The Plant Journal Salt tolerance (STO), a stress …Salt tolerance (STO), a stress-related protein, has a major role in light signalling Martin Indorf, Julio Cordero, Gunther Neuhaus,

Salt tolerance (STO), a stress-related protein, has a majorrole in light signalling

Martin Indorf, Julio Cordero, Gunther Neuhaus, and Marta Rodrıguez-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

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). Surpris-

ingly, 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). How-

ever, an interaction of STO with CONSTITUTIVE PHOTO-

MORPHOGENESIS 1 (COP1), a negative regulator of

photomorphogenesis in the dark, has also been reported

(Holm et al., 2001; Ma et al., 2002).

ª 2007 The Authors 563Journal compilation ª 2007 Blackwell Publishing Ltd

The Plant Journal (2007) 51, 563–574 doi: 10.1111/j.1365-313X.2007.03162.x

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. Here-

in 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. More-

over, 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 con-

taining the T-DNA insertion in the first intron of the STO

gene (SALK_067473). In addition, STO RNAi and overex-

pressor 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 ab-

sence, 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 wild-

type (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 rep-

resent the SE of > 45 plants. **Significant differ-

ences (P < 0.01) compared with the wild type.

564 Martin Indorf et al.

ª 2007 The AuthorsJournal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 51, 563–574

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 22�C 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-overexpress-

ing 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 (Fig-

ure 1e). Photomorphogenic mutants typically present recip-

rocal 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 regula-

ted.

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 con-

ditions. 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 regula-

tion pattern as that observed under light/dark conditions,

typical of genes regulated by the circadian clock. Downreg-

ulation 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 light-

regulated 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

STO, a new light signalling intermediate 565


significant upregulation of the transcript could be observed

under FR and B light treatments (Figure 3a). Also we ob-

served that the T-DNA and RNAi seedlings grown for 5 days

under FRc or Bc presented a higher anthocyanin accumula-

tion in the upper part of the hypocotyl (Figure 3b).

Accumulation of STO is light dependent

The subcellular localization of STO was analysed in trans-

genic 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, GFP-

containing 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 fluores-

cent signal was also analysed after transferring the seed-

lings 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

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 trans-

ferred 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 experi-

ment 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.



later. Whereas the seedlings kept in the light had an in-

creased 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 accumula-

tion 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 subcel-

lular compartment. Protein extracts of 5-day-old wild-type

plants, a transgenic line overexpressing eGFP and trans-

genic 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

Figure 4. Light-dependent accumulation of

STO:eGFP during seedling de-etiolation.

(a) Five-day-old dark-grown seedlings overex-

pressing STO:eGFP were analysed under fluor-

escence microscopy before (Dark) and after

being transferred to white light (WL) for different

time periods (as indicated). The pictures repre-

sent 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.



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 overex-

pression of COP1 causes recruitment of STO to the same

protein aggregations (Figure 5).

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 over-

expressing 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 har-

bouring 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 accu-

mulated GFP in the cell nuclei, as was previously observed

for the parental line (Figure 6). In the case of the siblings

exhibiting a cop1-4 phenotype, we did not observe a dra-

matic 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.

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)

Figure 5. Co-localization of salt tolerance (STO)

and CONSTITUTIVE PHOTOMORPHOGENESIS 1

(COP1).

Onion epidermal cells were transformed by

particle bombardment and analysed by fluores-

cence 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. Left-

hand 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 fluores-

cence and arrow heads point to cytoplasmic

aggregates.



longer hypocotyls than the cop1-4 mutant (Figure 7a), indi-

cating 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 loss-

of-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 con-

ditions, and the hypocotyls length of the progeny from two

different crosses was measured (Figure 7b). A clear reduc-

tion of the hypocotyls length was observed under the tested

light conditions in the cop1-4/sto double mutant, in com-

parison 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 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.

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) sto-

T-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).



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 hy-

pocotyl elongation during de-etiolation is mediated by dif-

ferent 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-of-

function 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 loss-

of-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 func-

tion 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; Tepper-

man 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 exclu-

sively 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 circa-

dian 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 accu-

mulation 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 wild-

type 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 pheno-

type, observed in a wild-type background for all light

conditions, surprisingly only appeared under R light condi-

tions in a cop1-4 mutant background. Interestingly, promo-

tion 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 light-

induced negative regulator of phyB signalling (Boccalandro

et al., 2004). Our data would strengthen the second hypo-

thesis. Under R light conditions, the STO-overexpression



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 photo-

morphogenesis. STO could act as the proposed phyB-

induced 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 depend-

ent 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 photomorph-

ogenesis 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 disappear-

ance 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 regula-

tors 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 illumin-

ation, 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 expres-

sion 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-ex-

pressed COP1 (Ballesteros et al., 2001; Jang et al., 2005;

Datta et al., 2006), or depends on the presence of co-

expressed COP1 (Ang et al., 1998; Holm et al., 2002), as also

observed for STO. However, the relevance of the co-local-

ization 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 studywere in the Columbia background and were compared with wild-type Col-0 in all analyses. The sto-T-DNA line (SALK_067473) wasobtained from the Nottingham Arabidopsis Stock Centre (http://arabidopsis.info). Seeds were placed on filter paper soaked in waterand kept for 48 h at 4�C in the dark for stratification. After exposureto WL (100 lmol m)2 sec)1) for 9 h to stimulate simultaneousgermination, seeds were transferred to different light conditions.De-etiolation experiments were performed in continuous R, FR andB light at different fluence rates. The light sources used aredescribed 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 mmwith a ruler or using ZEISS AXIOVISION 4.0 software. The absolutelength of at least 30 seedlings per line was estimated and theaverage 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 identifi-cation of an STO T-DNA line (Salk_067473) containing the insertionin the first intron of STO. The T-DNA was verified by PCR using theleft-border specific primer LBb1_5¢- GCGTGGACCGCTTGCTGCA-ACT-3¢ and an STO-specific reverse primer 5¢-GGGAAGCTTGAA-CAAAACTCAAACACAGACATTTGT-3¢. The specificity of thecorresponding PCR product was verified by sequencing.

Plasmid construction

STO full-length cDNA was amplified by PCR from an A. thalianacDNA library (Matchmaker; Clontech, http://www.clontech.com)using 5¢-CGGAATTCATCCCACCTACTTGTTCCCCACA-3¢ as theforward primer and 5¢-GGGAAGCTTGAACAAAACTCAAACAC-AGACATTTGT-3¢ as the reverse primer, and was cloned intothe plant binary vector pCambia 1390_35S. The STO RNAiplasmid was constructed by PCR amplification of a STO senseand 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



cloned first into the T-DNA cassette of the pHannibal vector beforeintroducing it into the plant binary vector pArt27, as described byWesley et al., 2001; The 35S:STO:eGFP vector was generated byPCR amplification of STO using the forward primer, 5¢-CGACCGGTATCCCACCTACTTGTTCCCCACA-3¢, and reverse pri-mer, 5¢-GAACCGGTATAGCTTTTAAGCCAAGATCAGGGACA-3¢. Theproduct was digested with Age I and cloned into the plant binaryvector pEGAD (Cutler et al., 2000).

For the co-localization construct, STO cDNA was amplified byPCR using the forward primer, 5¢-CGGGTACCATCCCACC-TACTTGTTCCCCA-3¢, and the reverse primer, 5¢-TTTACCCGGGAT-CAGGACAATGAAGTGTTCC-3¢. The PCR fragment was cloned inframe 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 tu-mefaciens 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 ger-minated on 0.5 · MS, 1% sucrose, 0.8% agar media supplied witheither 25 lg ml)1 hygromycin B or 50 lg ml)1 kanamycin. Plantscontaining the pEGAD constructs were grown on soil and after2 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 ExtractionReagent, according to the manufacturer’s protocol (Invitrogen, http://www.invitrogen.com). RNA-blot hybridizations were performedfollowing standard protocols (Sambrook et al., 1989). Specificprobes for the different genes were amplified from genomic DNAusing the following primer sets: CAB3-for, 5¢-CTTCGCAAC-CAACTTTGTTC-3¢; CAB3-rev, 5¢-TGAGTTTGATTAATGACAAATCA-TAC-3¢; CHS-for, 5¢-GTGCCATAGACGGACATTTGAG-3¢; CHS-rev,5¢-CACACCATCCTTAGCTGACTTC-3¢; RBCS-for, 5¢-GCACGGATTT-GTGTACCGTGAG-3¢; RBCS-rev, 5¢-GGTTCCGGATAGTCAACATTG-AATA-3¢, or were isolated from plasmids containing the STO cDNAor the 18S rRNA. Autoradiograms were scanned and quantifiedusing QUANTITYONE software (Bio-Rad, http://www.biorad.com).

Transient expression in onion epidermal cells

Expression was achieved by particle bombardment, performed asdescribed by Klein et al., 1987.

Epifluorescence microscopy

Epifluorescence and light microscopy on plant seedlings and epi-dermal stripes of onion cells was performed with an Axiovisionmicroscope with the appropriate filter settings (Zeiss, http://www.zeiss.com). Pictures were taken with a digital camera systemusing the AXIOVISION software 4.0 (Zeiss). Photographs weremounted 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 byhomogenizing 100 mg of seedlings in a potter using 250 ll ofextraction buffer (100 mM NaH2PO4, pH 7.2, 1 mM DTT, 7 mM

b-mercaptoethanol, 5 mM �-aminocaproate, 1 mM benzamidine).Samples were centrifuged for 10 min at 20 000 g in a microfuge andthe supernatants containing the protein extracts were quantifiedusing Bio-Rad Protein Assay Dye Reagent (Bio-Rad). SDS-PAGEsample buffer (5x) was added to the supernatant containing 50 lg oftotal protein and samples were heated at 95�C for 5 min. Totalproteins were separated on a 12% SDS-PAGE gel and transferredonto ImmobilonTM-P transfer membrane (Millipore, http://www.millipore.com). Inmunodetection of eGFP was performedusing rabbit polyclonal antibodies raised against the green fluor-escent protein or mouse monoclonal plant anti-actin (AtACT8)antibodies (Sigma, http://www.sigmaaldrich.com) as primary anti-bodies, and a peroxidase-coupled anti-rabbit or anti-mouse anti-serum (Sigma) as secondary antibodies. Detection of the proteinswas performed using the ECLTM Western Blotting DetectionReagents 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 Ulmfor providing some of the constructs, mutant seeds and the poly-clonal GFP-antibodies. We are thankful to Eberhard Schafer, StefanKircher, Tim Kunkel and Salim Al-Babili for helpful discussions. Wewould like to thank Eija Schulze for excellent technical assistance.We are in debt to Eberhard Schafer for the use of the light facilitiesof his lab. We thank BioMed Proofreading for English corrections.The work was supported by the GIF (GIF Grant No: 670) and by theDAAD.

Supplementary Material

The following supplementary material is available for this articleonline:Figure S1. Comparison of STO expression in wild type and differentmutants under different light conditions. STO transcription level of5-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 wasverified with an 18S RNA probe. The graphs show the STO/18Sexpression relative to that of the wild type from a representativeexperiment out of at least three repeats.

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The Plant Journal Salt tolerance (STO), a stress …Salt tolerance (STO), a stress-related protein, has a major role in light signalling Martin Indorf, Julio Cordero, Gunther Neuhaus,

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