FHY1 Mediates phyA Nuclear Import Figure 2. Subcellular localization of a constitutively localized phyA (phyA-NLS-GFP). (A)–(D) 3-day-old dark-grown phyA-211 seedlings complemented with either PPHYA:PHYA-GFP or PPHYA:PHYA-NLS-GFP were analyzed by fluorescence microscopy. The seedlings were analyzed directly (dark) or after 10 min irradiation with white light. The scale bars represent 250 mm. (E) and (F). 4-day-old dark-grown phyA-211 seedlings complemented with PPHYA:PHYA-NLS-GFP were analyzed by fluorescence microscopy. The preparation of the seedlings and the adjustment of the focal plane were done in safe green light. Then the fluorescence light (FL) was switched on for 5 s and a picture was taken (E). After 1 min incubation in the dark another picture was taken (F). The scale bars represent 10 mm. doi:10.1371/journal.pgen.1000143.g002 It was recently shown that FHY3 and FAR1, two closely related transcription factors, directly regulate the expression of FHY1 and FHL [31]. Given that phyA-NLS-GFP could rescue the fhy1 phenotype, we hypothesized that this construct may also be capable of rescuing fhy3 mutants, in which the major defect appears to be reduced FHY1 and FHL levels. We restricted our analysis to fhy3 mutants because FHY3 plays a significantly more important role for this response than FAR1 [31,34]. We thus crossed fhy3 with phyA- NLS-GFP plants and analyzed homozygous wild type and mutant fhy3 siblings. Our phenotypic characterization of the response to far- red light showed that while phyA-NLS-GFP rescued the fhy3 mutant phenotype phyA-GFP could not (Figure 6). Our results are thus consistent with the notion that the major function of FHY1 and FHY3 is to respectively operate a directly and indirectly control of phyA nuclear accumulation. FHY1 Is Important for Nuclear Import of phyA The only functionally important and widely conserved parts of FHY1 are the NLS and the phyA-interaction domain (Figure 1) [26,32,33]. Moreover, nuclear accumulation of phyA-NLS-GFP occurred independently of light and FHY1 (Figures 2 and 4). Taken together these data support the notion that FHY1 mediates light- dependent nuclear import of phyA upon interaction in the cytoplasm. A prediction of this model is that over-expression of either native or artificial FHY1 lacking the NLS should sequester phyA in the cytoplasm and thus result in a dominant negative phenotype. To test this hypothesis we omitted the SV40 NLS in the artificial FHY1 or replaced it by an NES and transformed the constructs (i.e. (NES-) YFP-FHY1 CT) into wild-type plants. As the fusion proteins encoded by the constructs are below the size exclusion limit of the nuclear pore [35] they can enter the nucleus by diffusion but do not accumulate there due to the absence of an NLS. The NES containing version, which is predicted to be actively exported from the nucleus, localized mainly to the cytosol (Figure 7B). As predicted by the nuclear import model, seedlings expressing these constructs were strongly hyposensitive to FR (Figure 7A). This phenotype is consistent with the previous finding that FHY1 containing a disrupted NLS does not complement the fhy1 phenotype but rather results in an almost complete loss of FR sensitivity [32]. Western blot analysis confirmed that the phyA levels were normal in seedlings expressing (NES-) YFP-FHY1 CT thus excluding the possibility that reduced amounts of phyA were responsible for the dominant negative phenotype (Figure S2). However, NES-YFP-FHY1 CT strongly inhibited phyA nuclear PLoS Genetics | www.plosgenetics.org 4 August 2008 | Volume 4 | Issue 8 | e1000143