Journal of Experimental Botany, Vol. 58, No. 12, pp. 3113–3124, 2007
doi:10.1093/jxb/erm145 Advance Access publication 27 September, 2007
FOCUS PAPER
Light-regulated nucleo-cytoplasmic partitioning of
phytochromes
Eva Kevei1,2,*, Eberhard Schafer1 and Ferenc Nagy2
1
Biologie II/Institute fur Botanik, University of Freiburg, D-79104 Freiburg, Germany
2
Institute of Plant Biology, Biological Research Center, H-6726 Szeged, Hungary
Received 12 April 2007; Revised 25 May 2007; Accepted 30 May 2007
Abstract
Phytochrome photoreceptors regulate development,
growth, and fitness throughout the entire life-cycle
of plants, from seed germination to flowering, by regulating expression patterns of ~10–30% of the entire
plant transcriptome. Identification of components and
elucidation of the molecular mechanisms underlying
phytochrome-controlled signal transduction cascades
have therefore attracted considerable attention.
Phytochrome-controlled signalling is a complex cellular process; it starts with the light-induced intramolecular conformational change of the photoreceptor and
includes regulated partitioning and degradation of signalling components and of the photoreceptors themselves. In this review, the data available about light
quality- and quantity-dependent nucleo-cytoplasmic
partitioning of phytochromes is summarized and the
possible function of phytochrome-containing nuclear
complexes, termed nuclear bodies, in red/far-red lightinduced signalling is discussed.
Key words: Light signalling, nuclear import, phytochromes.
Introduction
Plants are sessile organisms which have established a considerable plasticity of development to respond to changes
in the natural environment. The highly variable environmental factor light is used not only as the main energy
source but also as an environmental cue to respond to
daily light/dark cycles and to compete with neighbouring
plants. To monitor changes in the ambient light environment, plants have evolved several classes of photoreceptors: the as yet unidentified UV-B photoreceptors
(Beggs and Wellman, 1994), the blue/UV-A sensing cryptochromes (CRY1 and CRY2) controlling plant development (Lin and Shalitin, 2003), the phototropins regulating
directional growth, chloroplast re-orientation, and stomatal
opening (Briggs and Christie, 2002), and the red/far-redabsorbing photoreceptors phytochromes, controlling plant
growth and development (Quail, 2002).
Phytochromes are synthesized in the cytosol as ;125
kDa monomers in their inactive, Pr form (red-lightabsorbing conformer, kmax¼660 nm). Each monomer
covalently binds one molecule of phytochromobilin,
a linear tetrapyrrole chromophore. Phytochromes exist
in vivo as dimers. Red light (R) induces an intramolecular
conformational change of the molecule resulting in the formation of the physiologically active Pfr conformer (far-red
light-absorbing form, (kmax¼730 nm). The photoreceptor
reverts back into its inactive Pr form with subsequent
far-red light (FR) treatment and this light quality- and
quantity-dependent conformational change enables phytochromes to act as light-sensitive molecular switches.
Higher plants contain various phytochromes, which differ in amino acid sequence by 50%. These different types
of phytochromes are selectively responsible for sensing
various light qualities. In the model plant Arabidopsis
thaliana this gene family consists of five genes, designated PHYA, PHYB, PHYC, PHYD, and PHYE (Clack
et al., 1994). Due to the overlapping absorption spectra,
a light quality-dependent photo-equilibrium is established,
making this photoreceptor system a very effective light
sensor, especially in the red/far-red range of the spectrum
(Smith, 2000).
Phytochrome proteins can be divided into two classes
on the basis of their mode of action and light stability.
Type I phytochromes show rapid proteolytic degradation
of the Pfr form, controlling Very-Low-Fluence-Responses
* To whom correspondence should be addressed. E-mail: [email protected]
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3114 Kevei et al.
(VLFR) and far-red High-Irradiance Responses (FR-HIR).
Type II phytochromes are light-stable and control Low
Fluence Responses (LFR) and red light High Irradiance
Responses (R-HIR). Analysis of mutants deficient in various phytochromes showed that (i) type I phytochromes
are encoded by the PHYA gene and type II phytochromes
by PHYB–E genes (Quail, 2002) and that (ii) different
members of the family have differential as well as overlapping physiological roles in controlling plant development (Smith et al., 1997; Franklin et al., 2003; Monte
et al., 2003).
The phytochrome molecule folds into two major domains separated by a protease-sensitive, flexible hinge
region: the N-terminal photosensory domain of ;70 kDa
and a C-terminal domain of ;55 kDa (for a recent review
see Rockwell et al., 2006). Recent studies established that
in all phytochromes the N-terminally located bilin-lyasedomain (BLD) is responsible for binding the chromophore
(for a review see Rockwell and Lagarias, 2006). It has
recently been shown that the very N-terminal part of
phytochromes plays a role in regulating the stability of the
Pfr conformer (Jordan et al., 1997; Sweere et al., 2001;
Casal et al., 2002; Ryu et al., 2005; Trupkin et al., 2007).
The phytochrome domain (PHY) is located toward the
C-terminal part of the molecule, adjacent to the BLD. The
PHY domain was shown to contribute to the integrity of
Pfr (Montgomery and Lagarias, 2002), thus deletion of
this domain resulted in substantial alterations in the
spectral properties of phytochromes (Cherry et al., 1993).
The first domain of the C-terminal part of the molecule
contains a core regulator region, termed Quail-box, which
is flanked by the so-called PAS1 and PAS2 subdomains
(Quail, 1997; Ponting and Aravind, 1997). The PAS1Quail-box-PAS2 region is adjacent to the dimerization
domains (Edgerton and Jones, 1993), and is followed by
the histidine-kinase related domain (HKRD) at the end
of the C-terminus. The HKRD domain displays homology
to bacterial two-component sensor kinases (Yeh and
Lagarias, 1998). Figure 1 shows a schematic view of phytochrome structure.
Recent structure–function studies have begun to reveal
the function of the various domains in initiating lightdependent signalling and showed that phyA–E regulate
various aspects of plant photomorphogenesis by independent or partially overlapping signal transduction cascades
(for recent reviews see Wang, 2005; Franklin et al., 2005;
Rockwell et al., 2006). In molecular terms, however, all
of these pathways have one common feature, i.e. light
in a quantity- and quality-dependent fashion alters the
nucleo-cytoplasmic distribution of phyA–phyE by inducing their import into and accumulation in the nucleus.
Consistent with this observation, it has been convincingly
documented that phytochromes interact both in vivo and
in vitro with a number of Phytochrome-Interacting-Factors
(PIFs) of nuclear localization in a light and, therefore,
Fig. 1. Schematic illustration of phytochrome’s structure. The ;125
kDa monomer PHY apoprotein folds into two major domains: the Nterminal domain, which determines the photosensory properties of the
receptor and the C-terminal, regulatory domain. Detailed description of
the function of different domains and motifs are in the text. CHR,
chromophore; BLD, bilin lyase domain; PHY, phytochrome domain;
DIM, dimerization domain; HKRD, histidine-kinase-related domain;
PAS, PER/ARNT/Sim domain, a motif, typified by the Drosophila
melanogaster clock protein PER, the mammalian aromatic hydrocarbon
receptor nuclear transporter ARNT, and the Drosophila single-minded
protein Sim; PRD, PAS-related domain.
conformation-dependent fashion. These data strongly suggest that the various phytochromes transmit the light
signal, at least partly, inside the nucleus.
Intracellular distribution of phytochromes in
etiolated seedlings and during the early
phase of photomorphogenesis
phyA
In etiolated seedlings the native phyA (McCurdy and
Pratt, 1986; Speth et al., 1986; Pratt, 1994) and the
35S:phyA::GFP fusion protein are distributed throughout
the cytosol (Kircher et al., 1999, 2002; Hisada et al.,
2000). A single, brief (;5 min) FR-, R- or blue-light
pulse (B) induces nuclear import of phyA and subsequent
formation of phyA-containing nuclear bodies (phyA NBs)
(as described by Hisada et al., 2000; Kim et al., 2000;
Kircher et al., 2002). In etiolated seedlings, the nuclear
import of phyA is a rapid process: it takes place within
a few minutes after the inductive light pulse. An R pulse
also promotes the rapid formation of phyA-containing
cytosolic spots, also referred to as sequestered areas of
phytochromes (SAPs) (Speth et al., 1986). The appearance of SAPs precedes nuclear transport of 35S:
phyA::GFP and is thought to be the place of ubiquitination and degradation of the photoreceptor. Continuous
FR light (cFR) also initiates nuclear transport and formation of phyA NBs in the nuclei. These data suggest that
nuclear import of phyA correlates with phyA-mediated
VLFRs and HIRs. Figure 2 shows the fluence ratedependent nuclear accumulation of 35S:phyA::GFP.
In addition to FR, white (W), R, and B light illumination of etiolated seedlings is also effective in inducing
Involvement of nuclear bodies in light signalling 3115
Fig. 2. Light intensity-dependence of 35S:phyA::GFP intracellular distribution. Transgenic Arabidopsis seedlings expressing the PHYA::GFP
transgene driven by the constitutive Cauliflower Mosaic Virus promoter (CaMV 35S) were grown in constant darkness for 2 d, then
transferred for 24 h to the fluence rate of FR light indicated below.
Epifluorescence-microscope images were taken on hypocotyl cells of
the irradiated seedlings (A, C–E). (B) A reference, light-microscope
image on hypocotyl cells. Fluence rates of FR-light used for irradiation
of the 2-d-old plants were (A, B) 0.002 lmol m2 s1; (C) 0.02 lmol
m2 s1; (D) 0.2 lmol m2 s1; (E) 2 lmol m2 s1; (F) 20 lmol
m2 s1. The bar in (A) represents 10 lm; nu, nucleus; nus, nucleolus.
nuclear translocation and rapid formation of 35S:phyA::GFP.
Although the pattern of redistribution of 35S:phyA::GFP
within the cell is similar in response to all of these light
treatments, the dynamics of protein movement is different.
The maximum number of phyA-containing nuclear bodies
is visible about 10 min after W light illumination, whereas
FR light-induced formation of phyA NBs reaches its
highest level approximately 2 h after irradiation (Kim
et al., 2000; Kircher et al., 2002).
phyB
The intracellular localization of phyB in etiolated seedlings is similar to that of phyA, thus the phyB::GFP fusion
protein, expressed from the 35S promoter, is also found
mainly in the cytosol (Gil et al., 2000; Kircher et al.,
2002). It should be noted, however, that in the case of
phyB (in contrast to phyA, which is exclusively localized
in the cytosol), a weak, diffuse nuclear staining is always
visible in etiolated seedlings. Whether this difference has
any biological significance for phyB-controlled signalling
and is caused by different retention/import mechanisms
for phyA and phyB, or is simply due to the relatively
higher over-expression of phyB is not yet understood.
Apart from this, it is safe to say that light, in a qualityand quantity-dependent fashion, induces translocation of
35S:phyB::GFP into the nucleus. However, import of
phyB is at least one order of magnitude slower than that
of 35S:phyA::GFP and it was also shown that, in contrast
to phyA, single pulses of R, FR or B light cannot induce
detectable nuclear transport of 35S:phyB::GFP. The Pfr
conformer of phyB is photostable, and continuous R (cR)
(3 h) irradiation was as effective as white light in inducing
the nuclear import of 35S:phyB::GFP. cB only induced
low-level phyB accumulation in the nuclei, whereas cFR
light was completely ineffective in initiating the nuclear
transport of phyB. Nuclear import of phyB, similarly to
that of phyA, is also followed by the accumulation of the
photoreceptor into nuclear bodies, termed phyB NBs,
visible under the microscope as discrete intranuclear
complexes, often called speckles. Formation of phyB
NBs depends not only on light quality but also on
the intensity and duration of the light used for induction
(Gil et al., 2000).
Generally speaking, it can be stated that in cR these
phyB-containing sub-nuclear complexes are stable and
their size and number change in a fluence rate-dependent
fashion. When plants are moved to darkness after the
inductive light treatments, these phyB NBs start to
dissolve; the half-life of the R-induced phyB NBs in
darkness is around 3 h and they all disappear in about
12 h. Disappearance of 35S:phyB NBs is accompanied by
the emergence of diffuse nuclear staining, which gradually
declines and in about 20–24 h decreases to the level
detectable in etiolated seedlings. Although phyB transport
into the nuclei cannot be initiated by either pulse or cFR,
FR has a significant effect on 35S:phyB::GFP import
when applied after the inductive R treatment. R-induced
nuclear transport of 35S:phyB::GFP is prevented by treatment of plants with FR light directly after R illumination.
Therefore, nuclear transport of 35S:phyB::GFP resembles
R/FR reversibility of phyB-controlled low fluence responses (LFRs) (Gil et al., 2000) and it is characteristically different from that of phyA, which shows features of
either the non-reversible VLFRs or the FR-HIRs (Kim
et al., 2000). By using a glucocorticoid receptor-based
fusion protein system, Huq et al. (2003) provided conclusive evidence that photo-activation and nuclear translocation are necessary and sufficient for the biological
function of phyB. Data provided by these authors indicate
that signal transfer from photoactivated phyB to its primary signalling partners takes place in the nucleus and
suggest the absence of a cytosolic pathway from photoactivated phyB to light-responsive genes.
phyC, phyD, and phyE
Similarly to 35S:phyB::GFP, 35S:phyC–E::GFP are
mainly localized in the cytosol, but are also found in the
nuclei of etiolated seedlings, displaying diffuse, weak
staining (Kircher et al., 2002). The intensity of nuclear
staining (the amount of fluorescent protein within the
nuclei) varied among the different photoreceptors and was
obviously affected by the experimental protocol applied
(e.g. light or GA-induced germination). The Pfr form of
phyC–E is thought to be photostable, similarly to phyB
3116 Kevei et al.
Pfr. Thus it is assumed that the low level of these phytochromes in the nuclei of etiolated seedlings may reflect
transient activation of the nuclear import mechanism
during germination. White light irradiation, however,
clearly increases the concentration of these phytochrome
species in the nuclei and induces subsequent formation of
35S:phyC–E-containing nuclear bodies. The kinetics of
the formation of 35S:phyC::GFP and phyE::GFP NBs is
comparable with that of 35S:phyB NBs, i.e. the maximum
number of phyC and phyE NBs is detectable within 2 h
after white light irradiation. Interestingly, although the
35S:phyD::GFP is obviously detectable in nuclear complexes, the number of these phyD::GFP NBs does not
increase significantly during the first 8 h after irradiation
with white light (Kircher et al., 2002). In contrast to phyA
and phyB, available data about the light quality- and
quantity-dependent nuclear import of phyC–E are fairly
limited, thus very little is known about the detailed kinetics of R-, FR-, and B-induced changes in the intracellular distribution of these phytochrome species.
Intracellular distribution of phytochromes in
plants grown under light/dark cycles
With the exception of phyB, surprisingly little is known
about the intracellular distribution of phytochromes in
young plantlets or mature plants grown under light/dark
cycles. In the past, studies addressing this problem by
using classical approaches, such as immunolocalization,
faced several technical problems. phyA is known to be
photolabile, thus the low level of phyA made detection
problematic; expression levels of the photostable phyB–E
are generally low, which once again hindered reliable data
being obtained. With the advent of fluorescent protein tags
these problems could only partially be overcome. Because
of the relatively high level of autofluorescence generated
by chlorophyll molecules, reliable detection of 35S:phy::YFP,
35S:phy::GFP, and especially 35S:phy::CFP fusion proteins still requires relatively high level expression of these
proteins, which in turn makes interpretation of the data
somewhat problematic (for example, the detection of
low-level diffuse staining in the cytosol and/or nuclei).
Thus it is not surprising that in most of these studies the
appearance of phytochrome-containing nuclear bodies was
used as a tool to detect phytochromes localized in the
nucleus. Interestingly, it was reported that in young
plantlets grown under short-day conditions (8/16 h light/
dark) all phys form nuclear bodies and the emergence of
these nuclear complexes follows a diurnal pattern (Kircher
et al., 2002). However, rhythmic appearance of these
NBs required irradiation with distinct qualities/intensities
of light. Accordingly, the number and appearance of
35S:phyA NBs were monitored in plants grown under FR/
dark cycles, whereas 35S:phyB-E NBs were detected in
plants grown under WL/dark cycles. More importantly, it
was also found that these nuclear complexes appeared
before the light-on signal and started to decrease before
light-off (Kircher et al., 2002). Anticipation of light/dark
transitions indicates that this process is not simply regulated by the change in the environmental light conditions,
but is controlled by the circadian clock. Interestingly, Toth
et al. (2001) showed that transcription of PHYA–E genes
is also regulated by the circadian clock, thus it appears
that the circadian clock regulates both the expression and
the activity of PHY genes and phy proteins. Since phytochromes have been shown to regulate light input pathways
to the clock (Somers et al., 1998) these data indicate the
existence of a feedback loop to control phytochromemediated photo-transduction to the clock.
What is the biological significance of
phytochrome-containing nuclear bodies?
The first papers reporting on light-induced translocation
of 35S:phyA::GFP and phyB::GFP into the nucleus
(Sakamoto and Nagatani, 1996; Nagatani, 1998, 2004;
Kircher et al., 1999) documented that (i) formation of
phyA::GFP NBs was as fast as the import of the photoreceptor, i.e. they were easily detectable within minutes
after light treatments and that (ii) formation of phyB::GFP
NBs was detectable only in about 1.5–2 h after irradiation.
More importantly, however, it was also reported that
native phyA and phyB also accumulated in electrodense
subnuclear structures, excluding the possibility that the
accumulation of 35S:phy::GFP fusion proteins is mediated
by the fluorescence tag (Hisada et al., 2000; Kircher
et al., 2002).
With the advance of microscopic techniques, however,
it became obvious that not only phyA but also phyB::GFP
and phyD::GFP can form nuclear bodies very rapidly (2–3
min) after the light treatment of etiolated seedlings
(Kircher et al., 2002). These early 35S:phyB-D NBs were
transient, generally smaller in size, but more numerous, as
compared with the late appearing 35S:phyB-D NBs, and
they dissolved and became undetectable within 10–15
min. Interestingly, after these early NBs disappeared, in
cR new 35S:phyB-D NBs formed in 2–3 h, but these were
fewer and larger. More importantly, Bauer et al. (2004)
reported that PIF3, a bHLH-type transcription factor colocalized with phyA, B, and D only in the early NBs and
it was not detectable in the newly appearing, late NBs.
These data provided the first evidence that components of
the various 35S:phy NBs are likely to be different.
In an independent line of experiments, Chen et al.
(2003) demonstrated convincingly that the heterogenity of
35S:phyB::GFP NBs regarding their size and number is
fluence rate-dependent and that the detectable formation
of late phyB NBs (appearing hours after the R treatment)
Involvement of nuclear bodies in light signalling 3117
is not required for phyB signalling at low fluence rates.
These authors showed that, at low fluence rates of cR,
35S:phyB::GFP is imported into the nucleus as a Pfr-Pr
heterodimer and phytochrome signal transduction is
initiated (measured by hypocotyl growth inhibition), but
the appearance of phyB NBs cannot be detected. They
also showed that by increasing the intensity of the inductive R treatment an increasingly higher percentage of
phyB molecules are converted to and exist in Pfr conformation, which is paralleled by the appearance of
various 35S:phyB::GFP-containing nuclear bodies. Chen
et al. (2003) divided fluence rate-dependent phyB NB
patterning into four stages. At stage I, phytochrome is
found in both cytoplasm and nuclei, but there is no
obvious phyB NB formation within the nuclei. At stage I,
no more than 10% of total phyB is in the Pfr form. Gil
et al. (2000) reported similar results by studying fluence
rate-dependent nuclear translocation of a tobacco
35S:phyB::GFP fusion protein. At stage II, where ;15%
of total phyB is Pfr, numerous small phyB NBs appear in
the nuclei after irradiation. The size and number of these
35S:phyB::GFP NBs resemble the so-called early
35S:phyB::GFP NBs detected by Bauer et al. (2004).
However, these phyB NBs were monitored 3 d after the
onset of R, whereas Bauer et al. (2004) detected the early
phyB NBs, in which phyB co-localizes with PIF3, within
minutes after the R treatment. Experiments to find answers
for these vexing questions, i.e. whether or not these small
phyB NBs have identical composition (does PIF3 colocalize with phyB in both of them?) and/or similar
function (do both types mediate light-induced degradation
of PIF3?) are yet to be performed. Stage III is marked
by the presence of about 24% Pfr of total phyB and
the presence of larger but fewer nuclear bodies, although
a number of small phyB NBs are still visible within the
nuclei. When roughly half or more of total phyB is in the
active Pfr form, only a few large NBs are observed with
little fluorescence in the nucleoplasm. 35S:phyB::GFP
NBs detectable at this stage (stage IV) are very similar to
the so-called late speckles detected by Bauer et al. (2004)
at higher intensities of R. Figure 3 illustrates the fluence
rate-dependent formation of phyB NBs. As increasing
fluence rates of R not only induced a change in the
nuclear patterning of phyB but also enhanced inhibition of
hypocotyl elongation, these results also indicated that the
formation of phyB NBs plays a role in the regulation of
phyB-mediated signal transduction, at least at higher
fluence rates (Chen et al., 2003).
These observations by Bauer et al. (2004) and Chen
et al. (2003), however, provoked additional questions. First,
is the translocation of 35S:phyA::GFP, 35S:phyB::GFP,
and 35S:phyD::GFP into the nucleus fast enough to
explain co-localization of these phys with PIF3 in the socalled early NBs detected minutes after light treatment?
Translocation of phyA into the nucleus is very fast, even
Fig. 3. Red light fluence rate dependency of 35S:phyB::GFP speckle
formation/morphology. Transgenic Arabidopsis seedlings expressing
the PHYB::GFP transgene driven by the constitutive Cauliflower
Mosaic Virus promoter (CaMV 35S) were grown at 0.5 lmol m2 s1
(A), 1 lmol m2 s1 (B), 2 lmol m2 s1 (C), or 8 lmol m2 s1 (D)
of red light for 4 d and then the intracellular distributon of phyB::GFP
fusion protein was examined in hypocotyl cells under epifluorescent
microscopy. According to calculations by Chen et al. (2003), under
0.5 lmol m2 s1 continuous red light 10% of total phyB is in the Pfr
form (A). 1 lmol m2 s1 cR light induces the formation of 16%
Pfr (B), 2 lmol m2 s1 induces 24% (C), whereas under continuous
red light irradiation of 8 lmol m2 s1 R light more than 46% of total
phyB is in Pfr form (D). The bar in (A) represents 5 lm.
a single R pulse induces nuclear import of phyA, thus
the appearance of phyA NBs correlates well with the
appearance of phyA-PIF3-containing NBs. However, in
contrast to phyA, translocation of 35S:phyB::GFP and
especially 35S:phyD::GFP into the nucleus is considerably slower even at higher light intensities. In addition,
the frequently detected weak, diffuse staining in the
nucleus by 35S:phyB::GFP and 35S:phyD::GFP in etiolated seedlings makes it difficult to determine that
minimal increase in nuclear levels of phyB and phyD
which can be caused by a few minutes of irradiation. A
brief R pulse is not sufficient to induce phyB translocation
into the nucleus and phyB-mediated signalling. However,
at least theoretically, the same short R pulse can convert
the nuclear phyB Pr, visible as weak, diffuse staining in
dark-grown seedlings, into phyB Pfr, thus promoting the
formation of early 35S:phyB::GFP NBs in which phyB
co-localizes with the PIF3 protein. If so, is it possible that
these very early steps of phyB-mediated signalling are
mediated by phyB constitutively localized in the nucleus,
rather than by the freshly translocated photoreceptor? An
equally vexing problem is to explain the relative stability
of the large 35S:phyB::GFP NBs in the dark or after FR
irradiation. It has been shown that phyB is present in the
nuclear bodies in its Pfr form, since PHYB molecules
3118 Kevei et al.
lacking chromophore never show NB formation (Kircher
et al., 1999) and FR alone is also not inductive (Gil et al.,
2000; Kircher et al., 2002). Yet the disappearance of
phyB NBs in the dark, even after FR treatment, is a slow
process that takes hours. The amount of FR applied in
these experiments is sufficient to convert all phyB Pfr
into Pr, thus this FR treatment should result in the rapid
decomposition of phyB NBs, which is obviously not
the case.
Molecular mechanism(s) for nucleo-cytoplasmic
trafficking of phytochromes
The transport of proteins from the cytoplasm into the
nucleus exclusively occurs through the Nuclear Pore
Complexes (NPCs) that span the nuclear envelope and
allow the passage of molecules between the two compartments (for reviews see Stoffler et al., 1999; Merkle, 2003;
Lim and Fahrenkrog, 2006; Tran and Wente, 2006).
Smaller molecules below the size of 40 kDa can diffuse
through these pores passively. The transport mechanism
by which large proteins or their complexes are moved to
the other side of the nuclear envelope is called facilitated
translocation; in contrast to diffusion, it can operate
against the concentration gradient (Ribbeck and Gorlich,
2001).
Phytochromes, these large dimeric proteins are transported to the nuclei via this active, energy-consuming
process, which requires the action of importins. Nuclear
translocation is normally initiated by the recognition of the
target protein by the transport receptor (e.g. importin b-like
receptors), followed by docking of the substrate to the
nuclear envelope and its translocation through the nuclear
membrane. Importins have two subclasses, namely importin a-like and importin b-like proteins (Merkle, 2003).
Importin b-like receptors are components of the nuclear
transport that anchor the transportable protein to the NPC
by direct binding of the nucleoporins and the target protein
itself. Being hydrophobic but soluble proteins, the importin
b-like receptors can facilitate the transport of large proteins
or their complexes through the nuclear pores. Within the
nuclei importin b interacts with the GTP-binding form of
RanGTPase and releases the target protein. The importin
b receptor-RanGTP complex recycles back to the cytoplasm, where GTP is hydrolysed by Ran and the complex
dissociates. Import of proteins carrying classical basic
nuclear localization signals (NLS) needs the action of
another type of importin molecule, importin a, which
functions as the cytoplasmic NLS receptor (Gorlich et al.,
1995). In contrast to the transport receptor importin b,
importin a cannot leave the nuclear compartment on its
own. Recycling of importin a to the cytoplasm therefore
requires an additional transport receptor, which specifically
exports importin a from the nucleus (Kutay et al., 1997).
Do PHYs have NLS for nuclear targeting?
If phytochromes contain putative NLS signals, importins
can be involved in targeting these photoreceptors into or
out of the nucleus. Sakamoto and Nagatani (1996)
examined the nuclear localization of 35S:phyB::GUS
fusion protein. They showed for the first time that the Cterminal part of phyB is transported into the nucleus,
which suggested that an active NLS(s) is present in this
part of the molecule. Additional studies showed that,
although the carboxy terminus of phyB translocates into
the nuclei and forms NBs, yet it fails to complement phyB
null mutants (Matsushita et al., 2003). However, it has
also been shown that nuclear transport of the C-terminal
part of PHYB and the formation of NBs thereof occurs
constitutively, irrespective of light conditions (Nagy et al.,
2000, 2001; Nagy and Schafer, 2002). These data suggested that the transport machinery is able to recognize
and target the PHYB C-terminal part into the nuclei, but
light dependence of the nuclear transport requires the
presence of the N-terminal region of phyB. On the other
hand, it was found that the N-terminal half of phyB does
not translocate into the nuclei and fails to form NBs and
to complement phyB null mutants, indicating that the
nuclear transport machinery is unable to recognize the
truncated phyB protein.
If so, what region of phyB is responsible for initiating
light-induced signalling? To answer this question,
Matsushita et al. (2003) constructed a chimeric gene that
contained the N-terminal half of phyB fused to the GUS
reporter and a functional NLS or NES (nuclear exclusion
signal) and expressed this fusion protein in the phyB-5
null mutant. Their results convincingly showed that (i) the
N-terminal part of phyB is sufficient to initiate phyBdependent light signalling, but only when it is localized in
the nuclei and (ii) the C-terminal part is responsible for
dimerization, nuclear translocation, and the formation of
late, stable phyB NBs. Interestingly, they also showed that
a missense mutation located in the C-terminal region
prevents efficient translocation of the full-length phyB
into the nucleus.
By this time, it was firmly established that translocation
of phyB into the nucleus is conformation dependent, that
chromophore minus PHYB molecules are not imported
into the nucleus, and FR treatment applied immediately
after R induction blocks nuclear transport (Nagy and
Schafer, 2002), which requires at least the formation of
a phyB Pfr/Pr heterodimer (Chen et al., 2003). One way to
interpret these data is to assume that in the inactive Pr
conformation the N-terminal half of the phy molecule
masks the NLSs of the C-terminal part, whereas in the Pfr
form the NLS is unmasked and becomes accessible for the
transport machinery. Findings reported by Chen et al.
(2005) support this hypothesis. These authors showed that
the N-terminal and the C-terminal parts of phyB can
Involvement of nuclear bodies in light signalling 3119
interact in yeasts, and this interaction is weakened by light
irradiation which induces the conformational change of
the molecule, leading to Pr-Pfr transition. In vitro pulldown assays showed that, when co-expressed with PCB,
the interaction between the N- and C-terminal parts of the
molecule is much stronger than in the inactive Pr form.
Thus, reduction of the intramolecular interaction between
the two parts of the molecule may lead to the presentation
of a hidden NLS in the C-terminal region and ultimately
to nuclear translocation of the photoreceptor.
To narrow down the region which may contain nuclear
targeting signals, a series of different C-terminal truncations of the Arabidopsis phyB molecule were created and
the nuclear transport of YFP fusions of these molecules
was examined by the same authors. Chen et al. (2005)
reported that (i) the very C-terminal HKRD domain alone
is not sufficient for mediating translocation of the fusion
protein into the nuclei, whereas (ii) the PRD::YFP protein
translocates into the nuclei upon light illumination but
forms no stable NBs. These data indicated that the PRD
domain is required for nuclear transport, but the precise
mechanism responsible for importing phyB into the nucleus remained largely elusive for the following reasons.
Sequence analysis of phytochromes revealed no canonical
NLS within the PHYB. The N-terminal half of the
molecule fused only to GUS (which ensures dimerization
of the fusion protein) showed limited, although not lightregulated nuclear localization and partial complementation
of the phyB null mutant (Matsushita et al., 2003). Simple
diffusion can be excluded as the cause of the nuclear
localization of the phyB N-terminal-GUS monomer or
dimer proteins. Hence these data indicate that (i) the
N-terminal region also contains a weak NLS, (ii) the
nuclear transport of phyB is mediated by multiple NLSs
or (iii) it does not require an NLS or NLS-like domain.
Are FHY1 and FHL involved in mediating nuclear
import of PHYA?
The size- and light-dependent, very fast accumulation of
phyA in nuclei absolutely requires efficient active transport of the molecule into the nucleus. If so, the transport
machineries mediating nuclear import of phyA and phyB
are presumably different, since nuclear accumulation of
35S:phyB::GFP is a relatively slow process. Alternatively,
it can be hypothesized that phyA contains multiple NLSs
with higher affinity for recognition, or unmasking of these
NLSs occurs faster in vivo in the cytoplasm than in the
case of phyB. Recent observations published by Hiltbrunner
et al. (2005, 2006) suggest that phyA is imported into the
nuclei as part of a preformed protein complex rather than as
a single protein. These authors documented that the nuclear
accumulation of phyA requires at least two small, plantspecific proteins: FAR RED ELONGATED HYPOCOTYL
1 (FHY1) and its homologue FHY1 LIKE (FHL). FHY1
and FHL were shown to be positive regulators of phyA
signalling during de-etiolation (Desnos et al., 2001; Zhou
et al., 2005). Detailed analysis of the fhy1 mutant revealed
that nuclear accumulation of phyA during HIR and also
VLFR is strongly reduced in this mutant background
(representative fluorescent microscopic images are shown
in Fig. 4A). Recently it was shown that in the fhy1/fhl
double mutant the light-induced nuclear accumulation of
PHYA:phyA::GFP and or 35S:phyA::GFP is completely
abolished, suggesting an important role for these two
molecules in the nuclear transport mechanism of phyA
(Hiltbrunner et al., 2006).
Interestingly, each of FHY1 and FHL has a functional
monopartite NLS and a NES close by and it was shown
that FHY1 and FHL can form homo- or heterodimers via
binding through the C-terminal part of the molecule (Zhou
et al., 2005). Loss-of-function mutations of both molecules lead to a complete loss of sensitivity to FR light,
which indicates an indispensable role for these two
molecules in FR-light-induced, phyA-mediated responses
of Arabidopsis (Zhou et al., 2005) (Fig. 4B shows the
hyposensitive growth responses of fhy1 and fhy1/FHL
RNAi plants grown in cFR light). In addition, Zeidler and
co-workers (2004) showed that in dark-grown seedlings
the YFP::FHY1 fusion protein is present in both the nuclei
and the cytoplasm. A brief light treatment initiates
formation of 35S:YFP::FHY1 NBs, which is reminiscent
of the nuclear bodies formed by 35S:phyA::GFP. Similar
intracellular distribution was observed for 35S:YFP::FHL
(Hiltbrunner et al., 2006). Moreover, the presence of
the NLS on the N-terminal half of FHY1 was essential
for nuclear targeting of FHY1 and deletion of the
NLS resulted in the loss of function of FHY1 in FR-lightinduced, phyA-mediated inhibition of hypocotyl elongation (Zeidler et al., 2004).
Based on these observations, Hiltbrunner et al. (2005,
2006) performed a set of experiments that provided
evidence for the involvement of FHY1/FHL in facilitating
import of phyA into the nucleus. These authors showed
by in vitro pull-down assays that phyA interacts both with
FHY1 and FHL in a light-dependent fashion, preferentially in its Pfr form. The 406 amino acid-long N-terminal
fragment of phyA is sufficient for binding these two
factors and this interaction does not depend on the
presence of the very N-terminal region (first 100 amino
acid residues) of phyA. These data indicate that FHY1/
FHL binding is mediated by the chromophore-lyase
domain of phyA. Moreover, when etiolated plants are
irradiated with light, FHY1 and FHL co-localize with
phyA in nuclear bodies.
These data indicate that FHY1/FHL may mediate
nuclear accumulation of phyA by two different, though
not necessarily mutually exclusive mechanisms. Since no
obvious NLSs were found in the PHYA sequence, it is
3120 Kevei et al.
Fig. 4. Light-induced nuclear transport of PHYA::phyA::GFP. phyA nuclear accumulation is strongly dependent on the presence of functional
FHY1 and FHL molecules. 3-d-old, dark grown wild-type (WT), fhy1-1 mutant, and fhy1-1/FHL RNAi double mutant seedlings expressing the
PHYA promoter driven PHYA::GFP transgene were irradiated with 6 h FR light followed by 2 min of WL treatment (A). This irradiation causes
strong nuclear transport of phyA::GFP fusion protein in WT seedlings, in fhy1-1 plants, however, this response is severely reduced. In the double
mutant, phyA::GFP is restricted to the cytosol and does not accumulate to detectable levels in the nuclei. The aberrant nuclear localization of phyA in
the fhy1-1 and fhy1-1/FHL RNAi mutants leads to reduced sensitivity to far-red light, which is visible as a physiological response of the plants (B).
fhy1-1 seedlings show hyposensitivity to far red light when seedlings were grown for 5 d in cFR light of 13 lE intensity, whereas in fhy1-1/FHL
RNAi seedlings the inhibition of hypocotyl elongation is completely abolished. Based on experimental data the nuclear transport of phyA is initiated
by the light-induced conformational changes within the molecule (C). In this model, Pr–Pfr transition of phyA occurs in response to light irradiation,
then light probably unmasks the FHY1/FHL binding site on phyA, which in turn binds to the FHY1/FHL hetero- or homodimer. FHY1/FHL was
shown to exhibit functional NLS, which can be recognized by a cytosolic importin a protein. The importin a-FHY1/FHL-phyA(Pfr) complex is
transported through the nuclear pores to the nuclei, where importin dissociates from the complex. The bar in (A) represents 10 lm.
possible that phyA uses the NLS of FHY1/FHL for its
nuclear transport, via direct binding to homo- or heterodimers of these two proteins. In this case, light unmasks
the binding site for FHY1/FHL on phyA, and nuclear
transport occurs only after the complex had been formed
(Fig. 4C shows the possible mechanism of light-induced
nuclear transport of phyA::GFP). Another possible explanation is that FHY1/FHL works as a nuclear anchoring
factor rather than a nuclear import factor. In this model,
phyA possesses NLS and NES signals and phyA Pr
recycles fast between the cytoplasm and nucleus in the
dark. After light activation the Pfr form of phyA interacts
with FHY1/FHL. This interaction masks the NES signal of
phyA, thus the phyA-Pfr /FHY1/FHL complex is retained
in the nucleus. Consistently with these models, it is also
possible that FHY1 and/or FHL mediate(s) both import
and retention of phyA in the nucleus, thus they are also
involved in the light-induced degradation of phyA.
These models suggest that, similarly to phyB, the
N-terminal half of phyA may play a major role in phyAmediated signalling. Indeed, it was shown that a chimeric
protein containing the N-terminal half of phyA fused to
GFP-GUS-NLS can induce VLFR-type signalling but fails
to function in FR-HIR mode in a phyA null mutant cFR
(Mateos et al., 2006). However, it should be noted that
this short N-terminal fragment of phyA apparently
generates signals in constant darkness and causes hypocotyl shortening and cotyledon unfolding of etiolated
seedlings reminiscent of a weak Constitutive Photomorphogenesis 1 (cop1) allele (Mateos et al., 2006). Thus it is
possible that either the Pr form of the phyA N-terminal
fragment localized in the nucleus partially induces
photomorphogenesis by an unknown mechanism, or light
treatment given on the first day of seedling growth to
induce germination is enough to initiate the formation of
Pfr and its subsequent transport into nuclei. Independent of
Involvement of nuclear bodies in light signalling 3121
these explanations, this result indicates that, in contrast to
phyB, phyA requires additional domains of the molecule,
in addition to its N-terminus, to fulfil its role in signalling.
Does the phosphorylation status of phytochomes
play a role in the light-dependent nuclear
translocation of phytochromes?
The phosphorylation status of phytochromes can obviously influence transportability of the molecule. It was
shown that phyochromes can act as kinases and they are
also subjected to phosphorylation. Indeed, it was shown,
that oat phyA (McMichael and Lagarias, 1990) and
Mesotaenium caldariorum phytochrome are autophosphorylated on Ser/Thr residues (Yeh and Lagarias, 1998)
in a light-dependent manner. Therefore it is widely
assumed that all phytochromes are light-regulated atypical
Ser/Thr protein kinases, which autophosphorylate and
phosphorylate other proteins. It has been hypothesized
that (auto)phosphorylation of phytochromes may influence
the Pr to Pfr conformational change, therefore changes in
the autophosphorylation pattern of phytochromes could be
essential for the regulation of the intracellular distribution
of these photoreceptors. The Ser7 residue of phyA was
shown to be phosphorylated in vivo, however its phosphorylation occurs in both Pr and Pfr forms, whereas Ser17
was shown to be phosphorylated primarily in the Pr form
of the protein in the in vitro experiments. Phosphorylation
at these residues induces subtle conformational changes
within the molecule (Lapko et al., 1997, 1999). It was
shown by Kim et al. (2002) that the Arabidopsis
phosphatase 2A (FyPP) interacts with oat phyA in vitro
in a light-dependent manner. The over-expression of this
phosphatase leads to enhanced responses of plants to light
probably by causing hypo-phosphorylation of phyA,
whereas decreased expression of FyPP causes reduced
phy-A activity. However, cellular distribution of phyA has
not been investigated by the authors. In a more recent
report, Ryu et al. (2005) convincingly demonstrated that
the flux of light signal from Pfr-phytochromes is tightly
controlled by phosphorylation/dephosphorylation of the
photoreceptors. These authors showed that another phosphatase, designated type 5 protein phosphatase (PAPP5)
specifically dephosphorylates biologically active Pfrphytochromes (phyA and phyB) and enhances phytochrome-mediated responses by modulating phytochrome
stability and affinity for downstream transducers. Nuclear
import of phyA and phyB, however, was not significantly
affected by over-expressing PAPP5. In oat phyA, the
Ser598 residue located in the hinge region of the molecule
is preferentially phosphorylated in vivo in the Pfr form.
When Ser598 is mutated, the mutant oat phyA exhibits
hypersensitivity to FR light (Kim et al., 2004). These
authors also showed that phosphorylation at Ser598
inhibits interaction of phyA with two well-known signalling partners, NDPK2 and PIF3, thus inhibiting the
signalling from phyA localized in the nucleus, whereas
dephosphorylation of phyA at the same position by
a phosphatase could amplify the signals. Ser598 is not
autophosphorylated and is not required for kinase activity
of phyA (Kim et al., 2004), thus an as yet unidentified
protein kinase could play a regulatory role in phosphorylation of this residue. The same authors reported no
difference between the intracellular distributions of the
native and the mutant phyA, indicating that the Ser598
mutation does not affect nucleo-cytoplasmic distribution,
i.e. nuclear import of phyA. Taken together, these results
indicate that nuclear translocation of phyA is not
significantly affected by phosphorylation or dephosphorylation of the photoreceptor.
Mutations within phytochromes alter the
subcellular distribution of the photoreceptors,
but do they really affect nuclear import?
Nuclear import and intracellular redistribution of phyA is
specifically affected by point mutations identified in
various domains of the photoreceptor. For example, it
was shown that several missense mutations located in the
Quail-box of the Arabidopsis PHYA or PHYB genes
(Wagner et al., 1996) prevented high-level accumulation
and formation of stable nuclear bodies containing the
mutant photoreceptor proteins in the nuclei (Kircher et al.,
1999). It is worth noting that these mutants also displayed
hyposensitivity to cFR or cR, thus suboptimal physiological responses of these mutants correlated with the
aberrant intracellular distribution pattern of the photoreceptor. These results indicated that the formation of stable
phyA and phyB NBs may affect signalling by these
photoreceptors. The phyA-302 mutant, carrying a missense
mutation in the so-called PAS2 domain in the C-terminal
part of the photoreceptor retained normal VLFR but
lacked HIR. The 35S:phyA-302::GFP fusion protein
showed normal translocation from the cytosol to the
nucleus under cFR, but failed to produce nuclear bodies
containing the mutant photoreceptor. These observations
suggested that phyA NBs could be involved in mediating
HIR signalling (Yanovsky et al., 2002). It is thought that
the PAS1-Quail-box-PAS2 region of PHYA and PHYB is
involved in mediating the interaction of phyA and phyB
with their cognate signalling partners. Therefore, these
mutations may have a discernible effect on the nuclear
import process itself. The EID4 mutation is localized in
the chromophore binding domain (BLD) of phyA
(Dieterle et al., 2005). The mutant phyA protein is more
stable but displays normal photochemical properties. The
eid4 mutant was shown to form phyA NBs, the appearance of which is transient, i.e. they dissolve in 60–90 min
3122 Kevei et al.
after the onset of irradiation. In cFR light, a few hours
after the lights-on, larger nuclear bodies appear which are
stable for 8 h. By contrast with wild-type phyA, phyAEID4
can re-accumulate in these NBs even after a single pulse
of R. In dark-grown plants, the mutant phyA::GFP
showed normal cytosolic distribution. Upon R illumination, however, unlike the native phyA the phyAEID4 fails
to form cytosolic speckles (SAPs). The eid4 mutant is
hypersensitive in cFR, displays increased inhibition of
hypocotyl elongation and phyAEID4 appears in nuclear
bodies even after a brief light pulse. This can be explained
by the increased stability of the phyAEID4 Pfr form.
Alternatively, the mutant molecule stays for a longer time
in NBs (associates faster and dissociates slower) which
results in higher stability of the mutant photoreceptor. In
either case, more Pfr is available for a longer time in the
nuclei of eid4, which could explain the hypersensitivity of
the mutant.
Chen and co-workers (2003) performed a screen to find
mutants which show mislocalization of 35S:phyB::GFP
protein when seedlings are grown in cR. The mutants
were divided into two categories. The first group
contained mutants which had missense point mutations
within the 35S:PHYB::GFP transgene. The examination
of the translocation pattern of these molecules showed that
mutations in the hinge region and in the C-terminal PASrelated domain do modify intranuclear localization of
phyB. Although all mutant proteins were transported into
the nuclei in light-grown seedlings, they were distributed
homogenously within the nucleus and none of them
formed stable phyB NBs (Chen et al., 2003). The second
group of mutants, called Dsf (Deficient in Speckle
Formation) also showed nuclear transport of phyB and
were able to form early stage II and III but not stage IV
type phyB NBs. Since the formation of these late, large,
stage IV nuclear bodies is induced by high-fluence R
irradiation, the DSF proteins are presumably required to
mediate these responses. The identity and molecular
nature of these DSF proteins is not yet known.
Perspectives
phyA–E-mediated signalling requires nuclear translocation
of the photoreceptors and interaction with their cognate
signalling partners. Phytochromes interact with a number
of proteins and phytochrome-containing nuclear bodies
have been described as sites of co-localization of
phytochromes with CRY2 (Mas et al., 2000), COP1 (Seo
et al., 2004), and PIF3 (Bauer et al., 2004; Al-Sady et al.,
2006). The functional importance of these and other phycontaining nuclear bodies is a subject of intense discussion, but relevant experimental data to solve this problem
are not available. For example, it is not known whether
these apparently different NBs indeed represent structurally and functionally different nuclear complexes and/or
whether they contain, beside the few detected proteins,
additional specific and common components. Thus it
could be concluded that although it is tempting to
speculate about the possible function of phy NBs, genetic
(specific mutants specifically affecting NB formation) and
biochemical data (identification of components present in
different NBs) are needed to define the function of these
subnuclear complexes in phytochrome-mediated light
signalling.
Acknowledgements
Work in Hungary was supported by Hungarian Scientific Research
Fund (OTKA 60106) and Howard Hughes Medical Institute
International Research Scholar grants to FN. Work in Germany
was supported by a Humboldt Research Award grant to FN and an
SFB 746 grant to ES. Eva Kevei is supported by a grant from the
Humboldt Research Foundation. We thank Erzsebet Fejes for
critical reading of the manuscript and for providing unpublished
results for Fig. 3. Special thanks to Andreas Hiltbrunner for
providing the images and the figure describing the function of
FHY1 and FHL in light-induced nuclear transport of phyA and to
Stefan Kircher and Lana Kim for providing the fluorescent
microscopic images showing the 35S:phyA::GFP nuclear localization pattern under different light conditions.
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