression of the Arabidopsis thaliana Nia 2 gene in two nitrate reductase mutants of Nicotiana Plumbaginifolia

• Published in: Plant molecular biology Vol. 33; no. 6; pp. 953 - 964
• Main Authors: Kaye, C. (Georgia Univ., Athens, GA (USA). Dept. of Botany), Crawford, N.M, Malmberg, R.L
• Format: Journal Article
• Language: English
• Published: Netherlands Springer Nature B.V 01-04-1997
• Subjects: Arabidopsis - genetics; ARABIDOPSIS THALIANA; ARN MENSAJERO; ARN MESSAGER; Cell Line, Transformed; DNA, Plant - analysis; Enzymatic activity; Enzymes; EXPRESION GENICA; EXPRESSION DES GENES; GENE; GENE EXPRESSION; Gene Expression Regulation, Plant - genetics; GENES; Genes, Plant - genetics; GENETIC ENGINEERING; GENIE GENETIQUE; HAPLOIDIA; HAPLOIDIE; HAPLOIDY; INGENIERIA GENETICA; Irradiation; KANAMICINA; KANAMYCIN; KANAMYCINE; MESSENGER RNA; MUTANT; MUTANTES; MUTANTS; Mutation; Nicotiana - enzymology; Nicotiana - genetics; NICOTIANA PLUMBAGINIFOLIA; NITRATE REDUCTASE; Nitrate Reductases - genetics; Nitrate Reductases - metabolism; Nitrates; Nitrates - metabolism; NITRATO REDUCTASA; Nitrogen; PLANTAS TRANSGENICAS; PLANTE TRANSGENIQUE; Plants, Toxic; RNA, Messenger - biosynthesis; RNA, Plant - biosynthesis; Transformation, Genetic; TRANSGENIC PLANTS; Ultraviolet radiation
• ISSN: 0167-4412; 1573-5028
• DOI: 10.1023/A:1005710407155

We have isolated a haploid cell line of N. plumbaginifolia, hNP 588, that is constitutive and not inducible for nitrate reductase. Nitrate reductase mutants were isolated from hNP 588 protoplasts upon UV irradiation. Two of these nitrate reductase-deficient cell lines, nia 3 and nia 25, neither of which contained any detectable nitrate reductase activity, were selected for complementation studies. A cloned Arabidopsis thaliana nitrate reductase gene Nia 2 was introduced into each of the two mutants resulting in 56 independent kanamycin-resistant cell lines. Thirty of the 56 kanamycin-resistant cell lines were able to grow on nitrate as the sole nitrogen source. Eight of these were further analyzed 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 activity levels were not influenced by the nitrogen source in the medium. The eight lines examined expressed a constitutive, non-inducible 3.2 kb mRNA species that was not present in untransformed controls.