BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 250, 161–170 (1998) ARTICLE NO. RC989267 An Arabidopsis Gene Family Encoding DRE/CRT Binding Proteins Involved in Low-Temperature-Responsive Gene Expression Zabta K. Shinwari,* Kazuo Nakashima,* Setsuko Miura,* Mie Kasuga,* Motoaki Seki,† Kazuko Yamaguchi-Shinozaki,* ,1 and Kazuo Shinozaki† ,1 *Biological Resources Division, Japan International Research Center for Agricultural Sciences (JIRCAS), 1-2, Ohwashi, Tsukuba, Ibaraki 305-8686, Japan; and †Laboratory of Plant Molecular Biology, Institute of Physical and Chemical Research (RIKEN), 3-1-1, Koyadai, Tsukuba, Ibaraki 305-0074, Japan Received July 27, 1998 scribed recently, and their gene products are thought In higher plants, a cis-acting element, DRE/CRT, is to be involved in stress response and tolerance (1-4). involved in gene expression responsive to drought and It is important to know how plants sense low temper- low-temperature stress. To understand signal trans- ature and water deficit and process this information to duction pathways from the cold stress signal to gene alter gene expression. Promoter analyses have been expression, we characterized a gene family for DRE/ performed with many stress-inducible genes by drought, CRT-binding proteins DREB1A and CBF1 in Arabi- low temperature, and high salinity in a variety of dopsis thaliana. DREB1A and CBF1 were shown to be plants. ABA-independent as well as ABA-dependent involved in low-temperature-responsive gene expres- regulatory mechanisms have been shown to be involved sion. We screened an Arabidopsis genomic DNA library in transcription of those stress-inducible genes. Identi- with the cDNA fragment of DREB1A as a probe and fication of a cis-acting element involved in respon- isolated DREB1A and 2 related genes, DREB1B (Å siveness to drought, low temperature, and high salt CBF1) and DREB1C. These were arrayed in the order stress in Arabidopsis thaliana is an important advance B, A, C in an 8.7 kb region of Arabidopsis chromosome 4. Northern blot analysis using gene-specific probes in this regard. A 9-bp conserved sequence, TACCGA- showed that the 3 DREB1 genes are induced mainly by CAT, termed the dehydration-responsive element cold stress but not by osmotic stress in leaves, roots, (DRE), is essential for the induction of the rd29A gene and stems. Several conserved sequences were found (also known as cor78 and lti78) of Arabidopsis not only in the promoter regions of all 3 genes. The b-glucuroni- by osmotic stress caused by drought and high salinity dase (GUS) reporter gene driven by the DREB1 pro- but also by low-temperature stress (5). However, DRE moters was induced at transcriptional level by low is not involved in ABA-responsive gene expression. temperature in transgenic Arabidopsis plants.  1998 DRE-related motifs have been reported in promoters Academic Press of multiple genes regulated by osmotic and low-temper- ature stress, including kin1, cor6.6/kin2, and rd17/ cor47 in Arabidopsis (6, 7). A similar motif (C-repeat; CRT) was also reported in the promoter region of cold- Plants are inevitably confronted during their life and dehydration-inducible cor15A (8). The CCGAC core cycle with numerous environmentally determined sequence was shown to be important for cold-respon- stresses that can be detrimental to their survival. In sive gene expression in the 5 promoter region of the order to meet such challenges, plants not only possess structural physical barriers against environmental cold-inducible Brassica napus gene BN115 (9). stress but also have evolved inducible mechanisms that We isolated 2 types of cDNAs encoding DRE binding allow them to respond to environmental stimuli. A proteins (DREB1A and DREB2A) in Arabidopsis using number of genes that respond to desiccation and low yeast one-hybrid screening with cDNA libraries pre- temperature at the transcriptional level have been de- pared from dehydrated or cold-treated Arabidopsis plants (10). The deduced amino acid sequences of DREB1A and DREB2A showed no significant sequence 1 For correspondence: (Kazuko Yamaguchi-Shinozaki: Fax: /81 similarity except in the conserved DNA-binding do- 298 38 6643; E-mail: kazukoys@jircas.affrc.go.jp) or (Kazuo Shino- zaki: Fax: /81 298 36 9060; E-mail: sinozaki@rtc.riken.go.jp). main found in the EREBP and AP2 proteins. Both 0006-291X/98 $25.00 Copyright  1998 by Academic Press All rights of reproduction in any form reserved. 161