Department of Plant Molecular Biology Younousse Saidi and Pierre Goloubinoff The plant heat-shock response is controlled by specific calcium channels in the plasma membrane

The heat-shock response HSPs families: HSP100, HSP90, HSP70, HSP60, and small HSPs Under normal conditions: After temperature elevation: The heat-shock response is a conserved reaction to elevated temperatures, featured by the synthesis of heat shock proteins (HSPs). Contribute to the correct synthesis, subcellular targeting, or degradation of cellular proteins. 1- protect cells from severe damage ( protein denaturation, membrane hyperfluidity… ), 2- allow resumption of normal cellular and physiological activities, 3- lead to a higher level of thermotolerance.

What is the principle sensing mechanism leading to the activation of heat-shock genes? 1- Cellular protein denaturation ( Richard I. Morimoto, Genes & Dev. 1998) 2- The fluidity of the membrane 3- Evidences for the involvement of calcium ions and calmodulin in thermotolerance. (Gong et al. Plant Physiology 1998; Liu et al. Plant Cell Enviro 2006) (Torok et al. PNAS. 2003) Normal conditions Heat shock

Investigation of the heat sensing mechanism using the moss Physcomitrella patens  Size: µm - cm  Growth on liquid or solid medium  No multi-stratification, no vascular tissues and no cuticle  Growth as single cell-wide filaments (protonema) Physcomitrella patens is optimal for stress studies because:  Highly amenable for genetic manipulations We generated 3 different transgenic lines: Ubi-1Aequorinhsp17.3B GUS 35S Luciferase Ca 2+ Ca 2+  Genome sequence available  Represents a important step in the evolution of land plants

Anti-GUS (Saidi et al. 2005) The stress-inducible promoter hsp17.3B is highly sensitive to small variation of temperature GUS expression after 1h heat shock Immediatly after the HS 16h Recovery after the HS Fv/Fm ratio: the maximal photochemical efficiency of PSII hsp17.3B GUS

Environment Bio-monitoring: Screening for aromatic compounds that activate a heat- shock stress response (Saidi et al. 2007) hsp17.3B GUS

5X 0X Mild heat-treatment amplify chlorophenol effect and enhances bioassay sensitivity. 80X 4.5X 1 The synergistic effect between mild heat shock and TCP allows to detect lower concentrations of potential toxic compounds One hour exposure to different concentrations of trichlorophenol at 30 and 32°C (Saidi et al. 2007)

Mild heat-treatment amplify chlorophenol effect and enhances bioassay sensitivity to lower TCP concentrations. Mild heat treatment enhances bioassay sensitivity. One hour exposure to 100 µM of different compounds at 30°C The screen at 30°C allow to identify sulfonated anthraquinon as a co-activator of the heat shock response 1 One hour exposure to different concentrations of trichlorophenol at 25 or at 30°C (Saidi et al. 2007) hsp17.3B GUS

The heat-shock promoter is specifically activated by trichlorophenol (TCP) in a dose-dependant manner TCP DCP MCP DMSO time (h) (Saidi et al. 2007)

Induction kinetics at 38°C The nature of the heat shock signal is transient hsp17.3B GUS The re-setting of the HSR is chaperone-independent The full re-setting of the HSR requires about 5-7h at non-inducing temperature

Extracellular calcium ions are central to the heat- shock response Chelating the extracellular calcium by EGTA inhibits the heat-shock response hsp17.3B GUS

EGTA pre-treatment inhibits the heat-induced Ca 2+ influx Heat shock induces a transient elevation of cytosolic Ca 2+ concentrations Ubi-1Aequorin (°C) GUS activity The amplitude of temperature induced Ca 2+ influx correlates with subsequent levels of GUS

(BA) Benzyl alcohol (PCP) Pentachlorophenol (Cel) Celastrol Chemicals interacting with membranes induce Ca 2+ influx that precede the activation of heat-shock genes hsp17.3B GUS (Saidi et al. 2005; Saidi et al. 2007)

Pierre Goloubinoff Many thanks to Maude Murise and Peter Coenig Funding was from: