1 Peking-Yale Joint Center of Plant Molecular Genetics and Agrobiotechnology,
College of Life Sciences, Peking University, Beijing 100871, Peoples Republic
of China
2 Department of Molecular, Cellular and Developmental Biology, Yale University,
New Haven, CT06520-8104, USA
3 Department of Epidemiology and Public Health, Yale University School of
Medicine, New Haven, Connecticut 06520, USA
* Author for correspondence at address2 (e-mail: xingwang.deng{at}yale.edu)
Accepted 29 October 2002
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SUMMARY |
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Key words: Arabidopsis thaliana, COP/DET/FUS, Seeding development, Genome expression profile
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INTRODUCTION |
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Genetic screens for Arabidopsis mutant seedlings exhibiting a
light-grown phenotype when grown in darkness have resulted in the
identification of both pleiotropic and partially photomorphogenic mutations
(Chory et al., 1989;
Deng et al., 1991
;
Hou et al., 1993
;
von Arnim and Deng, 1996
). The
pleiotropic constitutive photomorphogenic (COP) or deetiolated (DET) mutants
were also defined in a FUSCA (FUS) mutant screen
(Misera et al., 1994
;
Wei and Deng, 1999
). Together
ten pleiotropic COP/DET/FUS mutant loci have been defined, including
cop1, det1, cop8, cop9, cop10, fus5, fus6/cop11, fus11, fus12 and
cop16 (Schwechheimer and Deng,
2000
). Recent biochemical characterizations of these 10 loci
define four biochemical entities: seven of the loci are required for the COP9
signalosome (CSN) biogenesis and three others, COP1, DET1 and COP10 are not
(Wei and Deng, 1999
;
Serino et al., 1999
;
Deng et al., 2000
). Mutations
in these 10 loci result in almost complete photomorphogenic development in
darkness, suggesting that their gene products act as light-inactivatable
repressors of photomorphogenesis (Wei and
Deng, 1999
; Osterlund et al.,
1999
). Recently, it was hypothesized that in darkness all of these
four defined functional entities are involved in promoting ubiquitination and
proteasome-mediated degradation of photomorphogenesis-promoting transcription
factors (Osterlund et al.,
2000
; Holm et al.,
2002
). Among them, COP1 and COP10 may constitute E3 and E2
activities and act together with the COP9 signalosome in targeting
transcription factors for ubiquitination and subsequent degradation
(Suzuki et al., 2002
).
Intriguingly, defects in a peroxisome protein were recently reported to
suppress weak mutations of both COP1 and DET1
(Hu et al., 2002
).
It has been generally assumed that the photoreceptors perceive and
interpret incident light and transduce the signals to modulate
light-responsive nuclear genes, which, in turn, direct appropriate
developmental responses during photomorphogenesis
(Terzaghi and Cashmore, 1995;
Puente et al., 1996
;
Ma et al., 2001
;
Tepperman et al., 2001
). In
our previous reports, we verified that the contrasting developmental patterns
are mediated primarily by coordinated changes in genome expression
(Ma et al., 2001
) and that a
large proportion of light-controlled genome expression can be achieved by
regulating nuclear COP1 activity (Ma et
al., 2002
). Thus logical questions to ask are whether all these
pleiotropic COP/DET/FUS loci are acting together to mediate the light
control of genome expression and whether they also have additional and/or
distinct roles in regulating plant development and genome expression.
The less pleiotropic or partially photomorphogenic mutants included those
with short hypocotyls and/or partially open and developed cotyledons. They
included mutations in COP2, COP3 and COP4 that result in
partial photomorphogenic cotyledon development
(Hou et al., 1993) and
mutations in DET2 and DET3 that result in partial
photomorphogenic hypocotyls as well as cotyledon development in darkness
(Chory et al., 1991
;
Cabrera y Poch et al., 1993
).
DET2 has been shown to encode an enzyme in the biosynthetic pathway
of the plant hormone brassinosteroid (Li and Chory, 1996). Interestingly,
mutants defective in response to the plant hormone auxin have also been
reported to have a partial photomorphogenic phenotype
(Dharmasiri and Estelle,
2002
). It is not clear exactly how these genes contribute to the
light regulation of seedling development and whether they can be considered
part of the light signaling pathway. It seems feasible that an analysis of
their effect on genome expression may provide some insight into their
contribution to photomorphogenesis.
However, we also found that the COP9 signalosome was involved in
multifaceted developmental processes besides photomorphogenesis
(Peng et al., 2001a;
Peng et al., 2001b
;
Schwechheimer et al., 2001
;
Schwechheimer et al., 2002
).
The COP9 signalosome directly interacts with SCF type E3 ubiquitin ligases and
regulates their activity in tagging substrates for proteasome-mediated
degradation (Schwechheimer et al.,
2001
; Schwechheimer et al.,
2002
). Clearly, the COP9 signalosome has a role beyond modulating
the hypothesized COP1 (E3) and COP10 (E2) pair in controlling
photomorphogenesis, but also in regulating other E3 and E2 pairs and, thus,
many additional cellular and developmental processes. An E2 also probably
associates with more than one E3 (Hellmann
and Estelle, 2002
), thus COP10 and COP1 may not necessarily work
together exclusively in controlling photomorphogenesis. Therefore a systematic
comparison of the genome expression profiles controlled by each of these
pleiotropic COP/DET/FUS activities may reveal the extent of overlapping, as
well as unique roles, in light regulated and other developmental
processes.
In a systematic effort to examine the overlapping and distinct roles of
these pleiotropic and partially photomorphogenic genes, we utilized a
previously described Arabidopsis cDNA microarray with 6126 unique
genes (Ma et al., 2001;
Ma et al., 2002
). A comparison
of their genome expression profiles to that of light-regulated genome
expression revealed the overlapping as well as distinct roles of the
pleiotropic COP/DET/FUS proteins. Furthermore, new insights into the
contribution of the partially pleiotropic photomorphogenic loci in overall
photomorphogenesis, as well as their specific function, can be derived from
this genomic analysis.
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MATERIALS AND METHODS |
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The microarray slide used in this study was described previously
(Ma et al., 2001;
Ma et al., 2002
). Each array
contains 9,216 EST clones that represented about 6,126 unique genes. For more
information, please see our web sites
(http://plantgenomics.biology.yale.edu
or
http://info.med.yale.edu/wmkeck/dna_arrays.htm).
RNA preparation, fluorescent labeling of probe, slide hybridization,
washing and scanning
Total RNA was extracted from the whole seedlings using the Qiagen RNeasy
Plant Mini Prep kit. For each treatment, at least two independent biological
samples were used for RNA preparations and probe synthesis. 50 µg total RNA
was first labeled with aminoallyl-dUTP (aa-dUTP; Sigma, St. Louis, Missouri)
by direct incorporation of aa-dUTP during reverse transcription as described
previously (Ma et al., 2002).
After 3-4 hours of incubation at 42°C, the reaction was stopped by adding
5 µl of 0.5 M EDTA and incubating at 94°C for 3 minutes. The RNA in the
mix was hydrolyzed by adding 10 µl of 1 M NaOH and incubating at 65°C
for 20 minutes. The reaction was neutralized by adding 6 µl of 1 M HCl and
2 µl of 1 M HCl-Tris (pH 7.5). The aa-dUTP-labeled cDNA was purified from
the unincorporated aa-dUTP molecules by adding 450 µl of water and spinning
through a Microcon YM-30 filter (Millipore, Bedford, MA) for 9 minutes at
11000 g. The flow-through cDNA solution was spun through the
same Microcon filter once more. The purified, labeled probe was concentrated
to a final volume of 5-7 µl. Then, the cDNA probe was further labeled with
fluorescent dye by conjugating aadUTP and monofunctional Cy-3 or Cy-5 dye
(Amersham Pharmacia Biotech, Piscataway, NJ) as follows: 1 µl cDNA solution
added to 0.1 volume 1 M sodium bicarbonate (Sigma, St. Louis, Missouri) and 1
µl Cy3 or Cy-5 dye (solved in DMSO). The mixture was incubated at room
temperature in the dark for 60 minutes. After incubation, the labeling
reaction was stopped by adding 1 µl 2 M ethanolamine (Sigma, St. Louis,
Missouri) and further incubated at room temperature for 5 minutes. The
dye-labeled probe was purified from the unincorporated dye molecules by
washing through a Microcon YM-30 filter (Millipore, Bedford, MA) three times,
as mentioned above. The probes from the designated sample pairs were combined
at the last washing. The purified, labeled probe was concentrated to a final
volume of 7 µl.
The protocols for hybridization to the Arabidopsis microarray,
microarray slide washing and scanning were as described previously
(Ma et al., 2001;
Ma et al., 2002
).
Data analysis
The general approaches were as described in our previous work
(Ma et al., 2001;
Ma et al., 2002
) with minor
modifications. Briefly, spot intensities were quantified using Axon GenePix
image analysis software (GenePix Pro 3.0). The channel ratio was measured with
the GenePix median of ratios method and was then normalized using the GenePix
default normalization factor. In order to merge the replicated GenePix output
data files in a reasonable way, we developed a computer program called GPMERGE
(http://bioinformatics.med.yale.edu/software.html).
With this program we pooled the four or more replicated data sets of each
experiment. Different quality control procedures were also conducted before
data points were averaged from the replicates. First, all spots that were
flagged Bad or Not Found by GenePix software, were not
included in the final data analysis. Second, a very simple outlier searching
algorithm was incorporated in GPMERGE; those spots that led to a large
difference between the ratio mean and the ratio median were defined as
outliers and eliminated from the analysis. Third, only those spots that met
both of the following two conditions were considered in further data analysis:
(1) signals were higher than the backgrounds for both channels; (2) the signal
was twofold higher than the background at least for one channel. For those
unique genes that have more than one EST clone, we developed a custom computer
program to extract a ratio for the gene as described previously
(Ma et al., 2002
).
Different kinds of expression pattern identification and pattern matching
were conducted within or across these experiment groups. Within each group a
hierarchical clustering analysis was performed as described by Eisen et al.
(Eisen et al., 1998). Only
those genes that had more than twofold changes in expression in at least one
of the experimental sets were used in the cluster analysis shown in the
figures.
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RESULTS |
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As in our previous studies (Ma et al.,
2001; Ma et al.,
2002
), at least two independent biological samples for each
treatment (6-day old dark or light-grown seedlings,
Fig. 1A and 1B) were used for
RNA preparations. Each RNA preparation was used to generate probes for
hybridization to at least two arrays. In this way we ensured that there were
at least four quality data sets for each experimental test. The average of the
mean value for each gene expression ratio was used for further analysis and
comparison. A commonly used clustering analysis
(Eisen et al., 1998
) was
employed to reveal the relatedness of distinct mutant genome expression, and,
thus, to extrapolate the functional relationship of the loci defined by the
mutations.
|
The mutations in pleiotropic COP/DET/FUS genes have a
similar influence over genome expression as light, but with clear
distinctions
We first analyzed the genome expression profile changes caused by the
selected mutations in darkness (Fig.
1A) and compared those to the light effect on genome expression in
wild-type seedling. All the genes that displayed a differential expression of
twofold or more in at least one experimental test were selected to undergo
clustering and relatedness analysis. Fig.
1C shows the relatedness of the genome expression changes across
these selected mutants and wild types for each experimental test based on the
calculation of distance matrices. The light-induced genome expression profiles
of the two most frequently used wild-type ecotypes (Col-O and WS) were quite
similar to each other (Fig.
1C). The genome expression patterns induced by the pleiotropic
cop/det/fus mutations were also similar. However, there were clear
differences conferred by the distinct pleiotropic loci and even the nature of
the mutations (viable versus lethal) from the same locus. Among the
representative pleiotropic cop/det/fus mutants, genome expression
profiles in darkness were most similar between those affected by the
cop1 and det1 mutations
(Fig. 1C). Both the viable
(weaker) mutants (cop1-1 and det1-1) and lethal mutants
(cop1-5, cop1-8 and det1-6) of COP1 and
DET1 showed very similar genome expression patterns. However, the
viable cop1 and det1 mutant patterns clustered much closer
to each other and exhibited significant differences from their respective
lethal mutant cluster. Consistent with our previous studies
(Ma et al., 2002), the viable
cop1 and det1 alleles triggered genome expression profiles
most closely resembling those of the white light-grown wild-type seedlings. In
contrast, the genome expression profiles caused by the three representative
lethal mutations in COP1 and DET1 in darkness were more
diverged from those of the wild type in white light.
The genome expression profiles of the csn mutants (fus6-1 or cop9-1) and cop10-1, each representing one of the other two functional units, exhibited distinct expression patterns from either the viable or lethal alleles of the COP1 and DET1 clusters. Both cop9-1 and fus6-1, which are lethal mutants in subunits 8 and 1 of the COP9 signalosome, induced similar genome expression patterns in darkness and light (data not shown). Thus, only one of them was used to represent lethal csn mutants in most of the data analysis. Among the representative lethal cop/det/fus mutants for the four functional units, the lethal csn mutant genome expression in darkness was most similar to that of white-light induction in wild-type seedlings (Fig. 1C). However, the csn1/fus6-1 genome expression profile in darkness is slightly more diverged from the white-light control of genome expression than the clade for the viable cop1 and det1 mutations. The genome expression profile of the lethal mutation in COP10 (cop10-1) in darkness was the most diverged from that of the white light control (Fig. 1C).
As anticipated, the less pleiotropic constitutive photomorphogenic loci, such as cop4 and det2 in darkness, triggered more diverged genome expression profiles than any of the pleiotropic cop/det/fus mutations when compared to the expression profile in white light [WT/WL vs. WT/D (WT, wild type; WL, white light; D, dark grown)]. Among the three examined, the mutation in COP4 resulted in an expression profile that was most similar, followed by det2, and tir1-1 produced the most divergent profile (Fig. 1C).
Functional relationship among pleiotropic COP/DET/FUS in
light-grown seedlings
We also carried out a similar genome expression relatedness analysis for
the pleiotropic cop/det/fus mutants in light and compared them with
the light control of genome expression (WT/WL vs. WT/D). As shown in
Fig. 1D, it was apparent that
all the representative lethal pleiotropic cop/det/fus mutations have
similar genome expression profiles and fall into one general clade. Within
this clade, the specific relationship of the genes representing the four
biochemical entities (Fig. 1D)
is similar to that revealed by their dark genome expression profiles
(Fig. 1C), e.g.,
det1-6 and cop1-8 were the most similar to each other, then
the COP9 signalosome representative cop9-1, and lastly the
cop10-1 mutant. However, the viable mutants of cop1 and
det1 showed similar genome expression profiles and were significantly
diverged from those of the lethal cop/det/fus mutants. In fact, the
genome expression profiles triggered by viable cop1 and det1
mutations in the light (mutant/WL vs. WT/WL) are closely related to those of
white-light regulation (WT/WL vs. WT/D). This implies that in light-grown
plants, the viable cop1 and det1 mutants may simply enhance
the control of genome expression by light.
The lethal mutations of pleiotropic COP/DET/FUS genes confer
distinct effects on different subgroups of light-regulated genes
To reveal what changes in genome expression are responsible for the
divergence between the lethal cop/det/fus mutations in darkness and
white-light control of genome expression
(Fig. 1C) and viable
cop1 mutations (Ma et al.,
2002), we examined genes belonging to 24 previously defined
metabolic pathways that are coordinately up- or down-regulated by light
(Ma et al., 2001
). As
summarized in Table 1, there
are clear differential effects of the lethal cop/det/fus mutations on
the expression of the genes in these pathways. For all pathways directly
linked to photosynthesis, including dark and light reactions, starch and
sucrose biosynthesis pathways, photorespiration and chlorophyll synthesis,
their gene expression in dark-grown lethal cop/det/fus mutants are
much less activated or even down-regulated, as compared to light activation in
wild-type seedlings. However, some pathways, such as phenylpropanoid
biosynthesis, cytoplasmic protein synthesis, and water transport across
tonoplast and plasma membranes, were more strongly regulated by the lethal
cop/det/fus mutations in darkness than by white light in wild-type
seedlings. For the remaining half of the pathways that are regulated by light,
their associated genes seem to be similarly regulated by the lethal
cop/det/fus mutations in darkness and by light.
|
The lethal mutations of pleiotropic COP/DET/FUS genes mimic
a high-intensity light stress effect on light regulated genes
To further examine the effect of the pleiotropic COP/DET/FUS group
of genes on light regulation of gene expression, we selected 15 genes involved
in photosynthetic light reactions and examined their expression levels in
various cop1 or det1 mutants.
Fig. 2A illustrates the
expression of 15 genes in the dark-grown viable (weak) mutants of
COP1 (cop1-6), DET1 (det1-1), a strong
cop1 mutant (cop1-1), lethal mutants of COP1
(cop1-8) and DET1 (det1-6). All these 15 genes were
induced to a certain extent in the dark-grown weak and strong mutants of
COP1 or DET1 when compared to the dark-grown wild-type
seedlings (lanes 2, 3 and 5), but were repressed in both cop1 and
det1 lethal mutants (lanes 4 and 6).
|
It has been shown previously that light can exert a quantitative control on
photomorphogenesis by proportionally inhibiting nuclear COP1 activity
(Osterlund et al., 2000). As
the lethal mutant seedlings accumulated high levels of pigments and resembled
Arabidopsis subjected to high light intensity stress, it is
reasonable to hypothesize that the null mutations of COP1 may mimic
the effect of an excess high intensity light environment even when mutants are
grown in darkness. To test this hypothesis, we examined whether extremely high
light intensity or excess light signaling can cause genome expression profiles
in wild-type similar to those of the lethal mutants.
To this end, we employed two experimental approaches. First, we grew wild-type seedlings under normal intensity of white light for 5.5 days, then transferred them to high-intensity white light (HWL; 2500 µmol/m2/second) for another 12 hours; the resulting genome expression pattern was analyzed. Indeed, the genome expression profile triggered by the high light intensity (WT/HWL vs. WT/D) is closely related to those of the dark-grown lethal mutants, falling between that of the fus6-1 mutant and those of the lethal mutants in COP1 or DET1 (data not shown). The genome expression profile triggered by high intensity white light (2500 µmol/m2/second) in wild type (WT/HWL vs. WT/WL) is also similar to those caused by the lethal cop/det/fus mutations in the light (Fig. 1D). This can be best illustrated by the expression profiles of the 15 selected photosynthetic genes, as their expression levels are all down-regulated by the high-intensity light compared to that of normal light-grown siblings (Fig. 2A, lane 8).
Second, we compared the effect of CRY1 over-expression (CRY1
OE) to that of the lethal cop/det/fus mutations on blue-light
regulation of genome expression (Ma et
al., 2001). In general, the genome expression profile triggered by
CRY1 over-expression is somewhat similar to that of the lethal
cop/det/fus mutations in darkness. In fact, all of the 15 selected
photosynthetic genes also exhibited a down-regulation as a result of
CRY1 over-expression when compared to wild-type siblings grown under
the same blue-light condition (Fig.
2A, lane 7).
We also analyzed the expression profiles of the 15 selected photosynthetic genes in cop1 or det1 mutants grown in normal white light. As shown in Fig. 2B, the light-grown cop1-6 mutants (the weakest allele) exhibited a slight enhancement of the light activation of all these genes. In the light-grown cop1-1 and det1-1 mutants (two other viable mutations), the expression for some genes was slightly enhanced, but most exhibited a slight decrease in light activation. In the lethal cop1-8 and det1-6 mutants, the light activation of all 15 genes was completely reversed in these mutants (Fig. 2B, lanes 4 and 6), an effect also caused by the high-intensity light treatment of the normal light-grown wild-type seedlings (Fig. 2B, lane 7).
Lethal cop1 or det1 mutations induce a new set of
genes in both darkness and light
Analysis of the genome expression profiles of light-grown lethal
cop/det mutants revealed a significant fraction of genes whose
expressions were oppositely regulated in comparison to that by light. For
example, the regulation of more than 250 genes in light-grown lethal
cop1 or det1 mutants (mutant/WL vs. WT/WL) was found to be
opposite that of the same genes from light grown wild-type plants
(Fig. 3). Again, the similarity
among the pleiotropic COP/DET/FUS mutations reflected their
relatedness as defined by their genome expression profiles
(Fig. 1). The genes that are
oppositely regulated in light-grown lethal cop1 and det1
mutants are essentially identical. Only a small fraction of genes exhibited
opposite regulation by either viable det1 or cop1 mutations
in light.
|
In addition to the oppositely regulated genes, there were also genes that were not regulated by light but exhibited significant variation in expression in the lethal cop/det/fus mutations. For example, about 200 genes that were not regulated by normal intensity white light in wild type, showed a twofold differential expression in light-grown lethal cop1 or det1 mutants (Fig. 3C). Interestingly, about 70% of those genes specifically affected by lethal cop/det/fus mutations in normal light are also up- or down-regulated in high-intensity white light in the wild type. This group of genes includes DREB2A (At5g05410), ADP-ribosylation factor (At5g17060) and phospholipase C (At4g38690).
Different light-regulated genes exhibit distinct light intensity
dependence in their regulated expression
The clear, distinct effects of the two light intensities on the genome
expression profiles described above prompted us to further examine the effects
of a large range of light intensities on the genome expression profiles. For
this purpose, the wild-type seedlings were grown under 1% (1.5
µmol/m2/second), 10% (15 µmol/m2/second) and
normal intensity (150 µmol/m2/second) white light for 6 days.
Furthermore, seedlings exposed to high-intensity white light (2500
µmol/m2/second) for 12 hours after growth in normal intensity
white light for 5.5 days were also included in this analysis. The morphology
of seedlings are shown in Fig.
4A, and a cluster analysis of their genome expression profiles is
shown in Fig. 4B. Among the
four increasing light intensities, about 7.7% (471), 15.3% (936), 30.0% (1838)
and 31.4% (1922) of genes showed differential expression (2-fold) when
compared to dark-grown siblings (Fig.
4C). Most of these genes showed the highest variation under normal
intensity white light. However, many distinct patterns of light intensity
dependence are evident. Twelve representative patterns, with the number of
genes following each pattern, are shown in
Fig. 5.
|
|
Genes involved in photosynthetic light and dark reactions, starch and
sucrose synthesis, photorespiration and chlorophyll synthesis pathways were
induced even at a very low intensity white light (1.5
µmol/m2/second). They fall into patterns A, C and F of
Fig. 5. Most of these genes
reached maximal induction at normal light intensity. Besides genes involved in
the metabolic pathways, expression of genes involved in signal transduction,
RNA splicing, the auxin-regulated pathway, peptide transport and transcription
regulation were also induced in very low intensity white light (1.5
µmol/m2/second) and some of them were repressed at high light
intensity (2500 µmol/m2/second). Among the 314 genes exhibiting
2-fold induction under low light intensity, only 14 of them showed the
highest induction at high light intensity (pattern G in
Fig. 5). About 24% of low light
intensity-induced genes were repressed by high light intensity (patterns
C,D,E,I in Fig. 5). The
expression of genes encoding phenylpropanoid synthesis and water transport
proteins (pattern G or H in Fig.
5) were not affected by very low light intensity (1.5
µmol/m2/second), but were affected at intermediate light
intensities, and reached maximal expression under high light intensity (2500
µmol/m2/second).
Comparison of the genome expression profiles regulated by different light intensities, together with the expression profiles of two lethal cop1-8 and det1-6 mutants (Fig. 4B) revealed different subclusters or expression patterns among different light conditions and mutants. Some of the genes showed similar expression patterns in all 6 tests, such as subclusters c and k. Some genes exhibited opposite expression patterns between low light intensity and lethal cop1-8 and det1-6 mutants, such as subclusters a, e, f, g, i and j. Other genes exhibited different expression patterns between light and the lethal mutations, such as b and h.
A fraction of transcription factor genes are regulated in light
intensity-dependent manners
Among the 333 putative transcription factors included in our array, 53
showed differential expression (twofold) in at least one light intensity
treatment. As shown in Fig. 6A,
the expression of the majority of these transcription factors was light
intensity dependent. For example, PAP3 was induced under two low
light intensities but was not affected by two higher light intensities. While
the expression of two WRKY protein genes was not affected under very low light
intensity, they were induced or repressed under higher light intensities. Some
transcription factor genes, such as Pcmyb1, were similarly regulated
by different light intensities, while other transcription factor genes (such
as the zinc finger protein At5g58620) were regulated differently in different
light intensities.
|
Interestingly, all these transcription factors also showed differential
expression (2-fold) in dark-grown cop1-6 mutant and wild-type
seedlings (Ma et al., 2002
).
The expression pattern triggered by the cop1-6 mutation in darkness
is very similar to that of normal intensity light-grown seedlings, with only
two exceptions (At3g59060 and At2g01530, both of which were induced in the
dark-grown cop1-6 mutant but not in normal light). We further checked
the expression profiles of these transcription factor genes in different
cop1 mutant alleles (N282, cop1-6, cop1-1 and
cop1-5 to represent very weak, weak, strong and lethal mutations of
COP1, respectively). As shown by the five representative genes in
Fig. 6B, the expression
patterns of most of the transcription factor genes behaved in a COP1
activity-dependent manner, and, in general, are consistent with their response
to increasing light intensities in wild type. This further substantiates a
previous conclusion that increasing the light intensity quantitatively
inactivates more COP1 activity, thus promotes stronger photomorphogenesis.
The COP10 mutation induces high expression of genes involved
in cytosolic translation
In comparing the effect of pleiotropic cop/det/fus mutations from
the four functional groups, we also observed some distinct expression patterns
among these groups. As shown in Fig.
3A, the number of distinctly regulated genes in these lethal
mutant seedlings is highly variable, with those of cop1-8 and
det1-6 being more similar to each other and with fus6-1 and
cop10-1 diverging. cop10-1 is, by far, the most diverged in
its genome expression profile (Fig.
1C). One striking example is illustrated in
Fig. 7, where almost all the
genes encoding 60S or 40S ribosomal proteins were much more sensitive to COP10
regulation (lane 5) than any other pleiotropic COP/DET/FUS proteins or to high
intensity light. The reason for this is not clear.
|
The majority of COP4-controlled genes are included within
light-regulated genes
The cop4 mutaant belongs to the less pleiotropic or partial
photomorphogenic mutants. Seedlings with mutations in the COP4 locus
exhibited open and enlarged cotyledons but long hypocotyls (Hou et al., 1933)
(Fig. 1A). Genome expression
profile analysis revealed that 496 genes showed twofold or higher differential
expression between dark-grown cop4 and wild-type seedlings, with 246
and 250 genes up- or down-regulated in the mutant, respectively. The genes
induced in the cop4 mutant encode proteins involved in photosynthetic
light reactions, carbon assimilation, starch synthesis, sucrose synthesis,
photorespiration, chlorophyll synthesis and the TCA cycle. Repressed genes in
the cop4 mutant encoded proteins that are involved in cell wall
degradation, water transport across tonoplast and plasma membranes, fatty acid
ß oxidation, glycoxylate cycle, nitrate assimilation and sulfate
assimilation. Most of these COP4-regulated genes are actually light-regulated
as well (Ma et al., 2001;
Ma et al., 2002
). However, the
number of differentially expressed genes controlled by COP4 is much fewer than
those of light or pleiotropic COP/DET/FUS protein.
To further characterize these COP4-regulated genes, we compared gene
expression patterns in cop4 with the differential gene expression
patterns of light-grown wild type and dark-grown cop/det/fus mutant
seedlings in a cluster analysis (Fig.
8A). The vast majority of the COP4-regulated genes showed
qualitatively similar expression in white light or the pleiotropic
cop/det/fus mutations. In fact, about 72% and 70% of the up-regulated
genes that displayed twofold or more differential expression in the
cop4 mutants were included in the white light- or
det1-1-induced genes, respectively. Likewise, about 73% and 72% of
the down-regulated genes displaying 2-fold differential expression in the
cop4 mutants were included in the white light or det1-1
repressed genes, respectively. Less than 5% of COP4-regulated genes show
opposite regulation in most pleiotropic cop/det/fus mutants, with
only the cop10-1 mutant exhibiting a higher percentage of opposite
regulation (Fig. 8C,D). These
results support a hypothesis that COP4 is a locus involved in
regulating a subset of photomorphogenic processes
(Hou et al., 1993
).
|
A BR synthesis mutation induces a partially overlapping set of
light-regulated genes in the dark
We also examined the genome expression profile of another representative
partially photomorphogenic locus, DET2, which encodes an enzyme
involved in brassinosteroid biosynthesis
(Li et al., 1996). Dark-grown
det2 mutants had a partially photomorphogenic phenotype with short
hypocotyls and small opened cotyledon without an apical hook
(Chory et al., 1991
)
(Fig. 1A). We observed that 253
genes in our array showed twofold or higher differential expression between
dark-grown det2 mutant and wild-type seedlings, while 156 and 97
genes were up- or down-regulated in dark-grown det2 mutant seedlings,
respectively. A comparison of the genome expression profiles between
det2 mutants and light or pleiotropic cop/det/fus mutants
showed that less than half of the DET2-regulated genes exhibited the same
expression pattern with light and pleiotropic COP/DET/FUS regulation. Those
genes regulated by both DET2 and light included the genes for the
photosynthetic light reaction, cell wall degradation, water transport across
the tonoplast, sulfate assimilation and fatty acid ß oxidation. Of the
approximately 50% of DET2-regulated genes in darkness that were not similarly
regulated by light or pleiotropic cop/det/fus mutations in the
darkness, a small fraction were oppositely regulated by light and/or
pleiotropic cop/det/fus mutations
(Fig. 8E,F). Our result
suggested that brassinosteroid also regulates the expression of other
unrelated genes in addition to a small subset of light-regulated genes.
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DISCUSSION |
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Pleiotropic COP/DET/FUS protein-regulated genome expression exhibits
a large overlap with that of light
Phenotypically, all pleiotropic cop/det/fus mutants showed an
almost complete light-grown response when they were grown in the dark
(Wei and Deng, 1999)
(Fig. 1A). In this genome
expression profiling study, we also found that individual pleiotropic
COP/DET/FUS proteins control a largely overlapping set genes; the majority of
COP/DET/FUS-regulated genes (more than 80%) overlapped with light-regulated
expression at seedling stage as judged by the gene expression profiles. The
gene products of the less pleiotropic COP/DET loci also controlled variable
fractions of the light-controlled genome expression. So, this genomic study
provides an explanation for why mutations in these loci lead to a pleiotropic
or partial light-grown phenotype when grown in the dark. This is because
mutations of an individual pleiotropic COP/DET/FUS locus will de-repress
almost the whole set of light-controlled genes, while mutations of a partial
COP/DET locus will de-repress a partial set of light-controlled genes. It is
not surprising that the former will have an almost completely light-grown
phenotype in the dark, while in the later case, the mutations will lead to a
partial light-grown phenotype in the dark. This result also supports the
notion that contrasting light-controlled developmental patterns are mediated
primarily by the change in light-regulated gene expression.
COP1 and DET1 control highly similar genome expression profiles and
may function in the close proximity in the pathway
The ten pleiotropic COP/DET/FUS loci represent four biochemical
entities: the COP9 signalosome, COP1, COP10 and DET1. Recently, it has been
proposed that COP1 functions as a putative component of an ubiquitin protein
ligase (E3) (Osterlund et al.,
2000). COP10 encodes an ubiquitin-conjugating enzyme (E2)
variant (Suzuki et al., 2002
)
and the COP9 signalosome is structurally similar to the lid of the 19S
regulatory particle of the 26S proteasome
(Wei and Deng, 1999
). While
DET1 encodes a novel nuclear-localized protein
(Pepper et al., 1994
), its
specific biochemical function is not known. These proteins appear to work
together to mediate the degradation of photomorphogenesis-promoting
transcription factors (Holm et al.,
2002
).
In this study, we found that among these pleiotropic mutants, cop1 and det1 mutant seedlings have the most closely related genome expression profiles both in dark and light growth conditions (Fig. 1C,D). This is true for both the viable mutations or the lethal mutations of COP1 and DET1. This genomic evidence supports the conclusion that COP1 and DET1 work closely together as observed in a previous genetic analysis (Ang et al., 1994). COP1 and DET1 may, therefore, act very closely in repressing photomorphogenesis. Thus signals perceived by photoreceptors act to negatively regulate COP1 and DET1 and relieve their repression of photomorphogenesis.
Light signals can quantitatively regulate the pleiotropic COP1 and
DET1 proteins and, thus, the degree of photomorphogenic response
Besides acting as signals to regulate many developmental processes, light
also serves as the source of energy for plant photosynthesis. Plants adjust
the structure and function of the photosynthetic apparatus in response to
changes in their growth environment. The change in the size of the chlorophyll
antennae associated with photosystem I and II is very sensitive to light
(Niyogi, 1999). Under limiting
irradiance conditions, the photosystems acquire large chlorophyll antennae and
more extensive thylakoid membrane to absorb more light; while under high
irradiance, especially under light stress, chlorophyll antennae sizes are
reduced in order to protect the photosynthetic apparatus from damage by
repressing LHC gene expression (Escoubas
et al., 1995
; Maxwell et al.,
1995
) and/or increasing LHC protein degradation
(Lindahl et al., 1995
). Also,
under light stress, plant cells express some genes encoding specific stress
proteins with possible protective functions
(Dunaeva and Adamska,
2001
).
COP/DET/FUS proteins act as repressors of light-controlled
Arabidopsis seedling development
(Wei and Deng, 1999;
Osterlund et al., 1999
). We
have found that light can largely achieve its control of genome expression by
negatively regulating COP1 (Ma et al.,
2002
) and DET1 protein (this work). Therefore, complete loss of
COP1 or DET1 function would mimic the action of light stress even in the dark.
To provide direct evidence for this, we first checked the expression of
representative genes encoding chlorophyll antennae and thylakoid membrane
proteins. We found that the expression of these genes was induced by normal
intensity light (and even at a very low intensity) and in a dark-grown weak
mutant of cop1 or det1, but was repressed in a dark-grown
null mutant of cop1 (cop1-8) or det1 (det1-6)
(Fig. 2A). The expression of
these genes was also dependent on a COP1 activity in weak, strong and null
mutants of cop1 (Fig.
2A). In addition, all these genes were repressed under high
intensity white light (light stress) (Fig.
2A). Second, a predominant portion (80%) of the genes that showed
opposite regulation in dark-grown cop1 or det1 null mutants
compared to normal light regulation also had a similar expression pattern to
that resulting from light stress. Some of the light stress-induced marker
genes, e.g. late embryogenesis abundant protein
(Dunaeva and Adamska, 2001
),
metallothionein (Dunaeva and Adamska,
2001
), HSP70 (Schroda et al.,
1999
), glutathione reductase
(Karpinski et al., 1997
) and
NAD(P)H dehydrogenases (Endo et al.,
1999
), were also induced in dark-grown cop1 or
det1 null mutants. Third, we found that the genes encoding
chlorophyll antennae and thylakoid membrane proteins began to be less induced
in weak cop1 mutants, repressed in light-grown strong cop1
or weak det1 mutants, and severely repressed in cop1 or
det1 null mutants (Fig.
2B). Again, more than 80% of the genes that showed opposite
regulation in light-grown cop1 or det1 null mutants to
normal light grown seedlings had a similar expression pattern under light
stress. Fourth, comparison of the whole genome expression profiles among
pleiotropic cop/det/fus mutants and normal and high intensity light
induction indicated that the genome expression pattern induced by high light
intensity is similar to that of lethal mutants of pleiotropic
cop/det/fus (Fig. 1
C,D). These genomic results are consistent with the physiological
observation that the very short hypocotyls and reduced cotyledon with
accumulation of anthocyanin (Fig.
1A) of the cop/det/fus mutants are also commonly observed
in the plants grown under high intensity light stress. The lethal (null)
mutants of COP/DET/FUS loci essentially exhibited light stress
responses even when grown in the dark.
The COP/DET/FUS loci have overlapping yet non-identical
roles in regulating Arabidopsis seedling development
For those genes that showed opposite regulation between light- and
dark-grown cop1 or det1 null mutants to regulation by light,
most of them do not show opposite regulation in dark-grown fus6-1,
cop9-1 or cop10-1 mutant seedlings. While in light fus6-1,
cop9-1 or cop10-1 mutant seedlings share similar
oppositely-regulated genes as the cop1 and det1 null mutants
or seedlings subjected to light stress, as compared to light regulation. For
example, in fus6-1, cop9-1 and cop10-1 mutants, the genes
encoding proteins involved in photosynthetic light reactions were induced in
the dark-grown mutant seedlings, whereas they were repressed in dark-grown
cop1-5, cop1-8 and det1-6. However, the expression of these
representative genes was repressed in all light-grown lethal pleiotropic
cop/det/fus mutant seedlings. All these characteristics that
fus6-1, cop9-1 or cop10-1 mutants exhibited were similar to
some viable cop1 or det1 mutants.
In addition, we found that individual pleiotropic COP/DET/FUS proteins also regulated distinct sets of genes. For example, cop10-1 mutants induced higher expression of cytosolic ribosomal proteins than other pleiotropic cop/det/fus mutants (Fig. 6). These results suggest that the pleiotropic COP/DET/FUS proteins, especially the COP9 signalosome and COP10, also function in other developmental processes besides seedling photomorphogenesis.
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ACKNOWLEDGMENTS |
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![]() |
Footnotes |
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