Department of Plant Molecular Biology

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

2. The heat-shock response The heat-shock response is a conserved reaction to elevated temperatures, featured by the synthesis of heat shock proteins (HSPs). HSPs families: HSP100, HSP90, HSP70, HSP60, and small HSPs Under normal conditions: Contribute to the correct synthesis, subcellular targeting, or degradation of cellular proteins. After temperature elevation: 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.

3. Heat shock 2- The fluidity of the membrane (Torok et al. PNAS. 2003) 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) What is the principle sensing mechanism leading to the activation of heat-shock genes? 1- Cellular protein denaturation (Richard I. Morimoto, Genes & Dev. 1998) Normal conditions

4. 2+ Ca We generated 3 different transgenic lines: hsp17.3B GUS 2+ Ca Ubi-1 Aequorin Luciferase 35S Investigation of the heat sensing mechanism using the moss Physcomitrella patens Physcomitrella patens is optimal for stress studies because: • Size: µm - cm • Growth as single cell-wide filaments (protonema) • Growth on liquid or solid medium • No multi-stratification, no vascular tissues and no cuticle • Highly amenable for genetic manipulations • Genome sequence available • Represents a important step in the evolution of land plants

5. 16h Recovery after the HS Immediatly after the HS Anti-GUS hsp17.3B GUS The stress-inducible promoter hsp17.3B is highly sensitive to small variation of temperature Fv/Fm ratio: the maximal photochemical efficiency of PSII GUS expression after 1h heat shock (Saidi et al. 2005)

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

7. 80X 5X 4.5X 0X 1 Mild heat-treatment amplify chlorophenol effect and enhances bioassay sensitivity. 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)

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

9. 0 1 2 4 20 20 20 20 time (h) TCP DCP MCP DMSO The heat-shock promoter is specifically activated by trichlorophenol (TCP) in a dose-dependant manner. (Saidi et al. 2007)

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

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

12. GUS activity 22 30 32 34 36 38 (°C) The amplitude of temperature induced Ca2+ influx correlates with subsequent levels of GUS Ubi-1 Aequorin Heat shock induces a transient elevation of cytosolic Ca2+ concentrations EGTA pre-treatment inhibits the heat-induced Ca2+ influx

13. hsp17.3B GUS Chemicals interacting with membranes induce Ca2+ influx that precede the activation ofheat-shock genes (PCP) Pentachlorophenol (BA) Benzyl alcohol (Cel) Celastrol (Saidi et al. 2005; Saidi et al. 2007)

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