Understanding water pathways in leaves…
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Transcript of Understanding water pathways in leaves…
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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
[email protected]@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
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Understanding water pathways in leaves…
Apoplasmic
Symplasmic
Gaseous
Mesopyll cell wall
Mesopyll cell symplasm
Evaporation in stomatal chambers
Xylem conduits in veins
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Understanding water pathways in leaves…
ApoplasmicSymplasmic
Gaseous
Xylem resistance
Mesophyll cell walls
Mesophyll cell symplasm
Stomatal resistance
Apoplasmic
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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
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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
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• 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 :
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Experimental SetupExperimental Setup
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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
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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%
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Can stomatal movement be responsible?
Leaves are infiltrated with water
The hydraulic conductance of the stomatal pore is also measured
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Light
Dark
Can stomatal movement be responsible? NO!
106
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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
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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
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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
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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
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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
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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
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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
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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?
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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 ?
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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
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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 ?
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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
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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
[email protected]@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