Post on 10-Jul-2015
description
The dark side of plant growth: e da s de o p a g oa role for phytochrome-interacting
transcription factorsp
PABLO LEIVARPABLO LEIVAR
XARXA DE REFERÈNCIA EN
BIOTECNOLOGIA
Barcelona, Juliol 2012
C t d RCentre de Recerca en Agrigenòmica (CRAG)
How do plants grow and develop in accordance to their ambient light/DARK conditions? g
- Introduction: phytochrome (phy) photoreceptors and PIF transcription factors- Research lines: Seedling deetiolation / Diurnal growth- Biotechnology potential
Lightgy p
Dark LightDark
*seedlings =plántulas
PHOTOMORPHOGENESIS=plántulas
photosynthesis environmentalphotosynthesisinformation
Light as Energy for photosynthesis and Light as Energy for photosynthesis and as a source of Information
Light and photomorphogenesis
DAY
Direction(phototropism)
Intensity(photosynthetic
adaptation)(phototropism) )
Color
(neighbor(deetiolation)(germination) ( gperception)
(shade avoidance) LIGHT(deetiolation)(germination)
DARK seed seedling juvenile adult
Darkness duration or photoperiod
(flowering)
NIGHT
Plant Informational PhotoreceptorsPlant Informational Photoreceptors
cryptochromes phototropins zeitlupes Phytochromes
RED / FAR-REDUVA / BLUE
The Red (R)/Far Red (FR) sensing phytochrome (phy) photoreceptorsphytochrome (phy) photoreceptors
VISIBLE SPECTRUMWavelength (nm)
Pr Pfr
phy act as molecular switches in response to R and FR
Red(660 nm)
PhysiologicalPhysiological responses
phy Pr phy Pfr
Synthesis
Far Red(730 nm)
phy Pr(Inactive)
phy Pfr(Active)
( )Slow Dark Reversion
How does the phy regulate plant morphogenesis in response to changes in ambient light/dark conditions?
Red(660 nm)
- SEEDLING DEETIOLATIONDARK/LIGHT
- DIURNAL GROWTHphy Pr phy Pfr
DARK/LIGHTTRANSITIONS
Far Red(730 nm)
phy Pr(Inactive)
phy Pfr(Active)
( )Slow Dark Reversion
Cotyledons
Seedling Deetiolation Diurnal Growth(day/night cycles)
Cotyledons (embryonic leaves)
Hypocotyl (young stem)
( y g y )
(young stem)
10 m
m
4 days seedlings after germination at 22º4 days seedlings after germination at 22º
Arabidopsis
0.5 mm
thaliana
Seedling Deetiolation Diurnal Growth(day/night cycles)
Etiolated growth
( y g y )10
mm
Deetiolation
DARK RED
phyB
(D) (R)
R
Pr Pfr
Eti l t dEtiolated growth
Seedling Deetiolation Diurnal Growth(day/night cycles)
Etiolated growth
SD-induced
( y g y )10
mm
SD-induced growth
Deetiolation
DARK RED WShort-Days (D) (R)
R
(SD: 16hD-8hW)
(day)RHow does the phy
Pr Pfr Pr PfrD
(night)
H t lEti l t d
transmit the primary light signal
information? Hypocotyl growth
Etiolated growth
information?
Seedling Deetiolation
Etiolated growth Seedling deetiolation as
d l t
10 m
m
Deetiolation
a model system to study responses to
Light/Dark
DARK RED(D) (R)
R How does the phy Pr Pfr
Eti l t d
transmit the primary light signal
information?Etiolated growth
information?
PIF3 and related PIFs are phy-interacting bHLH transcription factors that selectively bind the Pfr form of the phyfactors that selectively bind the Pfr form of the phy
Nuclear Localization Signal
APA/B
NLSg
bHLH
PIF3PIFs 1,4,5
phy Pfr Binding
APA/B bHLHBasic
Helix-loop-helix
, ,
GAD:PIF3Red
(660 nm)
phyB
Preferential binding to the DNA motif G-BOX:CACGTG
PIF3
GAD:PIF3
(Martínez-García et al., 2000, Science)
phy Pr(Inactive)
phy Pfr(Active)
+R +FR
(Ni et al., 1999, Nature)
Far Red(730 nm)
+ +
pifq mutants lacking PIFs 1, 3, 4 and 5 show a constitutively
Photoactivated phy triggers rapid proteolytic degradation of PIFs 1, y
photomorphogenic phenotypeproteolytic degradation of PIFs 1,
3, 4 and 5 in response to light
PIFs
D + R (min)0 10 20 30 40 50 60
R light
Etiolated
PIF3
0 10 20 30 40 50 60
(Al-Sady et al., 2006, Molecular Cell)
phyGrowth
PIFs
WT pifq (pif1pif3pif4pif5)
PIF3:CFP PHYB:YFP Overlay(Bauer et al, 2004, The Plant Cell)
(Leivar et al., 2008, Current Biology)
PIF1, 3, 4 and 5 accumulate in the dark and sustain etiolated growthand sustain etiolated growth
CYTOPLASM
PIF1
Dark
Pr PIF3PIF4PIF5
PIF regulated
NUCLEUS
G-boxPIF-regulated
genes
Transcriptionalnetwork
Etiolated Growth
PIF-regulatedtranscriptome1000~1000 genes
Initiation of seedling deetiolation through phy-induced degradation of PIFs 1 3 4 5 phy-induced degradation of PIFs 1,3,4,5
CYTOPLASM
? SE?
R
Red
RDark Light
PIF1PIF3
PIF1PPK
INA
SE?
PIF3
PIF1UbUbE3
LIG
AS 26S proteasome
induced degradation
PIF5PIF regulated
PfrPr PIF3PIF4PIF5
PIF3PIF4PIF5
PPP
PIF3PIF4PIF5
UbUbUb
G-boxPIF-regulated
genes
NUCLEUS
SignalPerception
& (Photomorphogenesis)
Seedling Deetiolation
Etiolated Growth
Transcriptionalnetwork
Transcriptional reprogramming
&Nuclear
translocation
(Photomorphogenesis)
The Dark Side of Plant
PIF
Side of Plant growth
R
Seedling Deetiolation
PIFs
Transcriptional
Pr Pfr
Transcriptional network
Etiolated Growth
How do plants grow in accordance to their ambient light/DARK conditions?
PIFR
Seedling Deetiolation
PIFs
Transcriptional
Pr Pfr
Transcriptional network
Etiolated Growth
RESEARCH LINE 1: Seedling growth in the Dark after germination: Identification of new regulators of deetiolation actingIdentification of new regulators of deetiolation acting downstream of the PIFs
1a- Functional profiling of PIF3-regulated genes
1b- Suppressor screen of pifq mutants
1a- Functional Profiling of PIF3-regulated genes
-1.35000 0 1.35000Z-score
I. Identification of PIF3-regulated MIDA(Misexpressed In DArk) genes
II. Selection candidate regulatory genes
13 MIDA genes selected
D0h_Rep1
D0h_Rep2D0h_Rep3
D1h_Rep1D1h_Rep2D1h_Rep3
D0h_Rep1D0h_Rep3D1h_Rep1D1h Rep3
WT
pif3
- 13 MIDA genes selected (MIDA1-MIDA13)
- Identification of insertional D0h_Rep2D1h_Rep2
D1h_Rep3
42 Induced Genes 40 Repressed Genes
e FC
pif3
e FC
1.21
0.8
2.52
1.5
mutants in public collections (mida1-mida13)
WT pif3
Ave
rag
Ave
rag
WT pif3
0.60.40.2
0
1.5
10.5
0
III. Phenotypic analysis of mida mutants.
(Sentandreu et al., 2011, Plant Cell)
1a- Functional Profiling of PIF3-regulated genes
PIF3-regulated transcriptional network implement organ-specific responses
during seedling deetiolationg g
Maria Sentandreu
Next:Characterization of the molecular function of the MIDAs during seedling deetiolation.g
(Sentandreu et al., 2011, Plant Cell)(Sentandreu et al., 2011, Plant Signaling and Behavior)
1b- Suppressor screen of pifq mutants
Guiomar Martín
WT pifq(pif1pif3pif4pif5)
1b- Suppressor screen of pifq mutants
Transformation with a Full-Length overexpression cDNA (FOX) library
(Ichikawa et al., 2006, Plant Journal 45: 974)
Screen in dark in the presence of hygromycin
WT pifq(pif1pif3pif4pif5)
pifqHygR
pifq HygR
Next:Putative sops
(suppressors-of-pifq)Validation of sop mutants.Identification of responsible genes.
How do plants grow in accordance to their ambient light/DARK conditions?
PIFR
Seedling Deetiolation Diurnal Growth(day)
RPIFs
Transcriptional
Pr PfrD
(night)
RPfr Pr
Transcriptional network
Etiolated Growth
Hypocotyl Growth
RESEARCH LINE 2: Seedling growth in the Dark in diurnal conditions (8h Light:16h Dark)
RESEARCH LINE 1: Seedling growth in the Dark after germination: Identification of new regulators of deetiolation acting downstream of the PIFs
2a- Characterization of PIF3 function
2b- Definition of PIF-regulated transcriptional
downstream of the PIFs
1a- Functional profiling of PIF3-regulated genesg p
networks implementing growth responses to light1b- Suppressor screen of pifq mutants
2a- Characterization of PIF3 function in diurnal growth
0h 8h 24h
Day Night0h 8h 24h
(Nozue et al 2007 Nature)
Hypocotyl growth rate peaks at the end of the night
(Nozue et al., 2007, Nature)
ate 0.2‐
0.25‐
WT
Gro
wth
Ra
(mm
h-1)
0.05‐
0.1‐
0.15‐
pif3
Judit Soy
Time (h)
0‐3 6 9 12 15 18 21 24 27
pif3
(Soy et al., 2012, Plant Journal)
2a- Characterization of PIF3 function in short-days
0h 8h 24h
I. PIF3 protein accumulates at 8
10-
vel
PIL1
II. PIF3 induces the expression of growth-related at the end-of the night
3 6 8 10 12 1614 2518 24232220 23
WT pif3
PIF3h:
pthe end of the night in short-days
2-
4-
6-
8-
Rel
ativ
e pr
essi
on L
ev PIL1WT
pif3PIF3n.sn.s
ponceauTime (h)
0-3 6 9 12 15 18 21 24
Ex
67
R ent
PIL1p
1
2
3
4
5
ChI
P qR
T-PC
old-
Enric
hme
PIF3PIF4/5
0C Fo
WT
NoAb Abpromoter
NoAb Abcontrolregion
NoAb Ab NoAb Ab
YFP-PIF3promoter
controlregion
phyB phyBphyA,phyB
PIF4/5PIL1
HFR1XTR7
phyB phyBphyB
Hypocotyl
phytochrome-Imposed oscillations of PIF3 abundance regulate hypocotyl growth
(Soy et al., 2012, Plant Journal)
yp yelongation
Day Night
under diurnal conditions in conjunction with PIF4/5 and the circadian clock
2b- Definition of PIF-regulated transcriptional networks implementing growth responses to light
(L i t l 2009 Pl t C ll)
Deetiolation(WT vs pifq)
(Leivar et al., 2009, Plant Cell)
Comparative transcriptomic
analysis (WT vs pifq)(839 genes)
729
analysis
Simulated shade
(WT vs pifq)
Diurnal(WT vs
2311 76
729
(WT vs pifq)(265 genes)
pif4pif5)(118 genes) 1866 148
(Nozue et al., 2011, Plant Physiology)(Leivar et al., 2012, Plant Cell)
Seedling deetiolation Diurnal conditions
D R SD(8hW-16hD)
Pr Pfr Pr PfrDayR
W
NightPr Pfr Pr Pfr
Pr
PIFs
Pr
PIFsG-box
cellular machinery that implementcellular machinery that implement different facets of growth
Seedling deetiolation Diurnal conditions
D R SD(8hW-16hD)
Pr Pfr Pr PfrDayR
W
Pf
NightPr Pfr Pr Pfr
The balance between phy and PIFs determines plant
PIFsPfr and PIFs determines plant
growth during light/dark transitions
G-box
cellular machinery that implementcellular machinery that implement different facets of growth
Aplicaciones y potencial biotecnológico del sistema del fitocromo
1. Obtención de variedades que muestran alteraciones en las respuestas a la luz para mejorar procesos agrícolas
- Revolución verde: Selección de variedades de cereales semi-enanas, portadoras de alelos que modulan la señalización por luz, y que muestran un crecimiento reducido a cambio de una mayor producción de semillas (p.ej. alelo Reduced height1 (Rht1) en arroz).
2- Ingeniería molecular basada en el fitocromo OFF ON2- Ingeniería molecular basada en el fitocromo2.1- Generación de interruptores moleculares, reversibles, y
modulables por luz:- Expresión génica
PIFX
PrY
+OFF
+R
+FR
PIFX
PfrY
ON
g- Splicing de proteínas - Señalización en dominios subcelulares discretos
2.2- Generación de nuevos marcadores fluorescentes
+FR
proceso celular
(Shimizu et al., 2002, Nat. Biotech.)(Tyszkiewicz et al., 2008, Nat. Methods) (Levskaya et al., 2009, Nature)
basados en el fitocromo que emiten en infrarrojo (Shu et al., 2009, Science)
3- Regulación por luz, fitocromo y PIFs del metabolismo lipídico g p , y pen semillas y plántulasPotencial aplicado: - Producción de ácidos grasos poli-insaturados con valor nutracéutico
Céline Diaz- Producción de aceites que actúen como biodiesel
Guiomar MartínCéline Diaz
Roger Miralles
El M tEl M t Céline DiazElena MonteElena Monte
Judit SoyMaria SentandreuSentandreu
At UC-BerkeleyPeter H. Quail
Old membersNahuel González
Peter H. Quail
At CNB-CSICSalomé Prat
FundingBeatriu de Pinós Fellowship (Generalitat de Catalunya)Marie Curie Actions IRG PIRG06-GA-2009–256420Mi i t i d Ci i I ió (MICINN)Ministerio de Ciencia e Innovación (MICINN)AGAUR 2009 SGR 206