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American Society of Plant Biologists Missense Mutation in the PAS2 Domain of Phytochrome A Impairs Subnuclear Localization and a Subset of Responses
a IFEVA, Facultad de Agronomía, Universidad de Buenos Aires, Av San Martín 4453, 1417-Buenos Aires, Argentina 4 To whom correspondence should be addressed. E-mail casal{at}ifeva.edu.ar; fax 54-11-45-14-87-30
Phytochrome A signaling shows two photobiologically discrete outputs: so-called very-low-fluence responses (VLFR) and high-irradiance responses (HIR). By modifying previous screening protocols, we isolated two Arabidopsis mutants retaining VLFR and lacking HIR. Phytochrome A negatively or positively regulates phytochrome B signaling, depending on light conditions. These mutants retained the negative but lacked the positive regulation. Both mutants carry the novel phyA-302 allele, in which Glu-777 (a residue conserved in angiosperm phytochromes) changed to Lys in the PAS2 motif of the C-terminal domain. The phyA-302 mutants showed a 50% reduction in phytochrome A levels in darkness, but this difference was compensated for by greater stability under continuous far-red light. phyA-302:green fluorescent protein fusion proteins showed normal translocation from the cytosol to the nucleus under continuous far-red light but failed to produce nuclear spots, suggesting that nuclear speckles could be involved in HIR signaling and phytochrome A degradation. We propose that the PAS2 domain of phytochrome A is necessary to initiate signaling in HIR but not in VLFR, likely via interaction with a specific partner.
Phytochrome is a small family of red light (R) and far-red light (FR) photoreceptors comprising five isoforms, phytochrome A through phytochrome E (phyA through phyE), in Arabidopsis (Neff et al., 2000  ). phyA and phyB are dimeric cytoplasmic proteins in dark-grown seedlings that migrate to the nucleus upon light activation (Kircher et al., 1999; Yamaguchi et al., 1999). The N-terminal domain bears an open chain tetrapyrrol chromophore. The C-terminal domain contains motifs that are important for interaction with the nuclear transcription factor PIF3 (Ni et al., 1998, 1999; Martinez-García et al., 2000), the phytochrome kinase substrate PKS1 (Fankhauser et al., 1999), nucleoside diphosphate kinase 2 (Choi et al., 1999), and cryptochrome (Ahmad et al., 1998).
The pattern of a response—graded, multiphasic, with a threshold, with biorhythmic oscillations, et cetera—depends on the architecture of the signaling network (Becskei et al., 2001
The architectural features of the phyA signaling network that give rise to the biphasic response have not been elucidated. Nevertheless, VLFR and HIR can be dissected genetically by the vlf1, vlf2, fhy3, and dim/dwarf1/eve1 loci that operate downstream of phyA (Yanovsky et al., 1997 Together, these observations are consistent with a model in which signaling downstream of phyA branches out into two cascades, depending on the mode in which the photoreceptor is activated. The point at which divergence is initiated has not been established. The aim of this work was to identify mutants selectively impaired in the HIR and not in the VLFR component to gain insight into the molecular differences between these two response modes.
Novel Alleles of phyA That Show Deficient Hypocotyl Growth Inhibition but Normal Seed Germination To obtain Arabidopsis mutants with impaired HIR but retaining VLFR, both germination of mutagenized seed and subsequent seedling growth were conducted under continuous FR (Figure 1) . phyA-mediated promotion of seed germination is a VLFR (Botto et al., 1996 ; Shinomura et al., 1996) because the response is saturated with hourly pulses of FR and continuous FR has no additional effect (Figure 2A)
. In contrast, phyA-mediated inhibition of hypocotyl growth is predominantly a HIR because continuous FR is significantly more effective than hourly pulses of FR.
Tall seedlings under continuous FR were selected from M2 seeds (Figure 1). Normal seedlings under pulsed R were selected from M3 seeds of these mutants to avoid phytochrome chromophore and nonphotomorphogenic hypocotyl growth mutants (Figure 1). Two independent lines (designated phyA-302-1 and phyA-302-2 based on the evidence described below), showing seed germination under FR (Figure 2A), normal inhibition of hypocotyl growth under R, and long hypocotyls under continuous FR (Figure 2B), were chosen for further studies. The two mutants failed to complement the null phyA-201 mutant (Figure 3) .
In etiolated seedlings, the levels of phyA detectable immunologically and by fluorescence signals were reduced by  50% in both mutant lines (Figures 4A and 4B)
. The fluorescence spectra (Figure 4C), the extent of Pr-to-lumi–R (the first photoproduct stable at low temperatures) conversion at 85K upon illumination with 639 nm (0.24 to 0.3), and the extent of Pr-to-Pfr conversion at 273K upon illumination with 680 nm (0.55 to 0.65) were not affected significantly by the phyA-302 mutations. Because the abundance of phyA normally is reduced by continuous FR compared with darkness or hourly FR pulses (Casal et al., 2000), immunoblot analysis also was conducted with dark-grown 3-day-old seedlings transferred to continuous FR for 8 h. Although phyA-302 mutants had less phyA in dark-grown control seedlings, the residual phyA was more stable than in the wild type (Figure 4D).
The Two Missense Mutations Occur in Amino Acid 777 of phyA The phyA genes of the two mutants and of the wild type were cloned and sequenced completely. The two independent mutants contain the same Guanidine-to-Adenine mutation that results in the change of amino acid 777 from Glu to Lys (Figure 5 ; amino acid numbering based on the chromophore attachment site at Cys-323).
phyA-302:Green Fluorescent Protein Fusion Proteins Fail to Produce Nuclear Speckles Null phyA mutant seedlings of Arabidopsis were transformed with the Arabidopsis PHYA:green fluorescent protein (GFP) or phyA-302:GFP genes under the control of a 35S promoter. The PHYA:GFP transgene fully rescues the phyA null mutant phenotype (Kim et al., 2000 ). The phyA-302:GFP transgene rescued the phyA null mutant phenotype under hourly pulses of FR (hypocotyl length [mm] in the wild type was 11.8 ± 0.2 in darkness and 10.8 ± 0.2 in pulsed FR [P < 0.05]; hypocotyl length in the null phyA mutant was 11.3 ± 0.4 in darkness and 11.2 ± 0.2 in pulsed FR [P > 0.1]; hypocotyl length in the null phyA mutant expressing the phyA-302:GFP transgene was 13.3 ± 0.3 in darkness and 12.2 ± 0.3 in pulsed FR [P < 0.05]). Continuous FR reduced hypocotyl length in the wild type dramatically (2.0 ± 0.1 mm), but it had no more effect than pulsed FR in the phyA-302:GFP transgenic plants (12.1 ± 0.3 mm). This pattern of response agrees with the behavior of the phyA-302 mutants (see below). The localization of phyA:GFP and phyA-302:GFP fusion proteins was investigated in transgenic seedlings grown in darkness and subsequently transferred to continuous FR.
In the seedlings that remained as dark controls, the nuclei showed no fluorescence associated with the presence of phyA (Figure 6)
. phyA:GFP or phyA-302:GFP was present in the nuclei of plants exposed to 1, 3, 9, or 24 h of continuous FR, indicating that the phyA-302 mutation did not affect nuclear translocation. However, although phyA:GFP was able to form speckles in >90% of the nuclei (Kim et al., 1999
Sustained and Transient Activation with FR Are Equally Effective in phyA-302 In the phyA-302 mutants, hypocotyl growth inhibition, which is dominated by the HIR component, was more affected than seed germination, which is dominated by the VLFR component. To compare VLFR and HIR of the same end point responses (hypocotyl growth and cotyledon unfolding), etiolated seedlings were allowed to germinate in darkness for 24 h after a saturating R pulse and transferred to either hourly pulses of FR, which saturate the VLFR but are insufficient to cause a HIR, or continuous FR at a fluence rate that is sufficient to initiate a HIR (Casal et al., 2000 ).
The wild type showed hypocotyl growth inhibition and cotyledon unfolding under hourly FR (i.e., VLFR), but continuous FR was significantly more efficient, suggesting a strong contribution of a HIR (Figure 7)
. In the phyA-302-1 and phyA-302-2 mutants, pulsed and continuous FR produced similar effects, indicating the absence of a HIR. The weak phyA-205 allele (Nagatani et al., 1993
We also investigated the effects of pulsed and continuous FR in the hy1 and hy2 mutants, which have severely reduced levels of phytochrome (Koornneef et al., 1980 ). These mutations affect chromophore synthesis genes and not apoprotein genes (Davis et al., 1999; Muramoto et al., 1999; Kohchi et al., 2001). Although photomorphogenesis under FR is very weak in these mutants, both showed stronger effects under continuous FR than under pulsed FR (angle between the cotyledons [°] in the wild type was 4 ± 1 in darkness, 108 ± 3 in pulsed FR, and 180 ± 0 in continuous FR; angle between the cotyledons in hy1 was 1 ± 1 in darkness, 3 ± 1 in pulsed FR, and 11 ± 2 in continuous FR; angle between the cotyledons in hy2 was 1 ± 1 in darkness, 5 ± 1 in pulsed FR, and 17 ± 5 in continuous FR). These results indicate that a reduction of photoactive phyA levels does not necessarily eliminate the HIR in Arabidopsis.
Weak Fluence Rate Dependence of the Effects of Continuous FR in phyA-302
Continuous FR Fails to Promote Anthocyanin Synthesis in phyA-302 In Arabidopsis, continuous FR promotes anthocyanin synthesis, but hourly pulses of FR have negligible effects on this process compared with dark controls (Yanovsky et al., 2000 ). This finding indicates that anthocyanin synthesis is dominated by the HIR component, whereas the VLFR component makes at most a weak contribution. If the phyA-302 mutations cause a general failure of HIR, the promotion of anthocyanin synthesis by continuous FR should be impaired severely in phyA-302-1 and phyA-302-2. This expectation was met by the data (Figure 2C). Thus, the phyA-302 mutants showed wild-type promotion of seed germination, intermediate inhibition of hypocotyl growth and cotyledon unfolding, and no promotion of anthocyanin by continuous FR (Figure 2). Across different processes, the effect of the mutations was inversely related to the effect of pulsed FR compared with continuous FR.
Inductive Responses in phyA-302
Analysis of the Interaction between phyA-302 and phyB Signaling The interaction between phyA and phyB signaling is synergistic when phyA operates in the HIR mode and antagonistic when phyA operates in the VLFR mode (Cerdán et al., 1999 ). To investigate whether the phyA-302 mutants retained these interactions, the seedlings were exposed daily to a brief pulse of R or FR in factorial combination with or without 3 h of FR (Figure 10A)
. The effect of R pulses compared with FR pulses is mediated by phyB (Cerdán et al., 1999). In seedlings exposed only to brief pulses, phyA operated in the VLFR mode, whereas in those seedlings exposed to 3 h of FR, phyA operated predominantly in the HIR mode. In the absence of 3-h FR exposures, wild-type seedlings failed to unfold the cotyledons in response to a daily pulse of R compared with FR (Figure 10B; the difference is shown in Figure 10C).
Because daily exposure to a pulse of R induces cotyledon unfolding in phyA null mutants but not in the phyA phyB double mutant, phyA signaling in the VLFR mode is predicted to antagonize phyB signaling (Cerdán et al., 1999 ). In wild-type seedlings exposed daily to 3 h of FR, a subsequent R pulse caused cotyledon unfolding. Both phyA and phyB were required for this amplification effect, indicating a synergistic action of both photoreceptors when phyA operates in the HIR mode (Cerdán et al., 1999). In the phyA-302 mutants, a pulse of R given alone was not effective (Figure 10). This finding indicates that in terms of the antagonistic effect, the mutated phyA molecule behaved like wild-type phyA. However, in contrast to what was seen in the wild type, daily exposure to 3 h of FR completely failed to amplify the response to a R versus FR test pulse (Figure 10). This finding indicates that in terms of the synergistic effect, the mutated phyA molecule lacks functional activity. In other words, in seedlings exposed to 3 h of FR followed by a R pulse, cotyledon unfolding was observed in the wild type (as a result of the amplification of the response by 3 h of FR) and in the phyA null mutant (in which the response to R is constitutive) but was very poor in the phyA-302 mutants (in which the constitutive response was minimal and amplification was null).
Novel phyA Alleles Affected in the PAS2 Domain Screening for mutants able to germinate but not to deetiolate normally under continuous FR resulted in the isolation of two independent phyA-302 plants carrying a missense mutation (Glu to Lys) at amino acid 777 (Figure 5). The traditional protocol for the screening of phyA mutants, which involves the induction of germination of chilled seeds by white light before transfer to FR (Nagatani et al., 1993 ; Parks and Quail, 1993; Whitelam et al., 1993), has yielded a number of null and leaky phyA alleles that do not coincide with the mutation reported here (Dehesh et al., 1993; Whitelam et al., 1993; Reed et al., 1994; Xu et al., 1995).
Several phyA and phyB mutations that do not alter phyA or phyB protein levels, spectral activity, or dimer formation are located between amino acids 632 and 768 (amino acid numbering based on the chromophore attachment site at Cys-323), particularly amino acids 715, 716, and 727 (Figure 5) (Quail et al., 1995
There is a second region within the C terminus, between amino acids 593 and 825, related to the His kinase domain of the cyanobacterial phytochrome Cph1 (Yeh and Lagarias, 1998
phyA-302 Mutants Retain VLFR but Lack HIR
The failure of HIR in phyA-302 is not a general (nonspecific) consequence of reductions in phyA signaling. The phyA-205 allele, which carries a missense mutation (Val to Met) at amino acid 631 (Reed et al., 1994
The loss of HIR in phyA-302 is not the consequence of reduced phyA levels. Although the phyA-302 mutation caused a 50% reduction in protein levels in dark-grown seedlings, this difference was compensated for by the greater stability of phyA under FR in phyA-302 than in the wild type (Figure 4D). Furthermore, reduced phyA levels do not eliminate HIR per se. The phyA-109 mutant has low protein levels but retains fluence rate–dependent inhibition of hypocotyl growth (Xu et al., 1995
The phyB-101 mutation involves a Glu-to-Lys change at the amino acid position equivalent to phyA-302, and the recombinant mutant protein produced by the phyB-101 allele shows enhanced Pfr-to-Pr dark reversion (Elich and Chory, 1997
The region between amino acids 730 and 821 serves as a homodimerization interface bearing two surface anti-parallel
The Glu-to-Lys mutation observed in phyA-302 mutants severely affected the fluence rate dependence of phyA action under FR (Figure 8), but the same mutation in the equivalent position of phyB did not impair fluence rate dependence under continuous R (Wagner and Quail, 1995
The wild type of Arabidopsis shows little morphological response to a daily pulse of R. This treatment, however, is effective in both phyA null mutants and wild-type seedlings exposed daily to a few hours of continuous FR before the R pulse. These observations have led to a model in which phyA in the VLFR mode (i.e., under R) negatively regulates phyB signaling, whereas phyA in the HIR mode (i.e., under continuous FR) positively regulates phyB signaling (Cerdán et al., 1999
phyA-302:GFP Fusion Proteins Fail to Produce Nuclear Speckles
In animal cells, many nuclear factors localize either partially or completely in distinct "bodies" or subnuclear compartments, some of which contain factors involved in the processing and transcription of RNA (Lamond and Earnshaw, 1998
Mutations that occur in the WD-40 repeat domain of COP1 (Stacey and von Arnim, 1999
Implications for phyA Signaling
Binding of nucleoside diphosphate kinase 2 to the C-terminal domain of phyA is disrupted by the phyA-103 mutation (Choi et al., 1999
Taking into account the fact that VLFR and HIR can be dissected genetically (Yanovsky et al., 1997
Plant Material Mutagenized seeds of Arabidopsis thaliana (ecotype Landsberg erecta) were purchased from Lehle Seeds (Round Rock, TX). For the mutant screening, the seeds were incubated in boxes (175 x 225 mm2 x 45 mm in height) containing 0.8% agar for 3 days at 7°C before transfer to the specific protocol conditions (Figure 1). The wild type was Landsberg erecta. phyA-201 (Nagatani et al., 1993 ), phyA-205 (Reed et al., 1994), and hy1 and hy2 (Koornneef et al., 1980; Davis et al., 1999; Muramoto et al., 1999; Kohchi et al., 2001) were included in some experiments for comparative purposes.
The construction of the 35S:PHYA:green fluorescent protein (GFP) chimeric gene containing the full-length PHYA cDNA from Arabidopsis in the binary vector pPCV 812 and its introduction in the phyA null background of Arabidopsis have been described (Kim et al., 2000
Experimental Setting Hypocotyl length was measured to the nearest 0.5 mm with a ruler, and the largest 10 seedlings from each box (i.e., one replicate) were averaged. The angle between the cotyledons was measured with a protractor in the same seedlings used for length measurements, and the 10 values obtained per box also were averaged before statistical analysis. For anthocyanin experiments, 50 seeds were sown per box according to the procedure used for hypocotyl growth experiments. After 3 days of light treatment, the seedlings were harvested for extraction with 1 mL of 1% (w/v) HCl methanol. Absorbance at 530 nm was measured and corrected for chlorophyll absorption by subtracting 0.25 absorbance at 657 nm. Hourly pulses of far-red light (FR; 3 min/h at 200 µmol·m-2·s-1) and continuous FR (10 µmol·m-2·s-1) were provided by incandescent lamps in combination with a water filter, a red acetate filter, and six 2-mm-thick blue acrylic filters (Paolini 2031; La Casa del Acetato, Buenos Aires, Argentina). The calculated Pfr/P was 10%. In the experiments in which responses were plotted against calculated Pfr/P, the seedlings were exposed to 3-min hourly light pulses (10 to 50 µmol·m-2·s-1). Incandescent lamps were combined with a water filter and an RG9 filter (calculated Pfr/P of 3%; Schott, Mainz, Germany), one red plus one green acetate filter (calculated Pfr/P of 33%), or one red acetate filter (calculated Pfr/P of 61%). R (calculated Pfr/P of 87%) was provided by light-emitting diodes.
Protein Extraction and Immunoblot Analysis The blot was incubated overnight at 7°C with 10 mL of this primary antibody at a dilution of 1:1000. After washing, the membrane was incubated with a 1:500 dilution of affinity-isolated alkaline phosphatase–conjugated antibody to mouse IgG developed in goat (Sigma). The bands were visualized by incubating the blots in 0.1 M Tris, pH 9.5, 100 mM NaCl, and 5 mM MgCl2 containing 0.165 mg/mL 5-bromo-4-chloro-3-indolyl phosphate (p-toluidine salt) and 0.33 mg/mL nitroblue tetrazolium (both Sigma).
Phytochrome Fluorescence For each experimental sample, three emission spectra usually were measured: (1) in the etiolated state (state 0); (2) after phototransformation induced by the saturating actinic light of Pr into the first photoproduct stable at low temperatures (lumi–R) (state 1); and (3) after Pr photoconversion into Pfr at 273K by monochromatic light at 680 nm (state 2). To obtain emission spectra of phytochrome, the spectrum of the phyA-201 phyB-1 Arabidopsis double mutant was subtracted from the experimental spectra of the samples after normalization at 660 nm (at which phytochrome emission is negligible), and the phytochrome difference spectrum was divided by the intensity of background emission at 660 nm to correct for sample mass. The noise level during the registration of the emission spectra was 5 to 7%, and the smoothing of the spectra was performed by eye.
DNA Sequencing
The mixture was heated to 94°C for 4 min and subjected to 35 cycles of 30 s at 94°C, 30 s at 55°C, and 210 s at 72°C. The reaction products were analyzed on 0.8% agarose gels. The size of the products was
Epifluorescence and Light Microscopy To minimize the nuclear import of fusion proteins induced by microscopic light, photographs of the GFP fluorescence were taken during the first minute of microscopic analysis. 4',6-Diamidino-2-phenylindole staining of nuclei was performed by incubating the seedlings in an aqueous solution containing 10% DMSO (v/v) and 10 µg/mL 4',6-diamidino-2-phenylindole. For optimal presentation, the images were processed using Adobe Photoshop 5.0 (Adobe Systems Europe, Edinburgh, UK).
We thank Peter Quail (University of California, Berkeley) for his kind provision of the phyA monoclonal antibody and the ABRC (Ohio State University, Columbus) for seed batches. This work was supported by grants from Fondo Nacional de Ciencia y Técnica (BID 1201/OC-AR-PICT 06739), the University of Buenos Aires (TG59), and the Fundación Antorchas (A-13622/1-40) to J.J.C., Consejo Nacional de Investigaciones Científicas y Técnicas Grant 4527/96 to R.J.S., a Howard Hughes Medical Institute International Scholar Fellowship, a Hungarian Foundation for Basic Science Grant (T-02584), and a Wolfgang Paul Award to F.N., Deutsche Forschungsgemeinschaft Grant SFB 592 to E.S., and Russian Foundation for Fundamental Research Grant 99-04-48730 to V.A.S.
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.000521.
1 Current address: Scripps Research Institute, La Jolla, CA 92037.
2 These authors contributed equally to this work.
3 Current address: INGEBI, Vuelta de Obligado 2490, 1428-Buenos Aires, Argentina. Received November 14, 2001; accepted March 19, 2002.
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Abstract
INTRODUCTION
-strands (amino acids 735 to 750 and 780 to 800) (Song, 1999