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. 2026 Sep 1;202(2):kiag647. doi: 10.1093/plphys/kiag647

Molecular characterization of capulet2 reveals the importance of ANAPHASE PROMOTING COMPLEX 6 maternal expression in endosperm development

Yuri S van Ekelenburg 1,#, Ida V Myking 2,#, Cathal Meehan 3, Morten P Hauger 4, Shinichiro Komaki 5,6, Keiko Sugimoto 7, José Gutierrez-Marcos 8, Paul E Grini 9,3,✉,4
PMCID: PMC13655448  PMID: 42679354

Abstract

Flowering plants are characterized by a double fertilization event, and the fertilized female gametes develop into the endosperm and embryo. Genomic imprinting promotes parental allele-specific gene expression in the endosperm by epigenetic modifications such as DNA methylation. Similarly, gametophyte maternal effects influence gene function in the female gametophyte that affects development of the endosperm and embryo postfertilization. While many imprinted genes do not display a seed phenotype upon mutation, gametophyte maternal effect mutants are characterized by distorted seed development upon maternal transmission of the mutant allele. Here, we have investigated the gametophyte maternal effect mutant capulet2 (cap2). We have established CAP2 to be encoded by ANAPHASE PROMOTING COMPLEX 6 (APC6), a subunit of the anaphase promoting complex/cyclosome (APC/C). Investigation of further cap2/apc6 alleles revealed female gametophyte maternal effects both in mutant segregation and seed phenotype, and both cap2 and apc6 phenotypes were rescued by an APC6 transgene. Furthermore, we have demonstrated that APC6 is a maternally expressed imprinted gene, in line with the observed female gametophyte maternal effect phenotype. To this end, we observed irregular nuclear division of the endosperm coenocyte in cap2/apc6 mutants, suggesting a role for the APC/C in early endosperm development. We have further demonstrated that similar endosperm defects are also produced by mutation of APC1, another subunit of the APC/C. Similar to CAP2/APC6, APC1 is imprinted and only expressed from the maternal allele, suggesting a maternal bias in the control of APC/C in the developing endosperm.

Introduction

Seed development in angiosperms is initiated by a double fertilization event forming the diploid embryo and triploid endosperm (Nowack et al. 2010). The embryo develops into the next generation and receives nutrients from the mother plant through the endosperm. Due to the triploid nature of the endosperm, a balanced parental gene expression is important (Barton et al. 1984; Birchler 1993). While most genes are expressed equally from both parental alleles (biparentally expressed genes [BEGs]), some genes have been found to be regulated in a parent-of-origin dependent manner (Köhler and Grossniklaus 2005; Nowack et al. 2010). Parent-of-origin specific gene expression, also called genomic imprinting, has been described in mammals, filamentous fungi, and flowering plants (Martienssen and Colot 2001; Feil and Berger 2007). In genomic imprinting, an epigenetic mark is established in the gametes, resulting in silencing of the allele postfertilization (Montgomery and Berger 2021).

Currently, 2 major epigenetic regulatory mechanisms are known to be involved in the establishment of imprinting, DNA methylation, and histone modification. Maternally expressed genes (MEGs) are mainly hypothesized to be regulated by DNA methylation and removal of methylated cytosines by DNA glycosylases. Several DNA methyltransferases have been identified in Arabidopsis thaliana (A. thaliana), including DNA METHYLTRANSFERASE 1 (MET1) (Finnegan and Dennis, 1993), CHROMOMETHYLASE 3 (CMT3) (Lindroth et al. 2001) and DOMAINS REARRANGED METHYLASE 1 & 2 (DRM1/DRM2) (Cao and Jacobsen 2002). It has been shown that DNA methylation marks established by MET1 (Xiao et al. 2003; Kinoshita et al. 2004; Gehring et al. 2006; Shirzadi et al. 2011), DRM1/DRM2 (Matzke and Mosher 2014; Hornslien et al. 2019) and CMT3, guided by histone H3 lysine nine di-methylation (H3K9me2) (Stroud et al. 2014; Moreno-Romero et al. 2019), can regulate the imprinting state of specific genes. In addition, RNA-directed de novo DNA methylation (RdDM) guided by small RNAs has been demonstrated (Vu et al. 2013; Hornslien et al. 2019; Satyaki and Gehring 2019; Batista and Köhler 2020).

Paternally expressed genes (PEGs) are mainly considered to be regulated by silencing of the maternal allele in the central cell by the FERTILIZATION INDEPENDENT SEED 2-Polycomb Repressive Complex 2 (FIS-PRC2) complex (Gehring 2013). Upon recruitment of FIS-PRC2 to DNA hypomethylated loci on the maternal allele, the histone methyltransferase subunit MEDEA (MEA) trimethylates histone H3 lysine 27 (H3K27me3), a mark for gene repression (Rodrigues and Zilberman 2015; Moreno-Romero et al. 2016). Several genes have been shown to exhibit maternal silencing by the FIS-PRC2 complex, including PHERES1 (PHE1), and display interaction between DNA and histone methylation (Köhler et al. 2005; Makarevich et al. 2008; Hornslien et al. 2019; Batista and Köhler 2020).

Similar to imprinted genes, gametophyte parental effect genes affect postfertilization seed development (St Johnston and Nüsslein-Volhard, 1992; Colombo et al. 1997). Although the outcomes of parental effect genes and imprinted genes are relatively similar, the genetic phenomena are distinct and have different molecular origins (Wolf and Wade 2009). The epigenetic marks for imprinted genes are established prior to fertilization and affect gene expression postfertilization (Montgomery and Berger 2021), whereas parental effects are caused by prefertilization products (ie mRNA and proteins) that become active and execute their effect postfertilization, irrespective of gene expression (St Johnston and Nüsslein-Volhard, 1992; Colombo et al. 1997).

Gametophyte maternal effect mutants have been characterized by normal gametophyte development, followed by disrupted embryo and/or endosperm development when the mutant allele is transmitted through the female parent (Yadegari and Drews 2004). Gametophyte maternal effect mutants have been identified in both Arabidopsis and maize (Evans and Kermicle 2001; Grini et al. 2002; Olsen 2004; Pagnussat et al. 2005; Gutiérrez-Marcos et al. 2006; Phillips and Evans 2011; Chettoor et al. 2016), including the capulet (cap) mutants (cap1 and cap2), which show developmental arrest in the early developing embryo and endosperm (Grini et al. 2002). The cap2 allele is only lethal when transmitted maternally, indicative of a gametophytic maternal effect mutant. Gametophyte maternal effects could be explained by maternally deposited RNA or protein or by genomic imprinting. However, it remains technologically challenging to identify the origin of transcripts in the endosperm directly after fertilization, ie whether transcripts come from the female central cell as maternal carryover or if they have been transcribed de novo (Evans and Kermicle 2001).

In this study, we have identified the molecular nature of the gametophytic maternal effect mutant cap2. Using whole genome sequencing and single nucleotide polymorphism (SNP) analysis, we identified the causative cap2 SNP to be located in an intron splice donor site of the ANAPHASE PROMOTING COMPLEX 6 (APC6), a subunit of the ANAPHASE PROMOTING COMPLEX/CYCLOSOME (APC/C). Two independent T-DNA insertion lines, apc6-2 and apc6-3, were thoroughly analyzed and phenocopied cap2, both in terms of seed phenotypes and frequency. Moreover, cap2, apc6-2, and apc6-3 could be rescued by molecular complementation using an APC6 transgene. In line with the gametophyte maternal effect phenotype, APC6 was shown to be a maternally expressed imprinted gene. Last, we demonstrated that mutation of APC1, another subunit of the APC/C, displays a female gametophyte phenotype and enhances the frequency of disrupted endosperm development in an apc6 genetic background. Similar to cap2/apc6, APC1 is imprinted and expressed only from the maternal allele, suggesting a maternal bias to the anaphase promoting complex (APC/C).

Results

Capulet2 causative mutation is located in APC6

The capulet2 mutant was identified in an ethyl methanesulfonate (EMS)-induced gametophytic mutant screen by a segregation distortion assay using the multiply marked chromosome 1 (mm1) line (Grini et al. 1999). The location of cap2 was determined between the flanking genetic markers ap1 and gl2 (Fig. 1a) at a genome interval between ADH1 and gl2 using molecular markers (Grini et al. 2002). To determine the causative mutation of cap2, we employed whole genome sequencing. For this purpose, gDNA was isolated from 12 cap2/CAP2 (cap2/+) and 12 CAP2/CAP2 (+/+) plants. We performed a nucleotide polymorphism analysis between pairs of cap2/+ and CAP2/CAP2 individuals, which revealed a clear linkage break on chromosome 1 (Figure S1). We found 100 to 180 candidate SNPs in the ADH1 and gl2 interval (Fig. 1b, Data S1). Candidate SNPs were further selected by their base alignment quality score (BAQ) of ≥70 (Li, 2011) and the presence of guanine to adenine conversion as the mutation arose in a population treated with ethyl methanesulfonate (EMS) with an allele frequency of 0.25 to 0.75 (Table S1a) and conserved across all 12 comparisons (Table S1b). We found 3 SNPs that met these requirements—1 in the ANAPHASE PROMOTING COMPLEX 6 (APC6), 1 in the STRUBBELIG RECEPTOR FAMILY 5 (SRF5), and 1 in an intergenic region.

Figure 1.

Visual representation of chromosomal location of candidate SNPs for capulet2, and precise loci for mutations in APC6.

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Identification of the causative cap2 mutation. a) Identification of candidate causative SNPs located on chromosome 1 between the visible mutant markers ap1 and gl2 of multiply marked chromosome 1 (mm1). Segregation analysis using molecular markers narrowed down the location of the cap2 mutation between ADH1 and gl2. b) SNP analysis of 12 independent comparisons between a cap2/CAP2 and a CAP2/CAP2 individual. Representation of identified SNPs, the type of mutation and allele frequency in a region of 1.06 Mbp of comparison 3. c) Molecular analysis of cap2 and apc mutant alleles: The cap2 G → A conversion (red) abolishes splicing and intron translation leads to a stop codon directly after the cap2 SNP. The apc6-2 T-DNA insertion, located in exon 12, has a 10 bp deletion and an in-frame stop codon 68 bp into the T-DNA. The apc6-3 T-DNA insertion located in intron 2 has a 12 bp deletion. cDNA analysis revealed a cryptic splice acceptor site present in the T-DNA 70 bp inwards, resulting in splicing of intron 2 (up till the T-DNA insertion site) and part of the T-DNA, followed directly by an in-frame stop codon. Both apc6-2 and apc6-3 have at least 2 truncated T-DNA insertions at the same locus consecutively in opposite orientations. Exons and introns are highlighted by color and letter size (blue uppercase and green lowercase, respectively). Dotted line indicates splice event. Underlined nucleotides represent a stop codon. LB = T-DNA left border.

APC6 mutants are defective in endosperm development

We obtained 3 homozygous T-DNA insertion lines for SRF5 and 2 T-DNA insertion lines flanking the intergenic candidate SNP and found that homozygous mutant plants for these insertions had normal seed sets and were indistinguishable from wild-type plants. Our genome sequencing analysis revealed that the cap2 mutant carries a splice site mutation in intron 8 of APC6, and sequencing of cap2 cDNA verified that this resulted in an unspliced transcript, introducing an early in-frame stop codon (Fig. 1c, Figure S2). We obtained 2 T-DNA insertion lines for APC6, 1 that we named apc6-2 (SAIL_442_F11) carrying an insertion located in exon 12 that results in the formation of a premature stop codon and a 2nd named apc6-3 (SALK_008789) that carries an insertion located in the 2nd intron, resulting in a cryptic splice acceptor site inside the T-DNA sequence leading to an in-frame stop codon (Fig. 1c). The predicted transcripts of both T-DNA lines were verified by sequencing of cDNA from the mutant alleles (Figure S2).

We found that when heterozygous apc6-2 and apc6-3 were crossed with wild-type (WT) pollen the resulting seeds displayed developmental defects that were apparent at 4 days after pollination (DAP) and that resembled the seed phenotype observed in cap2/+ (Fig. 2a). To further characterize the endosperm phenotypes, we crossed apc6-2/+ with an early endosperm (EE) green fluorescent protein (GFP) reporter line that shows GFP expression before endosperm cellularization and a total endosperm (TE1) GFP reporter line expressing GFP after endosperm cellularization (Bjerkan et al. 2023; van Ekelenburg et al. 2023). We found that a large proportion of seeds in apc6-2/+;EE-GFP plants had a reduced number of endosperm nuclei that were enlarged and marked by a strong EE-GFP signal at 4 DAP (Fig. 2a; Figure S3). In apc6-2/+;TE1-GFP plants, however, the postcellularization marker TE1-GFP was rarely detected in phenotypically mutant endosperm 4 DAP, suggesting that the mutant endosperm remain uncellularized (Figure S3).

Figure 2.

Light and confocal microscopy images of capulet2 and apc6 mutant seed phenotypes, and box plots illustrating the frequency of mutant seeds per silique in 3 different crosses for 3 different mutants.

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Seed phenotypes in cap2, apc6-2 and apc6-3 display gametophytic maternal effects. a) Endosperm phenotypes of cap2/+, apc6-2/+ and apc6-3/+ crossed maternally to Col-0 display arrested endosperm development at 4 days after pollination (DAP). A lower number of enlarged endosperm nuclei are observed compared to wild-type Col-0. Closed arrowheads indicate enlarged and abnormally shaped endosperm nuclei, arrows indicate smaller endosperm nuclei. A turquoise hue is added to highlight the endosperm, yellow hue highlights the embryo. Selfed EE-GFP compared to apc6-2/+;EE-GFP highlights enlarged and abnormally shaped endosperm nuclei. GFP-expressing seeds were counterstained with Propidium Iodide (red). Scale bar = 20 µm. b) Mutant seed phenotype frequencies for cap2/+, apc6-2/+ and apc6-3/+ at 4 DAP when selfed or crossed reciprocally to Col-0. n > 10 siliques per cross. Statistics was performed according to the Wilcoxon rank sum test; * P < 0.05, ** P < 0.01, **** P < 0.0001.

Notably, the Arabidopsis nomega mutant, caused by Ds-transposon insertion in APC6 (Kwee and Sundaresan 2003) and an APC6 mutant in rice (Awasthi et al. 2012) have both been reported to be female gametophytic mutants displaying prefertilization developmental defects. To investigate this discrepancy, we analyzed unfertilized ovules of wild-type, cap2/+, apc6-2/+, and apc6-3/+. We found that the embryo sac central cell and egg cell and the egg apparatus of mutant lines are indistinguishable from the WT (Table S2), and the 3 independent mutant alleles that we have identified for APC6 show only postfertilization seed defects.

We previously reported that cap2 is a female gametophyte mutant, because seed defects are only observed when the mutant allele is transmitted maternally (Grini et al. 2002). We carried out reciprocal crosses between cap2/+, apc6-2/+, and apc6-3/+ and WT plants and quantified defects in seed development (Fig. 2b, Data S2). We found that seeds failed to develop only when mutant lines were used as female parents (24.2%, 27.3%, and 27.5% for cap2/+, apc6-2/+, and apc6-3/+, respectively) whereas seeds developed normally when wild-type plants were crossed with pollen from mutant lines. As initially reported for cap2 (Grini et al. 2002), we did not find homozygous individuals in progenies of self-pollinated apc6-2/+ and apc6-3/+ plants (n = 47 and 42, respectively).

Collectively, these findings indicate that mutations on APC6 result in a maternal gametophyte effect on seed development.

Genetic complementation of mutants with APC6

To verify that cap2 is caused by mutation of APC6, we transformed cap2/+ and apc6-3/+ plants with a proAPC6:APC6-GFP (APC6-GFP) transgene. The transgene was also transformed into WT Ler-1 plants to confirm endosperm expression (Figure S4).

Plants from unique transformation events were genotyped for mutant alleles and APC6-GFP, and mutant seed phenotype frequencies were determined (Data S3). The frequency of mutant seeds was significantly reduced for the alleles tested, both in a hemizygous (APC6-GFP/+) or a homozygous (APC6-GFP/APC6-GFP) transgene background (Fig. 3; Figure S5). Notably, homozygous cap2 genotypes were identified in the T2 generation (Fig. 3; Figure S6a). Several homozygous apc6-3 individuals were detected in the T3 generation (Figure S6b) and no mutant seed phenotypes were observed (Figure S5). Homozygous apc6-3 mutants in a homozygous rescue construct background crossed to apc6-2/+ plants demonstrated several homozygous apc6-2 individuals in the F2, as well as trans-homozygous apc6-2/apc6-3 (Figure S6c).

Figure 3.

Box plot comparing seed phenotype frequencies in capulet2 mutants with different APC6 transgene backgrounds.

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Complementation of cap2 with an APC6 transgene restores seed phenotype. Individuals heterozygous for the cap2 SNP (cap2/+) were identified in both a hemizygous and homozygous transgenic background and homozygous individuals (cap2/cap2) were identified in a homozygous transgenic background. A significant reduction of the gametophyte maternal effect seed phenotype frequency is observed for cap2/+ in a hemizygous transgenic background. In a homozygous transgenic background, a significant reduction is observed for cap2/+ and cap2/cap2. Data from independent transgenic lines are merged for each box-plot: 4 independent rescue lines displayed significantly reduced phenotype frequency in a heterozygous APC6-GFP transgenic background, of which 3 segregated individuals with a homozygous construct background. Of these 3 lines, homozygous cap2/cap2 mutants were identified in 2. Statistics was performed according to the Wilcoxon rank sum test; **** P < 0.0001.

Collectively, these data show that all cap2/apc6 mutants can be rescued by a APC6-GFP transgene.

Maternal penetrance and transmission of mutant alleles

We found that when plants carrying the 3 different APC6 mutant alleles were pollinated with WT pollen, >50% of the seeds showed developmental abnormalities (Fig. 2b), suggesting that these mutations were not fully penetrant. Therefore, we investigated in more detail the parental transmission of the different mutant alleles. To this aim, we selfed and crossed cap2/+, apc6-2/+, and apc6-3/+ reciprocally with WT plants and determined the transmission of the mutant alleles by molecular genotyping (cap2 and apc6-3) or T-DNA herbicide resistance (apc6-2) of seedlings (Fig. 4, Data S4). Paternal transmission of cap2 (44.4%), apc6-2 (47.4%), and apc6-3 (46.7%) was not significantly different from the expected Mendelian transmission. Notably, the maternal transmission observed for cap2 (30.5%), apc6-2 (25.1%), and apc6-3 (20.2%) was significantly lower than the expected 1:1 ratio when crossed to WT and also significantly lower than the expected 3:1 ratio in self crosses.

Figure 4.

Transmission frequencies of apc6 mutant alleles from different crosses represented by bar plots, and compared to the expected Mendelian transmission of each cross.

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Genetic analysis shows reduced maternal transmission of cap2, apc6-2 and apc6-3. Mutant alleles were selfed and crossed reciprocally to Col-0 and transmission was determined by genotyping (cap2 and apc6-3) or using T-DNA herbicide resistance (apc6-2). Maternal transmission was significantly reduced while paternal transmission was not significantly different from the expected (50%, dashed line). Selfed transmission frequency for all mutant alleles was significantly lower than expected (75%, dashed line). Statistics was performed according to the Chi square test; ** P < 0.01, *** P < 0.001, **** P < 0.0001.

These data support the finding that maternal but not paternal cap2/apc6 alleles have significantly reduced transmission frequencies, suggesting a maternal bias in the role of APC6.

Expression of APC6 increases with endosperm cellularization

We investigated APC6 expression throughout seed development using publicly available RNA sequencing datasets (Bjerkan et al. 2020). As controls, we used several MADS-box transcription factors known to be expressed only in the endosperm, either bi-parentally or showing parental allelic bias (Colombo et al. 2008; Kang et al. 2008; Shirzadi et al. 2011). We found that APC6 was highly expressed between 1 and 2 DAP, decreasing between 3 and 4 DAP and increasing again at 6 DAP (Figure S7).

Because APC6 shows a dynamic pattern of expression during seed development, we carried out a phenotypic characterization at different stages of seed development (2, 4, 6, and 9 DAP) using cap2/+, apc6-2/+, and apc6-3/+ plants crossed with wild-type Col-0 pollen. Generally, we found that both embryo and endosperm development was delayed compared to wild-type plants (Figure S8) and in this phenotypic class endosperm proliferation was arrested after a few rounds of nuclear division. We also observed another phenotypic class in which the endosperm was not fully cellularized at late stages in seed development (6 to 9 DAP), while embryo development was normal, although delayed, compared to development in wild-type plants (Figure S8). The occurrence of seed phenotypes and collapsed seeds at 4 DAP and 6 DAP was highly similar in all APC6 mutants (Figure S8b, Data S5).

APC6 is a maternally expressed imprinted gene

Because all of the APC6 mutants identified showed maternal gametophyte effects on endosperm development, we hypothesized that this finding could be caused by uniparental expression of APC6 transcripts. To test this hypothesis, we performed reciprocal crosses between different Arabidopsis accessions (Ler-1, Col-0, C24, and Tsu-1) and analyzed the parental allele-specific expression of APC6 in developing seeds by taking advantage of SNPs between these accessions (Fig. 5a, Data S6). We found that at 4 DAP the expression of APC6 was exclusively from the maternal allele in most of the accessions tested. We quantified the maternal:paternal allelic ratios for the different reciprocal crosses and found that the maternal allele was consistently expressed between 8- and 16-fold higher than the paternal allele (Fig. 5b, Data S6).

Figure 5.

Allele specific assays presented for 4 different reciprocal crosses demonstrating maternal or biparental expression of APC6.

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APC6 is a maternally expressed imprinted gene. a) Imprinting analysis of APC6 using accession-specific restriction digestion of SNPs in reciprocal crosses of Col-0, C24, Ler-1 and Tsu-1. cDNA was generated from 4 days after pollination (DAP) crossed seeds. One replicate of homozygous wild-type controls is included as accession-specific cDNA reference and 3 biological replicates for each reciprocal cross are shown. One of 2 bioanalyzer replicates is presented and the accession of the maternal contributor is named first. In all directions, except for C24 as female parent, APC6 is maternally expressed. b) Molarity ratios of maternal and paternal digested fragments from bioanalyzer profiles in a). Arrows point from maternal to paternal ecotype parent. Parental ratios are determined from all biological replicates and both bioanalyzer technical replicates. sd, standard deviation.

To verify these results, we analyzed RNA-seq data of early developing seeds (1 to 6 DAP) from reciprocal crosses between Ler-1 and Col-0 (Shirzadi et al. 2011; Bjerkan et al. 2020). We identified several parental allele-specific SNPs, which we used to determine parental allele expression frequencies (Fig. 6, Data S7). As controls we used MADS-box transcription factors, known to be expressed exclusively in the endosperm and biparentally (AGL62), maternally biased (AGL36) or paternally biased (AGL23) (Colombo et al. 2008; Kang et al. 2008; Shirzadi et al. 2011). We found that during early stages of seed development (1 to 4 DAP), APC6 displays a strong maternal expression bias, similar to AGL36. The maternal bias of APC6 was highly concordant with the molarity ratios determined experimentally (11.1-fold and 8.4-fold, respectively; Data S7). However, whereas AGL36 expression remains maternally biased at 6 DAP, the paternal expression of APC6 increased, which could be attributed to an increasing contribution of the developing embryo.

Figure 6.

Bar graphs depicting maternal bias in APC6 expression in early seed development, compared to control maternally, paternally and biparentally expressed genes.

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Seed stage analysis of parent specific allelic expression of APC6 and imprinting control genes. Frequency of reads mapped to parental alleles of APC6, AGL36 (MEG control), AGL23 (PEG control) and AGL62 (BEG control) at different timepoints (one, 2, 3, 4 and six days after pollination (DAP)). Maternal:paternal read frequency ratios are plotted at respective timepoints. SNP analysis was performed with Geneious Prime version 2021.1.1 using 0.2 as variant (Ler-1) frequency threshold (except AGL23—0.05 variant frequency threshold) and SNPs were weighted according to their coverage.

We hypothesized that the silencing of the paternal APC6 allele may be caused by DNA methylation—a common characteristic of Arabidopsis MEGs (Gehring and Satyaki 2017; Hornslien et al. 2019; Batista and Köhler 2020). To test this hypothesis, we pollinated WT Ler-1 plants with pollen from met1-7/+ and drm1-2;drm2-2;cmt3-11 (ddc/+) plants (Figure S9a and b respectively). Due to possible DNA methyltransferase redundancy (Zhang and Jacobsen 2006), we also generated a quadruple met1-7;drm1-2;drm2-2;cmt3-11 (ddcm/+) mutant and used as pollen donor (Figure S9c). Depletion of DNA methylation in the pollen, using single, triple or quadruple mutants did not reactivate the silenced paternal expression of APC6 (Figure S9a–c). Notably, the maternal:paternal expression ratios for all methyltransferase crosses were similar to the WT controls (Fig. 5b, Figure S9d).

Collectively, these data suggest that APC6 is a maternally expressed imprinted gene and that imprinting of APC6 is not mediated by canonical DNA methylation.

Maternal expression of the APC/C

The APC/C is a large, multisubunit complex consisting of at least 14 subunits in Arabidopsis. These subunits can be divided into groups based on their structure and function, such as scaffolding, catalytic, substrate recognition and platform units (Eloy et al. 2015). APC6 is a scaffolding unit consisting of tetratricopeptide repeats (TPRs), such as APC3a and b, APC7, and APC8. In order to test whether members of other functional groups of the APC/C also display a maternal preference, we investigated APC1, a platform subunit containing proteasome-cyclosome (PC) repeats. APC1 is the largest subunit of the APC/C and has previously been described to be important for female gametogenesis and embryogenesis (Wang et al. 2013).

Heterozygous apc1-2/+ mutants displayed abnormalities in embryo sac formation, but no apparent reduction in seed set was found (Figure S10a, Data S8). When apc1-2/+ was crossed maternally to Col-0, gametophyte maternal effect endosperm phenotypes highly reminiscent of cap2/apc6 were found, both in single apc1-2/+ mutants and in an apc1-2/+;apc6-2/+ double mutant (Fig. 7a). Corresponding to mutants of apc6, apc1-2/+ mutants displayed endosperm defects only when crossed maternally to Col-0, with a phenotype frequency of 37.1% (Fig. 7b).

Figure 7.

Light microscopy images and bar plot comparing seed phenotype and phenotype frequencies of an apc6 and an apc1 mutant, and a panel showing an allele specific assay for APC1 in a reciprocal cross, demonstrating maternal expression.

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APC1 mutants display a cap2/apc6 endosperm phenotype and is a maternally expressed imprinted gene. a) Seed phenotypes of apc1-2/+ and apc1-2/+;apc6-2/+ double mutant at 4 days after pollination (DAP) when crossed maternally to Col-0. A turquoise hue is added to highlight the endosperm, yellow hue highlights the embryo. Arrows indicate small nuclei, arrowheads indicate large nuclei. Scale bar = 20 µm. b) Seed phenotype frequencies of apc1-2/+ crossed reciprocally to Col-0 and apc6-2/+ and apc1-2/+;apc6-2+ crossed maternally to Col-0 at 4 DAP. No seed phenotype is present when apc1-2/+ is crossed paternally, significantly different from when it is crossed maternally. The phenotype frequency of maternally crossed apc1-2/+;apc6-2/+ double mutants is significantly increased compared to both single mutants. Statistics was performed according to Wilcoxon rank sum test; **** P < 0.0001. c) Imprinting analysis of APC1 using accession-specific restriction digestion of SNPs in reciprocal crosses between Col-0 and Ler-1 at 4 DAP. One replicate of each homozygous wild-type control and 3 biological replicates for the reciprocal crosses are shown. One of 2 bioanalyzer replicates is presented and the accession of the maternal cross partner is named first. Mostly maternal APC1 expression was found in both directions of the reciprocal crosses shown.

When crossing the apc1-2/+;apc6-2/+ double mutant maternally to Col-0 we observed a synergistic epistatic effect in which the phenotype frequency of the double mutant (67.1%) was significantly increased compared to both single mutants (Fig. 7b). In addition, transmission of mutant alleles from a selfing apc1-2/+;apc6-2/+ double mutant was investigated. Using the phenotype frequencies of apc1-2/+ and the transmission frequency of apc6-2/+, we calculated the expected transmission frequencies to be 60.9% for the apc6-2 allele and 34.9% for the apc1-2 and apc6-2 alleles together (Data S8). Notably, transmission of the apc6-2 allele deviated from this expectancy, with a significant decrease in offspring carrying the mutant allele (57.4%; Figure S10b). However, the transmission of both mutant alleles together was not significantly different from our expectation.

Last, we investigated whether APC1 expression was maternally biased by genomic imprinting. To this end, we analyzed the parental allele-specific expression of APC1 in developing seeds using different Arabidopsis accessions (Ler-1, Col-0, C24, and Tsu-1) as described previously. In 4 DAP seeds, APC1 was exclusively expressed from the maternal allele in all accessions and reciprocal crosses (Fig. 7c, Figure S10c).

Collectively, these data suggest that both APC1 and APC6 are maternally expressed imprinted genes that are required maternally both in the scaffolding and platform units of the APC/C for early endosperm development, suggesting a maternal bias in the role of the APC/C complex.

Discussion

In this study, we identified that mutation in APC6 is associated with the female gametophyte maternal effect mutant capulet2 (cap2). Molecular characterization of cap2 has revealed that APC6 is a maternally expressed imprinted gene implicated in endosperm proliferation and cellularization. Our data reveals that the maternal contribution of the APC/C is critical for endosperm development in plants.

Capulet2 causative mutation is located in APC6

We have found that cap2 is caused by a splice donor mutation in APC6. We were able to functionally complement cap2, apc6-2 and apc6-3 mutants with an APC6 transgene, allowing the generation of homozygous cap2, apc6-3, and apc6-2/apc6-3 mutant plants. Previous studies have revealed that mutations on several APC/C subunits result in female gametophyte defects (Saleme et al. 2021), including Arabidopsis nomega, that carry a Ds-transposon insertion in APC6 (Kwee and Sundaresan 2003) and a mutant in rice OsAPC6 (Awasthi et al. 2012). We were unable to retrieve the original nomega mutant line and can therefore only speculate on the basis of the reported discrepancy. Under our growth conditions, 3 APC6 mutant alleles—cap2, apc6-2, and apc6-3—exhibit defects exclusively during early seed development. Female gametophytes carrying cap2, apc6-2, and apc6-3 were indistinguishable from the wild type. Although the nomega mutation introduces an earlier translational stop than the alleles characterized here, this mutation is unlikely to account for the phenotypic differences as we did not detect any variation in female gametophyte phenotypes throughout our allelic series. Because our experiments employed different accessions and growth temperatures than those used in the nomega study, we speculate that the discrepancy may arise from accession-specific genetic background effects or temperature dependent properties of the alleles. Both factors present promising avenues for future investigation into APC function.

APC6 is a maternally expressed imprinted gene

Our analysis, using both allele-specific assays and RNA-seq allele-specific expression quantification has revealed that APC6 is an imprinted, maternally expressed gene. However, since we used whole seeds in the analysis, transcripts from the maternal seed coat or embryo could affect this interpretation (Schon and Nodine 2017). To address this caveat, we analyzed publicly available expression data from early developing seeds, which showed that APC6 is 6-fold more highly expressed in the endosperm than the seed coat (Belmonte et al. 2013) and 2.7-fold more highly expressed in the endosperm than in all other seed tissues (Martin et al. 2026). Due to the different genotypes of the endosperm, embryo, and seed coat (2:1; 1:1; and 2:0 maternal:paternal, respectively), a whole seed maternal:paternal ratio of approximately 2:1 can be estimated if APC6 is equally expressed from both parental alleles. The maternal:paternal ratios in our analyses are at least >4-fold higher, ranging up to >50-fold higher, indicating that expression of APC6 is maternally biased in the endosperm. Our data also indicate that imprinting of APC6 is accession specific, a phenomenon that has been observed in previous studies of imprinted genes (Wolff et al. 2011; Pignatta et al. 2014).

It has been proposed that MEGs are regulated by DNA methylation through the activity of MET1 that silences gene expression in gametes and by DEMETER (DME) that removes methylation in the central cell and endosperm, thus restoring the expression of maternal alleles (Choi et al. 2002; Xiao et al. 2003; Gehring et al. 2006). Here, we have investigated the role of DNA methylation in imprinting of APC6 by crossing Ler-1 with various DNA methyltransferase mutants in a Col-0 background and performed SNP digestion analysis to determine if expression of the paternal allele is reactivated. The single, triple and quadruple mutants of met1-7, drm1-2;drm2-2;cmt3-11 and met1-7;drm1-2;drm2-2;cmt3-11 were used to reduce redundancy by other methyltransferases. None of the mutant combinations showed an increased paternal expression and thus we concluded that imprinting of APC6 is not caused by these canonical DNA methyltransferases. This finding is consistent with previous reports in which the majority of MEGs was not regulated by MET1 DNA methylation maintenance (Hornslien et al. 2019). Further analysis of the epigenomic landscape of APC6 is thus required to determine if APC6 imprinting is guided by histone modifications or RNA dependent DNA methylation.

It is experimentally demanding to distinguish de novo transcripts after fertilization from transcripts originating from the central cell as maternal carry over (Evans and Kermicle 2001). The APC6 expression profile is constructed from whole seed RNA (Figure S7), and RNA originating from the seed coat or the embryo is present as discussed above. Compared to the endosperm-specifically expressed AGL36, AGL23, and AGL62, overall expression of APC6 is higher at the early stages (1 and 2 DAP) of seed development and carry over of transcripts from the maternal central cell, as described on a general basis (Luo et al. 2014) cannot be excluded. However, at 4 DAP, allele-specific expression is mostly maternal, supporting the finding that APC6 is imprinted. Long-lived mRNAs in the endosperm have been described for endosperm maturation phase transcripts (Matilla 2022), but it seems unlikely that transcripts, originating from the maternal central cell, are abundant at the globular embryo stage. Moreover, microarray data (Belmonte et al. 2013) and recent single nuclei sequencing of seed tissue (Martin et al. 2026) indicated that expression of APC6 at the globular embryo stage is higher in the endosperm compared to the total of seed coat and embryo expression. Additionally, the increase in paternal expression at 6 DAP could be due to a stronger impact of the growing embryo, as this coincides with consumption of the endosperm (Nowack et al. 2010; Lafon-Placette and Köhler 2014). As such, late-stage expression of APC6 is directed toward a parental expression ratio closer to the 1:1 diploid genotype of the embryo. Altogether, the findings of this study indicate that APC6 is a maternally expressed imprinted gene in the endosperm.

The APC/C is maternally biased

The APC/C is a large protein complex consisting of at least 14 core subunits and is essential for gamete development (Saleme et al. 2021). Genetic analyses have revealed that several subunits of the APC/C are essential for megagametogenesis (APC2, APC3a/APC3b, and APC10 [Capron et al. 2003; Pérez-Pérez et al. 2008; Eloy et al. 2011]) and male gametogenesis (APC8 and APC13 [Saze and Kakutani 2007; Zheng et al. 2011]). Furthermore, overexpression of APC8 displayed reduced seed sets, suggesting that careful dosage regulation of APC subunits is essential for seed development (Zheng et al. 2011). Here, we have demonstrated that both APC6 and APC1 are imprinted and expressed predominantly from the maternal genome. When mutated, both APC/C loci lead to a gametophyte maternal effect phenotype in the developing endosperm, and double mutants suggest an additive effect on seed survival. The strong maternal expression bias of APC1 in all tested ecotypes further emphasizes the importance of maternal control of the APC/C during early seed development. Interestingly, and in addition to APC6 and APC1 mutants, APC4 and APC11 also show gametophyte maternal effect phenotypes (Kwee and Sundaresan 2003; Wang et al. 2012, 2013; Guo et al. 2016), in line with the notion of APC/C being under maternal control.

It remains unclear why subunits of such a crucial protein complex are subjected to parental allele-specific regulation, and the function of genomic imprinting in this regard remains to be determined. Our data support the gene dosage hypothesis for the evolution of genomic imprinting, which states that imprinting evolved as a mechanism to control and regulate gene expression levels (Dilkes and Comai 2004; Ferguson-Smith 2011). For subunits of the APC/C, in which too little or too much expression of a gene is detrimental for seed development, genomic imprinting could be a measure to balance gene expression contributing to this protein complex. Taken together, our observations propose a maternal bias to the control of endosperm proliferation.

Materials and methods

Plant material and growth conditions

Wild-type (WT) accessions Columbia (Col-0), Landsberg erecta (Ler-1), Tsushima (Tsu-1), C24 and T-DNA mutant lines were obtained from the Nottingham Arabidopsis Stock Centre (NASC; Scholl et al. 2000). Seedlings were grown on MS-2 (Murashige and Skoog medium with 2% sucrose) plates in a 16-h-light/8-h-dark cycle at 22 °C for 10 days prior to transferring to soil. Plants were further grown in a 16-h-light/8-h-dark cycle at 18 °C. The T-DNA mutant plant lines apc6-2 (SAIL_442_F11), apc6-3 (SALK_008789), apc1-2 (SALK_059826; g), SALK_002024C (SRF5), SALK_070429C (SRF5), SAIL_1280_D04 (SRF5), SALK_120562 (intergenic), SALK_033225 (intergenic), cmt3-11 (SALK_148381 [Chan et al. 2006]), met1-7 (SALK_076522 [Kanno et al. 2008]), and drm1-2;drm2-2 (N16383 [Chan et al. 2006]) were in the Col-0 accession background (except apc6-3; Col-3). The triple drm1-2;drm2-2;cmt3-11 mutant was crossed with the hemizygous met1-7 mutant to generate a hemizygous met1-7;drm1-2;drm2-2;cmt3-11 quadruple mutant. Methyltransferase mutants were maintained as hemizygous (Mathieu et al. 2007). Endosperm marker lines used in crosses with apc6-2, proAT5G09370>>H2A-GFP (EE-GFP) and proAT4G00220>>H2A-GFP (TE1-GFP) were in the Col-0 accession background (van Ekelenburg et al. 2023). For all crosses, closed flower buds were emasculated 2 days prior to crossing to avoid self-pollination.

Microscopy

Crossed siliques were manually dissected and seeds were mounted on a microscopy slide in a clearing solution of glycerol and chloral hydrate as previously described (Grini et al. 2002). Microscopic analyses were performed using an Axioplan2 Imaging microscope equipped with a Zeiss Axio cam HDR camera. For fluorescent phenotypic analyses, seeds were mounted on a microscopy slide in 2 drops of tap water or 0.1% Tween in 1 × phosphate buffered saline (PBS; pH 7.4) or 30% glycerol with 20 μg/mL propidium iodide (PI). Seeds were analyzed for GFP fluorescence using an Andor DragonFly Spinning Disk confocal microscope with a 488 nm wavelength diode laser (Oxford) and imaged with a Zyla4.2 sCMOS camera (Oxford).

DNA isolation and whole genome sequencing

Leaf and flower tissue from 12 individual cap2/CAP2 (cap2mut) and 12 CAP2/CAP2 (cap2wt) plants was collected in duplicate in a 2-mL round bottom tube containing a 5 mm metal bead and flash-frozen in liquid nitrogen. The frozen tissue was ground using a Retsch homogenizer for 1 min at 30 s−1. Genomic DNA (gDNA) was isolated as described in the ChargeSwitch gDNA Plant Kit (Invitrogen) manual and gDNA was eluted in 40 μL 10 mM Tris buffer (pH 8.5 at room temperature). Isolated gDNA was quality checked by Nanodrop and DNA concentrations were determined with the Qubit dsDNA BR Assay Kit (Life Technologies). DNA libraries were prepared using the KAPA Hyper Kit (Roche) with 24 Unique Dual Indexes (Illumina) and sequenced 150 bp paired-end over 2 lanes on the Illumina HiSeq 4000 system.

Identification of the cap2 causative single nucleotide polymorphism

Sequencing reads were preprocessed using fastp with default settings and the overrepresentation analysis parameter to remove reads with low quality score, irregular GC content, short length, and present sequencing adapters (Chen et al. 2018). Trimmed reads were then quality checked with outputs from fastp to ensure that trimming had been successful. Trimmed reads were mapped to the TAIR10 reference genome using bowtie2 (Langmead and Salzberg 2012). Variant calling was performed on aligned and sorted BAM files using samtools mpileup and piped to bcftools to produce VCF files (Li et al. 2009). VCF files were converted to SHOREmap format and analyzed using SHOREmap backcross and then annotated to output tables and plots of backcross SNPs (Sun and Schneeberger 2015).

RNA isolation and cDNA synthesis

RNA was isolated from plant tissue (seeds, seedlings or flowers) as described in the Spectrum Total Plant RNA Kit and On-Column DNaseI Digestion Set (Sigma Aldrich) manual. The tubes were shaken in a MagNA Lyser Instrument (Roche) at 7000 rpm for 15 sec, centrifuged at 13,000 rpm in 4 °C for 15 sec, and then placed at −20 °C for 2 min. This procedure was repeated 3 times before proceeding with the protocol. cDNA was synthesized from 2.5 µL RNA as described in the SuperScript III Reverse Transcriptase (Invitrogen) manual and cDNA was purified using Wizard SV Gel and PCR Clean-Up System (Promega). Verification of transcripts from mutant alleles was performed by sequencing of PCR products amplified from cDNA as specified in Table S3.

Single nucleotide polymorphism analysis

Single nucleotide polymorphisms (SNPs) between accessions were identified using GabiPD GreenCards (The GABI Primary Database) or Polymorph 1001 (1001 Genomes) and SNPs were verified by sequencing. Parental-specific expression based on these SNPs was determined by restriction digestion. For APC1, an SNP at position 6551 (G in Ler-1; A in Col-0, Tsu-1 and C24) gains a restriction site for AlwI in the Ler-1 accession. For APC6, an SNP at position 1297 (C in Ler-1; G in Col-0, Tsu and C24) results in the loss of the restriction recognition site for SacII for the Ler-1 accession. No restriction recognition site was identified covering the SNP at position 1516 (T in Col-0; A in C24), and therefore a recognition site for BgIII was introduced in the C24 accession using the Derived Cleaved Amplified Polymorphic Sequences (dCAPS) Finder 2.0 tool (Neff et al. 2002). The WT accessions Col-0, Ler-1, Tsu-1, and C24 were crossed reciprocally and Ler-1 was crossed maternally to met1-7/+, ddc/+, and ddcm/+. Siliques were dissected at 4 DAP using a stereomicroscope, and seeds harvested in MagNA Lyser Green Beads tubes (Roche) tubes were collected into liquid nitrogen. Seeds from 3 mother plants, 4 siliques each, were pooled for each biological replicate. RNA isolation and cDNA synthesis were performed as described above.

The SNP-containing DNA region was amplified by PCR with TaKaRa Ex Taq DNA Polymerase on 50 ng cDNA template using specific primers (Table S3), and PCR products were verified by sequencing. Restriction digestion was performed on a 400 ng PCR product with 10 U of enzyme in a final volume of 25 µL. Equal amounts of digested fragments were analyzed using DNA-1000-LabOnChip and 2100 Bioanalyzer (Agilent Technologies).

Molecular cloning and genotyping

Genotyping was performed using the KAPA3G Plant PCR kit (Roche) and gene- and T-DNA–specific primers (Table S3). PCR products were sequenced by Eurofins Genomic and analyzed using Geneious software. To construct the proAPC6:APC6-GFP plasmid, a genomic fragment of the APC6 gene, including 2 kb of the 5′-upstream sequence, was amplified by PCR and cloned between the AscI and SmaI sites of the pGFP_NOSG vector (Iwase et al. 2017). The resulting construct was then recombined into pGWB601 vector (Nakamura et al. 2010) using LR Clonase II. Genetic constructs were transformed into Agrobacterium tumefaciens C58 strain and introduced into capulet2, apc6-3 and WT accession Ler-1 using the floral dip method (Clough and Bent 1998).

RNA sequencing analysis

Raw reads from RNA Seq data were obtained from online resources (Bjerkan et al. 2020). Trimmed reads were obtained using Cutadapt version 1.18 (Martin 2011), version TrimGalore 0.6.2 (Krueger 2012), and mapped to the Araport11 CDS reference transcriptome (Cheng et al. 2017) using Bowtie version 2.3.5.1 (Langmead and Salzberg 2012) with the parameters –no-unal –no-mixed –no-discordant –sensitive –end-to-end -k 1. Libraries were normalized using DESeq2 version 1.30.1 (Love et al. 2014). Statistical analysis was performed using “plyr” version 1.8.6 (Wickham 2011) and visualized using RStudio with “ggplot2” version 3.3.5 (Wickham 2016).

For the analysis of parental ratios, mapped reads were extracted using SAMtools version 1.9 (Li et al. 2009), and SNP counting was performed in Geneious Prime version 2021.1.1 (http://www.geneious.com/) using 0.2 as the variant (Ler-1) frequency threshold (except AGL23—0.05 variant frequency threshold). Reads from all timepoints were merged and aligned to the Col-0 reference sequence. SNP counting was performed to present an overview of reliable SNPs between Ler-1 and Col-0 for each gene. The allele frequencies of each SNP for each timepoint (>10 coverage per SNP) were weighted for the coverage and visualized in Rstudio with “ggplot2” version 3.3.5 (Wickham 2016) and “ggpattern” version 0.2.0 (Mike and Davis 2022).

Image analysis and figure preparation

Images were processed using Fiji (Schindelin et al. 2012). Figures were assembled in Adobe Illustrator 2022 (Adobe Systems Incorporated, San Jose, USA).

Accession numbers

All sequences generated in this study have been deposited to the National Center for Biotechnology Information Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra) with project number PRJNA808889.

Supplementary Material

kiag647_Supplementary_Data
kiag647_supplementary_data.zip (45.2MB, zip)

Contributor Information

Yuri S van Ekelenburg, Section for Genetics and Evolutionary Biology, Department of Biosciences, University of Oslo, Oslo 0316, Norway.

Ida V Myking, Section for Genetics and Evolutionary Biology, Department of Biosciences, University of Oslo, Oslo 0316, Norway.

Cathal Meehan, School of Life Science, University of Warwick, Coventry CV4 7AL, United Kingdom.

Morten P Hauger, Section for Genetics and Evolutionary Biology, Department of Biosciences, University of Oslo, Oslo 0316, Norway.

Shinichiro Komaki, Nara Institute of Science and Technology, Division of Biological Science, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan; School of Biological Science and Technology, College of Science and Engineering, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan.

Keiko Sugimoto, RIKEN, Center for Sustainable Resource Science, Yokohama, Kanagawa 230-0045, Japan.

José Gutierrez-Marcos, School of Life Science, University of Warwick, Coventry CV4 7AL, United Kingdom.

Paul E Grini, Section for Genetics and Evolutionary Biology, Department of Biosciences, University of Oslo, Oslo 0316, Norway.

Author contributions

P.E.G. designed the research; Y.S.vE., I.V.M., M.P.H., S.K. & C.M. performed the experiments; Y.S.vE., I.V.M., C.M., J.G.-M., and P.E.G analyzed and discussed the data; Y.S.vE., I.V.M., J.G.-M., and P.E.G. wrote the article. All authors revised and approved the article.

Supplementary material

Supplementary material is available at Plant Physiology online.

Funding

This work was supported by the Norwegian Research Council (FRIPRO grant no. 276053 to P.E.G.).

Data availability

All sequences generated in this study have been deposited to the National Center for Biotechnology Information Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra) with project number PRJNA808889.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

kiag647_Supplementary_Data
kiag647_supplementary_data.zip (45.2MB, zip)

Data Availability Statement

All sequences generated in this study have been deposited to the National Center for Biotechnology Information Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra) with project number PRJNA808889.

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