these conditions the biologically active Pfr conformer represents ,20 and .80% of the total phyB amount, respectively (for a detailed explanation for the calculation of the Pfr/Ptot ratio see Material and Methods). PHYB:YFP displayed diffuse staining and was detectable in the nuclei and cytoplasm in dark-grown seedlings (Figure 4A). Figure 4B shows that illumination of PHYB:YFP seedlings with weak cR induces, in a low percentage of cells, the formation of a few small NBs. Irradiation with higher light intensity clearly promotes association of the PHYB:YFP fusion protein with NBs (Figure 4C). PHYB:YFP was still detectable in the cytoplasm after illumination with weak cR (Figure 4B), whereas the absence of cytoplasmic YFP signal in seedlings irradiated with high-intensity R light (Figure 4C) indicates an increased level of nuclear import of the PHYB:YFP. Intracellular distribution of PHYB-401:YFP was clearly different from that of PHYB:YFP. Figure 4E and F demonstrate that irradiations with low and/or high intensity cR uniformly induced formation of numerous large PHYB-401:YFP NBs, whereas, as shown in Figure 4D–F, cytoplasmic YFP fluorescence was detectable only in etiolated material. Taken together, these data indicate that nuclear import and formation of large PHYB- 401:YFP associated NBs display increased sensitivity in cR as compared to PHYB:YFP. Figure 4. Subcellular distribution of PHYB:YFP and PHYB- 401:YFP differs in seedlings irradiated with cR. Localization of PHYB:YFP (A, B, C) and PHYB-401:YFP (D, E, F) fusion proteins in 4-day- old seedlings grown in darkness (A, D), low-intensity (0.03 mmol m22 s21) (B,E) or high-intensity cR light (22 mmol m22 s21) (C, F) is shown. Below the fluorescence pictures of cells (A–F) the corresponding differential interference contrast microscopic images are also shown. doi:10.1371/journal.pone.0027250.g004 Regulation of Dark and Photoconversion of phyB-401 Kinetics of the formation, stability and photoreversibility of R pulse induced nuclear bodies containing PHYB- 401:YFP and PHYB:YFP First we tested the effectiveness of a single R pulse to induce the formation of phyB NBs. To this end we monitored cellular distribution of PHYB:YFP and PHYB-401:YFP in 4-days-old dark-grown seedlings that had been treated with a short R pulse (30 s, 70 mmol m22 s21) and then returned to darkness. In etiolated seedlings both PHYB:YFP (Figure 5A, D) and PHYB- 401:YFP (Figure 5B, C) showed weak, diffuse nuclear staining. The applied high-intensity R pulse induced rapid formation of numerous, small NBs associated with PHYB:YFP or PHYB- 401:YFP already within 2 minutes after the treatment. These NBs were short-lived and transient, as they were not detectable later than 1 hour after the R pulse (Figure 5A and B). As for PHYB:YFP, we could not detect NBs in samples monitored 5 h, 16 h, 24 h and 48 h after the treatment: the PHYB:YFP fusion protein remained uniformly distributed in the nucleoplasm (Figure 5A). In contrast, the PHYB-401:YFP-associated NBs reappeared in the nuclei within 5 h after the R pulse (Figure 5B). These newly formed, late NBs differed from the transient NBs detected shortly after the light treatment, i.e. they were less numerous but larger and, surprisingly, they were detectable and got even larger up to 48 h of darkness (Figure 5B). As only the Pfr conformer of phyB is known to be able to associate with NBs, these observations imply that the Pfr form of PHYB-401:YFP is more stable than that of PHYB:YFP. Furthermore the disappearance of early speckles and appearance of late NBs after 2–3 h in complete darkness, indicates that PHYB-401:YFP remains in the Pfr state in darkness for long time and that formation of PHYB containing late NBs takes several hours even in the continuous presence of Pfr. This slow process can either be due to the slow modification of pre-existing NBs to allow interaction with PHYB Pfr or a slow modification of PHYB Pfr to facilitate interaction with preexisting NBs. We also tested far-red light reversibility of this process. For these assays we employed a similar experimental setup, i.e. 4-day-old- etiolated seedlings were treated with a short R pulse (30 s, 70 mmol m22 s21) directly followed by a 2 min RG9 pulse (40 mmol m22 s21) and transferred to darkness. We found that the RG9 treatment prevented the appearance of R-induced early, transient PHYB:YFP NBs (Figure 5D). In contrast, Figure 5C demonstrates that the formation of R-induced PHYB-401:YFP-associated NBs, early/transient as well as late/stable ones, was only partially affected by RG9. These nuclear complexes remained clearly detectable, although RG9 irradiation lowered the number of the early, transient and the newly appearing late PHYB-401:YFP NBs. As only the Pfr conformer of phyB is known to be able to associate with NBs, these experiments suggest that RG9-induced photo- conversion of PHYB-401:YFP Pfr holoprotein to Pr is significantly impaired as compared to PHYB:YFP. We conclude this despite the fact that in vivo spectroscopic measurements of the Pfr to Pr photoconversion -monitoring the photoconversion of the chromo- phore - does not show any significant alteration (Figure 3). Characterization of the photoreversibility of cR-induced PHYB:YFP and PHYB-401:YFP nuclear bodies Rausenberger et al. [23] demonstrated that light treatments reverting Pfr back to the Pr form led to a fast depletion of stable phyB NBs. Thus we concluded that monitoring the depletion kinetics of cR-induced PHYB-401:YFP NBs after an RG9 pulse provides an alternative method for testing photoreversibility of these nuclear complexes. To perform these studies, 3-day-old PLoS ONE | www.plosone.org 4 November 2011 | Volume 6 | Issue 11 | e27250