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XVII International
Botanical Congress
Vienna,
Austria Center , 17 - 23
July 2005
Light and temperature induced variable
leaf symplasmic hydraulic conductances
in Juglans regia L.
Are aquaporins involved?
H Cochard1, S Sakr1, A Nardini2
[email protected]
1
UMR PIAF, INRA France
2 Trieste University, Italy
Understanding water pathways in leaves…
Apoplasmic
Xylem conduits in veins
Mesopyll cell wall
Symplasmic
Mesopyll cell symplasm
Gaseous
Evaporation in
stomatal chambers
Understanding water pathways in leaves…
Mesophyll cell walls
Xylem
resistance
Stomatal resistance
~
~?
Mesophyll cell
symplasm
Apoplasmic
Symplasmic
Apoplasmic
Gaseous
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
Leaf conductance
Sack et al (2002) : light decreases leaf hydraulic resistance
Tyree et al (2005)
Objectives of this study :
• 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
Experimental Setup
Experimental Setup
Kleaf = Flow/LA / Pressure
mmol s-1 MPa-1 m-2
Temp
Bath
Air
pressure
F2
air
F1
P
tap
1400 µmol m-2 s-1
Pressure, MPa
A typical experiment
0.21
0.20
Pressure
0.19
Temp, °C
26
25
Temperature
LSC, mmol s-1 m-2 MPa-1
24
10
8
+400%
6
4
Leaf conductance
2
0
0
2
4
Time, hours
6
14
16
18
Can stomatal movement be responsible?
Leaves are
infiltrated with
water
The hydraulic
conductance of
the stomatal
pore is also
measured
Can stomatal movement be responsible? NO!
Light
Dark
106
Effect of light intensity on leaf hydraulic
conductance
3.0
2.5
Dark
Light
+ 126%
p<0.01
2.0
+ 24%
n.s.
-1
-2
Kleaf, e-4 kg s m MPa
-1
+ 129%
p<0.01
1.5
1.0
+ 30%
n.s.
a
+ 35%
n.s.
+ 77%
p<0.01
a
a
a
a
a
0.5
0.0
<6
50
300
600
PAR, mol m-2 s-1
1200
1800
Little effect on dark
conductance
(except at 35°C)
20
35°C
15
25°C
-1
-2
Kleaf at 20°C, mmol s m MPa
-1
Effect of bath temperature on leaf conductance
15°C
10
5
0
Col 2 vs Col 4
Col 8 vs Col 10
Col 14 vs Col 16
Col 20 vs Col 22
0
Strong effect on light
conductances
•Maximum values
•kinetics
5°C
1
Time, hours
2
Effect of bath temperature on leaf conductance
Light response of Kleaf is
strongly temperature
dependant
Dark
Light
Water fluidity
20
-2
LSC, mmol s m MPa
-1
25
-1
15
10
5
Not explained by water
fluidity under light
conditions
0
4
Q
10
3
1
Behavior of a biological
membrane
0
Implication of aquaporins
2
0
5
10
15
20
25
Temperature, °C
30
35
40
D
xylem
Bud
ND
Bark
leaf
flowers
F
M
root
PIP Aquaporins in Walnut
JrPIP2-1
JrPIP2-2
ARNr
Sakr et al Plant Phys 2003
JrPIP2 Aquaporins is a water channel
It increases the water permeability of a Xenopus ovocyte
JrPIP2-1
Pos (mms-1)
Water
RD28
240
220
200
180
160
140
120
100
80
60
40
20
0
Water
JrPIP2-2
RD28
Sakr et al Plant Phys 2003
3
10
8
-2
LSC, mmol s m MPa
-1
Do JrPIP2 transcripts accumulate with light ? YES
-1
6
4
4
2
2
1
0
0
Transcript of:
JrPIP2,1
JrPIP2,2
JrAct
2
1
T
4
Time, hours
2
Dark
3
Light
6
14
4
Dark
16
18
Can the gating of aquaporins alter leaf conductance?
HgCl2 (200 µM) : known to inhibit (some) aquaporins among other proteins!
1.75
b
Dark
Light
1.50
1.25
-1
-2
Kleaf, e-4 kg s m MPa
-1
c
1.00
0.75
a
a
0.50
0.25
0.00
Controls
Hg2+-treated
Can the gating of aquaporins alter leaf conductance?
DMSO dimethyl sulfoxide. Many effects, aquaporin inhibitor?
15
treated
12
-1
-2
K leaf, mmol s m MPa
-1
control
9
+ DMSO
3
- DMSO
+ DMSO
6
0
0
1
2
3
4
5
Time, hours
Strong and rapidly reversed inhibition of leaf conductance (minutes)
Osmotic effect? Effect on membrane polarity?
Does leaf light response is caused by the activation
or by the traduction (synthesis) of proteins ?
CHX (cycloheximide) : inhibits protein traduction in eukaryots
15
12
-1
-2
Kleaf, mmol s m MPa
-1
Control
Experimental
9
+ CHX
6
3
0
0.0
0.5
1.0
1.5
Time, hours
2.0
2.5
3.0
Does leaf light response is caused by the activation
or by the traduction (synthesis) of proteins ?
15
-1
12
-1
-2
LSC, mmol s m MPa
+ CHX
Control
Experimental
9
6
3
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Time, hours
The light response is reversed in less than 1 hour with CHX
The synthesis of at least one protein is required for the light response
This 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…
Mesophyll cell walls
Xylem
resistance
Stomatal resistance
Y xylem
Light
Transpiration
Temperature
Resistance
Y buffered
~
Y leaf
~
Leaf transpiration
Mesophyll cell
symplasm
Does variable symplasmic resistances buffer
leaf water status and favor stomatal function ?
XVII International
Botanical Congress
Vienna,
Austria Center , 17 - 23
July 2005
Light and temperature induced variable
leaf symplasmic hydraulic conductances
in Juglans regia L.
Are aquaporins involved?
H Cochard1, S Sakr1, A Nardini2
[email protected]
1
UMR PIAF, INRA France
2 Trieste University, Italy