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First published online August 19, 2004; 10.1105/tpc.104.022699

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The Plant Cell 16:2481-2498 (2004)
© 2004 American Society of Plant Biologists

Isolation and Functional Analysis of Arabidopsis Stress-Inducible NAC Transcription Factors That Bind to a Drought-Responsive cis-Element in the early responsive to dehydration stress 1 Promoter{fx1}

Lam-Son Phan Trana, Kazuo Nakashimaa, Yoh Sakumaa, Sean D. Simpsonb, Yasunari Fujitaa, Kyonoshin Maruyamaa, Miki Fujitac, Motoaki Sekic, Kazuo Shinozakic,d and Kazuko Yamaguchi-Shinozakia,d,e,1

a Biological Resources Division, Japan International Research Center for Agricultural Sciences, Tsukuba, Ibaraki 305-8686, Japan
b Genesis Research and Development Corporation, Parnell, Auckland, New Zealand
c Laboratory of Plant Molecular Biology, RIKEN Tsukuba Institute, Tsukuba, Ibaraki 305-0074, Japan
d Core Research for Evolution Science and Technology, Japan Science and Technology Agency, Honcho, Kawaguchi-shi, Saitama, 332-0012, Japan
e Laboratory of Plant Molecular Physiology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan

1 To whom correspondence should be addressed. E-mail kazukoys{at}jircas.affrc.go.jp; fax 81-298-38-6643.

The MYC-like sequence CATGTG plays an important role in the dehydration-inducible expression of the Arabidopsis thaliana EARLY RESPONSIVE TO DEHYDRATION STRESS 1 (ERD1) gene, which encodes a ClpA (ATP binding subunit of the caseinolytic ATP-dependent protease) homologous protein. Using the yeast one-hybrid system, we isolated three cDNA clones encoding proteins that bind to the 63-bp promoter region of erd1, which contains the CATGTG motif. These three cDNA clones encode proteins named ANAC019, ANAC055, and ANAC072, which belong to the NAC transcription factor family. The NAC proteins bound specifically to the CATGTG motif both in vitro and in vivo and activated the transcription of a ß-glucuronidase (GUS) reporter gene driven by the 63-bp region containing the CATGTG motif in Arabidopsis T87 protoplasts. The expression of ANAC019, ANAC055, and ANAC072 was induced by drought, high salinity, and abscisic acid. A histochemical assay using PNAC-GUS fusion constructs showed that expression of the GUS reporter gene was localized mainly to the leaves of transgenic Arabidopsis plants. Using the yeast one-hybrid system, we determined the complete NAC recognition sequence, containing CATGT and harboring CACG as the core DNA binding site. Microarray analysis of transgenic plants overexpressing either ANAC019, ANAC055, or ANAC072 revealed that several stress-inducible genes were upregulated in the transgenic plants, and the plants showed significantly increased drought tolerance. However, erd1 was not upregulated in the transgenic plants. Other interacting factors may be necessary for the induction of erd1 in Arabidopsis under stress conditions.




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