Molecular mechanisms of herbicide-inducible gene expression of tobacco CYP71AH11 metabolizing the herbicide chlorotoluron.
Tobacco cytochrome P450 (CYP) 71AH11 metabolized the herbicide chlorotoluron, and its mRNA level was increased in tobacco culture cells by the treatment of 2,4-D. In order to clarify molecular mechanisms of induced gene expression of CYP71AH11 by herbicide treatment, a 1574-bp 5'-flanking region of CYP71AH11 was cloned, ligated to the reporter β-glucuronidase (GUS) gene, and then transformed into tobacco plants. The GUS activity in the transgenic tobacco plants was highly induced by bromoxynil treatment, followed by 2,4-D. Chlorotoluron was slightly increased the GUS activity. The bromoxynil-increased GUS activity was partially repressed by the antioxidants, suggesting that reactive oxygen species may be involved in activation of the 5'-flanking region of CYP71AH11 by bromoxynil treatment. Deletion and mutation assays showed that the region CD (-1281 to -770bp from the start codon of CYP71AH11) was important, but not sufficient, for response to bromoxynil. Electrophoretic mobility shift assays and southwestern blotting revealed that the sequences AAAAAG, and GAACAAAC and GAAAATTC in the CD region were important for interaction to the nuclear proteins of <30 and ≈75 kDa, respectively. Particularly, interaction between AAAAAG and <30 kDa proteins was increased by bromoxynil treatment. These results gave a cue for understanding the bromoxynil-induced gene expression of CYP71AH11, which may contribute to herbicide tolerance and selectivity in crop plants.
Research Center for Environmental Genomics, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe, Hyogo 657-8501, Japan.
Keiko
K
Gion
Research Center for Environmental Genomics, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe, Hyogo 657-8501, Japan.
Hideyuki
H
Inui
Graduate School of Science and Technology, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe, Hyogo 657-8501, Japan.
Kazuyuki
K
Takakuma
Graduate School of Science and Technology, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe, Hyogo 657-8501, Japan.
Takashi
T
Yamada
Graduate School of Science and Technology, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe, Hyogo 657-8501, Japan.
Yumiko
Y
Kambara
Graduate School of Science and Technology, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe, Hyogo 657-8501, Japan.
Shuichi
S
Nakai
Graduate School of Science and Technology, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe, Hyogo 657-8501, Japan.
Hiroyuki
H
Fujiwara
Graduate School of Science and Technology, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe, Hyogo 657-8501, Japan.
Takashi
T
Miyamura
Research Center for Environmental Genomics, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe, Hyogo 657-8501, Japan.
Hiromasa
H
Imaishi
Research Center for Environmental Genomics, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe, Hyogo 657-8501, Japan. Electronic address: hideo.ohkawa@gmail.com.
Hideo
H
Ohkawa
D001483:Base Sequence; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D006540:Herbicides / Q000378:metabolism*; D008969:Molecular Sequence Data; D010671:Phenylurea Compounds / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
D006540:Herbicides; D010671:Phenylurea Compounds; D010940:Plant Proteins; D003577:Cytochrome P-450 Enzyme System; C007033:chlortoluron
Bromoxynil; Chlorotoluron; Cytochrome P450 71AH11; Reactive oxygen species