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Plant Physiol, December 2000, Vol. 124, pp. 1520-1524
SCIENTIFIC CORRESPONDENCE
The Roles of Photoreceptor Systems and the COP1-Targeted
Destabilization of HY5 in Light Control of Arabidopsis Seedling
Development1
Mark T.
Osterlund,
Ning
Wei, and
Xing Wang
Deng*
Department of Molecular, Cellular, and Developmental Biology, Yale
University, New Haven, Connecticut 06520-8104
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INTRODUCTION |
Arabidopsis seedlings display
contrasting developmental patterns depending on the light environment,
e.g. photomorphogenesis in the light and skotomorphogenesis
(etiolation) in darkness. Recent studies have implicated protein
degradation as a means of regulating this developmental switch
(Osterlund et al., 2000 ). For example, HY5, a positive photomorphogenic
regulator that acts to promote the light developmental pattern
(Koornneef et al., 1980; Oyama et al., 1997), is subject to the control
of negative photomorphogenic regulators such as COP1 and COP9
signalosome at the level of protein stability (Osterlund et al.,
1999, 2000). Light controls this process by modulating the subcellular
localization of COP1. COP1 is predominantly cytoplasmic in the light,
but accumulates in the nuclei in the darkness where it directly
interacts with HY5 (von Arnim and Deng, 1994; Ang et al., 1998). A
nuclear COP1-HY5 interaction is necessary for the degradation of HY5
(Osterlund et al., 2000).
HY5 is a basic Leu zipper-type transcription factor and is
localized in the nucleus constitutively (Oyama et al., 1997; Ang et
al., 1998). It has been shown that HY5 binds to the G-box motif of
multiple light-inducible promoters and is necessary for optimal expression of the corresponding genes (Ang et al., 1998; Chattopadhyay et al., 1998a, 1998b). The dark-dependent degradation of HY5
provides a reasonable means by which the activity of HY5 and ultimately HY5-mediated gene expression could be regulated in responsive to light.
We set out to examine HY5-mediated gene expression as a measure of its
activity in correlation with HY5 protein levels.
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THE ACTIVITY OF HY5 CORRELATES TO ITS CELLULAR ABUNDANCE |
Previous reports have shown that the abundance of HY5 directly
corresponds with an etiolated seedling development (Osterlund et al.,
2000). It is important to verify that the activity of HY5 correlates to
protein levels. To determine the activity of HY5, we used a previously
established promoter-reporter transgene (RbcS-GUS) that
expresses the  -glucuronidase reporter gene (GUS) from a Rubisco
small subunit gene (Rbcs) promoter. It has been shown that
light-regulated optimal expression of GUS in the RbcS-GUS transgenic seedlings is dependent on HY5 (Chattopadhyay et al., 1998a,
1998b). Consistent with the recent reports (Osterlund et al.,
2000), Figure 1A (bottom) shows that the
abundance of HY5 protein decreases with decreasing fluence of the
light. It is important to note that in transgenic seedlings
grown under various intensities of continuous white light, the RbcS-GUS
expression decreases proportionally with the level of HY5
and light fluence (Fig. 1A, top). This finding suggests that the
activity of the RbcS promoter correlates with the cellular level of
HY5, and that modulation of HY5 abundance by COP1 could directly
influence HY5-mediated gene expression.
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Figure 1.
Analysis of HY5 in mediating light activation of
its target promoter. A, Rbcs promoter activity as estimated via GUS
from RbcS-GUS transgenic seedlings grown in continuous white
light for 5 d (top). Seedlings were grown in decreasing light
intensities from left to right. The fluence rates were 127.5, 19.0, 8.7, 2.8, and 0.6 µmol s 1
m2, respectively (Osterlund et al., 2000). GUS
activity is measured in arbitrary units.  HY5 western on the
seedlings used for the GUS assay is shown at the bottom. B, The
RbcS-GUS activity from wild type and the hy5-1 seedlings
grown in continuous darkness (D), white light (W), red light (R), blue
light (B), and far-red light (FR). The reporter GUS activity is
measured in arbitrary units. All error bars indicate
SD between four samples.
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MULTIPLE WAVELENGTHS OF LIGHT REGULATE HY5 ACTIVITY |
It has been shown that HY5 acts as a positive regulator of
photomorphogenesis in all light conditions, confirmed by the long hypocotyl phenotype of hy5 mutants in white, red, blue, and
far-red light (Koornneef et al., 1980; Ang and Deng, 1994). As a
transcription factor, HY5 must act downstream of multiple
wavelength-specific signal transduction pathways to regulate the
expression of its target genes (Ang et al., 1998; Chattopadhyay et al.,
1998). We recently showed that HY5 abundance is regulated by
multiple photoreceptor systems (Osterlund et al., 2000). To extend this
study, we examined the effect of an hy5-null mutation on the
downstream target gene expression under the light wavelengths that
selectively activate individual photoreceptors. The Rbcs-GUS
reporter transgene was introduced into the null hy5-1
mutant and its expression under various wavelengths of light was
examined (Fig. 1B). It is clear that the activity of the Rbcs promoter
is reduced in an hy5 mutant background relative to the wild
type in all light conditions. This result supports the notion that the
binding of HY5 to the RbcS promoter is essential for optimal expression
under all light conditions. This result also implied that signaling
pathways, initiated by different photoreceptors, converge to regulate HY5.
Although HY5 is required for optimal light activation of the RbcS
promoter, we noted that in no light environment has the RbcS promoter
activity in the hy5-1 mutant reduced to that of dark-grown
seedlings (Fig. 1B). This finding suggests that additional positive regulators other than HY5 must contribute to the expression of
the light-inducible gene. This finding is consistent with the partially
etiolated phenotype of the hy5-null mutant (Oyama et al.,
1997) and a previous finding that overexpression of HY5 alone has no
obvious phenotypic effect in darkness (Ang et al., 1998).
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ISOLATION OF NEW hy1 AND
hy2 ALLELES AS ENHANCER MUTATIONS OF AN
hy5-NULL MUTANT |
In an effort to identify additional factors that act together with
HY5 to promote photomorphogenesis, we employed genetic screening
for extragenic mutations that enhance the partially etiolated phenotype
of a null hy5 mutant (hy5-ks50; Oyama et al., 1997). Using ethyl methanesulfonate as the mutagen, 31 double mutants exhibited elongated hypocotyls (compared with the
hy5-ks50 parent) with either open or closed cotyledons (Fig.
2A). Seven mutants that exhibited
consistent and the most dramatic long hypocotyl phenotypes under white,
red, blue, and far-red lights were selected for further analysis.
Complementation tests revealed that these seven recessive mutants
belong to two complementation groups. The first group was represented
by a single allele, mutant 88.1 (Fig. 2A). In addition to
the seedlings' long hypocotyl phenotype, the 88.1 mutant
displayed a severe adult phenotype with small flowers
and dramatically reduced fertility (data not shown). The remaining
six mutations are all allelic. 134.1 displayed the most dramatic seedling phenotype (Fig. 2A).
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Figure 2.
Characterization of the hy5 enhancer
mutants. A, Enhancer mutants are shown in comparison with the parental
hy5-ks50 mutant. Seedlings were grown in continuous white
light for 5 d. Left, The hy5 seedling beside a
hy1/hy5 (88.1) double mutant. Right, The
hy5 seedling beside a hy2/hy5 (134.1)
double mutant. B, The representative F2
segregating seedlings from the hy2/hy5 double mutant crossed
to a wild-type plant. The HY5 western corresponding to the
segregating populations is shown in the bottom. The western verifies
the wild-type (WT), hy5, and hy2 genotypes in the
four types of segregants. C, Genomic map positions of the seven
hy5 enhancer mutations as determined by PCR-based
mapping.
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To further characterize the enhancer mutations, we first
outcrossed mutant 134.1 to wild-type plants. In the
F2 generation, we noted that seedlings carrying
the homozygous 134.1 mutation exhibited a long
hypocotyl phenotype on its own (Fig. 2B), and segregated as a single
recessive trait. The white light-grown seedlings containing the
134.1 mutation in a wild-type background resembled hy5
mutants with slightly closed cotyledons (Fig. 2B). To confirm the
genotypes of the segregated populations (wild type, hy5,
134.1, and hy5/134.1), F2
seedlings were allowed to mature and their offspring were used for an
HY5 western blot. Figure 2B (bottom) shows that HY5 is present in the
134.1 mutant but not in the hy5 or
hy5/134.1 mutants. Therefore, hy5 and
134.1 have additive effects that result in a highly
etiolated seedling phenotype in the white light.
To determine if 134.1 is allelic to any known long hypocotyl
mutants (Koornneef et al., 1980), the location of the mutation was
mapped using PCR-based markers and located close to marker nga126, on the upper arm of chromosome three (Fig. 2C). This position was confirmed by the mapping positions of four additional allelic mutants isolated from this screen (174.2, 92.1,
208.2, and 1.1). Of the known long hypocotyl
mutants, hy2 is located within 5 centimorgans of nga126. A
complementation test revealed that 134.1 is allelic to
hy2, indicating that the 1.1, 1.3, 92.1, 134.1, 174.2, and 208.2 mutations defined six new alleles of
the HY2 locus (data not shown).
Past studies has revealed that the HY2 protein defines a biochemical
step in the biosynthesis of the phytochrome chromophore, phytochromobilin from heme (Parks and Quail, 1991; Goto et al., 1993).
Thus the hy2 mutants are expected to have reduced activity of all phytochromes. Therefore, it is possible that the other nonallelic 88.1 mutant might be related to HY1,
the only other known gene in addition to HY2 that is
involved in phytochrome chromophore biosynthesis (Parks and Quail,
1991; Davis et al., 1999). Fine mapping of the 88.1 mutation
located it close to the HY1 locus. To confirm an allelic
relationship between 88.1 and hy1,
88.1/hy5 double mutants were crossed to hy1
plants. Although the F1 seedlings were shorter
than the 88.1/hy5 parent, they are all quite similar to
hy1 mutants (data not shown). Thus mutant 88.1 is
allelic to hy1. Therefore, all strong enhancers of
hy5 that were selected for the study disrupt
chromophore biosynthesis and phytochrome activity.
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GUS-COP1 IS A CONSTITUTIVE NUCLEAR LOCALIZED IN AN
hy1 MUTANT |
The identification of new hy1 and hy2
mutations as enhancers of hy5 indicates that the phytochrome
signaling pathways promote seedling photomorphogenic
development by both HY5-dependent and -independent mechanisms. This
finding is consistent with the speculation that other
transcription factors in addition to HY5 are involved in promoting
photomorphogenesis (Osterlund et al., 1999). In contrast, null
cop1 mutants completely lose the ability to undergo
etiolation and affect expression of a wide range of genes (Deng et al.,
1991). It has been proposed that multiple photoreceptors regulate COP1 activity, whereas COP1 in turn regulates multiple transcription factors, including HY5, that promote photomorphogenesis (Hardtke et
al., 2000; Osterlund et al., 2000). Because COP1 targets proteins for
degradation (Osterlund et al., 2000), mutations that increase the
nuclear abundance of COP1 could also decrease the
activities of these transcription factors, causing etiolated
phenotypes. Therefore, we investigated whether the enhancement of the
hy5 phenotype by the hy1 and hy2
mutants might be attributable to increased nuclear accumulation of
COP1. To test this, the subcellular localization of COP1 fusion protein
was analyzed in an hy1 mutant background. A translation
fusion between the GUS and COP1 (GUS-COP1) has been shown to
represent the subcellular distribution of COP1 (von Arnim and Deng,
1994). After crossing the GUS-COP1 transgene with the
hy1-1 (20.84N) mutant (Koornneef et al., 1980), we analyzed the subcellular localization of COP1.
To determine the abundance of GUS-COP1 in the hypocotyl nuclei, a
semiquantitative analysis of nuclear GUS staining (Osterlund and Deng,
1998) was used. In this analysis, hypocotyl cells showing strong
nuclear staining were given a value of three plus signs (+++), those
with weak but obvious nuclear staining were given a value of two plus
signs (++), and those with undetectable nuclear staining were given a
value of one plus sign (+). This analysis was conducted with the
hy1 seedlings that were grown in various wavelengths
of light for 5 d. As shown in Figure
3, GUS-COP1 accumulates to high levels in
the hypocotyl nuclei of hy1 seedlings grown in all light
conditions tested. This result reinforces the notion that COP1
activity is under the control of upstream photoreceptors (Osterlund and
Deng, 1998) and further indicates that the phytochrome system is
required not only for the red and far-red light control of COP1 nuclear
localization, but it is also essential for the blue-light control of
COP1 nuclear abundance. Therefore, it represents another example of
functional interplay between phytochrome and cryptochrome systems
(Ahmad and Cashmore, 1997; Chattopadhyay et al., 1998a, 1998b;
Neff and Chory, 1998). Taken together, the enhancement of the
hy5 phenotype by hy1 and hy2 might
be a result of the increased nuclear localization of COP1,
which might down-regulate HY5 and, more importantly, other
photomorphogenesis-promoting factors.
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Figure 3.
Effect of hy1 mutation on GUS-COP1
nuclear localization. A, Subcellular localization of GUS-COP1 in
representative wild-type and hy1 seedlings. a, Typical
cytoplasmically localized GUS-COP1 in a hypocotyl cell from wild-type
seedlings grown in continuous white light. b, Typical nuclear-localized
GUS-COP1 in a hypocotyl cell from wild-type seedlings grown in
continuous darkness. c, Nuclear-localized GUS-COP1 in a hypocotyl cell
from hy1 seedlings grown in continuous white light. Arrows
indicate the locations of the nuclei. B, The subcellular localization
of GUS-COP1 in wild-type and hy1 seedlings grown in white
light, red light, blue light, and far-red light. Seedlings showing
strong nuclear staining of GUS-COP1 received a value of +++, seedlings
with slight but obvious nuclear staining received a value of ++, and
seedlings displaying no observable nuclear staining received a value of
+.
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CONCLUDING REMARKS |
In an extension of our recent report (Osterlund et al., 2000), we
verified that the abundance of HY5 does correspond with expected HY5
activity. There is a strong correlation between the activity of
HY5-dependent RbcS promoter and the level of HY5 protein under changing
light intensity. Further, optimal activation of the RbcS promoter under
different light regions also requires HY5, correlating well with the
reported HY5 abundance under those light conditions (Osterlund et al.,
2000). These data together support the notion that COP1-targeted
degradation of HY5 can in fact act as a means to regulate HY5-mediated
gene expression. In addition, the likely influence of COP1 on multiple
transcription factors, along with the light-regulated subcellular
localization of COP1, provides an attractive model to explain the
molecular switch between photomorphogenic and etiolated development.
The isolation of new alleles of hy1 and hy2 as
enhancers of the hy5-ks50 mutation revealed additional
insights about the role of photoreceptors, COP1, and HY5 in mediating
light control of Arabidopsis development. The hy1 and
hy2 mutations, which are defective in all phytochromes,
likely disrupt the normal activation of parallel
photomorphogenic-promoting factors that act in coordination with HY5 to
promote photomorphogenic development. As COP1 is constitutively localized in the nuclei in the hy1-null mutants, it
seems possible that the increased nuclear accumulation of COP1
negatively regulates activities of multiple
photomorphogenesis-promoting transcription factors, resulting in the
enhanced etiolation of the hy1/hy5 or hy2/hy5
double mutants.
It is tempting to speculate why all the dramatic enhancer mutations of
hy5 in our screen are new hy1 or hy2
alleles. Because the functions of individual photoreceptors are
partially overlapping, mutations in individual phytochromes or
cryptochromes might only cause weak or subtle enhancement of the
hy5 mutant phenotype, whereas the chromophore deficiency
would result in a defect in all phytochromes and lead to dramatic
enhancement of etiolation. We cannot rule out that the limited nature
of our screen might simply miss the identification of parallel
transcription factors that act together with HY5 to promote
photomorphogenesis. However, an alternative explanation is that
mutations in any individual transcription factor beside HY5 would cause
weak or subtle effect on the seedling development and would not be
included in this dramatic enhancer collection. In the latter case, the
future characterization of the remaining weak hy5
enhancer mutations from our initial screen could prove quite valuable.
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FOOTNOTES |
Received August 9, 2000; modified August 16, 2000; accepted September 5, 2000.
1
This work was supported by grants from the
National Institutes of Health (to X.W.D.) and the U.S. Department of
Agriculture (to N.W.). X.W.D. is a National Science Foundation
Presidential Faculty Fellow and M.T.O. was a National Institutes of
Health and U.S. Department of Education predoctoral trainee.
*
Corresponding author; e-mail xingwang.deng{at}yale.edu; fax
203-432-5726.
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TOP
INTRODUCTION
THE ACTIVITY OF HY5...