of phyA molecules after multiple transport cycles. In addition, the subcellular localization of phyA-NLS-GFP was not affected in the fhy1 mutant background (Figure 4), which is only compatible with the ‘‘nuclear import’’ model. The normal localization of phyA- NLS-GFP in fhy1 mutants is also supported functionally, given that this construct complements fhy1 (Figure 5). Moreover, western blot analyses show that FHY1 does not affect phyA protein levels in far-red light irrespective of whether phyA enters the nucleus using FHY1 [11]. Moreover the abundance of constitutively nuclear phyA-GFP was also unaffected in the fhy1 background (Figure S4). These data indicate that FHY1 does not act by protecting phyA from degradation once the photoreceptor entered the nucleus. Although phyA strongly accumulates in the nucleus in response to irradiation with FR in vivo spectroscopic measurements indicate that not significantly more than ,2% of the total phyA is in the Pfr form under such conditions [38]. This strongly suggests that in FRc the major fraction of nuclear phyA is in the Pr and not the Pfr form [12]. Furthermore, yeast two hybrid experiments show that the light-induced interaction of FHY1 and phyA is R/FR reversible, suggesting that the phyA/FHY1 complex rapidly dissociates upon conversion of Pfr to Pr (Figure S5). It is, however, inherent to the ‘‘FHY1 nuclear anchor’’ and ‘‘protection’’ models that FHY1 has to be bound to phyA to inhibit its export into the cytosol or protect it from degradation. Again, the only model compatible with our findings is the ‘‘import’’ model, where an interaction for a limited time period would be sufficient to allow accumulation of phyA in the nucleus. A constitutive interaction of phyA and FHY1 may even interfere with phyA nuclear accumulation as it may block recycling of FHY1. Once in the nucleus phyA would be trapped in the ‘‘import’’ model – irrespective of whether it is in the Pr or Pfr form – because it is too big to exit the nucleus by diffusion. Taken together our findings strongly support the import model (Figure S6). After accumulation in the nucleus phyA interacts with various transcription factors (e.g. PIFs) [20,21,24]. It is noteworthy that nuclear body formation is still light dependent for phyA-NLS-GFP (Figure 2). Moreover, formation of these subnuclear structures does not require FHY1 (Figure 4) although FHY1 and phyA have been found in light-induced nuclear bodies (Figure 1) [11,26,27]. The light-induced nuclear bodies may thus represent sites of phyA- PIF interaction as has previously been reported [39,40]. Complementation of the fhy1 mutant by phyA-NLS (-GFP) shows that the interaction of phyA and downstream signaling compo- nents does not require FHY1. Rather, binding of FHY1 may prevent the interaction of phyA and effectors. If dissociation of the phyA/FHY1 complex were a prerequisite to initiate downstream signaling this would be an additional argument against the ‘‘FHY1 nuclear anchor’’ and ‘‘protection’’ models. Answering these questions will provide a ‘‘molecular’’ link between phyA nuclear accumulation and initiation of the signaling cascade(s) leading to transcriptional regulation of 10–20% of the genes in the Arabidopsis genome [25,41,42]. Adding a strong NLS to phyA results in light- and FHY1- independent nuclear accumulation of the protein. Nevertheless, dark-grown seedlings expressing such constitutively nuclear localized phyA display a normal morphology in darkness and still show normal light responses (phyA-mediated VLFR and HIR) (Figure 3). The fluence-rate dependency and the need for sustained excitation are hallmarks of the HIR [1] and it is well established that nuclear accumulation per se is an HIR [23,43]. Yet, maximal hypocotyl growth inhibition and anthocyanin accumu- lation in seedlings expressing constitutively nuclear localized phyA are still fluence-rate dependent and require continuous irradiation (Figure 3). Thus, the ‘‘physiological HIR’’ does not derive FHY1 Mediates phyA Nuclear Import exclusively from the HIR characteristics of phyA nuclear accumulation, indicating that in wild-type plants more than only one step in phyA signaling is an HIR. The phenotype of plants expressing constitutively nuclear phyA is thus clearly distinct to the partial det/cop phenotype of a mutant expressing a constitutively Pfr-like phyA [44]. Thus, control of phyA nuclear accumulation does not seem to play an essential role to prevent initiation of downstream signaling in the absence of light, which is crucial for the highly sensitive VLFR. The different affinities of phyA in the Pr and Pfr forms for downstream signaling components such as PIF1 and PIF3 may be sufficient to inhibit the induction of a VLFR in the dark. Despite having a low total level of phyA (only around 25% of wild-type levels) inhibition of hypocotyl elongation and promotion of anthocyanin accumulation is very efficiently complemented in the phyA-NLS and phyA-NLS-GFP lines (Figure 3 and S1). These results suggest that nuclear phyA abundance (rather than total phyA levels) primarily controls these light responses. The strong phenotype of the fhy1 fhl and fhy3 far1 double mutants, which do not contain detectable levels of phyA in the nucleus, further supports this view [26,31,33]. Thus, nuclear accumulation of both phyA and phyB has been shown to be functionally important in Arabidopsis [26,27,29]. While these studies show that this is an important step of the signal transduction cascade for numerous phytochrome responses, they by no means exclude the possibility for cytoplasmic activities of the phytochromes. Cytoplasmic phytochrome responses are widely described in cryptogam species [45–47] and a recent paper indicates that cytoplasmic phyA may be required for the modulation of the phototropic response in Arabidopsis [11]. The vast majority of proteins enters the nucleus either passively or by active, importin-mediated transport [35]. However, there are nuclear localized proteins, which are too big to pass through the nuclear pore by diffusion but still do not contain an NLS. Similar to phyA many of these proteins use a piggyback mechanism and rely on the NLS of an interacting protein for nuclear transport [48–56]. Yet, in contrast to phyA, most of these proteins seem to interact with the NLS containing protein constitutively [48,49,51,52,56] or they are even part of a stable oligomeric complex with one of its components providing an NLS [53,55]. Often the NLS containing protein also performs an essential function besides nuclear transport [49–52,54]. Compared to the piggyback systems described above, the FHY1/phyA system is unique inasmuch as i) nuclear transport of the cargo protein is regulated by a conformational change of phyA [27] and ii) the NLS containing protein is dedicated exclusively to nuclear transport of the cargo protein given that FHY1 becomes dispensable in a strain where phyA possesses it own NLS (Figure 5). Materials and Methods Constructs, Transgenic Plants To obtain the PPHYA:PHYA-NLS-GFP5 construct (CF461), we inserted the following sequence AALQKKKRKVGGAAA be- tween phyA and GFP5 of CF161 [27] using standard molecular biology techniques (NLS is underlined). PPHYA:PHYA-NLS (CF460) is the same construct except that there is a stop codon directly after the last codon of the NLS sequence (i.e. does not contain GFP5). Transgenic plants expressing phyA-NLS (CF460) and phyA- NLS-GFP (CF461) under the control of the PHYA promoter were obtained by transforming the constructs (CF460, CF461) into phyA-211 mutants by Agrobacterium-mediated transformation [57]. Transgenic plants were selected on 0.56 Murashige & Skoog (MS) medium (Duchefa), 0.7% agar (Sigma) with 30 mg/ml kanamycin. PLoS Genetics | www.plosgenetics.org 9 August 2008 | Volume 4 | Issue 8 | e1000143