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 RoviraJ. F. Martínez García

July 2013

Crowded situations represent a challenge for p g

individual living organisms.

In plants, crowded situations result in competition for basic

resources, such as light.

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.

A single signal, low A 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)

Light and plant photoreceptors Light 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

omor

phog

enes

is

UVB

CAROTENOIDS

CHLOROPHYLLS

Phot

synt

hesi

s

CHLOROPHYLLS CHLOROPHYLLS

Pho

to CHLOROPHYLLS

in light-grown plants

high R:FR

ResponsesSynthesisR

FRPr

inactivelow R:FR Pfr

active

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

PfrB

genes 2ry

nucleus

P P

genes 1ry

B

cytoplasmcytoplasm

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

PfrPrTFTF

nucleusgenes 2ry

Pfr

IF IF

genes 1ry

cytoplasmPAR genes

P P

PHYB, ATHB4

cytoplasm

PAR (Phytochrome rApidly Regulated) genes[Roig-Villanova et al. (2006) Plant Physiol 141, 85-96]

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

nucleusPfr

IF IF

cytoplasm

P P

genes 1ry

cytoplasm

The R:FR ratio informs about plant’s competition

C.hirsutaA.thaliana d0 d3 d7 W

mm

) 6

5

WW+FR

low R:FR (ON)

yl le

ngth

(m

3

4

5

At Ch

Hyp

ocot

y

0

1

2

At Ch

shade-AVOIDANCEshade-TOLERANTWh 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?

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

PfrPrTFTF

nucleusgenes 2ry

Pfr

IF IF

genes 1ry

cytoplasmPAR genes

P P

PHYB, ATHB4

cytoplasm

PAR (Phytochrome rApidly Regulated) genes[Roig-Villanova et al. (2006) Plant Physiol 141, 85-96]

ATHB4 is a ATHB4 is a HomeoHomeo--Domain Domain 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)]

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

homozygotesATHB4

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]

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

ATHB4 function-structure:Which parts of ATHB4 are responsible for the biological activity?p p g y

• ATHB4 deletion fragmentsWL 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

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

Response to WL+FR treatment

Nt HD Zp GRCt pCS1935S

WWL+FR

Nt HD Zp GR pMG2335S

simulated shadeWL+FR

WL+FRd0 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.

Response to WL+FR treatment

Nt HD Zp GRCt pCS1935S

HD Zp GR pMG2435S

7,0

8,0pMG24W

WL+FR

5,0

6,0

th (m

m)simulated shade

WL+FR

d0 d2 d7

2,0

3,0

4,0

Hyp

. len

gt

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.

Which parts of ATHB4 are responsible for the biological activity?

• ATHB4 deletion fragmentsWL+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?

Which parts of ATHB4 are responsible for the biological activity?

+

Biological activity• ATHB4 deletion fragmentsWL+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 pMG4335SIs ATHB4 acting in a DNA binding independent manner?

AcknowledgementAcknowledgement

Jaime F. Martínez GarcíaChristianne ThenMercè Salla Martret

MªJosé MolinaNicolás Cifuentes Esquivel

Mercè Salla MartretAnahit GalstyanJordi Bou TorrentServices of CRAGServices of CRAG.

Ministry of Educationand Science of Spain (FPI)

Thank you for your attention!