FHY1 Mediates phyA Nuclear Import Figure 4. The subcellular localization of a constitutively localized phyA is not dependent on FHY1. (A)–(D) 3-day-old dark-grown phyA- 211 fhy1-1 seedlings expressing 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)–(H) 4-day-old dark-grown phyA-211 fhy1-1 seedlings expressing either phyA-GFP (E, F) or phyA-NLS-GFP (G, H) 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 and G). After 1 min incubation in the dark another picture was taken (F and H). The scale bars represent 10 mm. (A, B, E, F) phyA-211 fhy1-1 PPHYA:PHYA-GFP (Col6Ler) (C, D, G, H) phyA-211 fhy1-1 PPHYA:PHYA-NLS-GFP (Col6Ler). doi:10.1371/journal.pgen.1000143.g004 accumulation when crossed into plants expressing Cyan Fluores- cent Protein (CFP) tagged phyA (Figure 7B). This suggests that in the cytosol NES-YFP-FHY1 CT competes with endogenous FHY1/FHL for binding to phyA (-CFP) and thereby interferes with phyA (-CFP) nuclear transport. Discussion It was previously shown that FHY1 and its paralogue FHL are required for nuclear accumulation of phyA [26,27]. The analysis of mutants clearly demonstrates that FHY1 plays the predominant function for both phyA nuclear accumulation and phyA-mediated light responses [27,33]. This is presumably due to the roughly 15- fold higher level of FHY1 mRNA compared to FHL [33]. We therefore restricted our analysis to the fhy1 single mutant background, i.e. in the presence of functional FHL. Both FHY1 and FHL interact with light-activated phyA through a conserved C-terminal domain [26]. However, the mechanism, by which these proteins enable nuclear localization of phyA, remains to be established. Our phylogenetic analysis shows that, similarly to phyA, FHY1-related proteins are widely distributed among angiosperms (Figure 1A), suggesting conservation of this aspect PLoS Genetics | www.plosgenetics.org 6 August 2008 | Volume 4 | Issue 8 | e1000143