Regulation of Dark and Photoconversion of phyB-401 Figure 5. R pulse induced formation of PHYB-401:YFP nuclear bodies is not fully reversible by FR. (A) Localization of PHYB:YFP or (B) PHYB-401:YFP fusion proteins in 4-day-old seedlings grown in darkness, treated with a high intensity red light pulse (30 s, 70 mmol m22 s21) and returned to darkness. (C) Localization of PHYB-401:YFP or (D) PHYB:YFP fusion proteins in 4-day-old etiolated seedlings, irradiated with the same high- intensity red light pulse followed by a RG9 pulse (2 min, 40 mmol m22 s21) and then transferred to darkness. Localization of the fusion proteins was monitored at times after the light treatments indicated in the upper right corner of the pictures. Scale bar is shown. doi:10.1371/journal.pone.0027250.g005 etiolated seedlings were irradiated with cR light (3 mmol m22 s21) for 24 h and then either transferred to darkness, or irradiated with an additional RG9-pulse (2 min, 40 mmol m22 s21) prior to transfer to darkness. Localization of the PHYB-401:YFP fusion protein was analyzed immediately after the light treatments and during the following dark period, up to 6 days. Figure 6 (left panel) shows that without the RG9 pulse the PHYB-401:YFP NBs are stable in darkness, since their number and size do not change significantly up to 72 h. This figure also shows that treatment with RG9 pulse did not deplete the PHYB-401:YFP NBs up to 48 h after the pulse but led to the appearance of smaller, more numerous NBs. These observations are in sharp contrast to data reported by Rausenberger et al. [23], who showed that cR- induced PHYB:YFP NBs were no longer detectable after 9 h in darkness and RG9 treatment significantly accelerated depletion of these nuclear structures that became undetectable within 1 h after the RG9 treatment. Finally, we note that the RG9-pulse applied during these assays did not induce NB formation or any physiological response in seedlings expressing the PHYB- 401:YFP fusion protein. Taken together, these and the data shown in Figure 5C indicate that the RG9-generated Pr form of PHYB-401:YFP is still capable of associating with NBs, this Pr form is relatively stable and its properties resemble that of the biologically active Pfr form of PHYB:YFP. PLoS ONE | www.plosone.org 5 November 2011 | Volume 6 | Issue 11 | e27250