Light and temperature induced Light and temperature induced variable leaf symplasmic variable leaf symplasmic

hydraulic conductances in hydraulic conductances in Juglans regiaJuglans regia L. L.

Are aquaporins involved?Are aquaporins involved?

H H CochardCochard11, S Sakr, S Sakr11, A Nardini, A Nardini22

cochard@clermont.inra.frcochard@clermont.inra.fr

11 UMR PIAF, INRA France UMR PIAF, INRA France 22 Trieste University, Italy Trieste University, Italy

                

       

XVII InternationalBotanical Congress

Vienna, Austria Center , 17 - 23 July 2005

Understanding water pathways in leaves…

Apoplasmic

Symplasmic

Gaseous

Mesopyll cell wall

Mesopyll cell symplasm

Evaporation in stomatal chambers

Xylem conduits in veins

Understanding water pathways in leaves…

ApoplasmicSymplasmic

Gaseous

Xylem resistance

Mesophyll cell walls

Mesophyll cell symplasm

Stomatal resistance

Apoplasmic

Why should symplasmic resistances be variable?

Because water molecules cross cell membranes through a Nobel prize winning molecule :AQUAPORINS

Tajkhorshid, E., Nollert, P., Jensen, M.O., Miercke, L.J., O'Connell, J., Stroud, R.M., and Schulten, K. (2002). Science 296, 525-530

http://nobelprize.org/chemistry/laureates/2003/chemanim1.mpg

Recent evidences for variable leaf hydraulic resistances

Sack et al (2002) : light decreases leaf hydraulic resistance

Tyree et al (2005)L

eaf

con

du

cta

nce

• Confirm the light response Kleaf for walnut

• Confirm that stomatal movement was not involved

• Effect of light intensity

• Effect of temperature

• Molecular evidences for the implication of

aquaporins in this light response

Objectives of this study :

Experimental SetupExperimental Setup

F1

F2

P

TempBath

air

Air pressure

tap

Kleaf = Flow/LA / Pressuremmol s-1 MPa-1 m-2

1400 µmol m-2 s-1

Experimental SetupExperimental Setup

XYL’EM

Time, hours0 2 4 6 14 16 18

LS

C, m

mol

s-1

m-2

MP

a-1

0

2

4

6

8

10

Pre

ssur

e, M

Pa

0.19

0.20

0.21

Tem

p, °

C

24

25

26

A typical experiment

Pressure

Temperature

Leaf conductance

+400%

Can stomatal movement be responsible?

Leaves are infiltrated with water

The hydraulic conductance of the stomatal pore is also measured

Light

Dark

Can stomatal movement be responsible? NO!

106

Effect of light intensity on leaf hydraulic conductance

PAR, mol m-2 s-1

<6 50 300 600 1200 1800

Kle

af,

e-4

kg s

-1 m

-2 M

Pa-1

0.0

0.5

1.0

1.5

2.0

2.5

3.0

DarkLight

+ 30%n.s.

+ 35%n.s.+ 24%

n.s.

+ 77%p<0.01

a

a a

aa

+ 129%p<0.01

a

+ 126%p<0.01

Effect of bath temperature on leaf conductance

Col 2 vs Col 4 Col 8 vs Col 10 Col 14 vs Col 16 Col 20 vs Col 22

Time, hours

0 1 2

Kle

af a

t 20°

C, m

mol

s-1

m-2

MPa

-1

0

5

10

15

20

5°C

15°C

25°C

35°C

Little effect on dark conductance (except at 35°C)

Strong effect on light conductances

•Maximum values•kinetics

LSC

, mm

ol s

-1 m

-2 M

Pa-1

0

5

10

15

20

25

Temperature, °C

0 5 10 15 20 25 30 35 40

Q10

0

1

2

3

4

DarkLightWater fluidity

Light response of Kleaf is strongly temperature dependant

Not explained by water fluidity under light conditions

Effect of bath temperature on leaf conductance

Behavior of a biological membrane

Implication of aquaporins

flowers Bud

Bar

k

xyle

m

leaf

root

JrPIP2-1JrPIP2-2

ND DF M

ARNr

PIP Aquaporins in Walnut

Sakr et al Plant Phys 2003

JrPIP2 Aquaporins is a water channel

It increases the water permeability of a Xenopus ovocyte

020406080

100120140160180200220240

Water JrPIP2-2 RD28

Pos

(m

ms-1

)

JrPIP2-1 RD28Water

Sakr et al Plant Phys 2003

Time, hours0 2 4 6 14 16 18

LSC

, mm

ol s

-1 m

-2 M

Pa-1

0

2

4

6

8

10

1

3

24

1 2 3 4

JrPIP2,1

JrPIP2,2

JrAct

T Dark Light DarkTranscript of:

Do JrPIP2 transcripts accumulate with light ?YES

Can the gating of aquaporins alter leaf conductance?

HgCl2 (200 µM) : known to inhibit (some) aquaporins among other proteins!

Kle

af,

e-4

kg s

-1 m

-2 M

Pa-1

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75Dark Light

Controls Hg2+-treated

a a

b

c

Strong and rapidly reversed inhibition of leaf conductance (minutes)

DMSO dimethyl sulfoxide. Many effects, aquaporin inhibitor?

Can the gating of aquaporins alter leaf conductance?

Time, hours

0 1 2 3 4 5

K le

af, m

mol

s-1

m-2

MP

a-1

0

3

6

9

12

15

+ D

MS

O - DM

SO

+ D

MS

O

control

treated

Osmotic effect? Effect on membrane polarity?

CHX (cycloheximide) : inhibits protein traduction in eukaryots

Time, hours

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Kle

af, m

mol

s-1

m-2

MP

a-1

0

3

6

9

12

15ControlExperimental

+ C

HX

Does leaf light response is caused by the activation or by the traduction (synthesis) of proteins ?

Time, hours

0.0 0.5 1.0 1.5 2.0 2.5 3.0

LS

C, m

mol

s-1

m-2

MP

a-1

3

6

9

12

15

+ C

HX

ControlExperimental

Does leaf light response is caused by the activation or by the traduction (synthesis) of proteins ?

The light response is reversed in less than 1 hour with CHX

The synthesis of at least one protein is required for the light responseThis protein has a relatively high turnover

Conclusions

Leaf hydraulic resistance is strongly light and temperature dependant

Variables leaf resistances are located in the symplasmic pathway

Proteins (aquaporins) are involved in these responses

The synthesis or the activation of these proteins seems finely regulated according to leaf microclimate

WHY ?

Understanding water relations in leaves…

Xylem resistance

Mesophyll cell walls

Mesophyll cell symplasm

Stomatal resistance

Leaf transpiration

xylem leaf

Does variable symplasmic resistances buffer leaf water status and favor stomatal function ?

Light TemperatureTranspiration

Resistance

buffered

Light and temperature induced Light and temperature induced variable leaf symplasmic variable leaf symplasmic

hydraulic conductances in hydraulic conductances in Juglans regiaJuglans regia L. L.

Are aquaporins involved?Are aquaporins involved?

H H CochardCochard11, S Sakr, S Sakr11, A Nardini, A Nardini22

cochard@clermont.inra.frcochard@clermont.inra.fr

11 UMR PIAF, INRA France UMR PIAF, INRA France 22 Trieste University, Italy Trieste University, Italy

                

       

XVII InternationalBotanical Congress

Vienna, Austria Center , 17 - 23 July 2005