title: Multiple regulatory elements in the Arabidopsis NIA1 promoter act synergistically to form a nitrate enhancer.
abstract: To accommodate fluctuating nutrient levels in the soil, plants modulate their metabolism and root development via signaling mechanisms that rapidly reprogram the plant transcriptome. In the case of nitrate, over 1,000 genes are induced or repressed within minutes of nitrate exposure. To identify cis-regulatory elements that mediate these responses, an enhancer screen was performed in transgenic Arabidopsis (Arabidopsis thaliana) plants. A 1.8-kb promoter fragment from the nitrate reductase gene NIA1 was identified that acts as a nitrate enhancer when fused to a 35S minimal promoter. Enhancer activity was localized to a 180-bp fragment, and this activity could be enhanced by the addition of a 131-bp fragment from the nitrite reductase promoter. A promoter construct containing the 180- and 131-bp fragments was also induced by nitrite and repressed by ammonium, indicating that it was responsive to multiple nitrogen signals. To identify specific regulatory elements within the 180-bp NIA1 fragment, a transient expression system using agroinfiltration of Nicotiana benthamiana was developed. Deletion analysis identified three elements corresponding to predicted binding motifs for homeodomain/E-box, Myb, and Alfin1 transcription factors. A fully active promoter showing nitrate and nitrite enhancer activity equivalent to that of the wild-type 180-bp fragment could be built from these three elements if the spacing between the homeodomain/E-box and Myb-Alfin1 sites was equivalent to that of the native promoter. These findings were validated in transgenic Arabidopsis plants and identify a cis-regulatory module containing three elements that comprise a nitrate enhancer in the NIA1 promoter.
authors.affiliation: Division of Biological Sciences, University of California, San Diego, La Jolla, California 92093-0116, USA.
authors.forename: Rongchen
authors.initials: R
authors.lastname: Wang
authors.forename: Peizhu
authors.initials: P
authors.lastname: Guan
authors.forename: Mingsheng
authors.initials: M
authors.lastname: Chen
authors.forename: Xiujuan
authors.initials: X
authors.lastname: Xing
authors.forename: Yali
authors.initials: Y
authors.lastname: Zhang
authors.forename: Nigel M
authors.initials: NM
authors.lastname: Crawford
mesh_terms: D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics*; D020029:Base Pairing / Q000235:genetics; D001483:Base Sequence; D001681:Biological Assay; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D005966:Glucuronidase / Q000378:metabolism; D008969:Molecular Sequence Data; D050901:Nitrate Reductase / Q000235:genetics*; D009566:Nitrates / Q000378:metabolism*; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D017384:Sequence Deletion / Q000235:genetics; D014026:Nicotiana / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D009566:Nitrates; D000644:Quaternary Ammonium Compounds; D012097:Repressor Proteins; D050901:Nitrate Reductase; D005966:Glucuronidase