Ca2+ and Calmodulin Modulate DNA-Binding Activity of Maize Heat Shock Transcription Factor in Vitro

DNA-binding activity of a maize heat shock transcription factor (HSF) was induced by heat shock of a whole cell extract at 44 degrees C. Addition of the calcium ion chelator EGTA reduced the binding of the HSF to heat shock element (HSE) in vitro. Re-addition of CaCl(2) to the sample pretreated with EGTA restored the ability of the HSF to bind to DNA. DNA-binding activity of the HSF was also induced by directly adding CaCl(2) to a whole cell extract at non-heat-shock temperature, but not by MgCl(2). During HS at 44 degrees C, calmodulin (CaM) antagonists chlorpromazine (CPZ) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) inhibited DNA-binding activity of the HSF in a concentration-dependent manner, but N-(6-aminohexyl)-1-naphthalenesulfonamide (W5), an inactive structural analogue of W7, did not. Addition of antiserum specific to CaM reduced the binding of the HSF to HSE. Re-addition of CaM to the sample pretreated with antiserum could restore the binding activity of the HSF. DNA-binding activity of the HSF was promoted by directly adding CaM to a whole cell extract at 44 degrees C, but not by BSA. Moreover, at non-heat-shock temperature, DNA-binding activity of the HSF was also induced by directly adding CaM to a whole cell extract, but not by BSA. Our observations further confirm the role of Ca(2+) in activation of the HSF in plant and provide the first example of the role of CaM in regulation of DNA-binding activity of the HSF. These results suggest that Ca(2+) and CaM are involved in HSP gene expression likely through regulating the activity of the HSF.