GmNiR-1, a soybean nitrite reductase gene that is regulated by nitrate and light

Abstract

A genomic DNA clone encoding nitrite reductase (NiR; EC 1.7.7.1), GmNiR-1, was isolated from soybean [Glycine max (L.) Merr.]. It is composed of four exons and three introns carrying an open reading frame coding for a protein of 596 amino acids. The putative MW of GmNiR-1 is 67 kDa with theoretical pI of 6.95, but the measured MW is ca 64 kDa. The difference between putative and measured MW of GmNiR-1 lies in the presence of a transit peptide. Genomic DNA blot analysis suggested that soybean NiR gene family consisted of at least three genes. In 2-week-old plants grown in the nitrate-supplemented soil, GmNiR-1 was expressed in roots and leaves, but not in hypocotyls. Transcript level of GmNiR-1 in roots was not increased by light, but was increased by nitrate even in the dark. However, light and nitrate had a synergistic effect on the increased expression of GmNiR-1. A polyclonal antibody generated against the C-terminal of GmNiR-1 hybridized to a single (62 kDa), double (62 and 64 kDa), and three bands (62, 64 and 66 kDa) in roots, hypocotyls and leaves, respectively. In etiolated seedlings, NiR proteins in roots and hypocotyls were induced by simultaneous treatment with light and nitrate, but those in cotyledons were already present substantially without induction, implying the presence of at least two kinds of NiR genes (a constitutive one and an inducible one regulated by light and nitrate) in soybean seedlings.