Arabidopsis DDB1-CUL4 E3 ligase complexes in det1 salt/osmotic stress resistant germination

ABSTRACT

A key regulatory mechanism in plant growth, development, and stress signaling utilizes E3 ubiquitin ligases, which target a variety of substrates for degradation. DE-ETIOLATED 1 (DET1) forms a complex with DDB1 (DAMAGED DNA BINDING protein 1) and CUL4 (CULLIN 4), and negatively regulates light signaling. Another DDB1-CUL4 complex containing DWA1 and DWA2 (DWD hypersensitive to ABA 1 and 2) has been shown to negatively regulate abscisic acid (ABA) signaling. Since distinct DDB1-CUL4 complexes have been shown to influence each other, we analyzed genetic interactions between DET1 and components of DDB1-CUL4 complexes during seed germination under salt and osmotic stress conditions. det1 germination was resistant to salt and osmotic stress and dwa1 and dwa2 enhanced this phenotype. In contrast, ddb1a partially suppressed the det1 germination phenotype on both salt and mannitol, while ddb1b had no effect. Mutations in DDB2, a DDB1-CUL4 complex component involved in DNA repair, also partially suppressed the det1 germination phenotype while mutants in COP1, another light signaling component, completely suppressed the det1 resistant germination phenotypes. Taken together these data suggest that components of E3 ubiquitin ligase complexes have variable but significant effects on det1 salt/osmotic stress responses.

Abbreviations

ABA

abscisic acid

ABI5

ABSCISIC ACID INSENSITIVE 5

COP1

CONSTITUTIVE PHOTOMORPHOGENIC 1

CUL4

CULLIN 4

DDB1A/B

DAMAGED DNA BINDING PROTEIN 1A/B

DET1

DE-ETIOLATED 1

DWA1/2

DDB1 BINDING WD40 HYPERSENSITIVE TO ABA 1/2

HY5

LONG HYPOCOTYL 5

Ub

ubiquitin

Introduction

Eukaryotes use ubiquitination as a means of regulating protein function. Ubiquitination is a process by which the 76 amino acid conserved protein Ubiquitin (Ub) is covalently attached to a target protein. Monoubiquitination regulates protein trafficking or activity while polyubiquitination targets proteins for degradation via the 26S proteasome. Three major enzymes are involved in this process, namely Ub activating enzymes (E1), Ub conjugating enzymes (E2), and Ub ligases (E3). Ub E3 ligases play important roles in ubiquitination by providing substrate specificity. E3 ligases transfer Ub from the E2 to the target protein and position it properly for Ub conjugation.¹ E3 Ub ligase complexes have diverse roles in plants and animals including regulation of growth and development and response to abiotic and biotic stress. There are approximately 1400 E3 ligases in Arabidopsis thaliana.^2,3 During plant stress response, a large number of E3 ligases are implicated in the response to the stress hormone abscisic acid (ABA), regulating processes from biosynthesis to signaling. Thus E3 ligases play a critical role in ABA responses in plants.^3,4

Many E3 ligases employ one of the 4 CULLINs (CULs) as the scaffolding protein. CUL4 based E3 ligase complexes bind to a large number of substrates via the substrate adapter DAMAGED DNA BINDING protein 1 (DDB1), which interacts with a variety of substrate receptors.⁵ Arabidopsis has 2 homologues of DDB1, DDB1A and DDB1B.⁶ The substrate receptors in turn interact with specific substrates to be targeted for degradation. These substrate receptors are referred to as DDB1 BINDING WD40 (DWD) or DDB1-CUL4 ASSOCIATED FACTOR (DCAF) factors.⁵ Arabidopsis has 85 DWD proteins with the conserved 16 amino acid DWD motif.⁷

DE-ETIOLATED 1 (DET1), a central repressor of photomorphogenesis (light growth), interacts with DDB1, CUL4, and CONSTITUTIVE PHOTOMORPHOGENIC 10 (COP10) to form the CUL4-CDD complex.^6,8-10 CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), another central repressor of light signaling, also acts as an E3 Ub ligase. COP1 targets photomorphogenesis promoting transcription factors like LONG HYPOCOTYL 5 (HY5) for degradation.^11,12 COP1 interacts with SUPPRESSOR OF PHYTOCHROME A 1–4 (SPA1-4) and forms tetrameric COP1-SPA complexes that exhibit E3 ligase activity.¹³ COP1-SPA complexes act as E3 ligases alone in some instances and as part of CUL4-DDB1 E3 ligase complexes in others.¹⁴ COP1-SPA and CDD are distinct CUL4 complexes that do not interact directly with each other,¹⁴ however DET1 is required for COP1 nuclear localization¹⁵ and HY5 degradation, but the basis of this requirement is not known.^11,12

DAMAGED DNA BINDING protein 2 (DDB2) also interacts with CUL4-DDB1. The primary function of this E3 ligase complex is facilitating UV damaged DNA repair.^16,17 In an example of CUL4-DDB1 complexes interacting with each other, examination of the effect of det1 on CUL4-DDB1/2 complexes showed that DET1 is required for DDB2 degradation.¹⁸ In addition, while the Arabidopsis ddb2 single mutant has no significant developmental phenotypes, ddb2 modifies det1 phenotypes.¹⁹

During ABA signaling, another DDB1-CUL4 complex containing DWA1 and DWA2 (DWD hypersensitive to ABA 1 and 2) has been shown to regulate ABA response. DWA1 and 2 interact with each other as well as directly interact with the ABA response promoting transcription factor ABSCISIC ACID INSENSITIVE 5 (ABI5), targeting it for degradation.²⁰ Thus DWA1/2 negatively regulate ABA signaling. We have previously shown that DET1 interacts genetically with DWA1 during plant development. dwa1 affects det1 seedling growth, flowering time and fertility phenotypes. In contrast, dwa2 exhibited no consistent effects on det1 growth phenotypes. However both dwa1 and dwa2 partially suppressed det1 ABA sensitive germination.²¹ Therefore, these components of distinct DDB1-CUL4 E3 ligase complexes appear to interact with each other directly or indirectly during light signaling as well as in stress signaling.

Other DDB1-CUL4 ligase complexes have also been implicated in ABA signaling. DWA3 also forms DDB1-CUL4 complexes and is a negative regulator of ABA signaling, but its target is unknown. Although dwa3 mutants accumulate ABI5, no direct interaction was found between DWA3 and ABI5. Therefore it was suggested that DWA3 suppresses a negative regulator of DWA1 and 2.²² ABA HYPERSENSITIVE DCAF 1 (ABD1) also interacts with CUL4-DDB1 and forms another E3 ligase complex that targets ABI5 for degradation.²³ DET1-DDB1-ASSOCIATED 1 (DDA1) interacts with the CDD complex to target the ABA receptor PYRABACTIN RESISTANCE LIKE 8 (PYL8) for degradation.²⁴ Thus DDB1-CUL4 complexes are involved in many aspects of ABA signaling.

det1 exhibits ABA sensitive germination and dwa1, dwa2, ddb1a, ddb1b, and ddb2 have been shown to either partially or completely suppress this phenotype, while cop1 enhances it.²¹ In this study we showed that, in contrast, det1 mutants exhibit resistance to salt and osmotic stress induced inhibition of seed germination and examined the role of E3 ligase components in this phenotype.

Results

While det1 mutants have been shown to exhibit ABA sensitive germination,^21,24 they were in fact resistant to salt and osmotic stress induced inhibition of germination (Fig. 1). We have previously examined the role of DWA1, DWA2, DDB1A, DDB1B, COP1, and DDB2 in det1 ABA sensitive germination. Here we examined the role of these genes in det1 salt/osmotic stress resistant germination by generating double mutants and assessing germination on salt and mannitol containing media.

Figure 1.

Salt and osmotic stress resistant germination in det1 mutants. Germination of det1 mutants on (a) 0, (b) 100 mM NaCl (c) 200 mM Mannitol. Values are means ± SE of 2 replicates of 50–100 seeds, *= P ≤ 0.05 of det1 vs wildtype.

det1 dwa1 and det1 dwa2 salt/mannitol germination responses

Since we observed partial rescue of the det1 ABA sensitive germination phenotype in det1 dwa1 and det1 dwa2 double mutants,²¹ and plant responses to salt and mannitol often utilize ABA signaling, we examined the effect of dwa1 and dwa2 on det1 salt/mannitol resistant germination. The germination of the dwa1 single mutant was slightly delayed on mannitol containing media, as expected for the loss of function of a negative regulator of ABA signaling (Fig. 2).²⁰ On salt and 400 mM mannitol, dwa1 did not exhibit any consistent significant effect on det1 germination. On 200 mM mannitol, however, germination was enhanced in the det1 dwa1 double mutants, implying a possible role for DWA1 in det1 osmotic stress resistant germination. The dwa2 single mutant exhibited wildtype germination on both types of stress media (Fig. 3). dwa2 enhanced the det1 salt resistant germination phenotype on 100 mM salt, but no consistent significant effects were observed on mannitol or 200 mM salt. Thus while DWA1 is not required for det1 salt resistant germination but may be involved in osmotic stress response, DWA2 is involved in the det1 salt, but not osmotic, germination phenotype.

Figure 2.

Germination in det1 dwa1 double mutants. Germination (%) on (a) 0, 100 mM, 200 mM NaCl (b) 0, 200 mM, 400 mM Mannitol. Values are means ± SE of 2 replicates of 50–100 seeds. *= P ≤ 0.05 of single mutants vs wildtype, + = P ≤ 0.05 of doubles vs det1.

Figure 3.

Germination in det1 dwa2 double mutants. Germination (%) on (a) 0, 100 mM, 200 mM NaCl (b) 0, 200 mM, 400 mM Mannitol. Values are means ± SE of 2 replicates of 50–100 seeds. *= P ≤ 0.05 of single mutants vs wildtype, + = P ≤ 0.05 of doubles vs det1.

det1 ddb1a and det1 ddb1b salt/mannitol germination responses

Since DET1 interacts physically and genetically with both DDB1A and DDB1B,^6,10,25 and ddb1a completely suppresses and ddb1b partially suppresses the det1 ABA sensitive germination phenotype,²¹ we examined the role of DDB1A and DDB1B in det1 salt/osmotic resistant germination. Germination in the ddb1a single mutant was hypersensitive to both salt and mannitol (Fig. 4), consistent with previously reported ddb1a germination phenotypes.²⁵ The det1 ddb1a double mutant partially suppressed the det1 salt resistant germination phenotype on 200 mM salt on day 3. On mannitol, complete rescue was observed at 200 mM and an intermediate phenotype at 400 mM mannitol. Therefore, DDB1A contributes to the det1 salt and osmotic stress germination phenotypes. In contrast, the ddb1b single mutant did not show a phenotype on salt but was hypersensitive to mannitol, and the det1 ddb1b double mutants did not differ significantly from det1 (Fig. 5). Thus DDB1A but not DDB1B contribute to det1 stress resistant germination.

Figure 4.

Germination in det1 ddb1a double mutants. Germination (%) on (a) 0, 100 mM, 200 mM NaCl (b) 0, 200 mM, 400 mM Mannitol. Values are means ± SE of 2 replicates of 50–100 seeds. *= P ≤ 0.05 of single mutants vs wildtype, + = P ≤ 0.05 of doubles vs det1.

Figure 5.

Germination in det1 ddb1b double mutants. Germination (%) on (a) 0, 100 mM, 200 mM NaCl (b) 0, 200 mM, 400 mM Mannitol. Values are means ± SE of 2 replicates of 50–100 seeds. *= P ≤ 0.05 of single mutants vs wildtype, + = P ≤ 0.05 of doubles vs det1.

det1 ddb2 salt/mannitol germination responses

Although they are components of distinct DDB1-CUL4 complexes, DET1 and DDB2 have been shown to interact genetically during both development and DNA repair.^18,19 In addition, ddb2 completely suppressed the det1 ABA sensitive germination phenotype.²¹ Thus we examined the effect of ddb2 on det1 stress resistant germination. The ddb2 single mutant was slightly sensitive to salt inhibition of seed germination at 100 mM but exhibited wildtype germination on mannitol (Fig. 6). ddb2 partially rescued both salt and osmotic stress resistant germination in det1 mutants. Thus, as with the other phenotypes previously examined, DDB2 contributes to det1 stress resistant germination.

Figure 6.

Germination in det1 ddb2 double mutants. Germination (%) on (a) 0, 100 mM, 200 mM NaCl (b) 0, 200 mM, 400 mM Mannitol. Values are means ± SE of 2 replicates of 50–100 seeds. *= P ≤ 0.05 of single mutants vs wildtype, + = P ≤ 0.05 of doubles vs det1.

det1 cop1 salt/mannitol germination responses

DET1 and COP1 are both negative regulators of photomorphogenesis. Developmentally, det1 and cop1 tend to enhance each other phenotypes, such as reduced hypocotyl length and increased anthocyanin content, and the det1 cop1 double mutant is seedling lethal.^26,27 Therefore, det1 cop1 germination was scored in a segregating population of cop1 det1/+. cop1 enhanced det1 ABA sensitive germination and the cop1 det1 double mutant also exhibits reduced germination in control conditions.²¹ Here we found that cop1 single mutants were hypersensitive to both salt and mannitol stress, exhibiting the opposite phenotype to det1 (Fig. 7). In control conditions, the germination of cop1 det1 double mutants was delayed relative to both the det1 and cop1 single mutants, as previously described.²¹ On salt containing media, cop1 completely suppressed the det1 resistant germination phenotype, indicating that cop1 is epistatic to det1. On mannitol, cop1 again completely suppressed the det1 resistant phenotype. In fact, on 200 mM mannitol, germination in the double mutant was well below even that of the cop1 single mutant. Thus COP1 is required for det1 stress resistant germination.

Figure 7.

Germination in det1 cop1-4 double mutants. Germination (%) on (a) 0, 100 mM, 200 mM NaCl (b) 0, 200 mM, 400 mM Mannitol. Values are means ± SE of 2 replicates of 50–100 seeds. *= P ≤ 0.05 of single mutants vs wildtype, + = P ≤ 0.05 of doubles vs det1.

Discussion

Components of CUL4-DDB1 complexes have previously been shown to genetically interact with DET1 during Arabidopsis development and also play a role in det1 ABA sensitive germination.^19,21 Here we investigated whether these components have a function in det1 salt/osmotic stress resistant germination.

Role of DWA1 and DWA2 in det1 salt/mannitol resistant germination

dwa1 and dwa2 have been shown to exhibit salt sensitive root growth and ABA sensitive germination and dwa1 dwa2 double mutants exhibit enhanced phenotypes.²⁰ We observed that dwa1 enhanced det1 germination during osmotic stress, while dwa2 enhanced det1 germination under salt stress. DWA1 and DWA2 can interact with each other, suggesting they act as heterodimers, thus would be expected to have common loss of function phenotypes. However DWA1 and DWA2 can also interact with themselves, forming homodimers, and their loss of function phenotypes are additive, suggesting they also have independent functions.²⁰ Interestingly, eFP browser expression data indicates that in aerial tissues DWA1 is upregulated by osmotic stress, while DWA2 is upregulated by salt, consistent with the phenotypes we observed (Fig. 8).^28,29

Figure 8.

Relative expression levels of DWA1 and DWA2 under salt and osmotic stress. Relative expression levels of DWA1 and DWA2 in shoots of 18 d old plants following treatment with (a) 150 mM NaCl and (b) 300 mM Mannitol accessed via the Arabidopsis eFP browser.^28,29

Surprisingly the det1 dwa1 and dwa2 double mutants exhibited the opposite phenotype of what would be predicted based on the function of the CUL4-DDB1-DWA1/2 E3 ligase. In the presence of stress-induced ABA signaling, the absence of either DWA1 or DWA2 should increase ABI5 levels, repressing germination and thus rescuing the det1 resistant germination phenotype, but we observed increased germination in the double mutants. Interestingly we observed the same trend on ABA, the det1 dwa1/2 double mutants exhibited more germination than det1, in this case suppressing the det1 ABA sensitive germination phenotype.²¹ Thus even though det1 has opposite phenotypes on ABA and salt/mannitol, the effect of dwa1/2 was the same, increased germination. These results suggest that in the det1 background DWA1 and DWA2 act as positive, rather than negative, regulators of stress signaling. What might be the basis of this effect? Perhaps the downregulation of 2 CUL4-DDB1 complexes (DET1 and DWA1/DWA2) results in upregulation of other CUL4-DDB1 complexes that are negative regulators of ABA signaling, such as those containing DWA3, ABD1, or the recently described ALTERED SEED GERMINATION 2 (ASG2).^22,23,30 This would result in decreased ABA signaling and increased germination.

Alternatively this effect maybe due to transcriptional rather than post-transcriptional compensation. We have previously shown that DWA1 mRNA levels are higher in dwa2 mutants,²¹ thus perhaps dwa1 mutants have increased levels of DWA2. Due to the redundant nature of their function perhaps in the absence of one DWA the other compensates for it, resulting in reduced levels of ABI5 and enhanced germination. We have also previously observed variable levels of DWA1 in the det1 dwa2 double mutants, correlating with variable developmental phenotypes.²¹ This may also be the basis of the variable germination phenotypes observed here. Similarly, perhaps variable levels of DWA2 in the det1 dwa1 double mutants also result in the variation in germination phenotype observed.

Role of DDB1A/B in det1 salt/mannitol resistant germination

ddb1a and ddb1b were previously shown to suppress both det1 ABA sensitive germination and ABA resistant cotyledon emergence.²¹ Thus both DDB1A and DDB1B are required for these contrasting det1 ABA phenotypes. Under salt and osmotic stress conditions, ddb1a partially suppressed the det1 resistant germination phenotypes, while ddb1b did not. This could be due to the fact that ddb1b is a weaker allele than ddb1a.^25,31 Nonetheless, we find that DDB1A, but not DDB1B, is required for the det1 salt/osmotic resistant germination phenotypes. Although ddb1a and ddb1b generally suppress det1 germination phenotypes, they enhance the majority of det1 developmental phenotypes,^6,19,25 indicating that DDB1A and DDB1B act antagonistically to DET1 during germination.

Given that there are at least 5 DDB1-CUL4 complexes that negatively regulate ABA signaling (DWA1/2, DWA3, ABD1, DDA1, ASG2),^20,22-24,30 one would expect loss of DDB1A to result in increased ABA signaling and therefore less germination. This is in fact what we observed in salt and mannitol conditions in both the det1 and wildtype backgrounds. The det1 ddb1a salt and mannitol germination phenotypes could be interpreted as being additive, in that det1 still results in increased germination even in the ddb1a background. This is in contrast to the det1 ABA sensitive germination phenotype, which is completely suppressed by ddb1a.²¹ Thus a fraction of the det1 stress resistant germination phenotype is DDB1A independent. In contrast, ddb1b mannitol sensitive germination is completely suppressed by det1, thus ddb1b mannitol sensitive germination is DET1 dependent.

Role of DDB2 in det1 salt/mannitol resistant germination

Germination in the ddb2 single mutant was delayed on salt containing media but not on mannitol or ABA containing media.²¹ Interestingly, ddb2 suppressed det1 germination phenotypes on all the different stress media, completely suppressing det1 ABA sensitive germination and partially suppressing det1 salt and mannitol resistant germination. This suggests that DDB2 is required for det1 germination phenotypes under stress conditions. ddb2 suppresses not only det1 germination phenotypes but also a number of other det1 phenotypes, including chlorophyll content, anthocyanin content, and adult phenotypes,¹⁹ indicating that DDB2 and DET1 act antagonistically. Although DDB2s primary role is in UV damaged DNA repair, it also seems to be involved in numerous other growth, developmental, and stress phenotypes in the det1 background. The effect of ddb2 on det1 phenotypes does not seem to be a general effect of lack of one CUL4-DDB1 complex on another, since dwa1 and dwa2 do not exhibit the degree of suppression of det1 phenotypes that ddb2 does.^19,21 Castells et al.¹⁸ suggest that DET1 is required for DDB2 degradation. Perhaps in det1 mutants there are developmental consequences of this excess DDB2, which are rescued by the ddb2 mutant. The basis of this interaction requires further investigation.

Role of COP1 in det1 salt/mannitol resistant germination

COP1 is required for the det1 early germination phenotype in control conditions as well as in salt and osmotic stress conditions. On ABA, both cop1 and det1 exhibited ABA sensitive germination, and germination was further impaired in the double mutant, suggesting that DET1 and COP1 act in the same pathway in response to ABA.²¹ In contrast, cop1 and det1 showed opposite phenotypes during salt/osmotic stress, where det1 showed resistant germination while cop1 was sensitive. These results indicate that COP1 and DET1 act antagonistically during seed germination in salt/osmotic stress conditions. The det1 cop1 double mutant exhibited less germination than either single mutant on ABA and mannitol (Fig. 7 and ²¹). This result suggests that although cop1 and det1 exhibit opposite phenotypes during germination under stress conditions, det1 still requires COP1 to exhibit the resistant germination phenotype. In addition, both COP1 and DET1 are required to execute wild type germination in control conditions.

Recently Yu et al.³² showed that COP1 negatively regulates salt inhibition of seed germination by post-translational regulation of HY5. They showed that salt inhibits COP1 nuclear localization in both light and dark and thus stabilizes HY5 accumulation in the nucleus. HY5 had previously been shown to be a positive regulator of ABI5 transcription and therefore a negative regulator of germination.³³ Salt inhibition of germination in cop1 mutants was also shown to be via HY5 and ABI5.³² Previous studies indicate that both COP1 and DET1 are involved in negative regulation of HY5 levels in the cell.¹¹ We have previously shown that det1 ABA sensitive germination requires HY5 and ABI5.²¹ However other studies in our lab indicate that det1 salt resistant germination requires HY5 but not ABI5 (Fernando et al. in preparation). How cop1 and det1, both acting via HY5, result in different germination phenotypes during salt stress is currently unclear.

In conclusion, DET1 has genetic interactions with components of distinct E3 ligase complexes during salt/osmotic stress inhibition of seed germination. The interaction of DET1 with DWA1/2 showed the opposite effect of that expected, in that dwa1/2 enhanced the det1 salt/mannitol resistant germination phenotype. ddb1a partially suppressed the det1 resistant germination phenotype, while ddb1b did not show a significant effect on this response. The ddb2 single mutant was sensitive to salt, and ddb2 partially suppressed det1 resistant germination on both salt and mannitol. cop1 and det1 exhibit opposite phenotypes on stress media, but the absence of both COP1 and DET1 results in minimal germination, suggesting that COP1 and DET1 play important roles in stress signaling during seed germination. This study provides additional evidence of interactions between components of DDB1-CUL4 E3 ligase complexes.^18,19,21

Materials and methods

Plant materials

All Arabidopsis thaliana mutants in this study are in the Columbia-0 ecotype. det1-1, cop1-4, ddb1a, ddb1b, ddb2 and their respective det1 double mutants are as previously described.^{6,19,25,27,34} dwa1 and dwa2 and their respective det1 double mutants are as described in Lee et al.²⁰ and Fernando and Schroeder.²¹

Seed germination assays

Sterilized Arabidopsis seeds from the above genotypes were sown on Linsmaier and Skoog (LS) media (Caisson), with 0.86% Phytoblend (Caisson) and 0% sucrose, supplemented with 100 or 200 mM NaCl (Fisher Scientific) or 200 or 400 mM Mannitol (Fisher Scientific). Plates were stratified at 4°C for 2 d then transferred to 20°C and long day conditions (16 h light/8 h dark) supplied by fluorescent bulbs (100 μM photons m⁻² s⁻¹). Seed germination was scored every 24 hours as percentage of seeds with radical emergence for up to 5 d.³⁵ Germination assays for cop1 det1 and ddb1a det1 were done by visibly identifying double mutants in a population of segregating cop1 det1/+ and ddb1a det1/+ heterozygotes, because the doubles are lethal and infertile, respectively.^6,26,27 Specifically, plates were scanned and germination date of every seed monitored using a color coding system. Upon cotyledon emergence, distinctive purple cotyledons were used to identify the double mutants.

Statistical analysis

Each experiment included 2 replicates and every experiment was repeated at least 2 times. The results of a single representative experiment are presented here. Results are means ± SE compared using 2-tailed student t-test. P ≤ 0.05 was considered to be statistically significant.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) to D.F.S. and a University of Manitoba Faculty of Science Graduate Scholarship to V.C.D.F.