FOOTPRINTING OF THE SPINACH NITRITE REDUCTASE GENE PROMOTER REVEALS THE PRESERVATION OF NITRATE REGULATORY ELEMENTS BETWEEN FUNGI AND HIGHER-PLANTS

Nitrite reductase (NiR) is the second enzyme in the nitrate assimilato ry pathway reducing nitrite to ammonium. The expression of the NiR gen e is induced upon the addition of nitrate. In an earlier study, a 130 bp upstream region of the spinach NiR gene promoter, located between - 330 to -200, was shown to be necessary for nitrate induction of beta-g lucuronidase (GUS) expression in tissue-specific manner in transgenic tobacco plant [28]. To further delineate the cis-acting elements invol ved in nitrate regulation of NiR gene expression, transgenic tobacco p lants were generated with 5' deletions in the -330 to -200 region of t he spinach NiR gene promoter fused to the GUS gene. Plants with the Ni R promoter deleted to -230 showed a considerable increase in GUS activ ity in the presence of nitrate, indicating that the 30 bp region betwe en -230 to -200 is crucial for nitrate-regulated expression of NiR. In vivo DMS footprinting of the -300 to -130 region of the NiR promoter in leaf tissues from two independent transgenic lines revealed several nitrate-inducible footprints. Footprinting within the -230 to -181 re gion revealed factor binding to two adjacent GATA elements separated b y 24 bp. This arrangement of GATA elements is analogous to cis-regulat ory sequences found in the promoters of nitrate-inducible genes of Neu rospora crassa, regulated by the NIT2 Zn-finger protein. The -240 to - 110 fragment of the NiR promoter, which contains two NIT2 consensus co re elements, bound in vitro to a fusion protein comprising the zinc fi nger domain of the N. crassa NIT2 protein. The data presented here sho w that nitrate-inducible expression of the NiR gene is mediated by nit rate-specific binding of trans-acting factors to sequences preserved b etween fungi and higher plants.