Regulation of Dark and Photoconversion of phyB-401 Figure 8. Hypocotyl growth inhibition of PHYB-401:YFP seedlings induced by R pulses is conditionally photoreversible. (A) PHYB- 401:YFP and PHYB:YFP transgenic seedlings were grown for 4 days in darkness (black bars) or treated daily with a RG9 pulse (2 min, 40 mmol m22 s21, dark-red bars), a R light pulse (30 s, 70 mmol m22 s21, red bars), or immediately after the R pulse with a RG9 pulse (2 min, 40 mmol m22 s21, brown bars) and returned to darkness. (B) PHYB-401:YFP seedlings were grown for 4 days in darkness (black bar) or treated daily with a R light pulse (30 s, 70 mmol m22 s21, red bar) or after the R pulse were treated immediately with a RG9 pulse (2 min, 40 mmol m22 s21, brown bar) and returned to darkness. Alternatively, after the R pulse treatment seedlings were returned to darkness for 4 h (orange bar) or 8 h (yellow bar), then irradiated with a RG9 pulse, and again returned to darkness until the next pulse treatment. (C) Symbols illustrate the various light pulse treatments applied. Hypocotyl lengths of seedlings were measured and relative hypocotyl lengths are shown. Error bars illustrate the standard error of the mean. doi:10.1371/journal.pone.0027250.g008 Figure 9. FR induces CAB2 mRNA accumulation in phyB-401/ phyA-201seedlings. phyB-401/phyA-201 (black bars) and phyA-201 (grey bars) seedlings were grown for 4 days in darkness before onset of light treatments and were analysed by real time PCR for CAB2 and ACTIN mRNA accumulation after additional dark incubation of 6 h. 5 min FR light pulses of the indicated total fluences were applied before dark transfer. Relative transcript levels of CAB2/ACTIN are shown, the value of the phyB-401/phyA-201 dark sample was set to one. Error bars illustrate the standard error of the mean. doi:10.1371/journal.pone.0027250.g009 photoreversibility of hypocotyl growth inhibition to hourly R light pulses. This phenotype is unique among the numerous phyB intragenic mutants isolated to date yet it is still unclear how the G to E amino acid substitution at position 564 modifies phyB signaling at the molecular level. Here we report about a broad array of experiments performed with PHYB-401:YFP and GFP expressing transgenics that allowed us to confirm and extend results reported by Kretsch et al. [33]. We show that the 35S: PHYB-401:YFP and GFP transgenic lines mimic the phenotype of the original phyB-401 mutant, display extreme hypersensitivity to cR (Figure 1 and Figure S1A) and to cFR (Figure S1B). We also demonstrate that the PHYB-401:YFP fusion protein has a reduced dark reversion rate in vivo (Figure 2), whereas its in vivo photoconversion rates (Figure 3), its degradation in cR (Figure S3) and signalling efficiency under saturating light conditions (Figure 7) are identical to PHYB:YFP. Importantly, we found that hypocotyl growth inhibition induced by daily applied R pulses (in contrast to hourly R pulses) is photoreversible in PHYB-401:YFP seedlings (Figure 8A), which in turn allowed us to document, by increasing the interval between the inductive R and the inhibitory RG9 pulses, that signaling launched by the mutant photoreceptor is not faster than that initiated by wild-type phyB (Figure 8B). Monitoring light-induced changes of the nucleo/cytoplasmic distribution of PHYB and PHYB-401:YFP and/or GFP provided essential information for identifying a novel feature of the mutant photoreceptor and for suggesting a plausible explanation for the appearance of the unique phenotype. Our data clearly demon- strate that PHYB-401:YFP NBs are more stable in darkness (Figure 5B and Figure 6), RG9 light is ineffective to prevent appearance of R-induced PHYB-401:YFP NBs (Figure 5C) and/ or to induce rapid depletion of PHYB-401:YFP NBs formed in cR PLoS ONE | www.plosone.org 8 November 2011 | Volume 6 | Issue 11 | e27250