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. 2007 May;176(1):231–242. doi: 10.1534/genetics.107.070359

DDB2, DDB1A and DET1 Exhibit Complex Interactions During Arabidopsis Development

Wesam M Al Khateeb 1, Dana F Schroeder 1,1
PMCID: PMC1893029  PMID: 17409070

Abstract

Damaged DNA-binding proteins 1 and 2 (DDB1 and DDB2) are subunits of the damaged DNA-binding protein complex (DDB). DDB1 is also found in the same complex as DE-ETIOLATED 1 (DET1), a negative regulator of light-mediated responses in plants. Arabidopsis has two DDB1 homologs, DDB1A and DDB1B. ddb1a single mutants have no visible phenotype while ddb1b mutants are lethal. We have identified a partial loss-of-function allele of DDB2. To understand the genetic interaction among DDB2, DDB1A, and DET1 during Arabidopsis light signaling, we generated single, double, and triple mutants. det1 ddb2 partially enhances the short hypocotyl and suppresses the high anthocyanin content of dark-grown det1 and suppresses the low chlorophyll content, early flowering time (days), and small rosette diameter of light-grown det1. No significant differences were observed between det1 ddb1a and det1 ddb1a ddb2 in rosette diameter, dark hypocotyl length, and anthocyanin content, suggesting that these are DDB1A-dependent phenotypes. In contrast, det1 ddb1a ddb2 showed higher chlorophyll content and later flowering time than det1 ddb1a, indicating that these are DDB1A-independent phenotypes. We propose that the DDB1A-dependent phenotypes indicate a competition between DDB2- and DET1-containing complexes for available DDB1A, while, for DDB1A-independent phenotypes, DDB1B is able to fulfill this role.

PLANT development is dependent on environmental conditions. Because light is the energy source for plant growth, plants have evolved highly sensitive mechanisms for perceiving light. This information is used to regulate development and to maximize light utilization for photosynthesis. The transition from the vegetative to the reproductive stage is also regulated by light. Seedlings implement different developmental programs when grown in light or darkness. Light-grown seedlings undergo photomorphogenesis, exhibiting short hypocotyls, open and expanded cotyledons, and photosynthetically active chloroplasts. In contrast, seedlings grown under dark conditions are etiolated, having closed and unexpanded cotyledons, elongated hypocotyls, and undeveloped chloroplasts. This developmental pattern is known as skotomorphogenesis (Chen et al. 2004).

This developmental switch (etiolation/de-etiolation) is under the control of at least 10 genes (COP/DET/FUS). Molecular genetic studies in Arabidopsis indicate that these proteins function downstream of the photoreceptors to repress photomorphogenesis in the absence of light. Mutation of these genes results in seedlings with a de-etiolated (det) or constitutive photomorphogenic (cop) phenotype when grown under dark conditions. The null mutations of these genes are seedling lethal with high anthocyanin levels (fus) (Wang and Deng 2002). COP1 is a WD-40 and RING finger protein with E3 ubiquitin (Ub) ligase activity, which targets photoreceptors and downstream transcription factors for ubiquitination and subsequent degradation. The COP9 signalosome (CSN) is a multiprotein complex composed of eight subunits, which associates with and supports the activity of multiple cullin-containing E3 ubiquitin ligase complexes. In Arabidopsis, mutants in CSN components also exhibit the constitutive photomorphogenic/de-etiolated/fusca (cop/det/fus) phenotype (Schwechheimer et al. 2004).

De-etiolated 1 (det1-1) partial loss-of-function mutants exhibit short hypocotyls, open cotyledons, and high anthocyanin levels in the dark (Chory et al. 1989). Under light conditions, det1-1 seedlings are smaller and paler than wild type. In addition, they show reduced apical dominance, day-length-insensitive flowering (Pepper and Chory 1997) and defects in germination, expression of light-regulated genes, and chloroplast development (Chory and Peto 1990). Approximately 1000 genes are either up- or downregulated in det1-1 compared to wild type (Schroeder et al. 2002).

DET1 is a nuclear protein (Pepper et al. 1994) present in a complex with an approximate mass of 350 kDa. In tobacco BY2 cells, this complex includes the plant homolog of UV-damaged DNA-binding protein 1 (DDB1). In Arabidopsis, two homologs of DDB1 have been found: DDB1A and DDB1B, which are 91% identical at the amino acid level. Arabidopsis DDB1A matches tobacco DDB1 more closely than Arabidopsis DDB1B (Schroeder et al. 2002). DDB1A is expressed at almost twofold higher levels than DDB1B throughout the Arabidopsis life cycle in all organs studied (Figure 1A). ddb1a and ddb1b mutants in Arabidopsis were studied using T-DNA insertions. ddb1a mutants show no obvious phenotype in a wild-type background, but mutation of DDB1A in the det1 background enhanced det1 phenotypes. In contrast to ddb1a, ddb1b mutants are lethal, suggesting a crucial role for DDB1B during Arabidopsis development. DDB1 is evolutionarily conserved as Arabidopsis DDB1A is 83 and 46% identical at the amino acid level with rice and human DDB1, respectively (Schroeder et al. 2002).

Figure 1.—

Figure 1.—

Figure 1.—

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(A) Gene expression data as obtained from Genevestigator database (https://www.genevestigator.ethz.ch). (B) Schematic of 5′-end of the DDB2 gene (At5g58760). Transcription start sites (+1) are based on the GenBank accession nos. BX832566 and AK175124. Exons are shown as boxes and introns and upstream sequences as lines. Position of the T-DNA insertion (SALK_040408) is indicated, along with primers used for genotyping. (C) RT–PCR products for DDB2 and UBQ10 of 7-day-old seedlings grown under long-day conditions. (D) Quantification of DDB2 transcript level (DDB2 values normalized to UBQ10 levels, relative to Col-0). Data are shown as the means ± SE of three different technical repeats. Numbers above error bars indicate expression relative to DDB2 wild-type control.

The Arabidopsis DDB1 protein is homologous to human DDB1 (127 kDa), a component, along with DDB2 (48 kDa), of the damaged DNA-binding protein complex (DDB). DDB1 is present at higher levels than DDB2 in human cells (Liu et al. 2000). DDB1 is present in the cytoplasm, but upon UV irradiation, translocates to the nucleus. Loss of DDB2 function prevents accumulation of DDB1 in the nucleus (Shiyanov et al. 1999), whereas loss of DDB1 function has no effect on binding activity of DDB2 to the damaged DNA (J. Li et al. 2006). The suggested role of the DDB complex is to recognize DNA lesions, initiating nucleotide excision repair. In humans, the rare inherited disease Xeroderma pigmentosa group E (XPE) results from mutation of DDB2. XPE patients display an increased skin sensitivity to UV light and are at a highly elevated risk of developing UV-induced skin cancer (Cleaver 2005).

Recently, human DDB1 and DDB2 were found to be components of an E3 ubiquitin ligase. DDB1, along with CUL4 and ROC1, is a component of several types of E3 ligases, including one with DDB2 and another with the transcriptional coupled repair factor CSA. Both these E3 ligase complexes are regulated by the COP9 signalosome (Groisman et al. 2003). Subsequently, DDB1-CUL4 complexes were found to interact with many WD40-repeat proteins and use them as adaptors in recognizing substrates for proteolysis (Angers et al. 2006; He et al. 2006; Higa et al. 2006; Jin et al. 2006). Human DDB1 is also found in a complex with CUL4, ROC1, DET1, and COP1 (Wertz et al. 2004). Arabidopsis DDB1A and DET1 copurify with the E2 Ub conjugase variant COP10 (Yanagawa et al. 2004) and these proteins have recently been found to form a complex with AtCUL4 and RBX1 (ROC1) (Bernhardt et al. 2006; Chen et al. 2006). Thus DDB1 appears to be a central component of CUL4 E3 ligases.

The DDB complex is present not only in humans, but also in other organisms such as rice (Oryza sativa cv. Nipponbare) (Ishibashi et al. 2003). Arabidopsis DDB2 shows 59 and 30% identity with rice and human DDB2, respectively. In this study we examined the role of Arabidopsis DDB2 and its interaction with DDB1A and DET1 in light signaling. All combinations of double and triple mutants were generated to understand the genetic interaction between these genes. Plants were grown and analyzed at different developmental stages. Comparison between the mutants revealed complex interactions between these genes. In some cases, the modulation of det1 phenotypes by ddb2 was DDB1A dependent; in other cases, it was DDB1A independent. We interpret these results as consistent with a model whereby separate DET1- and DDB2-containing complexes compete for DDB1A, in the case of the dependent phenotypes, and where DDB1B is able to fill this role, in the case of DDB1A-independent phenotypes.

MATERIALS AND METHODS

Plant materials and growth conditions:

All mutations used in this experiment were in the Col background of Arabidopsis thaliana. The DET1 partial loss-of-function allele det1-1 and the ddb1a T-DNA mutant were described previously (Pepper et al. 1994; Schroeder et al. 2002), and the ddb2 allele (SALK_040408) was obtained from the Arabidopsis Stock Center (http://www.arabidopsis.org/). cop1-4 was kindly provided by X. W. Deng of Yale University. Seedlings were grown in a growth chamber at 20° and 50% relative humidity. Light was provided by fluorescent bulbs (100 μmol photons m−2 sec−1). Plants were grown in Sunshine mix number 1 (SunGro, Bellevue, WA). Short-day conditions corresponded to 10 hr light and 14 hr dark; long-day conditions consisted of 16 hr light and 8 hr dark.

Construction of double and triple mutants:

Double mutants:

The det1 ddb1a mutant was generated as described in Schroeder et al. (2002); cop1-4 ddb2, det1 ddb2, and ddb1a ddb2 double mutants were derived from genetic crosses of their corresponding single-parental mutants. Because all mutations analyzed were recessive, double homozygous plants were identified in the F2 generation, where they occur in a ratio close to 1:15. Putative double mutants in the F2 generation were selected on the basis of mutant phenotypes and PCR genotyping. For example, for the ddb2 × det1 cross, ∼100 F2 seeds were plated—segregating det1 homozygotes identified by their dwarf stature—transplanted, and genotyped for ddb2. In the F3 generation, several independent det1 ddb2 double-mutant lines consistently exhibited shorter hypocotyls and decreased anthocyanin in dark-grown seedlings and increased chlorophyll in light-grown seedlings relative to their det1 DDB2/− siblings (data not shown).

Triple mutant:

Pollen from plants homozygous for ddb2 was used to fertilize flowers of det1 ddb1a plants. As expected, all F1 plants showed wild-type phenotype. PCR was used to confirm the presence of the ddb2 insertion. Plants heterozygous for ddb2 and det1 ddb1a were selfed to produced F2 [DET1 and DDB1A are linked ∼10 cM apart (Schroeder et al. 2002)]. det1 ddb1a homozygotes were identified as extreme dwarfs and were then genotyped to identify ddb2 homozygotes. Due to the infertility of this triple mutant, stocks are maintained as a segregating population homozygous for ddb2 and heterozygous for det1 ddb1a.

PCR reactions were conducted to confirm the ddb1a and ddb2 insertions. DNA was extracted according to Weigel and Glazebrook (2002). ddb1a insertions were detected using LB2 (5′-TTG GGT GAT GGT TCA CGT AGT GGG CCA TCG-3′) and UV1.17 (5′-ACT GGG CTC AAC TAG AAA ATA TGG AAC AA-3′) while UV1.17 and UV1.1 (5′-GTC TTG ACT GTG CAT TTC AGA GTG CTT AT-3′) were used to detect the wild-type DDB1A. For ddb2, LB2 and DDB2.1 (5′-TTG GGT GAT GGT TCA CGT AGT GGG CCA TCG-3′) were used, while DDB2.1 and DDB2.3 (5′-ACG ACG TGT TTT GTC GGT GTG GAA GAA-3′) were used for wild-type DDB2.

RNA extraction and RT–PCR:

Total RNA was extracted from 7-day-old seedlings using the RNeasy plant mini kit (QIAGEN, Valencia, CA) according to the manufacturer's instructions. Quality and quantity of isolated RNA was checked by denaturing gel electrophoresis and by spectrophotometric analysis. cDNA synthesis and PCR amplification were performed in the same reaction using Access RT–PCR kit (Promega, Madison, WI) according to the manufacturer's instructions. DDB2-specific primers in exon 2 (5′-ACAGCCTGGCCATGAAGCTGGA-3′) and in exon 6 (5′-CCTGCCATCCATCAGGGTTGAG-3′) were used. cDNA synthesis was performed at 45° for 45 min, followed by PCR [5 min at 94°, 30 times (30 sec at 94°, 50 sec at 67°, 2 min at 72°), 2 min at 72°]. To detect relative differences in transcript levels, amplification was performed when the PCR product was accumulating exponentially with respect to cycle number (30 cycles). UBQ10 was used as an internal control (5′-GATCTTTGCCGGAAAACAATTGGAGGATGGT-3′ and 5′-CGACTTGTCATTAGAAAGAAAGAGATAACAGG-3′) [5 min at 94°, 22 times (1 min at 94°, 1 min at 64°, 1 min at 72°), 2 min at 72°]. PCR products were separated on 1% (w/v) agarose gels, and the intensities of ethidium-bromide-stained bands were determined using ImageJ software (1.36b National Institutes of Health).

Hypocotyl elongation assay:

Seeds were plated on Murashige and Skoog (MS) media (1× MS salts, 0.8% phytoagar, 2% sucrose) after sterilization and stratified at 4° for 2 days. Plates were transferred to a growth chamber and grown under either light (16 hr light 80 μmol m−2 sec−1) or dark conditions (after 6 hr light as germination enhancer). Seven days later, hypocotyl length was measured for at least 10 seedlings using National Institutes of Health image software.

Pigment analysis:

For chlorophyll measurement, 7-day-old seedlings were extracted with 80% acetone overnight, the A645 and A663 were determined in a spectrophotometer (model 2100 pro, Ultrospec), and chlorophyll content was calculated according to the method of Mackinney (1941). Anthocyanin was determined using standard technique (Fankhauser and Casal 2004). Experiments were repeated at least three times with four replicates per line in each experiment. Each replicate contained at least 50 mg of plant tissue.

Growth parameter measurements:

Seedlings were transplanted from plates to soil at 14 days old. For each line, 18 plants were used for growth parameter analysis. Flowering time was scored for each plant as the number of days until the first bud became visible. Also, the total number of rosette and cauline leaves on the main inflorescence was counted. Plants were moved regularly to random positions in the growth chamber to prevent any positional effects on plant growth. The following traits were also recorded: shoot fresh weight (4 weeks old), rosette diameter (4 weeks old), and total number of inflorescences and height (6 weeks old).

Statistical analysis:

All experiments were repeated at least three times. Results presented are means with 95% confidence intervals of 10–18 replicates. Means were compared by Student's t-test. Probabilities of 0.05 or less were considered to be statistically significant.

RESULTS

To examine the role of DDB2 in light signaling and interaction with DDB1A and DET1, we studied the physiological role of these proteins at different stages of Arabidopsis development. Gene expression data available at the Genevestigator website (https://www.genevestigator.ethz.ch) show that DDB2 is expressed in all Arabidopsis organs, as are DDB1A, DDB1B, and DET1 (Figure 1A). A T-DNA insertion in DDB2 (SALK_040408) was obtained from the Arabidopsis Stock Center (http://www.arabidopsis.org/). This insertion is located 180 bp upstream from the DDB2 ATG (Figure 1B). PCR genotyping was performed to confirm this insertion (data not shown). RT–PCR analysis showed that DDB2 expression was reduced by approximately twofold in ddb2 mutants compared to the wild type (Figure 1, C and D). Loss of function of DDB2 in other mutant backgrounds results in similar relative levels of expression (Figure 1, C and D). These data suggest that this T-DNA insertion results in partial loss of function of DDB2. This result is in contrast to Koga et al. (2006) who observed complete absence of DDB2 expression in the same T-DNA insertion line (SALK_040408). This variation in transcript level may be due to cosuppression.

For all the growth parameters measured in the following sections, no significant differences were observed among wild type, the single mutants ddb2 and ddb1a, and the double mutant ddb1a ddb2. Since we had previously shown that ddb1a exhibited no phenotype on its own, yet enhanced the phenotypes of the DET1 partial loss-of-function allele det1-1, we generated the det1-1 ddb2 double mutant. The triple mutant was generated to determine if det1-1 ddb2 phenotypes were DDB1A dependent or independent.

Dark-grown seedlings:

Hypocotyl elongation:

In the absence of light, wild-type plants show increased hypocotyl elongation, but det1 mutants lack this response and have short hypocotyls. Lines carrying mutations in DET1, DDB1A, and DDB2 singly or in combination were grown under dark conditions for 7 days to study the role of these proteins in de-etiolation and light signaling (Figure 2). Plants with partial loss of function of DDB2 in the wild-type or ddb1a background did not differ significantly from wild type. In contrast, mutation of DDB2 in the det1 background resulted in enhancement of the det1 short hypocotyl phenotype (P ≤ 0.001), resulting in 31% shorter hypocotyls. However, the triple mutant det1 ddb1a ddb2 did not differ significantly from the double mutant det1 ddb1a. Thus enhancement of the det1 short hypocotyl phenotype by ddb2 is a DDB1A-dependent phenotype.

Figure 2.—

Figure 2.—

Figure 2.—

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Phenotypic analysis of 7-day-old dark-grown seedlings. (A) From left to right: Col-0, ddb2, ddb1a, ddb1a ddb2, det1, det1 ddb2, det1 ddb1a, and det1 ddb1a ddb2. (B) Hypocotyl length. (C) Anthocyanin content (A530–A657/g fresh weight). Error bars indicate 95% C.I. **P ≤ 0.01 relative to DDB2 wild-type control.

Anthocyanin content:

We studied anthocyanin accumulation in seedlings, which ordinarily does not accumulate when plants are grown under dark conditions. Very low levels of anthocyanin were detected in Col-0, ddb2, ddb1a, and ddb1a ddb2 seedlings (Figure 2C). det1 showed higher levels of anthocyanin than the wild type (18-fold higher). The ddb2 mutation in the det1 background suppressed this increase in anthocyanin content, showing only 28% of the levels observed in the det1 single mutant. In contrast to ddb2 partial loss of function in the det1 background, ddb1a loss of function in the same background showed an increase in anthocyanin content, with 6.5-fold enhancement. The anthocyanin content of the triple mutant det1 ddb1a ddb2 exhibited no significant difference from the double mutant det1 ddb1a. Thus, ddb2 exhibits DDB1A-dependent suppression of det1 dark anthocyanin accumulation.

Light-grown seedlings:

Hypocotyl elongation:

In light, det1 seedlings are also shorter than wild type. Growth of wild type and ddb2, ddb1a, and ddb1a ddb2 mutants under long-day conditions for 7 days showed no significant differences (Figure 3, A and B). Loss of function of DDB2 in the det1 background did not affect the det1 short hypocotyl. In contrast, mutation of DDB1A in the same background reduced hypocotyl elongation (P ≤ 0.05). No significant differences were observed between det1 ddb1a and det1 ddb1a ddb2. Thus ddb2 has no effect on this phenotype.

Figure 3.—

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Figure 3.—

Figure 3.—

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Phenotypic analysis of 7-day-old long-day-grown seedlings. (A) From left to right: Col-0, ddb2, ddb1a, ddb1a ddb2, det1, det1 ddb2, and det1 ddb1a, det1 ddb1a ddb2. (B) Hypocotyl length. (C) Anthocyanin content. (D) Chlorophyll content (micrograms of chlorophyll/milligram fresh weight). Error bars indicate 95% C.I. *P ≤ 0.05 or **P ≤ 0.01, respectively, relative to DDB2 wild-type control.

Anthocyanin content:

When grown under light conditions, seedlings accumulate anthocyanin. Col-0, ddb2, ddb1a, and ddb1a ddb2 had similar levels while det1 showed more than a 60% increase compared to wild type (Figure 3, A and C). While no significant difference was observed between det1 and det1 ddb2, the increased anthocyanin content of det1 was dramatically enhanced in det1 ddb1a. However, ddb2 significantly suppressed (P ≤ 0.03) anthocyanin accumulation in the det1 ddb1a double mutant as levels in the triple mutant were only 76% that of the double mutant. Thus, ddb2 partially suppresses anthocyanin accumulation in light-grown det1 ddb1a seedlings but not in the det1 single mutant.

Chlorophyll content:

In light, det1 mutants accumulate less chlorophyll than wild type. Wild type, ddb2, ddb1a, and the double mutant ddb1a ddb2 all showed the similar levels of chlorophyll accumulation (Figure 3D). det1 showed a decrease compared to the previous genotypes with only 34% of wild-type chlorophyll content. Loss of function of DDB2 in the det1 background partially suppressed the pale color of det1 and resulted in 60% more chlorophyll in the double mutant. In contrast, loss of function of DDB1A in the det1 background showed a chlorophyll content similar to that of det1 alone. Similar to the effect of the ddb2 mutation on the det1 background, loss of function of DDB2 in the det1 ddb1a background resulted in twofold more chlorophyll accumulation in the triple mutant det1 ddb1a ddb2. This suggests that suppression of the det1 pale phenotype by ddb2 is DDB1A independent.

Adult plants:

Rosette diameter:

Adult det1 are also smaller than wild type, so after 1 month of growth under either long-day or short-day conditions, rosette diameter was measured for all genotypes. Analysis of rosette diameter for different genotypes showed similar trends when grown under either long- or short-day conditions. In general, plants grown under long-day conditions showed larger rosette diameter than those grown under short-day conditions. ddb2, ddb1a, and ddb1a ddb2 loss-of-function mutants did not differ significantly from wild type (Figure 4, A and B). This result is in contrast to Koga et al. (2006), who found that DDB2 mutation resulted in reduction in leaf length and width. Our weaker phenotype is consistent with the fact that we still observe ∼50% of wild-type DDB2 expression in our ddb2 mutants while Koga et al. (2006) used an RNA null. det1 showed smaller rosette diameter than wild type (Figure 4, A and B). The ddb2 mutation partially suppressed the det1 small rosette diameter in both long- and short-day conditions (P ≤ 0.01). In contrast, the ddb1a mutation significantly enhanced this phenotype in the det1 background, resulting in smaller rosette diameter. Mutation of ddb2 in the det1 ddb1a background has no significant effect under both conditions. Therefore, the partial suppression of the det1 small rosette diameter by ddb2 is DDB1A dependent.

Figure 4.—

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Adult phenotypes (A) From left to right: Col-0, ddb2, ddb1a, ddb1a ddb2, det1, det1 ddb2, det1 ddb1a, and det1 ddb1a ddb2 grown in long-day conditions. (Top) Rosette diameter at 4 weeks. (Bottom) Height at 6 weeks. (B) Rosette diameter of 4-week-old plants. (C) Flowering time (in days). (D) Height (in centimeters) of adult plants. Error bars indicate 95% C.I. *P ≤ 0.05 or **P ≤ 0.01, respectively, relative to DDB2 wild-type control.

Flowering time:

To examine the role and interaction of these proteins in controlling Arabidopsis flowering time, we compared wild type, single mutants, double mutants, and the triple mutant grown in short and long days. Arabidopsis is a facultative long-day plant, so flowering in wild type is accelerated in long day. We found that wild type, ddb2, ddb1a, and ddb1a ddb2 start flowering after 26 and 62 days on average in long- and short-day conditions, respectively (Figure 4C). Loss of function of DET1 resulted in early flowering time compared to the wild type (18 or 22 days, respectively). The double mutant det1 ddb2 showed significant suppression (P ≤ 0.01) of det1 early flowering time (20 and 27 days, respectively). No significant difference was observed between det1 ddb2 and det1 ddb1a in both photoperiods. The triple mutant det1 ddb1a ddb2 showed significant (P ≤ 0.01) suppression of early det1 ddb1a flowering time, flowering at 24 and 32 days, respectively. Thus suppression of det1 early flowering time (in days) by ddb2 is a DDB1A-independent phenotype.

In addition, flowering time was measured by counting the number of rosette leaves plus cauline leaves on the main inflorescence. ddb2, ddb1a, and ddb1a ddb2 had the same number of leaves as wild type at flowering time. ddb2 suppressed significantly (P ≤ 0.01) the early flowering time of det1 in terms of number of leaves at flowering under long-day conditions but not under short-day conditions (data not shown). No significant differences were observed between the double mutant det1 ddb1a and the triple mutant det1 ddb1a ddb2 in either condition. Therefore, suppression of det1 early flowering time (as measured by number of leaves) by ddb2 is a DDB1A-dependent phenotype under long-day conditions but has no effect under short-day conditions.

Height:

After full adult height had been achieved under long- or short-day conditions, plant height was measured from the soil surface to the last flower on the inflorescence. ddb2, ddb1a, and ddb1a ddb2 did not differ from wild type (Figure 4, A and D). det1 mutants are 35% shorter than wild type. No significant difference was observed between det1 and det1 ddb2. In contrast, det1 ddb1a significantly enhanced the short det1 phenotype under both photoperiods. Loss of function of DDB2 in the det1 ddb1a background resulted in further enhancement of the short phenotype (P ≤ 0.01). det1 ddb1a ddb2 plants showing only 50% of det1 ddb1a height under long-day and 21% under short-day conditions. Thus ddb2 enhances the dwarf phenotype of the double mutant det1 ddb1a.

Fertility assessment:

The observation that the double mutant det1 ddb1a produced few and very small seed-containing siliques encouraged us to look thoroughly at floral development in the different genotypes. Long-day-grown wild type, ddb2, ddb1a, and ddb1a ddb2 did not have significantly different silique lengths (Figure 5). det1 exhibited shorter silique length, but mutation of ddb2 in this background partially suppressed the short silique phenotype (P ≤ 0.01). The double mutant det1 ddb1a showed very short siliques (4.75 mm). Loss of function of DDB2 in the det1 ddb1a background enhanced this short silique phenotype and resulted in 1.63-mm siliques on average. After dissecting at least six siliques from each genotype, we counted the number of seeds in each half. No significant differences were observed among wild type, ddb2, ddb1a, and ddb1a ddb2, with 29–33 seeds in each half-silique (Figure 5B). The det1 mutant showed lower seed numbers compared to the previous genotypes. Again, the ddb2 mutation in the det1 background suppressed this phenotype and resulted in more seeds. The det1 ddb1a siliques showed a lower seed number (9.17 seeds on average per half-silique). On the other hand, det1 ddb1a ddb2 siliques were found to be pale in color and dry; dissecting these siliques showed that none of them had developed seeds.

Figure 5.—

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Floral and fruit morphology. (A) Silique length (in millimeters). (B) Number of seeds per half-silique (n = 6). (C) (Top) Flower characteristics. From left to right: Col-0, ddb2, ddb1a, ddb1a ddb2, det1, det1 ddb2, det1 ddb1a, and det1 ddb1a ddb2. (Bottom) Dissected flower. From left to right: of Col-0, det1, det1 ddb2, det1 ddb1a, and det1 ddb1a ddb2. Error bars indicate 95% C.I. *P ≤ 0.05 or **P ≤ 0.01, respectively, relative to DDB2 wild-type control.

These observations led us to examine flowers of these genotypes. Early flowers of wild type, ddb2, ddb1a, and ddb1a ddb2 showed long petals and long stamens that are very close to the stigma in terms of height (Figure 5C). det1 flowers showed slightly shorter stamens than the wild type or the other single mutants. det1 ddb2 flowers were found to have longer stamens than det1, relatively similar to wild type. So ddb2 mutation in the det1 background suppressed the short stamens and subsequently facilitated the fertilization process. In contrast to det1 ddb2, mutation of DDB1A in the det1 background reduced stamen elongation. Dissecting det1 ddb1a flowers showed that the stamens exhibit approximately one-third the stigma length, as well as short petals. In later flowers, det1 ddb1a stamens were found to be longer and closer to the stigma, resulting in partial fertility. det1 ddb1a ddb2 flowers showed reduced size and petal length and short stamens throughout their life cycle. Thus, ddb2 suppresses the related det1 phenotypes of short silique length, reduced number of seeds, and short stamens. This suppression is not observed in the absence of DDB1A; in fact, these phenotypes are enhanced in the triple mutant.

Other growth traits were also assessed, such as number of inflorescences produced by each plant and fresh weight at 1 month of age under long- or short-day conditions. For these traits, loss of function of DDB2 had no effect in any background (data not shown).

Interactions with other de-etiolated mutants:

To determine if ddb2 interacts with det1 specifically or nonspecifically modifies the de-etiolated phenotype, we generated the cop1-4 ddb2 line. cop1-4 is a partial loss-of-function allele that is phenotypically similar to det1-1, exhibiting short hypocotyls when grown in the dark. However, loss of function of DDB2 in the cop1-4 background resulted in no significant effect on hypocotyl length under dark conditions (Figure 6, A and B) or on anthocyanin content at the seedling stage under either light or dark conditions (data not shown). Analysis of adult plants showed no significant differences between cop1-4 and cop1-4 ddb2 in flowering time, rosette diameter, height, number of inflorescences, and silique length (data not shown). Thus ddb2 appears to specifically modify det1, rather than the de-etiolated phenotype in general.

Figure 6.—

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(A) 7-day-old seedlings of wild type, ddb2, cop1-4, and cop1-4 ddb2 grown under dark conditions. Error bars indicate 95% C.I. (B) Hypocotyl length of 7-day-old dark-grown seedlings.

DISCUSSION

Arabidopsis DDB1A has been found in a complex with DET1 (Schroeder et al. 2002) and COP10 (Yanagawa et al. 2004). Also, DDB1 has been found in another complex with DDB2 involved in DNA repair (Cleaver 2005). Because DDB1 is involved in the formation of both complexes, we were interested in studying the interaction between DET1 and DDB2 through DDB1. To test this hypothesis, we generated the double and triple mutants of these genes. Our analysis indicates several modes of interaction among DDB2, DDB1A, and DET1.

Suggested models of interaction:

DDB1A dependent:

For some phenotypes, loss of function of DDB2 in the det1 background resulted in significant changes, while loss of function of DDB2 in the det1 ddb1a background showed no effect. This suggests that modulation of these phenotypes by ddb2 is DDB1A dependent. These phenotypes include dark hypocotyl elongation, dark anthocyanin content, and rosette diameter. Our hypothesis for the basis of this behavior is as follows. Due to the fact that the det1-1 mutation is not a null mutation, there are small amounts of this protein still active in the cell. This small portion of DET1 may compete with DDB2 for binding with DDB1A. Mutation of DDB2 may increase the availability of DDB1A inside the cell to interact with DET1. Since loss of DDB1A resulted in reduced DET1 activity, more DDB1A liberated from the DDB2 complex may increase DET1 activity. This model explains the basis of the suppression of det1 by ddb2 that we observed. However, for dark-grown hypocotyls, loss of function of DDB2 was found to enhance the det1 phenotype. In this case, perhaps the increase in DDB1A results in an increase in inactive complexes or increased degradation of ligase components. There is evidence that overexpression of E3 ligase components can result in loss-of-function phenotypes (e.g., Gray et al. 2002). Alternatively, both DET1-dependent and DDB2-dependent pathways may be required for optimal dark hypocotyl development.

DDB1A independent:

For some phenotypes, loss of function of DDB2 resulted in significant changes in both the det1 and det1 ddb1a backgrounds. This pattern of interaction suggests that regulation of these phenotypes by ddb2 is DDB1A independent. These phenotypes include light chlorophyll and anthocyanin content, flowering time (days), and height. Our hypothesis for the basis of this behavior is as follows. Arabidopsis is the only organism with two versions of the DDB1 protein (DDB1A and DDB1B). DDB1B is expressed at lower levels than DDB1A (Figure 1A). While DDB1A loss of function results in no obvious phenotype in wild-type background, DDB1B loss of function is lethal (Schroeder et al. 2002); thus little is known about the role of DDB1B in light signaling. Perhaps DDB2 interacts with DDB1B in the absence of DDB1A. Thus the DDB1A-independent phenotypes may be acting via DDB1B. If the redundant action of DDB1B results in DDB1A-independent phenotypes, this implies that DDB1B is unable to compensate for DDB1A-dependent phenotypes. For example, light-grown seedlings exhibit independent phenotypes (chlorophyll, anthocyanin) while dark-grown seedlings exhibit dependent phenotypes (hypocotyl, anthocyanin). Genevestigator data show that expression of DDB1A and DDB1B does not vary significantly between light and dark and that DDB1A is expressed at twice the level of DDB1B in both cases. Perhaps DDB1B is inactivated post-transcriptionally in dark-grown seedlings, resulting in DDB1A-dependent phenotypes.

These models suggest that both DET1 and DDB2 are able to interact with DDB1A and DDB1B. Support for this hypothesis is as follows. Arabidopsis DET1 and DDB1A interact genetically (Schroeder et al. 2002) as well as in a yeast two-hybrid assay (Bernhardt et al. 2006). In addition, DET1, DDB1A, COP10, CUL4, and RBX1 are able to form an active E3 Ub ligase complex (Chen et al. 2006). While no direct interaction between DET1 and DDB1B has been demonstrated, Bernhardt et al. (2006) showed that both DDB1A and DDB1B interact with At CUL4 in an in vitro pull-down assay, and myc-tagged DET1 immunoprecipitates with CUL4 from plant extracts. Residues required for interaction between human DET1 and DDB1 have recently been identified: DDB1 910-913 MALY in β-propeller C (Jin et al. 2006) and Arabidopsis DDB1A and DDB1B and rice DDB1 all contain the same variation of this sequence—LALY (Schroeder et al. 2002). With regard to DDB2/DDB1 interaction, Bernhardt et al. (2006) also showed that At DDB2 can interact with DDB1A in a yeast two-hybrid assay. No direct interaction between DDB2 and DDB1B has been demonstrated, nor have the human DDB1 residues required for interaction with DDB2 been identified, but competition experiments have shown that DDB2 competes with the viral protein SV5 for DDB1 interaction (Leupin et al. 2003) and SV5 has been shown to interact with the β-propeller C of DDB1 (T. Li et al. 2006). Several recent studies, however, have identified a WDXR motif at approximately residue 273 in human DDB2 and other DDB1-interacting WD40 proteins (DCAFs) that is required for interaction with DDB1 (Angers et al. 2006; He et al. 2006; Higa et al. 2006; Jin et al. 2006). This motif is also conserved in rice and Arabidopsis DDB2 (Ishibashi et al. 2003). Thus DDB1 appears to act as a scaffold to recruit specific factors, including DET1 and DDB2, to CUL4 E3 ligase complexes. Interaction with these specific factors appears to primarily occur via the β-propeller C domain of DDB1. In Arabidopsis, DDB1A appears to be expressed at nearly twice the level of DDB1B throughout development (Figure 1A), and both DET1 and DDB2 can interact with DDB1A in vitro (Bernhardt et al. 2006). Our genetic analysis suggests that this interaction is functionally important.

Modulation of dark hypocotyl elongation:

det1 seedlings show short hypocotyls when grown under dark conditions, and this phenotype is similar to light-grown wild-type seedlings (Chory et al. 1989). Photoreceptors mutations, which alone show long hypocotyls, in the det1 background exhibit the det1 phenotype. This suggests that DET1 is functioning downstream of the photoreceptors (Wang and Deng 2002).

Enhancers of the det1 dark hypocotyl phenotype include ddb1a (Schroeder et al. 2002) and a weak allele (cop1-6) of the WD-40 protein COP1. The double mutant cop1-6 det1-1 exhibits dark purple cotyledons, very short hypocotyls, and adult lethality (Ang and Deng 1994). Similarly, we have shown that loss of function of DDB2, another member of the WD-40 protein family, enhances the short hypocotyl phenotype of det1. All these enhancers appear to be acting near DET1 in the light-signaling pathway.

Suppressors of the det1 dark hypocotyl phenotype include CONSTANS-LIKE3 (col3). Dark-grown det1-1 col3 exhibits 55% longer hypocotyls than det1-1 alone (Datta et al. 2006). Loss-of-function alleles of the transcription factor HY5 also suppress the det1 dark phenotype (Chory 1992; Pepper and Chory 1997). Mutation of TED3 in the det1 background also completely suppresses the short det1 hypocotyl (Pepper and Chory 1997). TED3 encodes a peroxisomal protein (Pex2p) involved in Arabidopsis development (Hu et al. 2002). These suppressors act downstream of DET1 in light signaling.

Regulation of chlorophyll content:

Light-grown det1 seedlings exhibit a pale phenotype compared to wild type (Chory et al. 1989). This correlates with a decrease in CAB mRNA expression (Chory and Peto 1990). The region of the CAB2 promoter involved in DET1 upregulation of light expression has been mapped to the CAB upstream factor-1 element (CUF-1). The CUF-1 element is bounded by the transcription factor HY5, and hy5 mutants also underexpress CAB2 in the light (Maxwell et al. 2003). Other studies indicate that circadian regulation of CAB transcription by DET1 might be post-transcriptional. Degradation of the LHY (late elongated hypocotyl) factor is accelerated in det1-1 mutants (Song and Carre 2005).

Our results show that mutation of DDB2 in det1 or det1 ddb1a backgrounds significantly enhanced chlorophyll content of these seedlings (i.e., suppressed the det1 pale phenotype). It will be interesting to determine the basis of this suppression.

Regulation of anthocyanin content:

The anthocyanin biosynthetic pathway is well studied. Several transcription factors that regulate anthocyanin biosynthesis have been identified. In addition, many environmental conditions regulate this pathway. Light is an important factor that regulates anthocyanin accumulation in plant cells (Koes et al. 2005). As shown previously, det1 seedlings have more anthocyanin than wild type. Chory and Peto (1990) reported that the anthocyanin biosynthetic gene Chalcone synthase (CHS) is ectopically expressed in leaf mesophyll cells in det1 seedlings. Chory et al. (1989) studied CHS gene expression of dark-grown seedlings and found that det1 seedlings have more (20- to 50-fold) CHS mRNA than the wild type.

In light-grown seedlings, we observed no significant effect of ddb2 on det1 anthocyanin levels, but ddb2 suppressed anthocyanin accumulation in the det1 ddb1a double mutant. In contrast, in dark-grown seedlings, ddb2 suppressed anthocyanin accumulation in a DDB1A-dependent fashion. Interestingly, while ddb2 regulation of both anthocyanin levels and hypocotyl elongation was found to be DDB1A dependent in dark-grown seedlings, opposing effects were observed. Loss of function of DDB2 suppressed the high anthocyanin content of det1 but enhanced the det1 short hypocotyl phenotype. This opposing trend was also observed for another mutation. Loss of function of COL3 in the det1 background enhanced the high anthocyanin content of det1 but suppressed the short hypocotyl length of dark-grown seedlings (Datta et al. 2006).

Regulation of fertility parameters:

Mutation of DDB1A in the det1 background results in reduced floral development and seed production (Schroeder et al. 2002). Mutation of DDB2 suppressed the short stamens and reduced dehiscence, number of seeds, and silique length in det1 single mutants, but enhanced those phenotypes in the det1 ddb1a double mutant. These phenotypes were similar to those observed for loss-of-function alleles of jasmonic acid (JA) biosynthesis or signaling genes. JA, a lipid-derived signaling compound present in most plant species, has been found to play a major role in anther development. One model suggests that JA regulates water flow in the stamens and petals, which further regulates flower opening and stamen maturation. On the other hand, regulation of programmed cell death in the anther by JA as dehiscence proceeds has also been proposed (Scott et al. 2004). Similar phenotypes were also observed in an overexpression line of RBX1 (Gray et al. 2002). RBX1 (also known as ROC1) is a RING-domain protein and a component of cullin-containing E3 ubiquitin ligases, including the SCF-type complex COI1 involved in JA signaling (Schwechheimer et al. 2004) and the human DET1, COP1, DDB1, and CUL4A complex (Wertz et al. 2004).

DDB1 and DDB2 loss-of-function mutants in other organisms:

DDB1 and DDB2 were originally identified due to their role in nucleotide excision repair. Human XPE patients have loss-of-function alleles of DDB2 (Cleaver 2005). DDB2 knockout mice, although viable, also have increased tumor formation (Itoh 2004; Yoon et al. 2005). Decrease in leaf width and length and increase in UV sensitivity were observed in null ddb2 mutants in Arabidopsis (Koga et al. 2006). While good DDB2 homologs have been identified only in vertebrates and higher plants, DDB1 homologs also exist in Drosophila, Caenorhabditis elegans, and Schizosaccharomyces pombe. Recently, loss of function of DDB1 in human cells has been found to result in defects in UV-damage repair (J. Li et al. 2006) and increased DNA double-strand break accumulation throughout the genome (Lovejoy et al. 2006). DDB1 knockout flies are lethal, suggesting a crucial role for DDB1 in Drosophila development (Takata et al. 2004). RNAi screens in C. elegans have shown that loss of DDB1 results in embryonic and larval lethality (Kim and Kipreos 2006). Yeast (S. pombe) DDB1 was found to have a significant role in preventing mutation and genome stability (Holmberg et al. 2005), as well as a role in cell division and replication control (Zolezzi et al. 2002; Bondar et al. 2003). In tomato (Lycopersicon esculentum), High Pigment 1 (HP-1) is homologous to Arabidopsis and human DDB1. hp-1 mutants exhibit highly pigmented fruit and short hypocotyls like mutants in the tomato DET1 gene, HP-2 (Lieberman et al. 2004; Liu et al. 2004). Thus, the role of DDB2 appears to be specific to DNA repair, while DDB1 appears to have additional roles during development. This is consistent with biochemical evidence that DDB2 specifically recruits the DDB1/CUL4 E3 ligase to DNA damage (Kapetanaki et al. 2006), while DDB1/CUL4 can form complexes with multiple targeting factors, such as CSA (Groisman et al. 2003) and DET1 (Wertz et al. 2004; Bernhardt et al. 2006; Chen et al. 2006).

Here we show that the ddb2 and ddb1a single and double mutants exhibit no developmental phenotypes on their own, but in the det1-1 background, strong (ddb1a) or more subtle (ddb2) developmental effects can be observed. We propose that these effects are due to changes in the activity of the DET1 complex, either directly (ddb1a) or indirectly (ddb2) via modulations of the DDB1A/DDB1B pool.

Acknowledgments

We thank Mehdi Sefidgar, Chris Kuusselka, and Christine Yurkowski for technical assistance and Paul Mains and Jennifer Nemhauser for critical suggestions. The work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada.

References

  1. Ang, H., and X. Deng, 1994. Regulatory hierarchy of photomorphogenic loci: allele-specific and light-dependent interaction between the HY5 and COP1 loci. Plant Cell 6: 613–628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Angers, S., T. Li, X. Yi, M. Maccoss, R. Moon et al., 2006. Molecular architecture and assembly of the DDB1–CUL4A ubiquitin ligase machinery. Nature 443: 590–593. [DOI] [PubMed] [Google Scholar]
  3. Bernhardt, A., E. Lechner, P. Hano, V. Schade, M. Dieterle et al., 2006. CUL4 associates with DDB1 and DET1 and its downregulation affects diverse aspects of development in Arabidopsis thaliana. Plant J. 47: 591–603. [DOI] [PubMed] [Google Scholar]
  4. Bondar, T., V. Ekaterina, D. Ucker, W. Walden, S. Mirkin et al., 2003. Schizosaccharomyces pombe Ddb1 is functionally linked to the replication checkpoint pathway. J. Biol. Chem. 278: 37006–37014. [DOI] [PubMed] [Google Scholar]
  5. Chen, H., Y. Shen, X. Tang, L. Yu, J. Wang et al., 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. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen, M., J. Chory and C. Fankhauser, 2004. Light signal transduction in higher plants. Annu. Rev. Genet. 38: 87–117. [DOI] [PubMed] [Google Scholar]
  7. Chory, J., 1992. A genetic model for light-regulated seedling Arabidopsis. Development 115: 337–354. [Google Scholar]
  8. Chory, J., and C. Peto, 1990. Mutations in the DET1 gene affect cell-type-specific expression of light regulated genes and chloroplast development in Arabidopsis. Proc. Natl. Acad. Sci. USA 87: 8776–8780. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chory, J., C. Peto, R. Feinbaum, L. Pratt and F. Ausubel, 1989. Arabidopsis thaliana mutant that develops as a light-grown plant in the absence of light. Cell 58: 991–999. [DOI] [PubMed] [Google Scholar]
  10. Cleaver, J., 2005. Cancer in Xeroderma pigmentosum and related disorders of DNA repair. Nat. Rev. Cancer 5: 564–573. [DOI] [PubMed] [Google Scholar]
  11. Datta, S., G. Hettiarachchi, X. Deng and M. Holm, 2006. Arabidopsis CONSTANS-LIKE3 is a positive regulator of red light signaling and root growth. Plant Cell 18: 70–84. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fankhauser, C., and J. Casal, 2004. Phenotypic characterization of a photomorphogenic mutant. Plant J. 39: 747–760. [DOI] [PubMed] [Google Scholar]
  13. Gray, W. H., D. Sunethra and M. Estelle, 2002. Role of the Arabidopsis RING-H2 protein RBX1 in RUB modification and SCF function. Plant Cell 14: 2137–2144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Groisman, R., J. Polanowska, J. Kuraoka, J. Sawada, M. Saijo et al., 2003. The ubiquitin ligase activity in the DDB2 and CSA complexes is differentially regulated by the COP9 signalosome in response to DNA damage. Cell 113: 357–367. [DOI] [PubMed] [Google Scholar]
  15. He, Y., C. McCall, J. Hu, Y. Zeng and Y. Xiong, 2006. DDB1 functions as a linker to recruit receptor WD40 proteins to CUL4–ROC1 ubiquitin ligases. Genes Dev. 20: 2949–2954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Higa, L., M. Wu, T. Ye, R. Kobayashi, H. Sun et al., 2006. CUL4–DDB1 ubiquitin ligase interacts with multiple WD40-repeat proteins and regulates histone methylation. Nat. Cell Biol. 8: 1277–1283. [DOI] [PubMed] [Google Scholar]
  17. Holmberg, C., O. Fleck, H. Hansen, C. Liu, R. Slaaby et al., 2005. Ddb1 controls genome stability and meiosis in fission yeast. Genes Dev. 19: 853–862. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hu, J., M. Aguirre, C. Peto, J. Alonso, J. Ecker et al., 2002. A role for peroxisomes in photomorphogenesis and development of Arabidopsis. Science 297: 405–409. [DOI] [PubMed] [Google Scholar]
  19. Ishibashi, T., K. Seisuke, Y. Taichi, F. Tomoyuki, K. Takata et al., 2003. Rice UV-damaged DNA binding protein homologues are most abundant in proliferating tissues. Gene 308: 79–87. [DOI] [PubMed] [Google Scholar]
  20. Itoh, T., D. Cado, R. Kamide and S. Linn, 2004. DDB2 gene disruption leads to skin tumors and resistance to apoptosis after exposure to ultraviolet light but not a chemical carcinogen. Proc. Natl. Acad. Sci. USA 101: 2052–2057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jin, J., E. E. Arias, J. Chen, J. W. Harper and J. C. Walker, 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. [DOI] [PubMed] [Google Scholar]
  22. Kapetanaki, M. G., J. Guerrero-Santoro, D. C. Bisi, C. L. Hsieh, V. Rapic-Otrin et al., 2006. The DDB1–CUL4ADDB2 ubiquitin ligase is deficient in Xeroderma pigmentosum group E and targets histone H2A at UV-damaged DNA sites. Proc. Natl. Acad. Sci. USA 103: 2588–2593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kim, Y., and E. T. Kipreos, 2006. The Caenorhabditis elegans replication licensing factor CDT-1 is targeted for degradation by the CUL-4/DDB-1 complex. Mol. Cell. Biol. 27: 1394–1406. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Koes, R., W. Verweij and F. Quattrocchio, 2005. Flavonoids: a colorful model for the regulation and evolution of biochemical pathways. Trends Plant Sci. 10: 236–242. [DOI] [PubMed] [Google Scholar]
  25. Koga, A., T. Ishibashi, S. Kimura, Y. Uchiyama and K. Sakaguchi, 2006. Characterization of T-DNA insertion mutants and RNAi silenced plants of Arabidopsis thaliana UV-damaged DNA binding protein 2 (AtUV-DDB2). Plant Mol. Biol. 61: 227–240. [DOI] [PubMed] [Google Scholar]
  26. Leupin, O., S. Bontron and M. Strubin, 2003. Hepatitis B virus X protein and simian virus 5 V protein exhibit similar UV-DDB1 binding properties to mediate distinct activities. J. Virol. 77: 6274–6283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Li, J., Q. Wang, Q. Zhu, M. El-Mahdy, G. Wani et al., 2006. DNA damage binding protein component DDB1 participates in nucleotide excision repair through DDB2 DNA-binding and cullin 4A ubiquitin ligase activity. Cancer Res. 66: 8590–8597. [DOI] [PubMed] [Google Scholar]
  28. Li, T., X. Chen, K. Garbutt, P. Zhou and N. Zheng, 2006. Structure of DDB1 in complex with a paramyxovirus V protein: viral hijack of a propeller cluster in ubiquitin ligase. Cell 124: 105–117. [DOI] [PubMed] [Google Scholar]
  29. Lieberman, M., O. Segev, N. Gilboa, A. Lalazar and I. Levin, 2004. The tomato homolog of the gene encoding UV-damaged DNA binding protein 1 (DDB1) underlined as the gene that causes the high pigment-1 mutant phenotype. Theor. Appl. Genet. 108: 1574–1581. [DOI] [PubMed] [Google Scholar]
  30. Liu, W., A. Nichols, J. Graham, R. Dualan, A. Abbas et al., 2000. Nuclear transport of human DDB protein induced by ultraviolet light. J. Biol. Chem. 275: 21429–21434. [DOI] [PubMed] [Google Scholar]
  31. Liu, Y., S. Roof, Z. Ye, C. Barry and A. Van Tuinen, 2004. Manipulation of light signal transduction as a means of modifying fruit nutritional quality in tomato. Proc. Natl. Acad. Sci. USA 101: 9897–9902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lovejoy, C., K. Lock, A. Yenamandra and D. Cortez, 2006. DDB1 maintains genome integrity through regulation of Cdt1. Mol. Cell. Biol. 26: 7977–7990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mackinney, G., 1941. Absorption of light by chlorophyll solutions. J. Biol. Chem. 140: 315–322. [Google Scholar]
  34. Maxwell, B. B., C. R. Andersson, D. S. Poole, S. A. Kay and J. Chory, 2003. HY5, circadian clock-associated 1, and a cis-element, DET1 dark response element, mediate DET1 regulation of Chlorophyll a/b-Binding Protein 2 expression. Plant Physiol. 133: 1565–1577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Pepper, A., and J. Chory, 1997. Extragenic suppressors of the Arabidopsis detl mutant identify elements of flowering-time and light-response regulatory pathways. Genetics 145: 1125–1137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Pepper, A., T. Delaney, T. Washburn, D. Poole and J. Chory, 1994. DET1, a negative regulator of light-mediated development and gene expression in Arabidopsis, encodes a novel nuclear-localized protein. Cell 78: 109–116. [DOI] [PubMed] [Google Scholar]
  37. Schroeder, D., M. Gahrtz, B. Maxwell, K. Cook, M. Kan et al., 2002. De-etiolated 1 (DET1) and damaged DNA binding protein 1 (DDB1) interact to regulate Arabidopsis photomorphogenesis. Curr. Biol. 12: 1462–1472. [DOI] [PubMed] [Google Scholar]
  38. Schwechheimer, C., L. Irina and A. Villalobos, 2004. Cullin-containing E3 ubiquitin ligases in plant development. Curr. Opin. Plant Biol. 7: 677–686. [DOI] [PubMed] [Google Scholar]
  39. Scott, R., M. Spielman and H. Dickinson, 2004. Stamen structure and function. Plant Cell 16(Suppl): S46–S60. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Shiyanov, P., S. Hayes, M. Donepudi, A. Nichols, S. Linn et al., 1999. The naturally occurring mutants of DDB are impaired in stimulating nuclear import of the p125 subunit and E2F1-activated transcription. Mol. Cell. Biol. 19: 4935–4943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Song, H. R., and I. A. Carre, 2005. DET1 regulates the proteasomal degradation of LHY, a component of the Arabidopsis circadian clock. Plant Mol. Biol. 57: 761–771. [DOI] [PubMed] [Google Scholar]
  42. Takata, K., Y. Hideki, Y. Masamitsu and K. Sakaguchi, 2004. Drosophila damaged DNA-binding protein 1 is an essential factor for development. Genetics 168: 855–865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wang, H., and X. W. Deng, 2002. Phytochrome signaling mechanism, pp. 1–28 in The Arabidopsis Book, edited by C. Somerville and E. Meyerowitz. American Society of Plant Biologists, Rockville, MD.
  44. Weigel, D., and J. Glazebrook, 2002. Arabidopsis: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  45. Wertz, I., K. O'Rourke, Z. Zhang, D. Dornan, D. Arnott et al., 2004. Human de-etiolated-1 regulates c-Jun by assembling a CUL4A ubiquitin ligase. Science 303: 1371–1374. [DOI] [PubMed] [Google Scholar]
  46. Yanagawa, Y., J. Sullivan, S. Komatsu, G. Gusmaroli, G. Suzuki et al., 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. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Yoon, T., A. Chakrabortty, R. Franks, T. Valli, H. Kiyokawa et al., 2005. Tumor-prone phenotype of the DDB2-deficient mice. Oncogene 24: 469–478. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zolezzi, F., J. Fuss, S. Uzawa and S. Linn, 2002. Characterization of a Schizosaccharomyces pombe strain deleted for a sequence homologue of the human damaged DNA binding 1 (DDB1) gene. J. Biol. Chem. 277: 41183–41191. [DOI] [PubMed] [Google Scholar]

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