(Figure 6, left panel) as compared to PHYB:YFP (Figure 5A, Figure 6, right panel). As PHYB NBs were shown to contain phyB Pfr [22], and phyB-401 shows normal spectral photoconversion (Figure 3], the data described above indicate that FR treatment produces a PHYB-401:YFP P form, designated Pr*, that resembles Pfr. In contrast to this the in vivo spectroscopic measurements show that photoconversion of the chromophore - as measured by in vivo spectroscopy - is normal. This Pr* form is biologically active (Figure 9 and Figure S1B), it persists for some hours (Figure 5B and Figure 6) and it slowly falls back to Pr by a mechanism not yet elucidated. The kinetics of this process cannot be precisely estimated, however, our data show that the life time of Pr* must be longer than one hour as hourly red light pulses are not reversible by far-red light. The reversibility of daily pulses and the persistence of the nuclear bodies suggest that the life time of Pr* should be less than 8 hours. The precise calculation of the life time is not possible as the signaling efficiency of Pr* compared to Pfr is unknown. Our data demonstrate that such a Pr* signaling competent can also be produced by far-red light pulses. The level of P * will be r elevated with increasing fluence, whereas the Pfr level will remain constant after the photoequilibrium is reached. Data obtained by the in vivo photoconversion measurements (Figure 3) allowed us to estimate that 90% saturation of photoequilibrium will be reached at 500 mmol m22 s21. Thus the fluence rate dependence of CAB2 mRNA induction by far-red light pulses supports the hypothesis of the formation of a signaling Pr*. The hypersensitivity of inhibition of hypocotyl growth of PHYB-401:YFP under cFR also cannot be explained by simply altered dark reversion of the mutant photoreceptor, because even in the absence of any dark reversion far-red light can only produce Pfr level up to 2–3%. (Figure S1B). The strong fluence rate dependency of this response suggests that this unexpected sensitivity of phyB-401 mutant to continuous far- red light is mediated by photochemical cycling of the PHYB-401 photoreceptor. Under such conditions the Pr* will be accumulated in a fluence rate dependent manner. This hypothesis and the reduced dark reversion rate of the phyB-401 protein satisfactorily explain the extreme light sensitivity in weak cR and the loss of reversibility of hypocotyl growth inhibition to hourly R/FR pulses [33]. In weak cR, dark reversion of Pfr is the major regulator of Pfr levels. Under this condition only a low percentage of Pfr/Pfr PHYB homodimers are generated [6], thus the low dark reversion rate leads to an enhanced stability of the PHYB-401:YFP Pfr, which then results in an increased sensitivity. We note that dark reversion of PHYB-401:YFP could also generate a Pfr-like Pr* conformer, which then would further enhance the probability of the formation of the presumably active Pfr/Pr* dimers. This will also contribute strongly to the extreme enhanced responsiveness of PHYB-401:YFP or GFP to very low red light fluence rates under continuous irradiation (Figure 1 and 7). As far as the loss of photoreversibility of hypocotyl growth inhibition of the phyB-401 mutant is concerned [33], formation of the relatively long lived, biologically active phyB-401 Pr* (Figure 5C, Figure 6) readily explains why hourly given RG9 pulses are ineffective in blocking hourly R-induced hypocotyl growth inhibition and why the phyB-401 does respond strongly to far-red light (Figure 9 and Figure S1B). However, we note that the proposed Pr* form is likely to be less stable than the Pfr form. Data shown in Figure 8B support this conclusion by showing that hypocotyl growth inhibition of PHYB-401:YFP seedlings displays a significant, about 70% reversibility when R/RG9 pulses are applied at 24 h intervals. The phenotype displayed by phyB-401 mutant is sharply different from that of the constitutively active PHYBY276H mutant Regulation of Dark and Photoconversion of phyB-401 described by Su and Lagarias [35] and Hu et al. [36]. These authors showed that the universally conserved GAF domain Tyr residue, with which the chromophore is intimately associated, performs a critical role in coupling light perception to signal transduction. In other words, this particular amino acid substitu- tion confers a conformation that mimics the photoactivated Pfr form of phyB in the absence of light in a chromophore-dependent fashion. Consequently, the PHYBY276H mutant displays de- etiolated phenotype in dark and fluence rate independent hypocotyl growth inhibition in light, whereas the PHYB- 401:YFP seedlings display normal etiolated phenotype (Figure S2). It is of interest to understand at the structural level why the G to E amino acid change at position 564 in the PHY domain affects photoconversion of the Pfr form to Pr so drastically. Crystal structure analysis of the Synechocystis phytochrome (Cph1) [37] showed that the PHY domain contains an additional unique feature, a tongue-like protrusion that is present in all phytochrome classes. According to Essen and colleagues [37] the tongue lies on the protein surface, it interacts with the GAF domain, seals the chromophore pocket and thereby ensures close packing of the chromophore and stabilization of the photoactivated Pfr state of phytochromes. The authors postulate that the very close packing of the chromophore may play a role in passing the chromophore’s conformational change onto the protein, leading to the light- driven conformational change of phytochrome proteins and the transduction of the light signal. A sequence comparison of Arabidopsis phytochrome B and Cph1 shows that the G to E mutation of phyB-401 lies exactly in the conserved WGG motif of the tongue. Thus one can speculate that replacing a small G residue with a bulky polar E residue could disturb exactly that part of the phytochrome structure that couples the photoconversion of chromophore and protein. Thus it is possible that the mutation changes the conformation of the tongue-like protrusion in such a way that the first light switch from the inactive Pr to the active Pfr leads to a change in the holoprotein structure that reduces the dark reversion as well as the light-dependent conformational change back to the inactive state. If the chromophore’s conformational change is uncoupled from the protein after the first switch, then the phyB-401 mutant can hardly be switched off again by light. After reverting light treatments the mutant photoreceptor stays in an active form with the chromophore in its Pr state, while the protein possesses a Pfr-like structure, thus it is still capable of light signalling. Only a slow reversion back to the real inactive Pr state (Figure 8B) can switch off the signalling again. We have attempted to obtain direct biochemical evidence for demonstrating existence of the hypothesized phyB-401 Pr* conformer. As our data indicated above the Pr* conformer is clearly capable of signalling. Accordingly, we argued that this putative conformer should also be capable of binding to PIF3. To this end we have determined and compared binding of PIF3 to WT phyB and mutant phyB-401 Pfr and Pr proteins by yeast two- hybrid assays after different light treatments as shown in Figure S4. The ultimate goal of these studies was to demonstrate in vitro that FR treatment leads to the production of phyB-401 Pr* thus reversion of PIF3 binding to the Pfr conformer of phyB-401, in contrast to that of phyB, shall not be complete. However, Figure S4 shows that capacity of Pfr forms of phyB and phyB-401 to bind PIF3 is approximately identical and that FR irradiation applied after the inductive R pulse completely abolishes binding of PIF3. Judicious interpretation of the data however suggests that these results should not be considered as a decisive proof for the non- existence of the putative phyB-401 Pr* conformer. There are two major caveats of this experimental approach. First we perform these assays under saturating conditions and the sensitivity of such PLoS ONE | www.plosone.org 9 November 2011 | Volume 6 | Issue 11 | e27250