stable and control Low Fluence Responses (LFR) and red light High Irradiance Responses. Using mutant analysis, it was shown that type I phytochromes are encoded by the PHYA gene and type II phytochromes by the PHYB, PHYC, PHYD and PHYE genes [6,8,13]. Physiological experiments showed that all these phytochromes sense not only the quality but also the quantity of light. Light quality sensing is explained by the formation of different amounts of the active Pfr-form and for phyA, a light quantity sensing mechanism can also be envisaged. In this model the fast proteolytic degradation of phyA [14] and the EID1 dependent degradation of additional signal transduction components are likely to play major roles. EID1 is an F-box protein and mutations of the EID1 gene lead to light hypersensitivity and a shift of the action spectrum from the far-red peak to a red peak [15,16,17]. In the case of phyB the capacity to monitor light quantity is less obvious [18], as phyB-mediated responses cannot be provoked by a single R light pulse that establishes saturating Pfr levels, whereas continuous red light elicits strong responses [19,20]. Recent observations, however, suggest that dark-reversion [21], proteo- lytic degradation of the active phyB Pfr form [22] and formation of phyB-containing nuclear bodies [23] are important factors in mediating light quantity measurement by this photoreceptor. Accordingly, expression of phytochrome cDNA in yeast cells and reconstitution of the holo-photoreceptor by addition of the chromophore have shown that all tested phytochromes exhibit partial dark reversion of Pfr to Pr after phototransformation of Pr to Pfr [24]. Recently, a slower but almost complete dark reversion of phyB has been measured in vivo, in phyB over-expressing lines in a phyA background [21], whereas Oka et al. [25,26] reported that the N-terminal PHY domain of phyB is involved in regulating dark reversion of the photoreceptor in transgenic plants. Interestingly, it has been demonstrated [21,27] that dark reversion of phyB is at least partially regulated by its interaction with the response regulator ARR4. Thus it follows that regulation of dark reversion of phyB is likely to be a target for hormone-induced signalling pathways. In etiolated seedlings, phyB is predominantly localized in the cytosol [28]. After transfer to light, nuclear import of phyB and formation of intra-nuclear speckles, also termed as nuclear bodies (NBs) have been described [28]. Surprisingly, these reactions show a strong light quality and quantity dependence, specific for the individual photoreceptors [20,23,28,29,]. Although the precise biological function of the different types of phyB-associated NBs [29–32] is not fully understood, it is evident that the majority of phyB signalling mutants display aberrant NB formation [20,29,31]. Moreover, recent data described by Rausenberger et al. [23] suggest that, in contrast to phyB Pfr localized in the nucleoplasm, the NB-associated phyB Pfr conformers are prevent- ed from fast dark-reversion. The phyB-401 mutant isolated by Kretsch et al. [33] shows extreme hypersensitivity to red light and displays strongly reduced reversibility by far-red light. The same authors reported that the phyB-401 photoreceptor exhibits a nearly complete lack of dark reversion but normal photoreversibility in yeast cells. The reduced dark reversion could, in principle, explain the extreme hypersen- sitivity to R light, but it is not sufficient to account for the poor far- red light reversibility of the inhibition of hypocotyl growth. To interpret the complex phenotype of the phyB-401 mutant at molecular level we raised transgenic lines expressing the wild–type PHYB:GFP/YFP and mutant PHYB-401:GFP/YFP fusion pro- teins in phyB-9 background. Next we measured (a) the rate of dark reversion and light induced photoconversion of the mutant phyB- 401 and wild type phyB fusion proteins in planta and (b) analysed Regulation of Dark and Photoconversion of phyB-401 far-red reversibility of cellular and molecular events related to phyB action. Our data suggest that the complex phenotype of phyB-401 can be best explained by assuming that the G to E amino acid change at position 564 inhibits dark reversion and at least partially uncouples photoconversion of the chromophore from the Pfr-Pr conformational change of the holoprotein. Results We have generated several independent transgenic lines expressing the 35S:PHYB-401:YFP and 35S:PHYB-401:GFP fusion proteins in phyB-9 background. The primary transgenic plants were selfed, homozygous segregants were selected and lines displaying stable phenotype were multiplied for additional experiments. Next, the expression level of the PHYB-401:YFP or GFP fusion proteins was determined and compared to PHYB:GFP and PHYB:YFP in phyB-9 and endogenous PHYB in WT backgrounds, respectively. The transgenic lines expressing the 35S:PHYB:GFP or YFP fusion protein used in this study had been isolated previously, they display stable phenotypes and fully complement the phyB-9 mutant [20,28]. We performed a series of western-blot hybridization experiments and selected one line for further experiments from each of the 35S:PHYB-401:YFP, 35S:PHYB-401:GFP, 35S:PHYB:GFP and 35S:PHYB:YFP lines. The selected transgenic lines over-express the fusion proteins 3-8 times as compared to endogenous phyB (for details see Table S1). Experiments described below were performed using these 4 selected lines. Data obtained using the YFP and GFP fusion proteins were comparable and did not differ significantly. Fluence rate response analysis of hypocotyl growth inhibition Figure 1 shows the fluence rate response curves obtained for WT (Col), phyB-9 and transgenic PHYB-401:YFP and PHYB:YFP seedlings in cR. This figure illustrates that the transgenic line expressing the PHYB-401:YFP fusion protein displays extreme hypersensitivity to cR, since these seedlings reach saturation of the response already at approximately 0.001 mmol m22 s21. In contrast, the transgenic PHYB:YFP and the WT (Col) seedlings start responding at ,0.01 mmol m22 s21 and show saturation around 1 and 10 mmol m22 s21, respectively. Transgenic lines expressing the PHYB-401:GFP fusion protein displayed similar hypersensitivity to R (Figure 1 and Figure S1A). We note that all seedlings expressing PHYB-401 and PHYB fusion proteins displayed normal etiolated phenotype as compared to WT seedlings (Figure S2). Taken together, these fluence rate measurements are in good agreement with the data reported by Kretsch et al. [33] and indicate that the PHYB-401:YFP and GFP fusion proteins affect R induced inhibition of hypocotyl elongation as expected. In vivo dark reversion kinetics of PHYB-401:YFP after saturating red light treatment Data reported by Kretsch et al. [33] demonstrated that the phyB-401 photoreceptor exhibits a reduced dark reversion in vitro. To confirm and extend this observation, we measured the dark reversion kinetics of the PHYB-401:GFP fusion protein in planta. To this end, 4-day-old seedlings were irradiated for 3 h with R (3 mmol m22 s21) and the amount of Pfr was measured directly with a dual-wavelength ratio spectrophotometer (RatioSpec) during the following dark period. For detailed information about the RatioSpec measurement see Materials and Methods. Figure 2 shows that in vivo dark reversion of PHYB:GFP has a half life time of around 1 h, which is in good agreement with previously PLoS ONE | www.plosone.org 2 November 2011 | Volume 6 | Issue 11 | e27250