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Basic LEUCINE ZIPPER TRANSCRIPTION FACTOR67 Transactivates DELAY OF GERMINATION1 to Establish Primary Seed Dormancy in Arabidopsis

Fiona M. Bryant, David Hughes, Keywan Hassani-Pak, Peter J. Eastmond

Published June 2019. DOI: https://doi.org/10.1105/tpc.18.00892

Abstract

Seed dormancy governs the timing of germination, one of the most important developmental transitions in a plant’s life cycle. The DELAY OF GERMINATION1 (DOG1) gene is a key regulator of seed dormancy and a major quantitative trait locus in Arabidopsis (Arabidopsis thaliana). DOG1 expression is under tight developmental and environmental regulation, but the transcription factors involved are not known. Here we show that basic LEUCINE ZIPPER TRANSCRIPTION FACTOR67 (bZIP67) acts downstream of the central regulator of seed development, LEAFY COTYLEDON1, to transactivate DOG1 during maturation and help to establish primary dormancy. We show that bZIP67 overexpression enhances dormancy and that bZIP67 protein (but not transcript) abundance is increased in seeds matured in cool conditions, providing a mechanism to explain how temperature regulates DOG1 expression. We also show that natural allelic variation in the DOG1 promoter affects bZIP67-dependent transactivation, providing a mechanism to explain ecotypic differences in seed dormancy that are controlled by the DOG1 locus.

INTRODUCTION

Seed dormancy is a complex life history trait that plays an important role in local adaptation, as well as dispersal (Finch-Savage and Leubner-Metzger, 2006). Dormancy prevents seed germination when immediate environmental conditions are suitable, but the longer-term probability of survival is low (Finch-Savage and Leubner-Metzger, 2006). In the weedy annual species Arabidopsis (Arabidopsis thaliana), seed dormancy varies geographically, with strong dormancy being more prevalent where summers are long and dry, and weak dormancy being associated with short wet summers (Chiang et al., 2011; Kronholm et al., 2012). Alonso-Blanco et al. (2003) mapped several quantitative trait loci (QTL) that control dormancy using a biparental recombinant inbred population derived from a cross between the highly dormant ecotype Cape Verde Islands (Cvi-0) and a less dormant ecotype Landsberg erecta (Ler-0). The first of these QTL to be cloned was DELAY OF GERMINATION1 (DOG1; Alonso-Blanco et al., 2003; Bentsink et al., 2006). DOG1 is thought to act as a timer for seed dormancy release, because it is modified during after-ripening (Nakabayashi et al., 2012). Its mode of action has been the subject of several studies and remains to be fully elucidated (Nakabayashi et al., 2012, 2015; Graeber et al., 2014; Dekkers et al., 2016; Huo et al., 2016; Née et al., 2017). DOG1 expression is regulated by key environmental cues that control dormancy, such as temperature (Chiang et al., 2011; Kendall et al., 2011; Nakabayashi et al., 2012), and by allelic variation in DOG1, which appears to explain a substantial proportion of the phenotypic variation in dormancy observed in wild populations of Arabidopsis (Alonso-Blanco et al., 2003; Bentsink et al., 2006; Chiang et al., 2011; Kerdaffrec et al., 2016).

Genetic studies have shown that a network of transcriptional master regulators orchestrates the seed maturation program, of which the establishment of primary dormancy is a part (Vicente-Carbajosa and Carbonero, 2005; Santos-Mendoza et al., 2008). In Arabidopsis, four transcription factors (TFs) function as positive regulators of this process: LEAFY COTYLEDON1 (LEC1), ABSCISIC ACID INSENSITIVE3 (ABI3), FUSCA3 (FUS3), and LEC2 (Giraudat et al., 1992; Meinke, 1992; Keith et al., 1994; West et al., 1994; Lotan et al., 1998; Luerssen et al., 1998; Stone et al., 2001). LEC1 acts at the highest level in the regulatory hierarchy controlling the maturation phase (West et al., 1994; Santos-Mendoza et al., 2008) and encodes a homolog of the mammalian NUCLEAR TRANSCRIPTION FACTOR Y subunit B (NF-YB) of the trimeric CCAAT-box binding complex (CBC; Lotan et al., 1998; Lee et al., 2003). ABI3, FUS3, and LEC2 (known collectively as “AFL”) encode TFs containing a conserved B3 DNA binding domain, which is specific to plants (Giraudat et al., 1992; Luerssen et al., 1998; Stone et al., 2001). DOG1 is induced during seed maturation (Bentsink et al., 2006; Nakabayashi et al., 2012), and its expression is known to rely indirectly on LEC1 (Pelletier et al., 2017) and to require AFL (Braybrook et al., 2006; Mönke et al., 2012; Wang and Perry, 2013; González-Morales et al., 2016). Kendall et al. (2011) also showed that enhanced DOG1 expression in seeds matured at low temperature requires C-REPEAT BINDING FACTORS from the APETALA2-domain TF family. DOG1 is also regulated by alternative splicing, alternative polyadenylation, histone modifications, and a cis-acting antisense noncoding transcript (asDOG1; Bentsink et al., 2006; Müller et al., 2012; Graeber et al., 2014; Cyrek et al., 2016; Fedak et al., 2016).

Despite previous studies on DOG1 regulation, it is not known precisely which TFs bind to the DOG1 promoter and are responsible for driving its expression during embryo maturation, nor is it known how temperature (Chiang et al., 2011; Kendall et al., 2011; Nakabayashi et al., 2012) and allelic variation in the DOG1 promoter (Bentsink et al., 2006; Kerdaffrec et al., 2016) affect their ability to perform this function. We previously performed a reverse genetic screen on TFs that are induced by LEC1 during Arabidopsis seed maturation and identified basic LEUCINE ZIPPER TRANSCRIPTION FACTOR67 (bZIP67) as a regulator of several genes involved in seed storage reserve deposition (Mendes et al., 2013). Here, we show that bZIP67 is also a direct regulator of DOG1 expression, specifying LEC1’s action in the establishment of primary dormancy. We also show that temperature regulates bZIP67 at the level of protein abundance and that bZIP67-dependent transactivation of DOG1 is affected by natural variation in the gene promoter (Bentsink et al., 2006; Kerdaffrec et al., 2016).

RESULTS

bZIP67 Is Required for DOG1 Expression and Protein Accumulation

We previously characterized two Arabidopsis mutant alleles of bZIP67 in ecotype Col-0 (Mendes et al., 2013). Affymetrix ATH1 microarray experiments indicated that DOG1 expression may be reduced by as much as 13-fold in whole developing siliques of bzip67-1 (Mendes et al., 2013). To test whether bZIP67 is required for DOG1 expression during seed development, we performed quantitative (q)RT-PCR analysis of transcript abundance in wild-type, bzip67-1, and bzip67-2 plants grown in standard conditions (i.e. 22°C 16-h light/16°C 8-h dark cycle; Figure 1; Nakabayashi et al., 2012, 2015). DOG1 is alternatively spliced, producing five transcript variants, of which epsilon is the major form (Nakabayashi et al., 2015). A noncoding antisense DOG1 RNA (i.e. asDOG1) is also expressed independently of the sense transcripts (Fedak et al., 2016). Using a qRT-PCR primer pair that detects all sense transcripts, we determined that total DOG1 transcript abundance in wild type increased over the linear cotyledon, mature green (MG), and post MG (PMG) stages of embryo development (Pelletier et al., 2017) and then declined in freshly harvested dry seeds (DS; Figure 1A; Nakabayashi et al., 2012, 2015). However, in bzip67-1 and bzip67-2 seeds, total DOG1 transcript abundance was significantly (P < 0.05) lower, with the largest reduction (>10-fold) detected at the MG and PMG stages, when expression in wild type is strongest (Figure 1A). We also performed immunoblot analysis to quantify DOG1 in DS (Figure 1B), when the protein is most abundant (Nakabayashi et al., 2012). The antibody was raised against an N-terminal peptide (Cyrek et al., 2016) that is conserved in all Col-0 DOG1 isoforms (Nakabayashi et al., 2015). Although we could detect DOG1 protein in wild type, it was >7-fold less abundant in bzip67-1 and bzip67-2 (Figure 1B). DOG1 was also absent from the dog1-2 (Nakabayashi et al., 2012) negative control and was recovered to a wild-type level in the bzip67-1 mutant when complemented with a T-DNA construct expressing green fluorescent protein (GFP)-bZIP67 under the control of the bZIP67 promoter (Figure 1B; Bensmihen et al., 2005; Mendes et al., 2013). These data show that bZIP67 is required for DOG1 expression and protein accumulation in Arabidopsis seeds.

Figure 1.
Figure 1.

Effect of bZIP67 Disruption on DOG1 Expression and Protein Abundance in Arabidopsis Col-0 Seeds.

(A) DOG1 transcript abundance in developing seeds of wild type (WT), bzip67-1, and bzip67-2. qRT-PCR was performed on linear cotyledon (LCOT), MG, PMG, and DS stages, using a primer pair that detects all sense transcripts. Values are the mean ± se of measurements on five biological replicates (i.e. batches of seeds from separate plants) and are normalized to the geometric mean of three reference genes and expressed relative to wild-type DS. Asterisk denotes a significant difference from wild type (P < 0.05).

(B) DOG1 protein content in DS of dog1-2, bzip67-1, bzip67-2, wild type, and bzip67-1 ProbZIP67:GFP-bZIP67 (Comp.). The top panel is an immunoblot using anti-DOG1 and the bottom using anti-KAT2 as a loading control (LC). DOG1 abundance was measured by densitometry, normalized to LC, and is given below as a percentage of wild type.

bZIP67 Is Required for the Establishment of Primary Seed Dormancy

To investigate whether disruption of bZIP67 affects primary seed dormancy, we grew wild-type, bzip67-1, and bzip67-2 plants in standard conditions and scored the percentage of seeds that had germinated at 3 d after imbibition (Figure 2; Nakabayashi et al., 2012, 2015). Freshly harvested wild-type seed batches exhibited <20% germination, and an after-ripening period of >1 week was required before >90% germination was achieved (Figure 2A; Nakabayashi et al., 2012, 2015). By contrast, >90% of fresh bzip67-1 and bzip67-2 seeds germinated before after-ripening (Figure 2A). Seed batches from the complemented bzip67-1 mutant containing a T-DNA construct expressing GFP-bZIP67 under the control of the bZIP67 promoter (Bensmihen et al., 2005; Mendes et al., 2013) exhibit a wild-type dormancy phenotype (Figure 2A). We also grew the Col-0 dog1-2 mutant (Nakabayashi et al., 2012) and a bzip67-1 dog1-2 double mutant as controls and confirmed that they have a similar reduced dormancy phenotype to bzip67-1 (Supplemental Figure 1). These data show that bZIP67 is required for the establishment of primary seed dormancy and raise the possibility that bZIP67 functions within the same pathway as DOG1.

Figure 2.
Figure 2.

Effect of bZIP67 Disruption and Overexpression on the Dormancy of Arabidopsis Col-0 Seeds.

(A) Effect of dry storage period on germination of seed batches harvested from wild type (WT), bzip67-1, bzip67-2, and bzip67-1 ProbZIP67:GFP-bZIP67 (Complemented).

(B) DOG1 expression level in freshly harvested seeds of wild-type (WT) and three ProGLY:bZIP67 OE lines.

(C) Effect of dry storage period on germination of seeds overexpressing bZIP67. Values are the mean ± se of measurements on five biological replicates (i.e. batches of seeds from separate plants), and in (B) are normalized to the geometric mean of three reference genes and expressed relative to wild type (WT). Asterisk denotes a significant difference from wild type (P < 0.05).

Overexpression of bZIP67 Enhances Seed Dormancy

To determine whether overexpression of bZIP67 might cause a gain-of-function phenotype, we analyzed three independent overexpression (OE) lines in which bZIP67 is under the control of the strong embryo maturation-specific glycinin promoter (Figure 2B; Mendes et al., 2013). Disruption of bZIP67 reduced DOG1 expression in freshly harvested seeds (Figure 1A). By contrast, total DOG1 transcript abundance was significantly (P < 0.05) enhanced in freshly harvested seeds of the OE lines grown in standard conditions, as compared with wild type (Figure 2B). The freshly harvested OE seeds also exhibited deeper dormancy, requiring a longer period of after-ripening than wild type to achieve >90% germination (Figure 2C). However, when bZIP67 was overexpressed in the dog1-2 background, the enhanced seed dormancy was suppressed (Supplemental Figure 1). These data suggest that bZIP67 may contribute to the regulation of seed dormancy through the control of DOG1 expression.

bZIP67 Protein Abundance Is Increased in Seeds Matured in Cool Conditions

When Arabidopsis seeds mature in cool conditions, DOG1 transcript levels remain high right through to the end of seed desiccation (Kendall et al., 2011), contributing to a deeper state of dormancy (Chiang et al., 2011; Kendall et al., 2011; Nakabayashi et al., 2012; Murphey et al., 2015). To investigate whether bZIP67 function also affects dormancy in seeds matured in cool conditions, we grew plants to flowering in standard (22°C 16-h light/16°C 8-h) conditions and then transferred them to cool (16°C 16-h light/14°C 8-h dark) conditions (Figure 3; Nakabayashi et al., 2012). In ecotype Col-0, dog1 seeds are dormant when matured in cool conditions, but the level of dormancy is reduced relative to wild type (Kendall et al., 2011; Murphey et al., 2015). We observed a similar reduced dormancy phenotype for bzip67, where significantly (P < 0.05) fewer days of seed dry storage (DSDS) than wild type were required before >50% of bzip67 seeds germinated (Figure 3A; Murphey et al., 2015). Seed batches of bzip67-1 ProbZIP67:GFP-bZIP67 (Mendes et al., 2013) exhibited wild-type dormancy levels when matured in cool conditions (Figure 3A). We measured total transcript abundance in the mature seeds of this complemented line and found that, unlike DOG1 (Kendall et al., 2011; Nakabayashi et al., 2012), there was no significant increase (P > 0.05) in bZIP67 expression in cooler conditions (Figure 3B). However, immunoblot analysis performed using an anti-GFP antibody showed that GFP-bZIP67 protein quantity was increased ∼2-fold in cooler conditions (Figure 3C). These data suggest that bZIP67 is subject to posttranscriptional regulation by temperature; increased bZIP67 abundance could explain why DOG1 expression is enhanced in seeds matured in cool conditions (Chiang et al., 2011; Kendall et al., 2011; Nakabayashi et al., 2012).

Figure 3.
Figure 3.

Effect of Cool Conditions during Seed Maturation on bZIP67 Function.

(A) DSDS50. Wild type (WT), bzip67-1, bzip67-2, bzip67-1 ProbZIP67:GFP-bZIP67 (Complemented [Comp.]), and dog1-2 seeds were matured in a cool 16°C 16-h light/14°C 8-h dark (16/14) regime and germinated after increasing periods of dry storage.

(B) GFP-bZIP67 and DOG1 transcript and (C) GFP-bZIP67 protein abundance in freshly harvested Complemented seeds matured in a standard (22/16) or a cool (16/14) regime. In (A) and (B), values are the mean ± se of measurements on three biological replicates (i.e. batches of seeds from separate plants), and in (B) are normalized to the geometric mean of three reference genes and expressed relative to DOG1 22/16. Asterisk denotes a significant difference from wild type in (A) and 22/16 in (B) (P < 0.05). In (C), the top panel is an immunoblot using anti-GFP and the bottom using anti-KAT2 as a loading control (LC). Immunoblots are shown for three biological replicates (i.e. batches of seeds from separate plants). GFP-bZIP67 abundance was measured by densitometry, normalized to LC, and is given on the right as a fold increase in 16/14 versus 22/16.

Ectopic Expression of LEC1 Induces bZIP67 and DOG1 Expression

Ectopic expression of LEC1 in Arabidopsis triggers somatic embryogenesis and activates the transcriptional program for embryo maturation (Lotan et al., 1998; Pelletier et al., 2017). When LEC1 is expressed, it binds to and activates bZIP67, and lec1 embryos are also deficient in both bZIP67 and DOG1 expression (Pelletier et al., 2017). To test whether LEC1 also induces DOG1 expression, we transfected Arabidopsis mesophyll protoplasts with a LEC1 effector plasmid driven by the CaMV 35S promoter or with an empty vector control (EVC; Yamamoto et al., 2009; Mendes et al., 2013) and measured both bZIP67 and total DOG1 transcript abundance over 5 d (Figure 4). LEC1 expression led to a significant (P < 0.05) increase in bZIP67 transcript abundance by 2 d after transfection, and by 5 d, bZIP67 expression was >80-fold higher than in the EVC (Figure 4A). A significant (P < 0.05) increase in DOG1 expression was also detected after transfection with the LEC1 effector plasmid, but it occurred ∼1 d later than bZIP67, and by 5 d, DOG1 expression was >25-fold higher than in the EVC (Figure 4A). These data show that DOG1 is induced by LEC1 expression and that this occurs after the induction of bZIP67.

Figure 4.
Figure 4.

Induction of DOG1 by LEC1 Expression and DOG1 Promoter Binding by bZIP67.

(A) A time course of bZIP67 and DOG1 expression in wild-type protoplasts after transfection with Pro35S:LEC1. Values are the mean ± se of measurements on three biological replicates (i.e. three separate protoplast preparations) and are normalized to the geometric mean of three reference genes and expressed relative to the EVC.

(B) A diagram of the DOG1 locus showing the positions of amplicons (P1 to P6) used for ChIP-qPCR. GBL elements are marked as circles.

(C) Detection of DOG1 promoter binding by bZIP67 in protoplasts using ChIP-qPCR. Protoplasts from bzip67-1 ProbZIP67:GFP-bZIP67 and wild-type (WT) plants were transfected with Pro35S:LEC1 (or EVC) and anti-GFP antibodies were used for ChIP. ACT7 was used as a negative control. Values are expressed as a percentage of the input and are the mean ± se of measurements on three biological replicates (i.e. three separate protoplast preparations).

(D) DPI-ELISA assays quantifying in vitro bZIP67 binding to an equimolar concentration of wild type (WT) versus mutated/knocked out (KO) GBL1 and GBL2 oligonucleotides. Values are the mean ± se of five biological replicates (i.e. separate incubations). Asterisk denotes a significant (P < 0.05) difference from time zero in (A), ACT7 in (C), and wild type in (D).

bZIP67 Binds to the DOG1 Promoter

To determine whether bZIP67 binds to the DOG1 promoter, we performed chromatin immunoprecipitation (ChIP)-qPCR experiments on protoplasts from bzip67-1 ProbZIP67:GFP-bZIP67 and wild-type plants transfected with Pro35S:LEC1 (Mendes et al., 2013). We performed qPCR using primer pairs corresponding to six regions of DOG1 (Figure 4B) and also to ACTIN7 (ACT7) as a negative control. Amplicons P2, P3, and P4, spanning 0- to 1-kb upstream of the DOG1 transcriptional start site (TSS), were significantly (P < 0.05) enriched in bzip67-1 ProbZIP67:GFP-bZIP67 as compared with either the ACT7 or wild-type controls, and enrichment was strongest at P3 (−400 bp), suggesting that bZIP67 binds to this region of the DOG1 promoter (Figure 4C). No enrichment was observed using P3 when protoplasts were transfected with an EVC rather than Pro35S:LEC1 (Figure 4C). To confirm that bZIP67 binds to the DOG1 promoter in vivo, we also performed ChIP-qPCR experiments on developing (MG-PMG stage) seeds of bzip67-1 ProbZIP67:GFP-bZIP67 plants (Mönke et al., 2012; Pelletier et al., 2017) and detected a significant (P < 0.05) enrichment using P3 (Supplemental Figure 2).

We have previously shown that bZIP67 can bind to G box-like (GBL) cis-elements with the core sequence 5′-ACGT-3′ (Mendes et al., 2013). The DOG1 promoter contains multiple GBL elements (Nakabayashi et al., 2012), but only GBL1 and GBL2 (both 5′-CACGTA-3′) are present in the −400-bp region (Figure 4B; Supplemental Figure 3). To test whether bZIP67 can bind to GBL1 and GBL2, we performed a DNA–protein-interaction enzyme-linked immunosorbent assay (DPI-ELISA; Brand et al., 2010; Figure 4D). Epitope-tagged recombinant bZIP67 was incubated with immobilized double-stranded DNA oligonucleotides and binding was determined by immuno-detection (Mendes et al., 2013). When bZIP67 was applied to oligonucleotides containing GBL1 and GBL2, the ELISA signal was >30-fold stronger than when an equal concentration of the corresponding GBL oligonucleotides with mutated/knocked out (KO) 5′-ACGT-3′ cores (Mendes et al., 2013) were tested (Figure 4D). In competition experiments to define specificity, the addition of free GBL oligonucleotides also significantly reduced the ELISA signal from bound GBLs (P < 0.05), whereas the addition of free GBL KO oligonucleotides did not (Supplemental Figure 4). A combination of in vivo and in vitro experiments therefore suggests that bZIP67 binds to the DOG1 promoter.

Transactivation of DOG1 by bZIP67 Requires LEC1 Expression

Next, we tested whether bZIP67 expression is sufficient to transactivate DOG1 alone, or whether it requires LEC1 and/or other regulatory factors induced by LEC1 (Pelletier et al., 2017; Figure 5). We cloned a ∼600-bp region of the Col-0 DOG1 promoter containing GBL1 and GBL2 (Figure 5A) upstream of β-glucuronidase (GUS) and transfected the construct into bzip67-1 protoplasts in combination with Pro35S:bZIP67, Pro35S:LEC1, or EVC effector plasmids (Yamamoto et al., 2009; Mendes et al., 2013). Protoplasts from bzip67-1 were used to prevent induction by endogenous bZIP67 (Figure 4A; Kagaya et al., 2005; Mu et al., 2008). Cotransfection of ProDOG1:GUS with EVC resulted in minimal GUS reporter activity (Figure 5B), which is consistent with the finding that DOG1 expression is restricted to seeds (Bentsink et al., 2006; Nakabayashi et al., 2012). Cotransfection with Pro35S:bZIP67 did not enhance GUS activity significantly (P > 0.05). Cotransfection with Pro35S:LEC1 led to a <2-fold increase in GUS activity (Figure 5B). However, when Pro35S:bZIP67 was cotransfected together with Pro35S:LEC1, the level of GUS activity was enhanced by >18-fold (Figure 5B). These data suggest that bZIP67 is required for DOG1 expression, but is not sufficient, and that transactivation also relies on the expression of LEC1 (Pelletier et al., 2017). LEC1 induces the expression of several regulators of embryo maturation that may participate in DOG1 induction, including CBC components LEC1-like (L1L; Kwong et al., 2003) and NF-YC2 (Yamamoto et al., 2009) and AFL (Mu et al., 2008; Pelletier et al., 2017). There is also evidence that bZIPs form ternary complexes with AFL and CBC to regulate transcription during Arabidopsis seed maturation (Nakamura et al., 2001; Yamamoto et al., 2009; Mendes et al., 2013; Baud et al., 2016).

Figure 5.
Figure 5.

Transactivation of DOG1 in bzip67-1 Protoplasts by Coexpression of bZIP67 and LEC1.

(A) A diagram of the −600-bp DOG1 promoter showing the positions of GBL1, GBL2, and RYL elements.

(B) Effect of bZIP67 and LEC1 expression on ProDOG1:GUS reporter activity.

(C) Effect of GBL and RYL element mutations on ProDOG1:GUS reporter activity. Values are the mean ± se of measurements on three biological replicates (i.e. three separate protoplast preparations) and are expressed as a ratio with Pro35S:LUC. Asterisk denotes a significant (P < 0.05) difference from EVC in (B) and wild-type (WT) promoter in (C).

Transactivation of DOG1 by bZIP67 Requires GBL and RY-Like Promoter Elements

To determine whether transactivation of DOG1 requires the GBL1 and GBL2 cis-elements in the promoter, we mutated the 5′-ACGT-3′ core sequences in the ProDOG1:GUS construct (Mendes et al., 2013) and tested its ability to drive GUS expression in bzip67-1 protoplasts when cotransfected together with Pro35S:bZIP67 and Pro35S:LEC1. Disruption of GBL1 and GBL2 each reduced GUS reporter activity by >70%, and mutations in both blocked transactivation of GUS completely (Figure 5C). ChIP experiments have suggested that LEC1 does not bind DOG1 (Pelletier et al., 2017) and that CCAAT-box motifs that may be bound directly by CBC containing LEC1 or L1L (Gnesutta et al., 2017) are absent from the −600-bp DOG1 promoter. However, an overlapping RY-like (RYL) element (5′-GCATGC-3′) repeat exists between GBL1 and GBL2 (Figure 4A; Supplemental Figure 3). This putative cis-element could be bound by AFL (Braybrook et al., 2006; Baud et al., 2016). AFLs are induced by LEC1 (Pelletier et al., 2017) and are required for DOG1 expression (Braybrook et al., 2006; Mönke et al., 2012; Wang and Perry, 2013; González-Morales et al., 2016; Pelletier et al., 2017). Furthermore, ChIP experiments suggested that AFL FUS3 binds to DOG1 in vivo (Wang and Perry, 2013). Mutating the core sequence of the repeated RYL cis-element (Baud et al., 2016) eliminated GUS reporter activity driven by Pro35S:bZIP67 and Pro35S:LEC1 (Figure 5C). These data suggest that bZIP67-dependent transactivation of DOG1, induced by LEC1, relies on GBL and RYL cis-elements.

Natural Variation in the DOG1 Promoter Affects bZIP67-Dependent Transactivation

DOG1 was originally cloned by QTL mapping by exploiting the natural variation in dormancy observed between ecotypes Cvi-0 and Ler-0, and expression analysis revealed a positive correlation between DOG1 transcript abundance and dormancy in these (and many other) Arabidopsis accessions (Bentsink et al., 2006; Chiang et al., 2011; Kerdaffrec et al., 2016). Although allelic variation at the DOG1 locus explains a substantial proportion of phenotypic variation in seed dormancy, it is not known how cis-variation affects DOG1 expression (Bentsink et al., 2006; Chiang et al., 2011; Kerdaffrec et al., 2016). We therefore created ProDOG1:GUS constructs using −600-bp promoter regions from Cvi-0 and Ler-0 and tested their ability to drive GUS reporter expression when cotransfected together with Pro35S:bZIP67 and Pro35S:LEC1 in bzip67-1 protoplasts (Figure 6). GUS reporter activity driven by the Ler-0 promoter (Figure 6A) was >5-fold lower than when driven by that of Cvi-0 or Col-0 (Figure 6B). The intergenic region upstream of DOG1 in Ler-0 and Cvi-0 contains 15 sequence variants, including three within the −600-bp promoter region (Bentsink et al., 2006). None of these variants lie in GBL1, GBL2, or RYL, but a 285-bp insertion/deletion (INDEL) is present at −328 bp, situated between these cis-elements and the TSS (Figure 6A; Supplemental Figure 3).

Figure 6.
Figure 6.

Effect of Promoter Length on bZIP67-Dependent Transactivation of DOG1.

(A) A diagram of natural and synthetic DOG1 promoter variants showing the position of the 285-bp INDEL, GBL (solid circles), and RYL (open circles) elements relative to the TSS.

(B) Effect of the INDEL (+ or – I) on ProDOG1:GUS reporter activity in bzip67-1 protoplasts cotransfected with Pro35S:bZIP67 and Pro35S:LEC1. R = insertion of 285 bp of a randomly selected intergenic sequence from Arabidopsis. Values are the mean ± se of measurements on three biological replicates (i.e. three separate protoplast preparations) and are expressed as a ratio with Pro35S:LUC. Asterisk denotes a significant (P < 0.05) difference from Ler-0.

Deletion of this 285-bp insertion from the Ler-0 DOG1 promoter led to a >5-fold increase in GUS reporter activity, and conversely, its insertion into either the Cvi-0 or Col-0 DOG1 promoters led to a >5-fold decrease in GUS reporter activity (Figure 6B). These data suggest that the INDEL may be responsible for the allele-specific difference in DOG1 transcript abundance observed between Ler-0 and Cvi-0 (Bentsink et al., 2006). It is not clear precisely how the INDEL affects DOG1 expression. It might act by introducing repressive cis-elements into the promoter or by changing its length/context, i.e. the distance between existing cis-elements and the core promoter region most proximal to the TSS (Liu et al., 2014). To help distinguish between these possibilities, we replaced the Ler-0 insertion with an unrelated 285-bp intergenic sequence from Arabidopsis and found that this also suppressed bZIP67-dependent GUS reporter activity (Figure 6B). This result suggests that the INDEL could simply act as a spacer (Liu et al., 2014). Although we cannot discount the possibility that cis-elements might also exist in the insertion, they do not appear to be required to attenuate DOG1 expression.

Natural Variation in the DOG1 Promoter and GBL Elements Affect Dormancy

To test whether the INDEL and GBLs affect DOG1 function in vivo, we transformed the nondormant dog1-2 mutant with an ∼5-kb genomic construct containing Col-0 DOG1 (Nakabayashi et al., 2015) and also with variants of this construct containing either the 285-bp Ler-0 insertion or mutations in GBL1 and GBL2 (Figure 7). Analysis of freshly harvested seed batches from multiple homozygous transgenic lines grown in standard conditions showed that the Col-0 DOG1 genomic clone could complement dog1-2 (Nakabayashi et al., 2015), whereas the variant clones with either the 285-bp Ler-0 insertion or GBL1 and GBL2 mutations failed to restore wild-type levels of seed dormancy (Figure 7A). We also measured DOG1 expression in freshly harvested seeds of the transgenic lines using a qRT-PCR primer pair selective for the wild-type allele (Nakabayashi et al., 2012). Total DOG1 transcript abundance in dog1-2 seeds containing the Col-0 DOG1 genomic construct was >6-fold higher than in seeds containing the variant clones with either the 285-bp Ler-0 insertion or GBL1 and GBL2 mutations (Figure 7B). These data confirm the notion that GBL1 and GBL2 are necessary for in vivo DOG1 expression, and they indicate that the 285-bp INDEL also modifies DOG1 expression, and consequently, the strength of primary dormancy (Bentsink et al., 2006; Nakabayashi et al., 2012).

Figure 7.
Figure 7.

Effect of the 285-bp INDEL and GBL1 and GBL2 on Seed Dormancy and DOG1 Expression.

(A) Germination and (B) DOG1 expression in freshly harvested dog1-2 seed from three homozygous transgenic lines (a to c) containing either a Col-0 DOG1 genomic construct (Col-0) or a variant with either the Ler-0 285-bp insertion (Col-0+I) or GBL1 and GBL2 mutated (Col-0-GBL). Values are the mean ± se of measurements on five biological replicates (i.e. batches of seeds from separate plants) and in (B) are normalized to the geometric mean of three reference genes and expressed relative to wild type. Asterisk denotes a significant (P < 0.05) difference from dog1-2 in (A) and wild type in (B). WT, wild type.

DISCUSSION

In this study, we showed that the bZIP67 TF induces DOG1 expression during Arabidopsis seed development by binding to GBL cis-elements in the promoter of this gene and that it operates downstream of LEC1 and likely in concert with other central regulators of seed maturation from the CBC and AFL TF families (Figure 8). Models based on temporal and spatial transcriptional profiling have previously placed bZIP67 within the regulatory circuitry that governs seed maturation (Belmonte et al., 2013). bZIP67 expression relies on LEC1 and AFL (Kagaya et al., 2005; Braybrook et al., 2006; Mu et al., 2008; Mönke et al., 2012; Wang and Perry, 2013; González-Morales et al., 2016; Pelletier et al., 2017), and there is evidence that CBC and AFL form ternary complexes with bZIPs to transactivate seed maturation genes (Nakamura et al., 2001; Yamamoto et al., 2009; Baud et al., 2016). It remains to be determined whether CBC or AFL are direct regulators of DOG1 expression. Whole-genome ChIP experiments suggested that FUS3 binds to DOG1 (Wang and Perry, 2013), whereas Pelletier et al. (2017) and Mönke et al. (2012) did not detect DOG1 binding by LEC1 and ABI3, respectively. Unlike mutants in many of these central regulators of seed maturation, bzip67 lacks a morphological phenotype (Bensmihen et al., 2005; Belmonte et al., 2013). However, we previously found that DOG1 is one of just a few seed maturation-associated genes that are strongly downregulated in developing bzip67 siliques (Mendes et al., 2013), and we have shown here that, because bZIP67 is required for DOG1 expression in developing seeds, it is also required for the establishment of primary seed dormancy. It is noteworthy that bZIP67 maps adjacent to two dormancy genes (DOG6 and REDUCED DORMANCY1) whose identities are unclear, although they are unlikely to be synonymous, based on their contrasting phenotypes (Léon-Kloosterziel et al., 1996; Peeters et al., 2002; Alonso-Blanco et al., 2003).

Figure 8.
Figure 8.

A Model for Transcriptional Regulation of DOG1 Expression during Seed Maturation.

Expression of LEC1 is necessary and sufficient for the induction of bZIP67 and AFL (ABI3, FUS3, and LEC2), and ALF are also necessary for bZIP67 expression (Pelletier et al., 2017). Our data suggest that LEC1 expression also induces DOG1 in a bZIP67-dependent manner and that bZIP67 binds to GBL cis-elements in the DOG1 promoter, which are necessary for expression. Other LEC1-inducible proteins are also necessary for DOG1 expression, but it is not known whether AFL bind to RYL cis-elements in DOG1 directly (?). However, loss and gain of bZIP67 function in seeds is sufficient to reduce and increase DOG1 expression (and dormancy), respectively, and bZIP67 protein abundance is also increased by cool conditions during seed maturation that promote DOG1 expression (and dormancy). Our data also suggest that a 285-bp INDEL situated between GBL and the TSS is responsible for the difference in DOG1 expression found in ecotypes Ler-0 and Cvi-0.

Temperature is a key environmental regulator of DOG1 expression and seed dormancy in Arabidopsis (Chiang et al., 2011; Kendall et al., 2011; Nakabayashi et al., 2012). We demonstrated that cool conditions during seed maturation enhance bZIP67 protein (but not transcript) abundance and that overexpression of bZIP67 can increase DOG1 expression and cause dormancy to deepen. bZIP67 is one of four bZIPs from clade A, which are expressed in Arabidopsis seeds (Bensmihen et al., 2005). The best-characterized of these bZIPs is ABI5, which is also bZIP67’s closest homolog (Bensmihen et al., 2005). ABI5 functions in ABA signaling and regulates seed germination and early seedling growth in response to abiotic stress (Finkelstein and Lynch, 2000; Lopez-Molina et al., 2001; Skubacz et al., 2016). ABI5 is expressed later in seed development than bZIP67 (Bensmihen et al., 2002, 2005) and does not appear to be required for primary dormancy (Finkelstein, 1994), although it is regulated by DOG1 (Dekkers et al., 2016). Interestingly, ABI5 is subject to extensive posttranslational regulation (Skubacz et al., 2016), and key phosphorylation, ubiquitination, and s-nitrosylation sites identified in ABI5 are also within regions conserved in bZIP67 (Supplemental Figure 5). Group A bZIPs are also thought to form heterodimers (Deppmann et al., 2004). They can bind to similar (or identical) GBL cis-elements (Carles et al., 2002; Kim et al., 2002; Deppmann et al., 2004) and have negative, as well as positive, regulatory functions (Finkelstein and Lynch, 2000; Bensmihen et al., 2002). Further work will therefore be required to determine precisely how bZIP67 is regulated by factors such as temperature (Figure 8) and whether additional clade A bZIPs are also involved in DOG1 expression.

DOG1 also plays important roles in secondary dormancy and dormancy cycling (Finch-Savage et al., 2007; Footitt et al., 2011; Finch-Savage and Footitt, 2017). DOG1 expression is enhanced in secondary dormant Cvi-0 seed (Cadman et al., 2006; Finch-Savage et al., 2007). DOG1 expression correlates with other dormancy marker genes over the course of an annual cycle in the seed soil bank (Footitt et al., 2011), and the principal QTL for timing of emergence from the soil in a Cvi-0/Bur-0 (Burren-0) recombinant inbred mapping population also colocates with DOG1 (Finch-Savage and Footitt, 2017). However, bZIP67 does not appear to be expressed in secondary dormant Cvi-0 seed, based on published microarray data (Cadman et al., 2006; Finch-Savage et al., 2007). Therefore, it is possible that bZIP67’s role in transactivating DOG1 is restricted to seed maturation and that other TFs may control DOG1 (and asDOG1) expression postimbibition and through dormancy cycles in the seed soil bank (Finch-Savage and Footitt, 2017).

Functional allelic variation in DOG1 is believed to be widespread and to have considerable adaptive significance (Alonso-Blanco et al., 2003; Bentsink et al., 2006, 2010; Chiang et al., 2011; Kerdaffrec et al., 2016). Nakabayashi et al. (2015) previously identified a nonsynonymous substitution in DOG1 that affects protein function, but DOG1 expression also differs greatly between Arabidopsis accessions (Bentsink et al., 2006; Chiang et al., 2011). Cis-regulation of gene expression is common in Arabidopsis (Keurentjes et al., 2007), and Gan et al. (2011) previously reported that potential cis-acting sequence variants, associated with ecotypic differences in gene expression, are concentrated in the promoter regions, which are also hotspots for meiotic recombination (Choi et al., 2013). Here we showed that variation in promoter length, caused by a 285-bp INDEL, affects bZIP67-dependent transactivation of DOG1 (Figure 8), providing a molecular mechanism to explain how the DOG1 QTL contributes to the phenotypic difference in seed dormancy observed between ecotypes Cvi-0 and Ler-0 (Alonso-Blanco et al., 2003; Bentsink et al., 2006). Transcriptional activation is known to be modulated by promoter context, as well as response element-dependent specificity (Nagpal et al., 1992; Sanguedolce et al., 1997). Liu et al. (2014) previously showed that natural variation in FLOWERING LOCUS T promoter length, resulting from INDELs, is widespread in Arabidopsis and modulates the photoperiodic response of the floral transition.

The 285-bp insertion in the Ler-0 DOG1 promoter that confers low expression appears to be a duplication of an intergenic region from Chromosome 3, corresponding to 9,981,927 to 9,982,211 bp in Col-0. It is noteworthy that Ler-0 carries the erecta mutation, which is a product of x-ray mutagenesis (Zapata et al., 2016). This raises the following question: Is the 285-bp INDEL a natural polymorphism? The insertion is present in the chromosome-level assembly of the Ler-0 genome (Zapata et al., 2016), but it is not listed in Polymorph 1001 (http://tools.1001genomes.org/polymorph), which contains variants from de novo assembly of short-read sequencing of 1,135 Arabidopsis accessions (The 1001 Genomes Consortium, 2016). When we mapped the short-read data for 1,135 accessions (The 1001 Genomes Consortium, 2016) to Ler-0 DOG1, we found that the 285-bp insertion is also present in two accessions that are phylogenetically distinct from Ler-0. These accessions are Landsberg-0 (La-0) and Kazakhstan-13 (Kz-13). The 285-bp insertion is therefore a natural polymorphism, but it appears to be rare. However, DOG1 expression is also known to vary among ecotypes that lack the 285-bp insertion (Bentsink et al., 2006), and other cis-acting polymorphisms in noncoding regions of the gene are most likely to be causal in these instances (Bentsink et al., 2006; Kerdaffrec et al., 2016). Further work will be required to identify the mode of action of these polymorphisms, but it is likely that many also affect the efficiency of bZIP67-dependent transactivation.

METHODS

Plant Material and Growth Conditions

The Arabidopsis (Arabidopsis thaliana) bzip67 T-DNA insertion mutants (Mendes et al., 2013) were originally identified on the SIGnAL T-DNA Express web page (http://signal.salk.edu/cgi-bin/tdnaexpress) and seeds were obtained from the European Arabidopsis Stock Centre (University of Nottingham, UK). The dog1-2 mutant and ProbZIP67:GFP-bZIP67 reporter line used in this study have been described in Bensmihen et al. (2005) and Nakabayashi et al. (2012). For plant growth, seeds were sown on moist Levington F2 compost in 7-cm2 pots. The pots were stored in the dark at 4°C for 4 d before being transferred to a Gallenkamp 228 growth cabinet with T5 49-watt fluorescent tubes set to a “standard” 22°C 16-h light (photosynthetic photon flux density [PPFD] = 150 μmol m−2 s−1)/16°C 8-h dark regime (Nakabayashi et al., 2012) and 70% relative humidity. In some experiments, plants were then transferred at the onset of flowering to a “cool” 16°C 16-h light (PPFD = 150 μmol m−2 s−1)/14°C 8-h dark regime (Nakabayashi et al., 2012). During seed set, plants were monitored every day and seeds were harvested from individual siliques on the primary raceme, as soon as they became dehiscent. The seeds were used immediately for germination assays or were afterripened by dry storage at 22°C in the dark at 70% relative humidity.

Germination Assays

Approximately 50 freshly harvested or afterripened seeds from each individual plant were sown directly onto a 0.8% (w/v) agar plate prepared using deionized water and the plate was placed in a Gallenkamp 228 growth cabinet set to 22°C, 70% relative humidity (16-h light/8-h dark; PPFD = 150 µmol m−2 s−1). Afterripened wild-type and mutant seed batches exhibited >90% germination after 3 d of imbibition, and so this time point was used routinely for germination assays (Nakabayashi et al., 2012, 2015). Germination was scored as “radicle emergence” and was observed under a dissecting stereomicroscope. To determine DSDS50, we performed germination assays on seed batches every 7 d, until >90% germination was achieved (Murphey et al., 2015).

Gene Expression Analysis and Immunoblotting

DNase-treated total RNA was isolated from seeds at different morphological stages of development (Pelletier et al., 2017) and from protoplasts using the RNeasy kit from Qiagen, except that for seeds, the method was modified (Mendes et al., 2013). The synthesis of single-stranded cDNA was performed using SuperScript II RNase H- reverse transcriptase from Invitrogen. qqPCR was performed as described in Mendes et al. (2013), except that DOG1 or bZIP67 expression levels were normalized to the geometric mean of three reference genes. The reference genes (UBIQUITIN5, ELONGATION FACTOR-1α, and ACTIN8) were selected owing to their stable expression over the course of seed development (Gutierrez et al., 2008) and at different temperatures (Chiang et al., 2011; Nakabayashi et al., 2012). The primer pairs used for qPCR are listed in Supplemental Table 1. For analysis of DOG1 protein, seeds were homogenized in 50 mM of Tris-HCl buffer (pH 6.8) and the total protein was denatured and concentrated using chloroform/methanol precipitation (Wessel and Flügge, 1984). Protein quantification, SDS-PAGE, and immunoblotting were then performed as described in Craddock et al. (2015), except that anti-DOG1 (AS15 3032; Agrisera), anti-3-ketoacyl-CoA thiolase (KAT2; Germain et al., 2001), or anti-GFP antibodies (Roche) and anti-immunoglobulin G-horseradish peroxidase (Invitrogen) were used as primary and secondary antibodies at 1 in 1,000 and 1 in 10,000 dilutions, respectively, and HRP was detected using either an Enhanced Chemiluminescence kit (PerkinElmer) or colorimetric kit (Bio-Rad). Images were scanned, and band intensity was quantified using the software ImageJ (https://imagej.nih.gov/ij/).

Transient Expression in Arabidopsis Protoplasts

The −600-bp promoter region of DOG1 was amplified from Col-0, Cvi-0, and Ler-0 genomic DNA using primer pairs listed in Supplemental Table 1. The products were cloned into the entry vector pENTR/D-TOPO and then transferred to the destination vector pBGWFS7 (Karimi et al., 2002) using the Gateway LR Clonase enzyme mix from Invitrogen, following the manufacturer’s instructions. Additional versions of the DOG1 promoter containing GBL (5′-ACGT-3′ to 5′-AAGG-3′) and RYL (5′-CATG-3′ to 5′-CAAC-3′) cis-element mutations (Figure 6A; Mendes et al., 2013) and INDELs (Figure 7A) were created by gene synthesis and cloned into pBGWFS7 using the same procedure. Arabidopsis mesophyll protoplasts were prepared from the leaves of wild-type, bzip67-1 ProbZIP67:GFP-bZIP67, and bzip67-1 plants and transfected with effector plasmids as described in Yamamoto et al. (2009) and Mendes et al. (2013). After polyethylene glycol-calcium transfection with plasmid DNA carrying reporter gene constructs (ProDOG1:GUS and Pro35S:luciferase [LUC]) and/or combinations of effector plasmids (Pro35S:bZIP67 and Pro35S:LEC1), the cells were cultured for up to 5 d (Kim and Somers, 2010) before performing qPCR analysis of transcript abundance or LUC and GUS activity assays using methods described in Yamamoto et al. (2009) and Mendes et al. (2013).

ChIP and Protein-DNA Binding Assays

ChIP assays were performed using leaf mesophyll protoplasts (Mendes et al., 2013) and developing seeds (Mönke et al., 2012) from bzip67-1 ProbZIP67:GFP-bZIP67 and wild-type plants. Protoplasts were transfected with a Pro35S:LEC1 effector plasmid or an EVC, as described in Yamamoto et al. (2009), Kim and Somers (2010), and Mendes et al. (2013). After 5 d, the protoplasts were harvested, and ChIP-qPCR assays were performed following the procedures described in Mendes et al. (2013). Chromatin isolation from MG-PMG stage seeds (Pelletier et al., 2017) was performed by following the method described by Junker et al. (2012) and as adapted by Mönke et al. (2012). After chromatin was isolated, it was extensively sheared by sonication to obtain fragment sizes between 300 and 400 bp. Rat anti-GFP monoclonal antibodies (Roche) and Dynabeads Protein G magnetic beads (Invitrogen) were used to immunoprecipitate the genomic fragments. qPCR was performed on the immunoprecipitated DNA from bzip67-1 ProbZIP67:GFP-bZIP67 and wild-type plant material as described in Mendes et al. (2013) using primer sets corresponding to six regions of the DOG1 gene and to ACT7 as a control (Supplemental Table 1) and were corrected for their individual PCR amplification efficiencies (Mendes et al., 2013). Protein-DNA binding assays were performed using the DPI-ELISA method (Brand et al., 2010) as described in Mendes et al. (2013). Biotinylated complementary oligonucleotides for GBL1 and GBL2 cis-elements in the DOG1 promoter are listed in Supplemental Table 1.

Complementation of dog1

An ∼5-kb region of Col-0 DOG1, including ∼2.2 kb upstream and ∼1 kb downstream of the coding region, was amplified by PCR (Nakabayashi et al., 2015) and cloned into the entry vector pENTR/D-TOPO and then transferred to destination vector pEarlyGate 301 (Earley et al., 2006). Versions of Col-0 DOG1 containing the 285-bp Ler-0 insertion or GBL1 and GBL2 mutations were also created by overlap extension PCR (Heckman and Pease, 2007), using the Ler-0 or mutated Col-0 DOG1 promoter and Col-0 DOG1 genomic constructs as templates. Heat shock was used to transform the plasmids into Agrobacterium tumefaciens strain GV3101, and Arabidopsis transformation was then performed using the floral-dip method (Clough and Bent, 1998). Herbicide resistance was used to select >40 T1 primary transgenic lines per construct, and multiple homozygous T3 lines were subsequently recovered and analyzed.

Statistical Analysis

All experiments were performed using either three or five biological replicates, and the data are presented as the mean values ± se. We used analysis of variance to assess differences between genotypes or treatments (Supplemental Table 2). After significant (P < 0.05) F-test results, means were compared using the appropriate least significant difference value at the 5% (P = 0.05) level of significance, on the corresponding degrees of freedom. The GenStat (VSN International) statistical system was used for these analyses.

Accession Numbers

The TAIR accession numbers for the sequences of major genes mentioned in this study are as follows: bZIP67 (At3g44460), DOG1 (At5g45830), LEC1 (At1g21970), ABI3 (At3g24650), FUS3 (At3g26790), LEC2 (At1g28300), L1L (At5g47670), NF-YC2 (At1g56170), ABI5 (At2g36270), and FT (FLOWERING LOCUS T; At1g65480).

Supplemental Data

Dive Curated Terms

The following phenotypic, genotypic, and functional terms are of significance to the work described in this paper:

Acknowledgments

We thank Tsukaho Hattori for providing constructs, François Parcy and Sandra Bensmihen for the ProbZIP67:GFP-bZIP67 reporter line, and Steve Penfield for dog1-2 seed. Haolin Li assisted with bioinformatic analysis of the DOG1 promoter. This work was supported by the UK Biotechnology and Biological Sciences Research Council (grant BB/P012663/1).

AUTHOR CONTRIBUTIONS

P.J.E. designed research; F.M.B. and P.J.E. performed research; F.M.B., D.H., K.H.-P., and P.J.E. analyzed data; P.J.E. wrote the article.

Footnotes

  • www.plantcell.org/cgi/doi/10.1105/tpc.18.00892

  • The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Peter J. Eastmond (peter.eastmond{at}rothamsted.ac.uk).

  • ↵[OPEN] Articles can be viewed without a subscription.

  • Received November 26, 2018.
  • Revised March 15, 2019.
  • Accepted April 5, 2019.
  • Published April 8, 2019.

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Basic LEUCINE ZIPPER TRANSCRIPTION FACTOR67 Transactivates DELAY OF GERMINATION1 to Establish Primary Seed Dormancy in Arabidopsis
Fiona M. Bryant, David Hughes, Keywan Hassani-Pak, Peter J. Eastmond
The Plant Cell Jun 2019, 31 (6) 1276-1288; DOI: 10.1105/tpc.18.00892
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