THE DARK SIDE OF PLANT GROWTH: A ROLE FOR PHYTOCHROME-INTERACTING TRANSCRIPTION FACTORS - XRB Annual Meeting 2012

1. The da
e dark s de o p a
side of plant g o
growth:
a role for phytochrome-interacting
transcription factors
p
PABLO LEIVAR
XARXA DE REFERÈNCIA EN
BIOTECNOLOGIA
Barcelona, Juliol 2012
Centre de Recerca en
C t d R
Agrigenòmica (CRAG)

2. 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 p
gy potential
Dark Light
*seedlings
=plántulas
PHOTOMORPHOGENESIS

3. photosynthesis environmental
information
Light as Energy for photosynthesis and
as a source of Information

4. Light and photomorphogenesis
DAY
Intensity
Direction (photosynthetic
(phototropism) adaptation)
)
Color
( g
(neighbor
(germination) (deetiolation)
perception)
LIGHT (shade avoidance)
seed seedling juvenile adult
DARK
Darkness duration
or photoperiod
(flowering)
NIGHT

5. Plant Informational Photoreceptors
cryptochromes
phototropins
zeitlupes Phytochromes
UVA / BLUE RED / FAR-RED

6. The Red (R)/Far Red (FR) sensing
phytochrome (phy) photoreceptors
VISIBLE SPECTRUM
Wavelength (nm)
Pr Pfr

7. phy act as molecular switches in response to R and FR
Red
(660 nm)
Physiological
Synthesis
responses
phy Pr phy Pfr
(Inactive) (Active)
Far Red
(
(730 nm))
Slow Dark
Reversion

8. How does the phy regulate plant morphogenesis in response to
changes in ambient light/dark conditions?
Red
(660 nm)
- SEEDLING
DARK/LIGHT DEETIOLATION
TRANSITIONS
- DIURNAL
GROWTH
phy Pr phy Pfr
(Inactive) (Active)
Far Red
(
(730 nm))
Slow Dark
Reversion

9. Seedling Deetiolation Diurnal Growth
(day/night y
( y g cycles) )
Cotyledons
(embryonic leaves)
Hypocotyl
(young stem)
10 mm
4 days seedlings after germination at 22º
Arabidopsis
thaliana
0.5 mm

10. Seedling Deetiolation Diurnal Growth
(day/night y
( y g cycles) )
Etiolated
growth
10 mm
Deetiolation
phyB
DARK RED
(D) (R)
R
Pr Pfr
Etiolated
Eti l t d
growth

11. Seedling Deetiolation Diurnal Growth
(day/night y
( y g cycles) )
Etiolated
growth
SD-induced
growth
10 mm
Deetiolation
DARK RED Short-Days W
(D) (R) (SD: 16hD-8hW)
(day)
R How does the phy R
Pr Pfr transmit the primary Pr Pfr
D
light signal (night)
Etiolated
Eti l t d information? Hypocotyl
H t l
growth growth

12. Seedling Deetiolation
Etiolated
growth Seedling deetiolation as
a model system
d l t
10 mm
Deetiolation to study responses to
Light/Dark
DARK RED
(D) (R)
R How does the phy
Pr Pfr transmit the primary
light signal
Etiolated
Eti l t d information?
growth

13. PIF3 and related PIFs are phy-interacting bHLH transcription
factors that selectively bind the Pfr form of the phy
Nuclear
Localization Signal
g
NLS
PIF3
PIFs 1,4,5
, ,
APA/B bHLH
phy Pfr Basic
Binding Helix-loop-helix
Red
GAD:PIF3
(660 nm)
Preferential binding to
PIF3 the DNA motif G-BOX:
phyB CACGTG
GAD: (Martínez-García et al., 2000, Science)
PIF3
phy Pr phy Pfr
(Inactive)
+FR
(Active) +R
+
Far Red
+
(730 nm) (Ni et al., 1999, Nature)

14. Photoactivated phy triggers rapid pifq mutants lacking PIFs 1, 3,
proteolytic degradation of PIFs 1, 4 and 5 show a constitutively
y
3, 4 and 5 in response to light photomorphogenic phenotype
PIFs
R light
D + R (min)
Etiolated
0 10 20 30 40 50 60 Growth
phy
PIF3
(Al-Sady et al., 2006, Molecular Cell)
PIFs
PIF3:CFP PHYB:YFP Overlay
WT pifq
(Bauer et al, 2004, The Plant Cell)
(pif1pif3pif4pif5)
(Leivar et al., 2008, Current Biology)

15. PIF1, 3, 4 and 5 accumulate in the dark
and sustain etiolated growth
CYTOPLASM
Dark
PIF1
Pr PIF3
PIF4
PIF5
PIF-regulated
PIF regulated
G-box
genes
NUCLEUS
Transcriptional PIF-regulated Etiolated
network transcriptome Growth
~1000 genes
1000

16. Initiation of seedling deetiolation through
phy-induced degradation of PIFs 1 3 4 5
1,3,4,5
CYTOPLASM
SE?
E3 LIGAS
KINASE?
?
Red 26S proteasome
induced Dark Light
R P Ub degradation
R PIF1 PIF1 PIF1
P Ub
Pr PIF3 PIF3 PIF3
Pfr P Ub
PIF4 PIF4 PIF4
PIF5
P PIF5
Ub
PIF5
PIF-regulated
PIF regulated
G-box
genes
NUCLEUS
Signal Transcriptional Transcriptional Etiolated Seedling
Perception network reprogramming Growth Deetiolation
& (Photomorphogenesis)
Nuclear
translocation

17. The Dark
Side of Plant
Seedling Deetiolation growth
R
Pr Pfr PIFs
PIF
Transcriptional
network
Etiolated
Growth

18. How do plants grow in accordance
to their ambient light/DARK conditions?
Seedling Deetiolation
R
Pr Pfr PIFs
PIF
Transcriptional
network
Etiolated
Growth
RESEARCH LINE 1:
Seedling growth in the Dark after germination:
Identification of new regulators of deetiolation acting
downstream of the PIFs
1a- Functional profiling of PIF3-regulated genes
1b- Suppressor screen of pifq mutants

19. 1a- Functional Profiling of PIF3-regulated genes
I. Identification of PIF3-regulated MIDA II. Selection candidate
(Misexpressed In DArk) genes regulatory genes
-1.35000 0 1.35000
Z-score
- 13 MIDA genes selected
(MIDA1-MIDA13)
D0h_Rep1
D1h_Rep1
D1h_Rep2
D1h_Rep3
WT D0h_Rep3
- Identification of insertional
D0h_Rep2
D0h_Rep1
D0h_Rep3
D1h_Rep1
pif3 D1h_Rep3
D1h Rep3
mutants in public collections
D0h_Rep2
D1h_Rep2
42 Induced Genes 40 Repressed Genes (mida1-mida13)
2.5 1.2
Average FC
Average FC
2 1
1.5 0.8
0.6
1
0.4
0.5 0.2
0 0
WT pif3 WT pif3
III. Phenotypic analysis
of mida mutants.
(Sentandreu et al., 2011, Plant Cell)

20. 1a- Functional Profiling of PIF3-regulated genes
PIF3-regulated transcriptional network
implement organ-specific responses
during seedling deetiolation
g 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)

21. 1b- Suppressor screen of pifq mutants
Guiomar Martín
WT pifq
(pif1pif3pif4pif5)

22. 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)
pifq pifq HygR
Next:
HygR
Validation of sop mutants. Putative sops
Identification of responsible genes.
(suppressors-of-pifq)

23. How do plants grow in accordance
to their ambient light/DARK conditions?
Seedling Deetiolation Diurnal Growth
(day)
R
R
Pr Pfr PIFs
PIF Pfr Pr
D
(night)
Transcriptional
network
Etiolated Hypocotyl
Growth Growth
RESEARCH LINE 1: RESEARCH LINE 2:
Seedling growth in the Dark after germination: Seedling growth in the Dark in diurnal conditions
Identification of new regulators of deetiolation acting (8h Light:16h Dark)
downstream of the PIFs
2a- Characterization of PIF3 function
1a- Functional profiling of PIF3-regulated genes
2b- Definition of PIF-regulated transcriptional
g p
1b- Suppressor screen of pifq mutants networks implementing growth responses to light

24. 2a- Characterization of PIF3 function in diurnal growth
0h 8h 24h
Day Night
(Nozue et al 2007 Nature)
al., 2007,
Hypocotyl growth rate peaks at the end of the night
0.25‐
0.2‐ WT
ate
Growth Ra
(mmh-1)
0.15‐
0.1‐
Judit
0.05‐
Soy
0‐
pif3
3 6 9 12 15 18 21 24 27
Time (h)
(Soy et al., 2012, Plant Journal)

25. 2a- Characterization of PIF3 function in short-days
0h 8h 24h II. PIF3 induces the expression of
growth-related at the end-of the night
10-
I. PIF3 protein accumulates at
p
vel
Expression Lev
8
8- PIL1
the end of the night in short-days WT
Relative
6-
WT pif3 4-
h: 3 6 8 10 12 14 16 18 20 22 23 24 25 23 pif3
2-
PIF3
n.s 0-
3 6 9 12 15 18 21 24
n.s
Time (h)
ponceau 7
ent
6 PIL1p
ChIP qRT-PCR
old-Enrichme
5
4
3
PIF3 2
PIF4/5 1
Fo
C
0
PIL1 NoAb Ab NoAb Ab NoAb Ab NoAb Ab
XTR7 promoter control
region promoter
control
region
phyA, HFR1 WT YFP-PIF3
phyB phyB phyB
phytochrome-Imposed oscillations of PIF3
Hypocotyl
yp y abundance regulate hypocotyl growth
elongation under diurnal conditions in conjunction
Day Night with PIF4/5 and the circadian clock
(Soy et al., 2012, Plant Journal)

26. 2b- Definition of PIF-regulated transcriptional networks
implementing growth responses to light
(Leivar et al., 2009 Pl t C ll)
(L i t l 2009, Plant Cell)
Comparative
transcriptomic Deetiolation
(WT vs pifq)
analysis (839 genes)
729
11 76 Simulated
Diurnal 23
(WT vs
shade
(WT vs pifq)
pif4pif5) 66 148
18 (265 genes)
(118 genes)
(Nozue et al., 2011, Plant Physiology)
(Leivar et al., 2012, Plant Cell)

27. Seedling deetiolation Diurnal conditions
D R SD W
(8hW-16hD)
R Day
Pr Pfr Pr Pfr
Night
Pr
PIFs
G-box
cellular machinery that implement
different facets of growth

28. Seedling deetiolation Diurnal conditions
D R SD W
(8hW-16hD)
R Day
Pr Pfr Pr Pfr
Night
The balance between phy
Pfr
Pf and PIFs determines plant
growth during light/dark
PIFs transitions
G-box
cellular machinery that implement
different facets of growth

29. 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 ON
2.1- Generación de interruptores moleculares, reversibles, y
PIF Pr +R PIF Pfr
modulables por luz:
+
- Expresión génica
g X Y X Y
+FR
- Splicing de proteínas
(Shimizu et al., 2002, Nat. Biotech.)
- Señalización en dominios subcelulares discretos (Tyszkiewicz et al., 2008, Nat. Methods)
proceso
2.2- Generación de nuevos marcadores fluorescentes (Levskaya et al., 2009, Nature) celular
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 , p
en semillas y plántulas
Potencial aplicado:
- Producción de ácidos grasos poli-insaturados con valor nutracéutico
- Producción de aceites que actúen como biodiesel
Céline Diaz

30. Roger Miralles
Guiomar Martín
Elena Monte
El M t Céline Diaz
Maria Judit Soy
Sentandreu
Old members
Nahuel González At UC-Berkeley
Peter H. Quail
Funding
Beatriu de Pinós Fellowship (Generalitat de Catalunya) At CNB-CSIC
Marie Curie Actions IRG PIRG06-GA-2009–256420
Salomé Prat
Ministerio de Ciencia e I
Mi i t i d Ci i Innovación (MICINN)
ió
AGAUR 2009 SGR 206