Abstract
Heat shock (HS) is a common form of stress suffered by plants. It has been proposed that calmodulin (CaM) is involved in HS signal transduction, but direct evidence has been lacking. To investigate the potential regulatory function of CaM in the HS signal transduction pathway, T-DNA knockout mutants for AtCaM2, AtCaM3, and AtCaM4 were obtained and their thermotolerance tested. Of the three knockout mutant plants, there were no differences compared with wild-type plants under normal conditions. However, the AtCaM3 knockout mutant showed a clear reduction in thermotolerance after heat treatment at 45 degrees C for 50 min. Overexpression of AtCaM3 in either the AtCaM3 knockout or wild-type background significantly rescued or increased the thermotolerance, respectively. Results from electrophoretic mobility-shift assays, real-time quantitative reverse transcription-polymerase chain reaction, and western-blot analyses revealed that, after HS, the DNA-binding activity of HS transcription factors, mRNA transcription of HS protein genes, and accumulation of HS protein were down-regulated in the AtCaM3 knockout mutant and up-regulated in the AtCaM3-overexpressing transgenic lines. Taken together, these results suggest that endogenous AtCaM3 is a key component in the Ca2+-CaM HS signal transduction pathway.
Highlights
Heat shock (HS) is a common form of stress suffered by plants
The levels of CaM mRNA and protein increased during HS in wheat, and the increase of CaM mRNA for only 10 min preceded the expression of heat shock proteins (HSPs) genes, which was detected after 20 min of HS (Liu et al, 2003)
To determine whether AtCaM3 is involved in the HS signal transduction pathway, we first utilized a reverse genetic approach by screening Arabidopsis T-DNA insertion mutants effecting AtCaM family members
Summary
Heat shock (HS) is a common form of stress suffered by plants. It has been proposed that calmodulin (CaM) is involved in HS signal transduction, but direct evidence has been lacking. Results from electrophoretic mobility-shift assays, real-time quantitative reverse transcription-polymerase chain reaction, and western-blot analyses revealed that, after HS, the DNA-binding activity of HS transcription factors, mRNA transcription of HS protein genes, and accumulation of HS protein were downregulated in the AtCaM3 knockout mutant and up-regulated in the AtCaM3-overexpressing transgenic lines. Taken together, these results suggest that endogenous AtCaM3 is a key component in the Ca2+-CaM HS signal transduction pathway. To date, there is no direct genetic evidence for CaM involvement in HS signaling
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