arabidopsis cullin4-damaged dna binding protein 1

Arabidopsis CULLIN4-Damaged DNA BindingProtein 1 Interacts with CONSTITUTIVELYPHOTOMORPHOGENIC1-SUPPRESSOR OF PHYA Complexesto Regulate Photomorphogenesis and Flowering Time C W

Haodong Chen,a,b,c,1 Xi Huang,b,c,d,1 Giuliana Gusmaroli,b William Terzaghi,b,e On Sun Lau,b Yuki Yanagawa,b,2

Yu Zhang,a,c Jigang Li,a,b Jae-Hoon Lee,b Danmeng Zhu,a,b,c and Xing Wang Denga,b,c,3

a Peking-Yale Joint Center for Plant Molecular Genetics and Agro-biotechnology, National Laboratory of Protein Engineering and

Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, Chinab Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520-8104c National Institute of Biological Sciences, Beijing 102206, Chinad College of Life Sciences, Beijing Normal University, Beijing, 100875, Chinae Department of Biology, Wilkes University, Wilkes-Barre, Pennsylvania 18766

CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) possesses E3 ligase activity and promotes degradation of key factors

involved in the light regulation of plant development. The finding that CULLIN4 (CUL4)-Damaged DNA Binding Protein1

(DDB1) interacts with DDB1 binding WD40 (DWD) proteins to act as E3 ligases implied that CUL4-DDB1 may associate with

COP1-SUPPRESSOR OF PHYA (SPA) protein complexes, since COP1 and SPAs are DWD proteins. Here, we demonstrate

that CUL4-DDB1 physically associates with COP1-SPA complexes in vitro and in vivo, likely via direct interaction of DDB1

with COP1 and SPAs. The interactions between DDB1 and COP1, SPA1, and SPA3 were disrupted by mutations in the WDXR

motifs of MBP-COP1, His-SPA1, and His-SPA3. CUL4 cosuppression mutants enhanced weak cop1 photomorphogenesis

and flowered early under short days. Early flowering of short day–grown cul4 mutants correlated with increased

FLOWERING LOCUS T transcript levels, whereas CONSTANS transcript levels were not altered. De-etiolated1 and COP1

can bind DDB1 and may work with CUL4-DDB1 in distinct complexes, but they mediate photomorphogenesis in concert.

Thus, a series of CUL4-DDB1-COP1-SPA E3 ligase complexes may mediate the repression of photomorphogenesis and,

possibly, of flowering time.

INTRODUCTION

Light is an essential signal for plant development and is involved

in diverse processes throughout the life of a plant, ranging from

germination and seedling photomorphogenesis to flowering.

Light-grown seedlings exhibit short hypocotyls and opened

and expanded cotyledons, while dark-grown seedlings show

long hypocotyls, closed cotyledons, and apical hooks. The two

developmental strategies adopted in light and dark conditions

were named photomorphogenesis and skotomorphogenesis,

respectively (von Arnim and Deng, 1996). At the adult stage, the

transition from vegetative to reproductive development in plants

is regulated bymany cues, including photoperiod. InArabidopsis

thaliana, long days (LDs) accelerate flowering, whereas short

days (SDs) delay flowering (Liu et al., 2008).

Genetic screening for Arabidopsis mutants involved in

light-regulated seedling development followed by biochemical

analyses identified a group of pleiotropic Constitutive Photo-

morphogenic/De-etiolated/Fusca (COP/DET/FUS) proteins that

are central negative regulators of photomorphogenesis (Sullivan

et al., 2003; Yi and Deng, 2005). These proteins are components

of one of three biochemical entities: the COP1-SPA complexes,

the COP9 signalosome (CSN), or the CDD complex (COP10,

DDB1, and DET1), all of which are involved in proteasomal

degradation of photomorphogenesis-promoting factors (Serino

and Deng, 2003; Yanagawa et al., 2004; Yi and Deng, 2005; Zhu

et al., 2008).

COP1 is a conserved Ring finger E3 ubiquitin ligase that is

involved in multiple processes in many different organisms, in-

cluding plant development and mammalian cell survival, growth,

and metabolism. COP1 was first cloned and characterized in the

model plant Arabidopsis as a repressor of light-regulated plant

development (Deng et al., 1991, 1992). COP1 was shown to act

as an E3 ligase that targets several photomorphogenesis-

promoting proteins for destruction, including ELONGATED

HYPOCOTYL5 (HY5; Osterlund et al., 2000), HY5 Homolog

1 These authors contributed equally to this work.2 Current address: Plant Science Education Unit, Graduate School ofBiological Sciences, Nara Institute of Science and Technology, 8916-5Takayama, Ikoma-shi, Nara 630-0101, Japan.3 Address correspondence to [email protected] author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Xing Wang Deng([email protected]).CSome figures in this article are displayed in color online but in blackand white in the print edition.WOnline version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.109.065490

The Plant Cell, Vol. 22: 108–123, January 2010, www.plantcell.org ã 2010 American Society of Plant Biologists

Dow

nloaded from https://academ

ic.oup.com/plcell/article/22/1/108/6094855 by guest on 08 February 2022

(Holm et al., 2002), LONG AFTER FAR-RED LIGHT1 (Seo et al.,

2003), LONG HYPOCOTYL IN FAR-RED1 (Duek et al., 2004;

Jang et al., 2005; Yang et al., 2005), and Phytochrome A (Seo

et al., 2004). Recently, COP1 has also been identified as an E3

ligase involved in the regulation of flowering time by directly

targeting transcriptional activator CONSTANS (CO) for degrada-

tion (Jang et al., 2008; Liu et al., 2008). The expression of

FLOWERING LOCUS T (FT) is highly and rapidly increased in

response to CO expression (Samach et al., 2000). Also, COP1

can act with the substrate adaptor EARLY FLOWERING 3 (ELF3)

to modulate light input to the circadian clock by destabilizing

GIGANTEA (GI) (Yu et al., 2008).

SUPPRESSOR OF PHYA 1 (SPA1) was first identified as a

repressor of PHYA (Hoecker et al., 1998). In Arabidopsis, there

are three additional SPA1-like proteins: SPA2, SPA3, and SPA4

(Hoecker et al., 1998, 1999; Zhu et al., 2008). The quadruple spa

mutant displays a phenotype similar to that of strong cop1

alleles, and the SPA genes are partially redundant in mediating

light responses at both seedling and adult stages (Laubinger and

Hoecker, 2003; Laubinger et al., 2004, 2006). Biochemical anal-

ysis demonstrated that the SPA proteins can self-associate or

interact with each other, forming a heterogeneous group of SPA-

COP1 complexes in Arabidopsis (Zhu et al., 2008).

CULLIN4 (CUL4), a recently identified regulator of photomor-

phogenesis, links the three COP complexes (the COP1-SPA

complexes, CSN, and the CDD complex) in the ubiquitination/

proteasome-mediated degradation of key photomorphogene-

sis-promoting factors (Chen et al., 2006). CUL4 is an important

member of the Cullin family and serves as a scaffold in CUL4-

DDB1–based ubiquitin ligases to regulate many processes,

including cell proliferation, DNA repair, and genomic integrity

by ubiquitinating key regulators (Lee and Zhou, 2007). Recent

studies have identified a group of DDB1 and CUL4-Associated

Factors (DCAFs) or DDB1 binding WD40 proteins (DWD) as

substrate receptors that dictate the specificity of the ubiquitina-

tion process in different organisms (Angers et al., 2006; He et al.,

2006; Higa et al., 2006b; Jin et al., 2006; Lee et al., 2008; Zhang

et al., 2008). Furthermore, it has been demonstrated that several

of these DCAFs or DWD proteins interact with RING-BOX1

(RBX1)/Regulator of Cullins 1 (ROC1)-CUL4-DDB1 to form func-

tional E3 ligases. For example, CDT2 was shown to interact with

the CUL4-DDB1 complex to regulate S phase destruction of the

replication factor CDT1 in response to DNA damage (Higa et al.,

2006a; Jin et al., 2006; Ralph et al., 2006; Sansam et al., 2006). It

was further demonstrated that the CUL4-DDB1-CDT2 ubiquitin

ligase also targets the CDK inhibitor p21 for ubiquitination and

degradation (Abbas et al., 2008; Kim et al., 2008; Nishitani et al.,

2008). Similarly, DCAF1/VprBP, aWD40 protein, forms a nuclear

E3 ubiquitin ligase with CUL4-DDB1 and regulates DNA replica-

tion and embryonic development in both mammalian and plant

systems (McCall et al., 2008; Zhang et al., 2008). Related studies

identified the CUL4-DDB1-VprBP E3 ubiquitin ligase complex as

the downstream effector of lentiviral Vpr in arresting the cell cycle

in G2 phase (Hrecka et al., 2007; Le Rouzic et al., 2007).

Moreover, WD40 protein FBW5 (for F-box and WD repeat

domain containing 5) was shown to promote ubiquitination of

tumor suppressor TSC2 and regulate TSC2 protein stability by

associating with DDB1-CUL4-ROC1 ligase (Hu et al., 2008).

Similarly, in plants, DWD protein PRL1 was identified as the

substrate receptor of a CUL4-ROC1-DDB1-PRL1 E3 ligase

involved in the degradation of AKIN10 (Lee et al., 2008).

Our previous work showed that CUL4 may interact with the

CDD complex to form an E3 ligase and regulate photomorpho-

genesis. In addition, CUL4 associated with COP1, which sug-

gested that these two proteins may work together to regulate

photomorphogenesis (Chen et al., 2006). Recently, a detailed

biochemical characterization of the COP1-SPA complexes sug-

gested that the four endogenous homologous SPA proteins can

form stable complexes with COP1 in vivo regardless of light

conditions, although individual SPA proteins exhibited distinct

expression profiles in different tissues and light conditions (Zhu

et al., 2008). However, the mechanism by which CUL4-DDB1

works with the COP1-SPA complexes to mediate the light

regulation of plant development remains to be elucidated.

Here, we demonstrate that CUL4-DDB1 directly interacts with

the COP1-SPA complexes in vivo and in vitro and that this

interaction still exists in the absence of the CDD complex in

Arabidopsis. CUL4-DDB1 and COP1-SPA complexes may work

in concert to regulate photomorphogenesis and, possibly, flower-

ing time under SD conditions. Furthermore, we hypothesize that

CUL4-DDB1 may interact with COP1-SPA complexes to form a

new group of E3 ligases, which are distinct from the previously

described RBX1-CUL4-CDD complex but work in concert with

this complex to mediate light regulation of plant development.

RESULTS

Both COP10 and DET1 Are Required for the Normal

Accumulation of the CDD Complex

Previous data showed that COP10 is significantly reduced and

only present as monomers in det1-1 mutants (Pepper et al.,

1994; Yanagawa et al., 2004). To gain further insight into the

relationship between COP10 and DET1 proteins, we first mea-

sured the steady state abundance of DET1 in 6-d-old cop10-1

(Suzuki et al., 2002), det1-1, and wild-type Wassilewskija (Ws)

andColumbia (Col)Arabidopsis seedlings grown in darkness and

continuous white light. As shown in Figure 1A, DET1 abundance

is dramatically reduced in cop10-1 and absent in det1-1 under

both light and dark conditions. We next performed gel filtration

analysis to investigate formation of the DET1-containing com-

plex in 6-d-old light-grown wild-type, cop10-1, and 35S:flag-

COP10 (in cop10-1 mutant background) seedlings. As shown in

Figure 1B, DET1 was most abundant in the 200- to 300-kD

fractions in wild-type Arabidopsis seedlings, consistent with the

size of the CDD complex purified previously (Yanagawa et al.,

2004). Interestingly, the quantity of theDET1-containing complex

was reduced in the cop10-1 mutant (Figure 1B). The CDD

complex was restored to normal levels in the 35S:flag-COP10

transgenic line, which completely complements the hyperpho-

tomorphogenic phenotype caused by the absence of COP10 in

cop10-1. Thus, our results indicate that COP10 is essential for

normal CDD complex accumulation.

In contrast with these results with DET1, previous data showed

that the DDB1 gel filtration profile is very similar in the wild type

CUL4-DDB1 Associates with COP1-SPA Complexes 109

Dow



and cop10-1 mutants (Yanagawa et al., 2004). Similarly, we

found the CUL4 gel filtration profiles were quite similar in Ws

(wild-type), cop10-1, and flag-COP10, and the abundance of

CUL4 in cop10-1was almost unchanged as well (Figures 1A and

1B). Thus, CUL4-DDB1 is probably involved in other protein

complexes in addition to the CUL4-DDB1-CDD complex.

CUL4 and COP1 Interact with Each Other in the Absence of

the CDD Complex

The fact that RBX1-CUL4 can interact with the CDD complex to

form an E3 ligase (Chen et al., 2006) and that COP10 and COP1

can associate in vivo (Yanagawa et al., 2004) led us to further

study interactions between CUL4-DDB1 and COP1-containing

complexes. To test the in vivo interaction between CUL4

and COP1, we performed coimmunoprecipitation analyses on

6-d-old dark-grown wild-type, 35S:flag-CUL4, 35S:flag-CUL4/

cop10-1, and 35S:flag-CUL4/det1-1 seedlings, respectively. In

thedet1-1 background, all COP10was inmonomer form, and the

CDD complex was disrupted (Yanagawa et al., 2004). As shown

in Figure 1C, CUL4 associates with DDB1 and COP1 in the

absence of COP10 or DET1, showing that the existence of the

CDD complex is not necessary for CUL4 to interact with COP1.

DDB1 Interacts with COP1 and SPA Proteins in Vitro

Recently, several reports showed that a group of proteins

containing the WDXR motif (DWD or DCAF proteins) may work

Figure 1. CUL4 and COP1 Interact with Each Other in the Absence of the CDD Complex.

(A) The DET1 protein level is dramatically reduced in cop10-1mutants. Total protein extracts from 6-d-old continuous white light–grown (cW) and dark-

grown (cD) seedlings were examined by protein gel blot analysis using anti-CUL4, anti-DET1, and anti-Tubulin. Tubulin protein level was used as a

sample loading control.

(B) Protein gel blot analyses showing the gel filtration patterns of DET1 and CUL4 proteins in Ws (wild type), cop10-1, and flag-COP10 (in cop10-1

background), respectively. Molecular masses are indicated at bottom. T, total unfractionated extracts.

(C) Flag-CUL4 associates with DDB1 and COP1 in vivo in wild-type, cop10-1, and det1-1 backgrounds. Total protein extracts prepared from 6-d-old

dark-grown wild-type, 35S:flag-CUL4, 35S:flag-CUL4/cop10-1, and 35S:flag-CUL4/det1-1 transgenic Arabidopsis seedlings were incubated with anti-

flag antibody-conjugated agarose (a-flag). The precipitates and total extracts were subjected to immunoblot analysis with antibodies against flag,

DDB1, DET1, COP1, and RPN6.

110 The Plant Cell

Dow



as adaptors that interact with DDB1 in Cullin-based E3 ligases

and recruit the substrates for degradation in both mammalian

and plant systems (He et al., 2006; Higa et al., 2006b; Jin et al.,

2006; Lee et al., 2008). Sequence alignment analysis showed

that all known components of Arabidopsis COP1-SPA com-

plexes contain at least one WDXR motif (Figure 2A; Lee et al.,

2008). We therefore tested interactions of DDB1 with compo-

nents of COP1-SPA complexes by in vitro pull-down assays. In

these assays (Figure 2B), recombinant DDB1A and DDB1B

glutathione S-transferase (GST) fusion proteins were used to

pull down COP1 tagged with maltose binding protein (MBP) and35S Met-labeled his-tagged SPA proteins produced by coupled

in vitro transcription/translation reactions. As shown in Figure 2B,

GST-DDB1B could pull down MBP-COP1 and all His-tagged

SPA proteins. The amounts of MBP-COP1 and His-SPA proteins

pulled down by GST-DDB1A were lower, possibly due to the

instability and limited amount of GST-DDB1A in our system. To

further test whether DDB1 interacts with COP1 complex com-

ponents via the WDXR motif, the following amino acids were

mutated in the WDXR motifs of COP1, SPA1, and SPA3 (three

representatives showing clear interactions with GST-DDB1B in

Figure 2B): D534A, R536A, D576A, and K578A in COP1, D879A

andR881A inSPA1, andD689A andR691A inSPA3. Asexpected,

the mutations in COP1, SPA1, and SPA3 disrupted their interac-

tions with GST-DDB1B (Figure 2C; see Supplemental Figure

1 online), which demonstrates that the WDXR motifs of COP1

and SPAs are critically important for interaction with DDB1.

In Vivo Association between CUL4-DDB1 and

COP1-SPA Complexes

Arabidopsis has two isoforms of DDB1: DDB1A and DDB1B.

DDB1B is the predominant player in regulating embryogenesis

since knockout line ddb1b is embryo-lethal, while knockout line

ddb1a exhibits no obvious phenotype (Schroeder et al., 2002).

DDB1 works with CUL4 by directly interacting with both CUL4

and downstream adaptor proteins, and CUL4 and COP1 have

been observed to associate in vivo in Arabidopsis (Angers et al.,

2006; Chen et al., 2006; He et al., 2006; Higa et al., 2006b; Jin

et al., 2006; Lee et al., 2008). We therefore tested whether

DDB1B interacts with COP1-SPA complexes by performing

coimmunoprecipitation analyses using transgenic ddb1a seed-

lings transformed with full-length DDB1B fused to the flag

peptide and driven by the 35S promoter (35S:flag-DDB1B/

ddb1a) grown for 6 d in the dark or in continuous white light.

Transgenic flag-DDB1B pulled down CUL4, DET1, COP1, and

SPA1 (Figure 3A), which suggested that CUL4-DDB1 and COP1-

SPA complexes associate in vivo. Since the Arabidopsis SPA

family has four members, which have all been shown to interact

with COP1 in all light conditions and form heterogeneous com-

plexes (Zhu et al., 2008), we further tested the interactions

of DDB1 with SPA proteins using seedlings transformed with

SPA proteins fused to the tandem affinity purification (TAP)

peptide in the appropriate mutant background. All four TAP-SPA

proteins pulled down COP1 very effectively, confirming that

COP1 and SPA proteins may form tight complexes (see Supple-

mental Figure 2 online). TAP-SPA1, TAP-SPA3, and TAP-SPA4

also coimmunoprecipitated low levels of DDB1 protein (see

Figure 2. DDB1A and DDB1B Interact with COP1 and SPA Proteins in

Vitro.

(A) Alignment of the WDXR regions of COP1, SPA1, SPA2, SPA3, and

SPA4. The WDXR motif is highlighted in red. Pink, conserved D or E;

yellow, hydrophobic amino acids; gray, small amino acids.

(B) In vitro coimmunoprecipitation of COP1 and SPA proteins by recom-

binant DDB1A and DDB1B. Recombinant MBP-COP1 purified from

Escherichia coli and 35S-labeled His-SPA1, His-SPA2, His-SPA3, and

His-SPA4 generated by the TNT system were used as prey molecules

and incubated with unlabeled GST, GST-DDB1A, and GST-DDB1B,

respectively. Anti-GST antibody fused to agarose was used for immu-

noprecipitation. Pellet fractions were resolved by SDS-PAGE and visu-

alized by Coomassie blue staining (for GST fused proteins), anti-COP1

immunoblot (for MBP-COP1), and autoradiography using a phosphor

imager (for SPA proteins). Quantification was based on normalization

against the value of the background region. Band intensities of MBP-

COP1, His-SPA1, His-SPA2, His-SPA3, and His-SPA4 pulled down by

GST-DDB1B were each set to 100. Relative band intensities were then

calculated and are indicated by numbers below blots.

(C) Point mutations within the WDXR motifs of COP1 and SPA proteins

disrupt their interactions with DDB1. MBP-COP1mWDXR contains the

following mutations in the WDXR motifs: D534A, R536A, D576A, and

K578A; His-SPA1mWDXR contains mutations D879A and R881A; His-

SPA3mWDXR contains mutations D689A and R691A. Wild-type and

mutated recombinant MBP-COP1 purified from E. coli, and wild-type

and mutated 35S-labeled His-SPA1, His-SPA3 generated by the TNT

system were used as prey molecules and incubated with GST and GST-

DDB1B, respectively. Anti-GST antibody fused to agarose was used for

immunoprecipitation. Pellet fractions were resolved by SDS-PAGE and

visualized by anti-GST immunoblot (for GST fused proteins), anti-MBP

immunoblot (for wild-type and mutated MBP-COP1), and autoradiogra-

phy using x-ray films (for wild-type and mutated SPA proteins). Quan-

tification was performed for each pair of wild-type and mutated prey

proteins. Band intensities of MBP-COP1, His-SPA1, and His-SPA3

pulled down by GST-DDB1B were set to 100. Relative band intensities

were then calculated and are indicated by numbers below blots.


Dow



Supplemental Figure 2 online). The low level of DDB1 pulled

down by SPA proteins is probably due to the fact that DDB1 can

interact with a large number of different downstream adaptor

proteins (Lee et al., 2008; Zhang et al., 2008). TAP-SPA2 failed to

pull down DDB1, probably due to the low abundance of TAP-

SPA2 and the presence of truncated SPA2 in that line, which

likely competes with TAP-SPA2 for interaction with DDB1. Al-

though the interactions shown between TAP-SPA1, TAP-SPA3,

TAP-SPA4, and DDB1 are weak (see Supplemental Figure 2

online), this result does support the interactions between DDB1

and SPA proteins shown in Figures 2 and 3A. To further test the

interaction between CUL4-DDB1 and COP1 complexes, flag-

DDB1B transformed into the det1-1 mutant was used for coim-

munoprecipitation. As shown in Figure 3B, flag-DDB1B can still

pull down COP1 in the absence of DET1, similarly to flag-CUL4

(Figure 1C), further demonstrating that the interaction between

CUL4-DDB1andCOP1exists in the absence of theCDDcomplex.

CUL4-DDB1 and COP1-SPA Complexes Act Together to

Regulate Photomorphogenesis

Since both in vivo and in vitro data showed that there are bio-

chemical interactions between CUL4-DDB1 and the COP1-SPA

complexes, we decided to investigate the functional significanceof

this biochemical interaction in plant development. To this end, we

crossed a weak cop1 allele, cop1-4 (McNellis et al., 1994), with

cul4cs (for CUL4 cosuppression; Chen et al., 2006) and examined

the photomorphogenic phenotype. Some cul4cs plants (<5%)

opened their cotyledons and had short hypocotyls in the dark,

which are characteristics of weak constitutive photomorphogenic

mutants. We found that 6-d-old dark-grown cul4cs cop1-4 seed-

lings had opened cotyledons and extremely short hypocotyls,

which is similar to the cop lethal phenotype (Figure 4A). Moreover,

these double mutants accumulated large amounts of anthocyanin,

similar to the null mutant cop1-5 (McNellis et al., 1994). In addition,

25% of the F2 seeds of cul4cs crossed with cop1-4 were purple

(Figure 4B) due to the anthocyanin overaccumulation typically

found in cop lethal mutants (McNellis et al., 1994). Thus, reduction

of CUL4 function enhances the weak cop1-4 mutant phenotype,

supporting the hypothesis that CUL4-DDB1 works together with

COP1-SPA complexes to regulate plant photomorphogenesis.

CUL4 Regulates Flowering Time under SD Conditions

Previous data showed that both COP1 and SPA1 are involved in

regulating flowering time, and both cop1 and spa1 mutants

Figure 3. CUL4-DDB1 Interacts with the COP1-SPA Complexes in Vivo.

(A) Flag-DDB1B associates with CUL4, DET1, COP1, and SPA1 in vivo. Total protein extracts prepared from 6-d-old continuous white light–grown (cW)

and dark-grown (cD) wild-type and 35S:flag-DDB1B/ddb1a (abbreviated as flag-DDB1B) transgenic Arabidopsis seedlings were incubated with anti-

flag antibody-conjugated agarose (a-flag). The precipitates and total extracts were subjected to immunoblot analysis with antibodies against flag,

CUL4, DET1, COP1, SPA1, and RPN6. RPN6 was used as a control.

(B) Flag-DDB1B associates with COP1 in wild-type and det1-1 backgrounds. Total protein extracts prepared from 6-d-old dark-grown wild-type, 35S:

flag-DDB1B/ddb1a (abbreviated to be flag-DDB1B), and 35S:flag-DDB1B/det1-1 transgenic Arabidopsis seedlings were incubated with anti-flag

antibody-conjugated agarose (a-flag). The precipitates and total extracts were subjected to immunoblot analysis with antibodies against flag, DET1,

COP1, and RPN6. RPN6 was used as a control.

112 The Plant Cell

Dow



flower earlier under SDs (8 h light/16 h dark) (McNellis et al., 1994;

Laubinger et al., 2006; Jang et al., 2008; Liu et al., 2008). Since

CUL4 associates with COP1 and SPA1, we next asked whether

CUL4 is involved in regulating flowering time under SDs and

found that cul4cs floweredmuch earlier than its counterpart wild-

type Col under SDs (Figure 5A, Table 1). When the siliques of

cul4cs turned yellow, the wild-type plants had not yet bolted.

This result indicated that CUL4 is functionally involved in regu-

lating flowering time. By contrast, under LDs (16 h light/8 h dark)

cul4cs plants flowered at the same time as wild-type Col (Table

1). Thus, CUL4 regulates flowering time specifically under SDs.

In previous studies on photoperiodic flowering, CO was iden-

tified as a pivotal regulator for triggering flowering, which induces

the expression of the flowering time gene FT (Putterill et al., 1995;

An et al., 2004). Early flowering of SD-grown spa mutants

correlates with strongly increased FT transcript levels, whereas

CO transcript levels are not altered, and SPA proteins regulate

the stability of CO protein (Laubinger et al., 2006). COP1 regu-

lates CO function at both transcriptional and posttranslational

levels and reduces FT mRNA levels (Jang et al., 2008; Liu et al.,

2008; Yu et al., 2008). To investigate whether CUL4 also influ-

ences CO and FT, CO and FT transcript levels were examined in

Col and cul4cs grown under SDs and harvested at Zeitgeber (ZT)

16 (Figure 5B). There was no obvious difference in CO mRNA

abundance between cul4cs and its wild-type counterpart Col. By

contrast, FT transcript levels in 20-d-old cul4cswere higher than

in Col. We therefore studied the time course of FT transcription,

starting with young seedlings under SDs, by quantitative real-

time PCR. As shown in Figure 5C, compared with in Col, FT

transcription in cul4cs was elevated in the fourth day and

dramatically increased from the sixth day after they were sown

in soil. Thus, the decreased level of CUL4 protein resulted in a

significant increase in FT transcript level starting early in devel-

opment, indicating that CUL4 may play a negative role in regu-

lating FT transcription.

Since transcription of CO and FT shows circadian regulation,

we examinedCO and FT transcript levels in wild-type and cul4cs

plants to test whether CUL4 affects the circadian patterns of CO

and FT expression. As shown in Figure 5D, wild-type and cul4cs

plants showed very similar diurnal regulation of COmRNA levels

in SD-grown plants. Interestingly, FT transcript levels in cul4cs

were elevated through the whole day with two possible minor

peaks (Figure 5E). The pattern with a peak level of FT transcripts

in cul4cs around ZT 20 is similar to the patterns in cop1 and spa

mutants (Laubinger et al., 2006; Yu et al., 2008), which indicates

that CUL4 may function similarly to the COP1 and SPA proteins

to regulate FT transcription during the night phase. However, the

phenomenon that FT transcript levels increased during the day

phase with a possible minor peak level around ZT 8 is new. The

possible two-peak pattern of FT transcripts in cul4cs was con-

sistent in the two continuous days that we examined and was

repeatable in three independent experiments. Themechanism of

the regulation of FT transcripts by CUL4 is unclear.

DDB1 Is A Core Adaptor Protein

In CUL4-based E3 ligases, DDB1 plays an important role in

recruiting the adaptor proteins. DDB1 has three domains and

interacts with CUL4 via the BPB domain (Angers et al., 2006; Jin

et al., 2006). To test specific roles of the DDB1 domains in

Arabidopsis, we generated three groups of point mutations on

the surface of DDB1B’s three domains, which are predicted to be

responsible for mediating interactions with other proteins based

on structural information. The mutated amino acids and their

positions on the surface of DDB1B are shown in Figure 6A.

Mutant DDB1B coding sequences taggedwith the FLAGpeptide

and driven by the 35S promoter were transformed into ddb1a

plants, and the three resulting groups of transgenic plants were

named mBPA, mBPB, and mBPC, corresponding to the muta-

tions within the BPA, BPB, and BPC domains of DDB1B.

TransgenicmBPA andmBPBplants had phenotypes similar to

ddb1a plants and were used for coimmunoprecipitation analy-

ses. As shown in Figure 6B, themutations within the BPB domain

completely disrupted the interaction between CUL4 and DDB1,

while the mutations within the BPA domain did not affect the

interaction between CUL4 and DDB1. This is consistent with

results in mammalian cells (Angers et al., 2006; Jin et al., 2006).

Mutating the BPC domain had a very different effect: several

mBPC transgenic lines showed a severely dwarfed phenotype

(Figure 6C) that was stably transmitted to the next generation,

suggesting that the BPC domain is important for DDB1 function.

Although we are interested in studying the effects of the BPC

domain mutation on DDB1’s interactions with other proteins, we

could not collect enough material for further coimmunoprecipi-

tation analysis because each dwarf mBPC plant only yields a few

seeds. The observation that 75% of the progeny from a heter-

ozygous mBPC plant showed the identical severely dwarfed

phenotype indicates that one copy of the transgenic fragment is

Figure 4. CUL4 and COP1 Function Together to Regulate Photomor-

phogenesis.

(A) Repression of CUL4 enhances the seedling phenotype of the weak

cop1-4 allele in the dark. Different Arabidopsis lines (labeled at bottom)

were grown in complete darkness for 6 d. Bars = 1 mm.

(B) Repression of CUL4 increases anthocyanin accumulation in cop1-4

seeds. The cul4cs and cop1-4 seeds are homozygous, while the cul4cs

cop1-4 are F2 seeds from hybrid F1 plants.


Dow



sufficient to induce the phenotype. To test whether the dwarf

phenotype of mBPC is due to cosuppression of DDB1B, we

tested the levels of DDB1B mRNA and protein and found that

both DDB1B mRNA and protein levels, including endogenous

DDB1B and transgenic mutated DDB1B, are higher in mBPC

than in ddb1a (Figures 6D and 6E). Accordingly, the dwarf

phenotype may result from a dominant-negative effect of the

mutated DDB1B protein rather than DDB1B cosuppression.

No Obvious in Vivo Association between the CDD and

COP1-SPA Complexes

Although COP1-SPA complexes can interact with CUL4-DDB1

in the absence of the CDD complex, it is still possible that CUL4-

DDB1, COP1-SPA complexes, and the CDD complex all exist in

a super complex connected by DDB1’s multiple interacting

domains in planta. To test this, the interaction between DET1

and COP1, core components of the CDD complex and COP1-

SPA complexes, respectively, was examined by coimmunopre-

cipitation. Because native DET1 protein runs close to the IgG

heavy chain in SDS-PAGE and it is difficult to distinguish from the

IgG heavy chain, transgenic green fluorescent protein (GFP)-

DET1/det1-1 that rescued the det1-1 phenotype was used for

testing the interaction between DET1 and COP1. As shown in

Figure 7A, using seedlings grown under both white light and dark

conditions, antibodies against DET1 precipitated GFP-DET1

very well but did not pull down COP1 at all. Conversely, COP1

antibodies precipitated COP1 very well but did not pull down

GFP-DET1. At the same time, both DET1 and COP1 showed

interactions with DDB1. In addition, we used TAP-COP1 seed-

lings (transformed with COP1 fused to the TAP peptide) to

confirm the result that COP1 and DET1 have no obvious inter-

action. As shown in Figure 7B, TAP-COP1 could pull downDDB1

well but failed to pull down DET1, confirming that COP1 is

associated with DDB1 but not DET1 in planta. Thus, we did not

detect an in vivo association between DET1 and COP1 proteins,

indicating that CDDandCOP1-SPA complexesmay not exist in a

supercomplex.

We next performed gel filtration analysis to investigate forma-

tion of the DET1-containing complex in 6-d-old light-grown wild-

type and quadruple spa (Laubinger et al., 2006) seedlings. As

shown in Figure 7C, DET1 was found in similar fractions in

extracts from wild-type and spa1234 mutants, which suggests

that integrity of DET1-containing complexes is not dependent on

COP1-SPA complexes. Similarly, formation of COP1-containing

complexes in 6-d-old light-grownwild-type anddet1-1 seedlings

was tested by gel filtration. As shown in Figure 7C, COP1Figure 5. CUL4 Is Involved in the Regulation of Flowering Time

under SDs.

(A) cul4cs flowers early in SDs. cul4cs and its wild-type counterpart Col

were grown for 90 d under SDs (8 h light/16 h dark).

(B) Levels of CO and FT transcripts in cul4cs and wild-type Col. Total

RNA samples were collected from 20-d-old Col and cul4cs plants grown

under SDs (8 h light/16 h dark) and harvested at ZT 16. mRNA levels of

CO, FT, and ACTIN2 were tested by RT-PCR with three repeats. ACTIN2

transcripts were used as control.

(C) FT transcription starts very early in SD-grown cul4cs. Total RNA

samples were collected from 4-, 6-, 8-, 10-, 12-, and 14-d-old Col and

cul4cs plants grown under SDs (8 h light/16 h dark) and harvested at ZT

16. Levels of FT mRNA and 18S rRNA were tested by real-time PCR.

Relative amounts of FT transcripts were normalized to the levels of 18S

rRNA in the same sample.

(D) and (E)Circadian patterns ofCO (D) and FT (E)mRNA levels in cul4cs

and wild-type Col. The plants were grown under SDs (8 h light [white bar]/

16 h dark [black bar]) for 18 d and then RNA samples were collected

every 4 h, at the times shown, after dawn. Levels of CO and FT mRNA

and 18S rRNA were analyzed by real-time PCR. Relative amounts of CO

and FT transcripts were normalized to the levels of 18S rRNA. For (C) to

(E), mean and SD values of four replicates are shown. Some error bars are

too small to be shown in the figure.

114 The Plant Cell

Dow



complexes in the absence of DET1 are similar to those in the wild

type, which indicates that the COP1-containing complexes exist

in the absence of the DET1-containing complexes. These find-

ings support the hypothesis that CDD and COP1-SPA are

distinct complexes.

Given that flag-COP10 can coimmunoprecipitate COP1 in vivo

(Yanagawa et al., 2004), we then examined whether the in vivo

association between flag-COP10 and COP1 supports an inter-

action between the CDD complex and COP1-SPA complexes.

To do so, we performed gel filtration and coimmunoprecipitation

on 35S:flag-COP10/det1-1 extracts. The gel filtration profile of

flag-COP10 in det1-1 indicated that it existed solely as mono-

mers in this background, whereas it existed as both monomers

and in complexes in cop10-1 (Figure 7D). This is consistent with

the result that endogenous COP10 exists solely as monomers in

det1-1mutants (Yanagawa et al., 2004). Coimmunoprecipitation

showed that flag-COP10 still associates with COP1 when DET1

is disrupted (Figure 7E). Therefore, it is the COP10 monomer

rather than the CDD complex that associates with COP1. Ac-

cordingly, interaction between COP10 and COP1 does not

indicate that the CDD complex and the COP1-SPA complexes

interact. Instead, these data support a conclusion that both the

CDD complex and the COP1-SPA complexes can associate with

CUL4 in vivo but may form distinct complexes instead of a

supercomplex.

CDD and COP1-SPA Complexes Act Additively to

Regulate Photomorphogenesis

To characterize the functional relations between the CDD com-

plex and COP1-SPA complexes, the weak alleles cop10-4 and

det1-1 were introduced into the spa triple mutants. As shown in

Figure 8, reduction of COP10 and DET1 additively enhances the

phenotype of spa triple mutants, which is consistent with the

lethal phenotype of the double mutant det1-1 cop1-6 (Ang and

Deng, 1994). This shows that the components of the CDD

complex and the COP1-SPA complexes have genetic interac-

tions, implying that even without a direct association they may

work closely together in regulating photomorphogenesis.

DISCUSSION

This study provides a biochemical and functional analysis of

CUL4-DDB1 and COP1-SPA complexes. Our data show that

CUL4-DDB1 may directly interact with COP1-SPA complexes in

the absence of theCDDcomplex and form aheterogenous group

of E3 ligases that regulate multiple aspects of the light regulation

of plant development. Our previous data showed that the CSN

regulates the rubylation and derubylation cycle of CUL4, while

the CDD complex may bind CUL4 to form another E3 ligase

(Chen et al., 2006). How this CUL4-CDDE3 ligaseworks together

with CUL4-DDB1-COP1-SPA E3 ligases remains to be eluci-

dated.

CUL4-DDB1 and COP1-SPA Complexes Associate in Vivo

Previous data showed that CUL4 and COP1 proteins interact,

indicating that they may work in concert to mediate repression of

photomorphogenesis (Chen et al., 2006); here, we provide more

evidence to reveal the mechanism by which CUL4-DDB1 and

COP1-SPA complexes work together. Recently, several groups

independently reported that CUL4-based E3 ligases use the

adaptor protein DDB1 to interact with DCAF substrate recogni-

tion receptors (also called DWD or CDW proteins) and recruit the

substrates (Angers et al., 2006; He et al., 2006; Higa et al., 2006b;

Jin et al., 2006; Lee et al., 2008). Interestingly, core components

of COP1-SPA complexes, including COP1, SPA1, SPA2, SPA3,

and SPA4, all contain the WDXR motif, which was suggested to

be the determinant feature for binding DDB1. In vitro pull-down

experiments demonstrated that COP1 and SPA proteins may

directly interact with DDB1 (Figure 2), while in vivo coimmuno-

precipitation showed that the components of these COP1-SPA

complexes are associated with CUL4-DDB1 (Figure 3). Point

mutationswithin theWDXRmotifs ofMBP-COP1, His-SPA1, and

His-SPA3 disrupted their interactions with GST-DDB1B, which

suggests that associations between CUL4-DDB1 and COP1-

SPA complexes are via WDXR motifs. Regulation of these

interactions by different light conditions needs further study.

CUL4-DDB1 and COP1-SPA Complexes Work Together to

Regulate Multiple Aspects of the Light Regulation of

Plant Development

Since cul4cs, cop1, and spa mutants all show similar photo-

morphogenetic phenotypes (Deng et al., 1991, 1992; Laubinger

et al., 2004; Chen et al., 2006), it is expected that they may work

together to regulate the repression of photomorphogenesis.

Genetic interaction tests showed that reduction of CUL4 resulted

in enhancement of the phenotype of the weak cop1-4 mutant,

and the phenotype of cul4cs cop1-4 double mutants was iden-

tical to the lethal mutant cop1-5 (Figure 4). This is consistent with

previous data showing that CUL4 positively regulates the deg-

radation of HY5, a transcription factor enhancing photomorpho-

genesis regulated by COP1-SPA1 (Osterlund et al., 2000; Saijo

et al., 2003; Chen et al., 2006).We therefore conclude that CUL4-

DDB1 and the COP1-SPA complexes may work together to

regulate photomorphogenesis by regulating transcription factors

such as HY5.

As previously reported, cop1-4, spa1-7, and spa1 spa3 spa4

triple mutants exhibit early flowering phenotypes, and COP1 and

SPAs are involved in regulating flowering time under SDs

(McNellis et al., 1994; Laubinger et al., 2006; Jang et al., 2008;

Liu et al., 2008). Since CUL4 interacts with COP1 and SPA1, we

tested and found that cul4cs also flowers earlier than the wild

Table 1. Flowering Time of cul4cs and Col in SDs and LDs

Genotype Number of Leaves (SD) Number of Leaves (LD)

Col (wild type) 60.6 6 3.3 11.1 6 0.5

cul4cs 29.2 6 2.9 10.9 6 0.4

Plants were grown in SDs (8 h light/16 h dark) and LDs (16 h light/8 h

dark). Values shown are mean numbers of rosette leaves at flowering 6

SD. At least 15 plants were analyzed for each genotype. The cul4cs

mutation is in the Col ecotype.


Dow



Figure 6. Effects of Point Mutations on the Surface of the DDB1 Protein.

(A) Structural diagram depicting the locations of the three groups of mutations on the surface of DDB1. Mutated residues are indicated.

(B) Effects of DDB1 mutations on binding to CUL4 and COP1. Wild-type or point-mutated DDB1B coding sequences fused to the flag peptide and

driven by the 35S promoter were transformed into ddb1amutants, and the transgenic plants were used for coimmunoprecipitation. DDB1B, mBPA, and

mBPB indicate wild-type flag-DDB1B, flag-DDB1B with point mutations within the BPA domain, and flag-DDB1B with point mutations within the BPB

domain, respectively. Total protein extracts from 6-d-old light-grown Arabidopsis seedlings were incubated with anti-flag antibody-conjugated agarose

(a-flag). The precipitates and total extracts were subjected to immunoblot analysis with antibodies against flag, CUL4, and COP1.

(C) Phenotypes of 38-d-old ddb1a and mBPC plants under LDs (16 h light/8 h dark). The ddb1amutant is in the left pot, and the mBPC mutant is in the

right pot. mBPC contains flag-DDB1B with point mutations in the BPC domain transformed into the ddb1a mutant background.

(D) DDB1 transcript levels in ddb1a and mBPC. Total RNA samples were collected from 4-week-old ddb1a and mBPC plants, and DDB1B expression

levels were quantified by RT-PCR with three repeats. ACTIN2 transcripts were used as control.

(E) DDB1 protein levels in ddb1a and mBPC. Total proteins were extracted from 15-d-old ddb1a and mBPC plants and subjected to immunoblot

analysis with antibodies against flag, DDB1, and Tubulin. Tubulin was used as a sample loading control.

116 The Plant Cell

Dow



Figure 7. Biochemical Interactions between the CDD and COP1-SPA Complexes.

(A) GFP-DET1 and COP1 have no obvious in vivo interaction. Total protein extracts prepared from 6-d-old GFP-DET1/det1-1 transgenic Arabidopsis

seedlings grown in continuous white light (cW) or in the dark (cD) were incubated with anti-COP1 or anti-DET1 and then precipitated with Protein A

agarose. The precipitates and total extracts were subjected to immunoblot analysis with antibodies against COP1, DET1, DDB1. and RPN6.

(B) TAP-COP1 and DET1 have no obvious in vivo interaction. Total protein extracts prepared from 6-d-old Col and TAP-COP1 Arabidopsis seedlings

grown in the dark or continuous white light were incubated with IgG-coupled Sepharose. The precipitates and total extracts were subjected to

immunoblot analysis with antibodies against Myc, DDB1, DET1, and RPN6. RPN6 was used as a control.

(C) Protein gel blot analyses showing the gel filtration patterns of DET1 protein in Col (wild type) and spa1234, and COP1 protein in Col (wild type)

and det1-1. Molecular masses are indicated below the blot. Total: total unfractionated extracts.


Dow



type (Figure 5A). Molecularly, the FT transcript level is elevated in

cop1-4, spa1-7, and spa1 spa3 spa4 triple mutants (Laubinger

et al., 2006; Jang et al., 2008; Liu et al., 2008). Here, cul4cs early

flowering is ascribed to the accumulation of FT transcripts

(Figures 5B and 5C), suggesting that CUL4 participates in the

transcriptional regulation of FT and is involved in the negative

regulation of flowering.

As a floral output gene, FTmRNA abundance is dependent on

the level of COprotein (Valverde et al., 2004). It has been reported

that both COP1 and SPA1 interact with CO and shape its

temporal expression pattern by regulating its stability (Laubinger

et al., 2006; Jang et al., 2008; Liu et al., 2008). CO is a positive

regulator of flowering. The level of CO protein is elevated in spa1

mutants, whereas CO mRNA levels are unchanged, suggesting

that SPA proteins mediate CO protein degradation (Laubinger

et al., 2006). Two recent reports demonstrated that COP1 can

interact with CO directly and mediate its ubiquitination and

degradation to regulate flowering time (Jang et al., 2008; Liu

et al., 2008). Also, COP1 was shown to act with ELF3 to regulate

GI stability and modulate expression of CO (Yu et al., 2008).

Taken together, these data indicate that COP1-SPA complexes

modulate flowering time by regulating CO function at both

transcriptional and posttranscriptional levels (Yu et al., 2008). It

is of interest to examine CUL4’s role in the regulation of CO. As

shown in Figure 5D, CUL4 has no effect on CO transcript levels

under SDs, which is similar to the situation that CO transcript

levels are not altered in SD-grown spamutants (Laubinger et al.,

2006). By contrast, the onset of CO expression in cop1-4

mutants shifted 4 h earlier, leading to elevated CO expression

during daytime (Yu et al., 2008). This indicates that CUL4-DDB1

may work with COP1-SPA complexes to regulate the degrada-

tion of CO protein, and COP1 itself can interact with ELF3 to

modulateCO transcription through targeted destabilization ofGI.

Further examination of CO protein levels in cul4cs may provide

more information about how CUL4 regulates flowering time

under SDs.

A critically interesting result is the circadian pattern of FT

transcript levels in cul4cs, which shows general elevation with

possibly twominor diurnal peaks. The peak of FT transcript levels

around ZT 20 in SD-grown cul4cs is very similar to the peaks in

cop1 and spamutants (Figure 5E; Laubinger et al., 2006; Yu et al.,

2008). In this report, we demonstrated that CUL4-DDB1 asso-

ciates with COP1 and SPA proteins (Figures 1 to 3). It is possible

that CUL4-DDB1 interacts with COP1-SPA complexes to de-

grade CO protein and reduce FT transcripts in the night phase of

SDs. There is a possible second peak of FT transcript levels

around ZT 8 in SD-grown cul4cs (Figure 5E). As shown in Figure

5D, COmRNA levels are very low around ZT 8, which means CO

translation must also be low at this time. Also, the photoreceptor

phyB promotes the degradation of CO protein in the early day,

which means that CO protein accumulated in the night and early

morning will not persist until ZT 8 (Valverde et al., 2004; Jang

et al., 2008). Therefore, the increase in FT transcript levels around

ZT 8 in SD-grown cul4cs is not regulated by CO protein. This

conclusion is consistent with the reports that FT acts partially

downstream of CO and may also be mediated by other regula-

tors (Kardailsky et al., 1999; Kobayashi et al., 1999). The mech-

anism of CUL4’s repression of FT transcription in the day phase

in SD remains to be elucidated.

CUL4-DDB1-COP1-SPA Complexes and the CUL4-CDD

Complex Are TwoDistinct Groups of Complexes Involved in

Light Regulation of Plant Development

Our previous data showed that in Arabidopsis RBX1, CUL4 and

CDD form a complex that regulates photomorphogenesis (Chen

et al., 2006). Our data indicate that CUL4-DDB1 can interact with

COP1-SPA complexes in the absence of the CDD complex

Figure 7. (continued).

(D) Protein gel blot analyses showing the gel filtration patterns of flag-COP10 (F-COP10) in cop10-1 and det1-1.Molecular weights are indicated below

the blot. T, total unfractionated extracts. Arrow indicates the flag-COP10 complexes.

(E) Flag-COP10 monomer interacts with COP1 in vivo. Total protein extracts prepared from 6-d-old wild-type, flag-COP10/cop10-1, and flag-COP10/

det1-1 transgenic Arabidopsis seedlings grown in the dark were incubated with anti-flag antibody-conjugated agarose (a-flag). The precipitates and

total extracts were subjected to immunoblot analysis with antibodies against flag, COP1, DET1, and RPN6.

Figure 8. The CDD Complex and COP1-SPA Complexes Together

Mediate Plant Photomorphogenesis.

(A) cop10-4 enhances the seedling phenotype of spa1 spa2 spa4 triple

mutants in the dark.

(B) Reduction of DET1 in the spa1 spa2 spa3 triple mutant enhances the

seedling phenotype of spa1 spa2 spa3 in the dark. All plants (labeled at

bottom) were grown in continuous darkness for 6 d. Bars = 1 mm.

[See online article for color version of this figure.]

118 The Plant Cell

Dow



(Figures 1 to 3). In addition, the coimmunoprecipitation results

using GFP-DET1/det1-1 and TAP-COP1 plants showed that

there is no obvious association between DET1 and COP1 pro-

teins, which excludes the possibility that CUL4, CDD, andCOP1-

SPA all exist in a super complex (Figures 7A and 7B). Similar

DET1-containing complexes in quadruple spamutants and wild-

type plants and similar COP1-containing complexes in det1-1

and wild-type plants further support this conclusion (Figure 7C).

Both DET1 and COP1 can interact with DDB1 (Figures 3, 7A, and

7B), but they may do so in distinct complexes.

Since flag-COP10 can coimmunoprecipitate COP1 in Arabi-

dopsis, the CDD complex was considered to associate with

COP1 complexes in vivo (Yanagawa et al., 2004). However, our

data show that there is no obvious association between DET1

and COP1, which seems to contradict the previous conclusion.

Repeated experiments showed that there is interaction between

flag-COP10 and COP1 and that this interaction persists when

DET1 is disrupted (Figures 7D and 7E). This indicates that the

existence of the CDD complex is not necessary for interaction

between flag-COP10 and COP1 and that interaction between

flag-COP10 and COP1 does not represent interaction between

the CDD complex and COP1 complexes.

Inmammalian systems, DET1 andCOP1were shown to form a

dimer and work as CUL4-based E3 ligase adaptor proteins for

some substrates including c-Jun (Wertz et al., 2004). However, in

plant systems, much data suggest that DET1 and COP1 work in

distinct complexes based on their different subcellular locations

(Pepper et al., 1994; von Arnim and Deng, 1994; Ang et al., 1998;

Schroeder et al., 2002), protein complex sizes (Saijo et al., 2003;

Yanagawa et al., 2004), and reconstitution results (Chen et al.,

2006). The work reported here further showed that there is no

obvious in vivo association between DET1 and COP1. These

data support the conclusion that DET1 and COP1 exist in distinct

complexes.

Although the CDD and COP1-SPA complexes have no direct

interaction, they work together to regulate photomorphogenesis

(Figure 8).We propose that inArabidopsis, CUL4-DDB1 interacts

with COP1-SPA complexes to form a group of ligases distinct

from the previously identified CUL4-CDD complex (Figure 9).

These two groups of ligasesmaywork in concert tomodulate the

light regulation of plant development by targeting photomorpho-

genesis- and possibly flowering-promoting factors, such as HY5

and CO, for degradation. The function of all of these ligases

depends on the derubylation activity of the CSN. However, the

relationship between CUL4-CDD and CUL4-DDB1-COP1-SPA

complexes and themechanismbywhich the interaction between

the complexes is regulated still need further investigation. This

may require a combination of different strategies and studies

under different light conditions and development stages.

Another challenge is to determine the cooperation between

the RBX1-CUL4-DDB1 and COP1-SPA E3 ligase complexes. In

mammalian systems, c-Jun was thought to be ubiquitinated by a

CUL4-based E3 ligase using COP1 as an adaptor, but the RING

finger domain activity of COP1was found not to be necessary for

the ubiquitination (Wertz et al., 2004). For targeting and ubiquiti-

nating p53, COP1 itself can work as an E3 ligase, and its RING

activity is necessary for the ubiquitination (Dornan et al., 2004).

This indicates that COP1 may function as an E3 ligase for some

substrates and work as an adaptor protein of CUL4-DDB1 E3

ligases for other substrates. In Arabidopsis, complementation

experiments showed that COP1 (105-675) and COP1 (293-675),

two truncated COP1 proteins lacking the RING domain, could

weakly rescue the cop1-5 lethal allele (Stacey et al., 2000). This

suggests that the RING activity of COP1may not be essential for

targeting some substrates. This will be tested by introducing

COP1 (C52S, C55S, C86S, and C89A) with point mutations in the

RING motif shown to abolish the ligase activity (Seo et al., 2003,

2004) into the cop1-5 lethal allele to see whether it can partially

rescue the phenotype. In addition, interaction of COP1 (C52S,

C55S, C86S, and C89A) with CUL4-DDB1 and the COP1

Figure 9. A Working Model of How CUL4 and the Three COP/DET/FUS

Complexes Mediate Light Regulation of Plant Development.

RBX1-CUL4 and the CDD complex form a functional E3 ligase, while

RBX1-CUL4-DDB1 may interact with COP1-SPA complexes to form

another group of E3 ligases. Rubylation and derubylation of these CUL4-

based E3 ligases are regulated by the CSN complex, and these ligases

regulate the degradation of downstream factors to mediate light regu-

lation of plant development. The RBX1-CUL4-CDD complex and RBX1-

CUL4-DDB1-COP1-SPA complexes may have regulatory relationships

(shown by broken arrow), which require further study. DDA1 (for DET1,

DDB1 associated 1) is shown as a small-sized component of the CDD

complex. SPAx and SPAy represent homogenous or heterogeneous SPA

proteins. A, B, and C represent the BPA, BPB, and BPC domains of

DDB1, respectively. Ub represents Ubiquitin. E1 represents a ubiquitin-

activating enzyme, and E2 represents a ubiquitin-conjugating enzyme.


Dow



substrate degradation pattern will be examined. These investi-

gations, together with other genetic, biochemical, and cell bio-

logical studies, will reveal how the RBX1-CUL4-DDB1 and

COP1-SPA complexes cooperate.

METHODS

Plant Material and Growth Conditions

Wild-type Arabidopsis thaliana plants used in this study were Col-0, Ws,

and RLD ecotypes. Mutants and transgenic lines previously described

are as follows: cul4cs (Chen et al., 2006), cop10-1 (Wei et al., 1994),

cop10-4 (Suzuki et al., 2002), det1-1 (Chory et al., 1989), ddb1a

(Schroeder et al., 2002), cop1-4 (McNellis et al., 1994), spa1-3 (Hoecker

et al., 1998), spa1 spa2 spa3 and spa1 spa2 spa4 (Laubinger et al., 2004),

GFP-DET1/det1-1 (Schroeder et al., 2002), 35S:flag-DDB1B/ddb1a (Lee

et al., 2008), 35S:TAP-SPA1 (Saijo et al., 2003), 35S:TAP-SPA2, 35S:

TAP-SPA3, 35S:TAP-SPA4 (Zhu et al., 2008), 35S:TAP-COP1 (Rubio

et al., 2005), 35S:flag-CUL4 (Chen et al., 2006), and 35S:flag-COP10

(Yanagawa et al., 2004).

Arabidopsis seeds were surface sterilized and cold treated at 48C for 2

to 3 d and were then placed on solid 13 Murashige and Skoog medium

supplementedwith 1% sucrose for biochemical assays and phenotypical

analysis. Cold-treated seeds were exposed to white light for 3 h and then

transferred to continuous white light (30 mmol·m22·s21) or continuous

darkness, unless otherwise stated. Flowering times were tested in a

chamber set for SDs (8 h light/16 h dark, 150 mmol·m22·s21) or LDs (16 h

light/8 h dark, 150 mmol·m22·s21).

Generation of Transgenic Arabidopsis Plants

The full-length open reading frame of DDB1B was amplified by RT-PCR

from wild-type Arabidopsis seedlings using the forward primer

59-TGGGGCCCGGAGGTGGCATGAGCGTATGGAACTACGCC-39 and

the reverse primer 59-ATGGTCGACTCAGTGAAGCCTAGTGAGTTCTT-

CAAC-39. The PCR product was cloned into the vector pCR2.1-TOPO

(Invitrogen), and three copies of the Flag peptide were fused in-frame to

the 59 end of DDB1B. Three groups of mutations within BPA, BPB, and

BPC domains were generated separately. BamHI/SpeI fragments con-

tainingmutated flag-DDB1Bwere subcloned into Gateway vector pENTR

1A (Invitrogen) and then recombined by the LR reaction into the plant

binary vector pGWB2 that includes both kanamycin and hygromycin

resistancemarkers and the 35S promoter of theCauliflower mosaic virus.

These constructs were then transformed into mutant ddb1a, and trans-

genic plants were selected with kanamycin (50 mg/mL; Sigma-Aldrich).

The three groups of transgenic plants with mutated flag-DDB1B were

named mBPA, mBPB, and mBPC, respectively. The anti-Flag antibody

(Sigma-Aldrich) was used to evaluate the level of fusion protein in total

protein extracts.

RT-PCR

To analyze DDB1B transcript levels, total RNA was extracted from the

plants using the Plant RNeasy Kit (Qiagen) according to the manufac-

turer’s instruction. The concentration of RNA was determined by spec-

trophotometric measurements. The cDNA was synthesized from 5 mg

total RNA using oligo(dT)18 primer and superscript II reverse transcrip-

tase enzyme (Invitrogen). DDB1B cDNAs were amplified for 30 cycles at

948C for 45 s, 558C for 45 s, and 728C for 1 min. The forward and reverse

primers for DDB1B were as follows: 59-GGGATAGGAATAAATGAA-

CAG-39and 59-CTTCTGAATGTCATCGATGGTACC-39. To analyze CO,

FT, and ACTIN2 transcript levels, total RNA was extracted from the green

parts of soil-grown plants using the Plant RNeasy Kit according to the

manufacturer’s instructions. The concentration of RNA was determined

spectrophotometrically. cDNA was synthesized from 5 mg total RNA

using oligo(dT)19 primer and superscript II reverse transcriptase (Invi-

trogen). cDNAs were diluted to 200 mL with water, and 5 mL of the diluted

cDNA was used for PCR amplification. A CO fragment was amplified

using primers CO53, 59-ACGCCATCAGCGAGTTCC-39, and COoli9,

59-AAATGTATGCGTTATGGTTAATGG-39 (Suarez-Lopez et al., 2001).

An FT fragment was amplified using primers FT-RTPCR-F, 59-AGAAGAC-

TTTAGATGGCTTCTT-39, and FT-RTPCR-R, 59-TTATCGCATCACACAC-

TATATAAG-39 (An et al., 2004). Twenty-five and 27 cycles were used for

CO and FT PCR, respectively. A fragment encoding ACTIN2 was ampli-

fied as an internal standard with primers described previously (Lee et al.,

2008). The PCR products were analyzed by agarose gel electrophoresis

using ethidium bromide staining. All RT-PCR analyses were repeated

three times, and one representative result is shown.

Quantitative Real-Time PCR

To quantify FT andCO transcript levels in wild-type and cul4cs plants, the

green parts of soil-grown plants were harvested for RNA extraction. Total

RNAwas extracted using the Plant RNeasy Kit. Reverse transcription was

performed using TaqMan reverse transcription reagents (Applied Bio-

systems) according to the manufacturer’s instructions. Quantitative PCR

was performed using the Applied Biosystems 7900HT real-time PCR

system with Taqman gene expression probes for FT, CO, and 18S rRNA.

Relative amounts of FT and CO transcripts were calculated using the

comparative CT method, which was normalized against 18S rRNA

expression from the same sample. Each experiment was repeated twice

with independent samples, and RT-PCR reactions were performed in

duplicate for each sample.

Antibodies and Immunoblot Assays

The primary antibodies used in this study were anti-CUL4 (Chen et al.,

2006), anti-DET1 and anti-DDB1 (Zhang et al., 2008), anti-COP1 (Saijo

et al., 2003), anti-SPA1 (Zhu et al., 2008), anti-flag, anti-Myc, and anti-

GST (Sigma-Aldrich), and anti-MBP (NEB).

For the immunoblot analyses, Arabidopsis tissues were homogenized

in an extraction buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl,

10 mM MgCl2, 0.1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride,

and 13 complete protease inhibitor (Roche). The extracts were centri-

fuged at 13,000g, and then protein concentrations in the supernatants

were determined by the Bradford assay (Bio-Rad). Protein samples

boiled in the sample buffer were run on SDS-PAGE, blotted onto

polyvinylidene difluoride membranes (Millipore), and probed with differ-

ent primary antibodies.

Gel Filtration Chromatography

For gel filtration analysis, 6-d-old Arabidopsis seedlings were homoge-

nized in lysis buffer: 50mMTris-HCl, pH 7.5, 150mMNaCl, 10mMMgCl2,

10 mM NaF, 2 mM Na3VO4, 25 mM b-glycerophosphate, 10% glycerol,

0.1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, and 13 com-

plete protease inhibitor cocktail (Roche). The extracts were centrifuged at

13,000g for 15 min at 48C and subsequently filtered through a 0.22-mm

syringe filter. Superose 6 and superdex 200 columns (Amersham Biosci-

ences) were used to fractionate the sample. After the void volume was

eluted, consecutive fractions were collected, concentrated using Stra-

taclean resin (Stratagene), and analyzed by protein gel blots.

In Vitro Pull-Down Assay

Arabidopsis DDB1A and DDB1B full-length coding sequences were

subcloned into the pGEX-4T-1 vector (Amersham). Arabidopsis full-length

120 The Plant Cell

Dow



SPA1, SPA2, SPA3, and SPA4 coding sequences were subcloned into

pET28a (Novagen). MBP-COP1 and MBP-COP1mWDXR were purified

as previously described (Saijo et al., 2003). MBP-COP1mWDXR was

generated using the QuikChange site-directed mutagenesis kit (Strata-

gene). Full-length DDB1A and DDB1B fused to GST were expressed and

purified from Escherichia coli strain RIL (DE3) (Stratagene) and induced

by isopropyl-b-D-thiogalactopyranoside at 10 to 138C. GST protein

was purified from E. coli BL21 (DE3) (Novagen) as a bait control. His-

SPA1, His-SPA2, His-SPA3, His-SPA4, His-SPA1mWDXR, and His-

SPA3mWDXR were synthesized by the TNT (Transcription/Translation)

coupled in vitro reaction system (Promega). His-SPA1mWDXR and His-

SPA3mWDXR were generated using the QuikChange site-directed mu-

tagenesis kit. Bait and prey proteins were incubated in reaction buffer

(50mMTris-HCl, pH 7.5, 150mMNaCl, and 0.1%Nonidet P-40) for 4 h at

48C, and anti-GST conjugated beads were then added to precipitate the

bait and bound prey proteins. Two micrograms of both bait and prey

proteins (wild-type and mutated MBP-COP1) were used for each pull-

down assay. The amounts of wild-type and mutated His-SPA proteins

used were according to the specifications in the TNT system protocol

(Promega). Immunoprecipitated proteins were resolved by SDS-PAGE

and detected by Coomassie Brilliant Blue staining, immunoblot, and

autoradiography. The quantification of the images was performed with

ImageJ (http://rsb.info.nih.gov/ij/). Relative band intensities were normal-

ized with the value of background region and then calculated.

In Vivo Coimmunoprecipitation Assay

About 1 mg total proteins extracted from Arabidopsis tissues were

incubated with antibody-conjugated agarose beads (Sigma-Aldrich) or

IgG-agarose beads (Amersham) for 4 h at 48C. Alternatively, total proteins

were incubatedwith anti-COP1 or anti-DET1 antibodies for 3 h at 48C, and

then protein A-agarose beads (Sigma-Aldrich) were added to each

sample and incubated for 1 h at 48C. Lysis buffer was the same as used

for gel filtration. Next, the beads were washed three times in the same

buffer. Following elution, protein blot analyses were performed as de-

scribed previously (Zhu et al., 2008).

Accession Numbers

Sequence data from this article can be found in the Arabidopsis Ge-

nome Initiative database under the following accession numbers: CUL4

(At5g46210), DDB1A (At4g05420), DDB1B (At4g21100), COP1

(At2g32950), SPA1 (At2g46340), SPA2 (At4g11110), SPA3 (At3g15354),

SPA4 (At1g53090), ACTIN2 (At1g49240), CO (At5g15840), and FT

(At1g65480).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. Mutated MBP-COP1, His-SPA1, and His-

SPA3 Are Expressed at the Same Level as the Wild-Type Proteins.

Supplemental Figure 2. DDB1 Associates with TAP-SPA1, TAP-

SPA3, and TAP-SPA4 in Vivo.

ACKNOWLEDGMENTS

We thank Ning Zheng for helping with design for the point mutations on

the surface of DDB1 protein, Tsuyoshi Nakagawa for providing the

binary vector pGWB2 vector, Liying Chen for participation in some

preliminary tests, Hui Cong and Hehe Chen for communication and

support, and other members of the Deng lab for their constructive

discussion and help. This research was supported by funds from

National Institutes of Health (GM47850) and the National Science

Foundation 2010 program and in part by the Ministry of Science and

Technology of China and Beijing Commission of Science and Technol-

ogy. H.C. was a Peking-Yale Joint Center Monsanto Fellow.

Received January 6, 2009; revised November 2, 2009; accepted Decem-

ber 14, 2009; published January 8, 2010.

REFERENCES

Abbas, T., Sivaprasad, U., Terai, K., Amador, V., Pagano, M., and

Dutta, A. (2008). PCNA-dependent regulation of p21 ubiquitylation

and degradation via the CRL4Cdt2 ubiquitin ligase complex. Genes

Dev. 22: 2496–2506.

An, H., Roussot, C., Suarez-Lopez, P., Corbesier, L., Vincent, C.,

Pineiro, M., Hepworth, S., Mouradov, A., Justin, S., Turnbull, C.,

and Coupland, G. (2004). CONSTANS acts in the phloem to regulate

a systemic signal that induces photoperiodic flowering of Arabidopsis.

Development 131: 3615–3626.

Ang, L.H., Chattopadhyay, S., Wei, N., Oyama, T., Okada, K.,

Batschauer, A., and Deng, X.W. (1998). Molecular interaction be-

tween COP1 and HY5 defines a regulatory switch for light control of

Arabidopsis development. Mol. Cell 1: 213–222.

Ang, L.H., and Deng, X.W. (1994). Regulatory hierarchy of photomor-

phogenic loci: Allele-specific and light-dependent interaction between

the HY5 and COP1 loci. Plant Cell 6: 613–628.

Angers, S., Li, T., Yi, X., MacCoss, M.J., Moon, R.T., and Zheng, N.

(2006). Molecular architecture and assembly of the DDB1-CUL4A

ubiquitin ligase machinery. Nature 443: 590–593.

Chen, H., Shen, Y., Tang, X., Yu, L., Wang, J., Guo, L., Zhang, Y.,

Zhang, H., Feng, S., Strickland, E., Zheng, N., and Deng, X.W.

(2006). Arabidopsis CULLIN4 forms an E3 ubiquitin ligase with RBX1

and the CDD complex in mediating light control of development. Plant

Cell 18: 1991–2004.

Chory, J., Peto, C., Feinbaum, R., Pratt, L., and Ausubel, F. (1989).

Arabidopsis thalianamutant that develops as a light-grown plant in the

absence of light. Cell 58: 991–999.

Deng, X.W., Caspar, T., and Quail, P.H. (1991). cop1: A regulatory

locus involved in light-controlled development and gene expression in

Arabidopsis. Genes Dev. 5: 1172–1182.

Deng, X.W., Matsui, M., Wei, N., Wagner, D., Chu, A.M., Feldmann,

K.A., and Quail, P.H. (1992). COP1, an Arabidopsis regulatory gene,

encodes a protein with both a zinc-binding motif and a G beta

homologous domain. Cell 71: 791–801.

Dornan, D., Wertz, I., Shimizu, H., Arnott, D., Frantz, G.D., Dowd, P.,

O’Rourke, K., Koeppen, H., and Dixit, V.M. (2004). The ubiquitin

ligase COP1 is a critical negative regulator of p53. Nature 429: 86–92.

Duek, P.D., Elmer, M.V., van Oosten, V.R., and Fankhauser, C.

(2004). The degradation of HFR1, a putative bHLH class transcription

factor involved in light signaling, is regulated by phosphorylation and

requires COP1. Curr. Biol. 14: 2296–2301.

He, Y.J., McCall, C.M., Hu, J., Zeng, Y., and Xiong, Y. (2006). DDB1

functions as a linker to recruit receptor WD40 proteins to CUL4-ROC1

ubiquitin ligases. Genes Dev. 20: 2949–2954.

Higa, L.A., Banks, D., Wu, M., Kobayashi, R., Sun, H., and Zhang, H.

(2006a). L2DTL/CDT2 interacts with the CUL4/DDB1 complex and

PCNA and regulates CDT1 proteolysis in response to DNA damage.

Cell Cycle 5: 1675–1680.

Higa, L.A., Wu, M., Ye, T., Kobayashi, R., Sun, H., and Zhang, H.

(2006b). CUL4-DDB1 ubiquitin ligase interacts with multiple WD40-

repeat proteins and regulates histone methylation. Nat. Cell Biol. 8:

1277–1283.


Dow



Hoecker, U., Tepperman, J.M., and Quail, P.H. (1999). SPA1, a WD-

repeat protein specific to phytochrome A signal transduction. Science

284: 496–499.

Hoecker, U., Xu, Y., and Quail, P.H. (1998). SPA1: A new genetic locus

involved in phytochrome A-specific signal transduction. Plant Cell 10:

19–33.

Holm, M., Ma, L.G., Qu, L.J., and Deng, X.W. (2002). Two interacting

bZIP proteins are direct targets of COP1-mediated control of light-

dependent gene expression in Arabidopsis. Genes Dev. 16:

1247–1259.

Hrecka, K., Gierszewska, M., Srivastava, S., Kozaczkiewicz, L.,

Swanson, S.K., Florens, L., Washburn, M.P., and Skowronski, J.

(2007). Lentiviral Vpr usurps Cul4-DDB1[VprBP] E3 ubiquitin ligase to

modulate cell cycle. Proc. Natl. Acad. Sci. USA 104: 11778–11783.

Hu, J., Zacharek, S., He, Y.J., Lee, H., Shumway, S., Duronio, R.J.,

and Xiong, Y. (2008). WD40 protein FBW5 promotes ubiquitination of

tumor suppressor TSC2 by DDB1-CUL4-ROC1 ligase. Genes Dev. 22:

866–871.

Jang, I.C., Yang, J.Y., Seo, H.S., and Chua, N.H. (2005). HFR1 is

targeted by COP1 E3 ligase for post-translational proteolysis during

phytochrome A signaling. Genes Dev. 19: 593–602.

Jang, S., Marchal, V., Panigrahi, K.C., Wenkel, S., Soppe, W., Deng,

X.W., Valverde, F., and Coupland, G. (2008). Arabidopsis COP1

shapes the temporal pattern of CO accumulation conferring a pho-

toperiodic flowering response. EMBO J. 27: 1277–1288.

Jin, J., Arias, E.E., Chen, J., Harper, J.W., and Walter, J.C. (2006). A

family of diverse Cul4-Ddb1-interacting proteins includes Cdt2, which

is required for S phase destruction of the replication factor Cdt1. Mol.

Cell 23: 709–721.

Kardailsky, I., Shukla, V.K., Ahn, J.H., Dagenais, N., Christensen, S.

K., Nguyen, J.T., Chory, J., Harrison, M.J., and Weigel, D. (1999).

Activation tagging of the floral inducer FT. Science 286: 1962–1965.

Kim, Y., Starostina, N.G., and Kipreos, E.T. (2008). The CRL4Cdt2

ubiquitin ligase targets the degradation of p21Cip1 to control repli-

cation licensing. Genes Dev. 22: 2507–2519.

Kobayashi, Y., Kaya, H., Goto, K., Iwabuchi, M., and Araki, T. (1999).

A pair of related genes with antagonistic roles in mediating flowering

signals. Science 286: 1960–1962.

Laubinger, S., Fittinghoff, K., and Hoecker, U. (2004). The SPA

quartet: A family of WD-repeat proteins with a central role in

suppression of photomorphogenesis in arabidopsis. Plant Cell 16:

2293–2306.

Laubinger, S., and Hoecker, U. (2003). The SPA1-like proteins SPA3 and

SPA4 repress photomorphogenesis in the light. Plant J. 35: 373–385.

Laubinger, S., Marchal, V., Le Gourrierec, J., Wenkel, S., Adrian, J.,

Jang, S., Kulajta, C., Braun, H., Coupland, G., and Hoecker, U.

(2006). Arabidopsis SPA proteins regulate photoperiodic flowering

and interact with the floral inducer CONSTANS to regulate its stability.

Development 133: 3213–3222.

Lee, J., and Zhou, P. (2007). DCAFs, the missing link of the CUL4-

DDB1 ubiquitin ligase. Mol. Cell 26: 775–780.

Lee, J.H., Terzaghi, W., Gusmaroli, G., Charron, J.B., Yoon, H.J.,

Chen, H., He, Y.J., Xiong, Y., and Deng, X.W. (2008). Characteriza-

tion of Arabidopsis and rice DWD proteins and their roles as substrate

receptors for CUL4-RING E3 ubiquitin ligases. Plant Cell 20: 152–167.

Le Rouzic, E., Belaidouni, N., Estrabaud, E., Morel, M., Rain, J.C.,

Transy, C., and Margottin-Goguet, F. (2007). HIV1 Vpr arrests the

cell cycle by recruiting DCAF1/VprBP, a receptor of the Cul4–DDB1

ubiquitin ligase. Cell Cycle 6: 182–188.

Liu, L.J., Zhang, Y.C., Li, Q.H., Sang, Y., Mao, J., Lian, H.L., Wang, L.,

and Yang, H.Q. (2008). COP1-mediated ubiquitination of CONSTANS

is implicated in cryptochrome regulation of flowering in Arabidopsis.

Plant Cell 20: 292–306.

McCall, C.M., Miliani de Marval, P.L., Chastain II, P.D., Jackson, S.

C., He, Y.J., Kotake, Y., Cook, J.G., and Xiong, Y. (2008). Human

immunodeficiency virus type 1 Vpr-binding protein VprBP, a WD40

protein associated with the DDB1-CUL4 E3 ubiquitin ligase, is es-

sential for DNA replication and embryonic development. Mol. Cell.

Biol. 28: 5621–5633.

McNellis, T.W., von Arnim, A.G., Araki, T., Komeda, Y., Misera, S.,

and Deng, X.W. (1994). Genetic and molecular analysis of an allelic

series of cop1 mutants suggests functional roles for the multiple

protein domains. Plant Cell 6: 487–500.

Nishitani, H., Shiomi, Y., Iida, H., Michishita, M., Takami, T., and

Tsurimoto, T. (2008). CDK inhibitor p21 is degraded by a proliferating

cell nuclear antigen-coupled Cul4-DDB1Cdt2 pathway during S

phase and after UV irradiation. J. Biol. Chem. 283: 29045–29052.

Osterlund, M.T., Hardtke, C.S., Wei, N., and Deng, X.W. (2000).

Targeted destabilization of HY5 during light-regulated development of

Arabidopsis. Nature 405: 462–466.

Pepper, A., Delaney, T., Washburn, T., Poole, D., and Chory, J.

(1994). DET1, a negative regulator of light-mediated development and

gene expression in arabidopsis, encodes a novel nuclear-localized

protein. Cell 78: 109–116.

Putterill, J., Robson, F., Lee, K., Simon, R., and Coupland, G. (1995).

The CONSTANS gene of Arabidopsis promotes flowering and

encodes a protein showing similarities to zinc finger transcription

factors. Cell 80: 847–857.

Ralph, E., Boye, E., and Kearsey, S.E. (2006). DNA damage induces

Cdt1 proteolysis in fission yeast through a pathway dependent on

Cdt2 and Ddb1. EMBO Rep. 7: 1134–1139.

Rubio, V., Shen, Y., Saijo, Y., Liu, Y., Gusmaroli, G., Dinesh-Kumar,

S.P., and Deng, X.W. (2005). An alternative tandem affinity purifica-

tion strategy applied to Arabidopsis protein complex isolation. Plant J.

41: 767–778.

Saijo, Y., Sullivan, J.A., Wang, H., Yang, J., Shen, Y., Rubio, V., Ma,

L., Hoecker, U., and Deng, X.W. (2003). The COP1-SPA1 interaction

defines a critical step in phytochrome A-mediated regulation of HY5

activity. Genes Dev. 17: 2642–2647.

Samach, A., Onouchi, H., Gold, S.E., Ditta, G.S., Schwarz-Sommer,

Z., Yanofsky, M.F., and Coupland, G. (2000). Distinct roles of

CONSTANS target genes in reproductive development of Arabidop-

sis. Science 288: 1613–1616.

Sansam, C.L., Shepard, J.L., Lai, K., Ianari, A., Danielian, P.S.,

Amsterdam, A., Hopkins, N., and Lees, J.A. (2006). DTL/CDT2 is

essential for both CDT1 regulation and the early G2/M checkpoint.

Genes Dev. 20: 3117–3129.

Schroeder, D.F., Gahrtz, M., Maxwell, B.B., Cook, R.K., Kan, J.M.,

Alonso, J.M., Ecker, J.R., and Chory, J. (2002). De-etiolated 1 and

damaged DNA binding protein 1 interact to regulate Arabidopsis

photomorphogenesis. Curr. Biol. 12: 1462–1472.

Seo, H.S., Watanabe, E., Tokutomi, S., Nagatani, A., and Chua, N.H.

(2004). Photoreceptor ubiquitination by COP1 E3 ligase desensitizes

phytochrome A signaling. Genes Dev. 18: 617–622.

Seo, H.S., Yang, J.Y., Ishikawa, M., Bolle, C., Ballesteros, M.L., and

Chua, N.H. (2003). LAF1 ubiquitination by COP1 controls photomor-

phogenesis and is stimulated by SPA1. Nature 423: 995–999.

Serino, G., and Deng, X.W. (2003). The COP9 signalosome: regulating

plant development through the control of proteolysis. Annu. Rev. Plant

Biol. 54: 165–182.

Stacey, M.G., Kopp, O.R., Kim, T.H., and von Arnim, A.G. (2000).

Modular domain structure of Arabidopsis COP1. Reconstitution of

activity by fragment complementation and mutational analysis of a

nuclear localization signal in planta. Plant Physiol. 124: 979–990.

Suarez-Lopez, P., Wheatley, K., Robson, F., Onouchi, H., Valverde,

F., and Coupland, G. (2001). CONSTANS mediates between the

122 The Plant Cell

Dow



circadian clock and the control of flowering in Arabidopsis. Nature

410: 1116–1120.

Sullivan, J.A., Shirasu, K., and Deng, X.W. (2003). The diverse roles of

ubiquitin and the 26S proteasome in the life of plants. Nat. Rev.

Genet. 4: 948–958.

Suzuki, G., Yanagawa, Y., Kwok, S.F., Matsui, M., and Deng, X.W.

(2002). Arabidopsis COP10 is a ubiquitin-conjugating enzyme variant

that acts together with COP1 and the COP9 signalosome in repres-

sing photomorphogenesis. Genes Dev. 16: 554–559.

Valverde, F., Mouradov, A., Soppe, W., Ravenscroft, D., Samach, A.,

and Coupland, G. (2004). Photoreceptor regulation of CONSTANS

protein in photoperiodic flowering. Science 303: 1003–1006.

von Arnim, A., and Deng, X.W. (1996). Light control of seedling

development. Annu. Rev. Plant Physiol. Plant Mol. Biol. 47: 215–243.

von Arnim, A.G., and Deng, X.W. (1994). Light inactivation of Arabi-

dopsis photomorphogenic repressor COP1 involves a cell-specific

regulation of its nucleocytoplasmic partitioning. Cell 79: 1035–1045.

Wei, N., Kwok, S.F., von Arnim, A.G., Lee, A., McNellis, T.W., Piekos,

B., and Deng, X.W. (1994). Arabidopsis COP8, COP10, and COP11

genes are involved in repression of photomorphogenic development

in darkness. Plant Cell 6: 629–643.

Wertz, I.E., O’Rourke, K.M., Zhang, Z., Dornan, D., Arnott, D., Deshaies,

R.J., and Dixit, V.M. (2004). Human De-etiolated-1 regulates c-Jun by

assembling a CUL4A ubiquitin ligase. Science 303: 1371–1374.

Yanagawa, Y., Sullivan, J.A., Komatsu, S., Gusmaroli, G., Suzuki, G.,

Yin, J., Ishibashi, T., Saijo, Y., Rubio, V., Kimura, S., Wang, J., and

Deng, X.W. (2004). Arabidopsis COP10 forms a complex with DDB1

and DET1 in vivo and enhances the activity of ubiquitin conjugating

enzymes. Genes Dev. 18: 2172–2181.

Yang, J., Lin, R., Sullivan, J., Hoecker, U., Liu, B., Xu, L., Deng, X.W.,

and Wang, H. (2005). Light regulates COP1-mediated degradation of

HFR1, a transcription factor essential for light signaling in Arabidopsis.

Plant Cell 17: 804–821.

Yi, C., and Deng, X.W. (2005). COP1 - From plant photomorphogenesis

to mammalian tumorigenesis. Trends Cell Biol. 15: 618–625.

Yu, J.W., et al. (2008). COP1 and ELF3 control circadian function

and photoperiodic flowering by regulating GI stability. Mol. Cell 32:

617–630.

Zhang, Y., Feng, S., Chen, F., Chen, H., Wang, J., McCall, C., Xiong,

Y., and Deng, X.W. (2008). Arabidopsis DDB1-CUL4 ASSOCIATED

FACTOR1 forms a nuclear E3 ubiquitin ligase with DDB1 and CUL4

that is involved in multiple plant developmental processes. Plant Cell

20: 1437–1455.

Zhu, D., Maier, A., Lee, J.H., Laubinger, S., Saijo, Y., Wang, H., Qu, L.

J., Hoecker, U., and Deng, X.W. (2008). Biochemical characteriza-

tion of Arabidopsis complexes containing CONSTITUTIVELY PHO-

TOMORPHOGENIC1 and SUPPRESSOR OF PHYA proteins in light

control of plant development. Plant Cell 20: 2307–2323.


Dow



arabidopsis cullin4-damaged dna binding protein 1

Documents