Light-independent phytochrome signaling mediated by dominant GAF domain tyrosine mutants of Arabidopsis phytochromes in transgenic plants

Abstract

The photoreversibility of plant phytochromes enables continuous surveillance of the ambient light environment. Through expression of profluorescent, photoinsensitive Tyr-to-His mutant alleles of Arabidopsis thaliana phytochrome B (PHYB(Y276H)) and Arabidopsis phytochrome A (PHYA(Y242H)) in transgenic Arabidopsis plants, we demonstrate that photoconversion is not a prerequisite for phytochrome signaling. PHYB(Y276H)-expressing plants exhibit chromophore-dependent constitutive photomorphogenesis, light-independent phyB(Y276H) nuclear localization, constitutive activation of genes normally repressed in darkness, and light-insensitive seed germination. Fluence rate analyses of transgenic plants expressing PHYB(Y276H), PHYA(Y242H), and other Y(GAF) mutant alleles of PHYB demonstrate that a range of altered light-signaling activities are associated with mutation of this residue. We conclude that the universally conserved GAF domain Tyr residue, with which the bilin chromophore is intimately associated, performs a critical role in coupling light perception to signal transduction by plant phytochromes.

Figure 1.

The Y²⁷⁶H Mutant of Phytochrome B Is Functionally Active in Vivo. (A) Five-week-old transgenic Ler plants expressing PHYB wild-type (B) and PHYB^Y276H (B^Y276H) mutant cDNA constructs grown under continuous W at a fluence rate of 50 μmol m⁻² s⁻¹ are hypersensitive to light. (B) PHYB^Y276H expression rescues the phenotype of the phyB-5 (phyB) null mutant under the same conditions as (A). Ler wild-type, untransformed phyB-5, and phyB-5 mutant transformed with the wild-type PHYB cDNA construct (B) or the PHYB-GFP chimera cDNA (B-GFP) (Yamaguchi et al., 1999) are shown for comparative purposes. (C) Six-day-old seedlings expressing cDNA and genomic constructs of wild-type PHYB (B) or the PHYB^Y276H mutant were grown under 20 μmol m⁻² s⁻¹ continuous R. One representative line from transformation of Ler wild-type (i.e., B#1 and B^Y276H#3 cDNAs), phyB-5 (i.e., B#7 and B^Y276H#10 cDNAs), and phyA-201/phyB-5 (i.e., B#2 and B^Y276H#3 cDNAs; and B#14 and B^Y276H#5 genes) is shown, along with R-grown untransformed parent and PHYB-GFP/phyB plant lines. (D) Immunoblot analysis of PHYB protein level in wild-type, phyB, phyA, phyA phyB mutants, and PHYB^Y276H-expressing transgenic plants using monoclonal anti-PHYB antibodies. Total protein extracts (40 μg) from 6-d-old dark-grown seedlings were loaded on each lane. Tubulin was used as loading control.

Figure 2.

COP Phenotypes of PHYB^Y276H-Expressing Transgenic Plants. (A) Six-day-old dark-grown seedlings expressing cDNA and genomic Y276H alleles of PHYB exhibit COP on sucrose-free Murashige and Skoog (MS) medium. The lines shown here are the same as in Figure 1. (B) Four-week-old dark-grown Ler plants expressing PHYB^Y276H exhibit light-grown development in darkness on MS medium containing 1% (w/v) sucrose. Shown for comparison are untransformed Ler and wild-type PHYB in Ler transgenic plant line. (C) phyA phyB double mutant plants with or without the PHYB^Y276H transgene were grown as in (B). Bars = 1 cm.

Figure 3.

Phytochrome Chromophore Biosynthesis Is Required for the Gain-of-Function Activity of PHYB^Y276H. (A) Transgenic plants with or without the hy1 mutation were grown in darkness for 6 d on sucrose-free media. (B) Relative hypocotyl length of dark-grown seedlings in (A) normalized to that of Ler shows the differential activity of PHYB^Y276H in chromophore-replete phyA phyB and chromophore-deficient hy1 phyA phyB backgrounds. Values are mean ± sd (n = 50). (C) Relative hypocotyl length of seedlings grown on sucrose-free media under 20 μmol m⁻² s⁻¹ continuous R. Mean hypocotyl lengths (± sd; n = 50) are normalized to those of dark-grown Ler seedlings. The shorter hypocotyl lengths of hy1/phyA, PHYB/hy1/phyA/phyB, and PHYB^Y276H/hy1/phyA/phyB plants compared with the hy1/phyA/phyB parent line indicate that sufficient bilin chromophore is present in the hy1 mutant background to maintain reduced but significant signaling activity of phyB and phyB^Y276H. (D) Immunoblot analysis of PHYB protein level was performed as in Figure 1.

Figure 4.

PhyB^Y276H Localizes to the Nucleus, Forms Nuclear Bodies (Speckles), and Activates the Expression of Light-Regulated Genes in Darkness. (A) Comparative subcellular localization of phyB-GFP and PHYB^Y276H-derived phyB proteins in 5-d-old dark- and continuous W-grown (80 μmol m⁻² s⁻¹) seedlings was performed by fluorescence confocal microscopy. PhyB-GFP (B-GFP) was visualized using GFP optics (green), phyB^Y276H (B^Y276H) was visualized using Texas Red optics (red), and nuclei were identified by 4′,6-diamidino-2-phenylindole (DAPI) staining (blue). Merged images represent overlapping DAPI and GFP images (for phyB-GFP) or overlap of the DAPI with Texas Red images (for phyB^Y276H). Bars = 10 μm. (B) Light-independent expression of CAB and CHS transcripts was observed in dark-grown PHYB^Y276H plants by RT-PCR. Ethidium bromide–stained agarose gels contain 10 μL of PCR product per lane. ACT transcript levels are shown as a control.

Figure 5.

Phenotypic Analysis of Other Light-Grown Y^GAF Mutants of Phytochrome B Reveals Both Gain-of-Function and Loss-of-Function Phenotypes. (A) Transgenic plants expressing PHYB^Y276I, PHYB^Y276Q, and PHYB^Y276R mutant alleles of PHYB in the phyA phyB double mutant were grown on sucrose-free media for 6 d under 20 μmol m⁻² s⁻¹ of continuous R irradiation. One representative line from each transformation is shown. (B) Immunoblot analysis of PHYB protein levels was performed as in Figure 1. (C) Transgenic plants expressing Y276H, Y276I, Y276Q, and Y276R alleles of PHYB were grown for 5 weeks under continuous white light (W) at the fluence rate of 50 μmol m⁻² s⁻¹.

Figure 6.

PhyB^Y276Q Is a Constitutively Active Phytochrome, whereas phyB^Y276I Requires Light for Function and phyB^Y276R Is Inactive. (A) Comparative phenotypic analysis of various lines expressing Y^GAF alleles of PHYB in 6-d-old dark-grown seedlings on sucrose-free MS medium. One representative line from each transformation is shown. (B) Comparative fluence response curves for hypocotyl growth indicate that phyB^Y276H-mediated growth suppression is red light independent, while the growth suppression activities of phyB^Y276Q and phyB^Y276I are fluence rate dependent and phyB^Y276R is inactive. Each data point represents the mean of 50 seedlings ± sd. (C) Mean hypocotyl lengths (± sd; n = 50) of 6-d-old seedlings grown under 20 μmol m⁻² s⁻¹ of continuous red light (24h R) or 8-h-R/16-h-dark photoperiods are shown.

Figure 7.

Signaling Activities of phyB^Y276H and phyB^Y276Q Are Not Inhibited by Continuous FR Illumination. (A) Comparative analysis of seedling development of 6-d-old plants grown under 20 μmol m⁻² s⁻¹ continuous FR. One representative line from transformation of phyA/phyB is shown. (B) Comparative fluence response curves for hypocotyl growth indicate that phyB^Y276H-mediated growth suppression is FR light independent. Each data point represents the mean of 50 seedlings ± sd. (C) Comparative analysis of seed germination phenotypes of the wild type and phyA phyB mutant transformed with genomic PHYB^Y276H or PHYB^Y276Q alleles.

Figure 8.

Gain-of-Function Activity of the Y^GAFH Mutant of Phytochrome A. (A) Comparative morphogenesis of 6-d-old dark-grown seedlings with or without wild-type PHYA or PHYA^Y242H transgenes. Plants were grown as in Figure 2. (B) Comparative morphogenesis of 6-d-old seedlings grown under 20 μmol m⁻² s⁻¹ FR. (C) Immunoblot analysis of PHYA protein levels was performed as in Figure 1 using monoclonal anti-PHYA antibody. (D) Comparative fluence response curves for hypocotyl growth indicate that PHYA^Y242H confers gain-of-function FR-independent signaling activity to the phyA-deficient phyA-201 genotype and interferes with the FR high irradiance response of endogenous phyA in the Ler background. Each data point represents the mean of 50 seedlings ± sd.

Figure 9.

Proposed Mechanism of Light-Independent Light Signaling by Y^GAFH Mutant Phytochromes. (A) A proposed model of phytochrome protein conformational changes is shown. In Y^GAFH PHYB (or PHYA) apoproteins and the Pr form of the wild type (left), the photosensory domains (PSDs) are tightly associated with the C-terminal regulatory domains (CTRDs). This association masks a cryptic nuclear localization signal (NLS) located in the PAS repeat region within the CTRD that is specific to plant phytochromes (Chen et al., 2005). Activation occurs by Pr-to-Pfr photoconversion for the wild-type (green arrow) or by assembly of the Y^GAFH mutant apoprotein (YH) with phytochromobilin (PΦB) to produce the activated holoprotein species YH*. This results in release (or uncoupling) of the CTRD domain from the PSD by chromophore-mediated allosteric changes within the GAF domain that potentially are transduced via the PHY subdomain of the PSD. We envisage that this conversion exposes the CTRD-localized NLS, triggering nuclear translocation of phytochrome. For the wild type, this conversion is metastable and can be reversed both by FR irradiation (red arrow) or by dark reversion. (B) A cellular model for phytochrome signaling. For wild-type phytochromes, nuclear migration, nuclear body (speckle) formation, and transcription of light-regulated genes require both PΦB chromophore binding (black arrows on left) and red light activation (green arrows). While the relationship of nuclear body (nb) formation to PIF-dependent transcription, proteosome-mediated protein turnover of these factors, and COP1- and DET1-dependent repression pathways remains unresolved, all of these phyB-mediated signaling processes are reversed by FR light (red arrows) and/or by dark reversion (black dashed arrows). By contrast, PΦB binding is sufficient for light-independent activation of the Y^GAFH mutant of phytochrome B, since YH* activates these processes in the absence of light (solid black arrows). The superscript “c” and “n” refer to cytoplasmic and nuclear localization, respectively.