The Mediator Complex Subunit PFT1 Interferes with COP1 and HY5 in the Regulation of Arabidopsis Light Signaling^{<a href="#fn1" rid="fn1" class=" fn">1</a>,}^{<a href="#fn2" rid="fn2" class=" fn">[C]</a>}^{<a href="#fn3" rid="fn3" class=" fn">[W]</a>}^{<a href="#fn4" rid="fn4" class=" fn">[OA]</a>}

Isolation of the eid3 Mutant

To identify specific mutants in phyA signaling in Arabidopsis, ethyl methylsulfonate-treated phyB-5 seedlings were treated with a multiple pulse program consisting of 20 min of strong red light followed by 20 min of strong far-red light for 3 d after germination induction (Büche et al., 2000). In phyB-5 seedlings, preirradiation with red light pulses reduces the amount of phyA, which in turn decreases the HIR mediated by subsequent treatments with far-red light. The isolated phyB-5 eid3 mutant was able to avoid this red light-dependent reduction of HIR (Fig. 1A). Compared with its phyB-5 background, the mutant exhibited reduced hypocotyl elongation, open cotyledons, and increased anthocyanin accumulation under screening conditions. Except for the opening of hypocotyl hooks, phyB-5 eid3 remained etiolated in darkness (Fig. 1B). Segregation analyses of backcrosses with phyB-5 and the corresponding Landsberg erecta (Ler) wild type revealed that the eid3 mutant behaved like a dominant monogenic locus under selective light conditions.

Figure 1.

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 ^{s; C), and under weak red light (1 µmol m s; 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.

Epistasis between eid3 and phyA and phyB Loss-of-Function Mutants during Seedling Development

In order to test the influence of phyA and phyB on the expression of the Eid3 phenotype, an eid3 mutant in a PHYB wild-type background, a phyA-201 eid3 double mutant, and a phyA-201 phyB-5 eid3 triple mutant were created. The eid3 single and the phyB-5 eid3 double mutants exhibited an approximately 10-fold increase in light sensitivity compared with the wild type and phyB-5 under continuous far-red light (Fig. 1, C and E). In contrast, phyA-201 eid3 and phyA-201 phyB-5 eid3 seedlings did not respond to far-red light, similar to their phyA-201 and phyA-201 phyB-5 background lines.

Seedlings of the eid3 single mutant exhibited an extremely enhanced sensitivity to red light compared with the wild type (Fig. 1, D and F). Studies with phyA-201 eid3 and phyB-5 eid3 double mutants demonstrated that phyA is mainly responsible for the expression of the hypersensitive phenotype at photon fluence rates below approximately 0.01 µmol m ^{s, whereas the increased red light sensitivity can mainly be attributed to the presence of the phyB photoreceptor at higher photon fluence rates. The} phyA-201 phyB-5 eid3 triple mutant exhibited a slightly increased red light response compared with its phyA-201 phyB-5 background.

Blue light is not only sensed by cryptochrome and phototropin photoreceptors but can also induce the formation of the active Pfr conformation of phytochromes. Among the phytochromes, phyA is the most potent blue light receptor and, correspondingly, its loss resulted in a clear reduction of blue light responses in phyA-201 eid3 compared with the eid3 mutant background (Fig. 1G). In contrast, the lack of phyB caused only a weak reduction in blue light responses in phyB-5 eid3 and phyA-201 phyB-5 eid3 with respect to background lines. Comparison of fluence rate response curves between phyA-201 phyB-5 and phyA-201 phyB-5 eid3 indicated that blue light sensitivity in the triple mutant background is only slightly increased compared with its phyA-201 phyB-5 background.

In contrast to the strong photomorphogenic phenotype during seedling development, the eid3 mutation caused only a mild alteration during vegetative growth. Rosettes of eid3, phyA-201 eid3, phyB-5 eid3, and phyA-201 phyB-5 eid3 plants exhibited a slightly decreased diameter and more rounded leaves compared with the wild type and the corresponding mutant backgrounds, indicative of a reduced SAR (Supplemental Fig. S1). The eid3 mutation also did not alter phyA degradation or subcellular localization (Supplemental Fig. S2).

Genetic Interaction with EID1 and COP1 Loss-of-Function Mutants and the phyA-401 Gain-of-Function Allele

In order to test the interaction between eid3 and negatively acting components of the light signaling cascade, the mutant was crossed with the strong eid1-1 loss-of-function allele and the cop1^eid6 allele, which does not exhibit a constitutive photomorphogenic phenotype in the absence of light (Dieterle et al., 2001, 2003). Additionally, double mutants were generated with eid3 and the phyA-401 (eid4) gain-of-function allele. The extreme light sensitivity of eid3 single and double mutants hinders standard procedures used to compare light responses under continuous irradiation: it was nearly impossible to apply nonsaturating doses of light. To overcome this problem, a 3-d light program was used that consisted of hourly 5-min light pulse treatments interrupted by 55 min of darkness. Wavelengths that adjust the levels of the physiologically active form of phytochromes [Pfr/(Pfr + Pr)] were used and resulted in relative ratios of approximately 0.87 (659-nm filter), approximately 0.5 (692-nm filter), approximately 0.05 (719-nm filter), and below 0.001 (760-nm filter; Dieterle et al., 2005).

In accordance with the proposed role of EID1 as a specific regulator of phyA-dependent HIR (Dieterle et al., 2001; Zhou et al., 2002), a significant difference between wild-type and eid1-1 seedlings was only detectable under multiple 719-nm pulse treatments, which are known to be maximally effective at inducing HIR light responses (Fig. 2). Correspondingly, a strong synergistic effect was seen in the eid3 eid1-1 double mutant upon the application of multiple 719-nm pulses. Weaker synergistic effects also became detectable under pulses with 692- and 659-nm light. In addition, eid3 eid1-1 double mutants exhibited clear alteration in the morphology of rosette leaves of adult plants compared with background lines (Supplemental Fig. S3).

Figure 2.

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).

The phyA-401 missense allele leads to an enhanced light sensitivity under continuous red and far-red light (Dieterle et al., 2005), but the mutant did not exhibit significant differences in light responses compared with the wild type under hourly light pulse treatments (Fig. 2). In contrast, eid3 phyA-401 double mutants showed an increased light response compared with their corresponding background lines and the wild type under all tested wavelengths. The eid3 phyA-401 double mutant did not show any further alteration in the shape of the rosette compared with the eid3 line (Supplemental Fig. S3).

The most severe synergistic effects were obtained with the eid3 cop1^eid6 double mutant. Even though cop1^eid6 and eid3 background lines remained almost completely etiolated in darkness, the double mutant exhibited a strong constitutive photomorphogenic phenotype (Fig. 2, A and A′). The eid3 cop1^eid6 double mutant also showed an extremely enhanced light sensitivity compared with both background lines and the other eid3 double mutant lines. Seedlings were extremely short, had open and expanded cotyledons, and accumulated very high levels of anthocyanin under all applied pulse treatments (Fig. 2). The eid3 cop1^eid6 mutant also showed a severe reduction in rosette size compared with the wild-type, eid3, and cop1^eid6 background lines (Supplemental Fig. S3).

Epistatic Analyses with Positively Acting Factors Involved in Arabidopsis Light Signaling

In order to test for the interdependency between eid3 and the positive phyA-dependent light signaling effectors HFR1, FHY3, FAR1, and HY5 (Oyama et al., 1997; Fairchild et al., 2000; Wang and Deng, 2002; Hudson et al., 2003), double mutants were isolated to perform epistatic analyses with 4-d-old seedlings that were grown under continuous far-red light of variable photon fluence rates.

A full epistatic effect was only obtained with hy5 eid3 double mutants (Fig. 3A). The hy5 loss-of-function mutant reduced light responses to the level of the hy5 mutant background line under all applied far-red light intensities. Loss of FHY3 caused a clear reduction in light sensitivity at all applied photon fluence rates, but far-red light sensitivity was still increased compared with fhy3-1 under light intensities normally inducing HIR (Fig. 3B). The lack of HFR1 and FAR1 had only a weak influence on the expression of the Eid3 phenotype at low fluence rates of far-red light, whereas far-red light responses remained more or less unaltered at high light intensities (Fig. 3, C and D). This finding contrasts with the results obtained for the hfr1 and far1 single mutants, which showed strongest loss-of-function phenotypes at high photon fluence rates.

Figure 3.

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.

Isolation of the Mutated Gene

For mapping analyses, phyB-5 eid3 was crossed with a phyB-9 loss-of-function mutant in the Columbia ecotype (Col). Analyses of seedling phenotypes under the screening program revealed that all F1 seedlings exhibited a clear eid3 mutant phenotype, indicative of a dominant mutant. The F2 generation exhibited a segregation of seedling phenotypes under selective light conditions (Eid3:wild type = 88:39) that was consistent with the expected 3:1 segregation of a dominant mutation (P < 0.05, χ ^{test). Because} eid3 has a dominant genetic inheritance, the recessive phenotype of the wild-type allele was used to isolate homozygous plants. The observed linkage with markers on chromosome 1 indicated that eid3 might be a novel allele of PFT1 (Supplemental Fig. S4). Sequence analyses of amplified genomic fragments revealed that eid3 carries a point mutation in the PFT1 gene (Fig. 4A). The eid3 mutation results in the replacement of Thr-650 of PFT1 with a Met residue in a highly conserved motif in the herpesvirus protein16 (VP16)-like domain, which is thought to be involved in specific recognition of transcription factors bound to promoter elements (Fig. 4B). The base pair exchange enabled the design of a specific derived cleaved-amplified polymorphic sequence (dCAPS) marker for the mutated allele. Analyses with the eid3dCAPS marker exhibited a perfect cosegregation with the hypersensitive Eid3 phenotype after examination of 15 homozygous phyB-5 eid3 plants. Taken together, these findings strongly indicate that the dominant eid3 mutation is caused by a missense mutation in PFT1.

Figure 4.

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 pft1-2T-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^-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.]

To further analyze the effect of PFT1 on light signaling, a transferred DNA (T-DNA) insertion line was isolated from the SALK collection (SALK_129555) that is identical to the published pft1-2 mutant (Kidd et al., 2009). The T-DNA was inserted into the fifth exon, which encodes for the van Willebrand factor type A domain (Fig. 4A). Reverse transcription (RT)-PCR analyses using primers downstream of the insertion site exhibited clear reduction in transcript accumulation in the pft1-2T-DNA insertion line (Fig. 4C). With respect to transcript accumulation, pft1-2 resembles pft1-1, which accumulates reduced levels of a truncated transcript and carries a T-DNA insertion in the fourth intron (Cerdán and Chory, 2003).

To test whether the reduction of PFT1 gene expression leads to alterations in light sensitivity, the pft1-2 mutant was subjected to the multiple red/far-red pulse treatment used for the screening of eid mutants and to continuous irradiation with variable fluence rates of red and far-red light. The T-DNA line showed only a very weak hypersensitive response under the red/far-red pulse treatment compared with the Col wild-type control (Fig. 4D). No difference in light response was seen under continuous far-red light with pft1-2, which contrasts with the strong hypersensitive response of eid3 (Fig. 4E). The pft1-2 line exhibited an increase in light sensitivity under strong continuous red light (Fig. 4F), similar to published results for the pft1-1 allele (Cerdán and Chory, 2003).

To further prove whether the mutated form of PFT1 is responsible for the hypersensitive eid3 phenotype, genomic fragments spanning the promoter region and all exons and introns of PFT1 and PFT1^eid3 were cloned in front of a Yellow Fluorescent Protein (YFP) cassette, and constructs were then introduced into the pft1-2 mutant. This approach was chosen because overexpression of PFT1 and PFT1^eid3 causes the cosuppression of protein function in transgenic lines (Cerdán and Chory 2003; data not shown). Different homogenous lines were isolated that carried single T-DNA integrations. Analyses of light responses under the screening program demonstrated that the introduced Pro_PFT1-PFT1^-YFP constructs restore the hypersensitive Eid3 phenotype (Fig. 4G). The analyzed transgenic lines exhibited an increased sensitivity toward continuous red and far-red light compared with the Col wild type and the pft1-2 background (Fig. 4, E and F). The increase in light sensitivity was stronger in PFT1^-YFP2 compared with PFT1^-YFP1, even though transcript levels were lower in PFT1^-YFP2 (Fig. 4H). This finding, together with the increased red light sensitivity compared with the pft1-2T-DNA line, argues against a dominant negative effect of PFT^eid3 and further supports the notion that the eid3 missense mutation created a hyperactive transcriptional coregulator.

Temporal Expression Pattern of Light-Regulated Marker Genes during Seedling Deetiolation

PFT1 was identified as a component of the Mediator transcriptional coregulator complex (Bäckström et al., 2007). In order to gain insight into the function of PFT1^eid3 during light-regulated gene expression, temporal transcript accumulation patterns were analyzed by RT-PCR of different light-regulated marker genes and ACTIN1 (ACT).

HY5 and PHYTOCHROME KINASE-SUBSTRATE1 (PKS1) become rapidly increased upon light treatment and respond to even single pulses of red light (Peschke and Kretsch, 2011); they accumulated to very low levels in etiolated wild-type seedlings. In contrast, etiolated eid3 seedlings exhibited increased transcript accumulation for both genes. The red light pulse induced a rapid increase in HY5 and PKS1 transcript levels in the wild type, with maximum levels at 1 h (Fig. 5A). The eid3 mutant exhibited the same temporal accumulation pattern for both transcripts, but transcript levels were enhanced at all analyzed time points. Quantitative RT-PCR measurements verified the strong influence of eid3 on HY5 and PKS1 transcript accumulation in dark-grown and pulse-treated seedlings (Fig. 5B). The eid3 mutant exhibited increased transcript levels compared with the Ler wild type for both marker genes. In contrast, marker gene expression was reduced in pft1-2 compared with the corresponding Col wild type. Thus, eid3 behaves like a gain-of-function mutant, whereas pft1-2 shows characteristics of a loss-of-function allele.

Figure 5.

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.

Transcripts of SUBUNIT E OF PHOTOSYSTEM I (PSAE) and CHALCONE SYNTHASE (CHS) accumulate late compared with HY5 and PKS1 (Fig. 5A). PSAE gene expression is very light sensitive, whereas strong CHS expression is only obtained under prolonged irradiation with strong continuous blue and far-red light or upon UV-B pulse treatment (Peschke and Kretsch, 2011). In contrast to the wild type, eid3 seedlings accumulated enhanced transcript levels for PSAE, even in darkness. The red light pulse caused a weak preliminary accumulation of PSAE transcripts in eid3, but no major differences in signal intensities were detectable after 4 h, when maximum transcript levels were reached in wild-type and mutant seedlings (Fig. 5A). CHS transcripts remained below the detection level in etiolated wild-type seedlings and were faintly induced by red light pulse treatment. In striking contrast, eid3 seedlings exhibited enhanced CHS transcript levels in the dark and strong induction of CHS by a single red light pulse.

Constitutive Expression of Light-Regulated Marker Genes in Complete Darkness

All tested light-regulated marker genes exhibited weak constitutive expression in etiolated eid3 seedlings (Fig. 5, A and B), which had received 2 h of red light to induce homogeneous seed germination. Since eid3 exhibits an extremely enhanced light sensitivity, seeds were sown on 10 µm GA to enable germination and seedling development in complete darkness. The lack of red light pretreatment for germination induction did not alter the enhanced dark expression of light-regulated marker genes in eid3 (Fig. 5C). Furthermore, GA treatment did not alter HY5, PKS1, and CHS transcript accumulation in the wild type or eid3 upon the application of a red light pulse. HY5 transcript accumulation was also tested in both dark-grown phyA-201 phyB-5 eid3 seedlings and in GA-induced, dark-grown seedlings that had received pulses of extreme far-red light (red glass9 filter light) to photoconvert any remaining Pfr molecules back to the inactive Pr form. Neither the lack of the two dominant phytochromes nor the reduction of Pfr remaining from seed development resulted in a reduction of HY5 transcript levels in etiolated seedlings of eid3 mutant lines (Supplemental Fig. S5). Taken together, the data clearly indicate that the eid3 mutation of PFT1 enables the expression of light-regulated marker genes independent of any light input.

Transcript Accumulation Patterns in Dark-Grown eid3 Seedlings

To obtain broader insight into the influence of eid3 on gene expression during skotomorphogenic development, transcript accumulation patterns were analyzed using Agilent 44K Arabidopsis gene expression microarrays, which include approximately 28,900 nuclear genes, 66 plastidic genes, and 67 mitochondrial genes. Three independent RNA samples were isolated from 4-d-old etiolated eid3 or wild-type seedlings and subjected to microarray analyses. Transcript levels were only regarded as differentially regulated between the wild type and eid3 if they exhibited both a 2-fold or greater change in signal intensities (up or down) and a statistically significant difference in expression values (t test, P < 0.05) adjusted for a false discovery rate of Q < 0.05 (Benjamini and Hochberg, 1995).

In etiolated eid3 plants, 542 genes exhibited significantly enhanced and 718 genes exhibited significantly reduced transcript levels compared with etiolated wild-type seedlings, indicating a dual role for PFT1 as a transcriptional activator and suppressor. The list of up-regulated genes includes PSAE and CHS marker genes, which exhibited enhanced transcript levels with RT-PCR analyses. Twelve percent of the up-regulated genes (66) encode for genes related to photosynthesis and chloroplast development (Fig. 6A; Supplemental Table S1). High numbers of the up-regulated genes belong to the group’s transcriptional regulators (50 of 542) and signaling components (62 of 542). Among these 112 genes, 34 are known to be related to light signaling (10), hormone function (13), leaf development (four), or regulation of meristematic activity and cell differentiation (six; Supplemental Table S2). Up-regulated genes involved in light signaling include ATTENUATED FAR-RED RESPONSE1, LAF1, and HY5 HOMOLOG, all of which function as important positive regulators of photomorphogenesis. Among the up-regulated genes related to hormone function, five genes encode for negative regulators of cytokinin that function by either inactivating the hormone (CYTOKININ OXIDASE5) or at the level of transcriptional regulation (ARABIDOPSIS RESPONSE REGULATOR7/15/16; BEL1-LIKE HOMEODOMAIN3). Up-regulated genes also included ALTERED MERISTEM1, which functions as a regulator of photomorphogenesis (cop2 mutant) and transition to flowering.

Figure 6.

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: ^,Peschke and Kretsch (2011); ^,Leivar et al. (2009); ^{this study; ,}Jiao 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&amp;M, ions and nutrition; CM, carbohydrate metabolism; LM, lipid metabolism; Met, metabolism miscellaneous; Str, stress; Mis, miscellaneous; Uk, unknown.

High numbers of the down-regulated genes encode for transcriptional regulators (63 of 718), signaling components (107 of 718), and factors related to cell wall metabolism (26 of 718; Fig. 6A; Supplemental Table S3). Many of the down-regulated genes encoding for signaling and transcription factors are related to cell cycle control (15), meristem organization (five), leaf development (four), and differentiation of epidermal cells (six; Supplemental Table S2). Down-regulated genes also included repressors of the floral transition, such as FLOWERING LOCUS C (FLC), AGAMOUS-LIKE42, and MADS AFFECTING FLOWERING1. These findings indicated that the eid3 mutation induces strong alterations in the expression of genes related to growth and development in the absence of light.

Furthermore, the eid3 mutant caused down-regulation of a high number of genes related to diverse metabolic processes (Fig. 6A; Supplemental Table S3). Among these genes, a high proportion encode for enzymes and proteins involved in the activation of storage compounds. Of the 28 genes involved in carbohydrate metabolism, 12 have been annotated as hydrolases. Of the 27 genes involved in lipid metabolism, 12 are lipases and 10 are lipid transfer/seed storage proteins. Furthermore, 17 genes encode for proteases (Supplemental Table S3).

Supplementary Material