calculation of relative transcript quantities [42]. Data were normalized to the corresponding ACTIN1 mRNA levels; phyB- 401/phyA-201 relative dark levels were set to one. Each time point was measured at least in two biological replicas, error bars indicate standard error of technical replicas. Sequences of primers and probes used for Real-Time RT-PCR were as follows: ACTIN1 (forward: 59- GGCTCCAAGCAGCAT- GAAG-39; reverse: 59- ACCCTCCAATCCAGACAGAGTATT- 39; probe: 59JOE- CAAAGTCGTTGCCCCTCCAGAGAGG -39 BHQ1) and CAB2 (forward: 59- GAGAGGCCGAGGACTTGC- TT-39; reverse: 59- CTCTGGGTCGGTAGCCAAAC-39; probe: 59FAM- ACCCCGGTGGCAGTTTCGACC -39TAMRA). Yeast two-hybrid assay Quantitative yeast two-hybrid assay was performed as previ- ously described [43]. Supporting Information Figure S1 Fluence rate dependent hypocotyl growth inhibition of seedlings expressing PHYB and PHYB-401 fusion proteins. Wild-type Col-0 (filled diamonds), phyB-9 mutant (empty squares) and transgenic seedlings expressing PHYB:GFP or PHYB:YFP (empty triangle) and PHYB-401:GFP or PHYB-401:YFP (empty circle) fusion proteins in phyB-9 (empty circle) were grown for 4 days under different fluence rates of cR (A) or cFR (B) light. Hypocotyl lengths were measured and relative hypocotyl lengths are shown. Each time point was measured at least three times, error bars indicate standard error. (TIF) Figure S2 Overexpression of the PHYB-401:YFP fusion protein does not affect skotomorphogenesis of the transgenic seedlings. Arabidopsis seedlings were germinated according to the standard protocol (after 3 days stratification at 4uC seeds were imbibed and treated with 4 h white light) and then grown for 4 days in darkness. Phenotypes of seedlings expressing the PHYB:YFP (A) and PHYB-401:YFP (B) fusion proteins in phyB-9 background and those of the wild-type Col-0 (C) and phyB- 401/phyA-201 (D) double mutant are shown. (TIF) Figure S3 Degradation kinetics of the mutant and wild- type phyB do not differ significantly in cR. Accumulation levels of phyB and phyB-401 in phyA-201 background were References 1. Rizzini L, Favory JJ, Cloix C, Faggionato D, O’Hara A, et al. (2011) Perception of UV-B by the Arabidopsis UVR8 protein. Science 332: 103–106. 2. Beggs C, Wellmann E (1994) Photocontrol of flavonoid biosynthesis. In Kendrick RE. Kronenberg GHM, eds. Photomorphogenesis in Plants (2nd Ed.). Dordrecht, Boston, London: Kluwer Academic Publishers 733-751. 3. Lin C, Robertson DE, Ahmad M, Raibekas AA, Jorns MS, et al. (1995) Association of flavin adenine dinucleotide with the Arabidopsis blue light receptor CRY1. Science 269: 968–970. 4. Briggs W, Christie J (2002) Phototropin 1 and phototropin 2: Two versatile plant blue-light receptors. Trends Plant Sci 7: 204–210. 5. Christie J, Swartz T, Bogomolni R, Briggs W (2002) Phototropin LOV domains exhibit distinct roles in regulating photoreceptor function. 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(TIF) Figure S4 Interaction of PIF3 with phyB and with phyB- 401 is identical. Liquid overnight cultures of yeast cells co- expressing the indicated proteins fused to the GAL4 activation (AD) or DNA-binding (BD) domain were treated with 5 min red light pulse (R) which was followed by a 5-min-long far-red light pulse (FR) in the indicated cases. After the given light pulses the cultures were incubated in the dark for the indicated time (between 2-6 h). Subsequently the ß-Galactosidase activity was determined. Error bars represent standard error of the mean. (TIF) Table S1 Comparison of the expression levels of PHYB and PHYB-401 fusion proteins to the endogenous phyB and phyB-401 photoreceptors. Accumulation levels of phyB, phyB-401 and those of the PHYB and PHYB-401 fusion proteins were analyzed by western blot hybridization using the monoclonal antibody B3-B6 [41]. 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Zhou Y, Dieterle M, Bu¨che C, Kretsch T (2002) The negatively acting factors EID1 and SPA1 have distinct functions in phytochrome A-specific light signaling. Plant Physiol 128: 1098–1108. PLoS ONE | www.plosone.org 11 November 2011 | Volume 6 | Issue 11 | e27250