Download The Phototropic Response is Locally Regulated Within the Topmost

The Phototropic Response is Locally Regulated Within the
Topmost Light-Responsive Region of the Arabidopsis
thaliana Seedling
1
Department of Botany, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan
Graduate School of Science and Technology, Niigata University, Nishi-ku, Niigata, Japan
3
Present address: Technical Division, Technology Development Office, MIRAI CO., LTD., Chiba, 270-2218 Japan.
4
Present address: Division of Environmental Photobiology, National Institute for Basic Biology, Okazaki, 444-8585 Japan.
*Corresponding author: E-mail, [email protected]; Fax, +81-75-753-4126.
(Received August 1, 2013; Accepted November 30, 2013)
2
Phototropism is caused by differential cell elongation between the irradiated and shaded sides of plant organs,
such as the stem. It is widely accepted that an uneven
auxin distribution between the two sides crucially participates in this response. Plant-specific blue-light photoreceptors, phototropins (phot1 and phot2), mediate this
response. In grass coleoptiles, the sites of light perception
and phototropic bending are spatially separated. However,
these sites are less clearly distinguished in dicots.
Furthermore, the exact placement of the action of each
phototropic signaling factor remains unknown. Here, we
investigated the spatial aspects of phototropism using spotlight irradiation with etiolated Arabidopsis seedlings. The
results demonstrated that the topmost part of about
1.1 mm of the hypocotyl constituted the light-responsive
region in which both light perception and actual bending
occurred. In addition, cotyledons and the shoot apex were
dispensable for the response. Hence, the response was more
region autonomous in dicots than in monocots. We next
examined the elongation rates, the levels of phot1 and the
auxin–reporter gene expression along the hypocotyl during
the phototropic response. The light-responsive region was
more active than the non-responsive region with respect to
all of those parameters.
Keywords: Arabidopsis thaliana Auxin Blue light Phototropin Phototropism Spotlight irradiation.
Abbreviations: ABCB, ATP-binding cassette subfamily
B-type; AGC, protein kinase A/protein kinase G/protein
kinase C; AUX1, AUXIN RESISTANT 1; BTB/POZ, BROAD
COMPLEX, TRAMTRACK, AND BRIC A BRAC/POX VIRUS
AND ZINC FINGER; CPT1, COLEOPTILE PHOTOTROPISM
1; CUL3, CULLIN3; DR5rev::GFP, GFP driven by the synthetic
auxin responsive promoter; GFP, green fluorescent protein;
LAX, like-AUX; LED, light-emitting diode; LOV, light, oxygen
and voltage; NPH3, NONPHOTOTROPIC HYPOCOTYL 3;
Regular Paper
Kazuhiko Yamamoto1,3, Tomomi Suzuki1, Yusuke Aihara1,4, Ken Haga2, Tatsuya Sakai2 and
Akira Nagatani1,*
P1::P1G, full-length phot1 fused to GFP under control of
the PHOT1 promoter; P2::P1G, full-length phot1 fused to
GFP under control of the PHOT2 promoter; phot, phototropin; PIN, PIN-FORMED; PKS, PHYTOCHROME KINASE
SUBSTRATE; RPT2, ROOT PHOTOTROPIOSM 2.
Introduction
Plants exhibit various physiological responses to changing environmental factors, such as light, temperature and moisture.
Light is one of the most important factors, as it drives photosynthesis in plants. Phototropism describes the phenomenon in
which plants change their growth direction toward the light
source to increase the light capturing efficiency.
Phototropism has garnered attention from numerous scientists for a long time (Whippo and Hangarter 2006). In 1880,
Charles Darwin and his son Francis reported that the grass
seedling sensed the light direction at the tip region and bent
in the region several millimeters below the tip (Darwin 1880).
They concluded that there was an unidentified substance transmitted from the tip to the lower region of the coleoptile to
trigger the bending. This transmittable substance was named
auxin, which was later identified as IAA (reviewed in Whippo
and Hangarter 2006). Phototropism is caused by differential cell
elongation between the irradiated and shaded sides of the plant
organs (Iino and Briggs 1984, Orbović and Poff 1993). It is widely
accepted that an uneven auxin distribution crucially mediates
this differential growth both in monocots (Iino 1991, Haga et al.
2005, Matsuda et al. 2011) and in dicots (Esmon et al. 2006,
Haga and Iino 2006).
The sites of light perception and phototropic bending are
spatially separated in monocot coleoptiles (Darwin 1880).
Uneven auxin distribution established at the coleoptile tip in
response to the light stimulus moves downwards to cause differential growth in a lower part of the coleoptile (Briggs et al.
Plant Cell Physiol. 55(3): 497–506 (2014) doi:10.1093/pcp/pct184, available online at www.pcp.oxfordjournals.org
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1957, Iino 1991, Iino 1995). In dicots, the perception and bending sites in the hypocotyl are less clearly distinguished. At least
the tip and cotyledons are dispensable for the light perception
(Franssen and Bruinsma 1981, Kato-Noguchii and Hasegawa
1992). Hence, the response might be more region autonomous
in dicots. However, an uneven distribution of the auxin
response has been observed above the bending site in deetiolated Arabidopsis seedlings (Christie et al. 2011), implying
that the perception site may reside above the binding site.
The phototropic response is mediated by plant-specific
blue-light photoreceptor phototropins (Huala et al. 1997,
Christie et al. 1998). They are light-activated Ser/Thr protein
kinases of the protein kinase A/protein kinase G/protein kinase
C (AGC) kinase group (Galván-Ampudia and Offringa 2007).
They harbor two photosensory domains [light, oxygen, voltage
1 (LOV1) and LOV2] at the N-terminus and a kinase domain at
the C-terminus. This C-terminal kinase activity is repressed by
the LOV2 domain in the dark. Upon light absorption, the LOV2
domain is released from the kinase domain to transduce the
signal (Matsuoka and Tokutomi 2005, Kong et al. 2007, Okajima
et al. 2011). Arabidopsis harbors two types of phototropin molecules, phototropin 1 (phot1) and phot2. Phot1 regulates the
phototropic response to a wide range of blue light intensities,
whereas phot2 only mediates the response to high-intensity
blue light (Kagawa et al. 2001). This sensitivity difference is
determined by an N-terminal moiety (Aihara et al. 2008).
Consistent with the involvement of auxin in phototropism,
several auxin transporters, such as PIN-FORMEDs (PINs), ATPbinding cassette subfamily B-type (ABCB) transporter and the
AUXIN RESISTANT (AUX) 1/LIKE-AUX (LAX) family proteins,
are required for normal phototropism. The pin3 mutant exhibits a slightly decreased phototropic response (Friml et al.
2002, Ding et al. 2011). More recently, Haga and Sakai (2012)
have demonstrated that PIN1, 3 and 7 additively participate in
pulse-induced phototropism. Conversely, the abcb19/pgp19
mutants display an enhanced phototropic response in
the hypocotyls of both etiolated and de-etiolated seedlings
(Noh et al. 2001, Nagashima et al. 2008, Christie et al. 2011).
The results indicate that ABCB19 negatively regulates phototropism and is directly phosphorylated by phot1 (Christie et al.
2011). Genetic analysis indicates that the amino acid/auxin:
proton symport permease AUX1/LAX family of genes is also
involved in phototropism in etiolated seedlings (Stone et al.
2008).
Genetic and biochemical approaches have identified another class of factors required for phototropism (Motchoulski
and Liscum 1999, Sakai et al. 2000), such as
NONPHOTOTROPIC HYPOCOTYL 3 (NPH3) and ROOT
PHOTOTROPIOSM 2 (RPT2), which harbor BROAD
COMPLEX, TRAMTRACK, BRIC A BRAC/POX VIRUS AND
ZINC FINGER (BTB/POZ) domains at their N-terminal region
and a coiled-coil domain at the C-terminus. COLEOPTILE
PHOTOTROPISM 1 (CPT1), a homolog of NPH3 in rice, has
been demonstrated to be necessary for light-dependent auxin
re-distribution in the coleoptiles tip region (Haga et al. 2005).
498
The PHYTOCHROME KINASE SUBSTRATE (PKS) family consists of four genes (PKS1–PKS4) that harbor no recognizable
domains. Among them, PKS1, PKS2 and PKS4 transduce phototropic signals (Lariguet et al. 2006).
In the present study, we aimed to identify the site of each
phototropism signaling step, such as photoreceptor activation,
photoreceptor interactions with downstream components and
auxin re-distribution, in the seedling by performing spotlight
irradiation experiments. We also investigated the spatial patterns of phot1 activation induced by whole seedling irradiation.
Results
The phototropic response is locally regulated in
Arabidopsis etiolated seedlings
The tip and cotyledons are dispensable for the phototropic
response in dicot seedlings (Franssen and Bruinsma 1981,
Kato-Noguchi and Hasegawa 1992). Hence, we first asked
whether those organs were dispensable for the phototropic
response in Arabidopsis (Fig. 1). As demonstrated in the
figure, decapitation with a razor blade did not abolish the
phototropic bending in the Arabidopsis seedlings. Hence,
unlike the grass coleoptiles, the most apical region was dispensable for the response in the Arabidopsis hypocotyl.
Next, we examined how the local light stimulus was processed by the hypocotyl (Fig. 2). To assess this response, we
assembled equipment with an acryl-fiber ( = 0.25 mm) connected to a blue-light-emitting diode (LED) bulb fixed on an
XYZ stage for adjusting the irradiation point (Supplementary
Fig. 1 Phototropic response in decapitated seedlings. Three-day-old
etiolated seedlings were decapitated, and irradiated with a unilateral
blue light at 2.5 mmol m 2 s 1 from the side opposite the cotyledon
attachment side in the presence of a background red light at
3.0 mmol m 2 s 1. The bending angle was determined after 4 h of
irradiation. The values represent the mean ± SD of at least 14
seedlings.
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Spatial analysis of Arabidopsis phototropism
Fig. 2 Phototropic responses to spotlight irradiation. (A) Three-day-old etiolated seedlings were irradiated with a blue spotlight at
2.5 mmol m 2 s 1 in the presence of a background red light at 3.0 mmol m 2 s 1. The blue light was guided through an acryl-fiber
( = 0.25 mm). The irradiation was administered from the side opposite the cotyledon attachment side (Supplementary Fig. 1). Bar = 2 mm.
(B) Bending angles after the spotlight treatment in the topmost 2.13 mm region of the seedlings. The seedlings were irradiated at the indicated
positions on the x-axis for 2 h. The dashed line indicates the bending angle after the whole seedling irradiation. The values represent the
mean ± SD of at least seven seedlings. (C) Elongation profile in the top part of seedlings in darkness. The 4 mm (open circle) and 1.5 mm (filled
circle) topmost regions were divided into four and six segments, respectively. The lengths of each segment were then determined before and after
2 h dark incubation (Supplementary Fig. 4). The lengths after incubation relative to those before incubation are shown. The values represent the
mean ± SD of at least 11 seedlings. (D) Relationship between the sites of irradiation and the curvature (upper) or the bending position (lower)
within the light-responsive region. The diagonal line indicates that the hypocotyl bends exactly at the site of irradiation. The dashed lines indicate
curvature and bending position after the whole seedling irradiation. The values represent the mean ± SD of at least five seedlings.
Fig. S1A). As shown in Fig. 2A, the spotlight did not spread very
much within the seedling. Three-day-old etiolated seedlings
that were grown along the vertical agar surface were irradiated
with blue light at 2.5 mmol m 2 s 1 from the side opposite
the cotyledons in the presence of background red light at
3.0 mmol m 2 s 1, which was used as the observation light
(Fig. 2A). The fiber center was defined as the point of spotlight
irradiation for descriptive purposes.
When the spotlight irradiation was administered at 0.13,
0.63 or 1.13 mm from the top of the hypocotyl, phototropic
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bending was observed, whereas irradiation administered below
1.63 mm barely elicited a response (Fig. 2B). Hence, the nonresponsive region begins somewhere between 1.13 and
1.63 mm from the top. Since irradiation at 1.13 mm elicited a
50% response compared with the whole seedling irradiation
(Fig. 2B), the region above 1.1 mm was arbitrary defined as
the light-responsive region.
Because phototropic bending is a consequence of differential growth, we examined the elongation profile in this region in
darkness (Fig. 2C). Consistent with a previous report
(Gendreau et al. 1997), the elongation rate was higher toward
the topmost part of the seedling. Therefore, the light-responsive region defined above corresponded to the faster elongating
region. However, the upper part of the non-responsive region
also displayed some elongation (at 2.13 mm, for example). Thus,
there might be a minimum elongation rate to indicate a significant phototropic bending during the time frame of the
assay.
Relationship between the sites of light perception
and phototropic bending
We examined the site of bending in detail within the lightresponsive region. When the hypocotyl was irradiated at 0.63
and 1.13 mm, the observed maximal curvature was at 0.68 and
0.96 mm, respectively (Fig. 2C). Thus, the light stimulus was
processed quite locally. In contrast, the bending site appeared
to shift downward slightly when the spotlight was administered
at the topmost part at 0.13 mm (Fig. 2C). It should be noted
here that the hypocotyl length increased by 0.4–0.5 mm during
the irradiation period (Supplementary Fig. S2). Hence more
precise analysis is required to draw a definite conclusion.
The expression profile of phot1
The sites of light perception and bending were confined within
a topmost region of approximately 1.1 mm in the Arabidopsis
hypocotyl (Fig. 2). To elucidate how this region was molecularly
defined, we first hypothesized that the phot1 expression level
was too low in the non-responsive region because the response
to 2.5 mmol m 2 s 1 blue light is mediated predominantly by
phot1 (Kagawa et al. 2001). Hence, we employed a line expressing a fusion protein between phot1 and green fluorescent protein (GFP) under the control of the PHOT1 promoter (P1::P1G)
in the phot1-5 phot2-1 double mutant background (P1::P1G 24;
Aihara et al. 2008). First, we performed an immunoblot assay to
confirm that phot1–GFP was expressed at an endogenous level
(Fig. 3A). Analysis of GFP expression in the P1::P1G 24 seedlings
revealed that phot1–GFP expression gradually decreased
toward the bottom of the hypocotyl (Fig. 3B).
Next, the phototropic response in P1::P1G 5, which expressed phot1–GFP at a lower level than P1::P1G 24, was examined (Aihara et al. 2008; Fig. 4) along with several P2::P1G lines
in which phot1–GFP was expressed at lower levels under the
PHOT2 authentic promoter (P2::P1G 3 and 26) (Aihara et al.
2008). Consequently, all of the lines exhibited a normal
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Fig. 3 Expression profile of phot1. (A) A 40 mg aliquot of total protein
from three-day-old etiolated seedlings was subjected to immunoblot
analysis with an anti-phot1 polyclonal antibody (Kong et al. 2006).
Molecular mass markers in kDa are presented on the left of the blot.
Black and white arrowheads indicate the phot1–GFP and endogenous
phot1, respectively. The WT samples were serially diluted (1, 1/2, 1/4).
WT, wild-type; p1p2, phot1phot2 double mutant. (B) The confocal
image (left) and GFP fluorescence quantification (right) in a threeday-old etiolated P1::P1G 24 seedling. The relative GFP fluorescence
intensities per area are presented.
phototropic response (Fig. 4B). Hence, it is unlikely that the
non-responsive region failed to respond to the light simply
because the expression level of phot1 was inadequate.
The relationship between the light-responsive
region and phot1 internalization
Phot1 internalizes into the cytoplasm in response to blue light
in an autophosphorylation state-dependent manner (Sakamoto and Briggs 2002, Kaiserli et al. 2009). Therefore, we compared the response at different positions (Fig. 5). The seedlings
were assessed after 30 min of irradiation according to the literature (Han et al. 2008). As reported, phot1–GFP internalization was observed independently of position. Thus, phot1
appeared to be activated in both the light-responsive and
non-responsive regions.
Uneven auxin—reporter gene expression caused
by spot-light irradiation
After unilateral irradiation with blue light, an uneven auxin
distribution is observed in the hypocotyl of both monocots
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Spatial analysis of Arabidopsis phototropism
Fig. 4 Hypocotyl phototropism in transgenic seedlings expressing
phot1–GFP at different levels. (A) Confocal microscopic images of
etiolated seedlings expressing phot1–GFP at different levels.
Bar = 200 mm. (B) Bending angles in three-day-old etiolated seedlings
treated with a unilateral blue light (2.5 mmol m 2 s 1) and a background red light at 3.0 mmol m 2 s 1 for 2 h. The values represent
the mean ± SD of at least nine seedlings. The GFP fluorescence intensities relative to that in P1::P1G 24 in darkness are presented (blue
symbols). The values represent the mean ± SD of three seedlings.
and dicots (Iino 1991, Haga et al. 2005, Esmon et al. 2006, Haga
and Iino 2006). Consistently, the GFP reporter gene driven by a
synthetic auxin-responsive promoter (DR5rev::GFP) is expressed
differentially between the two sides of the Arabidopsis hypocotyl (Christie et al. 2011, Ding et al. 2011, Haga and Sakai 2012).
Hence, we examined whether the spotlight irradiation affected
the auxin–reporter gene expression in the DR5rev::GFP seedlings (Fig. 6).
When the irradiation was administered from the opposite
side of the cotyledons, the blue light effect on auxin could not
be monitored because of the strong GFP signal in and below the
concave region of the hook (Haga and Sakai 2012). Hence, we
irradiated two-day-old seedling perpendicular to the hook
plane according to the literature (Supplementary Fig. S1B).
Because of the changes in experimental conditions, we first
confirmed that the response was essentially the same as that
observed in other experiments (Supplementary Fig. S3). The
Fig. 5 Blue light-induced phot1–GFP internalization. GFP fluorescence in the cortical cells at the indicated positions in etiolated
P1::P1G 24 seedlings. The seedlings were grown along the vertical
agar surface. They were treated with (B, D, F, H) or without (A, C, E,
G) unilateral blue light (2.5 mmol m 2 s 1) for 30 min and transferred
onto the glass slide without changing the orientation for microscopic
observation. The light treatment was given from the left side in the
figures. The white arrowheads indicate internalized phot1–GFP.
Bars = 20 mm.
wild-type seedlings bent at similar angles under the new condition. It was also confirmed that the response was locally
regulated as in the other experiment (Fig. 2), although the
light-responsive region was a little smaller under the new condition. The DR5rev:GFP seedlings were then grown in darkness
for 2 d perpendicular to a horizontal agar surface and irradiated
with blue spotlight 2.5 mmol m 2 s 1 for 90 min. After an additional 90 min dark incubation to allow GFP to accumulate, GFP
fluorescence was examined (Fig. 6).
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Fig. 6 Effect of spotlight irradiation on auxin distribution. Confocal microscopic observation of DR5rev::GFP seedlings treated with a unilateral
spotlight. The spotlight treatment was given from the left side in the figures. The white arrowheads indicate a higher fluorescence than on the
opposite side of the hypocotyl. White bars = 200 mm. (A) Two-day-old etiolated seedlings grown on a horizontal agar plate were pre-treated with
red light and subjected to the spotlight irradiation with blue light at 2.5 mmol m 2 s 1 for 90 min as shown in Supplementary Fig. 1A. After an
additional 90 min dark incubation, seedlings were mounted on a glass slide to direct the side of the hypocotyl opposite to the cotyledons upward.
(B) Heat map representation of the GFP signals show in (A).
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Spatial analysis of Arabidopsis phototropism
When whole seedlings were irradiated, the GFP signal on the
shaded side was stronger in the upper (0.25–0.85 mm) and
lower (2.0–2.6 mm) regions of the hypocotyl, although the
signal was weaker at 2.0–2.6 mm (Fig. 6). The spotlight irradiation at 0.13 mm induced an extremely similar response to that
observed after whole plant irradiation. This finding was consistent with the observation that irradiation at the same position
induced a full phototropic response (Fig. 2; Supplementary
Fig. S3). When irradiation at 1.13 mm was administered,
which caused a weak phototropic response (Fig. 2), an
uneven GFP expression in the 2.0–2.6 mm region but not in
the 0.25–0.85 mm region was observed. Therefore, the signal
was transmitted toward the hypocotyl base but not the tip.
Consistent with the lack of a phototropic response, irradiation
at 2.13 mm failed to induce uneven GFP expression in any part
of the seedling.
In summary, phototropin in the light-responsive region affected the auxin–reporter gene expression beyond the site of
irradiation. In contrast, phototropin in the non-responsive
region failed to affect the expression even at the site of irradiation. Hence, some signaling components and/or conditions
are probably missing in the non-responsive region and thus
preclude changes in the auxin–reporter response. Notably,
the uneven GFP expression was observed even in the nonbending region between 2.0 and 2.6 mm when the light was
administered at the light-responsive region (Fig. 6). Hence, differential auxin–reporter expression did not always induce the
bending.
Discussion
Phototropic response in Arabidopsis seedlings is
region autonomous
We performed spotlight irradiation experiments to identify the
light-responsive region for the phototropic response in
Arabidopsis etiolated seedlings. Our results identified an approximate region confined within the topmost 1.1 mm of the
hypocotyl (Fig. 2). Both the light perception and bending regions reside within this region. Furthermore, the decapitation
experiment indicated that the shoot apex and cotyledons were
dispensable for the response (Fig. 1). These results are consistent with earlier studies using green sunflower seedlings
(Franssen and Bruinsma 1981) and radish etiolated seedlings
(Kato-Noguchi and Hasegawa 1992). Therefore, the phototropic response in Arabidopsis seedlings was region autonomous as is the case with other dicot seedlings.
In addition, the bending site appeared to shift downwards
slightly when the spotlight was administered at the topmost
region at 0.13 mm. However, it was difficult to draw a definite
conclusion. For one thing, the size of the spot and some scattering from the irradiated site should be considered.
Furthermore, the hypocotyl length increased by 0.4–0.5 mm
during the irradiation period (Supplementary Fig. S2).Hence,
the actual size of the irradiated region remained obscure. More
detailed study is needed to draw a clear conclusion about the
downward signaling.
In grass seedlings, which are commonly used for phototropic
studies, the light perception occurs at the tip of the coleoptile,
whereas the bending site is located several millimeters below
(Darwin 1880). In contrast, the shoot apex and cotyledons are
dispensable for the response in dicots (Franssen and Bruinsma
1981, Kato-Noguchi and Hasegawa 1992; Fig. 1). Furthermore,
the downward signal, if any, traveled only for a short distance in
Arabidopsis etiolated seedlings (Fig. 2). Hence, the response
appeared more region autonomous in Arabidopsis than in
grass plants. Nevertheless, the basic mechanisms that mediate
phototropic bending might not drastically differ between these
two seedlings types. Notably, the elongation zone was restricted
in the upper hypocotyl in Arabidopsis (Gendreau et al. 1997;
Fig. 2B), whereas it resides in the lower to middle part in the
grass coleoptile (Darwin 1880, Furuya et al. 1969, Iino 1996). The
apparent differences could be due to quantitative differences in
the distances between the tip and the elongation zone.
Auxin distribution within the light-responsive
region
An uneven auxin distribution between the irradiated and
shaded side is known to occur before or during the bending
response (Iino 1991, Haga et al. 2005, Esmon et al. 2006, Haga
and Iino 2006, Matsuda et al. 2011). We indirectly examined the
site of auxin re-distribution using the auxin–reporter line
DR5rev::GFP (Christie et al. 2011, Ding et al. 2011, Haga and
Sakai 2012). As expected, uneven GFP expression was observed
when the light-responsive region was locally irradiated (Fig. 6).
Additionally, the differential expression propagated basipetally
from the irradiated site. This result is consistent with the view
that auxin is transported along the hypocotyl toward the basal
end (Haga et al. 2005). It should be noted here that we waited
for 3 h to observe the uneven GFP expression. Since the hypocotyl starts to bend earlier, we need a more sensitive method to
connect the auxin levels directly with the differential cell
growth.
In Arabidopsis de-etiolated seedlings, gibberellin has been
recently demonstrated to be required for auxin-dependent
elongation (Chapman et al. 2012) and to participate in the
phototropic response (Tsuchida-Mayama et al. 2010).
Furthermore, gibberellin is unevenly distributed in root gravitropism (Löfke et al. 2013). Hence, gibberellin rather than auxin
might define the elongation and/or bending zones. Future studies should assess the distribution patterns not only auxin of
but also of gibberellin.
Phototropin expression region and the activity
As previously reported (Sakamoto and Briggs 2002, Wan et al.
2008), phot1 levels in the etiolated hypocotyl gradually
decreased toward the basal end of the hypocotyl (Fig. 3). It
should be noted that the fluorescence intensity per area might
not reflect the exact concentration of phot1–GFP on the
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plasma membrane. Since cells are smaller and more numberous
in the upper part, the apparent fluorescence could become
higher. In any case, expression of phot1–GFP at below endogenous levels restored an almost normal phototropic response in
the phot1 phot2 mutant background (Fig. 4). Hence, the phot1
expression level in the non-responsive region was unlikely to be
insufficient to mediate the response.
Although phot1–GFP expression was lower in the nonresponsive region, blue light induced phot1–GFP internalization in both the photo-responsive and non-responsive regions
(Fig. 5). Autophosphorylation of Ser851 in the phot1 kinase
domain is known to trigger the internalization (Kaiserli et al.
2009). Furthermore, this autophosphorylation is crucial for the
activation of phot1 kinase function and all tested phot1mediated responses (Inoue et al. 2008). Hence, the initial signaling step was thought to proceed equally in both the regions.
Signaling mechanism in the light-responsive and
non-responsive regions
The results in this study suggest that the phot1 expression level
was high enough and that the initial signal transduction step
was not prohibited in the non-responsive region. Thus, we hypothesize that some downstream factor is missing in the nonresponsive region. Although PIN1, 3 and 7 additively function in
the pulse-induced phototropism, these PINs do not participate
in the phototropic response to continuous blue light (Haga and
Sakai 2012). Likewise, PINOID, a regulator of PIN localization, is
not involved in the phototropism under continuous blue light
(Haga and Sakai 2012). Hence, it is unlikely that those factors
distinguish between the light-responsive and non-responsive
regions.
The NPH3 family proteins including RPT2 are involved in the
phototropic response (Motchoulski and Liscum 1999, Sakai
et al. 2000). Although the detailed expression patterns of
NPH3 and RPT2 in the hypocotyl are not known yet in
Arabidopsis, an NPH3-like gene in maize, which shows high
similarity to Arabidopsis NPH3, is exclusively expressed in the
top part of the coleoptiles (Matsuda et al. 2011). Nevertheless, it
is less likely that NPH3 and/or RPT2 are the missing factors in
the non-responsive region in Arabidopsis. The phot1–GFP internalization is mediated by clathrin-dependent endocytosis
(Kaiserli et al. 2009), and the NPH3 protein has been suggested
to participate in this process (Roberts et al. 2011). Because
phot1–GFP was internalized in response to the light treatment,
NPH3 may also function in the non-responsive region (Fig. 5).
Likewise, the RPT2 protein, which physically interacts with both
phot1 and NPH3 (Inada et al. 2004), might be functioning in the
same way.
PKS proteins are attractive candidates for a factor defining
the light-responsive region. Among them, PKS1 expression is
induced by white light in the elongation zone of both hypocotyls and roots (Lariguet et al. 2003). PKS4 is predominantly
expressed in the hypocotyl elongation zone under any light
conditions (Schepens et al. 2008) and is directly phosphorylated
504
by phot1 (Demarsy et al. 2012). Therefore, low PKS expression
might cause a lack of responsiveness in the non-responsive
region. To confirm this hypothesis, the expression levels of
PKS proteins should be carefully examined.
Materials and Methods
Plant materials and growth conditions
The wild-type (gl1, Colombia) and phototropin double mutant,
phot1-5 phot2-1 (Kinoshita et al. 2001) were used. Plants expressing phot1 fused to GFP under the control of the PHOT2
authentic promoter (P2::P1G) or the PHOT1 authentic promoter (P1::P1G) in phot1-5 phot2-1 mutants were previously
described (Aihara et al. 2008). A Columbia strain harboring
DR5rev::GFP was obtained from the Arabidopsis Biological
Resource Center (ABRC). The seeds were planted on 1.5 %
(w/v) agar plates containing Murashige and Skoog (MS)
medium with 1% (w/v) sucrose. The seedlings were grown in
darkness for 2 or 3 d along the vertical agar surface of the plate.
To observe the GFP signals in the DR5rev::GFP seedlings, the
seeds were sown in 0.2 ml plastic tubes filled with 1.5% (w/v)
agar medium (Haga and Sakai 2012) and stored at 4 C for 3–5 d.
Following irradiation with an overhead white light for 1 h, the
plants were grown at 23 C for 2 or 3 d in darkness. The experiments were performed under a dim green safe light to avoid any
light responses.
Spotlight irradiation experiment
The three-day-old etiolated seedlings grown along the vertical
agar surface were placed in the irradiation device together with
the agar block (Supplementary Fig. S1A). A blue light at
2.5 mmol m 2 s 1 was administered from opposite the cotyledon attachment side with a background red light at
3.0 mmol m 2 s 1, which was used as the observation light.
The blue light from a blue-light LED bulb (TOYODA GOSEI)
was guided through an acryl-fiber ( = 0.25 mm, MITSUBISHI
LAYON). The fiber was fixed on an XYZ stage (CHUO
PRECISION INDUSTRIAL) to adjust the irradiation point.
Seedling pictures were captured every 5 min using a CCD
camera (WAT-508, Watec). To prevent the seedlings from
touching the fiber, the fiber tip position was checked continually and moved away from the seedling in a horizontal direction if necessary. The bending angle was measured after 2 h of
spotlight irradiation using ImageJ software (http://rsbweb.nih.
gov/ij/).
To observe the GFP signals in the DR5rev::GFP seedlings,
2-day-old seedlings were placed in the spotlight irradiation
equipment (Supplementary Fig. S1B) covered with an acryl
box to maintain high humidity. Because a strong GFP signal was
observed in and below the concave region of the hook, the blue
light was administered perpendicular to the hook plane
(Supplementary Fig. S1B). The seedlings were first irradiated
with an overhead red light at 3.0 mmol m 2 s 1 (660 nm, ISL150X150RB, CCS) for 2 min. After 2 h in the dark, the hypocotyls
Plant Cell Physiol. 55(3): 497–506 (2014) doi:10.1093/pcp/pct184 ! The Author 2013.
Spatial analysis of Arabidopsis phototropism
were irradiated with a blue spotlight for 90 min and incubated
in darkness for 90 min. In this experiment, infrared light
(940 nm, M-Light, Uniel-denshi) was used as a background
light during image capture. We selected seedlings with hypocotyl lengths of 3–5 mm and that grew straight and vertically
during the experiment.
Unten-Kobayashi (Kyoto University, Japan) for technical
support.
Disclosures
The authors have no conflicts of interest to declare.
Curvature analysis
The hypocotyl curvature was analyzed using the ‘straighten
curved objects’ plugin for the ImageJ software (Kocsis et al.
1991). The pictures of seedlings irradiated for 2 h were
analyzed, and the hypocotyl curvatures on the center line
were measured.
Elongation analysis
The seedling surfaces were marked with copier toner particles
and photographed before and after 2 h incubation in darkness
(Supplementary Fig. S4). Points at certain intervals (0.25 or
1 mm) at 0 h were traced after 2 h dark incubation using toner
particles as reference, and relative elongation rates were
determined.
Laser scanning confocal microscopy
The specimens were observed under a confocal laser scanning
microscope (FV300 + BX60; Olympus). A band-pass filter (510–
530 nm) was used to observe GFP fluorescence, which was
quantified using the ImageJ software. To assess the GFP fluorescence in DR5rev::GFP seedlings, the signals were observed
opposite the cotyledon attachment side. The images were
scanned along the Z-axis for 60 mm at 5 mm intervals. To
assess the phot1–GFP internalization, the images were scanned
along the Z-axis for 16 mm at 0.8 mm intervals.
Supplementary data
Supplementary data are available at PCP online.
Funding
This work was supported by the Ministry of Education, Culture,
Sports, Science and Technology, Japan [a Grant-in-Aid for
Scientific Research on Priority Areas (No. 17084002), a Grantin-Aid for the Global COE Program ‘Formation of a strategic
base for biodiversity and evolutionary research: from genome to
ecosystem’ (A06), a Grant-in-Aid for Scientific Research on
Innovative Areas (No. 22120002) and the Japan Society for
the Promotion of Science [a Grant-in-Aid for Scientific
Research (B) (No. 21370020) to A.N.].
Acknowledgments
We would like to thank Dr. T. Kozuka and Dr. N. Mochizuki
(Kyoto University, Japan) for helpful discussion, and Ms. J.
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