Comparison of the signalling efficiency of PHYB:GFP and PHYB-401:GFP Beside the decreased rate of dark reversion, the G to E change at position 564 may influence interaction of the mutant photoreceptor with components of the signal transduction cascade and thereby contribute to the dramatic hypersensitivity of the phyB-401 mutant and the PHYB-401:GFP seedlings as compared to WT (Figure 7). Since it has been shown that the total amount of phyB is the limiting factor for maximal signal strength [23], comparison of fluence rate responses of lines containing different levels of total phyB can provide additional information concerning the mechanism underlying the hypersensitive phenotype of the phyB-401 mutant. To achieve this, we measured fluence rate dependent inhibition of hypocotyl growth in lines containing different amounts of phyB (in phyA-201 and 35S:PHYB:GFP in phyB-9, respectively) and mutant phyB (in phyB-401/phyA-201 and 35S:PHYB-401:GFP in phyB-9 backgrounds, respectively). Over- expression levels are shown in Table S1. Figure 7 shows the relation between the amount of the photoreceptor and the maximal response/signal for the mutant phyB (in phyB-401/ phyA-201 and PHYB-401:GFP in phyB-9) and wild-type phyB (in phyA-201 and PHYB:GFP in phyB-9). This figure demonstrates that under saturating light conditions (10 mmol m22 s21 red light) the amount of phyB or phyB-401 Pfr neither in phyA-201 nor in phyA-201/phyB-401 background is sufficient to induce maximal physiological response in contrast to the transgenic lines expressing the PHYB:GFP and PHYB-401:GFP in phyB-9 background. Because at 10 mmol m22 s21 fluence rate the amounts of phyB and phyB-401 and PHYB:GFP and PHYB-401:GFP Pfr are pairwise identical we conclude that the phyB-401 Pfr molecule is not more efficient than the wild-type photoreceptor. However, the extreme hypersensitivity for red light induced physiological responses displayed by the phyA-201/phyB-401 and Figure 7. Signalling efficiency of phyB-401 does not differ from phyB in saturating cR. Mutant phyA-201 (black diamonds, dashed line), phyA-201/phyB-401 (grey triangles, dashed line) as well as transgenic PHYB:GFP (black squares) and PHYB-401:GFP (grey circles) seedlings in phyB-9 background were grown for 4 days under different intensities of cR. Hypocotyl lengths were measured and the relative hypocotyl lengths are shown. Experiments were repeated three times, error bars are shown. doi:10.1371/journal.pone.0027250.g007 Regulation of Dark and Photoconversion of phyB-401 PHYB-401:GFP seedlings makes it difficult to asses the signalling efficiency under non-saturating conditions. To determine this by using the hypocotyl growth inhibition as a read-out we have to know the exact amount of phyB and phyB-401 Pfr present. This parameter we can estimate only with large uncertainty because of the altered dark reversion and finding shown in Figure 6 which indicates that the cycled Pr form of phyB-401 may display biological activity. To overcome this difficulty we compared signalling efficiency of the wild type and the mutant phyB-401 photoreceptors by other experimental approaches described below. Kinetics of signalling initiated by PHYB:YFP and PHYB- 401:YFP As RG9 pulses were shown to revert hourly R pulses in WT but not in the phyB-401 mutant [33] we tested the reversibility of daily applied R pulses. Figure 8A shows that under these conditions seedlings expressing the wild-type PHYB:YFP display a weak hypocotyl growth inhibition response to daily applied R pulses, whereas the PHYB-401:YFP seedlings show an almost saturated growth inhibition. We note that the observed strong effect of daily red light pulses is in good agreement with the reduced dark reversion kinetics of PHYB-401 and clearly demonstrates the physiological consequence of the increased stability of the Pfr form. The same figure, however, also shows that (i) the effect of daily R pulses on hypocotyl growth of the PHYB-401:YFP seedlings can be reverted (to about 80%) by an additional RG9 pulse (ii) which alone was not inductive. We also determined the kinetics of the reversibility of PHYB-401:YFP-controlled inhibition of hypocotyl growth. To this end we grew PHYB-401:YFP seedlings as described above but included 4 and 8 h dark intervals between the inductive R and the reverting RG9 pulses. Figure 8B illustrates that the PHYB-401:YFP-induced response can be partially reversed by far- red pulses applied leading to 60% and 50% reduction of hypocotyl length, respectively. These data fit well the kinetics of the loss of reversibility of wild-type phyB published recently by Hennig et al. [34]. Taken together, we conclude that (i) the kinetics of signal transduction initiated by the mutant phyB-401 photoreceptor and wild-type phyB do not differ significantly, and that (ii) the loss of photoreversibility of the R pulse induced hypocotyl growth described by Kretsch et al. [33] is not caused by an accelerated signal transduction operating in the phyB-401 seedlings. Fluence rate dependence of light pulse induced accumulation of CAB2 mRNA To compare the sensitivity of PHYB and PHYB-401 photore- ceptors at the molecular level we tested the CAB2 mRNA accumulation after an inductive far red light pulse. 4-day-old etiolated seedlings were irradiated for 5 min with far red light of variable fluence rates to establish fluences between 104 and 13350 mmol m22. Figure 9 shows that these fluences were not inductive in phyA-201 but they caused significant elevation of CAB2 mRNA levels in phyA-201/phyB-401. This figure also shows that maximal induction of CAB2 mRNA was detected at 3418 mmol m22 fluence. These data demonstrate the hypersensitivity of the PHYB-401 because at least at the higher fluences the same Pfr levels should be established for PHYB and PHYB-401. The unexpected fluence dependence of the CAB2 mRNA induction by a far-red light pulse indicates that the photochemical cycling of the PHYB-401 photoreceptor produces an active signalling component. Discussion The intragenic phyB-401 point mutant reported by Kretsch et al. [33] displays extremely enhanced light sensitivity and loss of PLoS ONE | www.plosone.org 7 November 2011 | Volume 6 | Issue 11 | e27250