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.
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 beta-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 P(NAC)-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.
Biological Resources Division, Japan International Research Center for Agricultural Sciences, Tsukuba, Ibaraki 305-8686, Japan.
Lam-Son Phan
LS
Tran
Kazuo
K
Nakashima
Yoh
Y
Sakuma
Sean D
SD
Simpson
Yasunari
Y
Fujita
Kyonoshin
K
Maruyama
Miki
M
Fujita
Motoaki
M
Seki
Kazuo
K
Shinozaki
Kazuko
K
Yamaguchi-Shinozaki
D000040:Abscisic Acid / Q000494:pharmacology; D000064:Acclimatization / Q000235:genetics; D000251:Adenosine Triphosphatases; D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence / Q000235:genetics; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D060365:Chloroplast Proteins; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003681:Dehydration / Q000235:genetics / Q000378:metabolism* / Q000503:physiopathology; D008969:Molecular Sequence Data; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D012462:Saline Solution, Hypertonic / Q000494:pharmacology; D037941:Silencer Elements, Transcriptional / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015854:Up-Regulation / Q000235:genetics
D000040:Abscisic Acid; D000251:Adenosine Triphosphatases; D029681:Arabidopsis Proteins; D060365:Chloroplast Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D012462:Saline Solution, Hypertonic; D014157:Transcription Factors; C493659:NAC019 protein, Arabidopsis; C493660:ANAC055 protein, Arabidopsis; C493661:ANAC072 protein, Arabidopsis; C000629270:ClpD protein, Arabidopsis