calmodulin-binding protein kinase 3 is part of heat-shock signal transduction in Arabidopsis thaliana

• Published in: The Plant journal : for cell and molecular biology 2008-09, Vol.55 (5), p.760-773
• Main Authors: Liu, Hong-Tao, Gao, Fei, Li, Guo-Liang, Han, Jin-Long, Liu, De-Long, Sun, Da-Ye, Zhou, Ren-Gang
• Format: Article
• Language: English
• Subjects: Arabidopsis; Arabidopsis - enzymology; Arabidopsis - genetics; Arabidopsis Proteins - genetics; Arabidopsis Proteins - metabolism; Arabidopsis thaliana; Biological and medical sciences; Botany; Calcium-Calmodulin-Dependent Protein Kinases - genetics; Calcium-Calmodulin-Dependent Protein Kinases - metabolism; calmodulin; calmodulin-binding protein kinase; Cell physiology; DNA, Bacterial - genetics; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Fluorescence; Fluorescence Resonance Energy Transfer; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Plant; Genes, Plant; Genetic Complementation Test; Genetic recombination; Heat Shock Transcription Factors; heat tolerance; Heat-Shock Proteins - genetics; Heat-Shock Proteins - metabolism; Heat-Shock Response; Hot Temperature; Kinases; Molecular and cellular biology; Mutagenesis, Insertional; Phosphorylation; phosphorylation of HSF; Plant Proteins - genetics; Plant Proteins - metabolism; Plants, Genetically Modified - enzymology; Plants, Genetically Modified - genetics; Protein Binding; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; thermotolerance; Transcription Factors - genetics; Transcription Factors - metabolism; Transcription, Genetic; Two-Hybrid System Techniques
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1111/j.1365-313X.2008.03544.x

Based on our previous findings, we proposed a pathway for the participation of Ca²⁺/calmodulin (CaM) in heat-shock (HS) signal transduction. The specific mechanism by which CaM regulates activation of heat-shock transcription factors (HSFs) is not known. CaM-binding protein kinases (CBK) are the most poorly understood of the CaM target proteins in plants. In this study, using a yeast two-hybrid assay, we found that AtCBK3 interacts with AtHSFA1a. Fluorescence resonance energy transfer was used to confirm the interaction between AtCBK3-YFP and AtHSFA1a-CFP. Furthermore, we demonstrate that purified recombinant AtCBK3 phosphorylated recombinant AtHSFA1a in vitro. We also describe the results of both downregulation of AtCBK3 expression and ectopic overexpression in Arabidopsis thaliana. The T-DNA insertion AtCBK3 knockout lines had impaired basal thermotolerance, which could be complemented by transformation of plants with the native gene. Overexpression of AtCBK3 resulted in plants with increased basal thermotolerance. Results from real-time quantitative PCR and protein gel-blot analyses suggest that AtCBK3 regulates transcription of heat-shock protein (HSP) genes and synthesis of HSPs. The binding activity of HSF to the heat-shock element (HSE), the mRNA level of HSP genes and synthesis of HSPs were upregulated in AtCBK3-overexpressing lines after HS, but downregulated in AtCBK3 null lines. These results indicate that AtCBK3 controls the binding activity of HSFs to HSEs by phosphorylation of AtHSFA1a, and is an important component of the HS signal transduction pathway.