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-1 Aequorinhsp17.3B GUS 35S Luciferase

Ca2+

Ca2+

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.

0 1 2 4 20 2020 20TCP

DCPMCP

DMSO

time (h)

(Saidi et al. 2007)

Induction kinetics at 38°C

The nature of the heat shock signal is transienthsp17.3B 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 Ca2+ influx

Heat shock induces a transient elevation of cytosolic Ca2+ concentrations

Ubi-1 Aequorin

22 30 32 34 36 38 (°C)

GU

S a

cti

vity

The amplitude of temperature induced Ca2+ influx correlates with subsequent levels of GUS

(BA) Benzyl alcohol

(PCP) Pentachlorophenol

(Cel) Celastrol

Chemicals interacting with membranes induce Ca2+ influx that precede the activation of heat-shock

geneshsp17.3B GUS

(Saidi et al. 2005; Saidi et al. 2007)

Pierre Goloubinoff

Many thanks to

Maude Murise and Peter Coenig

Funding was from: