1. Healthcare

CAPITULO 2
-Percepción del ABA/ABA-BSA al nivel de
la membrana plasmática (PM), corrientes
de iones y expresión de genes. Caso de los
ROS
– Concepto de receptores ABA al nivel
celular (extracelular y intracelular)
-Receptores externos (PM) : CTC/GCR/…
-Receptores internos : CHLH / PYR / PYL /
RCAR/…
- Establecimiento de vías de señalización
de ABA
-proteínas quinasas y fosfatasas
-SnRKs;
-Ca2 +-dependiente de las proteínas quinasas
-OST1; CPK23
JP Rona, 2014, Université Paris Diderot
JP Rona, Univ. Paris 7
1
Se muestra en electrofisiología
vegetal que hay receptores
ABA al nivel de la
membrana plasmática (RMP)
que son involucrados en la
estimulación
de la actividad de canales
iónicos para controlar el
movimiento de agua y inducir la
expresión de genes
JP Rona, 2014, Université Paris
Diderot
2
1
Plant cells – channels and pumps
acting in concert
(mA)
Ca2+
(pA)
VOC
VAC.
SOC
CYT.
K+in
H+
K+out
(mA)
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Courants K+ sortants
Sauts de potentiels imposés
entre - 40 & +100 mV
+100 mV
K+
Vh=-40 mV
4
cytoplasme
Current (nA)
Whole cell
+100mV
3
+ 80mV
2
+ 60mV
+ 40mV
1
+ 20mV
0
-1
0
1000
2000
Time (ms)
3000
JP Rona, 2014, Université Paris
Diderot
4
2
Corrientes
K+ : percepción extracelular
5
y identificación de ABA-Receptores
ABA suelto
SCOR / GORK family
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5
BSA
BSA
ABA-BSA, que no entra el las células,
tiene la casiJPla
misma actividad que el
Rona, 2014, Université Paris
JP Rona,Diderot
Univ.
Paris 7
ABA
suelto
6
3
Current (nA)
Activation, puis
inactivation des courants Cl-
2
0
-2
-4
-6
-8
Time (s)
4
6
8
10
12
14
0
Sauts de potentiels
entre + 40 to -200mV
JP Rona, 2014, Université Paris
Diderot
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Current (nA)
Effect of Extracellular ABA on the
Plasma Membrane anion currents of
intact A. thaliana Suspension Cells.
0
ECLAMP : – 200 mV
-2
Control
-4
ABA
-6
0
1
2
Time (s)
4
-200 -160 -120 -80
2
-40
0
Control
-2
ABA
-4
Current (nA)
E (mV)
Plasma membrane
currents
resulting
from voltage
JP Rona,
2014, Université
Paris
8
pulses from Diderot
- 200 to + 20 mV
4
Effect of ABA-BSA on the plasma
membrane anion whole cell currents
of intact A. thaliana suspension cells.
+ABA-BSA
+ABA-BSA
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9
An anion channel inhibitor
(9-AC) prevent the effect of
the ABA-BSA
Current (nA)
2
2) 9-AC
3) 9-AC+ABA-BSA
1) Control
0
-2
-4
-6
-8
0
2
4
6
Time (s)
8
ECLAMP : – 200 mV
JP Rona, 2014, Université Paris
Diderot
10
5
ABA contribute to the ROS
increase (H2O2)
ABA
O2 + H2O2
PM
CYT.
DPI
NADPH oxidase
ABA
DPI
ROS
DPI (diphenyleneiodonium), a
specific inhibitor of NADPH
oxidases.
JP Rona, 2014, Université Paris
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ABA produced a rapid and transient release
in reactive oxygen species (ROS) via NADPH
oxidases.
25
A
ABA
ROS release (arbitrary unit)
20
15
Control
10
5
0
-2
0
2
4
6
8
10
Time (min)
JP Rona, 2014, Université Paris
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6
Plasma membrane anion currents Ca2+ sensitive
recorded in A. thaliana cells under oxydative
stress
Current (nA)
2
0
-2
-4
-6
-8
4
6
8
10
12
14
Time (s)
Current (nA)
2
0
-2
-4
+ external H2O2 (10 mM)
-6
-8
4
6
8
10
14
12
Time (s)
Potential (mV)
2
1
-200
-160
-120
-80
-40
1
-1
-2
Current (nA)
0
∆ H2O2
0
-1
-2
-3
4
6
JP Rona, 2014, Université Paris
Diderot
8
10
12
14
Time (s)
13
A - Plasma membrane depolarization
observed in response to 10 µM ABA
-
50mV
Membrane potential (mV)
A
-
-
40mV
control
30mV ABA
1 min
-
+ DPI
35mV
-
25mV
-
15mV
B
ABA
H2O2
1 min
B- The plasma membrane depolarization
induced by ABA is prevented by DPI,
(no ROS synthesis), but not by H2O2
JP Rona, 2014, Université Paris
added after
Diderot
14
7
Conclusion :the anion current
activation promoted by ABA was
dependent on ROS production
A
B
Control
DPI
DPI+ABA
ABA
2 nA
1s
2
2
-120
-40
0
Control
-1
-2
ABA
Current (nA)
1
-200
1
-200
-120
-40
0
DPI
-1
DPI+ABA
-3
-2
-3
Potential (mV)
Corresponding I/V curves
JP Rona, 2014, Université Paris
Diderot
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Is the current activation promoted
by ABA necessary for RAB 18
expression?
+ 9-AC
FEBS Letters, 2000, 474, 43-47
1-Adding the anion channel
blockers 9-AC with ABAinhibited the
ABA-induced RAB18 expression
Northern blot analysis of total RNA from
cells. Similar RNA loading was checked by
16
hybridizationJP Rona,
with
an Arabidopsis 18S
2014, Université Paris
Diderot probe.
16
ribosomal
8
2-Adding the anion channel
blockers niflumic acid with ABA
inhibited the ABA-induced RAB18
expression
RAB 18
JP Rona, 2014, Université Paris
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17
3-Induction of RAB18 gene
expression triggered by an
extracellular ABA perception is
independent of K+ channel activities
in A. thaliana suspension cells.
JP Rona, 2014, Université Paris
DELLIS Olivier, FEBS, 2000
Diderot
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9
4-The Depolarizing Effect of
Extracellular ABA-BSA on the Plasma
Membrane Potential of Cells After
the H+ Pump was Inhibited.
Membrane potential (mV)
-20 mV A
-30 mV
+ABA-BSA
-40 mV
+VAN
-20 mV B
-30 mV
-40 mV
+ABA -BSA
+DES
1 min
Vanadate 200µM (VAN)
Diethyl stilbestrol 100µM (DES)
Medium alkalinization (∆
(∆pH)
JP Rona, 2014, Université Paris
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0.08
19
C
ABA-BSA
0.04
0.00
VAN+ABA-BSA
0
20
40
60
Time (min)
5-El ABA libre (o ABA-BSA) puede inducir
una alcalinización solamente cuando la bomba
H+ no esta ya inhibida
ABA-BSA
VAC.
CYT.
(-)
ABA
H+
JP Rona, 2014, Université Paris
Diderot
Brault et al. 2004 Plant Physiol, 135, 231-243
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10
Conclusión : la percepción del ABA al
nivel exterior de la membrana
plasmática muestra que hay un
receptor-ABA que juega un papel en
la regulación de flujos de iones
necesaria para inducir la expresión de
RAB 18
ABA (ABA-BSA)
Rc
CYT.
N
?
RAB 18
+
-
H
A
activación
JP Rona, 2014, Université Paris
Diderot
Ghelis et al. 2000, FEBS Letters, 474,
43-47
inhibición
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2 – Concepto de receptor al nivel
de las membranas : identificación de
ABA-R(s), los SAR y MAR
3 candidatos intracelulares de receptores de
ABA son bien descritos, pero parece que
hay muchos más.
También hay receptores adicionales que
fueron encontrados en la membrana
plasmática para explicar la percepción
extracelular de ABA.
Soluble ABA Receptors
Membrane ABA Receptor
JP Rona, 2014, Université Paris
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11
-Las proteínas-receptores de ABA
identificadas hasta el momento
sugieren que el ABA intracelular
(núcleo, citoplasma, cloroplasto )
y su percepción extracelular
pueden controlar muchos
fenotipos iguales o diferentes.
Estos sitios (receptores) son muy
importantes para manejar,
al mismo tiempo, las diferentes
respuestas fisiológicas controladas
por el ABA interno y externo
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Concepto de ABA-receptores
Reported ABA binding proteins
FCA
RCAR -Regulatory Components of ABA Receptor
PYR -Pyrabactin Resistance Protein
PYL -PYR-Like proteins
GTG -plasma membrane–localized GPCR type G-proteins
GCR : G-prptein coupled receptor
CHLH -(Mg-chélatase H); ABAR : ABA-binding protein
-(Mg-chélatase H);
FCA : Flowering-time
Control protein ABA
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Univ. Paris 7
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-(Mg-chélatase H); JP Rona,Diderot
12
GCR2, a G protein-coupled
receptor-like receptor, is localized
at the plasma membrane
Also, plants use ABAR in the
chloroplast ; FCA as ABA receptors to
integrate the ABA signal with other
stress signals that affect chloroplast
function and flowering-time control to
adapt to various environmental
conditions.
FCA is localized in the nucleus and is
involved in the regulation of mRNA
stability, which is required for
flower induction.
Chloroplast function and floweringtime control are crucial for
plant
JP
Rona, 2014, survival.
Université Paris
Diderot
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The ABA signal is received by (at least) three
ABA receptors: GCR, GTG ABAR and FCA.
GTG/GCR2
Ca2+
Shinozaki, K, 2007.
In the ABA signaling pathway, Ca2+ , second
messengers (ROS), protein kinases, protein
phosphatase 2Cs JPand
transcription
factors form a
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web-like
network.
13
The core complexes of the better
established ABA signalling path-ways.
ABA Controls Rapid Drought Adaptive Responses by
Modification of Selective Transport Across
the Plasma Membrane and in the cytoplasm
ABA EXT.
Receptor ABA
GCR
GTG
H2O2
SnRK2
ABA INT.
Soluble ABA Receptors
D’après Leung, 2011, Gif-sur-Yvette
SnRK2: SNF1- related protein kinase : The sucrose non-fermenting 1related protein kinases (SnRKs) are protein kinases containing catalytic domains
with sequences similar to SNF1 (sucrose non-fermenting1).
RCAR -Regulatory Components of ABA Receptor
PYR -Pyrabactin Resistance Protein
PYL -PYR-Like proteins
GTG -plasma membrane–localized GPCR type G-proteins
GCR- G protein-coupled receptor-like receptor
PP2Cs: protein phosphatases
type Paris
2C
JP Rona, 2014, Université
Diderot
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Relation tree of the SnRK2 family from
Arabidopsis. and rice.
Arabidopsis SnRK2 proteins activated by hyperosmotic
conditions can be divided into three groups, depending on
additional activation by ABA.
G2
G1
G3
Boudsocq M .Plant Physiol. 2005;
Group 1 (G1) corresponds to kinases
strongly activated by ABA,
Group 2, SnRK2s are not responsive to
ABA.
Group 3 includes kinases poorly or not
JP Rona, 2014, Université Paris
activated by ABA.
Diderot
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14
a-Chloroplast : ABA-CHLH
CHLH est un régulateur
positif dans la signalisation de l’ABA.
Le gène CHLH (chélatase H) code pour une
protéine chloroplastique, la sous-unité H de la
Mg-chélatase, déjà connue pour être impliquée
dans la biosynthèse de la chlorophylle et dans
la signalisation entre le plaste et le noyau.
JP Rona, 2014, Université Paris
JP Rona,Diderot
Univ. Paris 7
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b-proteína G-receptores
acoplados (PM)
GPA1 modulates the GTPase activity
of the GTGs (PLC
IP3?)
PLC/IP3
Ca2+
GPA1 : G Protein α subunit Arabidopsis 1 proteins
GPCR :G-protein coupled receptor
GTG proteins = GPCR-type G
GTG -plasma membrane–localized
type G-proteins
JP Rona, 2014, Université GPCR
Paris
JP Rona,Diderot
Univ. Paris 7
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15
b.1-proteína-receptor nuclear
FCA is localized in the nucleus and is
involved in the regulation of mRNA
stability and Ca2+ mobilization, which is
required for flower induction.
ABA int.
PYR
FCA : Flowering-time JPControl
protein ABA-(Mg-chélatase H);
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PYR -Pyrabactin Resistance Protein
31
FCA is an ABA receptor
It binds target RNA via an interaction with its 3' endprocessing factor FY that leads to promoting flowering
with in a positive regulation of FLC (floral repressor).
ABA binding prevents this interaction, required to reduce
production of FLC and full-length FCA, maintaining a
flowering repression.
flowering repression
FCA : Flowering-time Control protein ABA-(Mg-chélatase H)
JP Rona, 2014, Université Paris
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c-Solubles PYR / PYL / RCAR
A new model for ABA action has been
proposed and validated, in which the soluble
PYR/PYL/RCAR receptors function at the
apex of a negative regulatory pathway
to directly regulate PP2C phosphatases,
which in turn directly regulate
ion channels-SnRK2 kinases.
R
R is free : SnRK2 is
INACTIVATED
by PP2C
R is bound to
ABA and PP2C :
SnRK2 is free
SnRK2 can
ACTIVATES ABF
ABF- transcription factor
PP2Cs: protein phosphatases type 2C
RCAR -Regulatory Components of ABA Receptor
SnRK family: SNF1- related protein kinase
JP Rona, 2014, Université Paris
PYR-Like proteins
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Structural mechanism of ABA action
R is free :
SnRK2 is
inactivated
by PP2C
R is first bound
to ABA
P
SnRK2
R -ABA bound PP2C
: SnRK2 is free
W
D’après Sheard et Zheng, 2009
ABA initiates an allosteric open-to-close
transition of the gating loop, allowing it to
approach the second entrance loop and
sequester ABA in the pocket.
The phosphatase PP2C binds to the
hydrophobic site on the gating loop, inserting
a conserved tryptophan (W) next to the
JP Rona, 2014, Université Paris
gating loop
and Diderot
locking it closed.
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17
In conclusion :
ABA receptors PYR1, PYL1 and PYL2 are
allosteric switches controlled by ABA binding.
PYR/PYL receptors inhibit PP2C phosphatases,
which leads to activation of SnRK2 kinases.
+ ABA
SnRK2s phosphorylate downstream target
proteins to regulate
ABA
responses.
JP Rona, 2014, Université Paris
JP Rona,Diderot
Univ. Paris 7
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Summary of abscisic acid (ABA
signaling factors.
)
D’après Cutler et al., 2010
RCAR -Regulatory Components of ABA Receptor
PYR -Pyrabactin Resistance Protein
PYL -PYR-Like proteins
GTG -plasma membrane–localized GPCR type G-proteins
SnRK2: SNF1- related protein kinase
PP2Cs: protein phosphatases
type Paris
2C
JP Rona, 2014, Université
JP Rona,Diderot
Univ. Paris 7
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18
2.2 - Establecimiento de vías de
señalización de ABA en los estomas
a-proteínas quinasas y fosfatasas
SnRKs;
Ca2 +-dependiente de las proteínas
quinasas
OST1; CPK23
OPEN
CLOSED
ABA effects :increase of outward A- /
K+ /H202 fluxes, then stomatal guard
cells are closed
JP Rona, 2014, Université Paris
JP Rona,Diderot
Univ. Paris 7
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Receptores ABA específicos
de los estomas
En las células de guardia, el receptor
ABA está formado por el complejo
heteromérico PP2C como ABI1 y
RCAR que se puede unir al ABA
ABA
Negative action without ABA
OST
Signalling pathways
OST : Open Stomata gene : serine–threonine protein kinase
RCAR -Regulatory Components of ABA Receptor
ABI1 gene encodes a member of the 2C class of protein
serine/threonine phosphatases (PP2C)
JP Rona, 2014, Université Paris
JP Rona,Diderot
Univ. Paris 7
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19
Action of the protein kinases OST1
Mutations in the OST1 gene
OST1
OST1
OST1
Mutations in the OST1 gene (Open Stomata 1),
encoding a serine–threonine protein kinase,
render Arabidopsis thaliana guard cells
insensitive to abscisic acid, such that stomata
remain open
in the presence of this
JP Rona, 2014, Université Paris
Diderot
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phytohormone
Sin ABA, la actividad de la fosfatasa
PP2C inhibe la acción de las proteínas
quinasas (presentadas en verde) OST1
(por ej., actividad de la H +-ATPasa) y
de las SnRKs - asociadas (canales
anicónicos lentos) y, posiblemente, de
las Ca2+ - CPKs (como la CPK23
40 que es
dependiente del calcio).
PP2C
ION JPCHANNELS/PUMPS
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JP Rona,Diderot
Univ. Paris 7
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20
En las células de guarda, las llaves
principales son las actividades de los
canales iónicos SLAC1 (activación) y
KAT1 (inhibición) : se activan y se
inhiben respectivamente por la acción
de OST1.
E. Grill, Cell, 2010.
In the presence of ABA, the phosphatase
activity of the receptor is blocked and
OST1 is activated increasing the inhibition
of IK+in and JPthe
stimulation of IA-out
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JP
Rona,
Diderot
Univ. Paris 7
41
(I: current)
Effect of an anion channel
inhibitor on inward currents
elicited by 10 µM ABA.
100
ABA+9-AC
0
ABA
50
Control
Inward currents (%)
150
200 µM anthracene-9-carboxylic acid
(9-AC)
JP Rona, 2014, Université Paris
Diderot
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21
Mecanismo de acción y cadena de pasos del ABA en las
células oclusivas de estomas
(Membranas plasmática y vacuolar)
JP Rona, 2014, Université Paris
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JP Rona, 2014, Université Paris
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