Cf. Hwang et al., SEQUENCES NECESSARY FOR NITRATE-DEPENDENT TRANSCRIPTION OF ARABIDOPSIS NITRATE REDUCTASE GENES, Plant physiology, 113(3), 1997, pp. 853-862
Nitrate increases the transcription of the two Arabidopsis thaliana ni
trate reductase genes. We demonstrated previously that 238 and 330 bp
of the 5' flanking regions, designated as NP1 and NP2, of the two nitr
ate reductase genes NR1 and NR2, respectively, are sufficient for nitr
ate-dependent transcription (Y. Lin, C.-F. Hwang, J.B. Brown, C.-L. Ch
eng [1994] Plant Physiol 106: 477-484). Here we identify the cis-actin
g elements of NP1 and NP2 that are necessary for nitrate-dependent tra
nscription by linker-scanning (LS) analysis. In transgenic plants one
LS mutant of NP1 and two LS mutants of NP2 exhibited significantly low
er nitrate-induced reporter gene chloramphenicol acetyltransferase act
ivity. To distinguish which of these three mutants lost nitrate induci
bility, competitive reverse-transcriptase polymerase chain reaction wa
s used to measure the chloramphenicol acetyltransferase mRNA levels be
fore 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' 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.