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. 2008 Aug 1;4(8):e1000143.
doi: 10.1371/journal.pgen.1000143.

FHY1 Mediates Nuclear Import of the Light-Activated Phytochrome A Photoreceptor

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Free PMC article

FHY1 Mediates Nuclear Import of the Light-Activated Phytochrome A Photoreceptor

Thierry Genoud et al. PLoS Genet. .
Free PMC article

Abstract

The phytochrome (phy) family of photoreceptors is of crucial importance throughout the life cycle of higher plants. Light-induced nuclear import is required for most phytochrome responses. Nuclear accumulation of phyA is dependent on two related proteins called FHY1 (Far-red elongated HYpocotyl 1) and FHL (FHY1 Like), with FHY1 playing the predominant function. The transcription of FHY1 and FHL are controlled by FHY3 (Far-red elongated HYpocotyl 3) and FAR1 (FAr-red impaired Response 1), a related pair of transcription factors, which thus indirectly control phyA nuclear accumulation. FHY1 and FHL preferentially interact with the light-activated form of phyA, but the mechanism by which they enable photoreceptor accumulation in the nucleus remains unsolved. Sequence comparison of numerous FHY1-related proteins indicates that only the NLS located at the N-terminus and the phyA-interaction domain located at the C-terminus are conserved. We demonstrate that these two parts of FHY1 are sufficient for FHY1 function. phyA nuclear accumulation is inhibited in the presence of high levels of FHY1 variants unable to enter the nucleus. Furthermore, nuclear accumulation of phyA becomes light- and FHY1-independent when an NLS sequence is fused to phyA, strongly suggesting that FHY1 mediates nuclear import of light-activated phyA. In accordance with this idea, FHY1 and FHY3 become functionally dispensable in seedlings expressing a constitutively nuclear version of phyA. Our data suggest that the mechanism uncovered in Arabidopsis is conserved in higher plants. Moreover, this mechanism allows us to propose a model explaining why phyA needs a specific nuclear import pathway.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
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 . 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 P35SYFP-FHY1 or P35SNLS-YFP-FHY1 167202 (artificial FHY1) in fhy1-1 background were grown for 5 days in the dark or in weak far-red light (0.9 µmol m−2 s−1). #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 P35SYFP-FHY1 or P35SNLS-YFP-FHY1 167202 (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 µm. (D) Artificial FHY1 colocalizes with phyA. fhy1-1 P35SNLS-YFP-FHY1 167202 was crossed into phyA-201 PPHYAPHYA-CFP. F1 seedlings were grown for 3 days in the dark, irradiated for 6 h with FR (15 µmol m−2 s−1) and used for microscopic analysis. The scale bar represents 10 µm.
Figure 2
Figure 2. Subcellular localization of a constitutively localized phyA (phyA-NLS-GFP).
(A)–(D) 3-day-old dark-grown phyA-211 seedlings complemented with either PPHYAPHYA-GFP or PPHYAPHYA-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 µm. (E) and (F). 4-day-old dark-grown phyA-211 seedlings complemented with PPHYAPHYA-NLS-GFP 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). After 1 min incubation in the dark another picture was taken (F). The scale bars represent 10 µm.
Figure 3
Figure 3. A constitutively localized phyA is functional but does not trigger constitutive photomorphogenesis.
(A) FR-HIR for inhibition of hypocotyl elongation. Col, phyA-211 and phyA-211 seedlings expressing phyA-NLS, phyA-NLS-GFP or phyA-GFP (two independent lines each) were grown in the dark (D) or in FR (0.3, 3 or 15 µmol m−2 s−1). 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. Col, phyA-211 and cop1-4 as well as the transgenic lines described in (A) were grown in the dark or in FR (5 µmol m−2 s−1). After 4 days the anthocyanin content was measured. The mean value (A530–A647/seedling) of three replicates and the SD are indicated. (C) VLFR for inhibition of hypocotyl elongation. Col, phyA-211 as well as the lines described in (A) were grown for one day in the dark and then exposed for 3 days to either continuous FR (20 µmol m−2 s−1) or 3 min FR pulses (20 µmol m−2 s−1) with different dark intervals (27, 57 and 127 min). At the end of the FR treatment the hypocotyl length was measured. Error bars indicate the SEM (n = 11).
Figure 4
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 PPHYAPHYA-GFP or PPHYAPHYA-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 µm. (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 µm. (A, B, E, F) phyA-211 fhy1-1 PPHYAPHYA-GFP (Col×Ler) (C, D, G, H) phyA-211 fhy1-1 PPHYAPHYA-NLS-GFP (Col×Ler).
Figure 5
Figure 5. A constitutively nuclear localized phyA can compensate for the absence of FHY1.
(A) FR-HIR for inhibition of hypocotyl elongation. phyA-211 PPHYAPHYA-GFP and PPHYAPHYA-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 µmol m−2 s−1). 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 µmol m−2 s−1). After 4 days the anthocyanin content was measured. The mean value (A530–A647/seedling) of three replicates and the SD are indicated.
Figure 6
Figure 6. A constitutively nuclear localized phyA can compensate for the absence of FHY3.
(A) Morphology of seedlings grown for 5 days in continuous FR (15 µmol m−2 s−1) 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.
Figure 7
Figure 7. Cytoplasmically localized FHY1 CT induces a dominant negative phenotype.
(A) Morphology of seedlings expressing FHY1 CT. Wild-type (Ler), fhy1-1, and phyA-201 seedlings as well as transgenic lines expressing different FHY1 167–202 ( = FHY1 CT) constructs in wild-type background were grown for 5 days in the dark or in FR (15 µmol m−2 s−1). #2590, #2619, #2643; Ler P35SYFP-FHY1 167202 (Ler YFP-FHY1 CT) #4520, #4527; Ler P35SNLS-YFP-FHY1 167202 (Ler NLS-YFP-FHY1 CT, i.e. artificial FHY1) #4578, #4597; Ler P35SNES-YFP-FHY1 167202 (Ler NES-YFP-FHY1 CT) (B) Cytoplasmically localized FHY1 CT inhibits phyA nuclear accumulation. Ler P35SNES-YFP-FHY1 167202 was crossed into phyA-201 PPHYAPHYA-CFP. F1 seedlings were grown for 3 days in the dark, irradiated for 6 h with FR (15 µmol m−2 s−1) and used for microscopic analysis. The scale bars represent 10 µm.

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