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Dissecting the Heat Stress Response in Chlamydomonas by Pharmaceutical and RNAi Approaches Reveals Conserved and Novel Aspects

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Molecular Plant 2013年第6期6卷 1795-1813页

作者： Stefan Schmollinger Miriam Schulz-Raffelt daniela strenkert Daniel Veyel Olivier Vallon Michael Schroda Molekulare Biotechnologie und Systembiologie TU Kaiserslautern PauI-Ehrlich-Str. 23 D-67663 Kaiserslautern Germany Max-Planck-lnstitut for Ilolekulare Pflanzenphysiologie Am MQhlenberg 1 D-14476 Potsdam-Golm Germany CEA-CNRS-Universite Aix Marseille Institut de Biolog~e Environnementale et Biotechnologie UMR 7265 CEA (:adarache F-13108 Saint-Paul-lez-DuranceFrance Institut de Biologie Physico-Chimique UMR 7141 CNRS/Universite Pierre et Marie Curie F-75005 Paris France

To study how conserved fundamental concepts of the heat stress response （HSR） are in photosynthetic eukaryotes, we applied pharmaceutical and antisense/amiRNA approaches to the unicellular green alga Chlamydomonas reinhardtii. The Chlamydomonas HSR appears to be triggered by the accumulation of unfolded proteins, as it was induced at ambient temperatures by feeding cells with the arginine analog canavanine. The protein kinase inhibitor staurosporine strongly retarded the HSR, demonstrating the importance of phosphorylation during activation of the HSR also in Chlamydomonas. While the removal of extracellular calcium by the application of EGTA and BAPTA inhibited the HSR in moss and higher plants, only the addition of BAPTA, but not of EGTA, retarded the HSR and impaired thermotoler- ance in Chlamydomonas. The addition of cycloheximide, an inhibitor of cytosolic protein synthesis, abolished the attenu- ation of the HSR, indicating that protein synthesis is necessary to restore proteostasis. HSP90 inhibitors induced a stress response when added at ambient conditions and retarded attenuation of the HSR at elevated temperatures. In addition, we detected a direct physical interaction between cytosolic HSP90A/HSP70A and heat shock factor 1, but surprisingly this interaction persisted after the onset of stress. Finally, the expression of antisense constructs targeting chloroplast HSP70B resulted in a delay of the cell＇s entire HSR, thus suggesting the existence of a retrograde stress signaling cascade that is desensitized in HSP7OB-antisense strains.

关键词： heat shock factor unfolded protein response heat stress response inhibitor feeding Chlamydomonas rein-hardtii HSP90 HSP70 protein folding calcium geldanamycin canavanine.

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