Download Salt tolerance (STO), a stress-related protein, has a major role in

The Plant Journal (2007) 51, 563–574
doi: 10.1111/j.1365-313X.2007.03162.x
Salt tolerance (STO), a stress-related protein, has a major
role in light signalling
Martin Indorf, Julio Cordero, Gunther Neuhaus, and Marta Rodrı́guez-Franco*
Department of Cell Biology, University of Freiburg, Freiburg D-79104, Germany
Received 20 December 2006; revised 4 April 2007; accepted 13 April 2007.
*
For correspondence (fax +49 761 2032675; e-mail [email protected]).
Summary
The salt tolerance protein (STO) of Arabidopsis was identified as a protein conferring salt tolerance to yeast
cells. In order to uncover its function, we isolated an STO T-DNA insertion line and generated RNAi and
overexpressor Arabidopsis plants. Here we present data on the hypocotyl growth of these lines indicating that
STO acts as a negative regulator in phytochrome and blue-light signalling. Transcription analysis of STO
uncovered a light and circadian dependent regulation of gene expression, and analysis of light-regulated genes
revealed that STO is involved in the regulation of CHS expression during de-etiolation. In addition, we could
show that CONSTITUTIVE PHOTOMORPHOGENESIS 1 (COP1) represses the transcription of STO and
contributes to the destabilization of the protein in etiolated seedlings. Microscopic analysis revealed that
the STO:eGFP fusion protein is located in the nucleus, accumulates in a light-dependent manner, and, in
transient transformation assays in onion epidermal cells, co-localizes with COP1 in nuclear and cytoplasmic
aggregations. However, the analysis of gain- and loss-of-function STO mutants in the cop1-4 background
points towards a COP1-independent role during photomorphogenesis.
Keywords: light signalling, phytochrome, blue light, CONSTITUTIVE PHOTOMORPHOGENESIS 1, B-box,
Zn-finger protein.
Introduction
Plants as sessile organisms must cope with, and adapt to, a
number of environmental cues during their whole life cycle.
However, light may be the most important factor controlling
and influencing plant development (Franklin et al., 2005). The
well-modulated responses of plants to environmental factors
is a consequence of established signalling networks, where
different molecules are members of one or more pathways
that converge, diverge, and interact according to specific
environmental conditions and/or developmental stages
(Ludwig et al., 2005; Suzuki et al., 2005; Nakagami et al.,
2005). For example, it is well documented for abiotic stress
that a coordinated crosstalk amongst drought, cold and high
salinity pathways exists (Glombitza et al., 2004; Narusaka
et al., 2004; Chinnusamy et al., 2004; Mahajan and Tuteja,
2005); however, much less is known about the interplay
between light and other environmental signalling pathways.
Salt tolerance protein (STO) is a B-box type Zn finger
protein with sequence similarities to CONSTANS (Putterill
et al., 1995; Lagercrantz and Axelsson, 2000; Griffiths et al.,
2003). It was first identified through a screening approach
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd
using a yeast calcineurin mutant. Thus, yeast null mutants in
the catalytic subunit genes (cna1cna2), or in the regulatory
subunit gene (cnb1), present a salt sensitive phenotype that
can be rescued with STO (Lippuner et al., 1996). Surprisingly, in Arabidopsis plants STO gene expression seems not
to be induced by salt treatment (Lippuner et al., 1996;
Nagaoka and Takano, 2003), although it has been shown
that overexpression enhances root growth tolerance to high
salinity (Nagaoka and Takano, 2003). In addition, STO
interacts with CEO1/RCD1, an Arabidopsis protein that
complements an oxidative stress-sensitive yeast strain
(Belles-Boix et al., 2000) and negatively regulates a wide
range of stress-related downstream genes (Fujibe et al.,
2004). CEO1/RCD1 has been recently identified as a new
component in the plant salt-stress response, through the
interaction with SOS1 (Katiyar-Agarwal et al., 2006). However, an interaction of STO with CONSTITUTIVE PHOTOMORPHOGENESIS 1 (COP1), a negative regulator of
photomorphogenesis in the dark, has also been reported
(Holm et al., 2001; Ma et al., 2002).
563
564 Martin Indorf et al.
In an attempt to isolate genes involved in general calcium
signalling and regulation in plants, we established a
complementation screening analysis using a yeast L-type
calcium-channel (CCH1) knock-out mutant. This mutant
exhibits the same growth arrest in a medium containing
high salt concentrations as yeast calcineurin mutants
(Paidhungat and Garrett, 1997). Several proteins from
Arabidopsis were able to complement this cch1 salt-sensitive
phenotype. Amongst them STO appears not only to increase
the growth of the knock-out strain in high-salt medium, but
also conferred tolerance to higher salt concentrations in the
wild-type yeast strain.
To further investigate the function of the STO protein in
plants, we isolated a T-DNA insertion line and generated
RNAi and overexpressor Arabidopsis transgenic lines. Herein we show that the expression of STO mRNA is light
regulated, and that the protein shares a regulatory function
in phytochrome and blue-light signalling pathways. Moreover, the STO:GFP fusion protein is actively degraded in the
dark in a COP1-dependent manner, whereas light promotes
the accumulation of the protein in the nucleus. Additional
data shedding light on STO function independent of COP1
are presented.
Results
STO is involved in R, FR and B light signal transduction
The screening of the Arabidopsis T-DNA Salk database
(Alonso et al., 2003) led to the identification of a line containing the T-DNA insertion in the first intron of the STO
gene (SALK_067473). In addition, STO RNAi and overexpressor lines were generated and plants homozygous for the
transgene were selected for STO mRNA analysis. The RNAi
and T-DNA lines exhibited a drastic reduction, if not absence, of STO mRNA, whereas a high constitutive STO
transcript level was observed in the overexpressor lines
(Figure 1a).
Bearing in mind the interaction of STO and COP1 shown
by a two-hybrid system assay (Holm et al., 2001), and that
Figure 1. Phenotypic analysis of STO transgenic
lines.
(a) Transcript levels of STO in 3-week-old wildtype (Col-0) and transgenic lines grown under
long-day conditions. Equal RNA loading was
verified with an 18S rRNA-specific probe.
(b, c, d) Hypocotyl length of 5-day-old wild type,
sto-T-DNA and 35S:STO at different fluence rates
of continuous (b) red light, (c) far-red light and (d)
blue light. Error bars represent the SD of > 30
plants.
(e) Hypocotyl length of 5-day-old wild-type and
STO gain- and loss-of-function transgenic lines
in dark, continuous red (Rc; 30 lmol m)2 sec)1),
far-red (FRc; 0.5 lmol m)2 sec)1) and blue (Bc;
1.5 lmol m)2 sec)1) light conditions. Error bars
represent the SD of > 30 plants.
(f) Relative cotyledon area of 5-day-old wild-type
and STO gain- and loss-of-function transgenic
lines in Rc (30 lmol m)2 sec)1). Error bars represent the SE of > 45 plants. **Significant differences (P < 0.01) compared with the wild type.
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Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 51, 563–574
STO, a new light signalling intermediate 565
COP1 is also a major regulator of light signal transduction
(Ma et al., 2002), we investigated whether STO is also
directly involved in light signalling by fluence rate response
experiments.
Seeds from homozygous T-DNA, RNAi and overexpressor
plants were germinated on filter paper and grown for 5 days
at 22C under continuous red (Rc, 0.042–31 lmol m)2 sec)1),
far-red (FRc, 0.017–0.56 lmol m)2 sec)1) or blue light (Bc,
0.1–10 lmol m)2 sec)1).
RNAi and T-DNA lines exhibited a pronounced inhibition
of hypocotyl growth compared with the wild type under all
the light conditions tested. However, the STO-overexpressing lines, including GFP fusion overexpressors (data not
shown), developed longer hypocotyls than the wild type
under the above described conditions (Figure 1b–e). These
results provide evidence that STO is directly involved in light
signalling, and suggest a role as a negative regulator of R, FR
and B light-mediated hypocotyl elongation. No obvious
differences in hypocotyl length were observed when the
different homozygous lines were grown in the dark (Figure 1e). Photomorphogenic mutants typically present reciprocal growth responses of hypocotyl and cotyledon cells
induced by light signals (Khanna et al., 2006). We measured
the cotyledon area of gain- and loss-of-function transgenic
lines grown for 5 days in Rc (30 lmol m)2 sec)1). Both the
T-DNA and the RNAi lines exhibited larger cotyledons than
the wild type, whereas the overexpressors presented a
reduced cotyledon area, providing further evidence for STO
function in phytochrome signalling (Figure 1f).
Expression of STO is controlled by light and the circadian
clock
In subsequent experiments the transcription pattern of STO
mRNA under different light conditions in wild-type plants
was investigated. Five-day-old wild-type seedlings grown in
the dark were exposed to Rc (10 lmol m)2 sec)1), FRc
(10 lmol m)2 sec)1) and Bc (10 lmol m)2 sec)1) light for
3 h, following which STO transcript levels were analysed by
Northern blot. The results showed a significant increase of
the transcript abundance under all light conditions tested
(Figure S1), as compared with dark conditions. These data
show that the transcription of the gene is also light regulated.
To unravel the controlling pathway of the light-dependent
induction of STO, mRNA expression was investigated in
phyA (phyA-211), phyB (phyB-9) and phyA/phyB double
mutant lines during de-etiolation under different light conditions. Five-day-old etiolated seedlings were irradiated for
3 h under the above conditions with Rc or FRc light and the
STO expression pattern was analysed by Northern blot.
After R light treatment, induction levels of STO were
reduced in the phyA, phyB and the phyA/phyB double
mutant, by comparison with the wild-type, revealing a
contribution of both photoreceptors in the induction of the
gene (Figure S1). Under FR treatment, the expression of STO
in the wild type reached similar levels as under R light. Also,
a clear induction was observed under these conditions in the
phyB mutant, in contrast to the phyA and phyA/phyB double
mutant, where the transcript levels of STO were reduced
(Figure S1). These results suggest that regulation of STO
expression under R and FR light is controlled by phyA and
phyB. Similar levels of induction were observed for STO
after B light treatment in the wild type, phyA, cry1/cry2 and
phot1/phot2 double mutants (Figure S1).
Additional experiments to analyse the level of expression
of STO in the dark were performed with the cop1-4 mutant
line. In contrast to the basic constitutive level of expression
present in the wild type, the cop1-4 mutant clearly exhibited
an increased level of transcript (Figure S1), indicating that
functional COP1 is required to maintain the low transcript
level of STO in etiolated seedlings.
We monitored STO transcript levels in adult plants grown
under short-day (8-h light/16-h dark) conditions. STO mRNA
was present at the end of the dark period and increased
during the light phase. In addition, a decrease of the
transcript was observed at the beginning of the dark period
(Figure 2a). As many light-regulated genes also follow a
circadian regulation (Schaffer et al., 2001), we investigated
whether the circadian clock might control the transcription
of STO. Genes under control of the circadian clock keep on
cycling in the absence of external stimuli (Schoning and
Staiger, 2005), therefore experiments under continuous light
were performed. Three-week-old plants entrained in 12-h
light/dark cycles were transferred to continuous light at the
end of the daily dark phase, and samples were taken for RNA
analysis at different time points during the subjective day/
night cycle. STO followed the same transcriptional regulation pattern as that observed under light/dark conditions,
typical of genes regulated by the circadian clock. Downregulation of the gene at the end of the subjective light phase
and at the start of the subjective dark period, followed by an
upregulation at the end of the subjective dark period and
during the subjective light phase, were indicative of a
circadian regulation of the gene (Figure 2b).
The expression of CHS is regulated by STO
In order to uncover effects of STO on light-inducible genes,
an analysis of the expression of well-characterized lightregulated genes (RBCS1b, CAB3 and CHS) was carried
out. For this purpose, wild-type, sto-T-DNA and 35S:STO
seedlings were grown for 5 days in the dark and exposed
to light treatments of 24 h for R (30 lmol m)2 sec)1), FR
(0.5 lmol m)2 sec)1) or B (1.5 lmol m)2 sec)1). The results
showed no marked variation of the transcript level of CAB3
and RBCS1b in the transgenic lines in comparison with
the wild type. However, in the sto-T-DNA line, for CHS, a
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Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 51, 563–574
566 Martin Indorf et al.
Figure 2. Diurnal and circadian regulation of
STO expression.
(a) Analysis of STO transcription in 6-week-old
plants growing under short-day conditions (8-h
light/16-h dark) at different time points during the
dark or light phase.
(b) Transcript levels of STO in 3-week-old plants
grown under 12-h dark/light cycles and transferred to continuous light. Total RNA samples
were taken at different time points during the last
dark period or after transfer to continuous light.
The subjective light/dark cycle is indicated
below. Equal loading of the samples was verified
with an 18S RNA probe. The graphs show the
STO/18S expression of a representative experiment from at least three repeats.
Figure 3. Expression of light-regulated genes in wild-type, STO-overexpressor and T-DNA transgenic lines.
(a) Transcript levels of CHS, CAB3 and RBCS in 5-day-old dark-grown seedlings of wild-type (wt), sto-T-DNA (sto–) and STO-overexpressor (ox) lines before (D) or
after illumination for 3 h with red (R), far-red (FR) or blue (B) light. Equal loading of the samples was verified with an 18S RNA probe. The graphs show the
quantification of a representative blot, out of at least three repetitions, as relative expression of the genes/18S compared with the wild-type line.
(b) Representative pictures of 5-day-old wild-type (WT), sto-T-DNA, RNAi and overexpressor lines grown under continuous far-red (FR; 1.35 lmol m)2 sec)1) or blue
(B; 37 lmol m)2 sec)1) light.
significant upregulation of the transcript could be observed
under FR and B light treatments (Figure 3a). Also we observed that the T-DNA and RNAi seedlings grown for 5 days
under FRc or Bc presented a higher anthocyanin accumulation in the upper part of the hypocotyl (Figure 3b).
Accumulation of STO is light dependent
The subcellular localization of STO was analysed in transgenic lines overexpressing STO:eGFP fusions under the
control of the CaMV35S promoter. Five-day-old etiolated
seedlings were analysed by fluorescence microscopy
immediately after the period of growth in the dark, or after
transfer to white light (WL) for several hours. In etiolated
seedlings no GFP fluorescence was detected (Figure 4a).
However, in the seedlings transferred to the light, GFPcontaining nuclei started to appear in the hypocotyls after
1–1.5 h of light exposure, and the number increased during
prolonged illumination of up to 5 h. In roots and cotyledons
fluorescent nuclei appeared after only 3 h of illumination
(Figure 4a). In cotyledons the fluorescence signal decreased
after 5 h of illumination; in the root it was still visible after
7 h (Figure 4a). After 24 h of continuous light exposure, the
GFP signal was barely detectable. The decay of the fluorescent signal was also analysed after transferring the seedlings to the dark, preceded by different periods of
illumination. The plants were exposed to light for 1.5 h and
transferred to the dark or kept in the light and analysed 1.5 h
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Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 51, 563–574
STO, a new light signalling intermediate 567
Figure 4. Light-dependent accumulation of
STO:eGFP during seedling de-etiolation.
(a) Five-day-old dark-grown seedlings overexpressing STO:eGFP were analysed under fluorescence microscopy before (Dark) and after
being transferred to white light (WL) for different
time periods (as indicated). The pictures represent images taken from cotyledon, hypocotyl
and root cells.
(b) Protein gel-blot analysis of crude extracts for
the detection of STO:eGFP fusions. Five-day-old
dark-grown seedlings of the transgenic
STO:eGFP overexpressing line, and a wild-type
(WT) and a 35S:eGFP line as negative and
positive (less loaded) controls, were used before
the light treatment (D) or after exposure for 4 and
24 h to WL. Polyclonal antibodies raised against
GFP or monoclonal anti-actin antibodies for
normalization were used for immunodetection.
later. Whereas the seedlings kept in the light had an increased number of GFP-containing nuclei, those transferred
to the dark did not show any fluorescence. Similar results
were obtained when transferring the seedlings to the dark
after irradiation for 3 h and examination again 2 h later (data
not shown). These results indicate that nuclear accumulation of the STO:eGFP fusion increases with time only if the
plants are kept under continuous light.
Western blot analysis was performed to investigate
whether the absence of GFP signal in the dark, and during
the longer exposure to light, was caused by degradation of
the fusion protein or re-localization into a different subcellular compartment. Protein extracts of 5-day-old wild-type
plants, a transgenic line overexpressing eGFP and transgenic seedlings overexpressing the STO:eGFP fusion were
isolated from dark-grown seedlings, or after exposure to WL
for either 4 or 24 h. A band of approximately 60 kDa,
corresponding to the STO:eGFP fusion protein, could only
be detected using polyclonal antibodies specific for GFP in
the sample taken after 4 h of light treatment, indicating that
during de-etiolation of the seedlings, light stabilizes the
fusion protein during the first hours in the cells (Figure 4b).
Co-localization of STO and COP1 in onion epidermal cells
The interaction between STO and COP1 in yeast two-hybrid
assays was previously described by Holm et al., 2001; In
order to analyse in which subcellular compartment the
interaction between STO and COP1 might take place in vivo,
localization of STO and COP1 proteins was analysed in
transient expression experiments. Expression vectors
containing the STO:CFP and YFP:COP1 translational fusions,
under the control of the CaMV35S promoter, were used for
either single transfection or co-transfection of onion
epidermal cells. Single transient transformation of each
construct revealed localization of the STO:CFP fusion protein
preferentially to the nucleus, although there was also some
CFP fluorescence detectable in the cytosol (Figure 5). The
fusion protein appeared in a diffuse distribution without any
recognizable structures. By contrast, the YFP:COP1 fusion
protein was found in aggregations of different sizes in the
cytosol, whereas in the nucleus the YFP fluorescence was
limited to speckles of more or less equal size (Figure 5), as
described previously (Stacey et al., 1999; Stacey and von
Arnim, 1999). However, co-expression of STO:CFP and
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Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 51, 563–574
568 Martin Indorf et al.
Figure 5. Co-localization of salt tolerance (STO)
and CONSTITUTIVE PHOTOMORPHOGENESIS 1
(COP1).
Onion epidermal cells were transformed by
particle bombardment and analysed by fluorescence microscopy after 24 h. The different panels
show images taken for the following constructs
analysed: single transformation by 35S:
STO:CFP, single transformation by 35S:YFP:COP1, and co-transformation by 35S:STO:CFP
and 35S:YFP:COP1; lower panels show a higher
magnification of a nucleus with speckles. Lefthand panels show images taken with the CFP
channel (STO:CFP distribution) and right-hand
panels are images taken with the YFP channel
(YFP:COP1 distribution). Insert panels represent
the DIC picture. Arrows point to nuclear fluorescence and arrow heads point to cytoplasmic
aggregates.
YFP:COP1 fusion proteins into onion epidermal cells led to
co-localization of both proteins in the same nuclear speckles
and cytoplasmic aggregations, indicating that the overexpression of COP1 causes recruitment of STO to the same
protein aggregations (Figure 5).
for the parental line (Figure 6). In the case of the siblings
exhibiting a cop1-4 phenotype, we did not observe a dramatic change of the GFP accumulation during the light
treatment (data not shown). These results indicate that COP1
is responsible for the short life of the protein fusion in the
etiolated tissues.
COP1 mediates STO:GFP degradation in the dark
The interaction of STO with COP1, together with the spatial–
temporal dynamics of STO localization in the cell, raised the
question whether COP1 is responsible for the degradation of
the protein. Crosses of cop1-4 with a transgenic line overexpressing the STO:eGFP fusion protein were performed,
and the F2 generation was analysed after growing the
seedlings for 5 days in the dark. The cop1-4 mutants harbouring the STO:GFP transgene exhibited GFP fluorescence
in nuclei of roots, hypocotyl and cotyledons cells to different
extents, whereas the dark-grown wild-type progeny had no
detectable GFP. After exposure to light, approximately 75%
of those seedlings presenting a wild-type phenotype accumulated GFP in the cell nuclei, as was previously observed
Overexpression of STO partially suppresses the cop1-4
phenotype under red light
In order to analyse the possible genetic interactions between
cop1-4 and STO, we performed crosses of the cop1-4 mutant
with two different transgenic lines overexpressing STO and
analysed the homozygous cop1-4_35S:STO lines. Seeds
were germinated and grown for 5 days in dark or under Rc
(30 lmol m)2 sec)1), FRc (0.5 lmol m)2 sec)1) and Bc
(1.5 lmol m)2 sec)1). Hypocotyl measurements showed
that the cop1-4 mutant did not differ in the hypocotyl length
from homozygous cop1-4 lines overexpressing STO when
grown under FRc or Bc. However, under Rc the seedlings
overexpressing STO presented slightly (but significantly)
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 51, 563–574
STO, a new light signalling intermediate 569
Figure 6. STO:eGFP accumulates in the cop1-4
mutant background in the dark.
F2 progeny from a cross between cop1-4 and a
line overexpressing STO:eGFP was grown for
5 days in the dark, and siblings presenting a
cop1-4 or wild-type phenotype were analysed
under fluorescence microscopy before (dark) and
after being transferred to light. Images are from
cotyledon, hypocotyl and root cells.
longer hypocotyls than the cop1-4 mutant (Figure 7a), indicating that STO functions independently or downstream of
COP1 in the regulation of the red-light mediated inhibition of
hypocotyl elongation. No significant differences were
observed amongst the dark-grown seedlings (data not shown).
STO loss-of-function mutants show enhanced light
sensitivity in the cop1-4 background
To investigate a putative COP1 dependency of the STO lossof-function effects, crosses between cop1-4 and sto-T-DNA
were performed and the F3 generation was analysed.
Homozygous cop1-4/sto double mutants were grown for
5 days in the dark or under the above-described light conditions, and the hypocotyls length of the progeny from two
different crosses was measured (Figure 7b). A clear reduction of the hypocotyls length was observed under the tested
light conditions in the cop1-4/sto double mutant, in comparison with the cop1-4 mutant line. This indicates that the
STO loss-of-function phenotype is also visible in the
absence of active COP1.
Discussion
The STO protein from Arabidopsis thaliana was previously
characterized as a protein conferring salt tolerance when
ectopically expressed in yeast cells (Lippuner et al., 1996).
We isolated STO by complementation of a yeast strain that
exhibits salt sensitivity, confirming the data obtained by
Lippuner et al. (1996). However, Nagaoka and Takano (2003)
Figure 7. Phenotype of salt tolerance (STO) loss- and gain-of-function
mutants in the cop1-4 background under different light conditions.
Seedlings of cop1-4 and the progeny from crosses with (a) 35:STO and (b) stoT-DNA were grown for 5 days in the dark or under continuous red (Rc;
30 lmol m)2 sec)1), far-red (FRc; 0.5 lmol m)2 sec)1) and blue (Bc;
1.5 lmol m)2 sec)1) light. Relative hypocotyl measurements of light versus
dark-grown seedlings are presented as means and SE of > 40 plants.
**Statistical significant differences (P < 0.01).
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Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 51, 563–574
570 Martin Indorf et al.
reported that overexpression of STO in Arabidopsis resulted
in an improved root growth of the seedlings in a medium
containing a high salt concentration. We performed salt
tolerance experiments using sto-T-DNA and RNAi lines, but
our results did not indicate a prominent role for STO in salt
stress, as the STO loss-of-function lines did not display any
distinguishable salt tolerance or sensitivity phenotype (data
not shown). However, our investigations using Arabidopsis
gain- and loss-of-function transgenic lines revealed that STO
participates in the light-signalling cascade. Inhibition of hypocotyl elongation during de-etiolation is mediated by different plant photoreceptors (Fankhauser and Casal, 2004),
and we could show in fluence-dependent irradiation
experiments that the STO transgenic lines were affected in
this response. These results indicate a significant role for
STO as negative regulator of light-mediated inhibition of
hypocotyl elongation. Furthermore, the gain- and loss-offunction alleles provoked coordinated reciprocal growth of
hypocotyl and cotyledon cells induced by light, providing
further evidence that the phenotypes observed are not a
consequence of a general cell growth effect of the mutation,
as discussed by Khanna et al. (2006). Interestingly, the lossof-function mutant of a close STO homologue, STH, also
presents a short hypocotyl phenotype under R and FR light,
but does not show a cotyledon size phenotype (Khanna
et al., 2006). Whether STH has a central regulatory function in light signalling, or is involved in specific hypocotyl
responses to light, or more general growth processes,
still has to be elucidated. In the dark, no marked differences
were observed within the T-DNA, RNAi and overexpressing
lines compared with wild type, indicating a lack of function
of STO during skotomorphogenesis. In addition, analysis
of CHS gene expression in the sto-T-DNA lines revealed
that STO is required for accurate regulation of the
light-dependent expression of this gene. Regulation of CHS
transcription in the T-DNA line was significantly altered
after FR and B light induction, revealing STO as a negative
regulator of CHS expression in B light and phyA dependent
FR signalling.
Our analyses of the expression of STO are in agreement
with results of microarray studies (Jiao et al., 2003; Tepperman et al., 2001, 2004, 2006). Normal induction of STO
transcription during de-etiolation requires functional phyA
and phyB, indicating that STO activity in light signalling is
downstream of the photoreceptors. Moreover, under B light
conditions, induction of STO gene expression is not exclusively dependent on functional phyA, cry1, cry2, phot1 or
phot2, suggesting that different photoreceptors might share
overlapping functions for the B light dependent expression
of this gene.
The analysis of STO transcription in adult plants revealed
that the gene is under the control of the circadian clock. The
circadian-regulated STO mRNA pattern is similar to that
observed for numerous light-regulated genes that typically
function in light. Microarray experiments have shown that
the wide majority of genes encoding enzymes of the
phenylpropanoid biosynthesis are regulated by the circadian clock to peak before dawn, whereas photosynthesis
genes peak near the middle of the day (Harmer et al., 2000).
In the present study it was shown that the STO transcript
level increases in the late dark phase, and remains elevated
during the light phase. Thus, the maximal transcript accumulation of genes of the phenylpropanoid biosynthesis
pathway is followed by a maximal STO transcript level.
Interestingly, it has been recently shown that transcription of
SUPRESSOR OF PHYA-105 (SPA1), a negative regulator of
phyA-mediated light responses in Arabidopsis (Hoecker
et al., 1998), is regulated by the circadian clock. SPA1 mRNA
levels increase at the end of the subjective night and
decrease towards the subjective dusk (Harmer et al., 2000;
Ishikawa et al., 2006). Similar to the STO loss-of-function
mutants, spa1 mutant seedlings accumulate anthocyanin to
higher levels compared with wild type under continuous FR
and B light (Hoecker et al., 1998; Yang et al., 2005). Thus, it is
tempting to speculate that both proteins might be part of a
negative regulatory network controlled by the circadian
clock, mediating a fine tuning of light-regulated gene
expression and thereby preventing an exaggerated light
response.
In the dark, COP1 represses STO transcription. COP1 is a
negative regulator of light signalling with an active role
during skotomorphogenesis (Yi and Deng, 2005). Microarray
experiments showed that expression profiles between wildtype seedlings grown under WL and cop1 mutants grown in
the dark are qualitatively very similar (Ma et al., 2002).
Regulation of STO expression by COP1 would suggest a role
for STO downstream of COP1. Nevertheless, STO does not
seem to have a function in skotomorphogenesis but only
functions during de-etiolation processes. This is supported
by the fact that dark-grown cop1-4 seedlings overexpressing
STO exhibit the same hypocotyl length as cop1-4 (data not
shown). However, a small but significant difference in the
hypocotyl length was observed when grown under Rc, albeit
that there was no difference (in the tested conditions) when
grown under FRc or Bc. The STO-overexpressing phenotype, observed in a wild-type background for all light
conditions, surprisingly only appeared under R light conditions in a cop1-4 mutant background. Interestingly, promotion of photomorphogenesis by COP1 under R light, but not
under FR or B light, has been observed using weak cop1
alleles, as well as COP1-overexpression lines (Boccalandro
et al., 2004; Khanna et al., 2006; Stacey et al., 1999). These
observations led to the suggestion of two models in
which COP1 would activate phyB-mediated transcription,
or alternatively would mediate degradation of a lightinduced negative regulator of phyB signalling (Boccalandro
et al., 2004). Our data would strengthen the second hypothesis. Under R light conditions, the STO-overexpression
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Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 51, 563–574
STO, a new light signalling intermediate 571
phenotype can be observed in a cop1-4 mutant background,
as STO function is activated through a COP1-independent
pathway and therefore displays a negative effect on photomorphogenesis. STO could act as the proposed phyBinduced repressor in the model of Boccalandro et al.
(2004). However, to the contrary, in B and FR light, the
negative regulatory function of STO seemed to be dependent on functional COP1. Interestingly, the analysis of the
cop1-4/sto double mutants revealed the same effect of the
STO loss-of-function in the cop1-4 mutant as in the wild
type, indicating that STO negatively regulates photomorphogenesis partially, if not completely, independently of COP1.
We analysed the light-dependent subcellular distribution
of STO in transgenic lines overexpressing an STO:eGFP
fusion protein. Accumulation of the chimeric protein in the
nucleus is a light-dependent process, whereas disappearance of the fusion protein occurs independently of the light
conditions. Moreover, accumulation of the protein in the
nucleus is not a result of subcellular redistribution but of
light-dependent stabilization.
We could also show that the degradation of the protein in
the dark is mediated by COP1. COP1 is a RING-type E3
ubiquitin ligase that directly interacts with positive regulators of the light signalling to mediate degradation of these
proteins. Modulation by regulated proteolysis is likely to be
a central theme for controlling the specificity and the
magnitude of STO function, as observed for other molecules
(Osterlund et al., 2000; Holm et al., 2002; Saijo et al., 2003;
Seo et al., 2003; Bauer et al., 2004; Duek et al., 2004; Park
et al., 2004; Seo et al., 2004; Jang et al., 2005; Shen et al.,
2005; Yang et al., 2005). Likewise with STO transcription,
STO protein abundance is tightly controlled by light. The
rapid accumulation of the fusion protein under WL illumination, and the subsequent disappearance after prolonged
light exposure, suggest that STO might be required for the
initial transition from skotomorphogenesis to light-adapted
development.
Holm et al. (2001) reported an interaction between COP1
and STO and the homologous protein STH. Our transient
expression experiments using STO:CFP and YFP:COP1
translational fusions indicated the presence of both proteins
in the nucleus, and in the cytosol with different pattern
distributions. Co-expression of the fusions resulted in the
co-localization of both proteins in the same aggregates,
indicating that overexpressed COP1 retains the STO protein
in these aggregates in all subcellular compartments. This,
together with the data published by Holm et al. (2001),
would suggest a direct interaction between COP1 and STO in
living plant cells. Several positive regulators of light signal
transduction co-localize with COP1 after transient expression in onion epidermal cells (Ang et al., 1998; Ballesteros
et al., 2001; Holm et al., 2002; Seo et al., 2003; Jang et al.,
2005; Datta et al., 2006). These proteins are exclusively
localized in the nucleus. Accumulation of the proteins in
nuclear speckles occurs either independently of co-expressed COP1 (Ballesteros et al., 2001; Jang et al., 2005;
Datta et al., 2006), or depends on the presence of coexpressed COP1 (Ang et al., 1998; Holm et al., 2002), as also
observed for STO. However, the relevance of the co-localization of STO and COP1 in cytosolic aggregates remains
elusive, and opens new perspectives for a possible function
of COP1 in the cytosol.
Experimental procedures
Plant material, growth and light conditions
All mutants (Reed et al., 1993; McNellis et al., 1994; Reed et al.,
1994; Mockler et al., 1999) and transgenic lines used in this study
were in the Columbia background and were compared with wildtype Col-0 in all analyses. The sto-T-DNA line (SALK_067473) was
obtained from the Nottingham Arabidopsis Stock Centre (http://
arabidopsis.info). Seeds were placed on filter paper soaked in water
and kept for 48 h at 4C in the dark for stratification. After exposure
to WL (100 lmol m)2 sec)1) for 9 h to stimulate simultaneous
germination, seeds were transferred to different light conditions.
De-etiolation experiments were performed in continuous R, FR and
B light at different fluence rates. The light sources used are
described in Kircher et al. (2002) and Kaiser et al. (1995).
Hypocotyl and cotyledon measurements
Hypocotyl length of seedlings was measured to the nearest 0.5 mm
with a ruler or using ZEISS AXIOVISION 4.0 software. The absolute
length of at least 30 seedlings per line was estimated and the
average value was used for comparison of the different lines.
Cotyledon area was measured with the same software package.
Statistical analysis was performed as described by Khanna et al.,
2006.
Identification of the sto-T-DNA line
Database research (http://www.arabidopsis.org) led to the identification of an STO T-DNA line (Salk_067473) containing the insertion
in the first intron of STO. The T-DNA was verified by PCR using the
left-border specific primer LBb1_5¢- GCGTGGACCGCTTGCTGCAACT-3¢ and an STO-specific reverse primer 5¢-GGGAAGCTTGAACAAAACTCAAACACAGACATTTGT-3¢. The specificity of the
corresponding PCR product was verified by sequencing.
Plasmid construction
STO full-length cDNA was amplified by PCR from an A. thaliana
cDNA library (Matchmaker; Clontech, http://www.clontech.com)
using 5¢-CGGAATTCATCCCACCTACTTGTTCCCCACA-3¢ as the
forward primer and 5¢-GGGAAGCTTGAACAAAACTCAAACACAGACATTTGT-3¢ as the reverse primer, and was cloned into
the plant binary vector pCambia 1390_35S. The STO RNAi
plasmid was constructed by PCR amplification of a STO sense
and antisense fragment using the following primers: sense-for,
5¢-AACTCGAGACCTGAGCCTTCCAACAACCA-3¢, and sense-rev,
5¢-TCGGTACCGGTCTCAAACCTCGGCTTCTT-3¢; antisense-for, 5¢AATCTAGAACCTGAGCCTTCCAACAACCA-3¢, and antisense-rev, 5¢TCAAGCTTGGTCTCAAACCTCGGCTTCTT-3¢. Both fragments were
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 51, 563–574
572 Martin Indorf et al.
cloned first into the T-DNA cassette of the pHannibal vector before
introducing it into the plant binary vector pArt27, as described by
Wesley et al., 2001; The 35S:STO:eGFP vector was generated by
PCR amplification of STO using the forward primer, 5¢CGACCGGTATCCCACCTACTTGTTCCCCACA-3¢, and reverse primer, 5¢-GAACCGGTATAGCTTTTAAGCCAAGATCAGGGACA-3¢. The
product was digested with Age I and cloned into the plant binary
vector pEGAD (Cutler et al., 2000).
For the co-localization construct, STO cDNA was amplified by
PCR using the forward primer, 5¢-CGGGTACCATCCCACCTACTTGTTCCCCA-3¢, and the reverse primer, 5¢-TTTACCCGGGATCAGGACAATGAAGTGTTCC-3¢. The PCR fragment was cloned in
frame with the CFP cDNA in the plasmid pMAV_35S:CFP.
Plant transformation and selection of transgenic lines
The plant binary vectors were introduced into Agrobacterium tumefaciens strain GV3101, and Arabidopsis wild-type plants (Col-0)
were transformed via the floral-dip method (Clough and Bent, 1998).
For selection of transgenic lines, surface-sterilized seeds were germinated on 0.5 · MS, 1% sucrose, 0.8% agar media supplied with
either 25 lg ml)1 hygromycin B or 50 lg ml)1 kanamycin. Plants
containing the pEGAD constructs were grown on soil and after
2 weeks were sprayed with Basta (240 lg ml)1, 0.005% Silvet L-77).
Northern blot analysis
Total RNA was isolated using Concert Plant RNA Extraction
Reagent, according to the manufacturer’s protocol (Invitrogen, http://
www.invitrogen.com). RNA-blot hybridizations were performed
following standard protocols (Sambrook et al., 1989). Specific
probes for the different genes were amplified from genomic DNA
using the following primer sets: CAB3-for, 5¢-CTTCGCAACCAACTTTGTTC-3¢; CAB3-rev, 5¢-TGAGTTTGATTAATGACAAATCATAC-3¢; CHS-for, 5¢-GTGCCATAGACGGACATTTGAG-3¢; CHS-rev,
5¢-CACACCATCCTTAGCTGACTTC-3¢; RBCS-for, 5¢-GCACGGATTTGTGTACCGTGAG-3¢; RBCS-rev, 5¢-GGTTCCGGATAGTCAACATTGAATA-3¢, or were isolated from plasmids containing the STO cDNA
or the 18S rRNA. Autoradiograms were scanned and quantified
using QUANTITYONE software (Bio-Rad, http://www.biorad.com).
b-mercaptoethanol, 5 mM -aminocaproate, 1 mM benzamidine).
Samples were centrifuged for 10 min at 20 000 g in a microfuge and
the supernatants containing the protein extracts were quantified
using Bio-Rad Protein Assay Dye Reagent (Bio-Rad). SDS-PAGE
sample buffer (5x) was added to the supernatant containing 50 lg of
total protein and samples were heated at 95C for 5 min. Total
proteins were separated on a 12% SDS-PAGE gel and transferred
onto ImmobilonTM-P transfer membrane (Millipore, http://
www.millipore.com). Inmunodetection of eGFP was performed
using rabbit polyclonal antibodies raised against the green fluorescent protein or mouse monoclonal plant anti-actin (AtACT8)
antibodies (Sigma, http://www.sigmaaldrich.com) as primary antibodies, and a peroxidase-coupled anti-rabbit or anti-mouse antiserum (Sigma) as secondary antibodies. Detection of the proteins
was performed using the ECLTM Western Blotting Detection
Reagents Kit (Amersham, http://www.amersham.com).
Acknowledgements
The sto-T-DNA line was obtained from NASC. We thank Ralf Welsch,
Andreas Hiltbrunner, Stefan Kircher, Tim Kunkel and Roman Ulm
for providing some of the constructs, mutant seeds and the polyclonal GFP-antibodies. We are thankful to Eberhard Schäfer, Stefan
Kircher, Tim Kunkel and Salim Al-Babili for helpful discussions. We
would like to thank Eija Schulze for excellent technical assistance.
We are in debt to Eberhard Schäfer for the use of the light facilities
of his lab. We thank BioMed Proofreading for English corrections.
The work was supported by the GIF (GIF Grant No: 670) and by the
DAAD.
Supplementary Material
The following supplementary material is available for this article
online:
Figure S1. Comparison of STO expression in wild type and different
mutants under different light conditions. STO transcription level of
5-day-old etiolated seedlings (Dark) or irradiated for 3 h with blue
(B), red (R) or far-red (FR) light. Equal loading of the samples was
verified with an 18S RNA probe. The graphs show the STO/18S
expression relative to that of the wild type from a representative
experiment out of at least three repeats.
Transient expression in onion epidermal cells
Expression was achieved by particle bombardment, performed as
described by Klein et al., 1987.
Epifluorescence microscopy
Epifluorescence and light microscopy on plant seedlings and epidermal stripes of onion cells was performed with an Axiovision
microscope with the appropriate filter settings (Zeiss, http://
www.zeiss.com). Pictures were taken with a digital camera system
using the AXIOVISION software 4.0 (Zeiss). Photographs were
mounted with ADOBE PHOTOSHOP (http://www.adobe.com).
Protein extraction and Western blotting
Seedlings were grown for 5 days in the dark and exposed to WL
(100 lmol m)2 sec)1) for either 4 or 24 h. Proteins were extracted by
homogenizing 100 mg of seedlings in a potter using 250 ll of
extraction buffer (100 mM NaH2PO4, pH 7.2, 1 mM DTT, 7 mM
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