Biochemistry, genetics and molecular biology of nitrite reduction in barley

Abstract

Nitrite reduction is the third step of the nitrate assimilation pathway in higher plants and is catalysed by nitrite reductase. The whole-plant barley mutants STA1010, STA2760 and STA4169 accumulate nitrite in the leaf after treatment with nitrate and, like the nir1 mutant STA3999 (Duncanson et al, 1993), lack detectable nitrite reductase cross-reacting material in the leaf and root. STA1010, STA2760 and STA4169 carry a recessive mutation in a single nuclear gene, identified as the Nir1 locus. RFLP analysis of the nir1 mutant STA3999 has allowed the Nir1 locus to be mapped to within 0.3cM of the nitrite reductase apoprotein gene, Nii. Studies to confirm the identity of the Nir1 locus as Nii, by establishing the full-length Nii cDNA sequences from STA3999 and from its wild-type cv Tweed for comparative purposes, were unsuccessful as attempts to isolate a Nii cDNA clone from a barley cv Tweed cDNA library yielded only partial-length Nii clones. These nirl mutants display greatly reduced nitrite reductase activity and increased NADH-nitrate reductase activity in the leaf, as compared to wild-type plants, suggesting a regulatory perturbation in the expression of the Nar1 gene. Northern analysis shows that the nir1 mutants possess nitrite reductase apoprotein (nii) transcript of wild-type size (2.3kb) and at approximately wild-type levels. Since nir1 mutants possess a phenotype that might be anticipated for a Nii mutant, it is likely that the nir1 mutation is present in the nitrite reductase apoprotein gene Nii and affects translation of the nii transcript. Studies of barley wild-type cv Golden Promise have demonstrated that nitrite reductase in leaf tissue is up-regulated by a coaction of nitrate and light which acts, at least partly, at the transcriptional level.