nuclear accumulation of the phytochromes

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1 Current Biology, Vol. 15, , December 6, 2005, ª2005 Elsevier Ltd All rights reserved. DOI /j.cub Nuclear Accumulation of the Phytochrome A Photoreceptor Requires FHY1 Andreas Hiltbrunner, 1 András Viczián, 1 Erik Bury, 1 Anke Tscheuschler, 1 Stefan Kircher, 1 Réka Tóth, 2 Ariane Honsberger, 3 Ferenc Nagy, 2 Christian Fankhauser, 3,4, * and Eberhard Schäfer 1, * 1 Institut für Biologie II/Botanik Albert Ludwigs Universität Schänzlestrasse 1 D Freiburg Germany 2 Plant Biology Institute Biological Research Center Temesvari krt. 62 H-6726 Szeged Hungary 3 Department of Molecular Biology 30 quai E. Ansermet CH-1211 Genève 4 Switzerland 4 Centre for Integrative Genomics University of Lausanne BEP CH-1015 Lausanne Switzerland Summary The phytochrome family of red/far-red (R/FR)-responsive photoreceptors plays a key role throughout the life cycle of plants [1 3]. Arabidopsis has five phytochromes, phya-phye, among which phya and phyb play the most predominant functions [1 3]. Lightregulated nuclear accumulation of the phytochromes is an important regulatory step of this pathway, but to this date no factor specifically required for this event has been identified [4]. Among all phya signaling mutants, fhy1 and fhy3 (far-red elongated hypocotyl 1 and 3) have the most severe hyposensitive phenotype, indicating that they play particularly important roles [5 7]. FHY1 is a small plant-specific protein of unknown function localized both in the nucleus and the cytoplasm [5, 6]. Here we show that FHY1 is specifically required for the light-regulated nuclear accumulation of phya but not phyb. Moreover, phya accumulation is only slightly affected in fhy3, indicating that the diminished nuclear accumulation of phya observed in fhy1 seedlings is not simply a general consequence of reduced phya signaling. By in vitro pulldown and yeast two-hybrid analyses, we demonstrate that FHY1 physically interacts with phya, preferentially in its active Pfr form. Furthermore, FHY1 and phya colocalize in planta. We therefore identify the first component required for light-regulated phytochrome nuclear accumulation. *Correspondence: christian.fankhauser@unil.ch (C.F.); eberhard. schaefer@biologie.uni-freiburg.de (E.S.) Results and Discussion Being sessile, photoautotrophic organisms, plants need to constantly adapt their growth and development according to the changing light environment. Higher plants possess several classes of photoreceptors, among which the phytochromes play important functions during all developmental transitions [1, 2, 8]. Upon light perception, the phytochromes translocate from the cytoplasm (where they reside in the dark) into the nucleus [4]. Light activation of the photoreceptor triggers conformational changes that presumably reveal cryptic NLS (nuclear localization signal) sequences or allow the interaction of the phytochrome with another protein that will enable nuclear import [4, 9]. In the nucleus, the phytochromes are found in nuclear bodies whose function is poorly understood [10 13]. In the case of the light labile phya, it has been proposed that such nuclear speckles may represent sites of degradation [14]. Photoactivated phytochromes mediate large changes in the gene expression profile [15]. Light-regulated gene expression depends on several classes of transcription factors, including some that physically interact with the lightactivated phytochromes [16]. Regulated nuclear import of the phytochromes is therefore a key step of this signaling cascade. Mutant screens have identified residues of the phytochromes required for normal translocation into the nucleus [13, 17 19]. However, to this date none of the factors required for light-dependent nuclear accumulation of phytochromes have been identified. We analyzed the subcellular localization of phya-gfp in previously identified phya-signaling mutants in order to test whether any of them is affected in light-regulated nuclear accumulation of phya. As a control, we used a line expressing PHYA promoter-driven phya-gfp in a phya null background. This line contains phya-gfp levels that are very similar to endogenous levels and complements the phya phenotype (see Figure S1 in the Supplemental Data available with this article online). For our studies we crossed this line with fhy1 and fhy3 mutants because these two mutants have the strongest hyposensitive phenotype of all phya-signaling mutants [5 7, 20, 21]. FHY3 is a member of a small gene family that has been suggested to act as a transcriptional activator [20, 22]. FHY1 codes for a plant-specific protein of unknown function. FHY1-GFP fusion proteins are found both in the cytoplasm and the nucleus [5, 6]. This localization requires an NLS and an NES (nuclear export signal) sequence, and nuclear localization is essential for FHY1 function [23]. The subcellular localization of phya- GFP was analyzed in siblings of crosses that were homozygous for the transgene, homozygous for the phya mutation, and either wild-type or mutant for the phyasignaling mutant. Immunoblot analysis confirmed that these lines expressed comparable levels of phya-gfp (data not shown). In dark-grown seedlings, phya-gfp was homogeneously dispersed in the cytoplasm as previously described [11, 24]. We did not observe any significant difference in the localization of phya-gfp in etiolated

2 Current Biology 2126 Figure 2. Subcellular phya-gfp Distribution in Epidermal Cells of Hypocotyls under VLFR Conditions 4-day-old, dark-grown seedlings expressing phya-gfp were irradiated with 10 min of weak FR light (1.8 mmol m 22 s 21 ) and analyzed with a specific GFP filter set. DIC images are shown in the left column, overlay images of GFP (green) and chlorophyll fluorescence (red) are shown in the right column. The GFP channel is scaled to [100, 700]. n, nucleus; pl, selected plastids; nb, nuclear bodies. Scale bar equals 10 mm. (A) phya-211 FHY1, PHYA:AtPHYA-GFP5 (Col x Ler). (B) phya-211 fhy1-1, PHYA:AtPHYA-GFP5 (Col x Ler). Figure 1. Subcellular phya-gfp Distribution in Epidermal Cells of Hypocotyls under HIR Conditions 4-day-old, dark-grown seedlings expressing phya-gfp were irradiated with 6 to 8 hr of FR light (20 mmol m 22 s 21 ) and analyzed with a specific GFP filter set. DIC (differential interference contrast) images are shown in the left column, overlay images of GFP (green) and chlorophyll fluorescence (red) are shown in the right column. The GFP channel is scaled to [100, 900]. n, nucleus; pl, selected plastids; nb, nuclear bodies. Scale bar equals 10 mm. (A) phya-211 FHY1, PHYA:AtPHYA-GFP5 (Col x Ler). (B) phya-211 fhy1-1, PHYA:AtPHYA-GFP5 (Col x Ler). (C) phya-211 FHY3, PHYA:AtPHYA-GFP5 (Col). (D) phya-211 fhy3-1, PHYA:AtPHYA-GFP5 (Col). seedlings of the different genetic backgrounds (Figure S2). phya signaling works in two partially distinct modes known as the VLFR (very low fluence response) and the HIR (high irradiance response) [8, 25]. To assess the localization of phya-gfp during the HIR, dark-grown seedlings were transferred into far-red light for several hours. Nuclear accumulation of phya-gfp was significantly reduced in fhy1 mutants (Figure 1). In the fhy3 mutant, the nuclear accumulation of phya-gfp was only slightly reduced compared to its wild-type control, indicating that diminished nuclear accumulation of phya observed in fhy1 seedlings is not simply a general consequence of reduced phya signaling (Figure 1 and Figure S3). It has previously been shown that FHY1 mrna expression is reduced in fhy3 mutants [5, 22, 26]. Since FHY1 is required for light-induced accumulation of phya in the nucleus, it is plausible that fhy3 mutants might also be partially defective in nuclear accumulation of phya. It is worth pointing out that although FHY1 mrna levels are reduced in the fhy3 mutant, the effect on protein level is currently unknown. Acting in its VLFR mode, phya is a broad-range light sensor responding to minute amounts of light [8, 25]. To trigger a VLFR, etiolated seedlings were treated with a pulse of far-red light. Under these conditions, phya nuclear accumulation was reduced in the fhy1 mutant background but normal in fhy3 mutants (Figure 2 and data not shown). Similar defects were also found when 35S:PHYA-GFP was transformed into fhy1 and compared to Ler-expressing 35S promoter-driven phya- GFP (Figure S4). These findings are one example of the good correlation between phya action and its nuclear accumulation and correlate well with photobiological studies. The fhy1 mutant is defective for the phya-mediated VLFR and the FR-HIR [27, 28] and, correspondingly, accumulation of phya-gfp in the nucleus is strongly reduced in the fhy1 background under both conditions (Figures 1 and 2).

3 FHY1-Dependent Nuclear Accumulation of phya 2127 Figure 3. Subcellular phyb-gfp Distribution in Epidermal Cells of Hypocotyls of Wild-Type and fhy1 Seedlings 5-day-old, dark-grown wild-type (A and C) and fhy1 (B and D) seedlings expressing 35S promotor-driven phyb-gfp were either analyzed directly (A and B) or exposed to 5 hr of red light (4 mmol m 22 s 21 ) prior to microscopy (C and D). A specific GFP filter set was used for microscopic analysis. DIC images are shown in the left column, overlay images of GFP (green) and chlorophyll fluorescence (red) are shown in the right column. The GFP channel is scaled to [100, 400] (A and B) and [100, 500] (C and D). n, nucleus; pl, selected plastids, nb, nuclear bodies. Scale bar equals 10 mm. (A) Ws, 35S:AtPHYB-GFP4, etiolated. (B) fhy1-1, 35S:AtPHYB-GFP4, etiolated. (C) Ws, 35S:AtPHYB-GFP4, 5 hr R. (D) fhy1-1, 35S:AtPHYB-GFP4, 5 hr R. The fhy1 mutant phenotype indicates that FHY1 is required for phya but not phyb signaling [6, 7, 26]. In good agreement with these physiological data, we found that light-induced nuclear accumulation of phyb-gfp is not affected in fhy1 plants (Figure 3). These data suggest that FHY1 specifically controls the subcellular localization of phya. To test for a direct interaction of FHY1 and phya, we used reconstituted photoactive phya for GST in vitro pull-down analysis. Vascular plant phytochromes contain covalently bound phytochromobilin (PFB) as chromophore that is indispensable for photoactivity [29]. However, phycocyanobilin (PCB), the chromophore in algal and cyanobacterial phytochromes, can subsitute for PFB [29]. Attachment of the chromophore to the phytochrome apoprotein occurs in an autocatalytic reaction [30]. We therefore incubated in vitro synthesized 35 S-labeled PHYA with PCB to allow reconstitution of photoactive phya [30, 31]. Reconstituted phya was then exposed to red light either followed by a far-red light pulse or not to obtain the inactive Pr and the active Pfr forms, respectively. In vitro pull down with recombinant GST-FHY1-H 6 showed that phya interacts with FHY1 in a light-regulated fashion, with red light increasing the binding of phya to FHY1 2- to 4-fold (Figure 4A). To further corroborate these findings, we analyzed the interaction of phya and FHY1 in a yeast two-hybrid based growth assay. Yeast cells expressing FHY1 and phya fused to the GAL4 activation (AD) and binding (BD) domains, respectively, were grown on selective plates. To allow reconstitution of photoactive phya in living yeast cells, the plates were supplemented with PCB [30, 32]. Growth was strongly dependent on red light (Figure 4C), suggesting that FHY1 interacts preferentially with the Pfr form of phya. However, yeast two-hybrid liquid b-galactosidase activity assays indicate that binding of phya to FHY1 also occurs after exposure to far-red light although at about 5- to 10-fold lower level than after red light treatment (Figure S5). It seems to be contradictory that far-red light induces nuclear accumulation of phya in plants, whereas in the pull-down and yeast two-hybrid assays, phya and FHY1 interact preferentially in red light, indicating a Pfr dependency on the interaction. Exposure of phya to far-red light results in about 3% of active phya (PfrA) that accumulates in the nucleus [4, 33]. This relatively low amount of active phya is, however, optimal to induce under continuous irradiation a saturating phya response in planta [25, 34]. Irradiation with red light results in more than 20 times higher levels of active phya [25, 34], suggesting that the in vitro assays are simply not sensitive enough to detect the interaction of FHY1 with the comparably low amount of PfrA obtained after a FR treatment. Consistent with this idea, red lightinduced phya nuclear accumulation is also dependent of FHY1 (data not shown). To confirm the interaction between FHY1 and phya in vivo, we cobombarded a 35S:YFP-FHY1 construct and a construct encoding 35S promoter-driven phya-cfp into etiolated mustard seedlings [35]. After 8 hr in the dark, the bombarded mustard seedlings were analyzed by microscopy. In good agreement with their physical interaction, FHY1 and phya colocalized in the nucleus and in particular in nuclear bodies induced by a short light treatment (Figure 5). phya has previously been shown to localize to nuclear bodies [10, 11, 33], but a similar localization has not been reported for FHY1. In order to verify that this subcellular localization is not an artifact due to our experimental system, the same

4 Current Biology 2128 Figure 4. Light-Regulated Interaction of FHY1 and phya In Vitro and In Yeast (A) In vitro synthesized 35 S-labeled phya was incubated with PCB to allow the covalent conjugation of PCB to phya. phya was then exposed to red light for 5 min, either followed by a 5 min far-red pulse (IVT phya Pr) or not (IVT phya Pfr), and incubated with recombinant GST-FHY1-H 6 or GST-H 6 (nonbinding control) bound to GSH sepharose. After washing, the sepharose beads were incubated with SDS-PAGE sample buffer for elution. The samples were separated by SDS-PAGE and transferred onto a PVDF membrane. A phosphorimager was used for signal detection. Lane 1 (IVT) contains 5% of the input used in lanes 2 5. Both the autoradiogram (top) and the Amido Black-stained membrane (middle) are shown. To confirm equal amounts of input, 2% of the nonbinding fractions were run on an SDS-PAGE and blotted onto PVDF membrane. The lower panel shows the Amido Black-stained membrane. The predominant band between 55 and 75 kda is BSA, which was added to the input (see Supplemental Experimental Procedures). (B) As a nonbinding control, recombinant GST-FHY1-H 6 bound to GSH sepharose was incubated with in vitro synthesized 35 S-labeled luciferase (IVT luciferase). The sepharose beads were washed and incubated with SDS-PAGE sample buffer for elution. The samples were separated by SDS-PAGE and transferred onto a PVDF membrane. For signal detection, a phosphorimager was used. Lane 1 contains 5% of the input used in lane 2. Both the autoradiogram (top) and the Amido Black-stained membrane (bottom) are shown. (C) Yeast (strain AH109) was transformed with the indicated plasmids. 5 ml of overnight cultures were spotted onto selective synthetic dropout plates (L-W-H-, containing 1 mm 3-aminotriazole) supplemented with 20 mm PCB (+PCB). The plates were incubated for 3 days in 1 mmol m 22 s 21 red light (R), 20 mmol m 22 s 21 far-red light (FR), or in the dark (D). As a control, equal amounts of overnight cultures were spotted onto selective (s) or nonselective (L-W-, ns) plates without PCB (2PCB). AD, GAL4 activation domain; BD, GAL4 DNA binding domain; FHY1-AD, FHY1-AD fusion protein; phya-bd, phya-bd fusion protein. 35S:YFP-FHY1 construct was introduced into the fhy1-1 background. This line exhibits a wild-type phenotype demonstrating that this fusion protein is functional in vivo (data not shown). Consistent with previous reports, microscopic examination demonstrated a diffuse YFP fluorescence in the cytosol and the nucleus when seedlings were grown in the dark (Figure 5) [5, 6, 23]. However, a short light pulse induced rapid formation of nuclear bodies reminiscent of phya speckles and similar to the ones observed in the cobombarded mustard seedlings (Figure 5). This result shows that similar to other phya signaling components, FHY1 localizes to nuclear bodies [36 39]. The functional significance of this localization will be the topic of future investigations. The fhy1 mutant is strongly impaired in nuclear accumulation of phya (Figures 1 and 2), suggesting that at least part of the severely reduced deetiolation response in fhy1 is due to reduced phya levels in the nucleus. Many genes required for phytochrome-mediated light perception have been identified, but their precise role and order of action in the pathway is poorly defined [40]. Our work, however, assigns a precise function to FHY1 in phya signaling. Although we cannot rule out that FHY1 has other functions as well, we identify FHY1 as the first factor essential for nuclear accumulation of phytochrome that is specific to plants. Two models, which are not mutually exclusive, can explain FHYdependent nuclear accumulation of phya. In the first model, FHY1 would work as an import facilitator for phya. It has been shown that the N- and C-terminal halves of phyb physically interact with each other and that this interaction is weakened after exposure to light [9]. Based on this finding, a model for nuclear import of phyb has been proposed that suggests that phyb contains an NLS in the PAS (Per/Arnt/Sim) domain. This NLS would be masked by the N-terminal half in the dark but revealed upon activation by light [9]. Although no NLS has been identified in phya so far, it is tempting to extend this model to phya. Instead of having an NLS of its own, phya may contain a binding site for FHY1 and use the NLS of FHY1 for nuclear translocation. The FHY1 binding site may be masked in the dark, preventing FHY1-dependent nuclear translocation of phya. Upon exposure to light, the FHY1 binding site may be

5 FHY1-Dependent Nuclear Accumulation of phya 2129 Figure 5. FHY1 Colocalizes with phya in the Nucleus (A and B) fhy1-1 seedlings complemented with 35S:YFP-FHY1 were grown for 5 days in the dark and analyzed directly (A) or exposed to white light (W) for 2 min before microscopy (B). A specific YFP filter set was used for microscopic analysis. Scale bar equals 2 mm. (C E) Constructs encoding 35S:AtPHYA-CFP and 35S:YFP-FHY1 were cobombarded into etiolated mustard seedlings. After bombardment, the seedlings were incubated for 8 hr in the dark and exposed to white light for 2 min before microscopy. Specific CFP (C) and YFP (D) filter sets were used for microscopic analysis. (E) shows the overlay of (C) and (D). Scale bar equals 5 mm. revealed and allow FHY1-mediated interaction of phya with the nuclear import machinery. In mammals, a mechanistically similar system has been described. mper3, which is involved in control of circadian rhythmicity, requires mper1 as adaptor protein providing an NLS for nuclear import [41]. Another example of a protein employing such a piggy back mechanism is cyclin B1, which relies on NLS sequences in cyclin F for nuclear transport [42]. In the second model, FHY1 does not work as an import facilitator but would rather prevent phya that has been imported into the nucleus from export to the cytosol. Binding of FHY1 to phya could mask an NES sequence in phya and thereby allow its accumulation in the nucleus. Although no NES has been identified in phya so far, it should, however, be noted that masking of NES sequences seems to be a quite common mechanism to induce nuclear accumulation of proteins. Nuclear localization of the tumor suppressors p53 and INI1 requires masking of NES sequences either by oligomerization or by an internal inhibitory domain [43, 44], and in Drosophila nuclear accumulation of the NES-containing protein Extradenticle (Exd) depends on Homothorax (Hth) [45]. Although FHY1 interacts preferentially with the Pfr form of phya, it is questionable whether masking and unmasking of a putative FHY1 binding site alone can account for the tight regulation of nuclear accumulation of phya by light. We therefore do not rule out that other factors contribute to the specificity in this process. FHL (FHY1-like), the only close homolog of FHY1 in Arabidopsis [5, 6, 26], is one obvious candidate that may contribute to nuclear accumulation of phya in response to light. Recently, the fhl-1 mutant containing a T-DNA insertion in the FHL coding region has been characterized. Compared to fhy1, this mutant has only a very weak phenotype in far-red light [26]. A line expressing a FHL RNAi construct in the fhy1 background, however, is indistinguishable from phya null seedlings when grown in farred light, and overexpression of FHL in the fhy1 background rescues the mutant phenotype [26]. Thus, it was concluded that FHY1 and FHL are at least partially redundant [26]. It will be interesting to determine whether this redundancy can also be seen at the level of nuclear accumulation of phya and whether FHL accounts for residual levels of nuclear-localized phya in the fhy1 background. Supplemental Data Supplemental Data include five figures, one table, and Supplemental Experimental Procedures and can be found with this article online at Acknowledgements This work was supported by grants from the Deutsche Forschungsgesellschaft to E.S. and S.K. (SFB 592), the Hungarian Science Foundation (T ) and the Howard Hughes Medical Institute (HHMI International Scholarship) to F.N., the Swiss National Science Foundation to C.F. (PP00A ), the Alexander von Humboldt Foundation to A.V. (IV-UNG/ STP), and the Human Frontier Science Program (HFSP) to A. Hiltbrunner (LT00631/2003-C). Purified PCB was kindly provided by Tim Kunkel, Institut für Biologie II/Botanik, Albert Ludwigs Universität, Freiburg, Germany. The empty binding domain vector and phya in the binding domain vector used for yeast two-hybrid assays as well as the vector used for in vitro synthesis of phya were a generous gift of Peter H. Quail, Department of Plant and Microbial Biology, University of California, Berkeley, CA. We thank Akira Nagatani (University of Kyoto, Japan) and Karin Schumacher (University of Tübingen, Germany) for antibodies against phya and DET3, respectively. We are grateful to Joanne Chory (Salk Institute, La Jolla, CA) in whose lab C.F. made the CF161 construct. The authors declare that they have no competing financial interests. 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