The Plant Journal (2007) 50, 429–438
doi: 10.1111/j.1365-313X.2007.03059.x
Phytochrome coordinates Arabidopsis shoot and root
development
Frances J. Salisbury1, Anthony Hall2, Claire S. Grierson3 and Karen J. Halliday1,*
Institute of Molecular Plant Sciences, Edinburgh University, Kings Buildings, Mayfield Road, Edinburgh, EH9 3JR, UK,
2
School of Biological Sciences, University of Liverpool, Bioscience Building, Crown Street, Liverpool, L69 7ZB, UK, and
3
School of Biological Sciences, Bristol University, University Rd, Bristol, BS8 1UG, UK
1
Received 8 December 2006; revised 12 December 2006; accepted 2 January 2007.
*For correspondence (fax þ44(0)131 650 6556; e-mail [email protected]).
Summary
The phytochrome family of photoreceptors are potent regulators of plant development, affecting a broad range
of responses throughout the plant life cycle, including hypocotyl elongation, leaf expansion and apical
dominance. The plant hormone auxin has previously been linked to these phytochrome-mediated responses;
however, these studies have not identified the molecular mechanisms that underpin such extensive
phytochrome and auxin cross-talk. In this paper, we show that phytochrome regulates the emergence of
lateral roots, at least partly by manipulating auxin distribution within the seedling. Thus, shoot-localized
phytochrome is able to act over long distances, through manipulation of auxin, to regulate root development.
This work reveals an important role for phytochrome as a coordinator of shoot and root development, and
provides insights into how phytochrome is able to exert such a powerful effect on growth and development.
This new link between phytochrome and auxin may go some way to explain the extensive overlap in responses
mediated by these two developmental regulators.
Keywords: phytochrome, auxin, signalling, shoot, root, Arabidopsis.
Introduction
In plants, sophisticated signalling pathways have evolved to
interconnect sensory input and developmental pathways.
These molecular channels coordinate development in the
natural environment where external parameters are in constant flux. Communication between the shoot and the root is
particularly important as these signals influence the relative
growth and development of aerial and underground structures. Key triggers for this inter-organ signalling are nutrient
and water availability, stress and temperature. However,
recent findings have demonstrated that light influences the
developmental course of the root, providing the intriguing
possibility that it acts via a long-distance signal.
In Arabidopsis, the light environment is monitored by at
least three major groups of photoreceptors, comprising red/
far-red (R/FR) light-absorbing phytochromes, UVA/blue
light-absorbing cryptochromes, and phototropins (Chen
et al., 2004). The phytochromes are well known for triggering seedling de-etiolation, but are also influential throughout the plant life cycle, controlling vegetative architecture,
apical dominance and the timing of reproductive developª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd
ment. In Arabidopsis, the phytochromes are encoded by a
small gene family, of which there are five members (PHYA–
PHYE) (Clack et al., 1994; Sharrock and Quail, 1989). The
levels and timing of expression for individual phytochromes
are subject to differential control by light and the circadian
oscillator (Hall et al., 2001; Sharrock and Clack, 2002; Toth
et al., 2001). Thus, the active pool of phytochrome is highly
dynamic and acutely responsive to the changing light
environment. Unique amongst light receptors, phytochromes exists as two photoconvertible isomers, Pr, which
absorbs maximally in the red regions, and Pfr, which
absorbs maximally in the far-red regions of the electromagnetic spectrum (Nagatani, 2004). Absorption of red light
promotes Pr conversion to Pfr, the ‘active’ form, whilst farred light reverses this process, converting Pfr back to the
inactive Pr form of the molecule. This property of phytochrome has enabled experimental manipulation of active
phytochrome levels (Franklin and Whitelam, 2004; Whitelam
et al., 1998). By providing seedlings grown under white light
with varying amounts of supplementary far-red light, or
429
430 Frances J. Salisbury et al.
end-of-day far-red light, the total seedling Pfr content can be
adjusted.
Recent studies have revealed that activation of phytochrome is accompanied by changes in the cellular location
of the molecule (Nagatani, 2004). Upon activation by light,
the phytochrome molecule undergoes a conformational
change that exposes nuclear localization signals in the PAS
domain and facilitates its nuclear translocation (Chen et al.,
2005). This is thought to be important for phytochrome
activity; indeed, red light-induced nuclear localization of
phyB–GFP was shown to be reversible by far-red light
(Kircher et al., 1999). Several studies have revealed that, in
the nucleus, phytochrome molecules aggregate in subnuclear foci (speckles), whilst speckling intensity has been
shown to correlate with the severity of the response (Chen
et al., 2003; Kircher et al., 2002). The precise function of the
sub-nuclear speckling is not yet fully understood, although it
has been proposed as the site where phytochrome regulates
downstream signalling events such as transcription. Several
lines of evidence support this view (Ang et al., 1998; Bauer
et al., 2004; Hardtke et al., 2000; Mas et al., 2000). Recent
work has demonstrated that speckle formation is not essential for all phyB responses, as biological activity has been
demonstrated for N-terminal phyB dimers that localize to the
nucleus but do not form speckles, and for diffuse phyB–GFP
nuclear staining that occurs at low light fluence rates (Chen
et al., 2003; Matsushita et al., 2003).
Whilst the role of phytochromes in the shoot has been
extensively studied, little attention has been directed to the
role of these photoreceptors in root development. However,
there is evidence for phytochrome activity within the
Arabidopsis root system. Roles have been identified for
phytochromes in the control of phototropism and gravitropism in roots (Correll et al., 2003; Kiss et al., 2003; Ruppel
et al., 2001). However, phytochrome action in roots does not
appear to be confined to the tropic responses. Work by Reed
et al. (1993) demonstrated a role for phyB in the control of
root hair elongation. More recently, phytochromes A, B and
D have been shown to control red light-mediated elongation
of the primary root (Correll and Kiss, 2005). The hy5 mutant,
known to be defective in phytochrome signalling, also has a
pleiotropic root phenotype, including altered lateral root
production (Cluis et al., 2004; Oyama et al., 1997). HY5 has
been shown to control these aspects of root growth by
altering signalling through the cytokinin and auxin pathways
(Cluis et al., 2004; Sibout et al., 2006). Thus, HY5 has been
proposed as a signal integration point in the light and
hormone signalling networks.
It is well established that auxin exerts a major influence on
root growth and development. Several studies have shown
that auxin is essential for lateral root production. aux1 auxin
efflux carrier mutants fail to develop auxin gradients, and
exhibit deficiencies in lateral root primordia production
(Marchant et al., 2002). Furthermore, mutants that are
defective in auxin transport, such as tir1 and tir3, produce
fewer lateral roots (Ruegger et al., 1997). Recent work has
demonstrated that seedling lateral root development is
dependent on a shoot-derived auxin pulse (Bhalerao et al.,
2002). In the young seedling, this auxin pulse appears to
promote lateral root growth and augment root auxin
production, which occurs predominantly in the primary
and lateral root tips (Ljung et al., 2005).
In this paper, we identify an important new role for
phytochrome in the coordination of shoot and root development. We present evidence for new phytochrome–auxin links
that contribute to inter-organ signalling. Our findings also
provide additional insights into how phytochrome is able to
exert such an extensive influence on plant development.
Results
Phytochromes are expressed in roots, and form nuclear
speckles in response to light
Previous studies have shown that phytochrome is expressed
in roots (e.g. Toth et al., 2001); however, the spatial expression pattern of root phytochrome has not been examined in
detail. To do this, we examined expression of PHYA-, PHYB-,
PHYC-, PHYD- or PHYE-promoter::LUC in seedlings grown
under a 16 h photoperiod for 10 days to allow lateral root
development. Under these conditions PHYC : : LUC was
poorly expressed in roots PHYA, PHYB, PHYD and PHYE::LUC activity was observed throughout the root, and, with the
exception of PHYB::LUC, higher bioluminescence was observed at the root tips of both primary and lateral roots
(Figure 1a). Interestingly, the root bioluminescence of PHYA
::LUC, PHYD::LUC and PHYE::LUC was relatively high and
PHYD::LUC appeared to be highly expressed throughout the
elongation zone of the primary root. The inset in Figure 1(a)
shows that similar levels of PHYD::LUC expression are
maintained after root tip excision, which establishes that this
expression pattern was not simply an artefact of light piping.
The apparent high levels of PHY::LUC expression in root tips
observed by us and other workers may reflect the increased
density of cells rather than a relatively high cellular expression in this area (Toth et al., 2001). Our experiments do not
distinguish between these two possibilities.
In Arabidopsis and tobacco shoots, the dynamic properties of phytochrome within the cell provide an activity
signature for the molecule. As phytochrome is expressed in
roots, we wished to determine whether individual phytochromes exhibited the same light-responsive cellular
dynamics in root and shoot cells. We analysed seedlings
expressing fusions of GFP with phyA–E under the control of
the CaMV35S promoter. In agreement with previous studies,
we observed cytosolic phyB–GFP expression in dark-grown
root epidermal cells (Figure 1b) (Yamaguchi et al., 1999).
Furthermore, as for shoot epidermal cells, we observed
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 50, 429–438
Phytochrome coordinates development 431
Figure 1. PHY::LUC expression and phy–GFP
cellular localization in roots.
(a) PHYA–E::LUC spatial expression patterns are
shown for 10-day-old seedlings. The inset shows
PHYD::LUC expression following excision of the
root tip.
(b) Cellular location of phyA–E–GFP in roots of 7day-old seedlings. Upper row: phyB–GFP localization patterns in cell epidermal cells of seedlings grown in darkness and 0.1 or
1 lmol m)2 sec)1 of red light, corresponding to
0, 1 and 16% Pfr, respectively, and sub-nuclear
speckling in a root hair cell (10 lmol m)2 sec)1 of
red light). Lower row: sub-nuclear speckling of
phyA–GFP, phyC–GFP, phyD–GFP and phyE–GFP
in root epidermal cells in response to far-red light
(phyA) or red light (phyC–E). Dotted lines outline
the nucleus.
(a)
(b)
PHYA::LUC
PHYB::LUC
PHYC::LUC
PHYD::LUC
0.1
0
PHYE::LUC
1.0
phyB-GFP
phyA-GFP
diffuse nuclear staining in seedlings grown in red light at
0.1 lmol m)2 sec)1 ( approximately 1% Pfr) and nuclear
speckling in seedlings grown at 1.0 lmol m)2 sec)1
(approximately 16% Pfr) (Chen et al., 2003). We noted the
formation of phyB–GFP sub-nuclear foci in a variety of cell
types, for example root hairs (shown in Figure 1b), epidermal and underlying cortical cells. We also observed far-red
light-mediated nuclear localization and speckling of phyA–
GFP, and red light regulation of these events for phyC–GFP,
phyD–GFP and phyE–GFP (Figure 1b). Collectively, our data
demonstrate that the phytochromes are expressed and
exhibit similar light-regulated intracellular dynamics in roots
as they do in shoots.
Phytochrome controls the rate of lateral root production
Whilst the role of phytochrome in the shoot has been well
characterized, its role in root development has received little
attention. Our analysis of seedlings null for individual
phytochromes demonstrates that phytochromes participate
in the regulation of lateral root production (Figure 2a). The
phyD mutant has slightly enhanced lateral root production,
and phyA, phyB and phyE mutants show reduced lateral root
production. Mutants deficient in phyA in addition to phyB
also exhibit a reduction in the rate of lateral root outgrowth
(Supplementary Figure S1). This indicates that phyA, phyB
and phyE promote lateral root production, and, as for the
shoot, phyB has the most prominent role. phyD appears to
antagonize this action. We and others have also shown that
reduced phytochrome activity impairs root gravitropism in
primary and lateral roots (data not shown; Correll and Kiss,
2005). Thus, in addition to their well documented roles in
shoot development, the phytochromes appear to play a
central role in controlling root development.
phyC-GFP
phyD-GFP
phyE-GFP
The phyB root phenotype is retained in soil-grown plants
Recent work has shown that, when directly irradiated with
red light, root elongation is inhibited (Correll and Kiss, 2005).
This suggests that phytochromes can act locally within the
root system to control its growth. It also raised the possibility that our observed root phenotypes were an artefact of
our experimental conditions where roots grown on agar
plates were exposed to light (Figure 2a). To test this, we
grew our phyB seedlings in a more natural situation. When
grown in a compost/sand mix, phyB seedlings produced far
fewer lateral roots than the Ler wild-type (Figure 2b,c). The
less bulky phyB roots had reduced total FW when compared
to wild-type roots (data not shown). Furthermore, when
expressed as percentage of shoot FW, root mass was proportionally lower in phyB seedlings compared with wildtype seedlings (Figure 2c). These observations demonstrate
that compost-grown phyB roots are phenotypically similar
to those of phyB seedlings grown on agar plates.
Root-localized phyB is not activated by axially conducted
light
Previous work has demonstrated that light is conducted
axially from the shoot to the root via the vascular tissue, with
wavelengths in the 710–940 nm range being transmitted
with the greatest efficiency (Sun et al., 2005). These findings
suggest that, in some species, light can penetrate a proportion of the root system to trigger phytochrome action.
We have shown that light induces nuclear localization and
speckling of root phy–GFP fusion proteins (Figure 1b). Furthermore, we have demonstrated that root-localized phyB–
GFP has comparable fluence rate-induced localization
properties to shoot-localized phyB (Chen et al., 2003). Thus,
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 50, 429–438
432 Frances J. Salisbury et al.
Lateral root number
(a) 25
(b)
phyD
Ler
20
phyA
phyE
15
phyB
10
5
Ler
phyB
0
7
(c)
8
9
10
Days after induction
11
Figure 2. Phytochrome regulation of root development in light- and dark-grown roots.
(a) Lateral emergence in phytochrome null
mutant seedlings. Lateral root number in Ler
(WT), phyA, phyB, phyD and phyE seedlings
counted between 7 and 11 days after germination. Data represent mean values from at least 30
seedlings. SE bars are shown.
(b,c) Root phenotypes (b) and mass (c) of 5-weekold, soil-grown Arabidopsis plants. Pie charts
show the root (white) expressed as a proportion
of the total seedling mass.
(d) Localization of phyB–GFP in soil-grown plants
with sub-nuclear speckling in guard cells of the
shoot, diffuse staining in the upper root, and
absence of staining in cells at the root tip.
(d)
Proportion of root
Shoot
Ler
phyB
Upper root
Root tip
light activation of root-localized phytochrome by conductance was a formal possibility. To establish whether this
occurred in Arabidopsis, we examined the localization of
phyB–GFP in the roots of soil-grown seedlings. We were
able to detect nuclear-localized phyB–GFP which aggregated
in sub-nuclear foci in hypocotyl and leaf epidermal cells.
Figure 2(d) illustrates this in leaf guard cells. In contrast, we
consistently observed diffuse nuclear phyB–GFP just below
the root–shoot junction, but never sub-nuclear foci (Figure 2d). We were unable to detect nuclear-localized phyB in
root cells that were more than 1 cm below the soil line.
These data suggest that, for the bulk of the root system,
axially conducted light is unable to trigger nuclear localization of phyB. This suggests that either phyB can act
independently of light to regulate root physiology or that
shoot-localized phyB exerts its control on the root through a
long-distance signal.
Phytochrome coordinates root and shoot growth by
controlling auxin transport and response
Recent work has provided evidence that seedling lateral root
emergence is triggered by a pulse of auxin derived from the
shoot (Bhalerao et al., 2002). We wished to establish whether phytochromes regulate lateral root growth by manipu-
lating this auxin pulse. To explore this possibility, we grew
seedlings expressing the auxin-responsive DR5::GUS construct in either high R:FR ratio or low R:FR ratio light, which
severely depletes active phytochrome (Pfr) levels. We found
that seedlings subjected to low R:FR ratio light had higher
levels of DR5::GUS in shoots, particularly at the base of the
hypocotyl, and lower levels in roots (Figure 3b,c). As these
seedlings had more elongated hypocotyls and produced
fewer lateral roots (Figure 3a,b), our data suggest that the
phytochromes may control hypocotyl elongation and lateral
root production either by altering seedling auxin distribution
and/or the response of auxin-regulated genes. We also
examined the influence of phyB on this response by assessing phyB (phyB-5) mutants expressing DR5::GUS. These
seedlings exhibited alterations in DR5::GUS expression that
were comparable to those of wild-type seedlings treated
with low R:FR ratio light, suggesting a contributing role for
phyB (Figures 3 and 4).
We next conducted auxin feeder experiments in which
NAA (freely diffusible) or IAA (which requires auxin transport) was applied in a band of agar to wild-type or phyB
shoots (Figure 4). We found that 0.1 lM NAA induced
expression of DR5::GUS in both wild-type and phyB roots.
The same concentration of IAA induced root DR5::GUS
expression in wild-type roots, but was quite ineffective in the
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 50, 429–438
Phytochrome coordinates development 433
(a)
High R:FR ratio
20
Low R:FR ratio
Lateral root number
Ler
15
Ler
Col
10
Col
5
0
7
(b)
9
10
8
Days after induction
High R:FR
11
Low R:FR
phyB null mutant, which exhibited only mild GUS staining at
the application site and at the root tip. It is possible that the
moderate levels of DR5::GUS expression at the application
site may reflect reduced IAA uptake in phyB. When combined with the data from Figure 3, these data support the
notion that phyB, and possibly other phytochromes, regulates auxin transport into the root system and/or reduces the
response of auxin-inducible genes.
To explore the possibility that phytochrome controls
auxin distribution, we conducted auxin transport assays
using tritiated IAA. In our experiments, whilst we always
observed small reductions in auxin transport in phyB null
seedlings when compared to the wild-type, the data were
not statistically significant (Figure 5). However, in phyA phyB mutants, we consistently observed significant reductions in auxin transport when compared with wild-type
seedlings (Figure 5). These data suggest that the combined
action of phyA and phyB controls the distribution of auxin
between the shoot and root.
IAA1 and IAA3 transcript levels are altered in phyA phyB
shoots and roots
MUG fluorescence
(c) 200
150
100
**
50
**
0
Shoot
Root
High R:FR
Shoot
Root
Low R:FR
Figure 3. Lateral root production and DR5::GUS expression patterns in
seedlings grown under low and high R:FR ratio light.
(a) Lateral root emergence in Ler (WT) and Col (WT) seedlings expressing
DR5::GUS and grown under high or low R:FR ratio light.
(b) DR5::GUS expression in seedlings grown under either high or low R:FR
ratio light.
(c) DR5::GUS expression (expressed in arbitrary units), quantified by
fluorometric assay, in shoots and roots of 5-day-old seedlings.
In all experiments, seedlings were grown under 16 h photoperiods at
18C. SE bars are shown. The difference between DR5::GUS levels in
roots treated with high or low R:FR ratio light is significant (**P < 0.022,
t = 2.42).
To further test the hypothesis that phytochrome moderates
auxin transport, we measured the mRNA levels of genes
known to be involved in auxin response. We focused on
IAA1 and IAA3/SHY2 as these genes have been previously
shown to be phytochrome-regulated (Devlin et al., 2003;
Tian et al., 2003). In these experiments, we compared transcript levels in shoot and root tissue. Figure 6 illustrates that,
when compared to wild-type, IAA1 and IAA3 are expressed
at higher levels in phyA phyB seedling shoots. In wild-type
root tissue, although we detected reduced expression in
roots, IAA1 and IAA3 transcript levels were moderately
lower in phyA phyB roots. These data demonstrate that loss
of phyB and phyA action enhances shoot IAA1 and IAA3
mRNA and leads to a modest reduction of IAA1 and IAA3
transcripts in the root. When combined with our auxin feeder data, these results support a role for phyA and phyB in
reducing auxin transport or availability in hypocotyl tissue,
and enhancing the transport of shoot-derived auxin into the
root.
Recent microarray experiments have suggested that
expression of the auxin efflux genes PIN3 and PIN7 is
controlled by phytochrome (Devlin et al., 2003). We have
shown that PIN3 and PIN7 mRNA levels are elevated in phyB
versus wild-type seedlings (Supplementary Figure S2). This
provides the possibility that phytochrome controls IAA1/
IAA3 transcription, at least in part, by regulating PIN3/PIN7
levels. Interestingly, the levels of PIN1 mRNA are similar in
phyB and wild-type seedlings (data not shown), indicating
that PIN1, a major regulator of shoot-to-root auxin transport
(see Friml and Palme, 2002), is not subject to control by phyB
at the transcriptional level.
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 50, 429–438
434 Frances J. Salisbury et al.
Control
NAA
0.1 µM
0.1 µM
1 µM
IAA
Figure 4. Auxin-induced DR5::GUS expression
in WT and phyB seedlings.
DR5::GUS expression in Col (WT) and phyB
seedlings following 24 h treatment with 0, 0.1
or 1 lM IAA or NAA applied in a strip of agar just
above the shoot–root junction. Representative
seedlings are shown (n = 30). The inset shows
DR5::GUS expression at the root tip of untreated
wild-type and phyB seedlings.
1 µM
Col
phyB
Col
phyB
Col
phyB
Col
phyB
Col
**
5
Radioactivity (Bq)
Col
phyB
and auxin signalling are connected, yet, until now, no firm
molecular link has been established. In this paper, we have
shown that phytochrome controls growth and development,
at least in part, by regulating auxin transport. This appears to
enable phytochrome to coordinate shoot and root development. It is presumably through this mechanism that shoot
and root development can be reciprocally adjusted to take
full advantage of the available light resources.
4
**
3
2
1
0
Ler
phyA
phyB
phyB
Figure 5. Auxin transport assay in WT, phyB and phyA phyB seedlings.
Auxin transport was measured in Ler (WT), phyB and phyA phyB seedlings.
0.1 lM 3H-IAA was applied in a strip of agar just above the shoot–root junction.
After 24 h, radioactivity (Bq) was assessed in 5 mm root tip sections. SE bars
are shown. The difference between WT and phyA phyB is statistically
significant (**P > 0.0045, t = 2.35). Similar results were also obtained 12 h
after 3H-IAA application (data not shown).
Relative expression
phyB
6
IAA1
IAA3
Ler
phyAphyB
*
*
4
2
0
*
*
0
Root
Shoot
Root
Shoot
Root
Shoot
Root
Shoot
Figure 6. Auxin-responsive gene expression in phyA phyB mutant seedling
shoots and roots.
Quantification of IAA1 and IAA3 mRNA levels in Ler (WT) and phyA phyB
shoots and roots. Relative transcript levels were determined by real-time
quantitative PCR and normalized using AtACT7 mRNA as a reference. SE bars
are shown. The differences between IAA levels in WT and phyA phyB roots
was significant for IAA1 (*P > 0.027, t = 2.13) and IAA3 (*P > 0.034, t = 2.13).
Discussion
Phytochrome and auxin are potent regulators of plant
development. Light regulates a broad range of responses
which include hypocotyl elongation, leaf expansion and
phototropism, responses that are also controlled by auxin. It
has been known for a number of years that phytochrome
phyB has a prominent role in controlling the rate of lateral
root production
In early seedling development, auxin synthesized in the first
leaves is transported to the root where it promotes the outgrowth of lateral roots (Bhalerao et al., 2002). Perturbation of
auxin transport or signalling can have dramatic effects on
lateral root production (Marchant et al., 2002; Santelia et al.,
2005). We have shown that phytochromes are important
components of this regulatory process, as loss of phyA and/
or phyB, of phyE or reduction of the Pfr:Pr ratio by low R:FR
ratio light, reduces the rate at which lateral roots emerge in
young seedlings. Of the monogenic phy null mutants, this
phenotype is most severe in phyB, indicating a prominent
role for phyB in this response.
Shoot-localized phytochrome controls root physiology
Previous work has shown that roots exposed to red light
exhibit increased inhibition of primary root elongation
(Correll and Kiss, 2005). It was therefore possible that the
root phenotypes of our agar plate-grown seedlings were
caused, at least partly, by local phytochrome action within
the root. However, we demonstrated that the phyB lateral
root phenotype was retained in compost-grown seedlings.
These findings support the notion that this response was
controlled from the shoot.
Recent work has demonstrated that, in some plant
species, light can penetrate the root through vascular
conductance. We have shown that root-localized phytochrome exhibits comparable light-regulated localization
characteristics to shoot phytochrome. However, we only
ever observed diffuse nuclear localization, and never
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 50, 429–438
Phytochrome coordinates development 435
speckling, of phyB–GFP in cells located just below the root–
shoot junction. We were unable to detect nuclear phyB–GFP
in root cells more than 1 cm below the soil line. This
illustrates that, under our light fluence rates (100 lmol m)2 sec)1), phyB activation by axially conducted light is unlikely
to play a significant role in shaping root development.
Combined, our data provide strong support for a role of phyB
and other phytochromes in modulating root growth in
response to light signals perceived in the shoot. Our experiments do not eliminate the possibility that cytosolic-localized
Pr plays a role in controlling this response. However, in
experiments where seedlings grown on sucrose plates were
transferred to darkness on day 3, prior to the early shoot–root
auxin pulse, lateral root production is arrested (data not
shown). This reinforces the importance of the shoot-derived
auxin pulse for the lateral root phenotype. It does not
preclude the action of Pr in roots following delivery of the
auxin to the root, but does suggest that primary control of
this response is achieved by manipulating the auxin pulse.
Phytochrome adjusts the distribution of auxin in the shoot
and root
Several reports have shown that phytochrome regulates a
subset of auxin-responsive genes, including IAA1, IAA3/
SHY2 (Devlin et al., 2003; Tian et al., 2002) and components
of the complex auxin transport machinery including MDR1/
PGP11, PGP1, PIN3 and PIN7 (Devlin et al., 2003; Lin and
Wang, 2005; Sidler et al., 1998). We have shown that subjecting seedlings to low R:FR ratio light, which reduces active
phytochrome (Pfr) levels, induces a change in the pattern of
DR5::GUS expression. These seedlings have enhanced
DR5::GUS expression in the lower third of the hypocotyl and
reduced expression in the roots. Furthermore, phyA phyB
null seedlings have higher levels of shoot IAA1 and IAA3
mRNA, whilst phyA phyB roots have slightly reduced levels.
DR5::GUS has been shown to be regulated by brassinolide in
addition to auxin, so this may reflect altered regulation of one
or both these pathways in seedlings exposed to low R:FR
ratio light (see Halliday, 2004). However, auxin transport in
hypocotyls has been shown to be phytochrome-dependent,
and mutants that are null for phyA and phyB have reduced
temperature-mediated hypocotyl extension (Jensen et al.,
1998; Mazzella et al., 2000). Furthermore, shoot-derived
auxin has been shown to be important for lateral root production, a response that is perturbed by treatment with low
R:FR ratio light (Bhalerao et al., 2002; Marchant et al., 2002).
Thus, our findings most likely reflect a role for phytochrome
in regulating auxin distribution. IAA feeder experiments
provide further support for this proposition. Our auxin
transport assays using tritiated IAA demonstrated that
depletion of both phyA and phyB attenuates shoot–root auxin
transport, whilst depletion of phyB alone does not. Interestingly, the lateral root phenotypes of phyB and phyA phyB are
similar, thus it is possible that our tritiated IAA transport assays were not sufficiently sensitive to detect subtle differences in auxin status. Indeed, auxin feeder experiments using
shoot-applied IAA were less effective at triggering DR5::GUS
expression in phyB roots than in wild-type roots. It is therefore possible that DR5::GUS, which comprises eight tandem
copies of the 11 bp natural AuxRE from the GH3 promoter,
may be a more sensitive indicator of auxin distribution (Ulmasov et al., 1997). Alternatively, phyB may also control lateral root production by an alternative mechanism.
Collectively, our results suggest that phytochrome effects at
least a subset of its responses by manipulating seedling
auxin distribution. This phytochrome–auxin link may account for a proportion of the extensive overlap between
phytochrome- and auxin-regulated responses.
What is the role of root-localized phytochrome?
Recent work has provided insights into roles for rootlocalized phytochrome in regulating phototropic responses
and primary root elongation growth (Correll and Kiss, 2005;
Correll et al., 2003). In support of these findings, we have
shown that phytochromes A, D and E are highly expressed in
primary and lateral root tips, and that phyD is expressed
throughout the elongation zone of the primary root.
Furthermore, we have demonstrated that root-localized
phy–GFP shows light-regulated cellular localization characteristics similar to those described for shoot phytochromes.
Indeed, the role of phytochrome in the root appears to be
similar to that in the shoot where it also regulates cell
elongation and phototropism. However, in contrast to the
shoot, phytochrome acts as a phototropic receptor in roots.
Given the role of shoot phytochrome in shaping root growth
via a long distance signal, it will be interesting to establish
whether root-localized phytochrome signals to the shoot.
Possible mechanisms of phytochrome–auxin cross-talk
This paper has demonstrated that the phytochromes act
collectively to control lateral root outgrowth. The phytochromes appear to regulate this response at least partly by
moderating the shoot–root auxin pulse in early seedling
development. These findings provide a means to coordinate
shoot and root development in response to the external light
environment. Interestingly, a recent study provides support
for a role of the cryptochromes in regulating aspects of root
growth by modulating auxin transport (Canamero et al.,
2006). This may therefore be a common control mechanism
for both the phytochromes and cryptochromes.
The extensive overlap between phytochrome- and auxininduced responses suggests their tight linkage in the
signalling network. At present, we do not know precisely
how the phytochromes may be acting to control auxin
transport; however, our work and recent studies provide
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 50, 429–438
436 Frances J. Salisbury et al.
some leads. In line with previous microarray studies, we
have shown that transcript levels of the auxin efflux effectors, PIN3 and PIN7, are phytochrome-regulated (Devlin
et al., 2003). Aspects of the pin3 mutant phenotype are
reported to be light-specific (Friml et al., 2002); furthermore,
we have shown that pin3 mutants have accelerated lateral
root production (Supplementary Figure S3). Therefore,
phytochrome may control auxin transport by altering the
levels and/or the cellular location of PIN proteins. Recent
work has implicated the p-glycoproteins PGP1 and MDR1/
PGP11 in auxin transport; indeed, PGP1 has been shown to
directly catalyse the active efflux of IAA (Geisler et al., 2005).
PGP1 and MDR1/PGP11 have previously been shown to be
regulated by phytochrome, and mdr1 and pgp1 loss-offunction mutants have light-specific hypocotyl phenotypes
(Lin and Wang, 2005; Sidler et al., 1998). Furthermore, MDR1
has been shown to operate in a phyA pathway in seedlings
grown under far-red light (Lin and Wang, 2005). These
observations suggest that MDR1/PGP1 activity is tightly
coupled to light signalling, and they are therefore good
candidates for phytochrome action in the manipulation of
shoot–root auxin distribution.
It is unlikely that phytochrome control of auxin signalling
is confined to manipulation of auxin gradients. We and
others have shown that phyB null mutants have elongated
root hairs (data not shown; Reed et al., 1993). This is the
antithesis of the expected phenotype for seedlings with low
root auxin levels (Pitts et al., 1998); instead, phyB root hairs
are suggestive of enhanced auxin signalling. Interestingly,
this is a phenotypic characteristic of hy5, a known phytochrome signalling component (Oyama et al., 1997). HY5 has
been shown to regulate transcription by binding to the core
G-box sequence CCACGTG (Ang et al., 1998). This binding
site is contained within the promoters of the SLR/IAA14 /
IAA28 and AXR2/IAA7 genes that exhibit reduced transcript
levels in hy5 mutants (Cluis et al., 2004). Furthermore, HY5
has been shown to bind the promoter of AXR2 in vitro. Thus,
HY5 appears to regulate auxin signalling by targeting
negative regulators of the pathway. It is therefore possible
that phyB moderates auxin signalling through the HY5
pathway. It is also feasible that phyB regulates AUX/IAA
activity by direct interaction. Pull-down assays identified an
interaction between phyB and SHY2/IAA3, a known auxin
and phyB signalling component (Tian et al., 2003). Further
analysis is required to establish whether this is occurs in vivo.
The data in this paper provide insights into how phytochrome coordinates shoot and root development. It appears
that phytochrome achieves this, at least in part, by reciprocally regulating auxin gradients in aerial and root structures.
It has been known for a considerable time that phytochrome
controls multiple developmental processes, yet the molecular basis of this control is not known. It may be through the
manipulation of auxin that phytochrome is able to influence
such a range of responses.
Experimental procedures
Plant materials and growth conditions
All studies were carried out using the Arabidopsis thaliana Landsberg erecta (Ler) or Columbia (Col) accessions. The phytochrome
mutant alleles used in this study were phyA-201 (Nagatani et al.,
1993), phyB-1 (Reed et al., 1993), and phyB-5, phyD-1 (Aukerman
et al., 1997) and phyE-1 (Devlin et al., 1998), all in the Ler background (Neff and Chory, 1998) in the Col background. Double mutants were created by genetic crossing as described previously
(Devlin et al., 1998, 1999). We used the PHYA–E promoter::LUC lines
and the lines expressing translational fusions of GFP with phyA–
phyE, as previously described (Kircher et al., 2002; Toth et al., 2001).
The DR5::GUS line, containing a synthetic auxin-responsive promoter fused to the GUS reporter gene, in the Col genetic background, has been described previously by Ulmasov et al. (1997).
For all experiments, seeds were surface-sterilized in 20% v/v
bleach for 5 min. After three washes in distilled water, seeds were
sown on plates containing Hoaglands No. 2 basal salts medium
(Sigma-Aldrich; http://www.sigmaaldrich.com/), pH 5.7, 1% w/v
sucrose and 0.5% w/v Phytagel (Sigma-Aldrich). Seeds were stratified in complete darkness for 4 days at 4C before transfer to
specific growth conditions. Plates were positioned vertically to
allow root growth along the gel surface. For plant growth, we used
plant growth cabinets (Snijders Scientific, www.snijders-tilburg.nl)
with a 16 h photoperiod (fluence rate of 100 lmol m)2 sec)1) and a
temperature of 18 0.5C. Experiments with high and low R:FR
ratio light were performed in climate-controlled growth rooms, also
set to 16 h photoperiods at 18 0.5C, with a photon fluence rate of
70 lmol m)2 sec)1. Supplementary FR light was supplied during
the photoperiod by light-emitting diode arrays to create a low R:FR
ratio of 0.126. Light quantity and quality were measured using a
StellarNet EPP2000 spectroradiometer (Astranet Systems, www.
astranetsystems.com).
Physiological analysis
Lateral roots were viewed with a stereomicroscope (MZFLIII, Leica
Microsystems, www.leica-microsystems.com) and were counted
daily between 7 and 11 days after transfer to light, following
stratification for 4 days. Assessment of root mass in soil-grown
seedlings was performed on 5-week-old plants grown in climatecontrolled growth rooms under white light (100 lmol m)2 sec)1)
with 16 h photoperiods at 20C. Seeds were sown directly on a
50:50 compost/sand mixture, and stratified for 4 days at 4C in
darkness before transfer to growth conditions.
Reporter gene analysis
Seedlings expressing PHYA–E::promoter::LUC were sprayed with
5 mM luciferin 5 min before analysis of the LUC expression pattern
using an intensified CCD camera (Hamamatsu VIM, www.
hamamatsu.com). Images were processed using Image J
(NIHimage, www.rsb.info.nih.gov/nih-image/).
The cellular location and characteristics of 35S::phyA–E–GFP
were assessed using an Eclipse confocal microscope (Nikon,
www.nikon.co.uk). Colour was artificially added using PHOTOSHOP
8 (Adobe Systems, www.adobe.com).
For histochemical analysis of GUS activity, Arabidopsis seedlings
were incubated overnight at 37C in GUS reaction buffer (0.5 mM
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 50, 429–438
Phytochrome coordinates development 437
5-bromo-4-chloro-3-indolyl-b-D-glucoronic acid in 100 mM sodium
phosphate, pH 7). Stained seedlings were cleared with 70% ethanol
overnight before being mounted in glycerol and viewed under a
DMLB stereomicroscope (Leica Microsystems). Representative seedlings were photographed using a Coolpix 4200 digital camera (Nikon).
GUS staining was quantified by analysis of MUG fluorescence.
Seedlings were severed at the shoot–root junction. Shoot and root
tissues were incubated in GUS extraction buffer [1 mM MUG
(Sigma-Aldrich), 50 mM sodium phosphate, pH 7, 10 mM EDTA,
0.1% SDS, 0.1% Triton X-100] at 37C for 6 h, before the reaction was
stopped with 1 M sodium carbonate. MUG fluorescence was
measured on a Fluorolite 1000 fluorometer (Dynatech Laboratories,
www.dynatechlaboratories.com).
Auxin feeder experiments
To assess auxin transport, a 1% agar band containing 0, 0.1 or
1.0 lM IAA or NAA was applied just above the shoot–root junction of
intact, light-grown seedlings expressing DR5::GUS in the Col or
phyB-9 backgrounds. Seedlings were treated for 24 h before staining as described above. Comparable results were obtained in three
replicate experiments using 15–20 seedlings.
In auxin transport assays using labelled IAA, 0.1 lM 3H-IAA was
applied in an agar band as described above. After 24 h, 5 mm
sections of the root tip were excised and rinsed twice in distilled
water to remove residual activity, before being soaked in 80 ll of
80% methanol for 60–90 min. Following this, 920 ll of sterile
distilled water and 10 ml of Optiscint HighSafe scintillation fluid
(PerkinElmer, www.perkinelmer.com) were added, and the radioactivity of samples was counted using a Beckman LS6500 scintillation counter (Beckman Coulter, www.beckmancoulter.com).
Comparable results were obtained in three replicate experiments
using 8–10 seedlings.
Quantitative PCR
Plant total RNA was extracted from 7-day-old, white-light-grown
seedlings using RNeasy Plant Mini kits (Qiagen; www.qiagen.com/),
according to the manufacturer’s instructions, including a DNase I
treatment step of column-bound RNA (Qiagen). cDNA was then
synthesized using a RevertAid first-strand synthesis kit (Fermentas,
www.fermentas.com). Quantitative PCR was performed in a RotorGene 3000 (Corbett Life Science, www.corbettlifescience.com)
using SYBR Green Jump-Start Taq ready mix (Sigma-Aldrich). The
primers used were: ACT7for (5¢-CAGTGTCTGGATCGGAGGAT-3¢),
ACT7rev (5¢-TGAACAATCGATGGACCTGA-3¢), IAA1for (5¢-ACATGTTCAAGTTCACAGTA-3¢), IAA1rev (5¢-TGCCTCGACCAAAAGGTGT-3¢), IAA3for (5¢-CTGTGGGAGAGTACTTTGAG-3¢), IAA3rev (5¢CATATGAACATCTCCCATGGA-3¢), PIN3for (5¢-CGAGACCAAAGCTGTAGCTC-3¢), PIN3rev (5¢-GTTTAGACCATTCTCGGCGT-3¢), PIN7for
(5¢-TTGCTTTCAGGTGGGATGTG-3¢) and PIN7rev (5¢-ACTCACCCAAACTGAACATTGC-3¢). Experiments were performed in
triplicate on two biological replicates.
Acknowledgements
We thank Professor Ottoline Leyser (University of York, UK) for
supplying seeds containing the DR5::GUS construct, and Professors
Ferenc Nagy (Biological Research Center of the Hungarian Academy
of Sciences, Szeged, Hungary) and Andrew Millar (University of
Edinburgh, UK) for supplying seeds of the PHYA–E::LUC and
phyA–E–GFP transgenic lines.
Supplementary Material
The following supplementary material is available for this article
online:
Figure S1. Regulation of lateral root emergence in the phyA phyB
mutant
Figure S2. PIN3 and PIN7 gene expression in phyB mutant seedlings
Figure S3. Lateral root emergence in the pin3 mutant
This material is available as part of the online article from http://
www.blackwell-synergy.com
References
Ang, L.H., Chattopadhyay, S., Wei, N., Oyama, T., Okada, K.,
Batschauer, A. and Deng, X.W. (1998) Molecular interaction between COP1 and HY5 defines a regulatory switch for light control
of Arabidopsis development. Mol. Cell, 1, 213–222.
Aukerman, M.J., Hirschfeld, M., Wester, L., Weaver, M., Clack, T.,
Amasino, R.M. and Sharrock, R.A. (1997) A deletion in the PHYD
gene of the Arabidopsis Wassilewskija ecotype defines a role for
phytochrome D in red/far-red light sensing. Plant Cell, 9, 1317–
1326.
Bauer, D., Viczian, A., Kircher, S. et al. (2004) Constitutive photomorphogenesis 1 and multiple photoreceptors control degradation of phytochrome interacting factor 3, a transcription factor
required for light signalling in Arabidopsis. Plant Cell, 16, 1433–
1445.
Bhalerao, R.P., Eklof, J., Ljung, K., Marchant, A., Bennett, M. and
Sandberg, G. (2002) Shoot-derived auxin is essential for early lateral root emergence in Arabidopsis seedlings. Plant J. 29, 325–332.
Canamero, R.C., Bakrim, N., Bouly, J.P., Garay, A., Dudkin, E.E.,
Habricot, Y. and Ahmad, M. (2006) Cryptochrome photoreceptors
cry1 and cry2 antagonistically regulate primary root elongation in
Arabidopsis thaliana. Planta 224, 995–1003.
Chen, M., Schwab, R. and Chory, J. (2003) Characterization of the
requirements for localization of phytochrome B to nuclear bodies.
Proc. Natl Acad. Sci. USA, 100, 14493–14498.
Chen, M., Chory, J. and Fankhauser, C. (2004) Light signal transduction in higher plants. Annu. Rev. Genet. 38, 87–117.
Chen, M., Tao, Y., Lim, J., Shaw, A. and Chory, J. (2005) Regulation
of phytochrome B nuclear localization through light-dependent
unmasking of nuclear-localization signals. Curr. Biol. 15, 637–642.
Clack, T., Mathews, S. and Sharrock, R.A. (1994) The phytochrome
apoprotein family in Arabidopsis is encoded by five genes: the
sequences and expression of PHYD and PHYE. Plant Mol. Biol. 25,
413–427.
Cluis, C.P., Mouchel, C.F. and Hardtke, C.S. (2004) The Arabidopsis
transcription factor HY5 integrates light and hormone signalling
pathways. Plant J. 38, 332–347.
Correll, M.J. and Kiss, J.Z. (2005) The roles of phytochromes in
elongation and gravitropism of roots. Plant Cell Physiol. 46, 317–
323.
Correll, M.J., Coveney, K.M., Raines, S.V., Mullen, J.L., Hangarter,
R.P. and Kiss, J.Z. (2003) Phytochromes play a role in phototropism and gravitropism in Arabidopsis roots. Adv. Space Res. 31,
2203–2210.
Devlin, P.F., Patel, S.R. and Whitelam, G.C. (1998) Phytochrome E
influences internode elongation and flowering time in Arabidopsis. Plant Cell, 10, 1479–1487.
Devlin, P.F., Robson, P.R., Patel, S.R., Goosey, L., Sharrock, R.A. and
Whitelam, G.C. (1999) Phytochrome D acts in the shade-avoidance syndrome in Arabidopsis by controlling elongation growth
and flowering time. Plant Physiol. 119, 909–915.
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 50, 429–438
438 Frances J. Salisbury et al.
Devlin, P.F., Yanovsky, M.J. and Kay, S.A. (2003) A genomic analysis
of the shade avoidance response in Arabidopsis. Plant Physiol.
133, 1617–1629.
Franklin, K.A. and Whitelam, G.C. (2004) Light signals, phytochromes and cross-talk with other environmental cues. J. Exp.
Bot. 55, 271–276.
Friml, J. and Palme, K. (2002) Polar auxin transport – old questions
and new concepts? Plant Mol. Biol. 49, 273–284.
Friml, J., Wisniewska, J., Benkova, E., Mendgen, K. and Palme, K.
(2002) Lateral relocation of auxin efflux regulator PIN3 mediates
tropism in Arabidopsis. Nature, 415, 806–809.
Geisler, M., Blakeslee, J.J., Bouchard, R. et al. (2005) Cellular efflux
of auxin catalyzed by the Arabidopsis MDR/PGP transporter AtPGP1. Plant J. 44, 179–194.
Hall, A., Kozma-Bognar, L., Toth, R., Nagy, F. and Millar, A.J. (2001)
Conditional circadian regulation of PHYTOCHROME A gene
expression. Plant Physiol. 127, 1808–1818.
Halliday, K.J. (2004) Plant hormones: the interplay of brassinosteroids and auxin. Curr. Biol. 14, R1008–R1010.
Hardtke, C.S., Gohda, K., Osterlund, M.T., Oyama, T., Okada, K. and
Deng, X.W. (2000) HY5 stability and activity in Arabidopsis is
regulated by phosphorylation in its COP1 binding domain. EMBO
J. 19, 4997–5006.
Jensen, P.J., Hangarter, R.P. and Estelle, M. (1998) Auxin transport
is required for hypocotyl elongation in light-grown but not darkgrown Arabidopsis. Plant Physiol. 116, 455–462.
Kircher, S., Kozma-Bognar, L., Kim, L., Adam, E., Harter, K., Schafer,
E. and Nagy, F. (1999) Light quality-dependent nuclear import of
the plant photoreceptors phytochrome A and B. Plant Cell, 11,
1445–1456.
Kircher, S., Gil, P., Kozma-Bognar, L., Fejes, E., Speth, V.,
Husselstein-Muller, T., Bauer, D., Adam, E., Schafer, E. and
Nagy, F. (2002) Nucleocytoplasmic partitioning of the plant
photoreceptors phytochrome A, B, C, D, and E is regulated
differentially by light and exhibits a diurnal rhythm. Plant Cell,
14, 1541–1555.
Kiss, J.Z., Mullen, J.L., Correll, M.J. and Hangarter, R.P. (2003)
Phytochromes A and B mediate red-light-induced positive phototropism in roots. Plant Physiol. 131, 1411–1417.
Lin, R. and Wang, H. (2005) Two homologous ATP-binding cassette
transporter proteins, AtMDR1 and AtPGP1, regulate Arabidopsis
photomorphogenesis and root development by mediating polar
auxin transport. Plant Physiol. 138, 949–964.
Ljung, K., Hull, A.K., Celenza, J., Yamada, M., Estelle, M., Normanly,
J. and Sandberg, G. (2005) Sites and regulation of auxin biosynthesis in Arabidopsis roots. Plant Cell, 17, 1090–1104.
Marchant, A., Bhalerao, R., Casimiro, I., Eklof, J., Casero, P.J.,
Bennett, M. and Sandberg, G. (2002) AUX1 promotes lateral root
formation by facilitating indole-3-acetic acid distribution between
sink and source tissues in the Arabidopsis seedling. Plant Cell, 14,
589–597.
Mas, P., Devlin, P.F., Panda, S. and Kay, S.A. (2000) Functional
interaction of phytochrome B and cryptochrome 2. Nature, 408,
207–211.
Matsushita, T., Mochizuki, N. and Nagatani, A. (2003) Dimers of the
N-terminal domain of phytochrome B are functional in the nucleus. Nature, 424, 571–574.
Mazzella, M.A., Bertero, D. and Casal, J.J. (2000) Temperaturedependent internode elongation in vegetative plants of Arabidopsis thaliana lacking phytochrome B and cryptochrome 1.
Planta, 210, 497–501.
Nagatani, A. (2004) Light-regulated nuclear localization of phytochromes. Curr. Opin. Plant Biol. 7, 708–711.
Nagatani, A., Reed, J.W. and Chory, J. (1993) Isolation and initial
characterization of Arabidopsis mutants that are deficient in
phytochrome A. Plant Physiol. 102, 269–277.
Neff, M.M. and Chory, J. (1998) Genetic interactions between
phytochrome A, phytochrome B, and cryptochrome 1 during
Arabidopsis development. Plant Physiol. 118, 27–35.
Oyama, T., Shimura, Y. and Okada, K. (1997) The Arabidopsis HY5
gene encodes a bZIP protein that regulates stimulus-induced
development of root and hypocotyl. Genes Dev. 11, 2983–2995.
Pitts, R.J., Cernac, A. and Estelle, M. (1998) Auxin and ethylene
promote root hair elongation in Arabidopsis. Plant J. 16, 553–560.
Reed, J.W., Nagpal, P., Poole, D.S., Furuya, M. and Chory, J. (1993)
Mutations in the gene for the red/far-red light receptor phytochrome B alter cell elongation and physiological responses
throughout Arabidopsis development. Plant Cell, 5, 147–157.
Ruegger, M., Dewey, E., Hobbie, L., Brown, D., Bernasconi, P.,
Turner, J., Muday, G. and Estelle, M. (1997) Reduced naphthylphthalamic acid binding in the tir3 mutant of Arabidopsis is
associated with a reduction in polar auxin transport and diverse
morphological defects. Plant Cell, 9, 745–757.
Ruppel, N.J., Hangarter, R.P. and Kiss, J.Z. (2001) Red-light-induced
positive phototropism in Arabidopsis roots. Planta, 212, 424–430.
Santelia, D., Vincenzetti, V., Azzarello, E., Bovet, L., Fukao, Y.,
Duchtig, P., Mancuso, S., Martinoia, E. and Geisler, M. (2005)
MDR-like ABC transporter AtPGP4 is involved in auxin-mediated
lateral root and root hair development. FEBS Lett. 579, 5399–5406.
Sharrock, R.A. and Clack, T. (2002) Patterns of expression and normalized levels of the five Arabidopsis phytochromes. Plant
Physiol. 130, 442–456.
Sharrock, R.A. and Quail, P.H. (1989) Novel phytochrome sequences
in Arabidopsis thaliana: structure, evolution, and differential
expression of a plant regulatory photoreceptor family. Genes
Dev. 3, 1745–1757.
Sibout, R., Sukumar, P., Hettiarachchi, C., Holm, M., Muday, G.K.
and Hardtke, C.S. (2006) Opposite root growth phenotypes of hy5
versus hy5 hyh mutants correlate with increased constitutive
auxin signaling. PLoS Genet. 2(11), e202.
Sidler, M., Hassa, P., Hasan, S., Ringli, C. and Dudler, R. (1998)
Involvement of an ABC transporter in a developmental pathway
regulating hypocotyl cell elongation in the light. Plant Cell, 10,
1623–1636.
Sun, Q., Yoda, K. and Suzuki, H. (2005) Internal axial light conduction in the stems and roots of herbaceous plants. J. Exp. Bot. 56,
191–203.
Tian, Q., Uhlir, N.J. and Reed, J.W. (2002) Arabidopsis SHY2/IAA3
inhibits auxin-regulated gene expression. Plant Cell, 14, 301–319.
Tian, Q., Nagpal, P. and Reed, J.W. (2003) Regulation of Arabidopsis
SHY2/IAA3 protein turnover. Plant J. 36, 643–651.
Toth, R., Kevei, E., Hall, A., Millar, A.J., Nagy, F. and Kozma-Bognar,
L. (2001) Circadian clock-regulated expression of phytochrome
and cryptochrome genes in Arabidopsis. Plant Physiol. 127, 1607–
1616.
Ulmasov, T., Murfett, J., Hagen, G. and Guilfoyle, T.J. (1997) Aux/
IAA proteins repress expression of reporter genes containing
natural and highly active synthetic auxin response elements.
Plant Cell, 9, 1963–1971.
Whitelam, G.C., Patel, S. and Devlin, P.F. (1998) Phytochromes and
photomorphogenesis in Arabidopsis. Philos. Trans. R. Soc. Lond.
[Biol.] 353, 1445–1453.
Yamaguchi, R., Nakamura, M., Mochizuki, N., Kay, S.A. and
Nagatani, A. (1999) Light-dependent translocation of a phytochrome B–GFP fusion protein to the nucleus in transgenic Arabidopsis. J. Cell Biol. 145, 437–445.
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 50, 429–438