1. EPSO Workshop Plant Productivity for Food-from Models to Crops Ghent, Belgium 7-8 September 2009 Shade Avoidance Response

2. Adapted from Jiao et al. Nat Rev Genet 2007

3. DARKLIGHT Elongation growth Adapted from Castillon et al. Trends in Plant Sci 2007

4. Adapted from Jiao et al. Nat Rev Genet 2007

5. Low High canopy density Smith 2000 Shade Avoidance Response Nicotiana Boonman et al 2006

6. Low R/FR as warning signal for future plant competitors Ballarè 1999

7. Biotech Single Cross Double Cross Open Pollinated Agronomic Practices ~ 40% 30 000 plants ha -1 80 000 plants ha -1  2-4-D  Narrower Rows  Atrazine Genetic Improvement ~ 60% Adapted from Troyer, 2006; Lee & Tollenaar, 2008. Maize Historic Yield Gains Average Corn Yield (Kg/ha)

8. Corn row spacing: 30" Shade Avoidance Response in Maize Courtesy of Clay Mitchell; Strip Maize-Soybean, Iowa. Alterations of Growth Patterns and Resource Partitioning Increased plant height Reduced vegetative branching (tillering) Elongated Internodes (increased lodging) Acceleration of flowering

9. Low R/FR Light PfrPr High R/FR Shade Avoidance Response in Arabidopsis

10. High R/FR Low R/FR Light PfrPr Shade Avoidance Response in Arabidopsis

11. 05 15 30 45 60 7580 105 120 135 min 90 Low R/FRHigh R/FR ATHB2 AtL18 ATHB2 is rapidly regulated by light quality changes Carabelli et al Proc Natl Acad Sci USA 1996

12. Wild type 35S::ATHB2 Seedlings overexpressing ATHB2 display constitutive shade avoidance response High R/FR Steindler et al Development 1999

13. ATHB2 acts as a positive regulator of shade avoidance response High R/FR athb2-2Col-0 ATHB2 AtL18 athb2-1Ws 0101h Low R/FR ATHB2 AtL18 athb2-2 Col-0 0101h Low R/FR athb2-1Ws Low R/FR

14. ATHB2 is transiently induced by low R/FR light ATHB2 AtL18 Sessa et al 2005 Genes Dev 19: 2811

15. Persistency of a low R/FR signal results in attenuation of shade avoidance response Hypocotyl length +15.6% +24.5% 0 0.5 1.0 1.5 2.0 2.5 3.0 0d (4d high R/FR) 1d2d3d days mm High R/FR Low R/FR +7.8%

16. Persistency of a low R/FR signal results in attenuation of shade avoidance response 1 st leaf area +173% +342% +175% +135% 0 0.02 0.04 0.06 0.08 0.10 0.12 0 (4d high R/FR) 23 days mm 2 High R/FR Low R/FR

17. Identification of genes involved in negative regulation of shade avoidance response Low R/FR wt sics mutant A two-step screen: 1. Identification of regulatory genes up-regulated in response to both brief and prolonged low R/FR exposure 2. Search for mutations in regulatory genes identified in step 1, causing an exaggerated elongated phenotype (slender) in shade Sessa et al Genes Dev 2005

18. Identification of genes involved in negative regulation of shade avoidance response Sessa et al Genes Dev 2005 A two-step screen: 1. Identification of regulatory genes up-regulated in response to both brief and prolonged low R/FR exposure

19. Putative G-box binders Putative non-G-box binders Putative non-E-box binders Putative non-DNA binders HFRI/SICS1 is a distantly related member of the PIF family PIF3 PIL1 PIL2/PIF6 SPT ALC UNE10 PIF7 PIL5/PIF1 bHLH023 bHLH119 bHLH056 bHLH127 PIF4 PIL6/PIF5 HFR1 bHLH109 bHLH108 All bHLHs

20. Mutations in HFR1/SICS1 cause exaggerated shade avoidance responses Col-0hfr1-4/ sics1-1 High R/FR Low R/FR hfr1-5/ sics1-2 Sessa et al Genes Dev 2005

21. HFR1/SICS1 is required to down-tegulate ATHB2 upon prolonged exposure to low R/FR Low R/FR Sessa et al Genes Dev 2005

22. HFR1/SICS1 is not required for the down-regulation of ATHB2 by high R/FR Sessa et al Genes Dev 2005

23. HFR1/SICS1 is required to switch off the signaling cascade induced by low R/FR 5 4 3 2 1 0 6 5 4 3 2 1 0 6 IAA Metabolism N. 9122379146 Percentage (%) 30 25 20 15 10 5 0 Col-o low R/FR up- regulated genes hfr1-4/sics1-1 low R/FR up-regulated genes Genomic Relative Transcript Level Col-0 hfr1-4/sics1-1 High R/FR Low R/FR

24. Hypocotyl length * * 3 0 7 5 mm * P < 0.01 vs phyB-9 phyB-9 35S::  NHFR1 Col-0 35S::  NHFR1 phyB-9 phyB-9 hfr1-4/sics1-1 hfr1-4/sics1-1 Relative transcript level AtL18 HFR1/SICS1 100 h Low R/FR Col-0phyB-9 AtL18 HFR1/SICS1 h Low R/FR AtL18 HFR1/SICS1 Col-0 phyA-211 0.54120 IAA29 PIL1 ATHB2 0 0 0 20 8 12 1 20 40 16 4 10 30 2 4 3 5 HFR1/SICS1 acts in the PHYB signaling pathway Sessa et al in preparation

25. HFR1/SICS1 is rapidly and reversibly regulated by low R/FR Mutations in HFR1/SICS1 cause exaggerated shade avoidance responses HFR1/SICS1 is required to down-regulate the signaling cascade induced by low R/FR HFR1/SICS1 acts in the PHYB signaling pathway

26. Low R/FR light HFR1/SICS1 ATHB4 PIL1 HLH1/PAR1 HFR1/SICS1 ATHB2 Shade avoidance PfrPr PIF TFs PHYB PHYD PHYE Type II phytochromes Adapted from Jiao et al. Nat Rev Genet 2007

27. Relative transcript level 0 2 4 8 6 Col-0 10 01221244822h2 LD 281 HFR1/SICS1 High R/FR Dark Low R/FR Relative transcript level 0 4 8 12 16 20 12212448 0 22h hfr1-4/sic1-1 Col-0 2 LD 24 28 1 ATHB2 HFR1/SICS1 is dynamically regulated during the day in low R/FR light Sessa et al in preparation

28. ?? TFs HY5 HYH PHYA Low R/FR light PIF TFs ATHB2 HFR1/SICS1 Shade avoidance PHYB PHYD PHYE Type II phytochromes ATHB2 ATHB4 PIL1 HLH1/PAR1 PfrPr Adapted from Jiao et al. Nat Rev Genet 2007

29. Mutations in PHYA cause exaggerated shade avoidance responses 0 2 4 6 8 10 Hypocotyl length (mm) Col-0 hfr1-4/sics1-1 phyA-211 0 0.4 0.8 1.2 1.6 Cotyledon area (mm 2 ) 3 mm Col-0 hfr1-4/sics1-1 phyA-211 Sessa et al in preparation

30. 0 2 4 6 8 10 0 2 1 3 4 5 Petiole length (mm) Leaf area (mm 2 ) Col-0 hfr1-4/sics1-1 phyA-211 2 mm Col-0 hfr1-4/sics1-1 phyA-211 Mutations in PHYA cause exaggerated shade avoidance responses Sessa et al in preparation

31. High R/FR Dark Low R/FR phyA-211 Col-0 Relative transcript level 0 4 8 12 16 20 12212448 0 22h2 24 28 1 ATHB2 PHYA is required to down-regulate genes early induced by low R/FR light phyA-211 Col-0 Relative transcript level 0 4 8 12 10 12212448 0 22h2281 IAA29 2 6 LD Sessa et al in preparation

32. 8 4 2 0 6 8 4 2 0 6 Relative transcript level Global profiling of phyA exposed to prolonged low R/FR Col-0 phyA-211 High R/FR Low R/FR ******* N. 9122379152 Percentage (%) 30 25 20 15 10 5 0 IAA Metabolism 1h 4h 1d Col-0 low R/FR up-regulated genes Genomic phyA-211 low R/FR up-regulated genes Sessa et al in preparation

33. Mutations in PHYA and HFR1/SICS1 are additive in low R/FR light Col-0 hfr1-4/sics1-1 phyA-211 hfr1-4/sics1-1 phyA-211 0 0.4 0.8 1.2 1.6 Cotyledon area (mm 2 ) 0 2 4 6 8 10 Hypocotyl length (mm) 3 mm Col-0 hfr1-4/sics1-1 phyA-211 hfr1-4/sics1-1 phyA-211 Sessa et al in preparation

34. Mutations in PHYA and HFR1/SICS1 are additive in low R/FR light Col-0 hfr1-4/sics1-1 phyA-211 hfr1-4/sics1-1 phyA-211 0 2 4 6 8 10 Petiole length (mm) 0 2 1 3 4 5 Leaf area (mm 2 ) 2 mm Col-0 hfr1-4/sics1-1 phyA-211 hfr1-4/sics1-1 phyA-211 Sessa et al in preparation

35. Mutations in PHYA and HFR1/SICS1 are additive in low R/FR light Sessa et al in preparation

36. PHYA and HFR1/SICS1 act additively in down-regulating genes early-induced by low R/FR light High R/FR Low R/FR 0 5 10 15 20 25 30 Relative transcript level ATHB2 0 5 10 15 20 25 30 Relative transcript level High R/FR Low R/FR IAA29 0 2 4 6 8 10 Relative transcript level High R/FR Low R/FR IAA19 0 2 4 6 8 10 Relative transcript level High R/FR Low R/FR PHYB Col-0 hfr1-4/sics1-1 phyA-211 hfr1-4/sics1-1 phyA-211 Sessa et al in preparation

37. Mutations in PHYA cause exaggerated elongation growth in low R/FR PHYA is required to down-regulate the signaling cascade induced by low R/FR PHYA acts independently from HFR1/SICS1 in low R/FR

38. Low R/FR light PfrPr PHYB PHYA HFR1/ SICS1 PIF TFs ? Shade-induced elongation growth

39. Global profiling of wild type and phyA seedlings exposed to prolonged low R/FR (1 d) Identification of genes acting downstream of PHYA in negative regulation of shade-induced elongation growth Search for regulatory genes up-regulated in wild type but not in phyA upon prolonged exposure to low R/FR Sessa et al in preparation

40. phyA-211 Col-0 Relative transcript level 12202848024 h 0 2 1 3 4 HYH 222 HYH Relative transcript level phyA-211 Col-0 0 3 1 5 7 HY5 12202848024 h 222 High R/FR Dark Low R/FR HY5 and HYH are late-induced by low R/FR light largely through the action of PHYA LD Sessa et al in preparation

41. 8 6 4 2 0 Hypocotyl length (mm) Ws hy5KS50 hy5 hyh 3d High R/FR 2 d High R/FR 3d High R/FR 2d Low R/FR 3d High R/FR ° * *P <0.01 vs hy5 hyh 3d High R/FR + 2d Low R/FR ° P <0.01 vs hy5 3d High R/FR + 2d Low R/FR ++ 3d High R/FR 2d Low R/FR 3d High R/FR + 2 d High R/FR + 300 200 0 100 300 200 0 100 Hypocotyl elongation (%) The hy5 hyh phenotype is enhanced by low R/FR light exposure Sessa et al in preparation

42. Relative transcript level 0 4 8 12 16 20 12212448 0 h21 ATHB2 LD High R/FR Dark Low R/FR ATHB2 hy5 hyh Ws HY5 and HYH are required to down-regulate ATHB2 in low R/FR light Sessa et al in preparation

43. HY5 and HYH are late-induced by low R/FR light largely through PHYA Mutations in HY5 and HYH cause exaggerated elongation growth in low R/FR HY5 and HYH are required to down-regulate the signaling cascade induced by low R/FR

44. ?? TFs HY5 HYH HY5 HYH PHYA Low R/FR light PIF TFs ATHB2 HFR1/SICS1 Shade avoidance PfrPr PHYB PHYD PHYE Type II phytochromes ATHB2 ATHB4 PIL1 HLH1/PAR1 Adapted from Jiao et al. Nat Rev Genet 2007

45. Valeria Cafardi Monica Carabelli Angela Ciarbelli Andrea Ciolfi Massimiliano Sassi Giovanna Sessa Valentino Ruzza Samanta Salvucci IBPM-CNR Rome - Italy Marco Possenti Giorgio Morelli INRAN Rome - Italy Collaborations Jian Xu Ben Scheres Utrecht University Teva Vernoux Jan Traas ENS-Lyon