The Plant Journal (2005) 41, 146–161
doi: 10.1111/j.1365-313X.2004.02286.x
A new type of mutation in phytochrome A causes enhanced
light sensitivity and alters the degradation and subcellular
partitioning of the photoreceptor
Monika Dieterle†, Diana Bauer, Claudia Büche, Martina Krenz, Eberhard Schäfer and Thomas Kretsch*
Institut für Biologie 2/Botanik, Universität Freiburg, Schänzlestrasse 1, 79104 Freiburg, Germany
Received 21 September 2004; accepted 13 October 2004.
*
For correspondence (fax þ49 761 203 2612; e-mail [email protected]).
†
Present address: Institut de Biologie Moleculaire des Plantes (IBMP du CNRS); 12, rue du General Zimmer; 67084 Strasbourg Cedex, France.
Summary
A specific light program consisting of multiple treatments with alternating red and far-red light pulses was
used to isolate mutants in phytochrome A-dependent signal transduction pathways in Arabidopsis. Because of
their phenotype, the mutants were called eid for empfindlicher im dunkelroten Licht, which means
hypersensitive in far-red light. One of the isolated mutants, eid4, is a novel semi-dominant allele of the
phytochrome A gene that carries a missense mutation in the chromophore-binding domain. The mutation did
not change the photochemical properties of the photoreceptor, but it leads to an increased stability under light
conditions that induce its rapid degradation. Fusion proteins with the green fluorescent protein exhibited clear
alterations in subcellular localization of the mutated photoreceptor: The fusion protein was impaired in the
formation of sequestered areas of phytochrome in the cytosol, which can explain its reduced light-dependent
degradation. In contrast, the mutation stabilizes nuclear speckles (NUS) that appear late under continuous farred light, whereas the formation of early, transiently appearing NUS remained more or less unaltered.
Keywords: phytochrome, light signaling, Arabidopsis, photomorphogenesis, nuclear localization, protein
degradation.
Introduction
Phytochromes comprise a small protein family that mainly
function as red and far-red photoreceptors (Møller et al.,
2002; Neff et al., 2000). In Arabidopsis, five isoforms have
been described named phytochrome A (phyA) through
phyE. phyA is light labile and accumulates to very high
levels in the dark. It is responsible for the very low fluence
responses (VLFR) and for the far-red-light-dependent high
irradiance responses (HIR). VLFR can be induced with extremely low photon fluences between 0.001 and 1 lmol m)2.
The extent of the HIR depends on the duration and the fluence rate of irradiation. The action spectra of HIR show a
maximum at approximately 720 nm, a wavelength that
inhibits responses of the other phytochromes. The other
phytochromes are more stable in the light. They predominantly regulate responses under continuous red and white
light. The light stable forms are also responsible for the low
fluence responses (LFR) that exhibit the classical red/far-red
photoreversible characteristics of phytochrome function
(Møller et al., 2002; Neff et al., 2000).
146
Phytochromes of higher plants are dimers of approximately 125 kDa subunits. Every monomer can be divided
into two large domains that are connected by a hinge region.
The N-terminal domain carries a single covalently linked
linear tetrapyrrole chromophore (phytochromobilin), attached via a thioether bond to a conserved cysteine residue.
This domain determines whether a phytochrome molecule
exhibits the functional characteristics of a light labile phyA
or a light stable phyB photoreceptor (Wagner et al., 1996).
The extreme N-terminus is rich in Ser (phyA) or Gly (phyB)
residues and displays strong structural alterations upon
photoconversion from the red-light-absorbing Pr to the farred light-absorbing Pfr form (Møller et al., 2002; Neff et al.,
2000). Mutated phyA constructs carrying Ser–Ala substitutions at the extreme N-terminus resulted in an increased
biological activity when overexpressed in plants, whereas
deletions reduced its activity (Jordan et al., 1996;
Stockhaus et al., 1992). Biochemical analyses with oat
phyA revealed that Ser7 and Ser14 are phosphorylated
ª 2004 Blackwell Publishing Ltd
A new type of mutation in phytochrome A 147
in vivo in dark-grown and red light-irradiated seedlings
(Lapko et al., 1999). The N-terminal domain of phyB interacts
with the response regulator ARR4. This interaction leads to a
modulation of red light signaling (Sweere et al., 2001).
Phosphorylation of Ser598 in the hinge region of oat phyA
was found only in red light-treated plants (Lapko et al.,
1999). The functional importance of the hinge region was
also underlined by the isolation of an extremely hypersensitive phyB allele that carries a point mutation in a highly
conserved motif (Kretsch et al., 2000).
The C-terminal domain is responsible for the dimerization
of the photoreceptor molecules (Edgerton and Jones, 1992).
It carries two histidine kinase-related domains (HKRD) and
two motifs with homology to PAS (PER-ARNT-SIM) domains
that are located in the first HKRD (Møller et al., 2002; Neff
et al., 2000). The region encompassing the first HKRD and
the PAS motifs form a hot spot for missense mutations that
lead to a reduction in light responses (Wagner and Quail,
1995; Xu et al., 1995). The C-terminal half is involved in the
interaction with nucleoside diphosphate kinase 2 (Choi
et al., 1999), phytochrome kinase substrate 1 (Fankhauser
et al., 1999), and the basic helix-loop-helix transcription
factor PIF3 (phytochrome-interacting factor 3; Ni et al., 1998,
1999).
The subcellular partitioning of phytochromes in higher
plants is regulated by light (Kim et al., 2000; Kircher et al.,
1999, 2002; Sakamoto and Nagatani, 1996; Yamaguchi et al.,
1999). Upon light irradiation all phytochromes are imported
into the nucleus, where they can form NUS. Nuclear
localization of phyB-GFP (green fluorescent protein) fusion
proteins is controlled by a red/far-red reversible low-fluence
response mode and exhibits a characteristic fluence rate
dependency (Kircher et al., 1999). In contrast, nuclear import
of phyA-GFP exhibits characteristic VLFR and HIR modes of
action (Kim et al., 2000). Thus, the nuclear import of
photoreceptors exhibits a high similarity to the welldescribed phyA and phyB modes of function. Mutations in
the first HKRD and the PAS domains of phyA-GFP and phyBGFP fusion proteins, shown to reduce light signaling, did not
alter nuclear localization but impaired formation of NUS
(Kircher et al., 2002; Yanovsky et al., 2002).
A powerful tool to identify components of light signaling
cascades has been the screening for mutants and the
cloning of the respective genes (Møller et al., 2002; Neff
et al., 2000). To identify new mutants of the phyA signal
transduction pathway, we established a specific irradiation
program consisting of repetitive cycles of alternating 20 min
red/far-red light pulses (Büche et al., 2000). The red light
pulses decrease the level of light labile phyA, which results
in a loss of far-red light-dependent HIR. Using this screening
program, several mutants were isolated that can overcome
red-light-induced suppression of HIR (Büche et al., 2000;
Dieterle et al., 2001, 2003; Zhou et al., 2002). Because of their
increased far-red light sensitivity, these mutants were called
ª Blackwell Publishing Ltd, The Plant Journal, (2004), 41, 146–161
eid for empfindlicher im dunkelroten Licht, which means
more sensitive in far-red light. In this paper, we report the
isolation of eid4 that carries a missense mutation in a novel
sub-domain of phyA. This mutation causes enhanced light
sensitivity and alters the degradation and subcellular partitioning of the photoreceptor.
Results
Isolation of the mutant
eid4 was screened from pools of ethylmethylsulfonatetreated phyB-5 seeds. The isolated eid4 phyB-5 mutant was
backcrossed with Landsberg erecta (Ler) wild type to obtain
eid4 mutants in a PHYB wild-type background. Both, the eid4
phyB-5 double and eid4 single mutants remained etiolated
in darkness similar to their background lines (Figure 1a).
Thus, eid4 clearly differs from the cop/det/fusca-class of
mutants that exhibit a de-etiolated phenotype even in darkness (Hardtke and Deng, 2000; Møller et al., 2002). Under
the alternating red/far-red light treatment used for screening and under continuous weak far-red light, eid4
mutants always exhibited a much stronger photomorphogenic response when compared with their background lines
(Figure 1b,c). In contrast, the mutant lines were undistinguishable from their background lines under continuous
strong far-red, red, and white light (Figure 1d–f).
The eid4 mutant exhibits an enhanced high-irradiance
response
To analyze the responsiveness toward continuous red and
far-red light in greater detail, fluence rate response curves
for hypocotyl elongation were determined (Figure 2a,b).
Relative hypocotyl lengths were calculated in relation to the
length of the respective dark controls.
Under continuous red light inhibition of hypocotyl elongation was always slightly increased in the eid4 phyB-5
double mutant compared with its phyB-5 background. The
difference between eid4 phyB-5 and phyB-5 remains more or
less constant over the whole range of the applied fluence
rates with the exception of the lowest fluence rate applied
(Figure 2a). The effect of phyB became first detectable at a
red light fluence rate of 0.008 lmol m)2 sec)1, when both
lines carrying the wild-type PHYB allele exhibited an
increased light response (Figure 2a). Again the fluence rate
response curves of eid4 and the Ler wild type run in parallel
over the whole range of fluence rates applied, whereby the
mutant always shows a stronger inhibition. In principle, the
shape of the eid4 fluence rate response curve can be
obtained by adding the increased light response of the
eid4 phyB-5 line to the fluence rate response curve of the Ler
background. Thus, the effect of the eid4 mutation in red light
seems to be independent of the presence of phyB.
148 Monika Dieterle et al.
Figure 1. Phenotypes of phyB-5, eid4 phyB-5,
Ler wild type, and eid4 seedlings.
(a–f) Comparison of 4-day-old phyB-5, eid4 phyB5, Ler and eid4 seedlings (from left to right)
grown in darkness (a), the screening program (b), continuous weak far-red light
[140 nmol m)2 sec)1 (c)], continuous strong farred light [14 lmol m)2 sec)1 (d)], continuous red
light [14,3 lmol m)2 sec)1 (e)], and continuous
white light [23 lmol m)2 sec)1 (f)]. Bars ¼ 2 mm.
Under continuous far-red light the eid4 phyB-5 and eid4
mutants exhibited a clearly increased light sensitivity (Figure 2b). The fluence rate response curves for eid4 phyB-5
and eid4 were almost identical again indicating that the
mutant phenotype is independent of the presence of phyB.
The eid4 lines showed a greater inhibition of hypocotyl
elongation even at the lowest fluence rates applied.
Additional fluence rate response curves at wavelengths
between 690 and 742 nm were measured for eid4 phyB-5
and phyB-5 to calculate action spectra for hypocotyl
elongation. The overall shape of these fluence rate
response curves was similar to the curve shown in
Figure 2b (data not shown). Fluence rates that led to a
relative hypocotyl length of 0.6 were determined using
linear regression in the log-linear range of the fluence rate
response curves. To construct the action spectra, the value
obtained for phyB-5 at 716 nm (0.137 lmol m)2 sec)1) was
set to 1 and relative photon effectiveness for the single and
double mutants at different wavelengths was calculated
accordingly.
The phyB-5 mutant exhibited a typical HIR action spectrum with a maximum at 716 nm (Figure 2c). Light sensitivity of the eid4 phyB-5 double mutant was higher at all
analyzed wavelengths. Although the maximum in light
sensitivity remained at 716 nm for eid4 phyB-5, the overall
shape of the action spectrum was changed. The differences
became more obvious by setting the maximum of the eid4
phyB-5 action spectrum also to 1 (Figure 2d). Compared with
the phyB-5 action spectrum, relative light sensitivity was
clearly more enhanced at wavelengths between 690 and
716 nm, whereas at wavelengths above 716 nm the influence of the eid4 mutation was weaker.
The Eid4 phenotype is caused by a missense mutation in a
conserved phytochrome A domain
Backcrosses with phyB-5 and analysis of the F1 and F2 generations demonstrated that eid4 is a semi-dominant mutation (data not shown). To create a mapping population, eid4
phyB-5 was crossed with the phyB-9 mutant from a Columbia (Col) background. The eid4 mutation was localized to the
top of chromosome 1 very close to the PHYA gene. No
recombination was detectable with a PHYA cleaved amplified polymorphic sequence (CAPS) marker after the analysis
of 100 plants (200 chromatids). Therefore, the PHYA gene
from eid4 was sequenced after amplification using polymerase chain reaction (PCR). Sequence analysis revealed a
single G–A nucleotide transition that leads to the exchange
of Glu229 by a Lys amino acid residue (Figure 3a). The sequence starting from Met219 to Met240 surrounding the
mutated Glu residue is completely conserved in all analyzed
PHYA genes of dicotyledonous species. Similar motifs were
detectable in the open reading frames of grass PHYA genes
and in the Arabidopsis PHYC gene (Figure 3b). The conserved Glu229 residue is missing in PHYB and the homologous PHYD and PHYE genes and the sequence around the
mutated residue is less conserved. The mutation localized in
the GAF domain (from cGMP-regulated cyclic phosphodiesterases, adenylyl cyclases, and the bacterial transcription
factor FhlA), which is probably involved in the binding of the
phytochromobilin chromophore (Figure 3c). Information of
the sequence analyses was used to create an eid4-specific
CAPS marker. Again no recombination between this CAPS
marker and eid4 was detectable after the analyses of 200
additional plants (400 chromatids).
ª Blackwell Publishing Ltd, The Plant Journal, (2004), 41, 146–161
A new type of mutation in phytochrome A 149
The mutation does not alter the photochemical properties of
phytochrome A
To analyze whether the eid4 mutation leads to alterations in
the photochemical properties of the photoreceptor, in vivo
photoconversion kinetics were measured under weak red
light (665 nm DEPIL interference light at 0.2 lmol m)2 sec)1).
Photoconversion in etiolated eid4 phyB-5 seedlings and
phyB-5 was very similar and the values of the individual
measurements were very often overlapping. Both mutant
lines reached photoequilibrium after about 7.5–10 min of
irradiation (Figure 4a).
Values for wavelength-dependent photoequilibria were
determined between 652 and 724 nm using in vivo spectroscopy. Differences in the photoequilibria of phytochromes
are most pronounced in this part of the light spectrum,
because the absorption of the Pr and Pfr forms overlap at
these wavelengths (Mancinelli, 1994). Thus, putative changes in the absorption of wild-type phyA and the mutated
eid4 phyA should be detectable under these light qualities.
Measurements of the photoequilibria showed no differences
for eid4 phyB-5 and phyB-5 seedlings (Figure 4b).
Dark reversion, the passive recovery of the Pr form from
the Pfr form without far-red light treatment, is another
property of the photoreceptor that might have been altered
by mutation (Eichenberg et al., 1999, 2000). Therefore, dark
reversion of phyA was measured after a single saturating red
light pulse. No dark reversion was detectable in the
mutant (Figure 4c) similar to its respective background line
(Eichenberg et al., 2000). Taken together, the data sets
presented in Figure 4 clearly indicate that the wild type
and the mutated phyA photoreceptor molecules have very
similar photochemical properties.
Phytochrome A degradation in the mutant is delayed under
light conditions that induce fast proteolysis of the
photoreceptor
One reason for the observed hypersensitivity of eid4 under
the screening program might be a slower degradation or an
Figure 2. Fluence rate response curves and action spectra for inhibition of
hypocotyl elongation under continuous light.
Hypocotyls were measured in 4-day-old plants. The relative hypocotyl lengths
were determined in relation to the length of the dark-grown seedlings for each
line. Hypocotyl length for dark controls were 9.6 0.2 mm for phyB-5,
8.2 0.1 mm for eid4 phyB-5, 9.8 0.2 mm for Ler wild type, and
8.4 0.1 mm for eid4 (mean SEM).
(a) Fluence rate response curves for continuous red light.
(b) Fluence rate response curves for continuous far-red light.
(c) Action spectrum for hypocotyl elongation. Fluence rate response curves
for different wavelengths were used to determine the fluence rate that induces
a relative hypocotyl length of 0.6. The value obtained for phyB-5 at 716 nm
was set to 1.
(d) Corrected action spectrum for hypocotyl elongation. The action spectrum
of (c) was recalculated by setting the maximum of the eid4 phyB-5 action
spectrum also to 1.
ª Blackwell Publishing Ltd, The Plant Journal, (2004), 41, 146–161
150 Monika Dieterle et al.
increased steady-state level of the mutated phyA photoreceptor. The phyA level per fresh weight was always reduced
in etiolated eid4 phyB-5 seedlings reaching about two-thirds
of the level of the respective phyB-5 background line [Figure 5a and Figure S1(a)]. Similar results were also obtained
using a phyA-specific antibody (data not shown). In darkness phyA levels remained more or less stable for at least
12 h, the maximum space of time, which was analyzed in
subsequent experiments [Figure 5a and Figure S1(b)].
Proteolysis of phyA was analyzed in 3-day-old etiolated
seedlings treated with different numbers of repetitive
20 min red/far-red light pulse cycles that were used for
screening of eid mutants. In phyB-5 seedlings phyA was
rapidly degraded and the amount fell below the detection
Figure 3. The eid4 mutant carries a missense mutation in the PHYA gene.
(a) Nucleotide and protein sequence of the wild type and the mutated PHYA
gene.
(b) The amino acid sequence in the region of the phyA(eid4) mutation is
compared with sequences of the wild-type phytochromes from Arabidopsis
thaliana (At), Nicotiana tabacum (Nt), Pisum sativum (Ps), Zea mays (Zm), and
Oryza sativa (Os). The proposed a-helix 3 of the GAF domain is indicated by
dashes (-). The mutated Glu residue in phyA(eid4) is underlined.
(c) Diagram of the Arabidopsis phyA protein. The GAF domain, the PAS
domains (PAS1 and PAS2), and the histidine kinase-related domain (HKRD) at
the C-terminus are indicated by boxes. The attachment site of the phytochromobilin chromophore (C323) is marked by a black triangle. Arabidopsis phyA
missense mutations in the GAF domain are given relative to their position in
the protein.
Figure 4. Analyses of the photochemical properties of phyA and phyA(eid4)
by in vivo spectroscopy.
(a) Photoconversion kinetics under continuous red light. Three-day-old
etiolated seedlings were irradiated with weak red light and the Pfr level in
relation to the total phyA level (Ptot) was determined at the given time points.
Pfr/Ptot at photoequilibrium was assumed to be 0.87 (Mancinelli, 1994).
(b) Relative amount of Pfr/Ptot at photoequilibrium. Three-day-old etiolated
seedlings were irradiated with saturating interference filter light on ice for
15 min. Each data point represents one individual measurement. At each
wavelength the Pfr level was at least detected twice for phyB-5 and eid4 phyB5. Under 665 nm light Pfr/Ptot was assumed to be 0.87 for the wild type
(Mancinelli, 1994). The solid line is obtained by fitting the measured Pfr/Ptot
values with the SigmaPlot 4.0 Regression Wizard (sigmoid, three parameters).
The dotted line gives the calculated photoequilibrium determined for purified
oat phyA (Mancinelli, 1994).
(c) Measurement of dark-reversion in the eid4 phyB-5 mutant. Ptot levels of 3day-old etiolated seedlings were set to 1. Maximum Pfr levels (assumed to be
0.87) were adjusted by treating 3-day-old etiolated seedlings with a saturating
red light pulse. Ptot and Pfr levels were measured at the given time points
after transfer to darkness. Pr levels were calculated as differences between
Ptot and Pfr levels. Data points were fitted with the SigmaPlot 4.0 Regression
Wizard (exponential decay, three parameters).
ª Blackwell Publishing Ltd, The Plant Journal, (2004), 41, 146–161
A new type of mutation in phytochrome A 151
Figure 5. The eid4 mutation leads to alterations in light-dependent phyA degradation.
Three-day-old etiolated seedlings were either kept in darkness or were exposed to different light treatments. Total phyA levels were measured spectroscopically at
the given time points and were normalized to the fresh weight of the samples. The symbols together with the error bars represent the mean SE for at least four
independent experiments. Data points were fitted with the SigmaPlot 4.0 Regression Wizard (exponential decay, two parameters for continuous red light treatment
and three parameters for continuous 705 nm and far-red light).
(a) PhyA levels of etiolated phyB-5 and eid4 phyB-5 seedlings. Levels of dark controls were determined during the measurements of degradation kinetics under
continuous 705 nm and far-red light.
(b) Degradation kinetics under multiple red/far-red light pulses. Dark-grown seedlings were irradiated with multiple pulses of 20 min red followed by 20 min of farred light that were used for the screening of eid mutants (Büche et al., 2001). Spectroscopical measurements were performed immediately after the last far-red light
treatment.
(c) Degradation kinetics under continuous red light (3.9 lmol m)2 sec)1).
(d) Degradation kinetics under continuous 705 nm light (1.9 lmol m)2 sec)1).
(e) Degradation kinetics under continuous far-red light (20 lmol m)2 sec)1).
level after six red/far-red light cycles. Although the phyA
content was lower in the mutant before the start of the light
treatment, it became higher after two red/far-red cycles. In
contrast to the wild-type protein phyA(eid4) levels remained
ª Blackwell Publishing Ltd, The Plant Journal, (2004), 41, 146–161
high up to at least six light pulse cycles [Figure 5b and
Figure S1(c)]. Thus, eid4 phyB-5 seedlings clearly exhibited
a reduced degradation rate and an increased level of the
photoreceptor under screening conditions.
152 Monika Dieterle et al.
A slower degradation rate for phyA(eid4) was also
detectable under continuous strong red light [standard red
light field; k (max) ¼ 650 nm; 3.9 lmol m)2 sec)1]. The
apparent half-life of phyA(eid4) was about 70 min compared
with 40 min for wild-type phyA [Figure 5c and Figure S1(d)].
Nevertheless, the photoreceptor molecule was still rapidly
degraded in the mutant and fell below the detection level
after about 4 h of continuous irradiation, about 1 h later
compared with its wild-type counterpart.
The difference in the degradation kinetics between the
wild type and the mutant was more pronounced under
continuous 705 nm light [1.9 lmol m)2 sec)1; Figure 5d and
Figure S1(e)]. The half-life of phyA(eid4) was clearly
enhanced (2.7 h compared with 1.3 h in the wild type)
and the apparent steady-state level reached about twice
the level observed for the wild-type protein.
Interestingly, phyA degradation rates exhibited nearly no
difference under strong continuous far-red light [standard
far-red light field; k (max) ¼ 730 nm; 20 lmol m)2 sec)1].
The half-life of the photoreceptor molecules (approximately
3 h) was quite similar in both lines [Figure 5e and Figure S1(f)]. Because of the nearly identical half-life of both
forms under continuous far-red light, the level of wild-type
phyA remained higher compared with its mutated form
(Figure 5e). Taken together, the degradation kinetics clearly
indicates that differences in the proteolysis between phyA
and phyA(eid4) are restricted to light qualities which form
high levels of Pfr and thus induce a rapid and nearly
complete degradation of the photoreceptor.
The results obtained for the degradation kinetics showed
that phyA(eid4) retains increased steady-state levels of the
photoreceptor only under certain light conditions. Because
increased phyA levels under continuous far-red light are
regarded as a prerequisite for the induction of HIR (Holmes
and Schäfer, 1981; Mancinelli, 1994), wavelength-dependent
phyA stability was examined in greater detail. To estimate
steady-state levels, 3-day-old etiolated seedlings were irradiated with continuous interference-filter-light of different
wavelengths at photon fluence rates of 1.9 lmol m)2 sec)1
for 8 h. The amount of photoreversible phyA was determined by in vivo spectroscopy immediately after the end of
the light treatments.
Photoreceptor levels fell below the level of detection in
eid4 phyB-5 and phyB-5 seedlings after 8 h of irradiation
with 690 nm light (Figure 6a) similar to the results obtained
for continuous red light treatments (Figure 5c). Between 699
and 708 nm, eid4 phyB-5 seedlings always maintained a
higher amount of phyA (Figure 6a and Figure S2). Under 716
and 718 nm light both lines exhibited very similar levels
(Figure 6a and Figure S2). At wavelengths above 718 nm
phyA(eid4) levels were always low compared with wild-type
phyA (Figure 6a), although the relative amounts were similar when calculated according to the respective dark
controls (Figure S2).
Figure 6. Phytotchrome A levels after prolonged irradiation with light of
different wavelengths.
Three-day-old seedlings were irradiated with strong interference filter light
(1.9 lmol m)2 sec)1) for 8 h before phyA levels were detected by in vivo
spectroscopy. The symbols together with the error bars represent the
mean SE of at least three independent measurements.
(a) D(DA) values are normalized to the fresh weight of seedlings.
(b) Calculated levels of the Pfr form. Pfr levels were calculated using the phyA
levels shown in (b) and the regression curve for Pfr/Ptot levels given in
Figure 4(b).
The data presented in Figures 4b and 6a were used to
estimate Pfr levels in eid4 phyB-5 and phyB-5 under steadystate conditions. According to these calculations maximum
Pfr levels are expected at 716 nm in phyB-5 seedlings,
whereas maximum Pfr levels should accumulate at wavelengths between 699 and 708 nm in the eid4 phyB-5 double
mutant (Figure 6b).
The phyA(eid4)-GFP fusion protein is a functional
photoreceptor that mediates an increased light sensitivity
To compare the subcellular partitioning of mutated and
wild-type phyA, GFP fusion proteins were expressed in wildtype plants under the control of a 35S promoter. All further
experiments were performed with phyA-GFP (Kim et al.,
2000; Kircher et al., 2002) and phyA(eid4)-GFP lines that
ª Blackwell Publishing Ltd, The Plant Journal, (2004), 41, 146–161
A new type of mutation in phytochrome A 153
exhibited comparable expression levels for the fusion proteins in dark-grown seedlings (Figure S3).
The clearest difference in the degradation kinetics
between wild type and mutant phyA was detected at
705 nm (Figure 5d). Therefore, degradation of phyA- and
phyA(eid4)-GFP was followed under this light quality using
immunoblots together with GFP and phyA antisera (Figure 7a). The results clearly demonstrate that the mutated
form of the fusion protein is more stable against lightinduced degradation similar to the results obtained with the
phyA(eid4) mutant. Furthermore, the data obtained with the
phyA antisera shows that, compared with the high levels of
the endogenous phyA, only very low levels of phyA- and
phyA(eid4)-GFP fusion proteins accumulated in the transgenic lines (Figure 7a).
To test whether phyA(eid4)-GFP is functional in transgenic
seedlings, fluence rate response curves were determined for
the inhibition of hypocotyl elongation under continuous 715
and 705 nm light. Under 715 nm light phyA-GFP and
phyA(eid4)-GFP lines exhibited a hypersensitive phenotype,
whereby the mutated version of the photoreceptor induced a
stronger effect (Figure 7b). In a second experiment 705 nm
light was used, because a very strong difference in light
sensitivity was detected between phyB-5 and eid4 phyB-5
seedlings under this wavelength (Figure 2c). Fluence rate
response curves of transgenic phyA-GFP lines and the wildtype background were nearly identical under continuous
705 nm light, which is most probably due to the low levels of
the expressed chimeric protein and the low sensitivity of the
HIR signaling system toward this wavelength (Figure 7c). In
contrast, phyA(eid4)-GFP lines exhibited a clearly enhanced
light sensitivity (Figure 7c). Taken together, the physiological data demonstrate that phyA(eid4)-GFP is a functional
photoreceptor which mediates an increased far-red light
sensitivity.
The eid4 mutation leads to alterations in the formation of
sequestered areas of phytochrome in the cytosol
Subcellular localization of wild-type phyA-GFP and the
mutated phyA(eid4)-GFP was analyzed in 3-day-old
etiolated seedlings that were irradiated with saturating red
light pulses or continuous 705 nm and 720 nm light.
Localization analyses were performed using the homozygous phyA-GFP and phyA(eid4)-GFP lines described above.
Most of the analyses were performed in cells of the upper
half of the hypocotyl, which accumulate a high level of the
photoreceptor in wild type.
In etiolated seedlings both GFP fusion proteins remained
in the cytosol and exhibited a diffuse staining (Figure 8a,a¢).
In phyA-GFP lines a high number of cytosolic speckles or
sequestered areas of phytochrome (SAP) formed upon red
light treatments (Figure 8b,c). The SAP disappeared when
seedlings were transferred back to darkness for 90 min after
ª Blackwell Publishing Ltd, The Plant Journal, (2004), 41, 146–161
Figure 7. Light-dependent degradation of phyA- and phyA(eid4)-GFP fusion
proteins and inhibition of hypocotyl elongation in transgenic lines.
(a) Degradation kinetics of phyA- and phyA(eid4)-GFP fusion proteins under
continuous 705 nm light (1.9 lmol m)2 sec)1). Extracts were isolated from 4day-old etiolated seedlings that were harvested before irradiation (D) or at the
given time points after the onset of irradiation. Fusion proteins were detected
using an antiserum against GFP (a-GFP) or phyA (a-phyA; Kircher et al., 2002).
WT, wild-type control. As a loading control the membrane of the immunoblot
was stained with Ponceau red (control). AGFP, phyA-GFP fusion protein. *,
unspecific side band.
(b) Inhibition of hypocotyl elongation under continuous 715 nm light.
Hypocotyls were measured in 4-day-old plants. The relative hypocotyl lengths
were determined in relation to the length of the dark-grown seedlings for each
line. Hypocotyl lengths for dark controls were 9.5 1.1 mm for wild type,
8.3 1.1 mm for phyA-GFP1, 8.0 0.9 mm for phyA-GFP2, 7.7 1.1 mm for
phyA(eid4)-GFP1, and 8.0 1.2 mm for phyA(eid4)-GFP2 (mean SE).
(c) Inhibition of hypocotyl elongation under continuous 705 nm light.
Hypocotyls were measured in 4-day-old plants. The relative hypocotyl lengths
were determined in relation to the length of the dark-grown seedlings for each
line. Hypocotyl lengths for dark controls were 9.4 1.0 mm for wild type,
8.7 1.1 mm for phyA-GFP1, 8.1 1.2 mm for phyA-GFP2, 9.4 1.2 mm for
phyA(eid4)-GFP1, and 7.5 1.1 mm for phyA(eid4)-GFP2 (mean SE).
154 Monika Dieterle et al.
Figure 8. Formation of sequestered areas of phytochrome (SAP) in the cytosol under different light treatments.
Transgenic seedlings expressing phyA-GFP and phyA(eid4)-GFP were etiolated for 3 days before the onset of irradiation. Pictures taken from characteristic
hypocotyl cells of phyA-GFP lines are marked by letters without dash (a–h), those from phyA(eid4)-GFP lines are marked by letters with a dash (a¢–h¢). Pictures from
seedlings of the different lines that obtained the same light treatment were given the same letters. Plastids (pl) are indicated. Bars ¼ 10 lm.
(a, a¢) Dark controls.
(b, b¢–d, d¢) Etiolated seedlings treated with continuous red light (14.3 lmol m)2 sec)1) for 1 min (b, b¢) and 5 min (c, c¢).
(d, d¢) Etiolated seedlings were treated with a 5-min red light pulse (14.3 lmol m)2 sec)1) to induce SAP formation before transfer to darkness for 90 min.
(e, e¢ and f, f¢) Etiolated seedlings were treated with continuous 705 nm light (1.9 lmol m)2 sec)1) for 5 min (e, e¢) and 90 min (f, f¢).
(g, g¢ and h, h¢) Etiolated seedlings were treated with continuous 720 nm light (1.9 lmol m)2 sec)1) for 5 min (g, g¢) and 90 min (h, h¢).
an inductive 5-min red light pulse (Figure 8d). In contrast to
phyA-GFP lines, no or only a very low number of SAP
became detectable with the phyA(eid4)-GFP construct upon
red light treatments (Figure 8b¢,c¢). Similar to the wild type
construct, the few remaining SAP disappeared after transfer
back to darkness (Figure 8d¢).
Under continuous 705 nm light, cytosolic SAP are formed
in phyA-GFP lines at a slightly reduced number due to the
lower Pfr level adjusted by this light quality (Figure 8e). SAP
completely disappeared after 90 min of continuous irradiation with 705 nm light (Figure 8f). In contrast, SAP were
detectable only in a few very rare cases with phyA(eid4)GFP under continuous 705 nm light (Figure 8e¢). Under farred light conditions no cytosolic speckles were detectable
with both constructs although a strong diffuse fluorescence
was visible in the cytosol (Figure 8g,h,g¢,h¢). Taken together, these data clearly indicate that the chimeric
phyA(eid4)-GFP protein has a reduced affinity toward
ª Blackwell Publishing Ltd, The Plant Journal, (2004), 41, 146–161
A new type of mutation in phytochrome A 155
cytosolic SAP, which are formed by the endogenous phyA
in the transgenic lines.
The eid4 mutation shows an altered subnuclear partitioning
of the photoreceptor
In etiolated seedlings phyA-GFP and phyA(eid4)-GFP proteins remained in the cytosol (Figure 9a). Both types of GFP
fusion proteins entered the nucleus under all applied light
qualities nearly immediately after the onset of irradiation,
where they formed NUS (Figure 9b,b¢,c,c¢,e,e¢,i,i¢). These
rapidly formed NUS accumulated only transiently and disappeared after about 60–90 min of continuous irradiation or
when the seedlings were transferred back to darkness after
the induction of speckle formation with a red-light pulse.
Although early NUS disappeared, a clear diffuse staining
always remained in the nucleoplasm (Figure 9d,f,f¢,j,j¢).
At wavelengths below 720 nm, speckles did not reaccumulate in the nucleus under prolonged irradiation in
the phyA-GFP lines (Figure 9g,h; unpublished results). Under
continuous far-red light, NUS were again formed with the
wild-type construct after 4 h and they remained stable for at
least 8 h (Figure 9k,l). In contrast to phyA-GFP, NUS carrying
phyA(eid4)-GFP re-appeared not only under continuous
irradiation with far-red light, but also at wavelengths
between 695 and 705 nm (Figure 9g¢,h¢,k¢,l¢; unpublished
results). With the mutated photoreceptor NUS also became
again detectable 90 min after an inductive red-light pulse
(Figure 9d¢). Thus, the data obtained for the wild type and the
mutated phyA-GFP fusion proteins clearly indicate that the
photoreceptor is able to form at least two different types of
NUS: an early transient type and another type, which appears
late under light conditions that allow the accumulation of
relatively high levels of phyA under continuous irradiation.
Discussion
The hypersensitive phenotype of the eid4 mutant is caused
by a missense mutation in the PHYA gene
In this study we describe the identification and characterization of eid4, a mutant that exhibits an increased light
sensitivity under light conditions, which promote
phyA-specific light responses. The phenotype is caused by
an amino acid transition in the phyA photoreceptor molecule, which was verified by mapping analyses, sequencing
of the respective gene, and the hypersensitive phenotype of
transgenic phyA(eid4)-GFP lines. The observed semi-dominant phenotype is also in good agreement with this finding,
because the effect of a hyperactive receptor should be
detectable even in the presence of its wild-type counterpart.
Except for phyA(eid4) only one additional phytochrome
allele (phyB-401) is known where one single amino acid
transition leads to a hypersensitive phenotype. The phyB401 amino acid exchange was localized to the flexible hinge
region, which connects the N-terminal and C-terminal subdomains of the protein. The mutated phyB-401 photoreceptor exhibits a loss of photoreversibility and a reduced dark
reversion (Kretsch et al., 2000). The substitution of the first
10 S by A residues at the extreme N-terminus of a rice phyA
gene also resulted in a hypersensitivity of the photoreceptor
in transgenic tobacco plants (Stockhaus et al., 1992).
The phyA(eid4) photoreceptor leads to an increased light
sensitivity although its level is reduced in etiolated seedlings. A similar reduction in phyA levels has been observed
for phyA-302 seedlings, which carry a missense mutation in
the PAS2 domain of phyA (Yanovsky et al., 2002). The
reduced amount of phyA(eid4) in darkness might be caused
by an altered localization of the molecule in the cytosol,
which might alter the accumulation of its Pr form. It also
cannot be excluded that the light treatment necessary for
germination induction already can reduce photoreceptor
accumulation in the hypersensitive mutant, because PHYA
gene expression is inhibited by phyA function in a negative
feedback loop (Canton and Quail, 1999).
The domain altered in phyA(eid4) is involved in the
regulation of phytochrome A-specific light responses
The mutation in phyA(eid4) leads to an exchange of Glu229
by a Lys residue. The mutated residue is localized to the GAF
domain, which is present in all known phytochromes (Ho
et al., 2000). The functional importance of the domain is
underlined by the high number of missense mutations that
cause a reduced light response in phyA (Arg279Ser,
Gly367Ser; Xu et al., 1995) and phyB lines (Cys349Thr;
Figure 9. Formation of nuclear speckles (NUS) under different light treatments.
Transgenic seedlings expressing phyA-GFP and phyA(eid4)-GFP were etiolated for 3 days before the onset of irradiation. Pictures taken from phyA-GFP lines are
marked by letters without dash (a–l), pictures taken from phyA(eid4)-GFP lines are marked by letters with a dash (a¢–l¢). Pictures from seedlings of the different lines
that obtained the same light treatment were given the same letters. Plastids (pl) are indicated. Bars ¼ 10 lm.
(a, a¢) Representative nuclei of dark controls. The position of the nuclei is marked by a hatched oval.
(b, b¢–d, d¢) Representative nuclei of etiolated seedlings treated with continuous red light (14.3 lmol m)2 sec)1) for 1 min (b, b¢) and 5 min (c, c¢).
(d, d¢) Representative nuclei of etiolated seedlings that were treated with a 5-min red light pulse (14.3 lmol m)2 sec)1) to induce NUS formation before transfer to
darkness for 90 min.
(e, e¢–h, h¢) Representative nuclei of etiolated seedlings that were treated with continuous 705 nm light (1.9 lmol m)2 sec)1) for 5 min (e, e¢), 90 min (f, f¢), 4 h (g, g¢),
and 8 h (h, h¢).
(i, i¢–l, l¢) Representative nuclei of etiolated seedlings that were treated with continuous 720 nm (1.9 lmol m)2 sec)1) light for 5 min (i, i¢), 90 min (j, j¢), 4 h (k, k¢), and
8 h (l, l¢).
ª Blackwell Publishing Ltd, The Plant Journal, (2004), 41, 146–161
156 Monika Dieterle et al.
(b)
(a)
(c)
(d)
NUS
nuc
cD
(a¢)
1¢
(b¢)
5¢
(c¢)
5¢ + 90¢D
(d¢)
nuc
pl
cD
(e)
1¢
(f)
5¢
(g)
5¢ + 90¢D
(h)
pl
pl
5¢
(e¢)
90¢
(f¢)
4h
(g¢)
8h
(h¢)
pl
5¢
(i)
90¢
(j)
5¢
(i¢)
4h
(k)
90¢
(j¢)
8h
(l)
4h
(k¢)
8h
(l¢)
NUS
5¢
90¢
4h
8h
ª Blackwell Publishing Ltd, The Plant Journal, (2004), 41, 146–161
A new type of mutation in phytochrome A 157
Bradley et al., 1996; Cys327Tyr, Ala372Thr; Chen et al.,
2003). The GAF domain overlaps with the chromophorebinding domain of phytochromes. According to the published structural model (Ho et al., 2000, 2001), Glu229 should
not directly participate in the interaction with the phytochromobilin chromophore. This interpretation is underlined
by the fact that the spectroscopic properties of wild-type
phyA and phyA(eid4) are almost identical. Both photoreceptor molecules exhibited similar photoconversion rates in
weak red light, similar Pfr/Ptot levels under different wavelengths, and no dark reversion.
The amino acid sequences upstream and downstream of
Glu229 are completely conserved in all analyzed dicot PHYA
genes and exhibited a high homology to PHYA genes from
monocot species and to Arabidopsis PHYC. The conserved
domain (Met219–Thr234) overlaps with a-helix 3 predicted
for GAF domains of phytochromes and several other
proteins (Ho et al., 2000). In PHYB and PHYB-homologous
genes the sequence of the proposed a-helix is less conserved and Ser or His residues replace the conserved Glu
amino acid. The proposed a-helix 3 is involved in the
formation of the outer layer of the structure and thus, it
should be accessible for interactions with other proteins.
The exchange of a negatively charged Glu by a positively
charged Lys clearly alters functions that are highly specific
for phyA, that is, the light-dependent degradation of the
photoreceptor and binding to SAP in the cytosol. Thus, the
respective domain might be responsible for some specificity
that separates the physiological functions of the phyA
photoreceptor from other phytochromes. This hypothesis
is consistent with the results of domain swap experiments,
where the N- and C-terminal halves of phyA and phyB were
exchanged (Clough et al., 1999; Wagner et al., 1996). In both
studies phyA chimera carrying the N-terminal half of the
protein with the GAF domain were light labile and exhibited
phyA functions, whereas phyB/A chimera were light stable
and behaved like a phyB photoreceptor.
The missense mutation in phyA(eid4) causes an altered
subcellular localization of the photoreceptor
In order to analyze the subcellular localization of phyA-GFP
and phyA(eid4)-GFP, transgenic lines were used that expressed only very low amounts of the introduced genes in a
wild-type background with normal phyA levels. The introduced phyA-GFP protein had only a very small positive effect on light sensitivity in the transgenic lines, which reduces
the possibility of artifacts that might be caused by extreme
overexpression of the chimeric photoreceptor. Furthermore,
microscopic analyses were restricted to hypocotyl cells that
contain high levels of phyA to avoid putative problems due
to ectopic expression of the transgene. In fact, earlier studies
and our own observations clearly have demonstrated that
the 35S-promoter::PHYA-GFP lines used in this study and
ª Blackwell Publishing Ltd, The Plant Journal, (2004), 41, 146–161
phyA null lines that carry PHYA-promoter::PHYA-GFP construct exhibited nearly identical results regarding their subcellular localization under different light treatments (Kim
et al., 2000; Kircher et al., 1999, 2002; unpublished results).
Thus, the analyzed phyA-GFP and phyA(eid4)-GFP lines
should be a versatile tool for the analyses of the subcellular
localization of the wild type and mutated form of the photoreceptor.
Both electronmicroscopic observations (McCurdy and
Pratt, 1986a,b; Speth et al., 1987) and analyses with phyAGFP fusion proteins (Kim et al., 2000; Kircher et al., 1999)
have shown that phyA forms cytosolic SAPs. Biochemical
data indicate that no other major component except phyA is
present in SAPs and that the Pfr form of phyA has a high
tendency of self-aggregation to form SAP-like structures
(Hofmann et al., 1991). The exchange of a negative Glu at the
surface of the protein by a positively charged Lys might be
responsible for the observed disturbance of SAP formation
in phyA(eid4), because such an alteration might severely
interfere with aggregation of Pfr.
Former studies have also demonstrated that phyA forms
NUS similar to other phytochromes (Kim et al., 2000; Kircher
et al., 1999, 2002). Detailed kinetic studies with phyA-GFP
and phyA(eid4)-GFP clearly indicate that the photoreceptor
forms at least two different types of NUS (Bauer et al., 2004;
this study) – an early and a late type. Early NUS aggregated
immediately after the formation of Pfr-A and showed a
transient accumulation with a half-life in the range of a few
minutes. This type of NUS also seems to contain other
phytochromes and the transcription factor PIF3 (Bauer et al.,
2004). In contrast, late and very stable NUS only became
detectable under light conditions that allowed the accumulation of a high level of phyA in the nucleus after prolonged
irradiation.
The formation of early NUS seems to be more or less
unaltered in phyA(eid4)-GFP lines. In contrast, a clear
difference has been detected for late NUS. With phyA-GFP
lines late NUS only accumulated under continuous far-red
light at wavelengths above 716 nm, whereas phyA(eid4)GFP also formed late NUS at wavelengths between 695 and
708 nm and in darkness after an inductive red light pulse.
Thus, in striking contrast to its negative influence on
cytosolic SAP formation and its weak effect on early NUS
formation, the mutation has a positive effect on the
formation of late NUS. These observations further confirm
the hypothesis that early and late NUS are different
compartments. Furthermore, our results indicate that the
formation of all three sub-types of phyA aggregates is
controlled by different mechanisms, because they are
altered in even opposite manners by the same mutation.
According to light requirements and structural features of
NUS, Chen et al. (2003) also postulated that phyB forms
different types of speckles with respect to the Pfr level
adjusted by light.
158 Monika Dieterle et al.
Phytochrome A degradation seems to be triggered by at
least two independent processes
This study and several former studies (Kim et al., 2000;
Kircher et al., 1999) have demonstrated that the number of
SAP formed correlates well with Pfr levels adjusted by different light treatments. Furthermore, several lines of evidence indicate that SAP formation and phyA degradation are
linked. SAP formation is extremely rapid and precedes phyA
degradation (MacKenzie et al., 1975; McCurdy and Pratt,
1986a,b). Immunoelectron microscopy in oat seedlings further demonstrated that phyA and ubiquitin co-localize in
cytosolic SAP in red light-treated plants (Speth et al., 1987).
Normally, specific complexes of ubiquitin ligases covalently
link the ubiquitin moieties to a Lys -amino group of target
proteins, which are then rapidly degraded in the 26S proteasome. Several studies demonstrated that phyA is ubiquitylated and that ubiquitylated phyA has a much shorter
half-life. Ubiquitylated photoreceptor molecules were also
enriched in the sequestered or pelletable fraction of phyA,
which is thought to represent the SAP fraction visible in the
microscopic studies (Clough and Vierstra, 1997). As predicted for phyA molecules impaired in cytosolic SAP formation,
phyA(eid4) exhibits reduced degradation rates under light
conditions, which normally induce rapid formation of a high
number of these subcellular compartments. In contrast,
degradation rates were very similar to wild-type phyA under
far-red light conditions, when no SAP accumulated in the
cytosol with phyA- and phyA(eid4)-GFP. Thus, our observations about phyA(eid4) degradation and its reduced
affinity to SAP clearly confirm the interpretation that these
subcellular compartments are involved in phyA degradation.
Our data also revealed strong evidence that multiple
pathways trigger phyA degradation. Although SAP formation is reduced in red light and nearly completely blocked in
705 nm light in the case of phyA(eid4)-GFP, the mutated
photoreceptor still becomes degraded. Furthermore, phyA
degradation still continues at time points, when SAPs were
no longer detectable in the cytosol with both the wild type
and the mutated phyA-GFP fusion protein. SAP-independent
degradation mechanisms should also dominate under continuous far-red light, because no cytosolic speckles were
detectable with both constructs. Taken together, these
findings clearly indicate that SAP formation is not the only
mechanism involved in phyA degradation.
Studies performed by Seo et al. (2004) have demonstrated
that COP1 can function as E3 ubiquitin ligase triggering the
rapid light-dependent degradation of phyA. Transient assays in onion cells showed a co-localization of COP1 and
phyA in NUS. This finding indicates that NUS might also be
involved in the proteolysis of phyA. Observations with the
phyA-302 missense mutation further support the idea that
NUS formation and phyA degradation are related. The
mutated phyA-302 molecule remained stable under continuous far-red light and its GFP fusion protein was impaired in
NUS formation under the same light conditions (Yanovsky
et al., 2002).
Because of their short half-life and their transient appearance, early NUS are good candidates for subnuclear compartments involved in phyA-degradation. In contrast,
several observations about late NUS argue against a major
role of these aggregates in the proteolysis of the photoreceptor. In phyA-GFP lines late NUS are formed under far-red
light conditions that allow the accumulation of relatively
high levels of the photoreceptor in the nucleus. Furthermore, a higher number of late NUS accumulated in
phyA(eid4)-GFP lines under prolonged light treatments that
normally induce a complete degradation of phyA in wild
type but not in the mutant. Taken together, these data
indicate that late NUS might either be involved in the
protection of phyA against proteolysis in the nucleus or that
they reflect the increased level of the photoreceptor in the
mutant. Alternatively, it has to be assumed that phyA(eid4)
is promoted with respect to formation of late NUS, but is
solely impaired in its interaction with a late NUS-specific
degradation machinery.
Phytochrome A levels and their influence on the expression
of high-irradiance responses
The eid screening program has been designed according to
former observations demonstrating that red-light reduction
of phyA levels leads to a reduction of HIR (Holmes and
Schäfer, 1981; Büche et al., 2000). These results clearly
indicate that a strong HIR is linked to a high steady-state
level of the active Pfr-A form. According to most published
models, Pfr-A should reach a maximum at about 720 nm,
when steady-state levels of phyA remain high and, when
about 5% of the photoreceptor are maintained in its active
form (Holmes and Schäfer, 1981; Mancinelli, 1994). In correspondence with this model, Pfr-A levels estimated for
wild-type phyA under prolonged irradiation fit quite well
with the action spectrum determined for the inhibition of
hypocotyl elongation under HIR conditions (Figure 2c,d
compared with Figure 6c).
For the phyA(eid4) mutant the situation is different.
According to the estimated Pfr levels, the peak of the HIR
action spectrum is expected at wavelengths between 699
and 710 nm, because the active Pfr-A form should reach
about two times higher levels compared with 716 nm light.
Nevertheless, maximum photon effectiveness for the inhibition of hypocotyl elongation was still observed at 716 nm
similar to the wild type. Only a small increase was detectable
at wavelength between 695 and 708 nm compared with the
shape of the action spectrum for the wild type and a
small decrease at wavelength above 720 as expected for
the somehow reduced Pfr levels at these wavelengths
ª Blackwell Publishing Ltd, The Plant Journal, (2004), 41, 146–161
A new type of mutation in phytochrome A 159
(Figure 2d compared with Figure 6c). Furthermore, the
mutant exhibited an increased light sensitivity under farred light conditions (Figure 2b), although phyA(eid4) levels
should be reduced under these conditions (Figure 5e, 6a).
Taken together, these data indicate that the extent of the HIR
is not strictly linked to the steady-state level of Pfr-A in the
mutant, but is controlled by further mechanisms. Interestingly, the unexpected low photon effectiveness in the
mutant at wavelengths between 695 and 708 nm correlates
with an increased level of late NUS in phyA(eid4)-GFP lines.
Thus, phyA molecules trapped in late NUS might have a
reduced activity in light signaling cascades controlling HIR.
Experimental procedures
Plant material and mutagenesis
For genetic crossing and physiological analyses, the following ecotypes and photomorphogenic mutants of Arabidopsis thaliana
were used: Landsberg erecta (Ler; Lehle Seed, Tucson, AZ, USA),
phyB-5 (ecotype Ler; Reed et al., 1993), phyB-9 (ecotype Columbia;
Nagatani et al., 1993). Propagation and mutagenesis were performed as described by Büche et al. (2000).
Seedling growth, light sources, and screening for mutants
Seeds were sown on four layers of Schleicher & Schüll 595 filter
paper circles (Schleicher & Schüll, Dassel, Germany), which were
placed in Greiner 94/16 petri dishes (Greiner, Kremsmünster,
Austria) supplemented with 4.5 ml distilled water. The standard
sowing procedure was followed by 2 days cold treatment at 8C in
the dark and 1 day of red light induction of germination at 25C
before onset of different light treatments for 3 days. Induction
of germination was performed with a standard light field
[k(max) ¼ 650 nm; 3.9 lmol m)2 sec)1; Heim and Schäfer, 1982].
For all other light treatments modified Leitz Prado 500-W universal
projectors (Leitz, Wetzlar, Germany) were used as light sources with
Osram Xenophot longlife lamps (Osram, Berlin, Germany). To
measure fluence rate response curves for the action spectra, light
was passed through narrow banded DIL and DEPIL interference filters (Schott, Mainz, Germany). Red light was obtained by passing the
light beam through KG65 filters [k(max) ¼ 650 nm; Balzers, Liechtenstein, Germany]. Far-red light treatments were performed with
715 nm DAL interference filters (Schott). Mutants were screened as
described by Büche et al. (2000). Hypocotyl lengths were measured
manually against a ruler. All data represent the mean of at least
40 seedlings analyzed in at least two independent experiments.
Mapping and isolation of the eid4 mutant
For mapping, phyB-5 eid4 plants were crossed with phyB-9. The
eid4 mutant was mapped by using PCR-based simple sequence
length polymorphisms and cleavable amplified polymorphic sequence (CAPS) markers (Bell and Ecker, 1994; Konieczny and
Ausubel, 1993). The PHYA gene was amplified as three overlapping
fragments, which were subsequently sequenced. For detection of
the eid4 mutation, a derived CAPS marker was used (Neff et al.,
1998). Genomic DNA was amplified by PCR using the oligonucleotides 5¢-AGCTGCTGGCTTACAATCATACAA-3¢ and 5¢-CCTGTCATACCCCGTGAGTTCAAAAAGCT-3¢. The PCR product was analyzed
ª Blackwell Publishing Ltd, The Plant Journal, (2004), 41, 146–161
for the presence (eid4) or absence (wild type) of a HindIII restriction
site. To isolate the eid4 mutation in a wild-type background, the
phyB-5 eid4 double mutant was crossed with Ler and F1 plants were
allowed to self-pollinate to obtain F2 seeds. F2 seedlings were preselected for normal red light sensitivity in the standard red light
field. The presence of the wild-type PHYB allele was further confirmed using a CAPS marker. PCR was performed using the
oligonucleotides 5¢-CGTGACTCGCCTGCTGGAATTGTT-3¢ and 5¢TCCATTGATGCAGCCTCCGGCA-3¢. BsaBI cannot cleave PCR
products from wild-type PHYB alleles.
Protein extraction and immunoblotting
Crude protein extracts and protein assays were performed as
described in Kircher et al. (1999). Aliquots containing 30 lg
of crude protein were separated on SDS-polyacrylamide gels
(Laemmli, 1970) and blotted to polyvinyldiflouride membranes
(Millipore, Schwalbach, Germany). Immunodetection of phyA:GFP
was performed using a GFP-specific antiserum (Kircher et al.,
1998; Convance, Freiburg, Germany) and an alkaline phosphatasecoupled anti-rabbit or anti-mouse antisera (Bio-Rad, München,
Germany).
In vivo spectroscopy
Photoreversible phyA was measured in a dual wavelength ratio
spectrophotometer at 5C as described by Gross et al. (1984).
Changes in absorbance D(DA) values were normalized to the
amount of fresh weight, which was determined immediately after
the measurements. If not stated otherwise, the data are the mean of
at least four independent experiments. All curves were fitted using
the SigmaPlot 4.0 Regression Wizard. Half-lives were determined by
using the values of the fitted curves.
To determine the photoconversion rates and the Pfr steady-state
levels at different wavelengths, 3-day-old etiolated seedlings were
transferred to an ice-cold metal plate for the light treatment to avoid
phyA degradation. Plants were transferred to cuvettes immediately
after light irradiation and were kept on ice in darkness or green safe
light (Heim and Schäfer, 1982) until the start of spectroscopic
measurements. Photoconversion rates were measured using red
light (665 nm DEPIL interference filter) with a photon fluence rate of
0.2 lmol m)2 sec)1. Steady-state levels of Pfr were obtained by
irradiating the seedlings with interference filter lights for 15 min at a
minimum photon fluence rate of 2.3 lmol m)2 sec)1 to reach
saturation. Photoequilibrium at 665 nm was assumed to be 0.87
(Mancinelli, 1994).
To measure degradation kinetics, dark-reversion, and phyA
levels after 8 h of irradiation, 3-day-old etiolated seedlings were
irradiated at 25C using different light sources. To measure
degradation kinetics and dark reversion the following filters and
light fields were used: Red light was obtained using a standard
red light field [k(max) ¼ 650 nm; 3.9 lmol m)2 sec)1; Heim and
Schäfer, 1982], 705-nm light was obtained using DEPIL
interference filters (1.9 lmol m)2 sec)1; Schott), far-red light was
obtained using a standard far-red light field [k(max) ¼ 730 nm;
20 lmol m)2 sec)1; Heim and Schäfer, 1982]. To follow dark
reversion, etiolated seedlings were treated with a 5-min red-light
pulse to reach maximum level of Pfr before transfer back to
darkness. To measure phyA levels after 8 h of irradiation, light
was passed through narrow-banded DIL and DEPIL interference
filters (Schott). Modified Leitz Prado 500-W universal projectors
(Leitz) were used as light sources with Osram Xenophot longlife
lamps (Osram).
160 Monika Dieterle et al.
Cloning of the PHYA(eid4):GFP construct and plant
transformation
Construction of the P35S:PHYA:GFP chimeric gene and the isolation
of transgenic lines carrying this gene have been described previously (Kim et al., 2000; Kircher et al., 2002). To generate the
P35S:phyA(eid4):GFP fusion, the EcoRI fragment close to the 5¢-end
of the wild-type PHYA cDNA was replaced by a mutated fragment
amplified by PCR from genomic eid4 DNA. Arabidopsis transformation was carried out as described by Clough and Bent (1998).
Hygromycin-resistant plants were grown to maturation, selfed, and
F2 seeds were tested for a 3:1 segregation of the Hygromycin
resistance gene and the expression of the phyA:GFP fusion protein.
F3 seeds of positive F2 lines were tested for a homozygous genetic
segregation of both marker genes. Seeds of homozygous F3 lines
were used for further experiments.
Epifluorescence and light microscopy
For epifluorescence and light microscopy, seedlings were transferred to glass slides under dim-green safe light and analyzed with
an Axioskop microscope (Zeiss, Oberkochem, Germany). The
nuclei were searched under dim-green safe light and only the first
pictures taken with a digital Axiocam camera system (Zeiss) are
presented. Excitation and detection of the fluorophors GFP was
performed with a GFP-filter set (AHF Analysentechnik, Tübingen,
Germany).
Acknowledgements
The authors thank Stefan Kircher, Tim Kunkel, Katia Marrocco, and
Kishore Panigrahi for helpful discussions about methodological
details, our data, and the manuscript. This research was supported
by the DFG grant ‘Signaltransduktionsmutanten der Photomorphogenese von Arabidopsis thaliana’ (SCHA 303/14-3) and by the
‘Arabidopsis Functional Genomics Network’ (KR 2020/1-3) program
of the DFG.
Supplementary Material
The following material is available from http://www.
blackwellpublishing.com/products/journals/suppmat/TPJ/TPJ2286/
TPJ2286sm.htm
Figure S1. The eid4 mutation leads to alterations in light-dependent
degradation of phyA.
Figure S2. Relative phyA levels after prolonged irradiation with
different wavelengths.
Figure S3. Levels of phyA- and phyA(eid4)-GFP fusion proteins in
dark-grown seedling of the transgenic lines used for microscopic
experiments.
References
Bauer, D., Viczian, A., Kircher, S., Nobis, T., Nitschke, R., Kunkel, T.,
Panigrahi, K.C., Adam, E., Fejes, E., Schäfer, E. and Nagy, F. (2004)
Constitutive photomorphogenesis 1 and multiple photoreceptors
control degradation of phytochrome interacting factor 3, a transcription factor required for light signaling in Arabidopsis. Plant
Cell, 16, 1433–1445.
Bell, C.J. and Ecker, J.R. (1994) Assignment of 30 microsatellite loci
to the linkage map of Arabidopsis. Genomics, 19, 137–144.
Bradley, J.M., Murphy, G.P., Whitelam, G.C. and Harberd, N.P.
(1996) Identification of phytochrome B amino acid residues mutated in three new phyB mutants of Arabidopsis. J. Exp. Bot. 47,
1449–1455.
Büche, C., Poppe, C., Schäfer, E. and Kretsch, T. (2000) eid1: A new
Arabidopsis mutant hypersensitive in phytochrome A-dependent
high-irradiance responses. Plant Cell, 12, 547–558.
Canton, F.R. and Quail, P.H. (1999) Both phyA and phyB mediate
light-imposed repression of PHYA gene expression in Arabidopsis. Plant Physiol. 121, 1207–1215.
Chen, M., Schwab, R. and Chory, J. (2003) Characterization of the
requirements for localization of phytochrome B to nuclear bodies.
Proc. Natl Acad. Sci. USA, 100, 14493–14498.
Choi, G., Yi, H., Lee, J., Kwon, Y.K., Soh, M.S., Shin, B., Luk, Z.,
Hahn, T.R. and Song, P.S. (1999) Phytochrome signalling is
mediated through nucleoside diphosphate kinase 2. Nature, 401,
610–613.
Clough, S.J. and Bent, A.F. (1998) Floral dip: a simplified method for
Agrobacterium-mediated transformation of Arabidopsis thaliana.
Plant J. 16, 735–743.
Clough, R.C. and Vierstra, R.D. (1997) Phytochrome degradation.
Plant Cell Environ. 20, 713–721.
Clough, R.C., Jordan-Beebe, E.T., Lohman, K.N., Marita, J.M.,
Walker, J.M., Gatz, C. and Vierstra, R.D. (1999) Sequences within
both the N- and C-terminal domains of phytochrome A are required for PFR ubiquitination and degradation. Plant J. 17, 155–
167.
Dieterle, M., Zhou, Y.-C., Schäfer, E., Funk, M. and Kretsch, T. (2001)
EID1, an F-box protein involved in phytochrome A-specific light
signaling. Genes Dev. 15, 939–944.
Dieterle, M., Büche, C., Schäfer, E. and Kretsch, T. (2003) Characterization of a novel non-constitutive photomorphogenic cop1
allele. Plant Physiol. 133, 1557–1564.
Edgerton, M.D. and Jones, A.M. (1992) Localization of protein–
protein interactions between subunits of phytochrome. Plant Cell,
4, 161–171.
Eichenberg, K., Kunkel, T., Kretsch, T., Speth, V. and Schäfer, E.
(1999) In vivo characterization of chimeric phytochromes in yeast.
J. Biol. Chem. 274, 354–359.
Eichenberg, K., Hennig, L., Martin, A. and Schäfer, E. (2000) Variation in dynamics of phytochrome A in Arabidopsis ecotypes and
mutants. Plant Cell Environ. 23, 311–319.
Fankhauser, C., Yeh, K.C., Lagarias, J.C., Zhang, H., Elich, T.D. and
Chory, J. (1999) PKS1, a substrate phosphorylated by phytochrome that modulates light signaling in Arabidopsis. Science,
284, 1539–1541.
Gross, J., Seyfried, M., Fukshansky, L. and Schäfer, E. (1984) In vivo
spectrophotometry. In Techniques in Photomorphogenesis
(Smith, H. and Holmes, M.G., eds). London: Academic Press, pp.
131–156.
Hardtke, C.S. and Deng, X.-W. (2000) The cell biology of the COP/
DET/FUS proteins: regulating proteolysis in photomorphogenesis and beyond? Plant Physiol. 124, 1548–1557.
Heim, B. and Schäfer, E. (1982) Light-controlled inhibition of hypocotyl growth in Sinapis alba L. seedlings. Planta, 154, 150–
155.
Ho, Y.-S.J., Burden, L.M. and Hurley, J.H. (2000) Structure of the
GAF domain, a ubiquitous signaling motif and a new class of
cyclic GMP receptor. EMBO J. 19, 5288–5299.
Ho, Y.-S.J., Burden, L.M. and Hurley, J.H. (2001) Corrigenda. EMBO
J. 20, 1483.
Hofmann, E., Grimm, R., Harter, K., Speth, V. and Schäfer, E. (1991)
Partial purification of sequestered particles of phytochrome from
oat (Avena sativa L.) seedlings. Planta, 183, 265–273.
ª Blackwell Publishing Ltd, The Plant Journal, (2004), 41, 146–161
A new type of mutation in phytochrome A 161
Holmes, M.G. and Schäfer, E. (1981) Action spectra for changes in
the ‘high irradiance reaction’ in hypocotyls of Sinapis alba L.
Planta, 153, 267–272.
Jordan, E.T., Cherry, J.R., Walker, J.M. and Vierstra, R.D. (1996) The
amino-terminus of phytochrome A contains two distinct functional domains. Plant J. 9, 243–257.
Kim, L., Kircher, S., Toth, R., Adam, E., Schäfer, E. and Nagy, F.
(2000) Light-induced nuclear import of phytochrome-A:GFP fusion proteins is differently regulated in transgenic tobacco and
Arabidopsis. Plant J. 22, 125–133.
Kircher, S., Ledger, S., Hayashi, H., Weisshaar, B., Schäfer, E. and
Frohnmeyer, H. (1998) CPRF4a, a novel plant bZIP protein of the
CPRF family: comparative analysis of light-dependent expression, post-transcriptional regulation, nuclear import and heterodimerisation. Mol. Gen. Genet. 257, 595–605.
Kircher, S., Kosma-Bognar, L., Kim, L., Adam, E., Harter, K., Schäfer,
E. and Nagy, F. (1999) Light quality-dependent nuclear import of
the plant photoreceptors phytochromes A and B. Plant Cell, 11,
1445–1456.
Kircher, S., Gil, P., Kozma-Bognár, L., Fejes, E., Speth, V., Husslstein-Muller, T., Bauer, D., Ádám, E., Schäfer, E. and Nagy, F.
(2002) Nucleoplasmic partitioning of the plant photoreceptors
phytochrome A, B, C, D, and E is regulated differentially by light
and exhibits diurnal rhythm. Plant Cell, 14, 1541–1555.
Konieczny, A. and Ausubel, F.M. (1993) A procedure for mapping
Arabidopsis mutations using co-dominant ecotype-specific PCRbased markers. Plant J. 4, 403–410.
Kretsch, T., Poppe, C. and Schäfer, E. (2000) A new type of mutation
in the plant photoreceptor phytochrome B causes loss of photoreversibility and an extremely enhanced light sensitivity. Plant J.
22, 177–186.
Laemmli, U.K. (1970) Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature, 227, 680–685.
Lapko, V.N., Jiang, X.Y., Smith, D.L. and Song, P.S. (1999) Mass
spectrometric characterization of oat phytochrome A: isoforms
and posttranslational modifications. Protein Sci. 8, 1032–1044.
MacKenzie, J.M., Coleman, R.A., Briggs, W.R. and Pratt, L.H. (1975)
Reversible redistribution of phytochrome within the cell upon
conversion to its physiological active form. Proc. Natl Acad. Sci.
USA, 72, 799–803.
Mancinelli, A.L. (1994) The physiology of phytochrome action. In
Photomorphogenesis in Plants, 2nd edn (Kendrick, R.E. and
Kronenberg, G.H.M., eds). Dordrecht: Kluwer Academic Publishers, pp. 51–59.
McCurdy, D.W. and Pratt, L.H. (1986a) Kinetics of intracellular
redistribution of phytochrome in Avena coleoptiles after its
photoconversion to the active far-red-absorbing form. Planta,
167, 330–336.
McCurdy, D.W. and Pratt, L.H. (1986b) Immunogold electron microscopy of phytochrome in Avena: identification of intracellular
sites responsible for phytochrome sequestering and enhanced
pelletability. J. Cell Biol. 103, 2541–2550.
Møller, S.G., Ingles, P.J. and Whitelam, G.C. (2002) The cell biology
of phytochrome signaling. New Phytol. 154, 553–590.
Nagatani, A., Reed, J.W. and Chory, J. (1993) Isolation and initial
characterization of Arabidopsis mutants that are deficient in
phytochrome A. Plant Physiol. 102, 269–277.
ª Blackwell Publishing Ltd, The Plant Journal, (2004), 41, 146–161
Neff, M.M., Neff, J.D., Chory, J. and Pepper, A.E. (1998) dCAPS, a
simple technique for the genetic analysis of single nucleotide
polymorphisms: experimental applications in Arabidopsis thaliana genetics. Plant J. 14, 387–392.
Neff, M.M., Fankhauser, C. and Chory, J. (2000) Light: an indicator of
time and place. Genes Dev. 14, 257–271.
Ni, M., Tepperman, J.M. and Quail, P.H. (1998) PIF3, a phytochromeinteracting factor necessary for normal photoinduced signal
transduction, is a novel basic helix-loop-helix protein. Cell, 95,
657–667.
Ni, M., Tepperman, J.M. and Quail, P.H. (1999) Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light. Nature, 400, 781–784.
Reed, J.W., Nagpal, P., Poole, D.S., Fururya, M. and Chory, J. (1993)
Mutations in the gene for the red/far-red light receptor phytochrome B alter cell elongation and physiological responses
throughout Arabidopsis development. Plant Cell, 5, 147–157.
Sakamoto, K. and Nagatani, A. (1996) Nuclear localization activity of
phytochrome B. Plant J. 10, 859–868.
Seo, H.S., Watanabe, E., Tokutomi, S., Nagatani, A. and Chua, N.H.
(2004) Photoreceptor ubiquitination by COP1 E3 ligase desensitizes phytochrome A signaling. Genes Dev. 18, 617–622.
Speth, V., Otto, V. and Schäfer, E. (1987) Intracellular localization of
phytochrome and ubiquitin in red-light-irradiated oat coleoptiles
by electron microscopy. Planta, 171, 332–338.
Stockhaus, J., Nagatani, A., Halfter, U., Kay, S., Furuya, M. and
Chua, N.-H. (1992) Serine-to-alanine substitutions at the aminoterminal region of phytochrome A result in an increase in biological activity. Genes Dev. 6, 2364–2372.
Sweere, U., Eichenberg, K., Lohrmann, J., Mira-Rodado, V., Bäurle,
I., Kudla, J., Nagy, F., Schäfer, E. and Harter, K. (2001) Interaction
of the response regulator ARR4 with phytochrome B in modulating red light signaling. Science, 294, 1108–1111.
Wagner, D. and Quail, P.H. (1995) Mutational analysis of phytochrome B identifies a small COOH-terminal-domain region
critical for regulatory activity. Proc. Natl Acad. Sci. USA, 92,
8596–8600.
Wagner, D., Fairchild, C.D., Kuhn, R.M. and Quail, P.H. (1996)
Chromophore-bearing NH2-terminal domains of phytochromes A
and B determine their photosensory specificity and differential
light lability. Proc. Natl Acad. Sci. USA, 93, 4011–4015.
Xu, Y., Parks, B.M., Short, T.W. and Quail, P.H. (1995) Missense
mutations define a restricted segment in the C-terminal domain
of phytochrome A critical to its regulatory activity. Plant Cell, 7,
1433–1443.
Yamaguchi, R., Nakamura, M., Mochizuki, N., Kay, S.A. and Nagatani, A. (1999) Light-dependent translocation of a phytochrome
B:GFP fusion protein to the nucleus in transgenic Arabidopsis. J.
Cell Biol. 145, 437–445.
Yanovsky, M.J., Luppi, J.P., Kirchbauer, D. et al. (2002) Missense
mutation in the PAS2 domain of phytochrome A impairs subnuclear localization and a subset of responses. Plant Cell, 14, 1591–
1603.
Zhou, Y.C., Dieterle, M., Büche, C. and Kretsch, T. (2002) The negatively acting factors EID1 and SPA1 have distinct functions in
phytochrome A-specific light signaling. Plant Physiol. 128, 1098–
1108.