an assay is obviously limited. Second, we do not have reliable information about the stability of the hypothetical Pr* in vitro as compared to in vivo. This latter one is especially a critical factor as the kinetics of accumulation of the hypothetical Pr* conformer is likely to be different in the cellular environment in planta and yeast two-hybrid assays. Taken together, data presented in this paper and elsewhere [24,25] adequately support the importance of the PHY domain in regulating stability, i.e. dark reversion of the Pfr form. The ultimate proof for the mechanism by which it mediates passing the conformational change of the chromophore onto the protein could, however, only be obtained after the resolution of the 3D structure of the full phytochrome-B. Materials and Methods Plant material and growth conditions Seeds were surface sterilized and sown on Petri-dishes (9 cm diameter) containing 4 layers of filter paper (Macherey-Nagel, Germany) and 4.7 ml distilled water. After stratification (three days at 4uC in darkness) uniform germination was induced by a 4 h white light treatment at 22uC. Afterwards seedlings were grown at 22uC in darkness for four days or subjected to various light treatments as described in the text. Transgenic plants expressing the 35S: PHYB:YFP and 35S:PHYB-401:YFP and GFP transgenes were generated in phyB-9 [38] backgrounds, as described [19,30]. The phyB-401 mutant was isolated in Landsberg erecta background [33]. Light treatments and hypocotyl length measurement Dark-grown seedlings were handled under dim-green safelight [19]. For light pulse treatments and fluence rate response curves modified Leitz Prado 500-W universal projectors (Leitz, Wetzlar, Germany) were used. Red light was obtained by using KG65 filters (lmax = 650 nm; Blazers, Liechtenstein) and far-red light was generated by filtering with an RG9 filter (lmax = 775 nm; Schott, Germany) which adjusts 99.9% Pr [33]. Unless otherwise noted, all other red light treatments were done in a red light field (3 or 22 mmol m22 s21) of fluorescent tube lamps (Philips TL 40 W/15) filtered with Plexiglas 501/3 (Ro¨hm und Haas, Darmstadt, Germany). Hypocotyl length of 4-day-old seedlings treated with various light programs was measured by using ImageJ software (http://rsb.info.nih.gov/ij). Epifluorescent and light microscopy For epifluorescent and light microscopy, seedlings were transferred to glass slides under dim-green safelight and analyzed with an Axioskop microscope (Zeiss, Oberkochem, Germany). Excitation and detection of YFP were performed with specific YFP filter (AHF Analysentechnik, Tubingen, Germany). Each exper- iment was repeated at least three times using at least five seedlings in which we monitored minimum 200 nuclei in each single experiment. Representative cells were documented by photogra- phy with a digital Axiocam camera system (Zeiss). Photographs were processed for optimal presentation using the Photoshop 7.0 (Adobe Systems Europe, Edinburgh, UK) and MS Office 97 (Microsoft, Redmond, WA) software packages. In vivo spectroscopy Dark reversion and photoconversion of transgenic PHYB-GFP and PHYB-401:GFP was measured in intact seedlings in a dual- wavelength ratio spectrophotometer (Ratiospect) [23,39]. Seed- lings were irradiated with saturating 3 h R to create maximal Pfr levels and to degrade light-labile phyA. The complete loss of phyA Regulation of Dark and Photoconversion of phyB-401 signal after this treatment was confirmed in phyB-9 mutant seedlings (data not shown). For measurements of dark reversion, seedlings were either directly analyzed in the Ratiospect or transferred to darkness for measurement of Pfr levels in prolonged dark incubation. For photoconversion analysis, seedlings were placed on ice during the last 15 min of the R treatment and all later steps to avoid dark reversion. After R irradiations and before measurements seedlings were subjected to 30 s, 60 s or 135 s far- red light pulses (720 nm; 6 mmol m22 s21). The total amount of phytochrome (Ptot) was measured by six times alternate irradiation with far-red and red light followed by absorption measurements that gave the relative amount of photo- convertible phytochrome D (DA) of the sample. The absorption difference in the sample between the baseline at the beginning of spectroscopy and the first far-red light treatment 25 in the Ratiospect gave the Pfr level of the sample relative to Ptot. For each measurement around 300 seedlings were used and fresh weight was determined directly before the measurement to normalize D (DA) between the samples. Each time point was measured at least three times, error bars indicate standard error. Calculation of Pfr/Ptot ratio under continuous irradiation Jabben et al [40] reported that the Pfr/Ptot ratio measured in etiolated mustard seedlings is fluence rate dependent. To account for this phenomenon they hypothesized that an unknown reaction inducing conversion of Pfr to Pr in dark must exist. This process called dark-reversion was shown to enhance conversion of Pfr to Pr, especially at low light. The ratio Pfr/Ptot is k1/(k1+k2+kr) whereby k1 and k2 are the rate constants for Pr to Pfr and Pfr to Pr photoconversion, respectively. kr is the rate constant for dark reversion. From Figure 3 k1 and k2 is estimated to be 0.008 s21 and 0.002 s21 at 6 mmol m22 s21. kr is calculated from Figure 2 (kr = 0.00013 s21) if a first order dark reversion reaction is assumed. Protein extraction, protein gel blotting and immunodetection To analyze cR-induced degradation of phyB 3-day-old dark- grown seedlings were irradiated up to 24 h in 3 mmol m22 s21red light and harvested together with a non-irradiated control at the end of irradiation period. Protein extraction and protein gel blotting was performed as described [30]. Immunodetection of phyB was performed using the monoclonal phyB antibody B6-B3 [41]. Horseradish-peroxidase-coupled anti mouse antiserum (Vector Laboratories) was used as secondary antibody. Develop- ment of the blot was done with the Phototrope Star Detection Kit (New England BioLabs). Quantification of phyB signals was carried out using the ImageJ software (http://rsbweb.nih.gov/ij/) and the gel analysis tools (described in the user manual: http.// rsbweb.nih.gov/ij/docs/user-guide.pdf). Linearity of the signal was ensured by the use of internal standards. Analysis of RNA accumulation by real-time RT-PCR Far-red light treatments were carried out in a far-red light field for 5 min with varying intensities to obtain the aimed total fluences. Total RNA from Arabidopsis seedlings was isolated with Plant RNA reagent (Invitrogen) according to the manufacturer’s protocol. Purification and on-column DNaseI digestion was performed by using the RNeasy mini Kit from Qiagen (Hilden, Germany). After first-strand cDNA synthesis, the RT-PCR was accomplished by using an ABI Prism7300 (Applied Biosystems). FAM- and JOE-reporter dyes (59end) were used for the TaqMan probes (see below). A standard curve method was used for the PLoS ONE | www.plosone.org 10 November 2011 | Volume 6 | Issue 11 | e27250