Respiratory metabolism is an important though poorly understood facet of plant adaptation to stress. Posttranslational modification of aconitase, a component of the tricarboxylic acid cycle (TCA), may be involved in stress tolerance. However, such stress-related transcriptional regulation and its mechanism remain unknown. In this study, we found that expression of the rice Aconitase gene OsACO1 is induced in a time-dependent manner by heat but not other typical abiotic stresses. To analyze the transcriptional regulation mechanism underlying the response to heat, the OsACO1 promoter (POsACO1) was isolated and characterized in transgenic rice. Using qualitative and quantitative analyses, we found that the expression of the GUS reporter gene responded to heat in different tissues and at different stages of development when driven by POsACO1. A series of 5' distal deletions of POsACO1 was generated to delineate the region responsible for heat-induced gene expression. Transient expression analyses in tobacco leaves identified a 322-bp minimal region between -1386 and -1065 as being essential and sufficient for heat-induced expression by POsACO1. We screened for known heat response-related cis-elements in this 322-bp region; however, sequences correlating with heat-induced gene expression were not identified in POsACO1. Therefore, truncations and successive mutagenesis analyses were performed in this 322-bp region. By comparing the activities of promoter fragments and their derivatives, our results indicated that the heat response element resided in a 9-bp region between -1132 and -1124, a sequence that contains a W-box motif. Additional site-directed mutagenesis analyses eliminated the heat response activity of POsACO1 via the W-box element, and an electrophoretic mobility shift assay (EMSA) indicated the binding of POsACO1 by factors in the nuclear extracts of heat-stressed rice seedlings in a W-box-dependent manner. Our results illustrate the expression pattern of a key component of the TCA response to abiotic stress and establish a putative regulatory pathway in the transcriptional modulation of rice respiratory metabolism genes in response to heat.
