Author Summary In response to changes in the environment, animals can take shelter while the sessile plants must adapt to the prevalent conditions. Great plasticity in growth and development are striking examples of how plants cope with a changing environment. In plants, light is both a source of energy and an essential informational cue perceived by several classes of photoreceptors. Phyto- chrome-mediated light signaling is particularly well studied, because these photoreceptors control all aspects of the plant life cycle. The phytochromes are cytoplasmic in the dark and must enter the nucleus upon light activation to initiate signal transduction. How this impor- tant light-regulated event is achieved is poorly under- stood. Here we describe the function of an evolutionary conserved protein called FHY1 for Far-red elongated HYpocotyl 1. We demonstrate that FHY1 interacts with a light-activated phytochrome in the cytoplasm, allowing the complex to be transported into the nucleus. Interest- ingly, if this phytochrome can enter the nucleus by another mechanism, FHY1 is no longer required for seedling development, indicating that a major function of FHY1 is to chaperone an activated phytochrome into the nucleus. Our experiments suggest that this mechanism uncovered in Arabidopsis is widely conserved among flowering plants. The subcellular localization of phytochromes is tightly regulated by light. They localize to the cytosol in the dark but translocate into the nucleus upon light activation, where they interact with several transcription factors (e.g. PIFs, phytochrome interacting factors) [20–24]. Given that light-activated phytochromes localize to the nucleus and interact with transcription factors, it is not surprising that 10–20% of the genes in Arabidopsis are subject to regulation by red and/or far-red light [25]. Consequently, nuclear accumulation of the photoreceptor is a key step in both phyA and phyB signaling [26–29]. The C-terminal half of phyB presumably contains an Nuclear Localization Signal (NLS), which is masked in the dark by the N-terminal half of the photoreceptor. Light triggers a conformational change, potentially unmasking the NLS and allowing nuclear transport of phyB [30]. This model predicts that the general nuclear import machinery is sufficient for phyB nuclear transport. In contrast, it has recently been shown that nuclear accumulation of phyA depends on two plant specific proteins called FHY1 and FHL [11,26,27]. Importantly, these proteins are not required for nuclear accumulation of phyB and for phyB signaling [26,27]. FHY3 and FAR1, two transposase-related transcription factors, directly control FHY1 and FHL transcription and thus indirectly affect phyA nuclear accumulation [31]. FHY1 and FHL are small proteins (202 and 181 aa, respectively) containing an NLS and a Nuclear Export Sequence (NES) [32,33]. High similarity between FHY1 and FHL is confined to the 36 most C-terminal amino acids. This small domain is necessary and sufficient for the light-regulated interaction with phyA in vitro and it is essential for function in vivo [26,32]. Our previous work has shown that FHY1 and FHL are essential for phyA nuclear accumulation but the molecular mechanism involved remains elusive [26,27]. Three models can explain the requirement of FHY1/FHL for light-regulated nuclear accumulation of phyA. i) FHY1/FHL may be essential for nuclear import of phyA and work as adapter proteins using their NLS and phyA binding-site to link phyA to the general nuclear import machinery. Alternatively, phyA would enter the nucleus indepen- dently of FHY1/FHL but ii) FHY1/FHL action may be required FHY1 Mediates phyA Nuclear Import to stabilize phyA and protect it from degradation or iii) to trap it in the nucleus and prevent it from being exported back into the cytosol. In this report we provide strong evidence for a model, in which FHY1 and FHL work as adaptor proteins facilitating nuclear transport of phyA. Our data reveal an intriguing system for regulated nuclear transport of a cargo protein that does not contain an NLS of its own. Results The NLS and the phyA-Interaction Domain Are the Only Functionally Important Parts of FHY1 The high degree of sequence conservation among phyA in angiosperms suggests that the same might be true for phyA signaling components, such as FHY1 and FHL [19]. Yet, the amino acid identity between them is below 30% although they are functional homologs [33]. The only motifs conserved in FHY1 and FHL are the NLS (and to a minor degree the NES) in their N- terminal region and the phyA binding-site at the C-terminus. A database search for FHY1/FHL homologs revealed the presence of FHY1-like proteins in numerous plant species. This is interesting given the key function of FHY1/FHL in phyA signaling in Arabidopsis. The only motifs conserved between all the FHY1-like proteins found in the database and Arabidopsis FHY1/FHL are the NLS and the C-terminal phyA binding-site (Figure 1A). In contrast, the ,150 aa linking the NLS and the motif essential for interaction with phyA are too diverse to be aligned. Together with the finding that the FHY1/FHL homologs from both rice and dandelion complement the fhy1 mutant phenotype (data not shown) this suggests that FHY1-like proteins may be defined as proteins containing an NLS and an ‘‘FHY1 type’’ phyA binding-site separated by a ,150 aa spacer. To test whether this definition holds true we generated an artificial FHY1 consisting of an SV40 NLS and the C-terminal 36 aa of Arabidopsis FHY1 (FHY1 167–202 = FHY1 CT) with Yellow Fluorescent Protein (YFP) as a spacer in between. fhy1 mutant seedlings expressing this artificial FHY1 under the control of the CaMV 35S promoter were hypersensitive to FR, similar to fhy1 seedlings complemented with P35S:YFP-FHY1 (Figure 1B). Fur- thermore, the artificial FHY1 accumulated in the nucleus and colocalized with phyA in light-induced nuclear speckles (Figure 1C, D) thus behaving like Arabidopsis FHY1/FHL [26,27]. We therefore conclude that the NLS and the phyA binding-site of FHY1/FHL are necessary and sufficient for phyA nuclear accumulation. A Constitutively Nuclear-Localized phyA Efficiently Rescues a phyA Mutant Given that both the NLS and the phyA-interaction domain of FHY1 are sufficient for FHY1 activity we tested whether adding the NLS to phyA directly would be enough to promote nuclear localization of phyA fused to the Green Fluorescent Protein (GFP). phyA mutants transformed with either PHYA-GFP (Figure 2A, B) or PHYA-NLS-GFP (Figure 2C–2F) driven by the PHYA promoter were analyzed microscopically. As previously described [23] nuclear accumulation of phyA-GFP was light-dependent (Figure 2A, B). In contrast, in lines expressing phyA-NLS-GFP nuclear localization was constitutive (Figure 2C, D). Nuclear bodies appeared extremely rapidly upon light excitation in phyA- NLS-GFP plants. When nuclei of etiolated phyA-NLS-GFP seedlings were imaged without a light treatment or immediately after a 5 sec red light pulse a smooth nucleoplasmic staining was observed (Figure 2E, data not shown). However as little as PLoS Genetics | www.plosgenetics.org 2 August 2008 | Volume 4 | Issue 8 | e1000143