How do light signals control nuclear genes for leaf & plastid development?

Can divide into 3 basic steps (or parts):1. Receiving the signal (photoreceptors) 2. Transmitting (and amplifying?) the signal

to the nucleus 3. Activating (de-repressing?) or repressing

transcription of genes associated with “greening” or “de-etiolation”

Transcriptional control of the pea rbcS3 gene by light:: The molecular approach

• Comparative analysis of 5’-upstream sequences of the rbcS gene family (pea) identified light-responsive elements (LRE)

Enhancer

______________________________________III* II* I II III VI V-330 -50 +1

• Several putative trans-acting factors for this promoter were identified based on their in vitro ability to bind to specific elements

- GT1, AF2 & AF3 binds to, or near, boxes II and/or III (and II* and/or III*) - AF1 binds box VI

• Present in both light and dark, however.• Some maybe regulated by phosphorylation-dephosphorylation

- Binding of AF3 to DNA is promoted by phosphorylation - Kinase may be a casein kinase 2 (CK2)

N-H. Chua

Postive and negative factors from a genetic approach in Arabidopsis

Another long hypocotyl mutant, Hy5, lacks a bZIP factor that promotes transcription from a number of genes with LREs

- Hy5 also responds to the blue (Cry) and red light (Phy) photoreceptors

bZIP proteins have basic (+) DNA-binding domain and a leucine dimerization zipper

COP/DET genes: Pleiotropic repressors of leaf and plastid development

• Pleiotropy- one gene affects many processes

• J. Chory identified a mutant DET1 that de-etiolates even in darkness– cotyledons are not green, but they do expand– plastids develop into chloroplast-like organelles– Many greening genes (rbcS and cab) are on

Det1 light Det1 dark WT dark

COP/DET/FUS genes: Pleiotropic repressors of leaf and plastid development

COP (constitutive photomorphogenesis) and FUS mutants similar to DET1

10 COP genes:- COP1, key repressor of photomorph.- 8 are subunits of a large complex, COP9 signalosome,- COP1 and COP9 complex also found in animal cells

- COP1 is a ubiquitin ligase, which triggers degradation of transcription factors (HY5) by the proteasome, also interacts with COP9 and DET1

COP1

Yi and Deng, 2005, TCB 15:618

PhyA, PhyB, Cry1, Cry2 inhibit COP1-mediated degradation of transcription factors that activate

photomorph. genes

Cry proteins are activated by BL, phosphorylated, and then bind to COP1

COP1 also moves to cytoplasm in light.

How do PhyA and PhyB inhibit COP1??

Phytochrome Interacting Factors (PIFs)

Another Model for Phytochrome Action.

Peter Quail, USDAEnamul Huq, UT

PhyB sees Red and PhyA, Far-red

D

Rc

FRc

WT phyA phyB Tepperman et al., 2004

Arabidopsisthalianamutants

Two-Hybrid Screening Strategy to get Phy Interacting Factors (PIFs)

GAL1 UAS

HIS3/lacZBD

phy PIFs AD

Monte et al (2004) PNAS

PIF3 (bHLH protein): Required for Full PhyB-Mediated Greening Response

Pho4 (bHLH) bound to DNA

Martinez-Garcia et al (2000) Science 288: 859CCA1 & LHY have G-box

Nagy and Schaefer (2000) EMBO J. 19: 157-163.

phyA

phyB

At least some PhyA and PhyB Translocate into Nucleus in Light

 

GFP-tagged Phy proteins

Postulated Direct Targeting of Light Signalto Promoter-Bound PIF 3

PIF3 PIF3 PICG-box TATA

PfrB

TATA

NUCLEUS

PrB

PIF3 PIF3G-box

FRPfrB

CYTOPLASM

PrB

PfrB

R

FR

Does PIF3 heterodimerize? A 2-Hybrid Screen with PIF3 as Bait

GAL1 UAS

HIS3/lacZBD

YPIF3 AD

** PIF1 and PIF4 are new bHLH proteins

What is/are the function of the heterodimer(s)?

SUMMARY

1. Interaction of DNA-bound PIF3 with Pfr form of PhyB may provide for direct regulation of gene expression in response to light.

2. PIF3 helps controls greening process, and interacts with other PIFs.

3. Phy signaling involves direct interaction with transcription factors (PIFs).