PLANT PHYSIOLOGY , Vol 113, Issue 3 853-862, Copyright © 1997 by American Society of Plant Biologists
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GENE REGULATION AND MOLECULAR GENETICS |
Sequences Necessary for Nitrate-Dependent Transcription of Arabidopsis Nitrate Reductase Genes
C. F. Hwang, Y. Lin, T. D'Souza and C. L. Cheng
Department of Biological Sciences, The University of Iowa, Iowa City, Iowa 52242
Nitrate increases the transcription of the two Arabidopsis thaliana nitrate
reductase genes. We demonstrated previously that 238 and 330 bp of the
5[prime] flanking regions, designated as NP1 and NP2, of the two nitrate
reductase genes NR1 and NR2, respectively, are sufficient for
nitrate-dependent transcription (Y. Lin, C.-F. Hwang, J.B. Brown, C.-L.
Cheng [1994] Plant Physiol 106: 477-484). Here we identify the cis-acting
elements of NP1 and NP2 that are necessary for nitrate-dependent
transcription by linker-scanning (LS) analysis. In transgenic plants one LS
mutant of NP1 and two LS mutants of NP2 exhibited significantly lower
nitrate-induced reporter gene chloramphenicol acetyltransferase activity.
To distinguish which of these three mutants lost nitrate inducibility,
competitive reverse-transcriptase polymerase chain reaction was used to
measure the chloramphenicol acetyltransferase mRNA levels before and after
nitrate induction. The single LS mutant in NP1 lost its response to
nitrate, whereas the two LS mutants in NP2 partially lost their response to
nitrate. A 12-bp sequence is conserved between the NP1 site and the two NP2
sites. This sequence motif is also conserved in the 5[prime] flanking
regions of other nitrate-inducible plant genes. Gel mobility shift
experiments indicate that these three regions bind to similar proteins. The
binding is constitutive with respect to nitrate treatment and was observed
in both nonphotosynthetic suspension cells and green leaves.
