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