Plants respond to proximity of other vegetation through shade avoidance syndrome (SAS). When plants detect low red (R) to far-red (FR) light ratios, which indicates nearby vegetation, they activate SAS even before being shaded. This single low R:FR signal regulates SAS throughout a plant's lifecycle, inducing responses like hypocotyl elongation and flowering time changes. The transcription factor ATHB4 is a direct target of phytochrome photoreceptors that perceive R:FR light. Studies on ATHB4 deletion fragments show that its N-terminal domain is required for SAS responses to low R:FR, suggesting it acts through DNA-binding to regulate gene expression and mediate shade avoidance.

1. Ab t l t t t ti i itAbout plant responses to vegetation proximity
Sobre les respostes de la planta a la proximitat d'altres vegetalsSobre les respostes de la planta a la proximitat d altres vegetals
Marçal Gallemí Rovira
J. F. Martínez García
July 2013

2. Crowded situations
represent a challenge forp g
individual living organisms.

3. In plants, crowded situations
result in competition for basic
resources, such as light.

4. The R:FR ratio informs about plant’s competition
l R FR
(ON)
high R:FR
low R:FR
(OFF)
high R:FR
Aft ti f l R FR ti b th h t h th h d idAfter perception of low R:FR ratio by the phytochromes, the shade avoidance
syndrome (SAS) gets activated, even before the plant is actually shaded.

5. A single signal, lowA single signal, low RR::FRFR, regulates SAS responses, regulates SAS responses
during plant life cycleduring plant life cycleg p yg p y
Arabidopsis thaliana
ONOFF
Adult plant
Seedling
(hypocotyl elongation)
Adult plant
(leaf expansion, flowering
time, plant architecture,
apical dominance, …)
ONOFF
Seed
(germination)
Adult plant
(fruit formation)

6. Light and plant photoreceptorsLight and plant photoreceptors
300 400 500 600 700 800 Light wavelength (nm)
uv-B uv-A blue green yellow RED FAR RED
300 400 500 600 700 800 Light wavelength (nm)
CRYPTOCHROMES
PHOTOTROPINS
PHYTOCHROMES
omorphogenesis
UVB
CAROTENOIDS
CHLOROPHYLLS
Photsynthesis
CHLOROPHYLLS CHLOROPHYLLS
Photo
CHLOROPHYLLS
in light-grown plants
high R:FR
ResponsesSynthesis
R
FR
Pr
inactive
low R:FR Pfr
active

7. Phytochromes are directly regulating gene expressionPhytochromes are directly regulating gene expression
hi h R
Phytochromes, in its active form, regulate the expression of primary (1ry) genes by binding to PIF
proteins.
high R:FR
PfrPr
IF
IF
Pfr
B
genes 2ry
nucleus
P
P
genes 1ry
B
cytoplasmcytoplasm

8. Phytochromes are directly regulating gene expressionPhytochromes are directly regulating gene expression
l FR
Focus on early transcriptional events after low R:FR perception to identify DIRECT target genes
of phytochrome action.
low R:FR
CHX
PfrPr
TFTF
nucleus
genes 2ry
Pfr
IF
IF
genes 1ry
cytoplasm
PAR genes
P
P
PHYB, ATHB4
cytoplasm
PAR (Phytochrome rApidly Regulated) genes[Roig-Villanova et al. (2006) Plant Physiol 141, 85-96]

9. Identification and functional analysis of SAS components:Identification and functional analysis of SAS components:
draculadracula ((dradra) mutants) mutants
l FR
dra2 is a nucleoporin and affect the first early molecular responses
(( ))
low R:FR
TFTF
Not related genes
PfrPr
nucleus
Pfr
IF
IF
cytoplasm
P
P
genes 1ry
cytoplasm

10. The R:FR ratio informs about plant’s competition
C.hirsutaA.thaliana d0 d3 d7
W
mm)
6
5
W
W+FR
low R:FR (ON)
yllength(m
3
4
5
At Ch
Hypocoty
0
1
2
At Ch
shade-AVOIDANCEshade-TOLERANT
Wh t i th l l d h i l i l b i f th diff(A. thaliana)(Cardamine hirsuta)What is the molecular and physiological basis of the differences
in response to shade between plant species?

11. Phytochromes are directly regulating gene expressionPhytochromes are directly regulating gene expression
l FR
Focus on early transcriptional events after low R:FR perception to identify DIRECT target genes
of phytochrome action.
low R:FR
CHX
PfrPr
TFTF
nucleus
genes 2ry
Pfr
IF
IF
genes 1ry
cytoplasm
PAR genes
P
P
PHYB, ATHB4
cytoplasm
PAR (Phytochrome rApidly Regulated) genes[Roig-Villanova et al. (2006) Plant Physiol 141, 85-96]

12. ATHB4 is aATHB4 is a HomeoHomeo--DomainDomain LeucineLeucine--Zipper (HDZipper (HD--Zip) transcription factorZip) transcription factor
athb4hat3Col-0
M. Salla-Martret
HD-Zip II
[Ariel et al. TRENDS in Plant Science (2007); Bou-Torrent J, Salla-Martret M et al. Plant Signal Behav. (2012)]

13. ATHB4 overexpresion presents a severe phenotype
Constitutive O/E O/E with inducible activity
ATHB435S ATHB4 GR35S
wt - DEX + DEX
wt
1 cm
wt
ATHB4-GR
homozygotes
ATHB4
O/E ATHB4 present strong dwarf phenotype and sterile adult plant.
We have to use an inducible line with the GR domainWe have to use an inducible line with the GR domain.
[Sorin et al. (2009) The Plant Journal 59(2):266-77]

14. ATHB4 is similar to ATHB2, the first studied member of the familyATHB4 is similar to ATHB2, the first studied member of the family
Nt
HomeodomainHD
L i i d iZIP Leucine zipper domainZIP
Ct
CPSCE Redox domain

15. ATHB4 function-structure:
Which parts of ATHB4 are responsible for the biological activity?p p g y
• ATHB4 deletion fragments
WL FR
Biological activity
WL+FR MolecularWL
Nt HD Zp GRCt pCS1935S
Nt HD Zp GR pMG2335S
HD Zp GR pMG2435S
Zp GR pMG2535S
GR MG2635S Z C GR pMG2635S Zp Ct
HD GR pMG2735S Zp Ct
HD GR pMG2835S Nt
pMG29HD GR35S

16. Phenotype and response to DEX in WL
Nt HD Zp GRCt pCS1935S
GNt HD Zp GR pMG2335S
HD Zp GR pMG2435S
- DEX + DEX
pMG24
- DEX + DEX
pMG23 pMG24

17. Response to WL+FR treatment
Nt HD Zp GRCt pCS1935S
W
WL+FR
Nt HD Zp GR pMG2335S
simulated shade
WL+FR
WL+FR
d0 d2 d7
- + - +- + - +- + - + - + - +DEX:
pMG23.2.5pMG23.1.4Col-0
(wild type)
pCS19
(ATHB4-GR
full lengh)
The Ct of ATHB4 is not important for the biological function, at least for shade responses.

18. Response to WL+FR treatment
Nt HD Zp GRCt pCS1935S
HD Zp GR pMG2435S
7,0
8,0
pMG24W
WL+FR
5,0
6,0
th(mm)
simulated shade
WL+FR
d0 d2 d7
2,0
3,0
4,0
Hyp.lengt
0,0
1,0
Col-0 pCS19 pMG24.5.2 pMG24.12.6- + - + - + - + - + - +- + - +DEX:
Col-0
(wild type)
pCS19
(ATHB4-GR
full lengh)
pMG24.12.6pMG24.5.2
The Nt of ATHB4 is necessary for the biological activity, al least for shade response.

19. Which parts of ATHB4 are responsible for the biological activity?
• ATHB4 deletion fragments
WL+FR Molecular
Biological activity
WL
+ +
WL+FR MolecularWL
+Nt HD Zp GRCt pCS1935S
+
-
+
-
+
-
Nt HD Zp GR pMG2335S
HD Zp GR pMG2435S
- n.a.-Zp GR pMG2535S
GR MG2635S Z C -
-
-
-
-
-
GR pMG2635S Zp Ct
HD GR pMG2735S Zp Ct
+
-
+
n a
+
-
HD GR pMG2835S Nt
pMG29HD GR35S
- n.a.-
Is ATHB4 acting in a DNA binding independent manner?

20. Which parts of ATHB4 are responsible for the biological activity?
+
Biological activity• ATHB4 deletion fragments
WL+FRWL
+Nt HD Zp GRCt pCS1935S
+
-
+
-
Nt HD Zp GRCt pCS1935S
Nt HD Zp GR pMG2335S
HD Zp GR pMG2435S
-
-
-
-
Zp GR pMG2535S
GR pMG2635S Zp Ct
-
+
-
+HD GR pMG2835S Nt
HD GR pMG2735S Zp Ct
-
?
+
-
?
+
pMG29HD GR35S
Nt GR pMG6035S
MG37 +
-
-
+
-
-
Nt HD Zp GRCt pMG3735S
Nt HD Zp GRCt pMG3835S
Nt HD Zp GRCt pMG4335S -
-
-
-Nt HD Zp GRCt pMG4435S
Nt HD Zp GRCt pMG4335S
Is ATHB4 acting in a DNA binding independent manner?

21. AcknowledgementAcknowledgement
Jaime F. Martínez García
Christianne Then
Mercè Salla Martret
MªJosé Molina
Nicolás Cifuentes Esquivel
Mercè Salla Martret
Anahit Galstyan
Jordi Bou Torrent
Services of CRAGServices of CRAG.
Ministry of Education
and Science of Spain (FPI)
Thank you for your attention!