FHY1 Mediates phyA Nuclear Import Figure 5. A constitutively nuclear localized phyA can compen- sate for the absence of FHY1. (A) FR-HIR for inhibition of hypocotyl elongation. phyA-211 PPHYA:PHYA-GFP and PPHYA:PHYA-NLS-GFP were crossed into fhy1-1. In the F2 generation seedlings homozygous for the transgene and the phyA-211 mutation and either wild-type (FHY1) or homozygous fhy1-1 at the FHY1 locus were selected. Col, Ler, phyA-211 and fhy1-1 seedlings as well as phyA-211 seedlings expressing phyA- NLS-GFP or phyA-GFP in FHY1 and fhy1-1 background were grown in the dark (D) or in FR (0.3, 3 or 15 mmol m22 s21). After 5 days the hypocotyl length was measured. The mean value and the SD are indicated with n.15. (B) FR-HIR for anthocyanin accumulation. The same seedlings as described in (A) as well as the cop1-4 mutant were grown in the dark or in FR (5 mmol m22 s21). After 4 days the anthocyanin content was measured. The mean value (A530–A647/ seedling) of three replicates and the SD are indicated. doi:10.1371/journal.pgen.1000143.g005 of phyA signaling. Moreover, this analysis shows that among FHY1-like proteins only the amino-terminal NLS, which is essential for the interaction with importin alpha (Figure S3), and the carboxy-terminal phyA-interaction domain are conserved. It has previously been shown that both these domains of FHY1 are necessary for function [32]. Our analyses now show that they are also sufficient for FHY1 activity and that the ,150 aa in between do not perform an essential function. The simplest model (hereafter termed ‘‘import’’ model) accounting for those results is that upon light excitation phyA interacts with FHY1 in the cytoplasm and that this complex enters the nucleus using the general nuclear import machinery (Figure S6). According to this model adding a strong (and exposed) NLS to phyA should render phyA nuclear accumulation both light- and FHY1-independent. Our experiments show that these predictions are fulfilled in plants expressing phyA-NLS-GFP (Figures 2 and 4). In addition, when an FHY1 variant lacking the NLS sequence is over-expressed in wild-type plants this construct sequesters phyA in the cytoplasm and results in a dominant-negative de-etiolation phenotype (Figure 7). These observations are fully consistent with the notion that FHY1 mediates light-regulated phyA nuclear import by binding selectively to the active Pfr form of phyA in the cytosol and, thereby, linking phyA in a regulated manner to the nuclear import machinery (Figure S6). Our findings indicate that Figure 6. A constitutively nuclear localized phyA can compen- sate for the absence of FHY3. (A) Morphology of seedlings grown for 5 days in continuous FR (15 mmol m22 s21) light. (B) FR-HIR for inhibition of hypocotyl elongation. Col, phyA-211 and fhy3-1 seedlings as well as phyA-211 seedlings expressing phyA-NLS-GFP or phyA-GFP in FHY3 and fhy3-1 background were grown as in (A). The mean value and the SD are indicated with n.15. doi:10.1371/journal.pgen.1000143.g006 during de-etiolation in far-red light the system essential for nuclear localization of phyA, i.e. FHY3 and FHY1, can be replaced by simply attaching an NLS to phyA. It is however highly unlikely that such plants do not show a decrease in fitness under more natural conditions. The complex system relying on FHY3/FAR1 and FHY1/FHL is highly conserved in evolution (Figure 1A) [31] and FHY1-like proteins from dandelion and rice can compensate for the absence of FHY1 in Arabidopsis (data not shown). The strict conservation of FHY1-like proteins in angiosperms (in the sense of proteins containing a phyA binding-site linked to an NLS) points to a common molecular mechanism of phyA nuclear import and underlines the importance for regulated subcellular localiza- tion of phyA. An obvious advantage of the FHY1/phyA system over targeting phyA to the nucleus using an NLS is that it allows for co-existence of nuclear and cytosolic phyA pools and that the pool sizes can be regulated. This may be especially important with regard to possible cytosolic functions of phytochromes as recently described [11]. Nuclear import of phyB does not rely on the FHY1/FHY3 pathway but is light regulated nevertheless [23,26,27,36,37]. The FHY1-mediated nuclear import described here may explain how phyA can be imported so rapidly in PLoS Genetics | www.plosgenetics.org 7 August 2008 | Volume 4 | Issue 8 | e1000143