A Calmodulin Binding Protein from Arabidopsis Is Induced by Ethylene and Contains a DNA-Binding Motif A. S. N. Reddy, 1 Vaka S. Reddy, and Maxim Golovkin Department of Biology and Program in Cell and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523 Received October 23, 2000 Calmodulin (CaM), a key calcium sensor in all eu- karyotes, regulates diverse cellular processes by inter- acting with other proteins. To isolate CaM binding proteins involved in ethylene signal transduction, we screened an expression library prepared from ethylene-treated Arabidopsis seedlings with 35 S- labeled CaM. A cDNA clone, EICBP (Ethylene-Induced CaM Binding Protein), encoding a protein that inter- acts with activated CaM was isolated in this screening. The CaM binding domain in EICBP was mapped to the C-terminus of the protein. These results indicate that calcium, through CaM, could regulate the activity of EICBP. The EICBP is expressed in different tissues and its expression in seedlings is induced by ethylene. The EICBP contains, in addition to a CaM binding domain, several features that are typical of transcrip- tion factors. These include a DNA-binding domain at the N terminus, an acidic region at the C terminus, and nuclear localization signals. In database searches a partial cDNA (CG-1) encoding a DNA-binding motif from parsley and an ethylene up-regulated partial cDNA from tomato (ER66) showed significant similar- ity to EICBP. In addition, five hypothetical proteins in the Arabidopsis genome also showed a very high se- quence similarity with EICBP, indicating that there are several EICBP-related proteins in Arabidopsis. The structural features of EICBP are conserved in all EICBP-related proteins in Arabidopsis, suggesting that they may constitute a new family of DNA binding proteins and are likely to be involved in modulating gene expression in the presence of ethylene. © 2000 Academic Press Key Words: calmodulin binding protein; ethylene; transcription factor; ankyrin repeat; DNA-binding do- main; Arabidopsis. Calcium (Ca 2+ ) is an important intracellular messen- ger in plants. In recent years, a large number of hor- monal signals, and biotic and abiotic stresses have been shown to elevate cytoplasmic Ca 2+ ([Ca 2+ ] cyt ) lev- els transiently (1–5). Four major families of proteins that sense changes in [Ca 2+ ] cyt have been identified in plants (5, 6). These include (i) calmodulin (CaM) and its isoforms, (ii) CaM-like proteins, (iii) Ca 2+ - dependent and CaM-independent protein kinases (CD- PKs) that are found only in plants and some protozo- ans, and (iv) Ca 2+ -binding proteins without EF-hand motifs (5– 8). Of these Ca 2+ sensors, CaM is highly conserved among all eukaryotes and contains four Ca 2+ binding EF-hand motifs (1, 6, 7). Activated CaM (Ca 2+ / CaM complex) interacts with a variety of unrelated proteins (CaM binding proteins, CBPs) and regulates their activity/function (5–7). To understand Ca 2+ me- diated signaling pathways in plants it is essential to isolate and identify CaM binding proteins. Screening of expression libraries with labeled CaM has resulted in isolation of a limited number of CBPs in plants (5, 9 –11). Interestingly, several plant CBPs have no ho- mologs in nonplant systems (5). Ethylene, a two-carbon gaseous plant hormone, reg- ulates diverse physiological processes during plant growth and development (12). Seed germination, seed- ling growth, fruit ripening, senescence, plant defense responses, abscission and sex determination are some of the processes that are regulated by ethylene (12, 13). Using genetic and molecular approaches significant advances have been made in elucidating the ethylene signal transduction pathway (13–16). Isolation of mu- tants that are insensitive to ethylene or show ethylene response in the absence of ethylene and cloning of corresponding genes from the model plant Arabidopsis have resulted in identification of many components of the ethylene signal transduction pathway (13–19). In addition to characterization of ETR/ERS and CTR gene products in the ethylene signal transduction 1 To whom correspondence and reprint requests should be ad- dressed. Fax: 970-491-0649. E-mail: reddy@lamar.colostate.edu. Biochemical and Biophysical Research Communications 279, 762–769 (2000) doi:10.1006/bbrc.2000.4032, available online at http://www.idealibrary.com on 762 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.