Omics-based identification of Arabidopsis Myb transcription factors regulating aliphatic glucosinolate biosynthesis
- Masami Yokota Hirai*,
- Kenjiro Sugiyama†,
- Yuji Sawada*,
- Takayuki Tohge*,
- Takeshi Obayashi‡,§,
- Akane Suzuki*,
- Ryoichi Araki*,¶,
- Nozomu Sakurai†,
- Hideyuki Suzuki†,
- Koh Aoki†,
- Hideki Goda*,
- Osamu Ishizaki Nishizawa*,¶,
- Daisuke Shibata†, and
- Kazuki Saito*,‡,‖
- *RIKEN Plant Science Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan;
- †Kazusa DNA Research Institute, 2-6-7 Kazusakamatari, Kisarazu, Chiba 292-0818, Japan;
- ‡Graduate School of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba, Chiba 263-8522, Japan;
- §Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Saitama 332-0012, Japan; and
- ¶Central Laboratories for Frontier Technology, Kirin Brewery Company, Ltd., 1-13-5 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-0004, Japan
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Edited by Marc C. E. Van Montagu, Ghent University, Ghent, Belgium, and approved February 13, 2007 (received for review December 28, 2006)
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Fig. 1.Coexpression analysis of aliphatic GSL biosynthetic genes and transcription factors. Yellow and red points indicate genes encoding enzymes and transcription factors, respectively. Transcripts from AtIMD1 and AtIMD3 and those from CYP79F1 and CYP79F2 were cross-hybridized to the same probe sets on an ATH1 microarray used in AtGenExpress and hence are indistinguishable. Lengths of the lines are valueless in these displays.
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Fig. 2.Expression of GSL biosynthetic genes and GSL contents in Myb28-knockout mutant myb28. (A) Heat map showing gene expression levels. Log ratio (base 2) of signal intensity in myb28 to wild type is shown by the color scale. The data represent three hybridizations. Transcripts of CYP79F1 and CYP79F2 were cross-hybridized to the same probe set on an ATH1 microarray because of similarity of their nucleotide sequences. (B) GSL contents of rosette leaves. The means and SD of three replicates are shown. Closed and open bars indicate aliphatic and indole GSLs, respectively. A statistically significant decrease in myb28 (Right) compared with wild type (Left) is shown as “a” (Welch's t test; P < 0.01) or “b” (P < 0.02) on the bar. 8MTO, 8-methylthiooctyl GSL; 8MSOO, 8-methylsulfinyloctyl GSL; I3M, indol-3-ylmethyl GSL; 1MOI3M, 1-methoxyindol-3-ylmethyl GSL. Note that the scale is logarithmic.
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Fig. 3.Gene expression in response to MeJA. The relative expression levels of Myb28, Myb29, CYP79F1, CYP79F2, and CYP83A1 normalized to the constitutive ubiquitin gene UBC9 are shown as means and SD of three replicates. Detailed methods of quantitative RT-PCR are described in SI Methods. Note scalar differences between regulatory and structural gene expression levels.
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Fig. 4.Expression of GSL biosynthetic genes and GSL contents in Myb28-overexpressing suspension cell cultures. (A) Heat map showing gene expression levels. Log ratio (base 2) of signal intensity in Myb28-overexpressing T87 cell suspension cultures (T87OX) to control lines (empty-vector transformed) is shown by the color scale. The data are of nine hybridizations. (B) GSL contents. The data of three independent transformants of control (empty-vector transformed) (Left) and Myb28-overexpressing suspension cells (T87OX) (Right) are shown as three bars for each type of GSL. The means and SD of three replicates are shown. Closed and open bars indicate aliphatic and indole GSLs, respectively. nd, not detected in all three control cell culture lines; 8MTO, 8-methylthiooctyl GSL; 8MSOO, 8-methylsulfinyloctyl GSL; I3M, indol-3-ylmethyl GSL; 1MOI3M, 1-methoxyindol-3-ylmethyl GSL. Note that the scale is logarithmic.
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Fig. 5.Regulatory networks model of GSL biosynthetic pathway in Arabidopsis accession Columbia. PMG1/Myb28 and PMG2/Myb29 are positive regulators of aliphatic GSL biosynthesis, whereas ATR1/Myb34 positively regulates indole GSL formation. PMG1/Myb28 is the essential and sufficient master regulator and thus supports the basal production of aliphatic GSLs. PMG2/Myb29 is an accessory factor that plays a role in response to MeJA signaling. MeJA also induces the ATR1/Myb34 cascade. Sulfur deficiency represses the expression of all three Myb factors. Previously unidentified structural genes can be mapped in the aliphatic GSL biosynthetic pathway by the present study. MTG, methylthioalkyl GSL; MSOG, methylsulfinylalkyl GSL.
Footnotes
- ‖To whom correspondence should be addressed. E-mail: ksaito{at}psc.riken.jp
- © 2007 by The National Academy of Sciences of the USA
