FHY1 Mediates phyA Nuclear Import Figure 1. An artificial FHY1 complements the fhy1 mutant phenotype. (A) Sequence alignment for FHY1-like proteins. The C-terminal 36 aa of Arabidopsis FHY1 were used as a query to search genomic and EST databases. Part of the sequences were assembled from overlapping EST clones. The alignment was done using MAFFT v6.240 (http://align.bmr.kyushu-u.ac.jp/mafft/software/) and Jalview [65]. The sequence Picea-Pinus was derived from EST clones of Picea glauca and Pinus taeda. At the bottom of the alignment the consensus sequence is shown. The accession numbers of the clones used for the alignment are listed in Table SI. (B) An artificial FHY1 complements the fhy1 mutant. Wild-type (Ler), fhy1-1, and phyA-201 seedlings as well as lines expressing either P35S:YFP-FHY1 or P35S:NLS-YFP-FHY1 167–202 (artificial FHY1) in fhy1-1 background were grown for 5 days in the dark or in weak far-red light (0.9 mmol m22 s21). #4313 and #4327 are independent T2 lines segregating into non-transgenic (fhy1-1) and transgenic (fhy1-1 artificial FHY1) individuals. (C) Artificial FHY1 behaves like native Arabidopsis FHY1. 3-day-old dark-grown fhy1-1 seedlings complemented with either P35S:YFP-FHY1 or P35S:NLS-YFP-FHY1 167–202 (artificial FHY1) were used for fluorescence microscopy. The seedlings were analyzed directly (D) or irradiated for 7 h with far-red light, either followed by a 1 min red light pulse (FR+R) or not (FR) prior to microscopic analysis. The scale bar represents 10 mm. (D) Artificial FHY1 colocalizes with phyA. fhy1-1 P35S:NLS-YFP-FHY1 167–202 was crossed into phyA-201 PPHYA:PHYA- CFP. F1 seedlings were grown for 3 days in the dark, irradiated for 6 h with FR (15 mmol m22 s21) and used for microscopic analysis. The scale bar represents 10 mm. doi:10.1371/journal.pgen.1000143.g001 1 minute after a 5 sec red light pulse nuclear bodies appeared in those nuclei (Figure 2F). The phenotypic consequences of expressing a constitutively nuclear version of phyA was evaluated by comparing wild type, phyA and phyA transformed either with a construct encoding PHYA- GFP, PHYA-NLS-GFP or PHYA-NLS. Western blot analysis of dozens of independent transgenics showed that while we obtained lines expressing wild-type levels of phyA-GFP at a reasonable frequency (10–20%) we never found lines expressing high levels of either phyA-NLS or phyA-NLS-GFP (data not shown). For our phenotypic analysis we used two homozygous single insertion lines for each construct. phyA-GFP line 1 expressed wild-type levels of phyA while phyA-GFP line 2 expressed phyA levels comparable to the highest expressing phyA-NLS-GFP lines we obtained (Figure S1). Despite the relatively low levels of phyA, the phyA-NLS-GFP lines rescued the FR-HIR phenotype of phyA mutants very efficiently for hypocotyl elongation and anthocyanin accumulation (Figure 3A, B). Moreover, the phyA-NLS and phyA-NLS-GFP lines also showed a normal phyA-mediated VLFR response for inhibition of hypocotyl elongation in response to pulses of FR light (Figure 3C). It should also be noted that, despite having constitutively nuclear phyA, phyA-NLS (-GFP) lines did not show a cop (constitutively photomorphogenic) phenotype, indicating that nuclear import of phyA is not sufficient to trigger a light response (Figure 3 and data not shown). FHY1 Is Dispensable in Plants Expressing a Constitutively Nuclear phyA The nuclear localization of phyA-NLS-GFP in darkness (Figure 2C), a condition, where there is much reduced phyA- FHY1 interaction, suggested that phyA-NLS-GFP nuclear accu- mulation did not require FHY1. In order to test this hypothesis genetically we crossed phyA phyA-NLS-GFP with fhy1 mutants and selected siblings in the F2 that were homozygous for phyA, fhy1 and the transgene. Microscopic analysis of such seedlings demonstrated that neither nuclear accumulation nor light induced formation of nuclear bodies of phyA-NLS-GFP required FHY1 (Figure 4C, D, G, H). In control experiments we confirmed that for phyA-GFP plants light-dependent nuclear import was strongly dependent on FHY1 (Figure 4A, B, E, F and data not shown) [26,27]. We concentrated our analysis on fhy1 mutants because fhy1 has a much stronger phenotype than fhl [33]. Given that nuclear accumulation of phyA-NLS-GFP did no longer require FHY1, we tested whether fhy1 mutants expressing phyA-NLS-GFP had a normal light response to continuous FR light. Interestingly, both the hypocotyl elongation and anthocyanin accumulation phenotypes of fhy1 mutants were efficiently rescued by phyA-NLS-GFP but not by phyA-GFP (Figure 5). Our data thus indicate that FHY1 becomes dispensable in seedlings expressing phyA-NLS-GFP, suggesting that during the FR-HIR FHY1 is only necessary to control nuclear accumulation of phyA. PLoS Genetics | www.plosgenetics.org 3 August 2008 | Volume 4 | Issue 8 | e1000143