Review
Blackwell Science, Ltd
Tansley review no. 136
The cell biology of phytochrome
signalling
Author for correspondence:
Simon G. Møller
Tel: +44 116 252 5302
Fax: +44 116 252 3330
Email: [email protected]
Simon G. Møller, Patricia J. Ingles and Garry C. Whitelam
Department of Biology, University of Leicester, University Road, Leicester, LE1 7RH, UK
Received: 17 September 2001
Accepted: 20 December 2001
Contents
Summary
553
V. Interactions with other signalling pathways
I. Introduction
554
II. Nucleus vs cytoplasm
556
Acknowledgements
583
III. The nucleus
562
References
583
IV. The cytoplasm
571
VI. Conclusions and the future
577
582
Summary
Phytochrome signal transduction has in the past often been viewed as being a
nonspatially separated linear chain of events. However, through a combination of
molecular, genetic and cell biological approaches, it is becoming increasingly evident
that phytochrome signalling constitutes a highly ordered multidimensional network
of events. The discovery that some phytochromes and signalling intermediates
show light-dependent nucleo-cytoplasmic partitioning has not only led to the
suggestion that early signalling events take place in the nucleus, but also that
subcellular localization patterns most probably represent an important signalling
control point. Moreover, detailed characterization of signalling intermediates has
demonstrated that various branches of the signalling network are spatially separated
and take place in different cellular compartments including the nucleus, cytosol,
and chloroplasts. In addition, proteasome-mediated degradation of signalling
intermediates most probably act in concert with subcellular partitioning events as an
integrated checkpoint. An emerging view from this is that phytochrome signalling is
separated into several subcellular organelles and that these are interconnected
in order to execute accurate responses to changes in the light environment. By
integrating the available data, both at the cellular and subcellular level, we should
be able to construct a solid foundation for further dissection of phytochrome signal
transduction in plants.
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I. Introduction
1. Light perception and signalling
Organisms respond to light in very different ways ranging
from simple signal perception and execution in single-cell
prokaryotes to complex signalling networks in multicellular
eukaryotes. In plants, light plays a very important role and
moreover plants need to be extremely adaptable to light due
to their sessile nature. Light not only acts as an energy source
for photosynthesis, but plants have to monitor the light
quality and quantity input in order to execute the appropriate
physiological and developmental responses. To achieve this
plants have evolved a complex set of photoreceptors, including
the blue/UV-A absorbing cryptochromes and the red/far-red
light-absorbing phytochromes, of which the latter are the best
understood (Furuya, 1993; Quail et al., 1995; Neff et al., 2000).
Our understanding of the roles of phytochrome action is
good, however, insight into how the perceived light signals are
transduced leading to morphological responses and altered
gene expression patterns has remained somewhat sparse.
Recently, tremendous efforts have been made in both dissecting these intermediate signalling events and understanding
how they are integrated into the overall phytochrome signalling network (Møller & Chua, 1999; McCarty & Chory,
2000; Neff et al., 2000). Early pharmacological approaches
suggested an involvement of G-proteins, cGMP and calcium
as mediators of light-regulated gene expression (Neuhaus
et al., 1993; Bowler et al., 1994) and these hypotheses have
now been strengthened genetically (Okamoto et al., 2001;
Guo et al., 2001). More recently, the isolation of mutants
disrupted in genes encoding phytochrome signalling intermediates have identified both positive and negative components
of the phytochrome signalling pathways. In addition, using
protein–protein interaction technology, such as yeast twohybrid and in vitro pull-down assays, there have been exciting
developments in understanding the plethora of physical
interactions that take place not only between phytochrome
and early signalling intermediates but also between bona fide
signalling components. An emerging view from the available
data is that phytochrome signal transduction is a highly
ordered but yet complex network of events with different
branches of the signalling network spatially separated into
different subcellular compartments. The notion that early
phytochrome signalling events take place in the nucleus
was prompted recently by the finding that nuclear localized
transcriptional regulators physically interact with the active
form of phyA and phyB (Ni et al., 1998; Ni et al., 1999;
Fairchild et al., 2000). This was further strengthened by the
demonstration that both phyA and phyB translocate to the
nucleus in a light-dependent fashion (Kircher et al., 1999a;
Gil et al., 2000; Kim et al., 2000). Similarly, several other
nuclear phytochrome signalling components have been
identified clearly implying that the nucleus is a hot-spot for
early signalling events (Choi et al., 1999; Hudson et al., 1999;
Büche et al., 2000; Ballesteros et al., 2001; Hicks et al., 2001).
Despite this, phytochrome signalling components have also
been identified in the cytoplasm (Fankhauser et al., 1999;
Bolle et al., 2000; Hsieh et al., 2000) and in chloroplasts
(Møller et al., 2001) demonstrating that the nucleus is not the
only place for phytochrome signalling action.
We have now accumulated a large number of components
that we need to integrate at the whole cell level to further
understand phytochrome signal transduction. It is clear that
multiple organelles are actively involved in phytochrome signalling and that these are interconnected. It is also evident
that the subcellular partitioning of both phytochrome and
signalling intermediates provide an elegant control mechanism. Likewise, the finely tuned degradation of signalling
components by the 26S proteasome is clearly an important
check point. In addition, the multitude of interactions that
occur between phytochrome signalling and other pathways is
fascinating and these interactions depend on controlled subcellular partitioning events.
This review focuses on the cell biology of phytochrome signalling. We have tried to describe the different signalling components in terms of their subcellular localization and how this
impinges on their intrinsic function as well as their overall role
in the phytochrome signalling network. We believe that one
of the keys to further dissect phytochrome signal transduction
is to use the spectrum of molecular and biochemical tools
available, not only to isolate new signalling components,
but to integrate our pool of phytochrome knowledge in a cell
biological context.
2. Phytochrome structure and function
Phytochromes were first described by Borthwick et al. (1952)
as the receptors responsible for red/far-red reversible plant
responses. It has since been shown that phytochromes belong
to a closely related family of photoreceptors, the apoproteins
of which are encoded by a small family of divergent genes. In
Arabidopsis thaliana five discrete apophytochrome-encoding
genes, PHYA-PHYE, have been isolated and sequenced
(Sharrock & Quail, 1989; Clack et al., 1994; Cowl et al.,
1994). Arabidopsis PHYB and PHYD polypeptides are
approx. 80% identical (Mathews & Sharrock, 1997) and are
more closely related to PHYE than they are to either PHYA
or PHYC (approx. 50% identity). Counterparts of PHYA,
PHYB and other PHY genes are present in most, if not all,
higher plants (Mathews & Sharrock, 1997).
All of the higher plant phytochromes appear to share
the same basic structure, consisting of a dimer of identical
c. 124 kDa polypeptides. Each monomer carries a single covalently linked linear tetrapyrrole chromophore (phytochromobilin), attached via a thioether bond to a conserved cysteine
residue in the N-terminal globular domain of the protein. The
C-terminal domain encompasses two histidine kinase related
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Tansley review no. 136
Review
Fig. 1 Structural features of phytochrome
B protein, indicating the positions of the
chromophore attachment site, serine
phosphorylation sites, histidine kinaserelated domains (HKRD) and the two PAS
domains.
domains (HRKD) and two motifs with homology to PAS
(PER-ARNT-SIM) domains (Lagarias et al., 1995; Kay,
1997). PAS domains are present in various signal transduction
molecules which sense environmental signals such us light
conditions, oxygen levels, and redox potential (Taylor &
Zhulin, 1999). They may also mediate protein–protein
interactions. See Fig. 1 for details of domains. The aminoterminal half of phytochromes can be considered as a lightsensing domain whilst the carboxyl-terminal half can be
regarded as the regulatory domain (Fig. 1).
Phytochrome can be classified into two groups based on
stability in light: type I (phyA) occurs in etiolated tissues in
large quantities and is subject to a high turnover, i.e. is light
labile and type II (phyB-phyE), are light stable. Phytochromes
undergo photoconversion between two stable states: the red
light absorbing form (Pr, synthesised in the dark) and the
far-red light absorbing form (Pfr). This Pr-to-Pfr transition is
due to a light induced double bond rotation in the chromophore and rearrangements of the protein backbone of the
apoprotein. For most responses Pfr is considered to be the
biologically active form. The Pr forms of (at least some)
phytochromes of higher plants localize to the cytoplasm,
whereas a proportion of the Pfr (active) isoforms localize to
the nucleus (Kircher et al., 1999a).
Many excellent reviews have previously covered phytochromemediated photomorphogenesis (e.g. Whitelam & Devlin, 1997;
Quail, 1998; Whitelam et al., 1998). To put phytochrome
signalling in context, we will briefly describe the principal
biological roles of phytochromes.
Phytochrome A Phytochrome A (phyA) is responsible
primarily for sensing prolonged far-red light in the far-red
High Irradiance Response (HIR) mode of phytochrome
action. This response mode operates in the regulation of many
aspects of seedling de-etiolation, including inhibition of
hypocotyl elongation, the expansion of cotyledons, changes in
gene expression and the synthesis of anthocyanin, etc. (Casal
et al., 1997; Whitelam et al., 1993; Barnes et al., 1996). These
responses to prolonged far-red irradiation are absent in
phyA null seedlings. Phytochrome A also mediates the
Very Low Fluence Responses (VLFR) of etiolated seedlings.
Additionally, in both young seedlings and in mature
Arabidopsis, phyA appears to be important for perception of
daylength (e.g. Johnson et al., 1994).
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Phytochrome B Phytochrome B (phyB) deficiency leads to
impaired de-etiolation responses in red light (Reed et al.,
1993), but not in prolonged far-red, thus, it is concluded that
for de-etiolation responses, phyA and phyB have discrete
photosensory activities. Phytochrome B also plays a major
role in the low fluence response (LFR) promotion of seed
germination, which is a red /far-red reversible response
(Shinomura et al., 1996). Phytochrome B is considered to be
the main phytochrome responsible for the shade avoidance
response (Smith & Whitelam, 1997) as phyB-deficient
mutants have the typical architecture of the mature lightgrown plant displaying shade avoidance responses (elongated
growth habit, reduced leaf area, increased apical dominance
and early flowering, Robson et al., 1993; Halliday et al.,
1994; Devlin et al., 1996). This indicates that phyB perceives
the low red:far-red signals, which result from the far-red-rich
light that is reflected from (or transmitted through) the leaves
of nearby plants. However, phyB null mutants still show
further shade avoidance responses to low red:far-red signals
(Devlin et al., 1996) indicating that one or more other
members of the phytochrome family are also involved in the
perception of red:far-red signals.
Phytochromes C, D and E From analyses of various phytochrome mutant combinations, it is clear that both phyD and
phyE are also mediators of shade avoidance responses such as
petiole elongation and flowering time, with phyE having
a specific role in regulating internode elongation (Devlin
et al., 1998). Phytochrome E also plays a role in the red/
far-red reversible promotion of seed germination and in the
promotion of germination by far-red light, a response
previously considered to be mediated solely by phyA (Hennig
et al., 2002). Studies of phyC function have previously relied
on analysis of transgenic plants that over-express PHYC
(Halliday et al., 1997; Qin et al., 1997) and analysis of the
phyAphyBphyDphyE quadruple mutant. These studies have
revealed that phyC may play a role in regulating leaf
expansion (Qin et al., 1997) and in the perception of
daylength (Halliday et al., 1997), but that phyC appears not
to play a major role in responses to low red:far-red ratio.
Photoreceptors talk to each other! Analyses of Arabidopsis
mutants containing null alleles of one or more phytochromes
have been used to try to dissect the individual roles of
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Tansley review no. 136
phytochromes. However, this is often complicated because as
well as having independent functions, phytochromes also
show redundancy of function and may modulate the action
of each other. Clearly, phytochromes also interact and coact
with other photoreceptors (Mohr, 1994)
Ahmad & Cashmore (1997) reported that cryptochrome
action, in the inhibition of hypocotyl elongation under blue
light, was dependent on the presence of phyA or phyB. However, it was later shown that cry1 had biological activity in a
phyAphyB null mutant background in blue light especially at
higher fluence rates (Neff & Chory, 1998; Poppe et al., 1998).
The reduced sensitivity of phyAphyB mutants to low fluence
rate blue light was accounted for on the basis of loss a phyA
contribution to blue light perception. Cryptochrome and
phytochrome also interact in phototropic curvature: prior
stimulation of phytochrome by red light enhances the bluelight mediated response, and this appears to be regulated
by phyA (Parks et al., 1996). Additionally, phyB and cry2 act
antagonistically in regulating flowering: phyB appears to
repress whilst cry2 stimulates floral induction (Guo et al.,
1998). In addition to these genetic studies indicating interactions between phytochrome and cryptochrome there is also
evidence that cry1 can physically interact with phyA in yeasttwo-hybrid assays (Ahmad et al., 1998) and more recently
that cry2 can interact with phyB (Mas et al., 2000).
Phytochrome signal transduction Upon photoconversion
from Pr to Pfr, phytochromes rapidly induce a cascade of
signalling events. Despite half a century of research on
phytochromes we are only beginning to understand how this
may be achieved. One approach relies on using a variety of
screens to find genes acting downstream of phytochrome that
mediate signal transduction. The following table introduces
some of the key players that have been identified so far and
addresses the possible subcellular compartmentalisation of
signalling events (Table 1).
II. Nucleus vs cytoplasm
In all eukaryotic cells the nucleus is separated from the
cytoplasmic compartment by the nuclear envelope. Although
the nucleus has a high degree of autonomy the nuclear
envelope is in intimate contact with the cytoplasm and
contains nuclear pore complexes to allow for the exchange of
macromolecules. The nucleo-cytoplasmic exchange highway
comprises a multitude of substrates such as histones and
transcription factors imported to the nucleus from the
cytoplasm and tRNA and rRNA molecules exported from the
nucleus to the cytoplasm. It is now becoming increasingly
clear that compartmentation not only aids in containment
of cellular activities but also acts as a control point for key
cellular events. Indeed, it has recently been shown that
phytochromes are imported into the nucleus in a light-quality
and light-quantity dependent manner, providing an early
upstream control point for phytochrome signal transduction
(Sakamoto & Nagatani, 1996; Kircher et al., 1999a; Gil et al.,
2000; Kim et al., 2000; Hisada et al., 2000).
1. Where is phytochrome?
The precise intracellular localization of phytochrome was
unknown for an extended period of time and conflicting data
was painting a cloudy picture. Interest into phytochrome
localization started to flourish in the early 1970s when various
research groups used physiological methods to show that
phytochrome is associated with various cellular compartments including rough endoplasmic reticulum (Williamson &
Morre, 1974), etioplasts (Welburn & Welburn, 1973), and
mitochondria (Manabe & Furuya, 1974). With the advent
of more sophisticated immunocytochemical and subcellular
fractionation techniques it was however, becoming largely
accepted that phytochrome (more precisely phyA) was mainly
cytosolic with a small proportion possibly bound to the
plasma membrane (Quail et al., 1973; Coleman & Pratt,
1974; Mackenzie et al., 1975; Speth et al., 1986, 1987).
Knowing that receptors are often membrane-bound upon
signal perception, the possibility of phytochrome being
associated with cytoplasmic membrane structures seemed
highly plausible. In addition, the red light-induced
sequestering of photoactive phyA into electron dense areas,
followed by reversible diffuse cytosolic distribution upon
conversion back to the Pr form (Mackenzie et al., 1975;
McCurdy & Pratt, 1986; Speth et al., 1986), indicated a lightdependent subcellular distribution pattern of phytochrome.
Taking a slightly different approach, Mösinger et al. (1987)
demonstrated that by adding oat phyA protein to isolated
barely nuclei, the transcription rate of genes encoding
chlorophyll a/b binding protein could be increased (Mösinger
et al., 1987). This suggested that phyA, or at least part of
phyA, is associated with the nucleus and affects the expression
dynamics of light-regulated genes. The concept of phyA being
nuclear-associated did not receive much credit and studies
performed by Nagatani et al. (1988) showed that phyA
associates with nuclei from dark-grown pea seedlings in a
nonspecific manner. The prevailing view that phyA was a
cytoplasmic protein were further substantiated by microinjection and pharmacological experiments (Section IV/1).
Using microinjection and pharmacological agents it was
speculated that the cytosolic form of phyA activates a
heterotrimeic G-protein either by Pfr-driven translocation to
the plasma membrane or by using a cytoplasmic intermediary
molecule to transduce the signal from Pfr to the G-protein
(Neuhaus et al., 1993; Bowler et al., 1994). Although it
seemed feasible that phyA may associate with the plasma
membrane, computational analysis revealed that phyA from
various species has no motif or structure suggestive of
membrane insertion. These studies were challenged by in vivo
microbeam irradiation experiments in lower plants
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Table 1 Phytochrome signalling components and their subcellular localization
Mutant
Interactions
Reference
Hypersensitive for red I
nduced de-etiolation
PIF3
Similarities to b-HLH transcription
factors; promoter insertion causing
increased levels of PIF3
phyA & phyB SN1
phyA & phyB in Y2H
Ni et al. (1998); Halliday et al. (1999);
Quail (2000)
gi-100
Impaired response to red
GIGANTEA
circadian clock-controlled gene that
regulates photoperiodic flowering
phyB SN
Huq et al. (2000b); Fowler et al. (1999)
rsf1/hfr1/rep1
rsf1:(reduced sensitivity to
far-red); hfr1:(long hypocotyl in
ar-red); rep1:(reduced
phytochrome signaling 1)
RSF1/HFR1/REP1
Similarities to b-HLH transcription
factors; high sequence
identity to PIF3
phyA SN
Spiegelman et al. (2000);
Fankhauser & Chory (2000);
Fairchild et al. (2000); Soh et al. (2000)
far1
Impaired response to far-red
FAR1
Putative coiled-coil domain
phyA SN
Hudson et al. (1999)
spa1
Isolated as a suppressor of a
weak phyA mutation
SPA1
WD repeat protein
phyA SN1
COP1 in Y2H
Hoecker et al. (1998, 1999, 2001)
elf3
Early flowering
ELF3
Ciracadian clock-regulated protein
phyB SN/synergistic to phyB?
Liu et al. (2001); Covington et al. (2001);
Hicks et al. (2001); McWatters et al. (2000)
hy5
Impaired responses to far-red,
red and blue light
HY5
bZIP transcription factor
COP1 in Y2H
Koorneef et al. (1980); Oyama et al. (1997);
Chattopadhyay et al. (1998); Ang et al. (1998)
eid1
Increased sensitivity to far-red
EID1
F-box protein leucine–1 zipper motif
phyA SN1
AKS1 and AKS2 in Y2H
Büche et al. (2000); Dieterle et al. (2001)
laf1
Impaired response to far-red
LAF1
R2R3 MYB-like transcription factor
phyA SN
Ballesteros et al. (2001)
fhy1
Impaired response to far-red
Novel light regulated protein
phyA SN
Whitelam et al. (1993); Desnos et al. (2001)
shy2
shy2–1D and shy2–2
suppressors of the longhypocotyl phenotype of hy2
and hy3, respectively
IAA3 Auxin-induced
transcription factor
coprecipitation with phyB protein
Kim et al. (1996); Reed et al.
(1998); Tian & Reed (1999)
cop/det/fus
Photomorphogenic phenotype
in the dark
COP1*: RING finger motif, coiled-coil
region and WD40 repeat domain.
DET1: novel nuclear localized protein
COP10
COP9 signalosome:
subunits CSN1-8
Epistatic analyses suggests
cop/det/fus loci act downstream
of phyA, phyB and CRY-1 SN.
COP1 interacts with CIP1,
CIP4, CIP7, CIP8 and HY5
Chory et al. (1989); Deng et al. (1991);
Misera et al. (1994); Pepper et al. (1994);
NDPK2*
Nucleotide diphosphate kinase 2
phyA & phyB SN
phyA & phyB inY2H
Choi et al. (1999)
Nucleus
poc1
ndpk2
Impaired response to red
and far-red
Wei & Deng (1996); Von Arnim & Deng (1994);
Deng & Quail (1999); Karniol & Chamovitz, 2000;
Yamamoto et al. (2001)
Review
Protein
Tansley review no. 136
Phenotype of mutant
557
Phenotype of mutant
Protein
Interactions
Reference
Cytosol
pat1
Insensitive to far-red
PAT1
VHIID/GRAS protein
phyA SN
Bolle et al. (2000)
fin219
Impaired response to far-red
FIN219
Auxin-inducible GH3 protein
phyA SN
FHY1 interaction by genetic
analysis
Hsieh et al. (2000)
pks1
PKS1 overexpressor: impaired
response to red; PKS1
antisense lines: no effect on
plant phenotype
PKS1
phytochrome kinase substrate 1
phyB SN
phyA & phyB inY2H
Fankhauser et al. (1999)
laf6
Impaired response to far-red
LAF6
ABC-like protein
PhyA SN
Møller et al. (2001)
gun5
Pale leaves: nuclear Lhcb1
expression in the absence of
chloroplast development
GUN5: ChlH subunit of
Mg-chelatase
HY1 and GUN5 metabolic
interaction
Mochizuki et al. (2001); Susek et al. (1993)
CHLOROPLAST
UNKNOWN SUBCELLULAR LOCATION
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psi2
Hypersensitive to red
and far-red
Not cloned
phyA & phyB SN1
Genoud et al. (1998)
pef1
Impaired response to red,
and far-red
Not cloned
phyA & phyB SN
Ahmad & Cashmore (1996)
pef2
Impaired response to red
Not cloned
phyB SN
Ahmad & Cashmore (1996)
pef3
Impaired response to red
Not cloned
phyB SN
Ahmad & Cashmore (1996)
red1
Suppressor of a phyB
overexpressor phenotype.
Impaired response to red
Not cloned
phyB SN
Wagner et al. (1997)
fhy3
Impaired response to far-red
Not cloned
phyA SN
Whitelam et al. (1993); Yanovsky et al. (2000)
shl
Hypersensitive to red,
far-red, and blue
Not cloned
PhyA-E, CRY
Pepper et al. (2001)
fin2
Impaired response to far-red
Not cloned
phyA SN
Soh et al. (1998)
bas1-D
Suppressor of phyB mutant
A cytochrome P450: activation
tagging causing increased
levels of CYP72B1
bas1-D is epistatic to phyB;
phyA is epistatic to bas1-D;
bas1-D partially suppresses
a cry1-null mutation
Neff et al. (1999)
SN, signalling network; COP1*, nuclear subcellular location in the dark, excluded in the light: constitutively nuclear in root cells. SN1, determined by epistasis of phytochrome mutation
NDPK2*: GFP-NDPK2 fusions show nuclear and cytoplasmic subcellular location. Y2H, yeast-two-hybrid screen.
Tansley review no. 136
Mutant
558 Review
Table 1 continued
Tansley review no. 136
demonstrating a very localized response and action dichroism
for phytochrome-mediated growth responses implying that
phyA is associated with the plasma membrane (Kraml, 1994).
Taken together these observations suggested that phyA is
mainly cytosolic but with some of the photoactive Pfr form
possibly localized to the plasma membrane via protein–
protein interactions.
2. Intracellular localization of phytochrome
A major, but surprising, breakthrough regarding phytochrome localization came when Sakamoto & Nagatani
(1996) fused the C-terminal region (dimerisation/protein–
protein interaction domain) of Arabidopsis phyB to βglucuronidase (GUS) and showed that the fusion protein
predominantly localizes to the nucleus in transgenic plants.
This indicated for the first time that phytochrome, more
specifically the C-terminal region of phyB, contains a
functional nuclear targeting signal (NLS). To corroborate the
localization pattern observed in transgenic plants, Sakamoto
& Nagatani (1996) isolated nuclei from light-grown wildtype Arabidopsis seedlings and showed that a large amount of
the total cellular phyB content localizes to the nucleus and
that this level decreases when seedlings are dark-adapted.
It was further inferred from these studies that since the
C-terminal fusion protein is constitutively nuclear localized,
the spectral form of phyB must be important for the lightdependent nuclear import or cytoplasmic retention.
Although the evidence for phyB nuclear localization was good
there was concern that the nucleus may not be the only site
of phytochrome action. This was largely based on the
microinjection (Neuhaus et al., 1993; Bowler et al., 1994)
and microbeam (Kraml, 1994) experiments indicating that
phyA is cytosolic and possibly membrane bound. This further
raised the intriguing possibility that although phytochromes
share common overall structural and molecular properties
they may function in very different ways.
Subcellular localization of phyB In order to fully dissect
the subcellular localization pattern of phyB and to gain
insight into how this may influence photoperception
and downstream signalling events, Nagatani and colleagues
(Yamaguchi et al., 1999) generated transgenic plants
overexpressing a fusion protein consisting of a full-length
Arabidopsis PHYB cDNA fused to GFP driven by the
CaMV35S promoter. One potential problem when analysing
intracellular localization patterns of fusion proteins is the risk
of mislocalization and loss of functionality due to the fusion
partner itself. Nagatani and colleagues (Yamaguchi et al.,
1999) addressed this elegantly by transforming the phyB/
GFP fusion construct into phyB-deficient Arabidopsis
seedlings (phyB-5; Reed et al. (1993)) and by doing so
generating transgenic seedlings exhibiting a typical phyB
overexpression phenotype in response to red light irradia-
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Review
tion (Wagner et al., 1991; McCormac et al., 1993). This
confirmed that the phyB/GFP fusion protein is biologically
and photochemically active and that the resulting localization
data must therefore reflect the real in vivo situation. As
observed with the C-terminal region of phyB (Sakamoto &
Nagatani, 1996), at least some of the full-length phyB/GFP
fusion protein localized to the nucleus in light treated
seedlings. However, at higher magnification it was evident
that the phyB/GFP localizes to subnuclear foci giving rise to
fluorescent speckles or spots within the nucleus. All cell types
examined had between 5 and 10 speckles/nucleus and they
were approx. > 1 µm in size. This phenomenon has now been
examined in more detail and it seems that the speckle number
varies depending on the fluence rate (Gil et al., 2000). In
darkness the full-length phyB cDNA/GFP fusion protein
localizes diffusely to the cytoplasm corroborating the previous
localization patterns (Sakamoto & Nagatani, 1996). In order
to further analyse the light-induced nuclear translocation
of photochemically active phyB, Yamaguchi et al. (1999)
examined the red-light induced translocation and speckle
formation over a period of 6 h. After 2 h of red light
irradiation the phyB levels in the nucleus increase, however, a
substantial part is still present in the cytoplasm. In addition,
very little speckle formation is observed. After 4 h the
translocation is almost complete showing the presence of a
large number of small speckles, whilst after a further 2 h GFP
fluorescence can only be observed in the nucleus in the form
of fewer but larger speckles. This time course clearly unveiled
that the translocation event and the speckle formation are
closely linked, both being part of a highly dynamic process.
These observations were corroborated and further extended
by Nagy, Schäfer and colleagues (Kircher et al., 1999a; Gil
et al., 2000) using a tobacco CaMV35S-phyB/GFP fusion in
transgenic tobacco plants. As shown for Arabidopsis (Yamaguchi
et al., 1999), the tobacco phyB/GFP fusion protein was able
to complement a phyB-deficient Nicotiana mutant (Kircher
et al., 1999a). Kircher et al. (1999a) demonstrated, as Yamaguchi
et al. (1999), that the accumulation of phyB/GFP in the
nucleus is slow, taking approx. 2 h to reach a fluorescence level
above the detection threshold. This could either represent a
real physiological phenomenon or an artifact due to the presence of the GFP fusion partner. Although the phyB/GFP
fusion protein was shown to complement a phyB-deficient
mutant, the amount of translocated nuclear phyB needed for
functional signalling to occur may be very little in which case
it is hard to determine if the slow import kinetics are in fact
real. Although fusion protein technology is a useful tool for
subcellular localization studies, kinetic measurements of fusion
protein translocation events do not necessarily reflect the
in vivo situation. More detailed in vivo immunolocalization
studies of the endogenous phyB would however, clarify this.
Kircher et al. (1999a) clearly showed that the phyB translocation event is highly dependent on the quality of light in
that neither continuous far-red light nor repeated pulses result
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in nuclear phyB accumulation or speckle formation. Taken
together these results indicate that phyB regulates its own
nuclear translocation, which is mediated by the low-fluence
response (LFR) of phytochrome (Kircher et al., 1999a). Further characterization also showed that red-light-induced
speckle formation is dependent on the fluence rate. Below a
fluence rate of 7 µmol m−2 s−1 the accumulation of nuclear
speckles follow a hyperbolic curve reaching saturation (c. 20
speckles/nucleus after 3 h red light irradiation) at higher
fluence rates (Gil et al., 2000). The authors also make the
observation that an equal number of photons at different time
points result in different kinetic and saturation properties,
indicating that nuclear import of phyB and speckle formation
is dependent on both the fluence rate and time.
Subcellular localization of phyA As for phyB, it was of great
interest to determine the dynamics of phyA translocation and
subcellular partitioning and it was partly expected that phyA
would behave differently from phyB due to their different
modes of action. As for phyB, Kim et al. (2000) generated a
full-length Arabidopsis PHYA cDNA /GFP fusion protein
driven by the endogenous PHYA promoter and showed that
this fusion protein is able to complement and restore wildtype characteriztics in a phyA-deficient Arabidopsis mutant
(phyA-201). Using these complemented lines it was shown
that the phyA /GFP fusion protein localizes to the cytoplasm
in darkness with no detectable nuclear fluorescence. The
intracellular localization of phyA is strongly affected by
irradiation showing rapid nuclear import upon brief exposure
to pulses of far-red, red, and blue light. It was also noted by
the authors that after the far-red pulse, but before the
translocation event, speckle formation was observed in the
cytoplasm reminiscent of phyA sequestration in monocots
(Speth et al., 1986; Speth et al., 1987) and rice phyA/GFP
speckle formation in tobacco plants (Kircher et al., 1999a).
Continuous red light irradiation for 5 h failed to induce
nuclear import and cytosolic speckle formation of phyA/GFP.
Conversely, continuous far-red and blue light irradiation for
5 h induced both nuclear import of phyA and speckle
formation whilst very little cytoplasmic speckling was
observed (Kim et al., 2000). Taken together these findings
suggest that the nuclear import of Arabidopsis phyA /GFP is
not only mediated by the very low fluence response (VLFR)
but also by the far-red high irradiance response (HIR). In
dicots the far-red HIR is diminished by red light pretreatment
and transgenic seedlings subjected to a red light pretreatment,
followed by far-red light irradiation, showed lack of phyA
nuclear import (Kim et al., 2000).
In tobacco, the rice phyA /GFP fusion protein translocates
to the nucleus in response to short pulses of red and far-red
light suggesting VLFR-mediated nuclear import (Kircher
et al., 1999a). This analysis was extended by generating transgenic tobacco harbouring the Arabidopsis phyA /GFP fusion.
Kim et al. (2000) found that in contrast to Arabidopsis phyA
in Arabidopsis and rice phyA in tobacco, Arabidopsis phyA
in tobacco does not show VLFR-mediated nuclear translocation. These results clearly demonstrate another intriguing
property of phytochrome; the differences in the spectral
sensitivity of Arabidopsis phyA are indicative of different
physiological roles depending on the cellular background.
The authors speculate that this is probably exerted at the level
of light-dependent degradation or by the mechanism mediating phyA retention or import into the nucleus (Kim et al.,
2000). A cautionary note from this is that localization data
from heterologous systems should be analysed carefully.
Subcellular localization of phyC, phyD and phyE It is becoming increasingly clear that the two major phytochromes,
phyA and phyB, exhibit very different subcellular localization
and translocation dynamics dependent on the quality and
quantity of light. To extend this analysis Nagy, Schäfer and
colleagues generated transgenic Arabidopsis and tobacco
plants harbouring phyC-E/GFP fusions proteins (Nagy et al.,
2001). Analyses of the transgenic plants showed that, phyCphyE are constitutively localized to the nucleus, that nuclear
staining in the dark is always diffuse and that speckle
formation can be induced by red light and reversed by far-red.
These findings indicate that phyC-phyE can be imported into
the nucleus in an inactive Pr form but yet speckle formation
is dependent on the active Pfr form. Interestingly, the phyCphyE speckle formation is much more heterogeneous than
that of phyA and phyB and can vary spatially within tissues
(S. Kircher, pers. comm.).
There are a number of obvious questions arising from the
accumulation of recent data. Why is the regulation and
translocation of the different phytochromes so very different?
Why, in contrast to phyA and phyB, are phyC-phyE localized
constitutively to the nucleus and do they have any biological
role in darkness? If phyC-phyE have a role in the nucleus
in darkness then why is speckle formation light-dependent
Indeed what is the significance of light-induced nuclear, and
occasional cytoplasmic, speckle formation?
3. All phytochromes localize to nuclear speckles
Nuclear speckling has been documented in animal cells
(Lamond & Earnshaw, 1998), however, the precise physiological role of speckle formation remains somewhat obscure.
In plants, several proteins have been shown to localize to
speckles including COP1 (constitutively photomorphogenic
1), all phytochromes, CRY2 (a blue light photoreceptor),
RPN6 (a component of the 26S proteasome) and LAF1 (long
after far-red 1) (von Arnim et al., 1997; Mas et al., 2000;
Ballesteros et al., 2001; Nagy et al., 2001; Peng et al., 2001).
Initially there was some concern whether speckle formation
was merely an artifact of phytochrome overexpression
(Neff et al., 2000). However, Kim et al. (2000) has clearly
shown that phyA forms speckles when expressed as a fusion
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Tansley review no. 136
protein from its own endogenous promoter. It is tempting to
hypothesise that speckle formation may represent general sites
of physical interaction between phytochromes, other light
receptors and nuclear signalling components. Indeed, recent
studies by Mas et al. (2000) clearly show, using fluorescence
resonance energy transfer (FRET) that phyB and cry2
physically interact in light-induced nuclear speckles. Similarly,
Quail and colleagues have convincingly shown that both
phyA and phyB can interact with nuclear proteins. The best
characterized of these is PIF3, a HLH-type protein, which
interacts with both phyA and phyB, albeit with different
affinities, in a photoreversible fashion (Ni et al., 1998; Ni
et al., 1999; Zhu et al., 2000). Moreover, it has been shown
that PIF3 together with phyB can bind, photoreversibly to
promoter elements of several light-regulated genes (MartinezGarcia et al., 2000). These results clearly indicate that for at
least some phyB-mediated responses the chain of signalling
events is very short (Section III/1). An attractive possibility
that may explain the variation in speckle formation between
the different phytochromes is that these may represent
distinct functional nuclear complexes. It is curious to note
that neither PIF3 nor HFR1 by themselves form speckles. To
this end it will be interesting to examine the phytochrome
translocation kinetics in various phytochrome signalling
mutant backgrounds.
Despite the compelling available evidence, nuclear speckle
formation may also serve a separate discrete role. Recently, it
was shown in onion epidermal cells, that COP1 and LAF1
speckles are different from phyA speckles indicating that neither COP1 nor LAF1 are in close physical proximity to phyA
(Ballesteros et al., 2001). Indeed, LAF1 does not interact with
phyA in yeast two-hybrid assays (Ballesteros et al., 2001). In
addition, the diffusion and complete loss of nuclear speckles
after light-to-dark transition may imply that speckle formation is involved in the controlled degradation of phytochrome. Speckle formation has also been implicated in
playing a role in the process of SUMO (small ubiquitin-like
modifier)-mediated protein protection and protein–protein
interactions via altered subcellular localization patterns
(Melchior, 2000; Muller et al., 2001). To this end Chua and
colleagues demonstrated recently that LAF1, a positive component of phyA signalling, contains a putative sumolation site
(KKQE) which if mutated (KRQE) abolishes in vivo nuclear
speckle formation (Ballesteros et al., 2001). This suggests that
the recruitment of LAF1 to subnuclear foci require this putative sumolation site. It will indeed be interesting to learn
whether SUMO interacts with LAF1 in yeast two-hybrid
assays. Likewise, colocalization studies in transgenic plants,
using both wild-type and mutant LAF1 fusion proteins, may
clarify more firmly the possible in vivo physical interaction
between LAF1 and SUMO. The potential role of protein
degradation and protection as part of controlling light
signal transduction will be covered in more detail in Section
III/4.
© New Phytologist (2002) 154: 553 – 590 www.newphytologist.com
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4. Possible partitioning mechanisms
It is now evident that the various phytochromes make up a
complex network of partitioning events involving both lightdependent and light-independent nuclear translocations.
However, questions such as ‘How are phyA and phyB retained
in the cytoplasm in darkness and how is this controlled?’
beg an answer. The Pr to Pfr conformation change is clearly
required for the translocation event in that a phyB mutant,
unable to bind its chromophore, is constitutively localized
to the cytoplasm (Kircher et al., 1999a). The simplest
explanation to this would be that either the Pr form ‘masks’
the nuclear import region or uncovers a region that is involved
in binding to cytoplasmic proteins. The long awaited threedimensional structure of phytochrome would clearly unveil
such possibilities. However, Nagy, Schäfer and colleagues have
noted that strong overexpression of both phyA and phyB can
result in weak nuclear staining in darkness indicating that the
Pfr conversion is not strictly necessary for the translocation
event to take place. This suggests that a separate, but not
mutually exclusive, mechanism exists. It is possible that the
Pr form of phyA and phyB are retained in the cytoplasm
in darkness by cytosolic ‘retention proteins’ and that upon
strong overexpression some of the phytochrome escapes the
retention mechanism due to ‘retention protein’ saturation.
Assuming that different phytochromes team up with different
‘retention proteins’ it is conceivable that these exist at distinct
endogenous levels reflecting the in vivo physiological amounts
of the different phytochromes. Since phyC, phyD, and phyE
are only present at low endogenous concentrations it is possible
that by overexpressing these they largely escape the retention
mechanism and therefore appear to be constitutively nuclear
localized. One way of addressing this would be to use the
endogenous phyC-phyE promoters for the localization studies
although this may present a problem due to low expression
levels. Equally conceivable is the possibility that the different
phytochromes have different affinities for the same ‘retention
protein’. It will be interesting to learn whether any cytosolic
phytochrome-interacting ‘retention proteins’ will be isolated
from yeast two-hybrid screens and if these prove to be
photoreversible in terms of their binding capacities?
Cytoplasmic retention of proteins as a control point for
cellular activity is a common process. For instance, the zincfinger protein BRAP2 binds to the NLS region of the tumour
suppressor protein BRCA1 with similar affinity as importin
α/karyopherin α thereby overriding the nuclear translocation
event and acting as a cytoplasmic retention protein (Li et al.,
1998). Similarly, the transduction of type 1 interferon is controlled by STAT2/p48 cytoplasmic complexes, which upon
stimulation dissociate leading to rapid nuclear translocation
of p48 (Lau et al., 2000). By substituting p48 with phyA in
the above scenario it is tempting to speculate that phytochrome is part of a cytoplasmic protein complex in darkness,
which rapidly dissociates when irradiated leading to rapid
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nuclear translocation. More recently, evidence has also
demonstrated the involvement of heterotrimeic G-proteins
in cytoplasmic retention and release. TUBBY, a transcription
regulator involved in maturity-onset of obesity, is released
from the plasma membrane by receptor-mediated activation of G-proteins via phospholipase C action resulting in
release and nuclear translocation of TUBBY to the nucleus
(Santagata et al., 2001).
Various studies have demonstrated that phyB has serine/
threonine kinase activity capable of both autophosphorylation and phosphorylation of other proteins (Section IV/2).
Limited evidence exists, but Park et al. (2000) has speculated
that phosphorylation of phytochrome may contribute to the
Pr to Pfr conformation change which may imply that phosphorylation patterns may be involved in the retention and
release of phytochromes. It is possible that PKS1 (phytochrome kinase substrate 1; Fankhauser et al., 1999a) may play
a role in this retention mechanism. Conversely, the phosphorylation patterns of retention proteins may be important. In
mammalian systems it has been shown that the phosphorylation of cytoplasmic proteins can either change binding
affinities or alternatively mask binding regions involved in
protein complex formation (Stanley, 1996). This may be the
case for phytochrome.
Signal transduction pathways cannot be viewed as linear
chains of events but should be viewed as an interconnected
network comprising a multitude of different pathways. There
are numerous interactions and intersections between light
signal transduction and other signalling pathways (Section V)
and these probably take place both in the cytoplasm and in the
nucleus (Møller & Chua, 1999). It is feasible therefore that
interaction among signalling cascades in the cytosol may
affect phytochrome translocation ultimately influencing
the regulation and transcription of light-regulated genes in
the nucleus. Indeed it was demonstrated very recently that the
potato photoperiod-responsive 1 (PHO1) locus, representing
a general component of the gibberellic acid (GA) pathway,
translocates rapidly to the nucleus in response to GA
application (Amador et al., 2001). It will be of great value to
examine the phytochrome translocation dynamics in mutants
affected in other signal transduction pathways.
III. The nucleus
The fairly recently discovered light-triggered nuclear
translocation of phyA and phyB, probably representing one of
the earliest check points in phytochrome signal transduction,
has created a lot of excitement and has opened up new ways
of viewing the intracellular light signalling pathways. The
nucleus undoubtedly plays a key role not only in recruiting
biologically active phytochrome but also in the subsequent
signal relay mechanisms from the photoreceptors to changes
in gene expression. Although the overwhelming importance
of the nucleus in light signalling is only now becoming
apparent, several approaches have been taken during the last
5–10 yr in efforts to identify nuclear localized phytochrome
signal transduction components. Although not selective for
nuclear proteins, various genetic screens have been employed
(Neff et al., 2000) resulting in the isolation of mutants with
genetic lesions in nuclear-localized phytochrome signalling
components. A more selective approach, using phytochrome
as bait in yeast two-hybrid screens, has also been employed.
From these two main approaches it is possible to divide the
identified nuclear proteins into phytochrome-interacting and
‘phytochrome-free’ components.
1. Phytochrome-interacting nuclear components
PIF3 The first bona fide phytochrome interacting partner
identified was PIF3 (phytochrome interacting factor 3) (Ni
et al., 1998). PIF3, a helix-loop-helix protein, was isolated
in a yeast two-hybrid screen using the nonphotoactive
C-terminal domain of the Arabidopsis phyB as bait (Ni et al.,
1998). It was subsequently shown that PIF3 could also bind
to the C-terminal domain of phyA suggesting that PIF3 may
act as a common interaction partner for both phyA and phyB
(Ni et al., 1999). The analysis was extended further and it
was shown that mutant versions of phyB (A776V, G793R,
E838K), that disrupt signal transfer, show dramatically
weaker binding to PIF3. To show that PIF3 is part of the
phytochrome signal transduction pathway, Quail and
colleagues generated Arabidopsis plants containing a PIF3
antisense transgene and demonstrated that reduced PIF3
levels result in reduced photoresponsivenss towards red light
irradiation but only minimally towards far-red light (Ni et al.,
1998). Although the effects in terms of hypocotyl length are
not striking these results show that PIF3 is a component of
both phyA and phyB signalling. Moreover, the weak PIF3deficient phenotype could indicate functional redundancy,
which is often observed due to the promiscuous behaviour of
transcriptional regulators. Is the binding of PIF3 to phyB
dependent on an active Pfr form? Quail and colleagues went
on to show, using in vitro pull-down assays, that PIF3 binds
tightly to the red-light activated full-length phyB in darkness
but dissociates rapidly upon conversion back to its Pr form
upon far-red light irradiation (Ni et al., 1999). More recently
it has also been shown that although phyA interacts with PIF3
in a photoreversible manner the apparent affinity for phyA is
approx. 10-fold lower than that for phyB (Zhu et al., 2000).
This could of course explain the weaker phenotype observed
in PIF3 antisense plants in response to far-red light irradiation
(Ni et al., 1998). In addition, in vitro pull-down assays and
deletion mapping has shown that a 37 amino acid stretch
present at the N-terminal region of phyB, but absent in phyA,
contributes to the stronger binding of phyB to PIF3 (Zhu
et al., 2000). It follows from this that PIF3 probably has a
more predominant role in phyB signalling with only a minor
role in phyA signalling.
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Martinez-Garcia et al. (2000) investigated whether, as
member of the helix-loop-helix transcriptional regulator family of proteins PIF3, can bind to promoter elements. Using a
random binding site selection procedure it was shown that
PIF3 can bind DNA in a sequence-specific fashion with the
core binding sequence being the palindromic hexanucleotide
G-box motif CACGTG found in many light-regulated genes
(Martinez-Garcia et al., 2000). It was subsequently shown
that in PIF3-deficient seedlings the expression levels of the
phytochrome-regulated genes CCA1 and LHY were reduced.
Interestingly, it has also been shown that the bZIP protein
HY5 binds to G-box elements in Arabidopsis (Chattopadhyay
et al., 1998) however, in the hy5 mutant CCA1 and LHY
show normal phyB-mediated expression dynamics. These
results suggest that G-box elements can discriminate between
different DNA-binding proteins. It is possible that PIF3,
through phyB, regulates genes such as CCA1 and LHY which
themselves encode MYB transcription factors suggesting
that PIF3 may act as a control point for different branches of
photomorphogenesis.
Another intriguing aspect of PIF3 biology is that the Pfr
form of phyB binds G-box-bound PIF3 forming a phyB/
PIF3/DNA complex as observed in gel retardation assays
(Martinez-Garcia et al., 2000). As for the PIF3/phyB binding
characteriztics, the Pr form of phyB does not interact with the
PIF3/DNA complex and moreover bound Pfr phyB rapidly
dissociates following far-red light irradiation. Taken together
this infers that PIF3 may acts as a recruitment agent, directing
incoming photoconversion-induced phytochrome to target
promoter sites and by doing so controlling the expression of
light-regulated genes.
The Arabidopsis genome contains a large number of
bHLH proteins. It has been well documented that bHLH
family members form both homodimers and heterodimer
combinations thereby modifying their DNA binding and
protein–protein interaction properties (Massari & Murre,
2000). If this is the case for phytochrome signal transduction,
different combinations of bHLH proteins may form,
depending on the light conditions, thereby generating a
vast array of different phytochrome- and DNA-binding
affinities.
Hfr1 The possibility of having a combinatorial network,
consisting of different bHLH proteins directing different
phytochromes to their target promoter sequences, has been
strengthened by the isolation of HFR1 (Fairchild et al.,
2000). HFR1 is an atypical bHLH protein, specific for phyA
signal transduction in Arabidopsis, which does not interact
with phyA or phyB directly but forms photoreversible
heterodimers with PIF3/phyA complexes. Since the mutant
phenotype of hfr1 is more prominent in response to far-red
light than that of PIF3-deficient seedlings it is clear that
different bHLH combinations have different effects under
different light regimes.
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Ndpk2 Another protein found to interact with the Cterminal domain of phyA is nucleoside diphosphate kinase 2
(NDPK2) (Choi et al., 1999). Using a quantitative yeast twohybrid assay Song and colleagues showed that NDPK2 has
reduced binding affinity towards phyA missense mutants as
seen for PIF3. Moreover, biochemical cross-linking/SDSPAGE studies showed that NDPK2 interacts preferentially
with the Pfr form of purified oat phyA with approximately
1.8-fold higher binding affinity than for the inactive Pr form.
The photoreversible binding characteriztics of NDPK2 is not
as clear-cut as for PIF3. To this end Song and colleagues tested
whether the enzymatic activity of NDPK2 is influenced by
the presence of photoactive phyA. It was shown that the
intrinsic γ-phosphate-exchange activity increased approx. 1.7fold upon incubation with the Pfr form of oat phyA whilst the
Pr form had no effect. From these data it is evident that
NDPK2 interacts directly with phyA and that this interaction
is stronger upon phyA activation.
Analysis of an ndpk2 loss-of-function mutant has shown
that NDPK2 is involved in the deetiolation process in Arabidopsis, displaying reduced responsiveness in both red- and
far-red-induced hook opening and cotyledon expansion (Choi
et al., 1999). Hypocotyl growth inhibition was however, unaffected. This does indicate that NDPK2 is involved in both
phyA and phyB signalling events. It would be interesting to
learn whether NDPK2 interacts with phyA or other phytochromes and whether these interactions are photoreversible.
NDPK2 localizes to both the nucleus and the cytoplasm
(Choi et al., 1999; Zimmermann et al., 1999). Mechanistic
data is still lacking but it is possible that NDPK2 acts as a transcriptional regulator. Zimmermann et al. (1999) has reported
that NDPK1a, which is in fact NDPK2, is capable of binding
to the HIS4 promoter in yeast, which is the target site for the
GCN4 transcription factor. Moreover, NDPK1a can fully
complement the gcn4 mutant (Zimmermann et al., 1999).
Although this may indicate that NDPK2 is involved in
nuclear transcriptional control, NDPK2 also localizes to the
cytoplasm. Whether the cytoplasmic localization data is
merely an artifact due to saturation of the nuclear import
mechanism or whether it proves to be real, it is conceivable
that NDPK2 has some functional overlap with PKS1 (phytochrome kinase substrate 1) (Fankhauser et al., 1999). NM23,
a mammalian NDPK, is postulated to be involved in a
phosphorelay/phosphotransferase mechanism (Engel et al.,
1995; Lu et al., 1996) and PKS1 has been postulated to be a
substrate for a phyA-mediated phosphotransfer reaction
(Fankhauser et al., 1999). PKS1 will be discussed in more
detail in Section IV/1.
Elf3 The elf3 mutant was initially characterized as being
photoperiod-insensitive early flowering, impaired in the
transduction of light signals to the circadian clock (Hicks
et al., 1996; Zagotta et al., 1996). Recently, Millar and
colleagues showed that ELF3 affects light input to the
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circadian oscillator (McWatters et al., 2000) and ELF3 has
received a lot of attention (Covington et al., 2001; Hicks
et al., 2001; Liu et al., 2001). The EL3 transcript is regulated
in a circadian fashion and the gene has now been cloned and
it encodes a novel 695 amino acid protein that may act as a
transcriptional regulator (Hicks et al., 2001). ELF3 is as
expected a nuclear protein and accumulates in a periodic
manner showing the highest levels just before the onset of
darkness during 24-h light/dark cycles (Liu et al., 2001). The
pattern of nuclear protein oscillation is almost identical to the
ELF3 transcript dynamics suggesting that ELF3 function is
transcriptionally regulated. Further, in a yeast-two hybrid
screen using ELF3 as bait, Liu et al. (2001) isolated ELF3 and
phyB, suggesting that ELF3 may act as a dimer interacting
with phyB. It was shown that ELF3 interacts with the Cterminal domain of phyB but not with phyA. Moreover, the
elf3 phenotype includes partial insensitivity towards red-light
induced hypocotyl growth inhibition implying that ELF3
may form a light-induced nuclear complex with phyB in vivo.
Conversely, genetic analysis shows that ELF3 controls
flowering independent of phyB. It remains to be shown
whether ELF3 regulates flowering by interaction with other
phytochromes or by regulating the circadian clock.
CRY1 and CRY2 Both genetic and physiological evidence
show that there is cross-talk between the phytochrome and
cryptochrome signalling pathways. Indeed it has been shown
in yeast two-hybrid assays that the C-terminal part of phyA
can interact with the C-terminal part of CRY1 (Ahmad et al.,
1998). To this end Cashmore and colleagues have shown that
oat phyA can in vitro phosphorylate recombinant CRY1 and
CRY2 specifically on serine residues. However, no difference
in phosphorylation patterns is observed between the Pr and
Pfr form of phyA. Conversely, using dark-adapted Arabidopsis
seedlings, phosphorylated CRY1 can be affinity purified
after a red light pulse whilst after a red/far-red pulse cycle
no phosphorylation occurs. These data suggest that the
photoactive form of phyA mediates in vivo phosphorylation
of CRY1 resulting in enhanced blue light activation (Ahmad
et al., 1998). More recently, Cashmore and colleagues ( Jarillo
et al., 2001) showed that the Arabidopsis circadian clock PAS
domain protein ADAGIO1, described originally as ZTL
(Somers et al., 2000), interacts with both CRY1 and phyB in
yeast two-hybrid and in vitro interaction studies. Knowing
that both phytochrome and cryptochrome are nuclear
localized it is feasible that there is functional, direct
interaction between these two photoreceptors resulting in a
coordinated nuclear network of red and blue light responses.
2. ‘Phytochrome-free’ nuclear components
Yeast two-hybrid screening, using phytochrome as bait,
has undoubtedly contributed enormously towards our
understanding of early phytochrome signalling events. How-
ever, by employing light-specific genetic screens a number of
nuclear-localized phytochrome signalling components have
been identified whose deficiencies result in either insensitivity
or hypersensitivity in terms of hypocotyl elongation.
Although these components do not physically interact with
phytochrome, their photomorphogenic phenotypes are
often more dramatic than those observed for the nullmutants of phytochrome-interacting components, clearly
implying an important role in phytochrome signalling. The
‘phytochrome-free’ nuclear components can be further
divided into three main classes: phyA-specific, phyB-specific
and phyA/phyB components, with phyA-specific mutants
being most abundant.
PhyA-specific The first bona fide phyA-specific signalling
mutants identified were fhy1 and fhy3 (Whitelam et al.,
1993). Apart from the photoreceptor mutants, they show
the strongest far-red insensitive phenotype amongst isolated
mutants to date, and maybe this is why fhy1 and fhy3 were the
first ones to be identified. The disrupted gene in fhy1 has very
recently been cloned and encodes a small (202 amino acids,
23 kDa), novel protein that is nuclear localized in dark-grown
seedlings (Desnos et al., 2001). Expression of the FHY1 gene
is down-regulated by light and phyA is involved in this
process, suggesting negative-feedback regulation of far-red
light signalling via phyA. However, additional photoreceptors
are also involved and the regulation of FHY1 mRNA levels by
photoreceptors other than phyA may reflect a cross-talk point
between the signalling pathways associated with different
photoreceptors.
The far1 mutant was isolated based on its partial insensitivity towards far-red light irradiation showing partial lack
of hypocotyl growth inhibition (Hudson et al., 1999). The
FAR1 gene was cloned by positional cloning and encodes a
novel nuclear localized protein with no known function. The
partial insensitivity towards far-red light irradiation may be a
result of functional redundancy amongst FAR1 family members since FAR1 is part of a multigene family in Arabidopsis.
Recently, Chua and colleagues isolated a phyA-specific
mutant, laf1, which is partially insensitive towards far-red
light irradiation in terms of hypocotyl growth (Ballesteros
et al., 2001). Moreover, a careful detailed physiological characterization of laf1 showed that far-red responses such as
apical hook opening, cotyledon expansion and gravitropism
were unaffected. LAF1 was cloned and encodes a 283 amino
acid MYB protein previously identified as atMYB18 (Kranz
et al., 1998) belonging to the two-repeat (R2R3-type) MYB
proteins of which there are approximately 130 members in
Arabidopsis. As for FAR1, the presence of this large R2R3-like
MYB family could explain the partial insensitivity towards
far-red light. Ballesteros et al. (2001) demonstrated using
yeast transactivation experiments that LAF1 can act as a transcriptional activator suggesting direct involvement in lightdependent gene regulation. The nuclear localization of LAF1
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was determined using a LAF1/GFP fusion protein in onion
epidermal cells. By contrast to the uniform nuclear staining
observed with other phytochrome signalling components,
LAF1 localizes to subnuclear foci or speckles in a timedependent manner. After the transfection (4 – 6 h) LAF1/GFP
fluorescence is observed diffusely throughout the nucleus with
speckle formation occurring only after 8–10 h. Following
this the GFP signal fades until it eventually disappears 4–8 h
later suggesting that speckle formation precedes degradation
of the protein. Domain mapping experiments demonstrated
further that the first 70 amino acids of LAF1 is sufficient for
nuclear translocation but not for speckle formation whilst an
84 amino acid region (amino acids 176–260) is responsible
for the speckle formation. Upon closer examination a putative
sumolation site (KK QE) was found in this region (amino
acids 257–260) of the protein and studies have shown that
proteins can localize to speckles when conjugated to SUMO
(Muller et al., 1998). To test whether sumolation could be
involved in LAF1 speckling, Ballesteros et al. (2001) mutated
the putative sumolation site (KK QE to KR QE), which
abolishes in vivo nuclear speckle formation. This suggests that
the recruitment of LAF1 to subnuclear foci requires SUMO
conjugation. Whether SUMO physically interacts with LAF1
remains to be determined. LAF1 does not interact with phyA
and phyA speckles do not colocalize with LAF1 speckles
(Ballesteros et al., 2001). It is of course tempting to speculate
that LAF1 may interact with PIF3 or some other phyAinteracting proteins or LAF1 may actually activate transcription of phyA-interacting components. Regardless of the
mechanism, these findings clearly indicate that transcriptional
activators that do not interact with photoreceptors are also
important for correct phytochrome signal transduction.
The hypersensitive mutants spa1 was identified from a suppressor screen using a weak phyA allele (Hoecker et al., 1998).
In wild-type plants the spa1 mutation causes hypersensitivity
(short hypocotyl) towards red and far-red light however,
genetic studies have shown that this hypersensitivity is lost in
a phyA null background. This implies a role for SPA1 specific
for phyA signalling and SPA1 must therefore encode a negative component of the phyA signal transduction pathways.
The spa1 locus has been cloned and SPA1 encodes a constitutively nuclear localized novel protein kinase containing a
WD-repeat motif (Hoecker et al., 1999). Interestingly, it has
been suggested that SPA1 may counteracts phy-mediated
growth inhibition during de-etiolation: phytochrome inhibits
elongation whilst SPA1 promotes elongation (Parks et al.,
2001). Recently, SPA1 was shown to interact with the coiledcoil domain of COP1 in a yeast two-hybrid screen and by
in vitro interaction studies (Hoecker & Quail, 2001). The
implications of this are that SPA1 may link phyA signalling to
the light signalling pathway of COP1.
Another phyA-specific hypersensitive mutant recently
isolated is eid1 (Büche et al., 2000). The eid1 mutant shows
hypersensitivity towards red and far-red light, but as for spa1
© New Phytologist (2002) 154: 553 – 590 www.newphytologist.com
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this hypersensitivity is abolished in a phyA null mutant. An
intriguing feature of the eid1 mutant is that it shifts the
responsiveness of the phyA-signalling pathway from far-red to
red wavelengths. EID1 has been cloned and encodes a novel
F-box protein (Dieterle et al., 2001). The N-terminal domain
of EID1 shows homology to F-box proteins that form part
of the SCF (Skp1, Cdc53 and F-box) complex. The SCF
complex functions as an ubiquitin-ligase involved in the
proteasome-dependent degradation of proteins (Craig &
Tyers, 1999) (Section III/4). The EID1 protein also contains
a leucine zipper domain, important for its function, which
may imply that EID1 acts as a homo- or heterodimer in vivo.
The presence of an F–box domain suggests interactions with
other members of the SCF complex. To this end Dieterle et al.
(2001) used EID1 as bait in a yeast two-hybrid screen and
isolated ASK1 and ASK2, two Arabidopsis homologs of the
yeast Skp1 protein (Gray et al., 1999). These interactions
were verified using in vitro pull-down assays. Moreover,
deleting part of the F-box domain and changing a conserved
N-terminal proline residue in EID1 abolished the ASK1 and
ASK2 interactions. EID1 is a constitutively nuclear localized
protein as shown by EID1/GFP fusion studies in protoplasts.
It is tempting therefore to speculate that EID1 may be
involved in the proteasome-mediated degradation of
phytochrome. However, a role for EID1 in phyA degradation
can be excluded because phyA levels are not altered in the
eid1 mutant. Therefore, EID1 is probably involved in the
degradation of positively acting phyA signalling components
(Section III/4) but this remains to be demonstrated.
PhyB-specific Mutants specific for phyB signalling are more
rare. This could partly be explained by the fact that phyBphyE show redundancy in terms of red light perception
(Devlin et al., 1998; Devlin et al., 1999). In addition, red
light plays multiple roles during development, separate
from phytochrome signalling, and it has therefore proven
problematic to isolate bona fide phyB-specific signalling
mutants.
Putative phyB-specific mutants have been isolated and
include red1, pef2, pef3, and srl1 (Ahmad & Cashmore, 1996;
Wagner et al., 1997; Huq et al., 2000a). However, the disrupted genes in these mutants have not yet been cloned so it
is difficult to assign any specificity as yet.
As far as we are aware there are only two mutants that have
been shown to be specifically disrupted in phyB signalling.
One of these is poc1 (photocurrent 1), which exhibits
enhanced responsiveness towards red light (Halliday et al.,
1999). Moreover, the poc1 mutant phenotype is abolished in
phyB-deficient seedlings demonstrating the phyB specificity
and suggesting a role of POC1 in enhancing phyB signalling.
Interestingly, the T-DNA insertion in poc1 is located in the
promoter region of PIF3 (Section III/1), which causes PIF3
overexpression in response to red light irradiation. The
mechanism by which the promoter insertion results in red
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light-induced overexpression is unknown but it is possible that
the T-DNA insertion disrupts a negatively acting red lightspecific PIF3 promoter region. It is interesting to note the
differences in hypersensitivity between wild-type Arabidopsis
seedlings overexpressing PIF3 (Ni et al., 1998) and poc1
(Halliday et al., 1999). Under identical red light fluence rates
(20 µmol m-2 s-1) the hypersensitivity of PIF3 overexpression
in wild-type seedlings is marginal whilst PIF3 overexpression
in poc1 results in a marked red-light induced hypersensitivity.
However, the reported differences may simply reflect differences
between endogenous promoter insertions and CaMV35Sdriven PIF3 overexpression or simply differences in ecotypes.
Recently, Quail and colleagues isolated a mutant, gi-100,
that is partially insensitive specifically towards red light irradiation (Huq et al., 2000b). Cloning of the mutant locus
revealed that the disrupted gene was the previously identified
GIGANTEA gene (Fowler et al., 1999). Since the mutant was
identified from an activation-tagged pool of mutants, Huq
et al. (2000b) tested whether the expression profiles of
GIGANTEA or any other neighbouring genes was affected in
gi-100. It was shown that the insertion results in a truncation
of the GIGANTEA transcript, with only marginal effects on
transcriptional activities of neighbouring ORFs. By contrast
to previous data indicating that GIGANTEA is a membrane
protein (Fowler et al., 1999), Huq et al. (2000b) showed
conclusively, using GIGANTEA/GUS fusions, that
GIGANTEA is constitutively nuclear localized. Taken
together these data indicate that GIGANTEA is involved in
the nuclear localized phyB-signalling pathway.
PhyA, phyB and cryptochrome components Due to the
interaction between the phyA and phyB signalling pathways
it is not surprising that there are mutants that show a
phenotype in both red and far-red light. To date there are four
described mutants that show red and far-red light induced
photomorphogenic phenotypes, pef1, psi2, dfl1, and shl
(Ahmad & Cashmore, 1996; Genoud et al., 1998; Nakazawa
et al., 2001; Pepper et al., 2001).
pef1 shows partial insensitivity towards red and far-red light
suggesting that PEF1 may represent a lesion in an early step
of the phytochrome signal transduction pathway which may
also be indicative of nuclear localization. This however,
remains to be shown.
The second dual phyA /phyB phytochrome signalling
mutant isolated is psi2. psi2 was identified based on elevated
activity of a chlorophyll a /b binding protein-luciferase
(CAB2-LUC) transgene in Arabidopsis (Genoud et al., 1998).
This mutant shows hypersensitive induction of lightregulated genes in the VLF range of red light and a hypersensitive hypocotyl growth response towards high fluence red light
irradiation. Double mutant analysis further demonstrated
that the psi2 phenotype is dependent on both phyA and phyB.
Surprisingly, at high fluence rates, psi2 shows light-dependent
development of spontaneous necrotic lesions. Recently, it
has also been shown that the putatively disrupted gene in
psi2 is constitutively and uniformly distributed in the nucleus
(S. G. Møller and N. H. Chua, unpublished). This suggests
that PSI2 may act as a nuclear negative component of phyA
and phyB signalling.
dfl1 exhibits hypersensitivity in terms of hypocotyl growth
under blue, red and far-red light conditions but also shows
auxin related phenotypes such as altered lateral root number
(Nakazawa et al., 2001). DFL1 encodes a GH3 homolog
providing a link between light signalling and auxin responses
(Section V/I).
Arabidopsis seedlings containing mutant shl alleles show
enhanced hypocotyl growth inhibition in red, far-red, blue,
and green light over a range of fluences indicating that the
SHL proteins act as negative regulators of photomorphogenic
responses in a downstream signalling cascade shared by
CRY1, PHYA, and PHYB and possibly CRY2, PHYC,
PHYD, and PHYE (Pepper et al., 2001). However, the
molecular evidence for this remains to be shown (Fig. 2).
3. cop/det /fus
The 11 recessive cop/det/fus (constitutive photomorphogenesis/
de-etiolated/fusca) mutants of Arabidopsis resemble lightgrown seedlings when grown in darkness, are pleiotropic in
nature and were identified from a number of genetic screens
(Chory et al., 1989; Deng et al., 1991; Wei & Deng, 1992;
Misera et al., 1994). As for light-grown wild type seedlings,
cop/det/fus mutants exhibit hypocotyl growth inhibition, open
cotyledons, and express light-regulated genes in darkness
implying that the disrupted proteins act as negative
components of light signal transduction. To date 5 cop/det/fus
mutant loci have been cloned and include COP1, FUS2/
DET1, COP8, COP9, COP11, and FUS5 (Schwechheimer &
Deng, 2000).
Cop1 The cop1 mutant was isolated back in 1991 (Deng
et al., 1991) and COP1 was the first cop/det/fus mutant locus
to be cloned and fully characterized (Deng et al., 1992).
COP1 encodes a soluble protein of 76 kDa and can be
divided into three structural domains: an N-terminal
zinc-finger domain, a putative coiled-coil domain and a
C-terminal WD-40 repeat domain with homology to the βsubunit of trimeric G-proteins. Several recessive cop1 mutants
have been identified which have highlighted the importance
of the different protein domains and also enabled further
analysis into COP1 function beyond the seedling stage. For
instance, in weak cop1 alleles both the phyA-mediated endof-day far-red response and the shade avoidance response
are impaired (Deng et al., 1991; McNellis et al., 1994). In
addition, weak cop1 mutants flower early under short day
conditions and also flower in the dark (McNellis et al., 1994).
COP1 is not regulated at the transcript or protein level
(Deng et al., 1992) but at the level of subcellular localization.
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Fig. 2 Phytochrome signalling in the nucleus. The figure illustrates the multitude of known phytochrome pathways and interactions that take
place in the nucleus. PhyA and phyB are translocated to the nucleus in response to light where they interact with various signalling components.
Note that phyA also interacts with CRY1 and that not all signalling components interact with the photoreceptors. Solid arrows represent
demonstrated steps whilst dashed arrows represent speculative steps.
In darkness COP1 accumulates in the nucleus but is excluded
when transferred to light. The cytoplasmic localization of COP1
is probably mediated through a cytoplasmic localization or
retention signal, which neutralizes the COP1 NLS in a lightdependent manner (von Arnim & Deng, 1996; Stacey & von
Arnim, 1999). Moreover, using various photoreceptor
mutants it has been shown that the subcellular localization
patterns are mediated by phyA, phyB and CRY1 implying
that COP1 acts downstream in at least three different light
signalling pathways (Osterlund & Deng, 1998). As for the
phytochromes and LAF1, COP1 localizes to subnuclear foci
both in onion epidermal cells and in transgenic Arabidopsis
plants (Ang et al., 1998; von Arnim et al., 1998) and more
detailed analysis has shown that a 58 amino acid stretch
between residues 120–177 confer speckle formation. Interestingly, mutations in the C-terminal WD-40 repeat domain are
phenotypically lethal and result in loss of speckle formation
implying that COP1 accumulation at subnuclear sites has a
functional role (Stacey & von Arnim, 1999).
© New Phytologist (2002) 154: 553 – 590 www.newphytologist.com
Another regulatory function of COP1 is mediated by
interactions to other nuclear proteins and several have been
isolated including CIP1, CIP4 and CIP7 (Matsui et al., 1995;
Yamamoto et al., 1998; Yamamoto et al., 2001). CIP1 was
isolated using biotinylated COP1 as a probe to screen an
Arabidopsis expression library. CIP1 encodes a cytoplasmic
protein probably associated with cytoskeletal structures in the
hypocotyl and cotyledons and it is possible that CIP1 regulates the nucleo-cytoplasmic partitioning of COP1 (Matsui
et al., 1995). Conversely, CIP4 is a nuclear protein and can act
as a transcriptional activator required for the promotion of
photomorphogenic responses (Yamamoto et al., 2001). CIP4
transcript levels are induced by light, regulated by COP1 and
CIP4 deficient plants show elongated hypocotyls in response
to light treatment. CIP7 is also a nuclear protein shown to act
as a transcriptional activator (Yamamoto et al., 1998) and as
for CIP4, CIP7 expression is induced by light and repressed
by COP1 in darkness. Furthermore, CIP7 antisense plants
show reduced expression of light-regulated genes but in
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contrast to CIP4 antisense plants exhibit no hypocotyl growth
inhibition defects. This clearly implies that COP1 can interact with a spectrum of proteins, which in turn regulate different light-dependent physiological responses.
Interestingly, it has been shown that the signalling
mechanism of Arabidopsis CRY1 and CRY2 are mediated
by the C-terminus and that plants overexpressing these
domains exhibit a COP1 phenotype (Yang et al., 2000).
This prompted Deng and colleagues (Wang et al., 2001) to
examine whether COP1 physically interacts with CRY1 and
CRY2 and indeed it has now been shown that CRY1 and
CRY2 repress COP1 activity via direct physical interactions.
The best studied COP1 interacting partner is the
bZIP transcription factor HY5 (Oyama et al., 1997). By contrast to COP1, HY5 localizes constitutively to the nucleus
(Chattopadhyay et al., 1998) but is more abundant in the
light than in darkness. Using GFP/HY5 fusion proteins it has
also been shown that COP1 can recruit HY5 to subnuclear
foci demonstrating in vivo physical COP1/HY5 interactions
(Ang et al., 1998). Interestingly, HY5 is subject to dark-specific
degradation by the 26S proteasome and physical interaction
between HY5 and COP1 is necessary for degradation to occur
(Osterlund et al., 2000). These findings imply that HY5
abundance is regulated by nuclear COP1. Moreover, COP1 is
a RING finger protein and it is possible that COP1 may assist
in the ubiquitin-mediated degradation of HY5 by the 26S
proteasome (Lorick et al., 1999) and this will be discussed in
more detail in Section III/4.
Cop9 COP9 is part of a 450–600 kDa protein complex
consisting of a number of subunits which are not present in
cop9 mutants nor in several other cop/det/fus mutants (Wei
et al., 1994; Chamovitz et al., 1996). Some of the genes
encoding these subunits (CSNs) have been cloned including
COP9/CSN8, COP8/CSN4, COP11/CSN1, and FUS5/
CSN7 whilst some still remain to be isolated (FUS8, FUS11,
FUS12). The finding that the loss of one subunit results in the
loss of the entire protein complex has enforced the notion that
the CSNs are involved in the COP9 complex assembly (Kwok
et al., 1998). The protein complex has now been named the
COP9 signalosome (Deng et al., 2000), which is thought
to act as the ‘lid’ subcomplex (19S regulatory particle) of
the 26S proteasome (Chamovitz et al., 1996). The COP9
signalosome subunits have orthologues in other eukaryotes
and both the mammalian and Drosophila COP9 signalosomes
have been copurified with the 26S proteasome based on their
homologies to the plant COP9 complex (Wei & Deng, 1998;
Wei et al., 1998).
The COP9 signalosome subunits have been shown to contain protein domains (PCI and MPN) present in the subunits
of the 26S proteasome and the translation initiation factor
eIF3 (Aravind & Ponting, 1998; Hofmann & Bucher, 1998).
Although the functions of these domains remain unknown
the similarity between the subunits of the COP9 signalosome
and the 26S proteasome is astonishing with all the COP9
CSNs showing extensive homology to the subunits of the 19S
regulatory particle (Glickman et al., 1998; Henke et al., 1999,
Wei & Deng, 1999). The 26S proteasome is probably the
main protein degradation apparatus in the cell (Fig. 3) and
consists of a central 20S proteasome core complex together
with a 19S regulatory particle, which act as lids (Hershko &
Ciechanover, 1998). Due to the extensive homologies it
possible that the COP9 signalosome can interact with the 20S
proteasome core complex forming an alternative type of
proteasome involved in the regulation of photomorphogenic
responses (Section III/4). Although this remains to be
demonstrated, Deng and colleagues have shown that Arabidopsis
CSN1 can interact with the 19S regulatory AtS9 subunit
(Kwok et al., 1999) suggesting that the COP9 signalosome
forms a supercomplex with the 26S proteasome (Fig. 3). The
26S proteasome and its implication in regulating photomorphogenic responses will be discussed in more detail below.
4. Degradation
It is becoming increasingly evident that the regulation of
cellular events is not only controlled by gene expression and
protein synthesis but also by targeted protein degradation. In
plants, protein degradation has been shown to be involved in
numerous processes including cell death, circadian rhythms,
defence responses, photosynthesis and photomorphogenesis.
Here we will describe recent findings demonstrating the role
of proteolysis as part of photomorphogenesis, with particular
emphasis on ubiquitination and SUMOlation.
Ubiquitin and the 26S proteasome Ubiquitin is a conserved
76 amino acid polypeptide that marks proteins for
degradation by the multisubunit 26S proteasome (Hershko
& Ciechanover, 1998; Pickart, 2001). The process of
ubiquitination, i.e. the marking of proteins destined for
degradation, consists of several critical steps (Hershko, 1996).
Briefly, the first step involves the activation of the C-terminal
glycine residue of ubiquitin by an ATP-dependent reaction
involving a specific ubiquitin-activating enzyme (E1). Once
activated, the ubiquitin is transferred to an active site cysteine
residue of an ubiquitin-carrier protein (E2). Following this,
a ubiquitin protein ligase (E3) catalyses the formation of an
amide isopeptide linkage between the ubiquitin C-terminus
and lysine residues on the protein to be degraded. Subsequently, activated ubiquitin is added to the previously conjugated
ubiquitin leading to the formation of a polyubiquitin chain.
Once containing polyubiquitin chains, the 26S proteasome
complex usually degrades the proteins by an ATP-dependent
reaction (Coux et al., 1996).
The 26S proteasome is a remarkable protein complex consisting of the 20S core particle, which harbours the proteolytic
sites, and the 19S regulatory particle, which make up the ‘lids’
(Fig. 3). The 19S regulatory particle contains a multitude of
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Fig. 3 Specific degradation of light signalling components by ubiquitination and the 26S proteasome. COP1 translocates to the nucleus in
darkness where it recruits HY5 for ubiquitination by the SCF complex and subsequent degradation by the 26S proteasome. EID1 may function
as an F-box protein in the SCF complex involved in the degradation of phytochrome signalling components (SP). Also, phyA itself may be
degraded by ubiquitination and the 26S proteasome. Note that the 19S regulatory complex of the 26S proteasome is in fact the COP9 complex.
Solid arrows represent demonstrated steps whilst dashed arrows represent speculative steps.
ATPases and other subunits probably involved in the specific
action of the 26S proteasome. The precise mode of action of
the 26S proteasome is not well understood, however, recent
evidence has started to unravel the functional role of the
COP9 signalosome/19S regulatory particle in plant signalling
events as discussed in sections below.
The E1-E2-E3 cascade is somewhat hierarchical. In most
organisms there are only one or very few E1 enzymes that are
responsible for all ubiquitin activation and because of this the
specificity of E1s is very low (McGrath et al., 1991). In contrast, there are several E2s, 36 isoforms already identified in
Arabidopsis (Callis & Vierstra, 2000), which have more specialised functions depending on the cellular localization and
their interaction with various E3s. Similarly, the E3 proteins
interact directly with the proteins to be degraded and are
therefore very diverse with varying specificities. E3s can be
further divided into HECT-domain proteins, Ubr-1-like E3s,
APC (anaphase-promoting complex), monomeric RING-H2
© New Phytologist (2002) 154: 553 – 590 www.newphytologist.com
E3s and SCF (Skp1/cullin/F-box-type) E3s. Although all
classes have been found in plants (Callis & Vierstra, 2000),
the involvement of SCF E3s in photomorphogenesis and in
the interaction with auxin signalling has recently been
demonstrated.
The SCF complex was originally identified in yeast and
consists of the subunits Skp1, Cdc53 or cullin and an F-box
protein, all making up the ubiquitin ligase protein complex
(Patton et al., 1998). The F-box protein gives the SCF-E3
complex its specificity, acting as the receptor, bringing E2s in
close proximity with the ubiquitinated proteins. Indeed several F-box proteins can interact with identical Skp1 and cullin
subunits (Patton et al., 1998). The first SCF complex identified in plants was SCFTIR1 (Transport Inhibitor Response 1),
which is most likely involved in the degradation of inhibitors
of the auxin response (Gray et al., 1999). It is possible, that
the substrates for SCFTIR1 are the very unstable nuclear
localized Aux/IAA proteins (Section V/1), which themselves
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can act as repressors of auxin-regulated genes (Ulmasov et al.,
1997). Indeed mutational analysis have identified IAA
domains that disrupt the auxin response by increasing protein
stability suggesting that the specific degradation of Aux /IAA
proteins are important for a proper auxin response (Leyser,
1998; Gray & Estelle, 2000). More recently, two SCF-type
E3s were isolated involved in circadian responses. Deficiency
of the Arabidopsis proteins ZTL (Somers et al., 2000) and
FKF1 (Nelson et al., 2000) result in impaired circadian
rhythm responses and both proteins have been shown to
contain an F-box implying that they are components of an
SCF complex. This further suggests that ubiquitin-dependent
degradation is important for correct circadian responses.
In terms of phytochrome signalling it has for a long time
been speculated, and almost assumed, that phyA is subjected
to ubiquitin-mediated degradation in its Pfr form ( Jabben
et al., 1989; Fig. 3). This has now been shown to be the case
demonstrating that both the N-terminal and C-terminal parts
of phyA are important for Pfr ubiquitination and breakdown
(Clough et al., 1999). However, only very recently was it
shown that phytochrome signalling intermediates are indeed
involved in ubiquitin-mediated proteolysis. The phyAspecific hypersensitive mutant eid1 has been isolated
(Büche et al., 2000) and as discussed in Section III/2, it has
been shown that EID1 contains homology to F-box proteins
(Dieterle et al., 2001). This does suggest that EID1 may interact
with other members of the SCF complex and indeed Dieterle
et al. (2001) demonstrated that EID1 can interact with ASK1
and ASK2, two Arabidopsis homologs of the yeast Skp1 protein (Gray et al., 1999). This interaction does seem specific
in that a mutant variant of EID1, lacking the F-box, can not
bind ASK1 and ASK2 (Dieterle et al., 2001). It is tempting to
speculate that EID1 may be involved in ubiquitin-mediated
processes in response to light. Since phyA levels are not altered
in the eid1 mutant, EID1 could be involved in the degradation of positively acting phyA signalling components (Fig. 3)
where the F-box moiety of EID1 confers light-specific SCF
ligase activity.
Apart from the HECT-domain E3s (Bates & Vierstra,
1999), the members of the different E3 classes harbour a subunit with a RING-finger motif. This motif is thought to interact with the E2 enzyme and by doing so mediating the
transfer of ubiquitin from E2 to the protein substrate. This is
of course not the case for SCF as discussed above. COP1 contains a RING-finger motif suggestive of E3 activity and it has
been shown by Deng and colleagues that COP1 most probably targets HY5 for degradation in darkness (Fig. 3; von
Arnim & Deng, 1994; Osterlund et al., 2000). In darkness,
COP1 translocates to the nucleus and interacts with HY5, a
bZIP protein that activates transcription of light-regulated
genes in the light, and this interaction results in HY5 degradation (Fig. 3). Conversely, in the light COP1 is excluded
from the nucleus resulting in HY5 stabilisation ultimately
leading to transcription of light-regulated genes. This point is
further underlined by the fact that HY5 is not degraded in
COP9 signalosome mutants (see below) and it has been demonstrated that COP1 does not translocate to the nucleus in
these seedlings (Osterlund et al., 2000). An additional HY5
control point involves a kinase, possibly a caseine kinase,
which phosphorylates HY5 in darkness resulting in a less
active form, that also binds COP1 less tightly (Hardtke et al.,
2000). HY5 activity is therefore regulated by two intertwined
mechanisms strongly dependent on nuclear COP1 activity.
As discussed previously, COP9 is part of a large protein
complex (COP9 signalosome), consisting of several subunits,
which are lacking in cop9 and in many of the constitutively
photomorphogenic cop/det/fus mutants. The eight subunits of
the COP9 signalosome are all paralogous to the subunits of
the 19S regulatory particle and therefore the COP9 signalosome probably interacts with the 20S core particle to form
alternative 26S proteasome complexes. Here there are two
possibilities: the COP9 signalosome interacts with the 20S
core particle directly forming a COP9 proteasome; or the
COP9 signalosome interacts with the 19S regulatory particle
forming a 26S proteasome/COP9 signalosome supercomplex. Both are possible and certainly not mutually exclusive.
The characteriztics of the various COP9 signalosome subunits (CSNs) are indeed interesting (cf. Wei & Deng, 1999)
and one subunit, which is different from the rest, CSN5,
not only acts as a COP9 signalosome subunit but also as a
functional monomer (Kwok et al., 1998). For instance, in
Arabidopsis COP9 signalosome mutants there is no protein
complex but CSN5 monomers can be detected. CSN5 has
been shown to affect the nucleo/cytoplasmic distribution of
several interacting proteins ultimately mediating proteolysis
implying that CSN5 does not only act as part of the COP9
signalosome. However, it still remains to be shown whether
the monomeric activity of CSN5 is related to the COP9
signalosome.
Although it is clear that the COP9 signalosome is part of
the 26S proteasome proteolytic pathway involved in the regulation of photomorphogenic responses, cop/det/fus mutants
are very pleiotropic suggesting that the affected proteins are
involved in other developmental processes. Using transgenic
plants with reduced CSN5 levels, Deng and colleagues
(Schwechheimer et al., 2001) found that COP9 signalosome
deficiency results in auxin related phenotypes typical of auxin
response mutants such as axr1–3 and tir1–1 (Lincoln et al.,
1990; Gray et al., 1999). As described earlier, auxin responses
are controlled by the SCFTIR E3 ubiquitin ligase, which
degrades Aux /IAA proteins (Gray et al., 1999; Gray & Estelle,
2000) and indeed decreased degradation of the pea IAA
protein PSIAA6 is seen in CSN5 transgenic plants compared
to wild type (Schwechheimer et al., 2001). In addition, the
cullin or Cdc53 subunit of various SCF complexes has been
shown to interact with the COP9 signalosome (Lyapina et al.,
2001) and it was therefore tempting to suggest that maybe
SCFTIR interacts with the COP9 signalosome. This is indeed
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Tansley review no. 136
the case (Schwechheimer et al., 2001) suggesting that the
COP9 signalosome may be important in mediating E3 ubiquitin ligase responses. It is of course tempting to speculate that
the pleiotropic nature of cop/det/fus mutants is a result of
impairment of several E3 ubiquitin ligase functions.
As part of a normal auxin response, the ubiquitin-related
protein RUB1, becomes conjugated to the atCUL1 subunit
of SCFTIR (del Pozo & Estelle, 1999) and it has been shown
that the COP9 signalosome promotes RUB1 deconjugation
(Lyapina et al., 2001). It is possible that RUB1 modifications
may affect COP9/SCFTIR interactions thereby regulating
the nucleo/cytoplasmic partitioning of SCFTIR. This would
be analogous to the hypothesis that COP9 signalosome is
required for the light-dependent subcellular distribution of
COP1 (von Arnim & Deng, 1994). Once again the importance of controlled subcellular distribution patterns on major
cellular processes is clear and it remains an exciting challenge
to dissect the network as part of developmentally controlled
proteolytic events.
SUMO There is a growing family of ubiquitin-related
proteins and one of these is the small-ubiquitin-relatedmodifier, or SUMO. SUMO resembles ubiquitin in many
ways in terms of structure, in its ability to ligate to proteins
and also in the mechanism of conjugation (Melchior, 2000).
However, in contrast to ubiquitination, SUMOlation does
not lead to protein degradation but rather protection. It has
indeed been speculated that SUMO acts as an antagonist to
ubiquitin and by doing so adding another level of control and
complexity in the degradation of specific proteins. How
SUMO exerts its role is largely unknown but there is evidence
that SUMOlation regulates both protein–protein interactions
as well as subcellular localization patterns. As described in
Section III/2, the phyA-specific signalling component LAF1
encodes a nuclear two-repeat MYB protein containing a
putative SUMOlation site (Ballesteros et al., 2001). Although
SUMOlation consensus sites are minimal (KKQE),
Ballesteros et al. (2001) demonstrated that by mutating the
conserved second lysine residue to an arginine the recruitment
of LAF1 to subnuclear foci was abolished suggesting a role for
SUMO in this process. If this is indeed the case it is possible
that SUMO conjugation recruits LAF1 to subnuclear foci, as
seen for HIPK2 (Kim et al., 1999), and by doing so brings
LAF1 in close proximity to other MYB factors or directly to
promoter elements of light-regulated genes. Transcription
factors/activators are often controlled by degradation and it is
possible that SUMOlation and speckle formation protects
LAF1 from the 26S proteasome. It would indeed be
interesting to learn whether LAF1/SUMO subnuclear speckle
formation and LAF1 stability is affected in COP9
signalosome mutants.
The identification of specific degradation of proteins in
response to both developmental and environmental cues has
added to the complexity of cellular regulation. Although
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recent research has provided exciting findings linking phytochrome signalling to protein degradation there still remains a
lot of uncharted territory. The recent identification of 37
multiubiquitin chain assembly factor (E4) proteins containing a U-box motif (Azevedo et al., 2001) and the finding that
Arabidopsis has 337 F-box containing and 358 RING finger
motif containing proteins (Estelle, 2001) does suggest that we
have a long way to go before we understand how phytochrome signalling and protein degradation are functionally
connected.
Clearly, there has been substantial recent activity centred
on the nucleus. The isolation of phytochrome interacting
proteins and the elucidation of the light-induced nuclear
translocation of phyA and phyB has certainly created a lot
of excitement. These data suggest that at least some of the
phytochrome signalling pathways are short and exclusively
nuclear localized. In addition, the characterization of EID1
and LAF1 has indicated the involvement of protein degradation and protection as being part of the nuclear phytochromesignalling network. Also, recent studies into the structure of
the COP9 signalosome have unveiled new insight into the
role of protein degradation as part of light signalling. To date,
the available data has probably only touched the tip of the iceberg regarding phytochrome signalling and regulation in the
nucleus. One would anticipate the future isolation of phytochrome interaction proteins involved in cytoplasmic retention, in the import/export process itself as well as components
of the ubiquitin and SUMO pathways. Regardless, it will now
be important to assemble all the ‘nuclear’ data and to expand
the findings and interpretations to the whole cell level.
IV. The cytoplasm
Traditionally, signal transduction pathways were generally
viewed as signal perception via plasma membrane-bound
receptors followed by signal relay through the cytoplasm
ultimately resulting in micro or macro morphological changes
underpinned by alterations in nuclear gene expression. The
vital role of the nucleus in phytochrome signal transduction
is undisputable, however, the cytosol and the organelles
suspended in it clearly form integrated compartments of
this network. For example, the cytosolic retention of phyA
and phyB in darkness, the isolation of cytosolic phytochrome interacting proteins, the possible involvement of
heterotrimeric G-proteins during early steps of phytochrome
signalling and the involvement of cytoplasmic organelles
in photomorphogenic responses all demonstrate that the
nucleus does not act alone in light signal transduction.
1. The cytosol
Dissecting secondary messengers: Ca2+/CaM, cGMP and
heterotrimeric G-proteins The activation of heterotrimeric
G-proteins and subsequent signal relay via secondary
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messengers have been implicated in signal transduction
cascades for a very long time. To this end Chua and colleagues
(Neuhaus et al., 1993; Bowler et al., 1994) investigated
whether this is the case for phytochrome signal transduction.
Using a microinjection approach it was demonstrated that
putative heterotrimeric G proteins might act as an upstream
component of the phyA signal transduction pathway (Bowler
et al., 1994; Neuhaus et al., 1993). Moreover, it was shown
that at least three different pathways, involving calcium
and/or cGMP, controlled phyA responses downstream of the
G protein complex. The data indicate that: cGMP stimulates
chalcone synthase (CHS) expression and anthocyanin
biosynthesis; that Ca2+/calmodulin (CaM) activates
chlorophyll a /b binding protein (CAB) expression and partial
chloroplast development; and that both cGMP and Ca2+
activates photosystem I (PSI) genes such as ferredoxin
NADP+ oxidoreductase (PETH) expression to produce fully
active chloroplasts. Using agonists and antagonists to disrupt
signal flow, cGMP and Ca2+/CaM were shown to exhibit
interpathway regulation where downstream regulators of one
pathway can negatively regulate another and vice versa, a
control mechanism termed reciprocal control (Bowler et al.,
1994). Although this approach yielded insights into phyA
signal transduction, in terms of cytoplasmic signal relay
mediated by G-proteins and secondary messengers, there is to
date very little genetic evidence supporting these findings.
Despite this and as discussed in Section II/4, there is new
evidence suggesting the involvement of heterotrimeric Gproteins in cytoplasmic retention and release mechanisms
(Santagata et al., 2001) implying that cytoplasmic G-proteins
may be involved in the cytoplasmic retention of photochemically inactive phytochrome. Direct genetic evidence of
heterotrimeric G-protein involvement in phytochrome signal
transduction came however, with the very recent finding that
overexpressed heterotrimeric G-protein α-subunit (Gα)
results in decreased hypocotyl cell elongation in response to
light (Okamoto et al., 2001). Deng and colleagues (Okamoto
et al., 2001) have shown that this hypersensitivity occurs in
response to far-red, red, and blue light irradiation implying a
general spectrum-wide role for Gα. However, more detailed
genetic studies demonstrate that the Gα-induced phenotype
requires functional phyA and FHY1 (Whitelam et al., 1993)
but not FHY3 (Whitelam et al., 1993) or FIN219 (Hsieh
et al., 2000). Since FHY1 has been shown to represent a
branch-point in the phyA signalling pathway (Barnes et al.,
1996), these results suggests that Gα is only involved in
certain branches of phyA signalling. The possible mechanism
of heterotrimeric G-protein activation via phytochrome still
remains to be unravelled. In Dictyostelium, G-proteins are
activated by NDPK (Bominaar et al., 1993) and it is tempting
to speculate that either NDPK2 activates Gα after it has been
activated by phyA or that Gα interacts with phyA directly.
In addition, the latest finding that PRA2, a small G-protein
in pea, may act as a cross-talk mediator between light and
brassinosteroid signalling in plants is exciting (Kang et al.,
2001).
To date, the involvement of the secondary messengers Ca2+
and cGMP in phytochrome signalling has little support
genetically. This may partially be due to the presence of such
a large cellular Ca2+ signalling network making isolation
of ‘Ca2+’-proteins specifically involved in phytochrome
responses difficult. However, it is assumed that protein kinases
and phosphatases, being interpreters of Ca2+ signals, will be
isolated from either forward or reverse genetic screens. To this
end Lin and colleagues (Guo et al., 2001) isolated an Arabidopsis photomorphogenic mutant, sub1, which is hypersensitive towards both blue and far-red light irradiation. Genetic
studies further showed that SUB1 functions as a signalling
component downstream of CRY1 and CRY2. Conversely, the
activity of phyA is not dependent on SUB1 indicating that
SUB1 may act as a modulator of phyA signalling. The cloning
of the mutant lesion in sub1 revealed that the disrupted gene
encodes a novel protein containing EF-hand-like motifs
suggestive of Ca2+ binding and indeed heterologously
expressed SUB1 can bind Ca2+. Moreover, SUB1 localizes to
the cytoplasm apparently enriched in the nuclear periphery.
Together with the proposed role of SUB1 being a cryptochrome signalling component that modulates phyA signalling, SUB1 probably defines a point of cross-talk between
cryptochrome and phyA and may play a role in monitoring
light-induced cytoplasmic changes in ion homostasis.
To try to dissect the involvement of Ca2+ in photomorphogenic responses it may be of value to use intracellular-directed
Ca2+ probes such as aequorin (Rizzuto, 2001) or CaMeleon
(Miyawaki et al., 1997) to monitor cytosolic subregions and
microdomains for changes in Ca2+ concentrations in response
to different light treatments.
Pat1 The first cytosolic phytochrome signalling component
to be isolated was PAT1 (Bolle et al., 2000). pat1 was isolated
in a genetic screen showing a far-red insensitive phenotype
almost as strong as that observed for fhy1 and fhy3. PAT1 is a
member of the GRAS protein family, which include members
such as SCR (Di Laurenzio et al., 1996), GAI (Peng et al.,
1997a) and RGA (Silverstone et al., 1998), all shown to be
involved in plant signal transduction. The T-DNA insertion
in pat1 leads to the production of a truncated protein and
the pat1 mutant phenotype can be recapitulated by
overexpression of the truncated PAT1 protein in wild-type
seedlings. These data indicate that the truncated version of
PAT1 acts as a dominant negative protein and since PAT1
does not interact with the C-terminal region of phyA or phyB
(S. G. Møller and N. H. Chua, unpublished), it suggests that
PAT1 may interact with other phyA signalling components.
In both onion epidermal cells and in transgenic plants, PAT1/
GFP fusions localize constitutively to the cytoplasm,
suggesting that PAT1 is involved in very early signalling
steps even before phyA is imported into the nucleus.
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Fin219 In a suppressor screen developed to identify genes
involved in the light inactivation of COP1, a repressor of
photomorphogenic development, Deng and colleagues
(Hsieh et al., 2000) identified a suppressor mutation that
showed loss of hypocotyl growth inhibition in response to
far-red light irradiation. The mutant locus, fin219, was
cloned and the disrupted gene encodes a 575 amino acid
constitutively cytoplasmic protein with very high similarity to
the GH3 family of proteins. The GH3 protein first identified
in soybean (Hagen et al., 1984) as being auxin inducible and
FIN219 shows the same auxin inducibility. To verify the
involvement of FIN219 in far-red light signalling, it was
further shown that FIN219 overexpression results in far-red
induced hypersensitivity. In addition, FIN219 interacts
genetically with FHY1, which has recently been shown to be
a nuclear protein, underlining the interaction between the
nucleus and cytoplasm as part of light signal transduction.
FIN219 may be involved in the far-red light mediated
inactivation of COP1 and although fin219 has no obvious
auxin phenotype it may represent an intersection between
light and auxin signal transduction.
Pks1 The first phytochrome kinase substrate identified
was phytochrome kinase substrate 1 (PKS1; Fankhauser et al.
1999). PKS1 was isolated in a yeast two-hybrid screen using
the C-terminal domain of phyA as bait and it was
subsequently shown that PKS1 binds to both the phyA and
phyB C-terminal regions. Unlike PIF3 and NDPK2, PKS1
binds with equal apparent affinity to both the Pr and Pfr
forms of phyA and phyB indicating no photo-selectivity of
phytochrome binding. PKS1 encodes a 439 amino acid novel
protein with no identifiable functional motifs. However,
purified oat phyA was able to phosphorylate a GST-PKS1
fusion protein and experiments showed that PKS1 was a
better substrate for the Pfr kinase form than for the Pr kinase
form of phyA. Also, immunoprecipitation experiments,
using phosphatase treatments, showed that PKS1 is a
phosphoprotein in vivo. In addition, both phytochrome
autophosphorylation and PKS1 phosphorylation is
stimulated approx. 2.5-fold upon red light irradiation
demonstrating that phyA shows light-regulated autophosphorylation and PKS1 phosphotransferase/phosphorelay
activity. Since PKS1 is shown to localize to the cytoplasm it
is possible, as discussed in Section II/4, that PKS1 is involved
in the cytoplasmic retention of phyA or phyB in the cytoplasm (Fig. 2). PKS1 may retain phyA and phyB in an
inactive state in the cytoplasm, however, this is difficult to
reconcile given the lack of photoreversible PKS1/phytochrome
binding characteriztics. To determine the role of PKS1
in photomorphogenesis, Chory and colleagues (Fankhauser
et al., 1999) generated transgenic seedlings overexpressing
PKS1 and showed that in red light PKS1 overexpression
results in partial insensitivity whilst far-red or blue light has no
effect. This suggests that PKS1 is a negative component of
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phyB signalling probably influencing phyB kinase activity or
maybe its subcellular partitioning pattern acting as a retention
protein. It will indeed be interesting to learn whether a PKS1
loss-of-function mutant has altered phyA or phyB nuclear
translocation patterns.
2. Is phytochrome a kinase?
At this point we should address the old issue of whether
phytochrome actually is a kinase. Historically, it was proposed
by Borthwick & Hendricks (1960), that phytochromes are
indeed light-regulated enzymes. Quail and colleagues showed
that phytochrome was a phosphoprotein by in vivo 32P
labelling experiments in 1978, but it was almost a decade later
that purified fractions of phytochrome were shown to have
protein kinase activity (Wong et al., 1986, 1989; McMichael
& Lagarias, 1990; Hamada et al., 1996). However, it was
unclear whether the kinase activity was due to phytochrome
itself or a copurified protein with kinase activity. This concern
was based on studies describing loss of kinase activity
following more rigorous downstream purification methods
(Grimm et al., 1989; Kim et al., 1989). Whether phytochrome
has bona fide kinase activity and if so what type of kinase is
still somewhat unclear (Elich & Chory, 1997; Cashmore,
1998; Fankhauser & Chory, 2000).
Bacterial ‘two component’ phosphorelay systems Work on
bacteriophytochromes, phytochrome-like photoreceptors
found in several bacteria, may provide relevant clues about
phytochrome action. The first bacteriophytochrome
identified, RcaE (response to chromatic adaptation E)
from the cyanobacterium Fremyella diplosiphon (Kehoe &
Grossman, 1996), shows homology at the N-terminus to
the chromophore-binding pocket of phytochromes and is
capable of binding a variety of bilins generating red/far-red
photoreversible chromoproteins. Significantly, RcaE contains
a region of homology to two component histidine kinase
systems, suggesting that bacterial photoreceptors can
transduce a light signal into a phosphorelay cascade.
Sequence searches with other bacteria, both photosynthetic and nonphotosynthetic, have revealed additional
phytochrome-like sequences showing similarities to plant
phytochromes at the amino terminus and a histidine kinase
domain (Vierstra & Davis, 2000).
The Synechocystis phytochrome Cph1 has also been shown
to be the photoreceptor of the two-component light sensory
system (Hughes et al., 1997; Yeh et al., 1997; Park et al.,
2000), exhibiting histidine kinase activity in vitro. The second
step in the phosphorelay reaction is the transfer of phosphate
to an aspartic acid residue in the Rcp1 response regulator.
Cph1 also shows red /far-red photoreversible absorbance
properties indicative of phytochrome activity. Interestingly,
the degree of histidine autophosphorylation observed in vitro
is greater in the Pr form compared to the Pfr form. It has been
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suggested that the Pr form has kinase activity whilst the Pfr
form may act as a protein phosphatase (Hughes & Lamparter,
1999).
Similarly, in a four-step phosphorelay model proposed by
Kehoe and Grossman (1997), RcaE is proposed to act as a
kinase in red light, phosphorylating RcaF, which then transfers the phosphate group to RcaC, whilst in green light RcaE
appears to act as an RcaF phosphatase.
This dual activity of bacteriophytochromes is in sharp contrast to plant phytochromes, where the Pr form is generally
considered to be inactive.
Sequence analysis shows that phytochrome seem to contain
two diverged histidine kinase related domains (HRKD) in a
tandem arrangement (Yeh & Lagarias, 1998). The first
HRKD domain also contains two motifs with homology to
PAS (PER-ARNT-SIM) domains (Lagarias et al., 1995; Kay,
1997). PAS domains are present in various signal transduction
molecules that sense environmental signals such us light, oxygen levels, and redox potential (Taylor & Zhulin, 1999). They
may also mediate protein–protein interactions (See Fig. 1 for
details of domains). Despite these sequence similarities, the
proposal that higher plant phytochromes are light-activated
kinases remains controversial because the histidine residues
critical for kinase activity in most bacterial sensors are not
conserved in all phytochromes (Shneider-PoetSchneiderPoetsch, 1992).
Autophosphorylation Purified oat phyA expressed in
accharomyces cerivisiae and Mesotaenium caldariorum
phytochrome expressed in Pichia pastroris form active
holophytochrome and display autophosphorylation activity
in a light-regulated fashion (Yeh & Lagarias, 1998). However,
in contrast to earlier findings, this appears to be a serine/
threonine kinase activity rather than histidine/aspartate
kinase activity. Mass spectroscopy revealed that phyA has
three phospho-acceptor sites, serine7, serine17 and serine598
(McMichael & Lagarias, 1990; Lapko et al., 1997, 1999).
Interestingly, serine7 phosphorylation is similar in both Pr
and Pfr, serine17 is phosphorylated in the Pr form by protein
kinase A in vitro whereas, serine598 is preferentially
phosphorylated in the Pfr form. Additionally, when the
amino acids shown to be important for histidine-kinase
activity in prokaryotic systems are mutated there is no effect
on phyA activity in transgenic plants (Boylan & Quail, 1996).
This suggests that phytochrome A may be a serine/threonine
kinase, that has diverged from a histidine kinase. This
suggestion is not without precedent; other prokaryotic-like
histidine kinases found in eukaryotes have been shown to
phosphorylate serine and threonine rather than histidine and
aspartate (Popov et al., 1993; Harris et al., 1997). Fankhauser
and Chory (1999), have also suggested that phytochrome
phosphorylation may proceed through less stable intermediates such as phospho-aspartate and phopho-histidine.
Taken together, it appears phytochrome is autophosphor-
ylated in both a light-dependent and a light-independent
manner, but what is the biological significance of this? In
studies where serine residues at the amino terminus of rice
phyA were mutated to alanines an increase in biological activity compared to wild type phyA can be observed (Stockhaus
et al., 1992; Jordan et al., 1997). From this observation it was
postulated that the light-independent phosphorylation of
serine7 down-regulates phytochrome activity.
Similarily, if serine598 is mutated to alanine, and the
mutated phytochrome is expressed in a phyA null background, hypersensitivity to far-red light is observed; suggesting that serine598 phosphorylation can also negatively
regulate phytochrome activity (Park et al., 2000). Serine598 is
positioned in the hinge region between the chromophorebinding N-terminal domain and the C-terminal regulatory
domain (Fig. 1). In a model described in Park et al. (2000),
serine/threonine phosphorylation is suggested to play a role in
photoinducible conformational changes of phytochrome and
to modulate interdomain signalling.
In this model, serine598 phosphorylation would serve to
desensitise Pfr, which could not then associate with phytochrome interacting factors (PIFs), whereas the unphosphorylated Pfr would be capable of binding PIFs. This is analogous
to the desensitising mechanism for the rhodopsin photoreceptor
in vision (Abdulaev & Ridge, 1998). It would be interesting
to examine whether a serine598 to alanine substitution in
phytochrome has an affect on nuclear transport vs. cytoplasmic
retention. It should be noted that no phosphatase activity has
been associated with phytochrome and so a dephosphorylation step would require an additional, as yet unidentified,
factor. Although the available evidence does suggest that
phytochrome is capable of autophosphorylation, the question
of whether phytochrome shows specific kinase activity still
remains unresolved. Various phytochrome-specific signalling
mutants and biochemical approaches do suggest that this
may be the case. However, most in vitro phytochrome kinase
assays described in the literature make use nonphysiological
conditions in terms of incubation times, rendering the results
somewhat difficult to interpret. If it is assumed that phytochromes do indeed have specific kinase activity it would be
expected that there are several proteins that are phosphorylated by phytochrome. Some of these have been discussed
including PKS1, CRY1, CRY2, and NDPK2, however, there
now is recent evidence suggesting that the phytochrome–
kinase boundary extends into other pathways such as auxin
signalling (Section V/1).
3. Chloroplasts
Chloroplasts are thought to have arisen from the engulfment
of free-living cyanobacteria-type organisms (Schwartz &
Dayhoff, 1978). Although chloroplasts contain their own
autonomous genome the nuclear genome in a plant cell
remains superior because it encodes most of the proteins
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(> 90%) necessary for chloroplast maintenance and function.
An important light-dependent developmental pathway in
plants involves the transition of nonphotosynthetically active
proplastids into photosynthetically active chloroplasts
(Kendrick & Kronenberg, 1994), a process that requires the
coordinated expression of both plastidic and nuclear genes.
This finely tuned developmental program requires precise
communication between the nucleus and chloroplasts and it
is now evident that the former senses the functional state
of the latter, via retrograde signalling, and orchestrates
subsequent morphological and molecular manifestations
(Oelmuller et al., 1986; Oelmuller, 1989; Susek et al., 1993;
Lopez-Juez et al., 1998; Streatfield et al., 1999). Indeed it
has been shown that the herbicide Norflurazon, which
impairs chloroplast functionality, results in reduced expression
of nuclear-encoded genes such as LHCB (chlorophyll a /bbinding protein) and RBCS (small subunit of ribulose
bisphosphate carboxylase) (Oelmuller, 1989; Susek et al.,
1993). The nature of the retrograde signalling pathway still
remains largely unknown. However, chlorophyll precursors
have been implicated and recent evidence has shown that
Mg-protoporphyrin IX and protoporphyrin IX can act as a
signal from the chloroplast to the nucleus.
Tetrapyrrole intermediates as retrograde signals Early studies
into retrograde signalling suggested that chlorophyll precursors,
such as protoporphyrin IX, could act as negative signalling
molecules in that they attenuate the expression of lightinducible nuclear genes in Chlamydomonas reinhardtii
(Johanningmeier & Howell, 1984; Johanningmeier, 1988).
Conversely, the Chlamydomonas brown mutants brs-1 and
brc-1, defective in Mg-protoporphyrin IX synthesis, are
unable to induce nuclear heat shock protein (HSP70) gene
expression in response to light, suggesting that Mgprotoporphyrin IX may act as a positive retrograde signalling
component (Kropat et al., 1997; Kropat et al., 2000).
Moreover, Mg-protoporphyrin IX-feeding experiments in
darkness show that this tetrapyrrole can substitute for the
light signal in a dose-dependent manner as shown by the
transient increase in HSP70 expression (Kropat et al., 1997).
Interestingly, protoporphyrin IX, protochlorophyllide and
chlorophyllide could not act as signalling molecules, at least
for HSP70 induction (Kropat et al., 1997; Kropat et al.,
2000), suggesting that specific tetrapyrrole intermediates play
different roles as part of the retrograde signalling network.
However, it is possible that nonphotosynthetic genes as
molecular probes may not represent the most accurate
measure of retrograde signalling.
Using a combination of biochemical and molecular techniques Grimm and colleagues (Grimm, 1998) have unravelled
numerous steps in the tetrapyrrole metabolic pathway (Grafe
et al., 1999; Lermontova & Grimm, 2000; Papenbrock et al.,
2000b, 2000a). For instance, it has been demonstrated that by
disrupting the Mg-chelatase protein complex, the branch-
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point of tetrapyrrole biosynthesis, there is a reduction of
protoporphyrin IX with a concomitant reduction in LHCB
expression (Papenbrock et al., 2000b; Papenbrock et al.,
2000a).
gun mutants The genomes uncoupled (gun) mutants were
isolated (Susek et al., 1993) by exploiting the fact that in the
presence of Norfluorazon chloroplasts become photooxidised
resulting in the repression of nuclear genes encoding
chloroplast proteins (Oelmüller, 1989). Chory and colleagues
(Susek et al., 1993) screened a battery of transgenic
Arabidopsis plants containing the CAB promoter fused to a
hygromycin-resistance gene in the presence of hygromycin
and Norflurazon with the idea that if the retrograde signalling
pathway is disrupted in some way, the damaged chloroplasts
will not relay information back to the nucleus and CAB
expression will remain high. Recently, Chory and colleagues
(Mochizuki et al., 2001) cloned the disrupted gene in gun5
and show that it encodes the Mg-chelatase H subunit located
on the inner plastid envelope. In contrast to the studies of
HSP70 expression in Chlamydomonas (Kropat et al., 1997;
Kropat et al., 2000), gun5 has reduced Mg-protoporphyrin
IX and protoporphyrin IX levels. Taken together these data
suggests that the Mg-chelatase H subunit might monitor the
flux through the chlorophyll biosynthetic pathway and
subsequently relay a positive signal to the nucleus or inhibit a
negative signal.
The finding that gun2 and gun3 are alleles of hy1 and hy2
(Vinti et al., 2000; Mochizuki et al., 2001) provide another
interesting twist to the retrograde signalling question. Both
hy1 and hy2, defective in photochromobilin synthesis, are disrupted in downstream components of the haem branch of the
tetrapyrrole biosynthetic pathways and they were originally
isolated based on their loss of hypocotyl growth inhibition.
The gun phenotype of hy1 and hy2 is most likely due to feedback inhibition in that downstream disruptions in the haem
branch can cause repression of early upstream steps ultimately
leading to decreased flux through the chlorophyll branch
(Terry & Kendrick, 1999). In fact, genetic studies have shown
that hy1 and gun5 affect the same retrograde signalling pathway (Vinti et al., 2000).
Clearly when dealing with complex biosynthetic pathways, such as chlorophyll and haem biosynthesis, it can be
complicated to dissect the precise role of the various components due to extensive and different feedback mechanisms
between the intermediates. However, the identification of
mutants, disrupted not only in components of the tetrapyrrole biosynthetic pathway itself, but also in components that
play an indirect role in the chlorophyll biosynthetic pathway
will aid in the understanding of the retrograde signalling
network.
Laf6 The laf6 mutant (Møller et al., 2001) was isolated in a
genetic screen designed to identify components of the phyA
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signal transduction pathway. laf6 seedlings display a partial
insensitivity towards far-red light irradiation and the cloning
of the disrupted gene revealed that it encodes an novel 557
amino acid ATP-Binding-Cassette (ABC) protein (atABC1).
Using atABC1/GFP fusion proteins in onion epidermal cells
and transgenic plants together with immunolocalization
studies, it was shown that atABC1 localizes to the envelope
region of chloroplasts (Møller et al., 2001). The disruption of
atABC1 in laf6 results in protoporphyrin IX accumulation
and in repression of light-regulated genes suggesting that the
accumulation of protoporphyrin IX in this case results in an
attenuation of nuclear gene expression. This again shows
that plastid-derived protoporphyrin IX is probably involved
in regulating nuclear gene expression. What is the role of
atABC1 or indeed protoporphyrin IX on hypocotyl
elongation? Clearly hy1 and hy2 show elongated hypocotyls in
response to light, however, laf6 has normal phyA levels and
cannot be rescued by chromophore feeding experiments
(Møller et al., 2001). To further investigate this it has been
shown that the long hypocotyl phenotype of laf6 and the
accumulation of protoporphyrin IX in the mutant can be
recapitulated by treating wild type seedlings with flumioxazin,
a protoporphyrin IX oxidase (PPO) inhibitor. In addition,
protoporphyrin IX accumulation and hypocotyl elongation
in flumioxazin-treated wild-type (WT) seedlings can be
reduced upon atABC1 overexpression. Knowing that ABC
proteins are most often involved in transport processes, these
observations suggest that atABC1 may be involved in the
transport and correct distribution of protoporphyrin IX. The
preferential specificity of laf6 towards far-red light suggest
further that elevated protoporphyrin IX levels may interact
with the cytosolic light-signalling pathway or trigger a
secondary pathway ultimately coordinating nuclear gene
expression patterns. Since atABC1 does contain putative
porphyrin-binding motifs (S. G. Møller and N. H. Chua,
unpublished) it will be interesting to learn whether atABC1
can bind protoporphyrin IX directly. Moreover, the role of
atABC1 in coordinating the interplay between the nucleus
and chloroplasts, ultimately modifying gene expression
affecting hypocotyl elongation, remains to be solved (Fig. 4).
Nontetrapyrroles as retrograde signals Despite the available
evidence demonstrating the involvement of tetrapyrroles as
retrograde signals, there are studies that suggest the presence
of nontetrapyrrole retrograde signalling pathways. Results
from studies using the green alga Dunaliella tertiolecta has
shown that by changing the redox state of the plastoquinone
pool in chloroplasts, nuclear CAB expression is affected
(Escoubas et al., 1995). By reducing plastoquinone at high
light intensities CAB expression is enhanced whereas CAB
expression is repressed upon inhibition of plastoquinone
oxidisation at low light intensities. This suggests that the
redox state of the plastoquinone pool in chloroplasts may
act as a photon-sensing system regulating nuclear CAB
expression. Interestingly, the accumulation of chlorophyll
at low-light intensities can be blocked by inhibition of
cytoplasmic phosphatases such as okadaic acid. This further
implies that CAB expression may be reversibly repressed by a
phosphorylated component, which is in turn coupled to the
redox state of plastoquinone, possibly via a chloroplast protein
kinase. Although an attractive model it still remains to be
shown that the signal from the plastoquinone pool is relayed
through a phosphorylation cascade.
The regulation of photosynthesis by carbon metabolite
feedback inhibition has been well established although the
cellular mechanism still remains largely unknown. For
example, CAB expression is increased upon sugar depletion.
However, by blocking photosynthetic electron transport,
sugar depletion does not lead to CAB induction (Oswald
et al., 2001). Taken together this suggests that the redox state
in the chloroplasts can outweigh the carbohydrate-regulated
expression of nuclear-encoded photosynthetic genes.
Recently, Lucas and colleagues (Provencher et al., 2001)
cloned the disrupted gene in the maize mutant sucrose export
defective 1 (sxd1), which encodes a novel chloroplast localized
protein of 488 amino acids. sxd1 has been shown to be defective in sugar transport, to accumulate anthocyanin and to
form occlusions of plasmodesmata between bundle sheath
cells. Many photosynthetic genes are suppressed by high sugar
levels through a hexokinase-mediated signalling cascade ( Jang
et al., 1997) however, in contrast to wild type, CAB expression
in sxd1 remains high despite the elevated sugar levels. It is
tempting to speculate that SXD1 may represent a chloroplast
protein required to integrate hexokinase-mediated signals
within the cells.
The chloroplast to nucleus retrograde signalling network is
unquestionably a more complex process than first anticipated.
Although by a largely unknown mechanism, chlorophyll
precursors can clearly act as ‘plastid signals’. However, there is
compelling evidence that both the plastoquinone redox state
as well as sugars can have a dramatic effect on nuclear gene
expression of light-regulated genes. There are clearly multiple
ways in which these ‘chloroplast retrograde signals’ are transduced to the nucleus. Moreover the role of these ‘signals’ and
indeed the role of the chloroplast in controlling photomorphogenic responses, such as hypocotyl elongation and
gene expression, remain unclear. The chloroplast undoubtedly
plays an important integrated role in photomorphogenic
responses along side the nucleus and the cytoplasm. It will
indeed be interesting to learn, using molecular in vivo chloroplast disruption techniques together with microarray analysis,
which nuclear genes are affected and whether the majority of
these encode photosynthetic genes.
V. Interactions with other signalling pathways
Signal transduction pathways have often been viewed as linear
chains of events but it is becoming increasingly clear that there
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Review
Fig. 4 Retrograde signalling from the plastid to the nucleus. The figure illustrates possible retrograde signalling events that take place between
plastids and the nucleus. Both LAF6 and GUN5 have been implicated in playing a role in this communication mechanism. It is still largely
unknown how retrograde signals are transduced to the nucleus in response to both internal and environmental cues.
is extensive cross-talk between different pathways (Møller &
Chua, 1999). The plethora of interactions that take place are
probably involved in modulating various pathway inputs so
that plants can develop in response to changing environmental and developmental cues. It has been conclusively
shown that the phytochrome signalling cascade does indeed
communicate and interact with various other signalling
pathways. The isolation of hormone signalling mutants and
the recent characterization of key regulatory processes have
given extensive insight into how light and hormone signalling
are integrated.
1. Light and hormones
It is not surprising that hormones contribute to photomorphogenic responses in that both light and hormones
generally act on similar cells and organs involving processes
such as cell elongation and expansion. For example cytokinin,
ethylene and abscisic acid (ABA) inhibit cell elongation whilst
© New Phytologist (2002) 154: 553 – 590 www.newphytologist.com
auxin, brassinosteroids, and gibberellic acid (GA) stimulate
cell elongation.
Auxin The roles of auxin during plant development are
several-fold. At the tissue/organ level, auxin is involved in root
elongation, lateral root development, meristem maintenance
and senescence whilst at a cellular level this hormone is
involved in cell division, cell differentiation and cell
elongation. Early studies into auxin signalling were mainly
focused on the identification of auxin responsive DNA
sequence elements (Guilfoyle et al., 1998). A number of
auxin-regulated genes have been isolated and the recent use of
Arabidopsis mutants has started to unravel the auxin pathway
and its interaction with phytochrome signalling.
Several auxin-regulated genes have been found to encode
proteins that localize to the nucleus. For instance parA, an
auxin-regulated gene in tobacco (Takahashi et al., 1995) and
IAA1 and IAA2, early auxin-induced genes in Arabidopsis
(Abel et al., 1994) are nuclear localized. Moreover, IAA1 and
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IAA2 are very short lived proteins (t1/2 4–8 min) and show
the presence of DNA binding sequence motifs suggesting that
very early primary responses in auxin signalling may involve
transcriptional control by IAA proteins (Abel et al., 1994).
More recent data has indeed shown that a vast number of IAA
genes encode short lived nuclear proteins that can homo- and
hetero-dimerise both in vivo and in vitro (Abel et al., 1994;
Kim et al., 1997). Does this have implications for phytochrome signal transduction? The semidominant mutant
shy2–1D (Kim et al., 1996) was isolated as a suppressor of the
long hypocotyl phenotype of hy2, a chromophore biosynthetic mutant. SHY2 has been cloned and encodes IAA3
(Tian & Reed, 1999), a member of the IAA auxin-inducible
gene family. Loss-of-function shy2 mutants consistently have
longer hypocotyls than wild type (Tian & Reed, 1999) whilst
gain-of-function mutations result in photomorphogenic
responses in darkness such as cotyledon expansion and
hypocotyl growth inhibition (Kim et al., 1997). From these
data it is clear that there is an interaction between phytochrome and IAA proteins. To this end Abel and colleagues
(Colon-Carmona et al., 2000) have demonstrated, by in vitro
interaction studies, that SHY2/IAA3, AXR3/IAA17 (Ouellet
et al., 2001), and Ps-IAA4 (Abel & Theologis, 1995) interact
with purified oat phyA. This suggests that one point of interaction between auxin and light signalling is direct physical
contact between early auxin inducible nuclear gene products
and phytochrome. Interestingly, it has now been shown that
phyA can phosphorylate IAA3, IAA17, IAA1, IAA9 and PsIAA4 in vitro independent of its Pr or Pfr form. In addition,
it has been demonstrated, using metabolic labelling and
immunoprecipitation that SHY2-2 is phosphorylated in vivo.
One potential problem with these data is that the in vitro
kinase assay was performed for 25 min, which is outside the
physiological time scale for a primary phyA signalling event.
Despite this, these data do suggest the possibility that a primary
interaction point between phytochrome and auxin signalling
resides in the nucleus and may involve phytochrome-mediated
phosphorylation of early auxin inducible proteins.
It is clear that auxin plays an important role during
hypocotyl elongation. For instance, when Arabidopsis seedlings are grown in the light at 29°C they show a dramatic
increase in hypocotyl elongation compared to seedlings
grown at 20°C (Gray et al., 1998). It was shown that seedlings
grown at elevated temperatures have increased free auxin
levels suggesting that temperature regulates auxin synthesis
and catabolism and by doing so mediates growth responses.
In addition, Estelle and colleagues ( Jensen et al., 1998) have
shown, using polar auxin transport inhibitors, that auxin
transport is required for hypocotyl elongation in the light
but not in darkness. Similarly, it has been shown that the
calossin-like protein BIG is required for polar auxin transport
and that BIG-deficiency results in altered expression of
light-regulated genes (Gil et al., 2001). Auxin transport has
also been implicated in playing a role in the shade-avoidance
response (Morelli & Ruberti, 2000). Steindler et al. (1999)
showed that by overexpressing ATHB-2, a homeodomain
leucine zipper protein that is down-regulated by red and
far-red light, hypocotyl elongation increases whilst in more
mature plants there are obvious auxin phenotypes. The
cross-talk between ATHB-2-mediated shade avoidance
response and auxin was further verified by IAA feeding
experiments which rescued the ATHB-2 phenotype. Similarly, HY5, a bZIP transcription factor acting downstream of
both phytochrome and cryptochrome, was identified as a
positive regulator of photomorphogenesis (Ang & Deng,
1994). Although hy5 shows light insensitivity, the pleiotropic
nature of this mutant suggests its involvement in other
aspects of plant development and auxin signalling has
been implicated as one of these (Oyama et al., 1997).
Recent evidence has also emerged that Arabidopsis seedlings
with reduced COP9 signalosome, a negative regulator of
photomorphogenesis, exhibits reduced auxin response similar
to loss-of-function E3 ubiquitin ligase SCFTIR plants
(Schwechheimer et al., 2001). In addition, it was shown that
COP9 interacts with SCFTIR in vivo and that COP9 is in fact
needed for the degradation of the pea IAA protein PSIAA6.
These results suggest that COP9 may play an important
role in mediating E3 ubiquitin-ligase responses involved the
degradation of IAA proteins (Section III/4).
Recently, the relationship between hypocotyl elongation
and auxin was further strengthened by studies showing that
the reduced auxin response mutant, axr1–12, and plants overexpressing iaaL, which conjugates free auxin to lysine, have
reduced hypocotyl lengths in the light (Collett et al., 2000).
Moreover, if auxin is added hypocotyl growth is promoted.
Conversely, the enhanced auxin response mutant axr3–1 does
not respond to exogenous auxin application. It has also been
shown that IAA7-deficient seedlings show longer hypocotyls
than wild-type in response to light (Nagpal et al., 2000).
Once again it is clear that the nucleus plays an important role,
not only as part of light signalling, but also as an interaction
point between different pathways.
The nucleus is not the only subcellular compartment that
contains auxin/light pathway integrators. Deng and colleagues (Hsieh et al., 2000) have shown that fin219 exhibits
far-red light induced hypocotyl elongation. fin219 was isolated as a suppressor of COP1 and the disrupted gene encodes
an auxin-inducible cytoplasmic protein with good homology
to GH3-like proteins. Similarly, Nakazawa et al. (2001) have
shown that a second GH3 protein, DFL1, is involved in
hypocotyl elongation. DFL1 overexpression results in
reduced hypocotyl length in response to red, far-red and blue
light but not in darkness implying again interaction between
auxin and photoreceptors.
Although it is still early days, it is clear that auxin plays a
major role in a variety of developmental processes, both at the
physiological and molecular level, and that a number of these
processes are modulated through phytochrome action.
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Brassinosteroids The possibility that brassinosteroids are
involved in the interaction between light and hormone
signalling has created a lot of excitement. This enthusiasm
was largely prompted by the finding that a number of dwarf
mutants such as det2, dwf1, dim1 and cpd, previously
identified as light signalling mutants, also exhibit defective
brassinosteroid biosynthesis (Ecker, 1997). Together with
the recent identification of the brassinosteroid insensitive
mutant bri1 (Li & Chory, 1997), the demonstration that
BRI1 is a brassinosteroid receptor (He et al., 2000), the
cloning of BAS1 (Neff et al., 1999), and the discovery that
light and brassinosteroids are integrated via a dark-induced
small G protein (Kang et al., 2001), has kept the enthusiasm
alive.
The pleiotropic mutant det2 shows a number of light
responsive traits in darkness including a short hypocotyl,
anthocyanin accumulation, expanded cotyledons and primary leaf buds (Li et al., 1996). Moreover, light responsive
genes show a 10–20-fold derepression. In contrast, det2 is
smaller and greener than wild type in the light with reduced
cell size, apical dominance and altered circadian rhythm
responses. The disrupted gene in det2 encodes a protein with
high similarity to mammalian steroid 5α-reductases which
catalyse the conversion of testosterone to dihydrotestosterone,
a key step in steroid biosynthesis. In plants, one of the early
reductive steps in brassinosteroid biosynthesis involves the
reduction of campesterol to campestanol, similar to the reaction catalysed by the mammalian steroid 5α-reductases. To
test the role of DET2 in brassinolide biosynthesis Chory and
colleagues (Li et al., 1996) demonstrated that the short
hypocotyl phenotype of det2 in darkness can be rescued by
10−6 M brassinolide. As for det2, the det3 mutant shows
photomorphogenic responses in darkness and exhibits reduced
responsiveness towards exogenous brassinosteroids. However,
in contrast to det2, the disrupted gene in det3 encodes subunit
C of a vacuolar H ± ATPase (Schumaker et al., 1999). These
results indicate that brassinosteroids are involved in several
processes, including modulation of light-regulated genes,
induction of cell elongation, floral initiation, and in leaf development, which in turn may be regulated by light or by other
hormones.
Another example illustrating the interaction between light
and brassinosteroid signalling is the cpd mutant, which displays deetiolation and derepression of light-regulated genes in
darkness. CPD has been cloned and encodes a cytochrome
P450 with good sequence homology to steroid hydroxylases.
As with det2, and the cpd mutant phenotype can be fully
reversed by feeding C23-hydroxylated brassinolide precursors
(Szekeres et al., 1996). Similarly, the Arabidopsis dim mutant
was shown to be impaired in brassinosteroid biosynthesis
(Klahre et al., 1998) resembling the det2 mutant phenotype
(Takahashi et al., 1995). However, in contrast to det2, dim1
mutant seedlings, grown in darkness, do not exhibit derepression of light-regulated genes.
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The Arabidopsis bri1 (brassinosteroid insensitive) mutant
was originally isolated based on its ability to elongate roots,
compared to wild type seedlings, on 10−7 M 24-epibrassinolide
(Clouse et al., 1996). The bri1 mutant also resembles the
det2 and cpd mutant phenotypes in light and darkness. BRI1
has been cloned and encodes a receptor-like protein which
belongs to a family of plant receptor-like transmembrane
kinases (RLK) known as leucine-rich repeat (LRR) kinases (Li
& Chory, 1997). Chory and colleagues (He et al., 2000) have
now shown that BRI1 is most likely a brassinosteroid receptor.
By fusing the extracellular leucine-rich repeat and transmembrane domain of BRI1 to the kinase domain of the rice disease
resistance receptor XA21, it was shown that transgenic plants
elicit a defence response when treated with brassinosteroids.
One interesting point to make is that, as far as we are aware,
the only ‘new’ brassinosteroid insensitive mutants isolated to
date, from numerous genetic screens, have proven to be new
bri1 alleles.
In a suppresser screen using a phyB mutant, Chory and colleagues (Neff et al., 1999) isolated what could be the closest
point of interaction between light and brassinosteroid signalling identified to date. bas1 suppresses the long hypocotyl of
a phyB mutant and has reduced levels of brassinosteroids
showing an accumulation of 26-hydroxybrassinolide during
feeding experiments. The bas1 mutant phenotype is due to
the overexpression of a cytochrome P450. The phenotype is
red-light specific, however, a partial suppression of a cryptochrome mutant is also observed. Conversely, reduced levels of
BAS1 renders seedlings hypersensitive towards brassinosteroids in a light-dependent fashion indicating that this P450
may represent a light/brassinosteroid control point.
Very recently another cytochrome P450 was shown to
play a role in coordinating light and brassinosteroid signalling. The small G protein PRA2 from pea is induced in
darkness and it has now been shown that PRA2 regulates
a cytochrome P450 (DDWF1) that in turn catalyses C-2
hydroxylation in brassinosteroid biosynthesis (Kang et al.,
2001). In fact PRA2 interacts with DDWF1 in vitro and this
interaction is strictly GTP-dependent. As for other brassinosteroid mutants, seedlings with reduced PRA2 levels show
dwarfism, which can be rescued by feeding exogenous
brassinosteroids. By contrast, overexpression of DDWF1
results in elongated hypocotyls in the light pointing towards
PRA2 acting as a mediator between light and brassinosteroids
signalling during etiolation.
The point of interaction between the light and brassinosteroid pathways lies probably within the ability of light to
modulate brassinosteroid signal transduction in target cells or
perhaps even by regulating their biosynthesis directly. This
however, remains to be conclusively shown. The situation is
however, more complicated because cytokinin addition can
cause a det2 phenotype in dark grown wild type Arabidopsis
seedlings (Chory et al., 1994) and moreover the CPD gene
is down-regulated by cytokinin, suggesting that cytokinin
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negatively controls brassinosteroid biosynthesis (Szekeres
et al., 1996).
Cytokinins Cytokinins regulate a number of developmental
processes including activation of cell division, suppression of
apical dominance and senescence, inhibition of root growth
and stimulation of de-etiolation. A major difficulty in the
elucidation of the cytokinin signalling pathway is the scarcity
of target genes that are induced specifically by the hormone.
To date, three genes, IBC6, IBC7 (Brandstatter & Kieber,
1998) and CycD3 (Riou-Khamlichi et al., 1999), appear to be
primary targets of cytokinin since their induction can occur
without new protein synthesis. Cytokinin and light can
clearly elicit similar physiological and biochemical responses
and recent progress has started to untangle the complexity of
this interaction.
In 1989 Chory and colleagues isolated the det1 mutant,
which constitutively displays many light-dependent characteriztics in darkness including leaf and chloroplast development, anthocyanin accumulation, and induction of several
light-regulated nuclear and chloroplast genes (Chory et al.,
1989). It was subsequently shown that by adding cytokinins
to wild-type plants in darkness the normal etiolation process
was disrupted resulting in a det1 phenotype including the
presence of intact chloroplasts and induction of lightregulated genes (Chory et al., 1991). These results probably
provided the first good link between cytokinin and light
signalling, suggesting that cytokinin is important during the
greening process in Arabidopsis.
More recently Sano and colleagues (Ikeda et al., 1999)
demonstrated that WPK4 is a protein kinase induced by light
and cytokinin but down-regulated by sucrose. This further
implies that sucrose overrides the effect of cytokinin in terms
of WPK4 expression similar to the antagonistic effect sucrose
has on wild-type Arabidopsis seedlings in response to far-red
light irradiation. Another example illustrating that cytokinin
can override light signals is the fact that red light-induced
loss of hypocotyl gravitropism in Arabidopsis is restored
upon addition of cytokinin (Golan et al., 1996). In addition,
cytokinin also replaces light in the inhibition of hypocotyl
elongation.
The finding that chloroplasts contain a wide range of
cytokinins and the enzymes needed for their metabolism
(Benkova et al., 1999) has also provided a link between light
and cytokinin signalling. Very recently, it has been shown that
cytokinin can rescue photomorphogenic responses in the pea
lip1 mutant of pea (Seyedi et al., 2001). By adding cytokinin
to both the lip1 mutant and wild type seedlings in darkness,
phyA levels were increased in the lip1 mutants and the high
levels of POR were reduced in both lip1 and wild type.
Taken together the results to date suggest a close connection between cytokinin and phytochrome signalling. With
the recent identification of the cytokinin receptor in
Arabidopsis (Inoue et al., 2001) and the concerted effort
in identifying more cytokinin-specific genes, additional
cytokinin–mediated interactions between light and this
hormone will undoubtedly surface.
Gibberellic acid There is a wealth of literature regarding
gibberellic acid (GA). However, the role of this plant growth
regulator in signalling and moreover its interaction with
phytochrome signal transduction, remains somewhat unclear.
Probably the best known GA signalling mutant, spy (spindly),
was isolated based on its light green leaves, increased
internode length and spindly growth phenotypes which
is reminiscent of wild type plants after repeated GA3
applications. The spy phenotype also resembles the phyB
mutant phenotype. SPY has been cloned and encodes a
TRP repeat protein with sequence homology to a serine
(threonine)-O-linked
N-acetylglucosamine
transferase
( Jacobsen et al., 1996) and SPY is most likely a repressor of
the GA response.
Conversely, the signalling mutant, gai (GA-insensitive),
shows reduced GA sensitivity (Peng et al., 1997a). GAI
encodes a protein with sequence homology to SCARECROW (SCR) (Di Laurenzio et al., 1996) at the C-terminal
region. However, the N-terminus of GAI contains a putative
nuclear localization signal, which is absent in SCR. Because
a null allele of gai confers a weak spy-like phenotype, it is
possible that GAI acts as a repressor of GA signalling. Also,
the suppression of gai, by strong alleles of spy, suggests
that SPY and GAI participate in the same pathway with SPY
acting upstream of GAI.
Silverstone et al. (1998) isolated a recessive suppressor of
the Arabidopsis ga 1–3 mutant that can restrain the effects of
GA deficiency. The disrupted gene is named RGA (repressor
of ga 1–3) and the RGA locus encodes a member of the GRAS
(VHIID) protein family, which includes SCR and GAI. RGA
is also shown to localize to the nucleus. Interestingly, the presence of leucine heptad repeats in these two proteins and the
sequence homology between them does suggest that they
may interact to form a regulatory complex to repress GA
signalling. In addition, PAT1, which is a member of the
GRAS (VHIID) protein family involved in phyA signalling,
has been isolated (Bolle et al., 2000) implying that the GRAS
protein family may have a global role in plants signal transduction. All members of the GRAS family also harbour structural motifs suggestive of protein–protein interactions, which
indicate that different pathways may intercept at the level of
common interacting partners as part of a signalling network.
Recent studies have shown more directly that phytochrome
and GA are indeed interlinked. For instance, the genes GA4
and GA4H, both encoding GA 3β-hydroxylase, are rapidly
induced in imbibed seeds within 1 h after a red light pulse
(Yamaguchi et al., 1998). Indeed, phyB promotes seed germination by increasing GA biosynthesis. Interestingly, GH4 but
not GA4H is induced in a phyB-deficient mutant, indicating
that the induction of different GA hydroxylases is mediated
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Tansley review no. 136
through different phytochromes. Similarly, double mutant
studies have shown that a functional GA signalling system is
necessary for the elongated hypocotyl phenotype observed in
phytochrome mutants; GA inhibits hypocotyl elongation by
reducing GA responsiveness (Peng & Harberd, 1997). It has
also been shown that endoreduplication levels in Arabidopsis
hypocotyls are under negative control of phytochrome
(Gendreau et al., 1998) whilst GA has a positive effect on the
endoreduplication (Gendreau et al., 1999).
GA also affects flowering, but in contrast to the phyBinduced delay in flowering, GA promotes flowering. However, phyB does of course also mediate flowering through a
GA-independent pathway (Blazquez & Weigel, 1999).
At this point in time the interaction between phytochrome
and GA signalling is somewhat hard to interpret. Nevertheless, it is clear that phytochrome and GA signalling pathways
do interact in order for plants to control such diverse developmental processes as seed germination and flowering.
Ethylene In darkness ethylene inhibits hypocotyl elongation
whilst in light ethylene promotes hypocotyl elongation
(Smalle et al., 1997). The cloning and characterization of the
Arabidopsis HOOKLESS1 (HLS1) gene has demonstrated
that ethylene promotes cell elongation in specific cells in the
apical hook region (Lehman et al., 1996). Interestingly, HLS1
is thought to control this elongation by regulating either the
transport or chemical modification of auxin (Smalle et al.,
1997). Moreover, auxin resistant mutants are ethylene
insensitive (Hobbie & Estelle, 1995). These results suggest
that there is a three-way interconnected pathway between
light, auxin and ethylene signalling.
By contrast to GA, ethylene has a positive effect on
endoreduplication events in the hypocotyl in the light and in
darkness (Gendreau et al., 1999). Although the ethylene
signal transduction pathway is probably the best understood
hormone pathway ( Johnson & Ecker, 1998) there is still
along way to go before we fully understand the integration of
ethylene and phytochrome signalling in plants.
2. Light and sugars
Sugars clearly have opposing effects on a broad range of genes.
For instance sugars stimulate the expression of genes involved
in anthocyanin biosynthesis, glycolysis, and defence responses
but at the same time inhibit the expression of genes involved
in chlorophyll biosynthesis, photosynthetic functions,
gluconeogenesis, starch degradation, and the glyoxylate cycle.
As end products of photosynthesis it is not surprising that
there is a plethora of interactions between light signalling and
sugars. In WT seedlings, sucrose antagonizes the effect of farred light with respect to inhibition of hypocotyl elongation
and cotyledon opening (Barnes et al., 1996). Also, the
addition of sucrose overrides the far-red light induced killing
effect (Barnes et al., 1996). Similarly, high levels of sucrose
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suppress the expression of photosynthetic genes during
development in the dark. A class of mutants (sun, sucroseuncoupled) have been isolated that show reduced repression
of photosynthetic genes in the presence of sucrose (Dijkwel
et al., 1997). More detailed analysis has revealed that sun7
displays a reduced response of sucrose-mediated repression
of cotyledon opening in far-red light whilst sun6 shows a
reduced response of the sucrose-mediated block of far-red
light-induced killing (Barnes et al., 1996). These results
clearly illustrate the myriad of interactions between sugar
sensing mechanisms and phytochrome signal transduction.
Recent studies have identified at least two sugar signalling
pathways: one appears to be regulated by hexokinase ( Jang
et al., 1997) whilst the other by SNF1-like protein kinases
(Nemeth et al., 1998). Arabidopsis seed germination, cotyledon expansion and greening, and the emergence of true leaves
are all arrested by 6% glucose. Sheen and colleagues have
demonstrated that the Arabidopsis hexokinases, HXK1 and 2,
play a key role in sensing this glucose signal ( Jang et al., 1997).
By overexpressing HXK1/2, plants become more sensitive
towards sugar, whereas antisense HXK1/2 plants are less sensitive compared to wild type.
The Arabidopsis AKIN10 and 11 genes can complement
the yeast snf1 deletion mutant, demonstrating that these genes
most likely encode SNF1 homologs (Bhalerao et al., 1999).
Using the yeast two hybrid system, the C-terminal domains
of AKIN10/11 can interact with the N-terminal domain of
PRL−1 and these interactions are sensitive to glucose. Koncz
and colleagues (Nemeth et al., 1998) have isolated the mutant
prl-1 (pleiotropic root locus), based on its glucose hypersensitivity and PRL-1, a WD-40 protein, most probably acts as a
negative regulator of glucose responsive genes. Sucrose phosphate synthase is a target of sugar regulation in plants and is
inactivated by phosphorylation at high sugar concentrations.
Purified AKIN10/11, were, able, to, phosphorylatea, peptide,
of, sucrose, phosphate, synthase, in vitro and this specific protein kinase activity was further inhibited by purified PRL-1.
Bhalerao et al. (1999) showed further that the kinase activity
in plants was stimulated by sucrose confirming the in vitro
finding that PRL-1 functions as a negative regulator of AKIN
10/11-kinase activity.
The cross talk between sugar sensing and phytochrome signalling can also be seen as an indirect interaction through the
action of ethylene. Glucose-mediated developmental arrest
and greening can be overcome by ethylene treatment (Zhou
et al., 1998) and this opposing effect of ethylene is not seen in
etr1–1, a receptor mutant that is insensitive to ethylene. Conversely, ctr1–1, a constitutive ethylene triple response mutant,
is not affected by glucose. These results indicate that the sugar
and ethylene signalling pathways work in opposition to
modulate the early stages of Arabidopsis seedling development,
maybe through the action of phytochrome.
In terms of positive sugar signalling components, Zhou
et al. (1998) isolated gin-1, a glucose-insensitive recessive
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mutant, which escapes the developmental arrest imposed
by 6% glucose. GIN1 is most probably a positive regulatory
element in transmitting the glucose signal for repression of
developmental processes such as seed germination, cotyledon
expansion and greening, and true leaf development. Glucose
is also known to delay flowering, which is relieved by the
gin-1 mutation, indicating that the glucose repressive effect
on flowering is mediated by GIN-1.
The interactions that take place between phytochrome
signalling and other signalling pathways are fascinating. One
way of gaining more insight into the interpathway regulation
will be to use microarray analysis to monitor global transcript
changes in response to specific pathway cues. For instance,
microarray technology has very recently shown that a number
of auxin-, GA-, and ethylene-regulated genes are repressed
after far-red irradiation (Tepperman et al., 2001). Clearly, the
interactions that take place play a very important role in coordinating the execution of key developmental processes and it
will be interesting to learn more about the multiple regulatory
aspect of this communication network.
VI. Conclusions and the future
The recent progress in phytochrome research has been
tremendous and at times it is hard to reconcile and integrate
all the incoming data into an overall picture. The view that
phytochrome signal transduction represents a linear chain
of events is now outdated and it is increasingly clear that
phytochrome signalling should be viewed as a multidimensional network. In this review we have attempted to
conceptionalise this complex network in terms of cell
biology, shedding light on recent and exciting findings with
particular emphasis on the role of the different subcellular
compartments in controlling phytochrome signalling cascades.
One emerging theme in phytochrome signalling is
undoubtedly that a wealth of signalling activities take place in
the nucleus. This is of course not entirely surprising given that
photomorphogenic responses, as for many physiological
responses, are usually underpinned by alterations in nuclear
gene expression dynamics. However, the demonstration that
phyA and phyB translocate to the nucleus in response to light
and that photoactive phytochrome can interact with nuclear
DNA-binding proteins to regulate gene expression, demonstrate that at least some branches of phytochrome signalling
are exceedingly short (Ni et al., 1998, 1999; Martinez-Garcia
et al., 2000). Evidence also shows that a number of signalling
intermediates interact directly with phytochrome, suggesting
that phytochrome may act as a nuclear ‘workhorse’, forming
protein complexes that execute appropriate signalling subpathways by physical contact to other nuclear proteins.
Maybe these complexes represent the observed subnuclear
foci or speckles? However, there are also a number of phytochrome signalling intermediates that are localized to the
nucleus that do not interact with phytochrome directly
(Hudson et al., 1990; Fairchild et al., 2000; Huq et al., 2000;
Ballesteros et al., 2001).
How is the nuclear signalling network controlled? One elegant way of controlling the signalling responses is at the level
of nuclear translocation. For instance, the inactive Pr form of
phyA and phyB are retained in the cytosol until activated by
light upon which they translocate to the nucleus (Sakamoto
& Nagatani, 1996; Kircher et al., 1999a; Yamaguchi et al.,
1999; Gil et al., 2000; Kim et al., 2000). Similarly, COP1 has
been shown to be cytosolic in the light but to translocate to the
nucleus in darkness (von Arnim & Deng, 1996; Osterlund
& Deng, 1998; Stacey et al., 1999). How are constitutively
nuclear proteins controlled? One mechanism clearly involves
the 26S proteasome whereby signalling components are
specifically targeted for degradation providing yet another
elegant way of controlling the signal flux (Wei et al., 1994;
Chamovitz et al., 1996; Wei & Deng, 1998; Wei et al., 1998;
Dieterle et al., 2001). In addition, the 26S proteasome may
also be involved in phyA degradation itself.
Although the nucleus seems to be the centre of attention,
the cytoplasm also plays an important role in phytochrome
signal transduction. The light-dependent nuclear translocation of phyA, phyB and COP1 clearly depends on the cytosol
for accurate reversible retention mechanisms. Likewise,
several phytochrome signalling intermediates are cytosolic
demonstrating that not all signalling events take place exclusively in the nucleus (Bolle et al., 2000; Hsieh et al., 2000;
Guo et al., 2001). In addition, the plethora of interactions
between phytochrome signalling and other signalling
pathways require both nuclear and cytoplasmic intersections
(Møller & Chua, 1999).
The chloroplast has often been on the sideline in phytochrome research, which is somewhat puzzling since active
holophytochrome depends on phytochromobibilin, generated in the chloroplast. However, it is becoming increasingly
clear that the chloroplast plays an important role, not only in
regulating nuclear gene expression via retrograde signals, but
also in controlling photomorphogenic responses (Mochizuki
et al., 2001; Møller et al., 2001).
Although our knowledge of phytochrome research is
expanding rapidly there are still numerous questions that
remain to be answered. Why do only some phytochromes
show light-dependent nuclear translocation? Why do some
nuclear components form speckles whilst others do not and
why are the speckles different between various classes of proteins? Are most bona fide phytochrome signalling cascades
short and nuclear localized and is the cytoplasm merely a
peripheral support component? How are retrograde signals
transduced from the chloroplast to the nucleus and what is the
precise role of chloroplasts in terms of photomorphogenic
responses? These are only some of the interesting questions
that beg an answer.
It is perfectly obvious that each cellular organelle has its
own specific role in terms of phytochrome signal transduction.
www.newphytologist.com © New Phytologist (2002) 154: 553 – 590
Tansley review no. 136
It is also clear that these multiple organelles are interconnected in order to execute the appropriate molecular and
physiological responses. Moreover, the extensive subcellular
network of interactions and intersections between light
signalling and other signalling pathways is very impressive.
How the numerous signals and pathways are coordinated
between cellular organelles and how this network is finetuned in response to changes in the light environment remain
exciting future challenges. One thing is however, clear: the
future is light!
Acknowledgements
We would like to thank Nam-Hai Chua and Stefan Kircher
for sharing results prior to publication.
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