Regulation of Dark and Photoconversion of phyB-401 Figure 1. Fluence rate dependent hypocotyl growth inhibition of PHYB:YFP and PHYB-401:YFP seedlings. Wild-type Col-0 (filled diamonds), phyB-9 mutant (empty squares) and phyB-9 transgenic seedlings expressing the PHYB:YFP (empty triangles) and PHYB-401:YFP (empty circles) fusion proteins were grown for 4 days under different fluence rates of cR light. Hypocotyl lengths were measured and the relative hypocotyl lengths are shown. The experiments were repeated three times, error bars are also shown. doi:10.1371/journal.pone.0027250.g001 published data [21,23]. As expected, dark reversion of the PHYB- 401:GFP expressing line displayed a different, strongly reduced reversion kinetics (Figure 2). This figure illustrates that after 12 h of darkness about 50%, whereas after 24 h darkness about 40% of the original amount of PHYB-401:GFP Pfr is still detectable. These data unambiguously demonstrate that the PHYB-401:GFP fusion protein, like the PHYB-401 protein in vitro, shows a strongly reduced dark reversion in vivo. This in turn results in an increased stability of the PHYB-401 Pfr form, which is especially important in continuous weak red light or after light pulses. Under these conditions, where only a low percentage of PHYB Pfr/Pfr homodimers are generated [6], the enhanced stability of the Pfr form of the PHYB-401:YFP fusion protein can indeed result in an increased sensitivity, but it has little to do with the loss of reversibility response described by Kretsch et al. [33]. In vivo photoconversion of PHYB and PHYB-401 Pfr to Pr Kretsch et al. [33] demonstrated that photoconversion of phyB- 401 does not significantly differ from that of the wild type phyB in vitro. To evaluate whether this statement is valid in vivo we measured far-red light induced photoreversibility of PHYB:GFP and PHYB-401:GFP fusion proteins in planta. Photoconversion of PHYB-401:GFP and PHYB:GFP Pfr to Pr was determined as follows. 4-day-old etiolated seedlings were treated with red light for 3 h (40 mmol m22 s21) to convert all phyB Pr into Pfr form and simultaneously to degrade phyA. During the last 15 min of red light treatment seedlings were placed on ice and afterward all additional steps were carried out on ice to avoid dark reversion of the generated phyB Pfr. After completing the red light treatment seedlings were irradiated with 6 mmol m22 s21 far-red light (720 nm) for 30 s, 60 s or 135 s and the Pfr and total phy levels were measured in dual-wavelength ratio spectrophotometer (Ratiospect). Figure 3 demonstrates that photoconversion of PHYB:GFP and PHYB-401:GFP Pfr to Pr is identical in vivo. Figure 2. In vivo dark reversion rate of PHYB-401:GFP is slower compared to PHYB:GFP. Dark reversion kinetics of PHYB:GFP and PHYB-401:GFP fusion proteins, measured in planta, is shown. 4-day-old etiolated seedlings were irradiated 3 h with R (3 mmol m22 s21), returned to darkness and the amount of PHYB:GFP (black squares) and PHYB-401:GFP (grey circles) Pfr relative to Ptot was measured, as indicated, during a 24 h period. doi:10.1371/journal.pone.0027250.g002 These data suggest that aberrant/impaired photoconversion of the phyB-401 Pfr to Pr cannot be accounted for the loss of far-red reversibility of phyB-401 mutant regulated responses. Light quantity dependent nucleo-cytoplasmic partitioning of the photoreceptor:YFP fusion proteins in seedlings grown in cR We examined the subcellular localization pattern of the PHYB- 401:YFP and PHYB:YFP fusion proteins in transgenic seedlings grown for 4 days in darkness or at 0.03 mmol m22 s21 and 22 mmol m22 s21 intensities of cR. It is estimated that under Figure 3. In vivo photoconversion. 4-day-old dark-grown PHYB:YFP and PHYB-401:GFP expressing seedlings were irradiated for 3 h with 40 mmol m22 s21 red light to convert PHYB and PHYB-401 to the Pfr form. Subsequently, seedlings were treated with 6 mmol m22 s21 far- red light (720 nm) on ice for 30 sec, 60 sec or 135 sec. Pfr and total phy content (Ptot) was measured in dual-wavelength ratio spectrophotom- eter. The ratio of Pfr/Ptot is plotted against the far red pulse duration. Error bars represent standard error. doi:10.1371/journal.pone.0027250.g003 PLoS ONE | www.plosone.org 3 November 2011 | Volume 6 | Issue 11 | e27250