- © 2019 American Society of Plant Biologists. All rights reserved.
Abstract
The control of seed dormancy by ethylene has been well studied, but the underlying molecular mechanisms are not fully understood. Here, we report the characterization of the Arabidopsis (Arabidopsis thaliana) mutant reduced dormancy 3 (rdo3) and the cloning of the underlying gene. We demonstrate that rdo3 is a loss-of-function mutant of the ethylene receptor ETHYLENE RESPONSE1 (ETR1). ETR1 controls seed dormancy partially through the DELAY OF GERMINATION1 (DOG1) pathway. Molecular and genetic analyses demonstrated that ETHYLENE RESPONSE FACTOR12 (ERF12) is involved in the regulation of seed dormancy downstream of ETR1. ERF12 interacts with TOPLESS (TPL) and genetically requires TPL to function. ERF12 and TPL repress the expression of DOG1 by occupying its promoter. Thus, we identified the dormancy pathway ETR1-ERF12-TPL-DOG1 and provide mechanistic insights into the regulation of seed dormancy by linking the ethylene and DOG1 pathways.
INTRODUCTION
Dormancy is an adaptive trait that helps plants optimize seed germination at the proper time, ensuring the completion of the plant life cycle within a suitable growing season. This trait is highly important for plant survival and crop yields. Factors inhibiting germination reside within the embryo and/or the surrounding structures, such as the seed coat and endosperm (Bewley and Black, 1994; Hilhorst, 2007). Dormancy is a quantitative trait whose depth and duration are regulated by genetic and environmental factors (Finkelstein et al., 2008; Penfield, 2017). The balance between abscisic acid (ABA) and gibberellins (GAs) plays a crucial role in the regulation of seed dormancy and germination. ABA plays a key role in the induction and maintenance of dormancy, and GAs are involved in dormancy release and germination (Finkelstein et al., 2008; Nambara et al., 2010; Graeber et al., 2012). In addition to ABA and GAs, other hormones such as ethylene play important roles in the control of seed dormancy and germination (Linkies and Leubner-Metzger, 2012; Arc et al., 2013; Corbineau et al., 2014).
Physiological studies have shown that exogenous ethylene reduces the level of seed dormancy and improves seed germination. Treatment with ethephon and 1-aminocyclopropane-1-carboxylic acid (ACC), two ethylene suppliers, breaks seed dormancy in Amaranthus retroflexus (Kepczynski et al., 1996). A high concentration of ethylene is required to break the physiological dormancy of fresh Townsville stylo (Stylosanthes humilis) seeds (Ribeiro and Barros, 2006); however, low levels of ethylene are able to stimulate Rhus coriaria seed germination (Ne’Eman et al., 1999). ACC treatment also improves germination speed in Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum) seeds (Siriwitayawan et al., 2003). These physiological findings indicate that ethylene plays a pivotal role in seed dormancy release and germination.
Genetic analysis of Arabidopsis mutant lines with altered ethylene biosynthesis and/or signaling revealed the molecular regulation of dormancy by ethylene. ACC synthase (ACS) and ACC oxidase are two key enzymes in the ethylene biosynthetic pathway (Adams and Yang, 1979; Linkies and Leubner-Metzger, 2012). The Arabidopsis loss-of-function ACC oxidase2 mutant exhibits impaired ACC-mediated reversion of the ABA-induced inhibition of seed germination (Linkies et al., 2009). The Arabidopsis ethylene-overproducer mutant eto3, an allele of ACS9 (Chae et al., 2003), shows decreased sensitivity to ABA during germination (Subbiah and Reddy, 2010). Another ethylene-overproducer mutant, eto1, shows slightly reduced seed dormancy (Cheng et al., 2009).
In Arabidopsis, ethylene responses are mediated by five membrane-associated receptors, including ETHYLENE RESPONSE1 (ETR1), ETHYLENE RESPONSE SENSOR1 (ERS1), ETR2, ERS2, and ETHYLENE INSENSITIVE4 (EIN4; Wang et al., 2002). These receptors modulate the activity of the rapidly accelerated fibrosarcoma-like kinase CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) to relieve its inhibition of EIN2. EIN2 activation triggers the activity of EIN3 and EIN3-like 1, which regulate the expression of ethylene response factors (ERFs; Riechmann et al., 2000). Seeds of the etr1 and ein2 mutants display enhanced primary dormancy compared with the wild type (Beaudoin et al., 2000; Chiwocha et al., 2005; Cheng et al., 2009), whereas the ctr1 mutant has a slightly enhanced rate of germination (Beaudoin et al., 2000; Cheng et al., 2009). ERF7-deficient RNA interference transgenic Arabidopsis lines show more sensitivity to ABA and increased seed dormancy compared with the wild type (Song et al., 2005). Overexpression of S. lycopersicum-ERF2 in transgenic tomato results in reduced seed dormancy (Pirrello et al., 2006). ERF1 promotes gibberellin biosynthesis to counter the inhibitory effects of ABA on germination in lettuce at high temperatures (Yoong et al., 2016). Ethylene might boost dormancy release and promote germination indirectly through interactions with GA and ABA (Ghassemian et al., 2000).
The Arabidopsis DELAY OF GERMINATION1 (DOG1) gene is a major regulator of seed dormancy (Bentsink et al., 2006). The accumulation of DOG1 protein in dry seeds is tightly correlated with the depth of dormancy, and the regulation of dormancy by DOG1 requires a functional ABA signaling pathway (Kendall et al., 2011; Nakabayashi et al., 2012). Two DOG1-interacting proteins, type 2C protein phosphatases ABA-HYPERSENSITIVE GERMINATION1 (AHG1) and AHG3, function as a convergence point of the ABA and DOG1 pathways (Née et al., 2017; Nishimura et al., 2018). DOG1 might also be involved in GA catabolism indirectly, since GIBBERELLIN2-OXIDASE6 expression showed a 10-fold decrease in dog1 mutants compared with the wild type (Kendall et al., 2011). In addition, DOG1 causes temperature-dependent alterations in seed GA metabolism by inhibiting the expression of GA-regulated genes encoding cell wall remodeling proteins (Graeber et al., 2014).
Four mutants with reduced dormancy (rdo) have been obtained in mutagenesis screens of the accession Landsberg erecta (Ler; Léon-Kloosterziel et al., 1996; Peeters et al., 2002). These mutants did not show altered ABA levels or altered sensitivity to ABA. The identification of the underlying genes of two of these mutants, RDO2 and RDO4/ HISTONE MONOUBIQUITINATION1 (HUB1), revealed that transcription elongation efficiency and histone H2B monoubiquitination are involved in the regulation of seed dormancy (Liu et al., 2007, 2011).
Here, we report the identification of the Arabidopsis rdo3 mutant and show that RDO3 encodes the ethylene receptor ETR1. Furthermore, we show that ERF12 plays a key role in the regulation of seed dormancy by interacting with TOPLESS (TPL) in the downstream pathway of ethylene signaling mediated by ETR1/RDO3. Moreover, DOG1 is a direct target of the ERF12-TPL repressor complex, thereby regulating seed dormancy mediated by the ethylene pathway.
RESULTS
RDO3 Encodes ETR1
The rdo3 mutant was originally isolated in a γ-irradiation mutagenesis screen of Ler based on its significantly reduced dormancy phenotype and was roughly mapped to chromosome 1 (Peeters et al., 2002). We first confirmed its reduced seed dormancy and its pleiotropic phenotypes, which were described as slightly darker leaves and a somewhat flattened top on inflorescence leaves by Peeters et al. (2002). We also noticed a slightly reduced plant height (Figure 1A; Supplemental Figure 1A). A segregating population from a cross between rdo3 and near-isogenic line LCN1-23 (Keurentjes et al., 2007) was used for fine mapping. The location of rdo3 was confined to a 50-kb region containing nine genes between markers T6JI9-1 and T27F4-2 (Supplemental Figure 1B). By sequencing these genes, ETR1 was identified as a candidate gene for RDO3 (Supplemental Figures 1B and 1C), because a 4-bp deletion was detected in the second exon of ETR1 in the rdo3 mutant (Supplemental Figure 1C). This gene had previously been identified as encoding an ethylene receptor (Schaller and Bleecker, 1995). The identity of RDO3 as the ETR1 gene was further confirmed by a complementation experiment. Two independent transformants (Pro35S:ETR1/rdo3-#1 and Pro35S:ETR1/rdo3-#2), which segregated for a single insertion event of the transgene, showed similar dormancy levels to those of the wild-type Ler plants (Figure 1B). The pleiotropic phenotypes of rdo3, including dwarfness and seed longevity, were also rescued, as shown in Figures 1A and 1C. Seeds of rdo3 showed enhanced resistance to the controlled deterioration test (CDT), but the complementation lines displayed the same sensitivity to a CDT treatment as the wild type (Figure 1C).
Complementation of rdo3 and Germination Phenotypes of etr1 Mutants.
(A) Visible phenotypes of rdo3 and the complementation lines 20 d after flowering.
(B) Germination rates after different periods of dry storage for rdo3, Ler, and two complementation lines containing a Pro35S:ETR1 insertion in the rdo3 background. Shown are means ± se of three independent batches of seeds per genotype.
(C) Seed longevity phenotypes of rdo3 and two complementation lines after a CDT. Shown are means ± se of three independent batches of seeds per genotype.
(D) Seed dormancy phenotypes of etr1 mutants in the Col background. Freshly harvested seeds were used, and radicle emergence was scored 7 d after sowing. Shown are means ± se of three independent batches of seeds per genotype.
(E) Phenotypes of seedlings after ACC (10 μM) treatment for rdo3 and the complementation lines.
(F) Hypocotyl growth in response to 10 μM ACC. The hypocotyl lengths of different lines are means ± se (n = 30). Asterisks in (C), (D), and (F) indicate a significant difference between the wild type and the mutant based on Student’s t test (*P < 0.05, **P < 0.01).
The deletion of four bp in the second exon causes a frameshift and an early stop codon after the mutation. Therefore, rdo3 represents a loss-of-function mutant of ETR1 in the Ler background. We also analyzed two loss-of-function mutants of ETR1 in the Columbia-0 (Col-0) background, that is, etr1-6 and etr1-7 (Hua and Meyerowitz, 1998). Both mutants showed reduced seed dormancy similar to that of rdo3 (Figure 1D), indicating that loss-of-function mutants of ETR1 cause reduced dormancy, unlike the gain-of-function mutant etr1-2, which shows increased seed dormancy (Chiwocha et al., 2005). Moreover, rdo3 showed enhanced sensitivity to ethylene (Figures 1E and 1F), which is consistent with the ethylene phenotypes of etr1-6 and etr1-7 (Hua and Meyerowitz, 1998).
ETR1 Requires DOG1 to Regulate Seed Dormancy
DOG1 is a key, specific regulator of seed dormancy (Bentsink et al., 2006). To explore the relationship between ETR1 and DOG1 in regulating seed dormancy, we crossed the dog1-2 mutant with etr1-2, which shows increased seed dormancy (Chiwocha et al., 2005). The double mutant etr1-2 dog1-2 showed a similarly low dormancy level to that of dog1-2 (Figure 2A). In addition, we crossed the rdo3 mutant with NILDOG1, an NIL containing a strong DOG1 Cape Verde Islands (Cvi) allele in the Ler background (Alonso-Blanco et al., 2003). The homozygous rdo3 mutant in the NILDOG1 background showed a higher dormancy level than Ler (Figure 2B). To investigate the relationship between ETR1 and DOG1, we also examined DOG1 expression in these lines. DOG1 showed strongly reduced expression in rdo3, the highest level of expression in NILDOG1, and a moderate level of expression in rdo3/NILDOG1 (Figure 2C); these results are consistent with seed dormancy levels of these lines. These results indicate that the regulation of seed dormancy by ETR1 requires DOG1, and they suggest that DOG1 might function downstream of ETR1.
ETR1/RDO3 Requires DOG1 to Regulate Seed Dormancy.
(A) Seed dormancy phenotypes of etr1-2, dog1-2, and the double mutant etr1-2 dog1-2.
(B) Seed dormancy phenotypes of rdo3, NILDOG1, and NILDOG1/rdo3. NILDOG1 is an NIL containing the Cvi-DOG1 fragment in the Ler background. Shown are means ± se of three independent batches of seeds per genotype.
(C) DOG1 expression in Ler, rdo3, NILDOG1, and NILDOG1/rdo3 during seed maturation. Siliques of plants at 6, 9, 12, 15, and 18 d after pollination were collected for RNA extraction. Shown are means ± se of three independent batches of siliques per genotype. Asterisks indicate significant differences from the wild type based on Student’s t test (*P < 0.05, **P < 0.01).
β-Glucuronidase (GUS) staining of DOG1 promoter (ProDOG1) ProDOG1:GUS transgenic lines show that DOG1 was expressed in the hypocotyls and cotyledons of imbibed seeds and early seedlings (Supplemental Figure 2). Therefore, we analyzed the triple responses of dog1-2, NILDOG1/rdo3, and etr1-2 dog1-2 as a measure of their sensitivity to ethylene. As shown in Supplemental Figures 3A and 3B, the level of ethylene sensitivity of NILDOG1/rdo3 was between that of rdo3 and NILDOG1 and similar to that of the wild-type Ler. Interestingly, compared with etr1-2, the double mutant etr1-2 dog1-2 showed the same sensitivity to ethylene, even though dog1-2 is more sensitive to ethylene than Col (Supplemental Figures 3C and 3D). These results indicate that the relationship between ETR1 and DOG1 in the regulation of seed dormancy is different from that in the regulation of ethylene sensitivity.
ERF12 Regulates Seed Dormancy Downstream of ETR1
To explore the role of ETR1 in the regulation of seed dormancy, we analyzed previously produced transcriptomic data from siliques of rdo3 and the wild-type Ler 18 to 19 d after pollination (Gene Expression Omnibus accession number GSE28446; Liu et al., 2011). At this stage, 113 genes were upregulated and 40 genes were downregulated in rdo3 compared with Ler (Supplemental Data Sets 1 and 2). Most of the upregulated genes are involved in stress-related and ethylene pathways, and some are involved in dormancy (Supplemental Figure 4). The downregulated genes play roles in cell wall modification, ethylene, and other hormone pathways (Supplemental Figure 4). ERF12, which encodes an ERF, showed significantly increased transcript levels in rdo3. The expression of ERF12 in rdo3 was 1.96 times higher than in the wild type (Supplemental Data Set 1).
Reverse transcription quantitative PCR (RT-qPCR) analysis confirmed the increased expression level of ERF12 in rdo3 (Figure 3A; Supplemental Data Set 1). By contrast, ERF12 transcript levels were similar to that of the wild type in the complementation lines (Figure 3A). We produced plants expressing ERF12 ectopically by placing ERF12 cDNA under the control of the 12S promoter, which confers seed-specific expression (Wang et al., 2016). Three independent single insertion lines showed reduced seed dormancy compared with the wild-type Col-0 (Figure 3B). These results indicate that ERF12 is a negative regulator of seed dormancy, which is consistent with the high ERF12 transcript levels in the nondormant rdo3 mutant. The erf12 single mutant did not show an altered seed dormancy phenotype. ERF12 is homologous to ERF3; we generated the erf3 erf12 double mutant, which showed slightly increased seed dormancy compared with the wild type (Figure 3C; Supplemental Figures 5A and 5B). This result confirms the notion that ERF12 and its homologous gene ERF3 play negative roles in the regulation of seed dormancy. Moreover, reduced seed dormancy was also observed when ERF12 was expressed under the control of the seed-specific ProDOG1 (Supplemental Figure 5C; Nakabayashi et al., 2012; Graeber et al., 2014; Née et al., 2017).
ERF12 Regulates Seed Dormancy via the ETR1 Pathway.
(A) Relative expression levels of ERF12 in freshly harvested seeds of rdo3 and the complementation lines. ACTIN8 was used as an internal control. Three independent experiments were performed with similar results. Values are means ± se (n = 3) from one representative experiment with three technical repeats. **P < 0.01, based on Student’s t test.
(B) to (D) Germination rates after different periods of dry storage for the Pro12S:ERF12 OE lines and the wild-type Col-0 (B); erf3, erf12, the erf3 erf12 double mutant, and Col-0 (C); etr1-2, transgenic line Pro12S:ERF12, containing Pro12S:ERF12 in the etr1-2 background, and Col-0 (D). Shown are means ± se of three independent batches of seeds per genotype.
To uncover the genetic relationship between ERF12 and ETR1, we crossed etr1-2 with the transgenic Pro12S:ERF12 line and obtained homozygous plants. The etr1-2/Pro12S:ERF12 seeds exhibited a significantly reduced rate of dormancy similar to the phenotype of Pro12S:ERF12 (Figure 3D), suggesting that ERF12 functions downstream of ETR1 in the regulation of seed dormancy. We also tested the sensitivity of Pro12S:ERF12 lines to GA and ABA by treating seed with the GA biosynthesis inhibitor paclobutrazol (PAC) and ABA (Supplemental Figure 6). The seed germination rate of the ERF12 overexpression (OE) lines was similar to that of the wild type in response to GA or ABA, suggesting that ERF12 regulates seed dormancy through pathways that are independent of GA and ABA.
ERF12 Interacts with the Corepressor TPL in the Nucleus
ERF12 physically interacts with the corepressor TPL, as revealed in a high-throughput yeast two-hybrid screening (Causier et al., 2012). We confirmed this finding by yeast two-hybrid analysis (Figure 4A) and using a chromogenic marker X-α-Gal assay (Figure 4A, Supplemental Figure 7). As the N terminus of TPL contains a protein interaction domain (Szemenyei et al., 2008), we also confirmed the protein interaction between the TPL N-terminal fragment and ERF12 (Figure 4B). When a mutated ERF-associated amphiphilic repression (EAR) motif [L/FDLNL/F(X)P] was introduced into ERF12, ERF12 and TPL no longer interacted (Supplemental Figure 7), indicating that the EAR motif is crucial for the interaction of ERF12 and TPL.
ERF12 Interacts with TPL in the Nucleus.
(A) Interaction of TPL and ERF12 in a yeast two-hybrid assay. Yeast cells were grown on synthetic dropout/−Leu/−Trp/−Ade/−His medium, and the interaction of ERF12 with TPL was assayed based on the expression of the reporter gene LacZ (X-Gal). RDO4-AD with RDO4-BD and AD with BD were used as positive and negative controls, respectively. Yeast cells diluted 1000 times were used for X-Gal staining. Ade, adenine.
(B) Interaction between the N-terminal part of TPL (TPL-N, 200 amino acids) and ERF12 in yeast. Yeast cells diluted 1000 times were used for X-Gal staining.
(C) Interaction of ERF12 and TPL, as determined by BiFC assay. Pro35S:nYFP-TPL was cotransformed with Pro35S:cYFP-ERF12 (middle panel) and Pro35S:nYFP-TPL-N with Pro35S:cYFP-ERF12 (bottom) into Arabidopsis protoplasts. ERF022, a member of the ERF family that lacks an EAR motif, was used as a negative control. Pro35S:nYFP-TPL and Pro35S:cYFP-ERF022 were cotransformed into Arabidopsis protoplasts (top). DAPI, 4′,6-diamidino-2-phenylindole. Bars = 10 μm.
To verify the interaction between ERF12 and TPL in planta, we performed bimolecular fluorescence complementation (BiFC) in Arabidopsis protoplasts as described previously (Abel and Theologis, 1994; Kovtun et al., 2000; Hwang and Sheen, 2001). Yellow fluorescent protein (YFP) fluorescence signals were observed in Arabidopsis protoplasts coexpressing the constructs Pro35S:nYFP-TPL and Pro35S:cYFP-ERF12 and the constructs Pro35S:nYFP-TPL-N and Pro35S:cYFP-ERF12 (Figure 4C), indicating that TPL can interact with ERF12 in vivo. When we used ERF022, which also belongs to ERF family but lacks an EAR motif, as does the fusion protein used to perform BiFC, YFP fluorescence was not observed (Figure 4C), which further demonstrates the important role of the EAR motif in the interaction of ERF12 with TPL.
Therefore, we reasoned that TPL might also be involved in regulating seed dormancy. Indeed, tpl-1, a dominant negative mutant of TPL (Long et al., 2002, 2006), showed an increased seed dormancy phenotype (Figure 5A), which is consistent with the phenotype of erf3 erf12. Two T-DNA insertion mutants, tpl-9 (SALK_034518) and tpl-10 (SALK_097230), also showed slightly increased seed dormancy (Supplemental Figures 8A and 8B). Accordingly, TPL OE lines driven by the 12S promoter showed reduced seed dormancy (Figure 5B). These results indicate that TPL indeed plays a role in seed dormancy. Genetic analysis showed that tpl-9 is completely epistatic to Pro12S:ERF12 (Figure 5C), suggesting that the promotion of seed germination by ERF12 requires TPL. Moreover, the tpl-9 dog1-2 double mutant showed reduced seed dormancy similar to dog1-2 (Figure 5D), suggesting that the regulation of seed dormancy by TPL depends on DOG1.
TPL Negatively Regulates Seed Dormancy.
(A) Germination rates after different periods of dry storage for tpl-1 and the wild-type Ler.
(B) Germination rates after different periods of dry storage for Pro12S:TPL OE lines and the wild-type Col-0.
(C) Seed dormancy phenotypes of Col-0, tpl-9, Pro12S:ERF12, and tpl-9/Pro12S:ERF12.
(D) Seed dormancy phenotypes of Col-0, dog1-2, tpl-9, and tpl-9 dog1-2.
Shown are means ± se of three independent batches of seeds per genotype.
ERF12 and TPL Promote Seed Germination by Repressing the DOG1 Pathway
ERF12 is a member of the ERF subfamily B-1 belonging to the ERF/APETALA2 transcription factor family, which regulate DRE-mediated transcription of cold- and/or drought-inducible genes by acting as repressors (Ohta et al., 2001). A putative dehydration-responsive element/C-repeat (DRE/CRT) element was found (−646 bp) in the promoter of DOG1, indicating the possible binding of ERF12 to the promoter of DOG1. To determine whether DOG1 is a target of ERF12, we crossed an ERF12 OE plant with a transgenic plant harboring the ProDOG1:GUS reporter construct; this construct was described previously (Nakabayashi et al., 2012). Homozygous embryos from the F3 generation lines were collected for GUS staining (Figure 6A), and fresh seeds were tested for GUS expression by RT-qPCR (Figure 6B). Compared with ProDOG1:GUS in the Col-0 background, the expression level of GUS was reduced in the ERF12 OE background (Figure 6B). This result indicates that the overexpression of ERF12 downregulates DOG1 promoter activity. The expression of DOG1 was also downregulated in the ERF12 OE lines, which is consistent with their reduced GUS staining (Figures 6A and 6C). Furthermore, RT-qPCR showed that DOG1 was downregulated in the TPL OE lines and upregulated in the tpl and erf mutants (Figure 6D). The overexpression of ERF12 and TPL in the respective transgenic lines was confirmed by RT-qPCR (Supplemental Figure 9). These results suggest that DOG1 is involved in the regulation of seed dormancy mediated by ERF12 and TPL.
ERF12 Inhibits Expression of DOG1.
(A) GUS staining of freshly harvested seeds of Col-0, Pro12S:ERF12, ProDOG1:GUS, and the double transgenic plant ProDOG1:GUS/Pro12S:ERF12. Seed coats and endosperm were removed from more than 20 freshly harvested seeds. The embryos were collected and incubated in GUS staining buffer for 24 h, followed by observation and under a microscope. Bar in the top panel = 100 μm; bar in the bottom panel = 2 mm. WT, wild type.
(B) Relative expression levels of GUS in ProDOG1:GUS and homozygous progeny of ProDOG1:GUS/Pro12S:ERF12. ACTIN8 was used as an internal control. Data are shown as means ± sd, different letters denote significant differences (n = 3, one-way analysis of variance test, P < 0.05).
(C) and (D) Relative expression level of DOG1 in Pro12S:ERF12 (C), Pro12S:TPL, the erf3 erf12 double mutant, and tpl-1 (D). The total RNA was extracted from freshly harvested seeds. ACTIN8 was used as an internal control. Three independent experiments were performed with similar results. Values are means ± se (n = 3) from one representative experiment with three technical repeats. *P < 0.05, **P < 0.01, based on Student’s t test.
We analyzed the physical interaction between ERF12 and the DRE/CRT element in the DOG1 promoter by electrophoretic mobility shift assay (EMSA). Adding ERF12 protein to biotin-labeled DRE/CRT elements caused reduced gel migration, indicating the formation of a DNA–protein complex (Figure 7A). The use of increased amounts of ERF12 protein led to enhanced amounts of the slower migrating band. However, when biotin-unlabeled DRE/CRT probe was added as a competitor, the amount of the slow-migrating DNA–protein band decreased with increasing concentration of competitor. Furthermore, we generated Pro12S:YFP-ERF12 transgenic lines, which showed reduced seed dormancy (Supplemental Figure 10).
ERF12 Binds Directly to the DRE/CRT Element in the DOG1 Promoter.
(A) EMSA of the oligonucleotide DRE/CRT with GST-tagged ERF12 protein. The 22-bp wild-type DRE/CRT fragment labeled with biotin probe was shifted by the GST-ERF12 protein, but this shift disappeared when unlabeled probe was added. The arrow shows the shifted band; free probe is distributed at the bottom. Biotin-labeled probe was used at a final concentration of 20 nM.
(B) Schematic diagram of the DOG1 promoter structure. The dotted line indicates the ∼2000-bp promoter sequence, and the black line between the vertical dashes represents the first 500 bp of the open reading frame. The relative positions of the PCR-amplified fragments (P1 to P5) for each tested region are depicted below the gene structure. P6 in a 211-bp fragment located in the untranslated region of AT5G45820, which is located 4397 bp away from ATG of DOG1 (AT5G45830); this fragment was used as the negative control. Bar represents 500 bp.
(C) ChIP-qPCR analysis of YFP-ERF12 levels on DOG1. Immunoprecipitates were obtained from freshly harvested seeds of Col-0 and Pro12S:YFP-ERF12 using YFP antibody. ACTIN8 was used as a negative control. The experiment was repeated three times with independent samples. The numbers on the x axis represent the PCR-amplified sites described in (B). **P < 0.01, based on Student’s t test.
(D) Relative activity of the ProDOG1:LUC reporter in Arabidopsis protoplasts cotransformed with the indicated effector constructs. M represents a mutated DRE/CRT element (GGC to TTC), which was introduced into ProDOG1:LUC. The activity of the LUC reporter driven by the DOG1 promoter was measured by assaying LUC/REN ratios × 1000. Data are shown as means ± sd, different letters denote significant differences (n = 3, one-way analysis of variance test, P < 0.05).
We performed chromatin immunoprecipitation (ChIP) experiments to investigate the interaction between ERF12 and the DOG1 promoter using an anti-YFP antibody. Six DNA fragments spanning the putative binding site in the DOG1 promoter (as shown in Figure 7B) were analyzed by RT-qPCR. Relative fold enrichment was calculated by normalizing the value of immunoprecipitated anti-YFP fragments to that of the control sample lacking antibody. Fragment P3, which contains the putative binding site with the G/ACCGAC sequence, showed the highest enrichment compared with the other fragments (Figure 7C), demonstrating a possible direct binding of ERF12 to this fragment in the promoter region of DOG1.
Next, to examine whether ERF12 and TPL repress DOG1 transcription, we transiently expressed ERF12 and/or TPL, together with the luciferase (LUC) reporter gene driven by the DOG1 promoter in Arabidopsis protoplasts. As shown in Figure 7D, ERF12 and TPL separately slightly inhibited LUC reporter gene expression. Remarkably, the coexpression ERF12 and TPL drastically suppressed the activity of the ProDOG1:LUC reporter, and this suppression recovered when the DRE/CRT element was mutated, suggesting that ERF12 and TPL act synergistically to bind to the promoter of DOG1 and suppress its activity via the DRE/CRT element. Finally, we generated the 5×DOG1 DRE/CRT-LUC reporter gene construct as described previously by adding five tandem copies of the DOG1 DRE/CTR sequence to the 35S promoter (Fujimoto et al., 2000; Song et al., 2005). Treatment with ERF12 resulted in a dramatically reduction in the expression of LUC, which was driven by 5×DOG1 DRE/CRT (Supplemental Figure 11). This observation supports the notion that ERF12 functions as a transcriptional repressor in Arabidopsis.
DISCUSSION
The phase from seed dormancy to germination is one of the major developmental transitions in the life cycle of plants. However, the control of germination by seed dormancy is still poorly understood at the molecular level. A mutagenesis screen for reduced dormancy in the Ler background yielded four mutants (Peeters et al., 2002). The underlying genes of two of these, hub1-2 and rdo2-1, were previously cloned (Liu et al., 2007, 2011). Here, we report the cloning and characterization of the underlying gene for the third mutant, rdo3. We demonstrate that RDO3 encodes ETR1, which regulates seed dormancy by a mechanism that integrates the ethylene response and DOG1 pathways.
RDO3 Encodes ETR1
The rdo3 mutant was selected in a γ-irradiation mutagenesis screen based on its dramatically reduced dormancy (Peeters et al., 2002). By combining precise mapping, sequencing, and complementation, we identified RDO3 as ETR1, which encodes an ethylene receptor. Sequencing revealed a 4-bp deletion in rdo3, which leads to a frameshift and early stop codon (Supplemental Figures 1B and 1C). The reduced seed dormancy and ACC hypersensitivity phenotypes of rdo3 were consistent with those of the loss-of-function mutants etr1-6 and etr1-7 in the Col background (Figures 1B, 1D to 1F; Hua and Meyerowitz, 1998). This observation indicates that loss of function of ETR1 causes reduced seed dormancy, and gain of function leads to increased seed dormancy (Figures 1B and 1D) (Chiwocha et al., 2005). Therefore, ETR1 plays a positive role in the establishment of seed dormancy. Five ethylene receptors have been identified in Arabidopsis: ETR1, ETR2, ERS1, ERS2, and EIN4 (Chang et al., 1993; Hua and Meyerowitz, 1998; Hua et al., 1998; Sakai et al., 1998). These receptors play overlapping roles in the control of many traits mediated by ethylene, but it was recently found that ETR1 and ETR2 play opposite roles in the control of seed germination under various inhibitory conditions such as high sodium chloride levels (Wilson et al., 2014; Bakshi et al., 2018), indicating that the roles of these receptors might be dependent or independent of ethylene signaling. It is not clear whether ETR1, ETR2, and other receptors play different roles in the regulation of seed dormancy; how the other receptors regulate seed dormancy should be investigated in the near future.
Seed longevity is a very important agronomic trait related to seed dormancy. We found that rdo3 had enhanced seed longevity compared with the wild type in a CDT (Figure 1C), which is consistent with the finding that seed longevity is negatively correlated with seed dormancy in Arabidopsis (Nguyen et al., 2012). However, mutants such as dog1, hub1, and snl1/snl2, which exhibit strongly reduced seed dormancy, show very poor seed longevity (Bentsink et al., 2006; Liu et al., 2007; Wang et al., 2013), which is different from rdo3. These results indicate that seed longevity and dormancy are independently regulated. The improved seed longevity of rdo3 might be related to its resistance to heat stress (Léon-Kloosterziel et al., 1996; Tamura et al., 2006).
ERF12 Is Involved in Regulating Seed Dormancy Mediated by ETR1
ETR1 is involved in seed dormancy by influencing ABA, auxin, cytokinin, and GA metabolic pathways (Chiwocha et al., 2005). Higher levels of ABA, auxin, cytokinin, and GA were found in etr1-2 seeds than in the wild type. However, the key molecular factors that function downstream of ETR1 are not clear. In this study, based on the results of phenotypic and genetic analysis of transgenic plants, we propose that ERF12 likely participates in the regulation of seed dormancy mediated by the ETR1 signaling pathway. Transgenic plants with enhanced ERF12 levels showed reduced seed dormancy consistent with that of rdo3 (Figures 3B; Supplemental Figure 5C). However, the erf12 T-DNA mutant had no obvious seed dormancy phenotype compared with the wild type (Figure 3C), which might be due to redundancy. ERF12 is a member of the ERF subfamily B-1, which includes 15 genes such as ERF3, ERF4, and ERF7 (Ohta et al., 2001; Nakano et al., 2006). Thus, we constructed the erf3 erf12 double mutant, which showed slightly increased dormancy (Figure 3C), indicating functional redundancy among the ERF members. To clarify the relationship between ERF12 and ETR1, we created an etr1-2/Pro12S:ERF12 homozygous line. This line showed a similar seed germination rate to that of Pro12S:ERF12 (Figure 3D), indicating that ERF12 functions downstream of ETR1 in the regulation of seed dormancy.
It is well established that seed dormancy is determined by the balance between ABA and GA, but the relationship between the ethylene pathway and ABA/GA signaling in seed dormancy is unclear. Although ABA levels were slightly different between rdo3 and the wild type, their levels of ABA sensitivity were similar. This finding indicates that an altered mode of ABA action is not the cause of the reduced dormancy in rdo3 (Peeters et al., 2002). We also tested the sensitivity of ERF12 transgenic lines to ABA and PAC, but did not observe any obvious phenotype (Supplemental Figure 6), which fits with the observations for rdo3. However, this result does not mean that ETR1 and ERF12 are not involved in ABA or GA pathways to regulate dormancy. The dog1 mutant also did not show obvious phenotypes in response to ABA treatment (Bentsink et al., 2006), but DOG1 functions by directly interacting with the ABA signaling proteins AHG1 and AHG3 (Née et al., 2017; Nishimura et al., 2018). Therefore, ETR1 and ERF12 might regulate seed dormancy through the DOG1 pathway.
It has not been clear how ethylene signaling mediated by ETR1 is transferred to ERF12. Some studies have indicated that ERF5, a member of the ERF B-3 subfamily, interacts with the kinases mitogen-activated protein kinase 3 (MPK3) and MPK6 in response to the pathogen defense reaction (Son et al., 2012), suggesting the possibility of an interaction between MPK family members and ERF12. Interestingly, transcriptome data and RT-qPCR analysis showed that MPK14 and mitogen-activated protein kinase kinase 9 (MKK9) were significantly upregulated in rdo3 (Supplemental Data Set 1; Supplemental Figure 4B), and their transgenic lines showed reduced seed dormancy (Supplemental Figures 5E and 5F). MKK9 is a member of the MPK kinase kinase family and has kinase activity. However, it is not known whether it binds directly to the receptor ETR1 to transmit a signal. A related study has shown that phosphorylation of EIN3 Thr174 by a mitogen-activated protein kinase cascade from MKK9 to MPK3/6 promotes the protein stability of EIN3. By contrast, phosphorylation of the Thr572 site accelerates the degradation of EIN3, possibly through an independent mitogen-activated protein kinase cascade involving CTR1 (Yoo et al., 2008). A weak interaction between MKK3 and MPK14 was reported using yeast two-hybrid assays, and MPK14 is activated by phosphorylation by MKK3 (Dóczi et al., 2007). Popescu et al. (2009) classified MKKs according to their target MPKs and found that MKK9 functions in a manner similar to MKK3 and MKK7, suggesting the possible interaction of MKK9 and MPK14. MPK14 might receive a signal from MKK9, but more evidence is needed to confirm a direct relationship between these proteins.
ERF12 Interacts with TPL to Regulate Seed Dormancy
ERF transcription factors are divided into two categories: transcriptional activation factors and inhibitory factors. ERF12 is a transcription inhibitor (Yang et al., 2005) containing an EAR motif that functions as a repression domain and is responsible for the interaction between transcription factors and TPL/TPL-related (TPR) corepressors (Ohta et al., 2001; Hiratsu et al., 2004; Szemenyei et al., 2008). The direct interaction between ERF12 and TPL was first demonstrated in a high-throughput yeast two-hybrid assay (Causier et al., 2012). In the current study, we confirmed the interaction between ERF12 and TPL using the chromogenic marker X-α-Gal assay in yeast two-hybrid analysis (Figures 4A and 4B; Supplemental Figure 7). Furthermore, we showed that ERF12 interacts with TPL in planta by BiFC (Figure 4B). Loss of function of TPL resulted in increased seed dormancy (Supplemental Figure 8B). Genetic analysis indicated that ERF12 requires TPL to regulate seed dormancy (Figure 5C), which is consistent with the interaction between ERF12 and TPL. The sensitivity of tpl mutants to PAC and ABA was not different from the wild type (Supplemental Figures 8D and 8E), suggesting that TPL might regulate seed dormancy through pathways independent of GA and ABA. TPL/TPR belongs to the Groucho/Tup1 corepressor family (Causier et al., 2012). These types of repressors do not bind to DNA directly; they must be recruited by a transcription factor to inhibit the expression of their target genes. These results suggest that ERF12 and TPL participate in the regulation of seed dormancy by functioning as a complex downstream of ETR1.
ERF12 Binds to the Promoter of DOG1 and Suppresses Its Expression
DOG1 was identified as a major quantitative trait locus controlling seed dormancy (Bentsink et al., 2006). Its expression is seed specific and shows a strong peak during seed dormancy establishment (Nakabayashi et al., 2015). DOG1 mRNA and protein levels are tightly correlated with the strength of seed dormancy (Bentsink et al., 2006; Nakabayashi et al., 2012; Fedak et al., 2016). Therefore, we measured the expression level of DOG1 by RT-qPCR and found that DOG1 displayed lower transcript levels in rdo3, Pro12S:ERF12, and Pro12S:TPL transgenic lines and higher levels in the erf3 erf12 double mutant and tpl-1 compared with the wild type (Figures 2 and 6; Supplemental Figure 4B). This indicates that DOG1 may be regulated by the ethylene pathway to control seed dormancy. The triple response test further showed that dog1-2 is more sensitive to ACC (Supplemental Figures 3C and 3D), implying that DOG1 plays a role in ethylene signaling since DOG1 protein is stable during seed imbibition (Nakabayashi et al., 2012) and DOG1 was expressed in the hypocotyls and cotyledons of imbibed seeds and early seedlings (Supplemental Figure 2). Moreover, genetic analysis demonstrated that DOG1 is required for etr1-2 to promote seed dormancy establishment (Figure 2A), and a strong allele of DOG1 from the Cvi accession rescued the seed dormancy of rdo3 (Figure 2B), suggesting that DOG1 functions downstream of ETR1.
We demonstrated that ERF12 is also involved in seed dormancy downstream of ETR1, which motivated us to study the relationship between ERF12 and DOG1. An analysis of the DOG1 promoter revealed a DRE/CRT (5′-RCCGAC-3′) element 646 bp upstream of the start codon of DOG1, which can be bound by ERF subfamily proteins (Hao et al., 1998; Xu et al., 2007). A previous study reported that ERF11, which also belongs to the ERF B-1 subfamily, binds to DRE regulatory regions of ACS genes and represses their transcription (Li et al., 2011). Furthermore, ERF53, a member of the ERF A-6 subfamily, binds to both GCC-box and DRE/CRT (5′-RCCGAC-3′) elements (Sakuma et al., 2002; Gong et al., 2008). ERF71 also binds to GCC-box or DRE/CRT elements to regulate root development and root cell elongation (Lee et al., 2015). Therefore, we speculate that ERF12 might bind to a DRE/CRT element to regulate DOG1 transcription.
In this study, we obtained several pieces of evidence to support this hypothesis. First, we showed that GUS staining signals and GUS expression were reduced in the ProDOG1:GUS/Pro12S:ERF12 homozygous lines (Figures 6A and 6B). Second, an EMSA showed that ERF12 directly binds to the DOG1 promoter fragment harboring a DRE/CRT element (Figure 7A). Third, binding of ERF12-YFP to a DRE/CRT element was demonstrated in vivo by ChIP-qPCR (Figures 7B and 7C). Finally, a transient transcriptional activity assay showed that coexpression of ERF12 and TPL significantly suppressed the activity of the ProDOG1:LUC reporter and that this suppression recovered when the DRE/CRT element was mutated (Figure 7D). These results indicate that DOG1 could be a direct target of the ERF12 transcription factor, suggesting that DOG1 partially takes part in regulating seed dormancy mediated by the ethylene pathway. Thus, ERF12 recruits TPL/TPR to the promoter of DOG1 to repress its transcription. We also found that DOG1 expression was downregulated in Pro12S:TPL lines and upregulated in tpl-1 (Figure 6D), implying that DOG1 plays a role in the regulation of seed dormancy mediated by TPL. The dehydration-responsive element binding proteins (DREB)/C-repeat (CRT) binding factors (CBF)-DRE/CRT regulon is associated with drought and cold responses (Sakuma et al., 2002). Here, we found that the ERF12-DRE/CRT regulon is associated with seed dormancy and might also be related to seed maturation, since DOG1 has effects on seed maturation (Dekkers et al., 2016).
Overall, in this study, we identified RDO3 as ETR1 and showed that ERF12 functions downstream of ETR1 to regulate seed dormancy. MPK14 and MKK9 might take part in the signal transduction pathway in which a signal is transmitted to ERF12. ERF12 forms a repressor complex with TPL and binds to the DOG1 promoter, thereby inhibiting its expression and regulating seed dormancy (Figure 8). ERF12 could represent a link between ethylene and the DOG1 pathway in the regulation of seed dormancy in Arabidopsis. Since ETR1 is an ethylene receptor, it is located at the beginning of the ethylene signal transduction pathway, and the crosstalk between the ethylene pathway and other pathways occurs via multiple layers with kinases and other regulators (Linkies and Leubner-Metzger, 2012; Arc et al., 2013; Corbineau et al., 2014). Therefore, we cannot exclude the possibility that ETR1 also regulates seed dormancy by linking with other pathways. This notion requires further study. However, our findings uncovered at least part of the molecular mechanism by which ethylene regulates seed dormancy through the ETR1/RDO3-ERF12-TPL-DOG1 module, which provides the possibility for breeders to control preharvest sprouting in crops in the future.
Hypothetical Model for the Regulation of Seed Dormancy Mediated by ETR1/RDO3.
ERF12 functions downstream of ETR1/RDO3 in the ethylene pathway. MPK14 and MKK9 might take part in this signal transduction pathway via an unknown mechanism. ERF12 recruits TPL to form a repressor complex and binds to the DOG1 promoter, thereby inhibiting the expression of DOG1 and leading to reduced seed dormancy. ER, endoplasmic reticulum.
METHODS
Plant Materials and Growth Conditions
The Arabidopsis (Arabidopsis thaliana) erf3 (Salk_200697), erf12 (Sail_873_D11), tpl-9 (Salk_034518), tpl-10 (Salk_097230), dog1-2 (Bentsink et al., 2006), etr1-2, etr1-6, and etr1-7 (Hua and Meyerowitz, 1998) mutants, Pro12S:ERF12, Pro12S:YFP-ERF12, ProDOG1:ERF12, Pro12S:TPL, and ProDOG1:GUS are all in the Col background. The rdo3 mutant, tpl-1, and NILDOG1 are in the Ler background. T-DNA insertion lines Salk_2009697, Sail_873_D11, Salk_034518, and Salk_097230 were obtained from the Arabidopsis Biological Resources Center; homozygous individuals were identified by PCR-based screening. Gene-specific primers, selected from the SALK SIGnAL database, were used in combination with T-DNA left border primers. RT-PCR with RNA isolated from leaves was performed to confirm the homozygous knockout lines. PCR was performed on a Biometra instrument with 35 cycles for ACTIN8, ERF3, ERF12, and TPL dependent on the primers’ annealing temperature. Primers used for PCR are listed in Supplemental Data Set 3. Double mutant and hybrid progeny (NILDOG1/rdo3, etr1-2 dog1-2, etr1-2/Pro12S:ERF12, erf3 erf12, tpl-9/Pro12S:ERF12, tpl-9 dog1-2) were obtained by genetic crossing, and homozygous lines were selected. Plants were grown in the greenhouse with a 16-h-light, under 80 to 90 μmol m−2 s−1 white light intensity/8-h-dark cycle at 22 ± 2°C, and seeds for dormancy phenotype analysis were harvested from the same experiment and stored under dry conditions at 25°C. The seeds were sown on half-strength Murashige and Skoog (1/2MS) medium after sterilization with 10% (w/v) sodium hypochlorite and washed with sterilized water at least three times. The plates were incubated at 4°C in darkness for 3 d to stratify and then transferred to a climate chamber with a cycle of 12 h of light, which was 80 to 90 μmol m−2 s−1 white light intensity, and 12 h dark at 25°C.
Dormancy and Germination Assays
The seed dormancy assay was performed as described by Alonso-Blanco et al. (2003). Freshly harvested seeds were plated on filter paper moistened with water in 5-cm Petri dishes. At least 200 seeds from independent seed batches were used for each genotype. The germination rate was determined after 7 d of incubation in a climate room, with a cycle of 12-h-light/12-h-dark at 25°C. For the seed germination sensitivity assay, the filter paper was soaked with mock solutions or solutions supplemented with PAC (Sigma) or ABA (Sigma). Seeds for each germination assay were harvested at the same time and stored under the same conditions.
Controlled deterioration test
Seeds without dormancy were used to perform the CDT treatment. A closed container with saturated potassium chloride solution to provide 82% RH was used to store the seeds. Seeds were equilibrated for 4 d at 25°C in the dark, transferred to 82% RH, and incubated at 43 ± 0.5°C in the dark for 2 d. After drying at room temperature for 1 d, the seeds were tested for germination (Bentsink et al., 2000).
ACC Treatment
Seeds were treated as described previously (An et al., 2010), with some modifications. Seeds were plated on 1/2MS medium, pH 5.8, containing 0.6% (w/v) agar with 10 μM ACC (Sigma Aldrich), which is a precursor of ethylene. The plates were incubated at 4°C in the dark for 3 d and transferred to a chamber set at 25°C in the dark. Seedling hypocotyls were measured as described by Hall et al. (1999). Each genotype was repeated at least three times with seeds harvested from plants grown simultaneously in the same tray and stored under identical conditions as a biological repeat.
Plasmid Construction and Plant Transformation
Total RNA was extracted from young Col-0 leaves using an RNeasy kit (Qiagen). First-strand cDNA was synthesized with SuperScript II reverse transcriptase (Invitrogen). The TPL construct pENTR233-TPL was ordered from Arabidopsis Biological Resources Center. ERF12 cDNA was amplified with Phusion DNA polymerase (NEB). All primers used in this study are listed in Supplemental Data Set 3. Pro12S:ERF12 (or TPL) was constructed by inserting ERF12 or TPL cDNA into the 12S:pLEELA vector as described previously (Wang et al., 2016). The constructs were transformed into the wild-type plants. All binary constructs were introduced by electroporation into Agrobacterium tumefaciens strain GV3101 carrying the helper plasmid pMP90RK, which was subsequently used for transformation by the floral dip method (Clough and Bent, 1998). Transformants were selected based on their ability to survive in 1/2MS medium with 10 mg/L dl-phosphinothricin. To select transgenic plants with a single T-DNA insertion, transformants were selected on 1/2MS medium with dl-phosphinothricin, and lines with 3:1 (resistant plants:sensitive plants) progeny segregation were selected. The homozygous single insertion lines were used for phenotypic analysis. The sequences of all constructs were confirmed by sequencing.
RNA Isolation and RT-qPCR Analysis
Total RNA was extracted from leaves of T-DNA insertion lines and the wild-type plants using TRIzol (Invitrogen) following the manufacturer’s protocol. Total RNA was extracted from freshly harvested seeds or imbibed seeds using an RNAqueous kit with plant RNA isolation aid (Ambion) and purified using an RNeasy mini kit (Qiagen). cDNA was synthesized with a QuantiTect reverse transcription kit (Qiagen). RT-qPCR was performed using SYBR Premix Ex Taq (Takara) by an Eppendorf instrument with 35 cycles. The relative expression level of each gene was calculated using a standard curve with a serial dilution of plasmid of known concentration, and ACTIN8 was used as an internal control (Chua et al., 2005; Czechowski et al., 2005; Graeber et al., 2011). All RT-qPCR analyses were performed with at least three biological replicates. Primers are listed in Supplemental Data Set 3.
Yeast Two-Hybrid Assay
The cDNAs of ERF12, TPL, and the TPL truncated fragment were amplified with primers ERF12-F and ERF12-R, TPL-F and TPL-R, and TPL-F and TPL-t-R, respectively, from Col-0 seeds and pENTR233-TPL. The ERF12-mEAR fragment was cloned using ERF12-mEAR-1, ERF12-EARm-2, and ERF12-mEAR-3 primers successively, as shown in Supplemental Data Set 3, followed the Fast Mutagenesis System (TransGen Biotech). The resulting fragments were fused in-frame with the GAL4 activation domain (AD) and the binding domain (BD) of the AD (pACT2-attR) and BD (pAS2-attR) vectors to generate prey and bait plasmids, respectively (modified from Clontech). These pairs of bait and prey plasmids were cotransformed into yeast AH109 cells using the lithium acetate method (Clontech) and analyzed for yeast growth on selective medium lacking His, Leu, Trp, and adenine at 30°C for 2 to 4 d. Colonies were transferred to filter paper, permeabilized in liquid nitrogen, and their β-galactosidase activity was tested with a solution containing an X-Gal substrate. The plates containing filter paper were incubated at room temperature for 3 h. Colonies producing β-galactosidase were recognized by their blue color.
BiFC Analysis
The cDNAs of ERF12 and TPL, and the TPL N-terminal fragment (200 amino acids) amplified with the same primers as for the yeast-two hybrid experiment and the coding sequence of ERF022 were fused in-frame into vectors pESPYNE and pESPYCE to generate Pro35S:nYFP-TPL, Pro35S:nYFP-TPL-N, Pro35S:cYFP-ERF12, and Pro35S:cYFP-ERF022. Construct Pro35S:nYFP-TPL with Pro35S:cYFP-ERF12, Pro35S:nYFP-TPL-N with Pro35S:cYFP-ERF12, and Pro35S:nYFP-TPL with Pro35S:cYFP-ERF022 were cotransformed into Arabidopsis protoplasts by polyethylene glycol–mediated transient transformation as described, with some modifications (Abel and Theologis, 1994; Kovtun et al., 2000; Hwang and Sheen, 2001). For microscopy, protoplasts were cultured for 12 h after transformation. 4′,6-Diamidino-2-phenylindole staining of protoplasts was performed with 1 μg mL–1 4′,6-diamidino-2-phenylindole in 10 mM phosphate-buffered saline buffer. A TCS SP5 confocal laser-scanning microscope (Leica) was used to detect the fluorescence signals.
Electrophoretic Mobility Shift Assay
Glutathione S-transferase (GST)-ERF12 recombinant protein was expressed in the Escherichia coli BL21 strain and purified with Glutathione Sepharose 4B (17-0756-01, GE Healthcare) according to the manufacturer’s instructions. EMSA was performed using a LightShift Chemiluminescent EMSA kit (20,148, Thermo Fisher Scientific) according to the manufacturer’s protocol.
ChIP Assay
Approximately 0.5 g of freshly harvested seeds was used for the ChIP assay. Chromatin preparation and immunoprecipitation were performed as described previously (Bowler et al., 2004). The seeds were fixed in 1% formaldehyde for 10 min under a vacuum. Gly was added to a final concentration of 0.125 M, and the reaction was terminated by incubation for 5 min under a vacuum. The seeds were rinsed three times with sterilized water and frozen in liquid nitrogen. After isolation, chromatin was sheared to 300- to 2000-bp fragments by sonication (Branson Sonifier 250). Immunoprecipitation was performed by adding specific antibodies (1:1000, ab290; Abcam) and protein G agarose/salmon sperm DNA (Millipore) to the extract. ChIP assays were performed using 15 μg of anti-YFP antibody (Abcam). After washing, immune complex was eluted from the protein G beads and reverse crosslinked by incubation at 65°C overnight. The samples were treated with Proteinase K for 1 h at 65°C. DNA was extracted in a final volume of 50 μL using a QIAquick PCR purification kit (Qiagen). A volume of 1 μL of DNA was used for each qPCR. The qPCR with SYBR FAST qPCR Master Mix (2×; KAPA) was performed on a real-time system (Eppendorf). Each sample was assayed in triplicate by PCR. ACTIN8 was used as a negative control in the ChIP assay (Chua et al., 2005; Wang et al., 2013, 2016). The primers used for the ChIP assays are listed in Supplemental Data Set 3.
LUC Activity Assay
For the transient transcriptional activity assay, a 721-bp DNA fragment of the DOG1 promoter was amplified by PCR and cloned into pGreenII0800 (Hellens et al., 2005), which harbors the Pro35S:REN (Renilla) cassette, to create the reporter ProDOG1:LUC. The coding sequences of ERF12 and TPL were, respectively, cloned into the pUC18-3×HA (hemagglutinin) vector as effectors (Chen et al., 2013). In addition, the reporter gene 5×DOG1 DRE/CRT-LUC, containing five tandem copies of DOG1 DRE/CTR sequence, was constructed as described previously (Fujimoto et al., 2000; Song et al., 2005). All primers used are listed in Supplemental Data Set 3. The ProDOG1:LUC, 5×DOG1 DRE/CRT-LUC reporter plasmid, effector constructs (Pro35S:ERF12, Pro35S:TPL), were cotransformed into Arabidopsis protoplasts. After transfection, the protoplasts were cultured at 22°C in the dark for 12 h. LUC and REN activities were measured with a luminometer (E1910, Promega) following the manufacturer’s instructions. LUC activity was calculated by normalizing to that of REN (LUC/REN × 1000).
GUS Activity analysis
ProDOG1:GUS (Nakabayashi et al., 2012) provided by Kazumi Nakabayashi was transformed into the wild-type Col-0, and three independent transformants were obtained. Transgenic plants were selected based on their ability to survive in 1/2MS medium with 50 mg/mL ammonia benzyl kanamycin (Sigma Aldrich) and 25 mg/mL hygromycin (Sigma Aldrich). To investigate whether ERF12 represses DOG1 activity, overexpressed ERF12 (Pro12S:ERF12) was introduced into ProDOG1:GUS transgenic plants by crossing and selection of homozygous lines. GUS expression levels were detected in freshly harvested seeds, and a GUS staining assay was performed using embryos extracted from freshly harvested seeds as described previously (Wang et al., 2016).
Accession Numbers
Sequence data from this article can be found in the GenBank/EMBL libraries under the following accession numbers: ETR1 (AT1g66340), DOG1 (AT5g45830), ERF12 (AT1g28360), ERF3 (AT1g50640), ERF022 (AT1g33760), MKK9 (AT1g73500), MPK14 (AT4g36450), and TPL (AT1g15750). The microarray data derived from the wild type and the rdo3 mutant have been deposited in the National Center for Biotechnology Information’s Gene Expression Omnibus database under accession number GSE28446.
Supplemental Data
Supplemental Figure 1. RDO3 encodes ETHYLENE RESPONSE1 (ETR1).
Supplemental Figure 2. GUS staining of ProDOG1:GUS transgenic lines.
Supplemental Figure 3. Triple responses of ETR1 and DOG1 mutants.
Supplemental Figure 4. Transcriptome analysis of rdo3.
Supplemental Figure 5. Identification of erf mutants and seed dormancy phenotypes of ERF12, MPK14, and MKK9 overexpression lines.
Supplemental Figure 6. Seed germination rates of the ERF12 overexpression lines in response to PAC and ABA.
Supplemental Figure 7. The mutated EAR motif in ERF12 leads to no interaction between ERF12 and TPL.
Supplemental Figure 8. TPL is involved in the regulation of seed dormancy.
Supplemental Figure 9. Relative expression of ERF12 andTPL in the overexpression lines (supports Figure 6).
Supplemental Figure 10. YFP signals in the Pro12S:YFP-ERF12 transgenic lines.
Supplemental Figure 11. Repression of reporter gene activity by ERF12.
Supplemental Data Set 1. Up-regulated Genes in rdo3 Compared with Ler.
Supplemental Data Set 2. Down-regulated Genes in rdo3 Compared with Ler.
Supplemental Data Set 3. Primers used in this study.
Supplemental File. The results of statistical analyses.
Dive Curated Terms
The following phenotypic, genotypic, and functional terms are of significance to the work described in this paper:
Acknowledgments
We acknowledge Kazumi Nakabayashi for providing the ProDOG1:GUS vector, Rongcheng Lin for the pUC18-3×HA and pGreenII0800 vectors, Dr. Lei Wang for the tpl-1 mutant seeds, and Elliot Meyerowitz for etr1-6 and etr1-7 mutant seeds. We thank Jingquan Li for her excellent technical assistance with confocal microscopy. This project was supported by the National Basic Research Program of China (973 Program; grant 2014CB943400) and the National Natural Science Foundation of China (grants 31071063, 31571257, and 31371242).
AUTHOR CONTRIBUTIONS
Y.X.L. and X.L. designed the research; X.L., and Z.W. performed the experiments; X.L. and Y.L. analyzed the data; T.C., Yu.L., Z.W., C.H., F.C., and W.L. contributed reagents/materials/analysis tools; C.H., Yu.L., W.L., Z.W., and W.J.J.S. contributed to the discussion and article editing; and X.L. and Y.X.L. wrote the article.
Footnotes
↵[OPEN] Articles can be viewed without a subscription.
- Received June 12, 2018.
- Revised January 31, 2019.
- Accepted March 2, 2019.
- Published March 5, 2019.
References
