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FHY1 mediates nuclear import of the light-activated phytochrome A photoreceptor.

Genoud T, Schweizer F, Tscheuschler A, Debrieux D, Casal JJ, Schäfer E, Hiltbrunner A, Fankhauser C - PLoS Genet. (2008)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Centre for Integrative Genomics, University of Lausanne, Lausanne, Switzerland.

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.

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Subcellular localization of a constitutively localized phyA (phyA-NLS-GFP).(A)–(D) 3-day-old dark-grown phyA-211 seedlings complemented with 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 µm. (E) and (F). 4-day-old dark-grown phyA-211 seedlings complemented with PPHYA∶PHYA-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.
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pgen-1000143-g002: Subcellular localization of a constitutively localized phyA (phyA-NLS-GFP).(A)–(D) 3-day-old dark-grown phyA-211 seedlings complemented with 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 µm. (E) and (F). 4-day-old dark-grown phyA-211 seedlings complemented with PPHYA∶PHYA-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.

Mentions: Given that both the NLS and the phyA-interaction domain of FHY1 are sufficient for FHY1 activity we tested whether adding the NLS to phyA directly would be enough to promote nuclear localization of phyA fused to the Green Fluorescent Protein (GFP). phyA mutants transformed with either PHYA-GFP (Figure 2A, B) or PHYA-NLS-GFP (Figure 2C–2F) driven by the PHYA promoter were analyzed microscopically. As previously described [23] nuclear accumulation of phyA-GFP was light-dependent (Figure 2A, B). In contrast, in lines expressing phyA-NLS-GFP nuclear localization was constitutive (Figure 2C, D). Nuclear bodies appeared extremely rapidly upon light excitation in phyA-NLS-GFP plants. When nuclei of etiolated phyA-NLS-GFP seedlings were imaged without a light treatment or immediately after a 5 sec red light pulse a smooth nucleoplasmic staining was observed (Figure 2E, data not shown). However as little as 1 minute after a 5 sec red light pulse nuclear bodies appeared in those nuclei (Figure 2F).


FHY1 mediates nuclear import of the light-activated phytochrome A photoreceptor.

Genoud T, Schweizer F, Tscheuschler A, Debrieux D, Casal JJ, Schäfer E, Hiltbrunner A, Fankhauser C - PLoS Genet. (2008)

Subcellular localization of a constitutively localized phyA (phyA-NLS-GFP).(A)–(D) 3-day-old dark-grown phyA-211 seedlings complemented with 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 µm. (E) and (F). 4-day-old dark-grown phyA-211 seedlings complemented with PPHYA∶PHYA-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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2483295&req=5

pgen-1000143-g002: Subcellular localization of a constitutively localized phyA (phyA-NLS-GFP).(A)–(D) 3-day-old dark-grown phyA-211 seedlings complemented with 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 µm. (E) and (F). 4-day-old dark-grown phyA-211 seedlings complemented with PPHYA∶PHYA-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.
Mentions: Given that both the NLS and the phyA-interaction domain of FHY1 are sufficient for FHY1 activity we tested whether adding the NLS to phyA directly would be enough to promote nuclear localization of phyA fused to the Green Fluorescent Protein (GFP). phyA mutants transformed with either PHYA-GFP (Figure 2A, B) or PHYA-NLS-GFP (Figure 2C–2F) driven by the PHYA promoter were analyzed microscopically. As previously described [23] nuclear accumulation of phyA-GFP was light-dependent (Figure 2A, B). In contrast, in lines expressing phyA-NLS-GFP nuclear localization was constitutive (Figure 2C, D). Nuclear bodies appeared extremely rapidly upon light excitation in phyA-NLS-GFP plants. When nuclei of etiolated phyA-NLS-GFP seedlings were imaged without a light treatment or immediately after a 5 sec red light pulse a smooth nucleoplasmic staining was observed (Figure 2E, data not shown). However as little as 1 minute after a 5 sec red light pulse nuclear bodies appeared in those nuclei (Figure 2F).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Centre for Integrative Genomics, University of Lausanne, Lausanne, Switzerland.

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.

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