The impact of simultaneous environmental stresses on plants and how they respond to combined stresses compared with single stresses is largely unclear. By using a transgene (RD29A-LUC) consisting of the firefly luciferase coding sequence (LUC) driven by the stress-responsive RD29A promoter, we investigated the interactive effects of temperature, osmotic stress, and the phytohormone abscisic acid (ABA) in the regulation of gene expression in Arabidopsis seedlings. Results indicated that both positive and negative interactions exist among the studied stress factors in regulating gene expression. At a normal growth temperature (22°C), osmotic stress and ABA act synergistically to induce the transgene expression. Low temperature inhibits the response to osmotic stress or to combined treatment of osmotic stress and ABA, whereas low temperature and ABA treatments are additive in inducing transgene expression. Although high temperature alone does not activate the transgene, it significantly amplifies the effects of ABA and osmotic stress. The effect of multiple stresses in the regulation of RD29A-LUC expression in signal transduction mutants was also studied. The results are discussed in the context of cold and osmotic stress signal transduction pathways.Plants grow in an inconstant environment that frequently imposes constraints on growth and development. Among the adverse environmental factors commonly encountered by land plants are extreme temperatures and osmotic stress that results from conditions of high salinity or periods of drought. Much research has been conducted to understand the molecular mechanisms underlying plant responses to these stresses (for review, see Bray, 1993;Guy et al., 1994;Thomashow, 1994;Ingram and Bartels, 1996;Zhu et al., 1997). In most of the laboratory studies a single environmental stress factor is imposed and the consequent responses are analyzed. However, in the natural habitats of plants the adverse environmental factors are almost never present alone. For example, osmotic stress caused by drought in the summer is often accompanied by hightemperature stress, and low temperature may also bring with it osmotic stress because it impairs water absorption and transport. Under conditions of simultaneous stresses, the negative effects on plants and plant adaptive responses may differ from those under a single adverse environmental condition such as that which prevails in laboratory research. It is necessary, therefore, to understand how plants respond to combined stress signals.The early events of plant adaptation to environmental stress are the sensing and subsequent signal transduction to activate various physiological and metabolic responses, including stress-responsive gene expression. Many genes can be induced by osmotic stress (Bray, 1993;Shinozaki and Yamaguchi-Shinozaki, 1997;Zhu et al., 1997) or low temperature (Thomashow, 1994). Osmotic stress and low temperature increase the cellular level of the phytohormone ABA (Zeevaart and Creelman, 1988;Chandler and Robertson, 1994), and th...