Epistatic analyses with phyA and phyB loss-of-function mutants. A to D, Photographs of Ler wild-type, phyA-201, phyB-5, phyA-201 phyB-5, eid3, phyA-201 eid3, phyB-5 eid3, and phyA-201 phyB-5 eid3 seedlings grown under different light conditions for 4 d after induction of germination. Seedlings were kept under the screening program (cycles of 20 min of red light followed by 20 min of far-red light; A), in darkness (B), under weak far-red light (0.1 µmol m⁻² s⁻¹; C), and under weak red light (1 µmol m⁻² s⁻¹; D). Bars = 5 mm. E to G, Fluence rate response curves for the inhibition of hypocotyl elongation of Ler wild-type, phyA-201, phyB-5, phyA-201 phyB-5, eid3, phyA-201 eid3, phyB-5 eid3, and phyA-201 phyB-5 eid3 seedlings under continuous far-red light (E), continuous red light (F), or continuous blue light (G). Relative hypocotyl lengths were calculated in relation to the length of dark-grown seedlings for each line. Each data point represents the mean ± se of two independent experiments with at least 30 seedlings.

The eid3 mutation cooperates with EID1 and COP1 to regulate photomorphogenic seedling development. Seedlings of Ler, eid1-1, eid3, phyA-401, cop1^eid6, eid3 eid1-1, eid3 phyA-401, and eid3 cop1^eid6 were grown for 3 d after induction of germination under repetitive pulse treatments consisting of 5 min of saturating light followed by 55 min of darkness. The light conditions used for the pulse treatment were darkness (A and A′), 760-nm light (B and B′), 719-nm light (C and C′), 692-nm light (D and D′), and 659-nm light (E and E′). A to E, Photographs of seedlings grown under the different multiple pulse treatment programs. Bars = 5 mm. A′ to E′, Absolute hypocotyl lengths of pulse-treated seedlings. Data represent means of two independent experiments ± se with 30 seedlings. +, significant difference from the Ler wild type; * significantly reduced hypocotyl lengths compared with the corresponding single mutant background lines (one-way ANOVA on ranks, P < 0.05).

Epistatic analyses with positively acting Arabidopsis light signaling factors. Fluence rate response curves for the inhibition of hypocotyl elongation are shown for seedlings grown under continuous far-red light for 3 d after germination induction. Relative hypocotyl lengths were calculated in relation to the lengths of dark-grown seedlings for each line. Each data point represents the mean of two independent experiments ± se with at least 30 seedlings. A, Ler wild type, eid3, hy5-1, and eid3 hy5-1 mutants. B, Ler wild type, Col wild type, eid3, fhy3-1, and eid3 fhy3-1 mutants. C, Ler wild type, eid3, hfr1-2, and eid3 hfr1-2 mutants. D, Ler wild type, Nossen (No) wild type, eid3, far1-3, and eid3 far1-3 mutants.

Characterization of the pft1-2 mutant and reconstitution of the Eid3 phenotype. A, Schematic structure of the PFT1 gene. Black boxes represent exons, lines represent introns, and white boxes show untranslated regions of the mRNA. The positions of the van Willebrand factor type A domain (vWFA) and the T-DNA insertion in the pft1-2 mutant are indicated. The position of the eid3 missense mutation is indicated by the asterisk. B, Schematic structure of the PFT1 protein, and amino acid sequence alignment of the plant-specific, highly conserved region containing the eid3 mutation. FH1, Formin homology 1 domain; VP16, VP16-like interaction domain; PolyQ, Gln-rich domain; aa, amino acids; At, Arabidopsis; Vv, Vitis vinifera; Pt, Populus trichocarpa; Os, Oryza sativa; Zm, Zea mays; Pp, Physcomitrella patens. C, PFT1 transcript levels were monitored by RT-PCR using 4-d-old etiolated seedlings of the pft1-2 T-DNA line, eid3, and the corresponding wild types. ACT transcript levels are presented as a constitutive control. Gels were stained with SYBR-safe DNA gel stain and are shown inverted. The gel shows the representative result of three independent experiments. D, Photograph of pft1-2 mutant and Col wild-type seedlings grown under the eid screening program (cycles of 20 min of red light followed by 20 min of far-red light) for 4 d after germination induction. Bar = 5 mm. E and F, Fluence rate response curves for the inhibition of hypocotyl elongation of eid3, Col wild type, pft1-2, and pft1-2 mutant lines transformed with Pro_PFT1-PFT1^eid3-YFP constructs. Seedlings were grown under continuous far-red light (E) or continuous red light (F). Relative hypocotyl lengths were calculated in relation to the lengths of dark-grown seedlings for each line. Each data point represents the mean ± se of two independent experiments with 30 seedlings. G, Photograph of pft1-2 mutant seedlings expressing PFT1^eid3-YFP under the control of the PFT1 promoter and grown under the eid screening program (cycles of 20 min of red light followed by 20 min of far-red light) for 4 d. Seedlings of two independent transgenic lines are shown together with the eid3 mutant and the pft1-2 mutant background. Bar = 5 mm. H, Expression levels of the PFT1^eid3-YFP transgene in seedlings of two independent transgenic lines and the corresponding pft1-2 mutant background line. Transcript levels were monitored as described for C. The gel shows representative results of three independent experiments. [See online article for color version of this figure.]

Expression pattern of light-regulated marker genes during seedling deetiolation. A, Expression of light-induced marker genes after a red light pulse. Ler wild-type and eid3 mutant seedlings were grown in darkness for 4 d after induction of germination. Expression of light-regulated genes was induced by a saturating 2-min red light pulse. The gel shows representative results of three independent experiments. HY5, PKS1, CHS, and PSAE transcript levels were monitored at the indicated time points after the red light pulse using RT-PCR. ACT transcript levels served as a constitutive control. Gels were stained with SYBR-safe DNA gel stain. Images are shown inverted. B, Quantification of transcript levels of light-induced HY5 and PKS1 marker genes in eid3, pft1-2, and the corresponding Ler and Col wild types. Seedlings were treated as described for A. Transcript levels were determined by quantitative real-time PCR analyses. Results of experiments were normalized according to the constitutively expressed ACT gene. Data represent means of three independent biological replicates ± se. Rp, 2-min red light pulse; cD, dark control. C, Light-independent expression of light-induced marker genes in the eid3 mutant and the Ler wild type. Germination was induced by either 2 h of continuous red light without GA (−GA) or without light treatment and application of 10 µm GA (+GA). Seedlings were grown in darkness for 4 d after germination induction. Expression of light-induced genes was initiated by a saturating 2-min red light pulse, and samples were harvested at 1 and 4 h or before light treatment (0 h). Transcript levels were monitored as described for A. The gel shows the representative result of two independent experiments.

Analyses of transcript accumulation patterns in dark-grown eid3 seedlings. A, Ler wild-type and eid3 seeds were grown in darkness for 4 d before extraction of RNA and determination of transcript accumulation patterns by microarray analyses. Pie charts show the distribution of more than 2-fold up- or down-regulated genes among functional categories. Distributions are expressed as percentages of 541 up-regulated and 718 down-regulated genes in etiolated eid3 compared with wild-type seedlings. B, Bar charts depict percentages of up- and down-regulated genes in etiolated eid3 seedlings that have been annotated as being light regulated in published data sets (Jiao et al., 2005; Leivar et al., 2009; Peschke and Kretsch, 2011) and upon red light pulse treatment of 4-d-old, etiolated Ler wild-type seedlings (this study). Ler wild-type seedlings received one saturating red light pulse before transfer back to darkness for 43 min. Transcript levels were compared with etiolated seedlings harvested at the same time. Genes exhibiting more than 2-fold up- or down-regulation in transcript levels were annotated as being light regulated: ¹Peschke and Kretsch (2011); ²Leivar et al. (2009); ³this study; ⁴Jiao et al. (2005). cD, Continuous darkness; cF, continuous far-red light; cR, continuous red light; cW/B/R/F, continuous white/blue/red/far-red light; Rp, red light pulse. C, Pie charts show the distribution among functional categories of up- and down-regulated genes in etiolated eid3 seedlings that have been annotated as being light regulated in other data sets. Distributions are expressed as percentages of 337 up-regulated and 179 down-regulated genes. Tr, Transcription; Sig, signaling; CW, cell wall; Pl, plastids; I&M, ions and nutrition; CM, carbohydrate metabolism; LM, lipid metabolism; Met, metabolism miscellaneous; Str, stress; Mis, miscellaneous; Uk, unknown.

The eid3 mutant affects the floral transition and the expression of flowering genes. A and B, Flowering time of eid3, eid3 phyA-201, eid3 phyB-5, eid3 phyA-201 phyB-5, and corresponding background lines under variable daylengths. Flowering time was determined in short days consisting of 8-h-light/16-h-dark cycles (A) and long days consisting of 16-h-light/8-h-dark cycles (B). Flowering time is presented as the sum of rosette and cauline leaves at the main stem. Data represent means of at least two independent experiments ± se with 20 or more plants. + Significant difference from the Ler wild type; ×, significant difference from eid3; * significant difference from the eid3 double and triple mutants to corresponding phyA, phyB, and phyA phyB mutant background lines (one-way ANOVA on ranks, P < 0.05). C, Diurnal transcript accumulation patterns of different flowering genes in Ler wild type, eid3, Col wild type, and pft1-2. Plants were grown under 12-h light/dark cycles for 2 weeks. On day 14, samples were taken every 4 h starting at the onset of light (DT 0 h). Transcript levels were determined by RT-PCR using identical cDNA samples. CO, FLC, and FT transcript levels were monitored at the indicated time points. ACT transcript levels served as a constitutive control. Gels were stained with SYBR-safe DNA gel stain. Images are shown inverted. The gel shows representative results of two independent experiments.

Models for the proposed function of PFT1 in light signaling during photomorphogenic seedling development and the regulation of flowering. A, Proposed function of PFT1 in light signaling downstream of different photoreceptors. PFT1 may function mainly by up-regulation of the HY5 basic Leu-zipper transcription factor gene, a central positive regulator of early light signaling. COP1-ULCs function as negative regulators that target positively acting factors like HY5 to degradation in the proteasome until COP1 inactivation by Pfr- and/or cry1/2-dependent mechanisms. COP1-ULC activity would act downstream of PFT1 by counteracting the protein accumulation of regulatory factors, for which transcription becomes up-regulated by the function of the respective subunit of a plant Mediator complex. PFT1 should also function as a negatively acting component in a phyA signaling cascade that inhibits phyB responses. PIFs, Phytochrome-interacting proteins. B, Proposed function of PFT1 in regulation of the floral transition. PFT1 is thought to mainly function downstream of phyA. As a component of the Mediator complex, PFT1 seems to increase the expression of the CO gene, a central regulator of the photoperiod pathway that is also involved in the acceleration of flowering under high far-red conditions sensed by phyB. CO protein levels are also regulated by phyA and cry1/2, both of which block COP1-dependent ubiquitin ligase activity in the light. Additionally, PFT1 might inhibit expression of the FLC gene, an important repressor of the FT florigen. The model further proposes that PFT1 inhibits phyB-dependent inhibition of SAR in a phyA-dependent manner.