1. 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”

2. 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

3. 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

4. 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

5. 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

6. 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??

7. Phytochrome Interacting Factors (PIFs) Another Model for Phytochrome Action. Peter Quail, USDA Enamul Huq, UT

8. PhyB sees Red and PhyA, Far-red D Rc FRc WTphyAphyB Tepperman et al., 2004 Arabidopsis thaliana mutants

9. Two-Hybrid Screening Strategy to get Phy Interacting Factors (PIFs) GAL1 UAS HIS3/lacZ BD phyPIFsAD

10. Monte et al (2004) PNAS PIF3 (bHLH protein): Required for Full PhyB- Mediated Greening Response Pho4 (bHLH) bound to DNA

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

13. 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

15. Does PIF3 heterodimerize? A 2-Hybrid Screen with PIF3 as Bait GAL1 UAS HIS3/lacZ BD YPIF3AD ** PIF1 and PIF4 are new bHLH proteins What is/are the function of the heterodimer(s)?

16. 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).