cdka;1 is a tool to identify key seed regulators

Here we have used cdka;1 as a tool to identify factors sensitive to the vital parental gene balance in the endosperm. In heterozygous cdka;1 mutants, the second pollen mitosis is either missing or is severely delayed. However, mutant pollen can successfully fertilize the egg cell while leaving the central cell unfertilized [40], [41]. A detailed analysis by Aw and colleagues has revealed that a second sperm cell is delivered to the central cell, but that karyogamy does not take place [42]. Although not properly fertilized, the majority of the central cells in cdka;1 fertilized ovules (70–90%) are triggered to initiate endosperm proliferation [40], [42], [43]. Thus, fertilization by cdka;1 sperm cells creates a unique situation where endosperm initially develops without any paternal contribution (in the following also referred to as cdka;1^P). The endosperm, however remains under-developed, and ultimately the seed aborts, further demonstrating the importance of the paternal contribution to the endosperm for proper seed development. Since activation of maternal alleles by loss of maternal FIS PRC2 could rescue seed lethality [43], we hypothesized that the disturbance of parental gene balance in the endosperm is the main cause leading to developmental arrest of cdka;1^P at 3–4 days after pollination (DAP).

To identify factors and mechanisms sensitive to such an imbalance in gene dosage in the endosperm and with that likely key regulators of seed development, we performed microarray transcript profiling of cdka;1 fertilized seeds at 3 DAP (Figure S1A). Due to the heterozygous nature of the cdka;1 mutant line used, a transcript that is absent in cdka;1^p seeds will lead to a reduction of maximal 50% in the genome profiling experiment. For example, genes that are only expressed from the paternal genome would show such reduced expression levels (Figure S1B). Likewise, maternally expressed genes that require activation by a paternally expressed gene(s) would be downregulated (Figure S1C), whereas genes that are acted upon by paternally expressed repressors were expected to be upregulated in the microarray screen (Figure S1D).

When we compared the transcriptional profiles of Ler x cdka;1 versus Ler x Col seeds 3 DAP, we detected 17223 nuclear genes that were expressed in all biological replicates of both mutant (cdka;1 set) and wild-type (WT set) seed profiles. Our result is in good agreement with a set of genes identified by Goldberg & Harada laboratories (GH) in globular stage seeds of Arabidopsis Ws-0 plants as 68% of our genes were also identified by GH, and our gene set included >90% of the GH globular seed gene set (Figure 1A; http://seedgenenetwork.net, [44]).

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Figure 1. Analysis of cdka;1 microarray profiles.

(A) Venn diagram representing overlap of genes expressed in globular stage seeds of Arabidopsis Ws-0 plants (red) and genes expressed in 3 DAP seeds from Ler plants pollinated with Col cdka;1 pollen (green). As high as 67.8% of the cdka;1 set and 90,7% of the GH set genes were found in the overlap. Gene numbers refer to the reference set of genes (see material and methods). GH: Goldberg & Harada laboratories (http://www.seedgenenetwork.net). (B) Boxplot showing the reduced relative expression of known maternally imprinted (blue background) and paternally imprinted (pink background) genes in the Ler x cdka;1 versus Ler x Col seeds. Calculations are based on values taken from three independent biological replicas. (C) GO functional classification of microarray expression data. Deregulated genes identified in the microarray experiment were functionally classified regarding their molecular function using the GO Slim classification system (http://www.arabidopsis.org/tools/bulk/go/). The total number of unique GO-term:locus assignments for each group is indicated (n). The functional classifications of all genes present on the microarray (On microarray) and all genes having a present call (All expressed) have been included for comparison. The cut-off for deregulation is ≤0.8 for the downregulated group, and ≥1.5 and ≥1.2 for the upregulated groups.

https://doi.org/10.1371/journal.pgen.1001303.g001

To further validate the quality of our dataset, we examined the expression pattern of genes known to be preferentially expressed from the paternal allele. To date, only three genes have been identified that show a predominant paternal expression pattern; PHE1, HDG3 and At5g62110, where all three genes were found to be downregulated in our arrays (Figure S1E), supporting our working hypothesis that paternally expressed genes can be detected amongst downregulated genes. In addition, out of seven imprinted maternally expressed genes present in our microarray sets, four were also detected as downregulated (Figure S1E). This could reflect required activation by paternal factors (Figure S1C), or be a result of more complex deregulation in response to change in gene dosage. To exclude array artifacts we tested all down-regulated genes by means of real-time PCR and could confirm their deregulation (Figure 1B).

Due to the background noise in the microarray experiment, modest but reproducible downregulation of arithmetic ratios (ar) ranging from 0.5 to 1.0 will produce False Discovery Rates (FDR, see materials and methods) with insignificant q values. Since the absence of paternally expressed genes was the simplest hypothesis to account for downregulation, we defined a functional limit for screening purposes that allowed us to detect two out of three known paternally expressed genes in the array. Both PHE1 and HDG3 are detected at q values of 0.35 and a downregulation cutoff of 0.8 (ar). Consequently these values were chosen and used to filter the microarray data.

Using these criteria, a set of 602 genes was extracted (q≤0.35 and ar ≤0.8), subsequently called Down 0.8. For upregulation, we worked with two gene sets. For the first set, Up 1.2, we used parameters equivalent to the downregulated set (q≤0.35 and ar ≥1.2), which resulted in a set of 1030 genes. For the second set, Up 1.5, resulting in 323 genes, we chose ar ≥1.5, a threshold for deregulation commonly used in genome-wide expression studies (Table S3).

To test whether the deregulated genes could preferentially be attributed to a certain seed structure, we compared our data to gene sets expressed in different seed regions and compartments of globular stage seeds using data generated by Goldberg & Harada (GH) laboratories available at http://seedgenenetwork.net [44]. The overlap between the upregulated gene sets and the GH embryo, seed coat and endosperm was significantly lower than expected for independent sets of genes, indicating that among the upregulated genes we preferentially find those that are below the detection limit of the GH analyses. However looking at the downregulated genes, the picture was different. While we found slightly less overlap than expected by chance for the GH embryo set, the overlap was clearly larger than expected by chance for GH seed-coat (1.2<2.7e⁻⁰⁷) and even more significant for the GH endosperm (rf  = 1.3, p<2.0e⁻¹³, Figure S2A, S2B).

Lack of the paternal genome results in the downregulation of a group of MADS-box Type-I Mγ transcription factors

In order to functionally classify the deregulated gene sets according to their molecular functions we used the GO Slim classification system (Figure 1C). Only for the GO Slim term “Transcription factor activity” we find a higher percentage and significant over-representation of both up- and down-regulated groups when compared to all genes on the array/all genes expressed. Since key regulators of seed development are likely to be transcription factors (TF), we analyzed this class in detail.

When comparing the fraction of deregulated genes among the different TF families, the Mγ MADS-box transcription factors clearly stood out with more than 60% of the seed expressed members being downregulated in Ler x cdka;1 arrays (Figure S3A, S3B). We therefore focused on this MADS Type-I class for further analysis. Searches in publically available expression databases (www.genevestigator.com, Figure S4) revealed that all identified genes were exclusively expressed in the seed and predominantly in the endosperm. From the identified Type-I Mγ MADS-box genes, we selected AGL36 for further in depth analysis (Figure S4). AGL36 was the previously undescribed Mγ candidate that interacted with the highest number of described AGLs in a Y2H screen performed by de Folter et al [45]. Both AGL36 and PHE1 have been shown to interact with AGL62, which plays a major role in endosperm development [45], [46]. Within the Mγ class, AGL36 clusters together with AGL34 and AGL90 [47], which are both also detected as downregulated in our microarray experiment (Figure S4). AGL36 shares 85.7% and 84% nucleotide identity with AGL34 and AGL90, respectively (Figure S8). On the amino acid level this results in of 80.2% similarity of AGL36 with AGL34 and 83.9% similarity with AGL90.

AGL36 is only expressed from its maternal allele

Real-time PCR measurement of AGL36 relative expression level three days after pollination (3 DAP) in Ler ovules fertilized with either Col or cdka;1 pollen confirmed that AGL36 expression was reduced in cdka;1 fertilized seeds, (27% when normalized towards ACT11, and 36% when normalized towards GAPA) compared to wild-type seeds (Figure 2A).

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Figure 2. AGL36 is only expressed from the maternal genome.

(A) Real-time PCR analysis showing AGL36 expression in Ler x cdka;1 relative to Ler x Col seeds 3 DAP. Gray bars represent Ler x Col expression levels, black bars represent the Ler x cdka;1 expression levels. Left section: AGL36 normalized to ACT11 levels. Right section: AGL36 normalized to GAPA levels. Average values from three independent biological replicas are shown. Error bars indicate standard deviation (STDEV). (B) Schematic overview of AGL36 SNP analysis. The presence of a SNP between Col and Ler ecotypes (C-T conversion respectively) allows the amplified AGL36 cDNA PCR product from the Col ecotype to be digested with AlwNI restriction enzyme, while the Ler ecotype remains undigested. (C) AGL36 is maternally expressed. Seeds obtained from Col x Ler and Ler x Col crosses were harvested at 3 DAP followed by AGL36 RT-PCR, AlwNI digestion and subsequent Bioanalyzer analysis. Genomic Col and Ler were included as controls (Left section, first two lanes). Digestion products of two independent biological replicas of maternal Col x Ler pollen crosses produced only Col bands, indicating maternal expression (Middle section). Similarly, the digestion products of two independent biological replicas of maternal Ler x Col pollen produced only Ler bands, indicating maternal expression (Left section). The intensities of the bands are represented as concentrations (nmol/L), and create a basis for comparison. 100 ng DNA was used as template for each PCR reaction.

https://doi.org/10.1371/journal.pgen.1001303.g002

To determine whether AGL36 has parental-specific expression, we took advantage of an AGL36 Single Nucleotide Polymorphism (SNP) existing between the Col and Ler ecotypes. This SNP allows the PCR product of Col cDNA to be digested by AlwNI, leaving the Ler cDNA PCR product intact (Figure 2B). We performed reciprocal crosses between Col and Ler ecotypes, and analyzed the digested RT-PCR fragments on an Agilent Bioanalyzer Lab-on-a-Chip, allowing accurate measurement of fragment sizes and their concentrations. When Col^maternal is crossed with Ler^paternal, we only detected the Col bands (165 bp+234 bp) after AlwNI digestion, indicating only maternal expression (Figure 2C). Similarly, in the reciprocal cross when Ler^maternal is fertilized with Col^paternal pollen, the cDNA PCR digest resulted only in an undigested band (399 bp) originating from Ler, indicative of maternal expression (Figure 2C). This testified that AGL36 was only expressed from the maternal genome after fertilization and thus identified as a novel imprinted gene.

AGL36 is imprinted throughout early seed development

AGL36 expression level in wild-type seeds (Ler x Col) at different stages of seed development was monitored over a period of 12 days after pollination. Initially, a low expression level was detected (1 DAP), followed by a rapid increase and subsequent peak in AGL36 expression at 4 DAP, when the embryo is at the late globular stage of development, before declining (Figure 3A). At the embryo heart stage, corresponding to 6 DAP, AGL36 expression had decreased to similar levels as 1 DAP. To address whether AGL36 imprinting is maintained throughout its expression cycle, we performed a SNP analysis of the RT-PCR product obtained from Ler x Col crosses harvested during 1 to 12 DAP (Figure 3B). We found that AGL36 expression is originating from the maternal genome (Ler) throughout the experiment. By plotting the molarities of the maternal band obtained by Agilent Bioanalyzer, an expression profile closely identical to the pattern obtained in the real-time PCR analysis was found (Figure 3C).

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Figure 3. AGL36 is imprinted throughout its expression cycle.

(A) AGL36 expression profile. Calculations were done using 3 DAP values as reference point, giving other obtained expression values relative to the 3 DAP expression level. Samples were taken at 1, 2, 3, 4, 6, 9, and 12 DAP. The graph represents the average relative expression values obtained from two independent biological parallels where the RNA from each biological sample gave rise to two independent cDNA syntheses (technical replica). The indicated STDEV is derived from the two independent biological parallels. The AGL36 transcript levels were normalized to ACT11 levels. (B) RT-PCR digest of the SNP containing region analyzed by the Bioanalyzer show that AGL36 imprinting is maintained throughout seed development. Samples were taken at time-points as indicated for each lane. A representative light micrograph of each DAP stage is shown. Only maternal (Ler) AGL36 expression was found when present. Genomic Ler and Col DNA were included as controls. 100 ng DNA/cDNA was used as template for each PCR reaction. The intensities of the bands are represented as concentrations (nmol/L). Note, weak paternal bands obtained at 2 DAP were below the detection limit for measurement on our instrument (0.1 ng/µl∼0.4 nmol/L). Intensities below the detection point of the instrument are indicated as b.d. The displayed SNP picture is representing one of four independent runs (2BR and 2TR). (C) Visual representation of the obtained intensities of maternal bands in B) represented as concentrations (nmol/L).

https://doi.org/10.1371/journal.pgen.1001303.g003

To rule out that the observed maternal expression is due to expression of AGL36 in the ovule integument, which is a maternal tissue, we generated a reporter construct consisting of 1752 bp of the AGL36 promoter region fused to a GUS reporter (pAGL36::GUS) (Figure 4A). Single-copy lines carrying this construct were used in reciprocal crosses with wild-type Ler and Col plants to examine GUS expression at 3 and 6 DAP. When inherited maternally, pAGL36::GUS expression in the seed was indeed found to be restricted only to the fertilization product (Figure 4B, Figure S7D). In the reciprocal cross, when pAGL36::GUS was inherited from the paternal genome, no GUS expression was detected, (Figure 4C, Figure S7E). Consistent with the SNP analysis, this demonstrated that AGL36 was imprinted and only maternally active throughout its expression cycle. Furthermore, the 1.7 Kb promoter fragment used in this analysis appears to be sufficient to confer parent-of-origin specific expression of the reporter.

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Figure 4. pAGL36::GUS is expressed only from its maternal allele (and only in the fertilization products).

(A) A pAGL36::GUS construct was generated using 1752 bp of the promoter region of AGL36 that spans the ATG start codon. Transposable element sequences (hAT, Helitron, Arnoldy, DNA transposons, MuDR and Pogo transposons) in 5′and 3′regulatory regions are color coded as indicated. The numbers indicate the positions on Chromosome 5 (http://gbrowse.arabidopsis.org). Note, Helitron and Arnoldy transposable elements in the pAGL36::GUS promoter region. Plants expressing the transgene were used either as maternal (B) or paternal (C) partners in crosses with wild-type plants. Samples were taken at 3 DAP (left panel) and 6 DAP (right panel). pAGL36::GUS is absent in the seed-coat and only maternally expressed in the endosperm. The pAGL36::GUS signal is increased in 6 DAP versus 3 DAP samples.

https://doi.org/10.1371/journal.pgen.1001303.g004

AGL36 is not required for seed survival

To further investigate the biological function of AGL36, we screened the Koncz T-DNA collection for insertions [48]. We identified a mutant line, agl36-1, harboring a single T-DNA insertion 16 bp upstream of the AGL36 ATG start codon (Figure S5A). The agl36-1 line showed Mendelian segregation of the T-DNA insertion, as 75% of the plants were resistant to Hygromycin (N = 1025, χ² = 0,83, Table S1).

To test the transmission through the male and female gametes directly, reciprocal crosses of both hemizygous and homozygous agl36-1 mutant plants with wild-type plants were performed (Table S1). In a reciprocal cross, a hemizygous mutant will segregate 50% of the T-DNA resistance marker if the disrupted gene is not vital for gametophyte transmission or function. Thus, gametophyte requirement can be scored directly as reduced frequency of resistant plants [49]. In reciprocal crosses with agl36-1, no transmission distortion through female or male gametophytes could be observed (N = 661, χ² = 0,13 and N = 1015, χ² = 0,00 respectively, Table S1).

The position of the T-DNA insertion in agl36-1 predicts AGL36 expression failure, and indeed real-time PCR analyses of 3 DAP seeds of homozygous agl36-1^−/− plants compared to Col wild-type indicate a 1000-fold AGL36 downregulation in the mutant seeds (Figure S5B). In line with an imprinted and maternal-only expression of AGL36, close to 50% reduction of the transcript level was observed in 3 DAP hemizygous agl36-1^+/− seeds (Figure S5B). We thereby concluded that agl36-1 represents a loss-of-function allele of AGL36.

Although depletion of AGL36 did not interfere with the fitness of the mutant allele in our experimental system, we have shown that AGL36 is specifically expressed from the maternal allele in the fertilization product, in a time frame between 2 and 6 DAP. To investigate whether this was reflected morphologically or developmentally in the developing seed, we compared embryo and endosperm development in wild-type and homozygous agl36-1^−/− seeds within the AGL36 expression time frame.

After fertilization of the egg and the central cell, the endosperm in Arabidopsis undergoes three syncytial rounds of nuclear divisions before the first asymmetric division of the zygote that creates the apical embryo proper and the basal suspensor that connects the embryo proper and the maternal tissue (Figure S5C). At the 2 DAP stage, no obvious difference could be observed between wild-type and agl36-1^−/− seeds, both typically harboring a 1–2 cell embryo proper and a 16–32 nucleated endosperm (Figure S5C, left section). The embryo continues to divide through radial, longitudinal and transverse divisions to produce the so-called globular stage at 4 DAP (Figure S5C, middle section). The endosperm also undergoes 3–4 syncytial nuclear divisions and remains uncellularized as cell proliferation at the upper half of the embryo forms the cotyledon primordia at the so-called heart stage at 6 DAP (Figure S5C, right section). Although the main AGL36 expression peak occurs during this time frame, no obvious deviation between wild-type and agl36-1^−/− could be observed at these stages. Similarly, using an endosperm specific pFIS2::GUS reporter [33], a wild-type endosperm division pattern was observed in agl36-1^+/− seeds (Figure S5D).

MET1 is required for AGL36 imprinting

The majority of imprinted, maternally expressed genes identified in Arabidopsis so far have been shown to be paternally silenced by mechanisms involving symmetric CG methylation, maintained by MET1 [11], [18], [19]. Although not directly linked to imprinting, methylation can also be directed by CHROMOMETHYLASE 3 (CMT3) that has specificity for CNG, and members of the DOMAINS REARRANGED METHYLTRANSFERASE (DRM) family; DRM1 and DRM2, that are mainly responsible for asymmetric CHH methylation [50]. In order to address the involvement of DNA methylation in the regulation of paternal AGL36 expression, we performed SNP analyses of 3 DAP ovules from reciprocal crosses with mutants that have been shown to be involved in DNA methylation. In the SNP RT-PCR analysis of mutant pollen crossed to wild-type, paternal AGL36 expression is expected if the tested mutants are involved in AGL36 imprinting.

CMT3 DNA methylation has been reported to be guided to specific sites by KRYPTONITE (KYP) H3K9 methylation [51]. When mutant cmt3-7 and kyp-2 pollen were crossed to Col wild-type plants, no difference in AGL36 expression was observed (Figure 5A). In the reciprocal cross with cmt3-7 also no difference could be detected compared to wild-type expression (Figure S6).

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Figure 5. The effect of DNA methylation on parental AGL36 expression.

(A,B) SNP analyses of 3 DAP seeds from crosses with DNA methylation mutants. The amplified SNP containing regions of AGL36 and FWA cDNA were digested with AlwNI and NheI, respectively, and analyzed in a Bioanalyzer. (A) Homozygous cmt3-7, kyp-2, and ago4-1 mutants in the Ler ecotype were used as pollen donors to pollinate Col plants, while homozygous drm1;drm2 and heterozygous ddm1-2 mutants in the Ws-2 and Col ecotype respectively, were used as male to fertilize Ler ovules. No paternal AGL36 expression could be detected in these crosses. (B) Upper panel: Hemizygous met1-4^+/− in the Col ecotype reciprocally crossed with Ler wild-type plants show that the paternal allele of AGL36 is expressed when met1-4 is crossed as male. No paternal bands are observed when met1-4 is used as the maternal cross partner. Using pollen from first generation met1-4^−/− homozygous plants in a Ler x met1-4^−/− cross gives rise to prominent bands of the digested paternal Col expression product. In the reciprocal met1-4^−/− x Ler cross no paternal expression can be detected. Lower panel: FWA control using the same tissue as above showing imprinted expression of FWA in dependence of MET1. Paternal FWA expression was observed when plants hemizygous and homozygous for met1-4 were crossed as male to Ler wild-type.

https://doi.org/10.1371/journal.pgen.1001303.g005

DRM1 and DRM2 are mainly responsible for asymmetric DNA CHH methylation [50] and rely on small interfering RNAs, processed by ARGONAUTE4 (AGO4), for target template guidance [52]. In our assays, fertilization by pollen lacking DRM1;DRM2 and pollen lacking AGO4 had no effect on the AGL36 expression pattern (Figure 5A). Likewise, AGL36 expression in the reciprocal cross was identical to wild-type (Figure S6).

DECREASE IN DNA METHYLATION1 (DDM1) is involved in maintenance of DNA methylation [53]. In our SNP RT-PCR analyses where mutant ddm1-2 pollen was used to fertilize wild-type ovules, paternal AGL36 expression was not activated (Figure 5A). In summary, CMT3, KYP, DRM1;DRM2, AGO4 and DDM1 appear not to be involved in the establishment nor maintenance of AGL36 imprinting (Figure 5A, Figure S6).

However, paternal AGL36 expression was detected when plants hemizygous for the met1-4 mutation were used as pollen donor in crosses with wild-type Ler (Figure 5B). In the reciprocal cross, using met1^+/− as the maternal partner, no AGL36 expression from the paternal genome could be observed (Figure 5B). Furthermore, we performed crosses using pollen from homozygous met1-4 parents. When first generation homozygous met1 plants were used as pollen donor on wild-type plants, prominent AGL36 expression from the paternal Col genome could be observed (Figure 5B). This strongly suggests that the repression of the paternal copy of AGL36 is lifted due to the met1-4 mutation, and that MET1 is required for maintaining paternal inactivation of AGL36. In the reciprocal crosses, only expression from the maternal genome could be detected, both in the heterozygous and the homozygous met1-4 situation, further substantiating the requirement of MET1 in the male germline in order to maintain AGL36 imprinting (Figure 5B). Maternal AGL36 expression levels using homozygous met1-4 as the maternal cross partner appeared to be equal to maternal levels in the reciprocal crosses (Figure 5B). This opens for the interpretation that DNA methylation is not required for the regulation of maternal AGL36 expression.

Silencing of vegetative AGL36 expression involves MET1

In public expression databases, AGL36 is reported to be expressed in the seed and more precisely in the endosperm [54] (Figure S4). In order to monitor AGL36 expression in vegetative tissues and its dependence on DNA methylation, we performed a real-time PCR experiment on vegetative tissues from reciprocal Ler x Col crosses and homozygous met1-4 tissues. In biological replicates of progenies from both reciprocal crosses, weak AGL36 expression ranging from 1–6% of the seed expression level could be detected in seedlings, leaves and flowers (Figure 6A). This showed that AGL36 was expressed throughout the plant life cycle, although at very low levels. In the same experiment, we monitored expression in met1-4 tissues. AGL36 expression levels were 50–90-fold higher in met1-4 leaves compared to seed expression levels (Figure 6A). In a direct comparison, expression levels were elevated 2000-fold in homozygous met1-4 leaves compared to wild-type Col x Ler leaves (Figure 6B). In flowers, the upregulation was more than 20-fold in met1-4 compared to wild-type Col x Ler flowers (Figure 6C). In conclusion, these data showed that silencing of AGL36 in vegetative tissues involves MET1, suggesting that the absence of maintenance DNA methylation elevates vegetative AGL36 expression beyond the maternal expression levels found in seeds.

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Figure 6. AGL36 expression is controlled by MET1 and DME.

(A) Real-time PCR of AGL36 expression levels in seeds, seedlings, leaves and flowers in F1 progenies of Ler x Col (gray bars), and Col x Ler (black bars) plants. AGL36 expression level in leaves and flowers of met1^−/− selfed plants in the Col ecotype are shown in the two rightmost bars. All expression levels shown are relative to the AGL36 expression levels in 3 DAP seeds. (B–C) Expression levels of AGL36 in leaves (B) and flowers (C) of met1^−/− plants (Col) relative to expression level in F1 progenies of Col x Ler plants. (A–C) represent the average relative expression values obtained from two independent biological parallels (BR) where each gave rise to four independent cDNA syntheses (TR). STDEV is derived from the two BRs. ACT11 is the reference gene used. (D) Parental dependence of AGL36 expression in seeds and flowers. SNP analyses of 3 DAP seeds (left) and F1 hybrid flowers (right) obtained from Col x Ler and Ler x Col crosses. The amplified SNP containing region of AGL36 was AlwNI digested and analyzed in a Bioanalyzer. Seeds express AGL36 only from the maternal genome (left). Flowers express AGL36 biparentally (Note both Ler and Col bands (right)). The electropherograms represent one BR. Peaks are representing the bands shown in the graph. Asterisk; digested Col product, Arrowhead; undigested Ler product. (E) Real-time PCR expression levels of FWA, FIS2 and AGL36 in dme-6 x Col vs. wild-type seeds 3 and 6 DAP. Graphs represent the average relative expression from four independent BRs. Values for FIS2 are calculated based on 3 BRs as the value for the fourth BR was clearly out of range. Samples used in the first BR gave rise to two TRs. STDEV is derived from the independent BRs. ACT11 is the reference gene used.

https://doi.org/10.1371/journal.pgen.1001303.g006

AGL36 is biparentally expressed in vegetative tissues

In order to investigate the parental expression pattern of AGL36 in vegetative tissues, we performed SNP analyses of flowers from F1 hybrids of Ler and Col reciprocal crosses. In both reciprocal crosses, AGL36 appeared to be expressed equally from the parental Ler and Col genomes, indicating biparental expression in flowers (Figure 6D). This indicates that parental-specific expression, i.e. imprinting of AGL36, as expected, only takes place in the seed and that a low basal biparental expression is present throughout the plant life cycle. Interestingly, biallelic expression in flowers suggests that further silencing of AGL36 takes place in the male germline before uniparental expression in the seed (Figure 6D).

AGL36 is controlled by DEMETER

According to our data, the action of MET1 suppresses AGL36 expression throughout the vegetative phase and this suppression is maintained in the fertilization product through the male germline. AGL36 imprinting thus requires specific activation of the maternal allele. DNA demethylation by DME has previously been shown to mediate maternal-specific gene expression in the endosperm [11], [18], [19], [24], and we therefore investigated AGL36 expression in dme-6 mutant plants. Since dme cannot be maintained in a homozygous state, we harvested siliques of dme-6^+/− heterozygous plants pollinated with Col pollen at 3 and 6 DAP. We monitored the relative expression by means of real-time PCR using FWA and FIS2 as controls. At 3 DAP, both controls were downregulated by 69^±0.09% and 53^±0.30% respectively (Figure 6E), in line with a lack of functional DME in 50% of the seeds in heterozygous dme-6^+/− plants. AGL36 was downregulated in a similar manner as FIS2 (41^±0.20%), suggesting that DME is indeed involved in early activation of the maternal AGL36 allele.

Expression of maternal AGL36 is regulated by the PRC2 FIS-complex

We also tested the expression of FWA and FIS2 in 6 DAP samples and found that their downregulation were sustained as predicted (Figure 6E). However, to our surprise AGL36 expression in dme-6^+/− seeds was elevated more than 50-fold (Figure 6E). This result was unexpected, and implicated a more intricate regulation of AGL36.

DME is required for the activation of MEA, the core histone H3K27 methyltransferase (HMTase) of the PRC2 FIS-complex [46], [55], [56]. To determine whether PRC2 FIS is involved in the regulation of AGL36, we analyzed the relative expression of AGL36 over time (1 to 12 DAP) in mea mutant seeds compared to wild-type (Figure 7A). While AGL36 expression in wild-type seeds was at its maximum at 4 DAP, we observed that AGL36 expression in mea seeds surpassed the maximum levels of wild-type at 4 DAP, and reached its highest levels at around 6 DAP. At this point, the AGL36 relative expression in mea mutant seeds was approximately 40-fold higher than wild-type expression at the same stage, and 7-fold higher than the maximum AGL36 level found in wild-type seeds at 4 DAP (Figure 7A). Our data thus indicate that the FIS-complex is indeed a repressor of AGL36 expression, and could also explain the elevated AGL36 expression level in 3 DAP dme-6^+/− seeds (Figure 6E). In line with these findings, we found highly elevated AGL36 relative expression levels in mutant seeds from three different mutant alleles of mea (Figure 7C). Similar results were also obtained with mutants of other components of the FIS PRC2 complex (FIS2, FIE and MSI1, data not shown).

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Figure 7. The maternal AGL36 allele is regulated by the PRC2 FIS-complex.

(A) Real-time PCR AGL36 expression profile in 1–12 DAP wild-type and mea mutant seeds. 3 DAP values were used as the reference point for calculations. Samples were taken at indicated time points. The graph represents average expression obtained from two BRs and subsequent two TRs. STDEVs are derived from biological parallels. ACT11 is the reference gene used. (B) The FIS-complex regulates the maternal allele of AGL36. The PCR product of AGL36 SNP region obtained from mea x Col fertilized seeds was AlwNI digested and analyzed. Genomic Ler and Col DNA were included as controls. The intensities of the represented bands (nmol/L), allows comparison between different time-points. Note, unsustainable weak paternal signals at 2 and 3 DAP are below the detection limit for measurement on our instrument (0.1 ng/µl∼0.4 nmol/L) and indicated as b.d. The chart represents the obtained concentrations from each sample. The displayed SNP picture is representing one of four different runs (2BRs and 2TRs). (C) AGL36 is regulated in three different alleles of mea but not in the E(z) MEA paralogues, clf and swn. Real-time PCR analysis showing AGL36 expression in (mea) fis1^−/−, mea-8^−/−, mea-9^+/−, swn-4^+/− and clf-2^−/− compared to wild-type. STDEVs are derived from two independent BRs. ACT11 is the reference gene used. (D) Real-time PCR expression level of FWA, FIS2, AGL36 and MPC in mea-9 x Col vs. wild-type seeds 3 and 6 DAP. Graphs represent the average relative expression values obtained from four independent BRs. Samples used in the first biological parallel gave rise to two TRs. STDEVs are derived from the independent BRs. The transcript levels were normalized to ACT11 levels.

https://doi.org/10.1371/journal.pgen.1001303.g007

To investigate whether FIS activity was exerted on the maternal and/or paternal allele of AGL36, we performed SNP analyses on the RT-PCR product of AGL36 obtained from mea mutant plants (in Ler background) pollinated with Col wild-type pollen. We found that AGL36 is expressed only from its maternal allele in the mea background throughout the duration of our experiment (Figure 7B). In comparison to the expression pattern in wild-type (Figure 3B), strong ectopic maternal expression was also observed at 9 and 12 DAP stages. No paternal expression could be observed in these stages. By plotting the molarities of the maternal band detected by the Agilent Bioanalyzer, an expression profile for the maternal allele could be generated (Figure 7B, lower panel). This demonstrated that in the absence of MEA, AGL36 expression continues to increase after 4 DAP, and although the intensity decreases from 6 DAP, high level of AGL36 is maintained at 12 DAP. Hence, the FIS-complex represses the maternal allele of AGL36 after the 3 DAP stage.

To further substantiate that maternal AGL36 expression is regulated by the maternal action of MEA, we crossed mea mutant plants with pollen expressing the pAGL36::GUS reporter line. Here, no obvious activation of the paternal transgene could be observed at 3 DAP (Figure S7A). Surprisingly, at 6 DAP, corresponding to embryo heart stage, weak expression of the paternal copy in the embryo could be found (Figure S7A). In addition, we performed reciprocal crosses with the pAGL36::GUS reporter line in mutant mea background. When the transgene was contributed from the female side in mea background, a GUS signal was found in 3 DAP stages that increased drastically up to 6 DAP (Figure S7B). In the reciprocal cross however, no expression could be observed (Figure S7C).

The E(z) class of H3K27 histone methyltransferases (HMTases) in Arabidopsis consists of MEA, SWINGER (SWN) and CURLY LEAF (CLF) that participate in different PRC2 complexes. To test whether AGL36 repression is a specific function of FIS^MEA PRC2, we analyzed AGL36 expression in homozygous swn-4 and clf-2 seeds. For mutants of both HMTases values similar to the wild-type situation were found, and in conclusion AGL36 appear to be specifically regulated by FIS^MEA PRC2 (Figure 7C).

In summary, maternal AGL36 expression appears to be repressed specifically by the maternal action of FIS PRC2.

PRC2 acts on a subset of MET1/DME–regulated genes

For all genes known to be imprinted by PRC2, the FIS-complex is involved in the repression of the silenced allele [25]-[27], [30], [56]. Our data suggest that silencing of the paternal AGL36 allele requires MET1 whereas the maternal allele is activated by DME. Modulation of female AGL36 expression by PRC2 thus represents a novel mechanism in this type of gene expression system, and adds an additional level of parent-of-origin specific gene expression to the scheme. In order to investigate if this regulation applies to other genes imprinted by the dual action of MET1/DME [11], [18], [19], we analyzed the relative expression levels of FWA, FIS2, AGL36 and MPC in a mea mutant. At 3 DAP expression levels were unchanged or slightly downregulated (0.40–0.99) for all genes tested (Figure 7D). However, while the expression of FWA and FIS2 remained stable at 6 DAP, AGL36 and MPC levels were elevated up to 80-fold (Figure 7D). Thus, genes imprinted by means of MET1/DME can be divided in two classes based on their dependence of FIS PRC2 for additional regulation of the expressed allele. Whereas one class appears not to be regulated by FIS PRC2, the other class depends on the action of the FIS-complex for developmental regulation of its expression.

AGL36 imprinting requires MET1

Here, we report that AGL36 is a novel imprinted gene that is only expressed from its maternal allele in the endosperm. Silencing of the paternal allele requires the action of MET1, as paternal expression is restored in met1 mutants.

In public high-density DNA methylation maps prepared from wild-type seedlings (http://signal.salk.edu), both the AGL36 transcribed region and the 5′and 3′regulatory regions are decorated by CG methylation. In line with this, AGL36 was expressed at very low levels in vegetative tissues. Transcript levels however, were highly elevated in the absence of MET1, in accordance with the virtual absence of CG methylation in met1 mutants (http://signal.salk.edu) [67].

AGL36 is expressed from both parental alleles at low levels in vegetative tissues, which show that AGL36 imprinting occurs specifically in the endosperm. Other imprinted genes in Arabidopsis have been shown to have biallelic expression in the embryo and other vegetative tissues [11], [34], [68]. However, for most imprinted genes this issue is not clarified [3]. Since paternal AGL36 expression is absent in the seed, it suggests that further silencing of AGL36 takes place by entry into the male germline. Moreover, silencing in the female germline must be lifted to allow AGL36 expression in the seed. Alternatively, maintenance methylation and further silencing do not take place on the AGL36 gene in the female gametophyte. The majority of previously described imprinted genes are regulated by a dual switch of methylation and demethylation involving MET1 and DME [11], [18]–[20], [35]. Here we have shown that AGL36 expression is reduced in a dme mutant, indicating that DME has an activating function towards AGL36. In accordance with this, mutants of CMT3, KYP, AGO4, DDM1 and DRM1/2 had no effect on paternal AGL36 expression suggesting that maintenance and repression by MET1 and activation by DME is sufficient for AGL36 imprinting.

In our SNP analyses, a weak paternal signal was observed only at the 2DAP stage. This was interpreted as an artifact since the signal was absent both before and after this stage. If this is a real paternal signal, it suggests an alternative hypothesis where silencing is achieved in the endosperm post fertilization. Further analyses are however required to support this.

In two recent studies, the genome-wide methylation profile of the seed was dissected by comparing cytosine methylation in wild-type embryos to wild-type and dme endosperm. This showed that endosperm development, and hence the activity of endosperm-specific genes, is marked by an extensive demethylation of the maternal genome, especially at specific transposon sequences [35], [69]. According to the Zilberman Lab Genome Browser (http://dzlab.pmb.berkeley.edu/browser/), such demethylation indeed takes place in the 5′and 3′regulatory regions of AGL36. Methylation patterns are regained in the dme mutant, supporting our data that AGL36 is maternally activated through the action of DME.

In an elegant approach by Gehring and colleagues, novel imprinted genes have recently been identified by the prediction of Differentially Methylated Regions (DMRs) between embryo and endosperm. In support of our findings, significant DMRs were also mapped to 5′and 3′region regions of AGL36 [35].

Imprinting could be demonstrated in transgenic pAGL36::GUS seeds, thus indicating that the 1752 bp promoter fragment used is sufficient for parent-of-origin specific expression. The genomic environment of imprinted genes is highly correlated with transposable elements (TE), and imprinting has been postulated to be an evolutionary byproduct of silencing of invading transposons [23], [69], [70]. For instance, methylation of a SINE-related tandem repeat structure in the 5′-region correlates with FWA expression [32], [71], and DMRs in MEA, PHE1, HDG3 and HDG9 map to TE [35]. In line with this, a variety of remnants of TE reside in both the 5′and 3′ regulatory regions of AGL36 (Figure 4A). The 1752 bp pAGL36::GUS promoter fragment harbors remnants of Helitrons and parts of an Arnoldy TE. An 800 bp DMR maps immediately (78 bp) upstream of the AGL36 transcriptional start site overlapping the Helitron TEs ([35], Mary Gehring, personal communication). Clearly, the 1752 bp 5′region is sufficient for basal AGL36 imprinting, and similar to the abovementioned examples, AGL36 DMRs map to TE. Further investigations will be needed to elucidate the role and the mechanisms of additional 5′and 3′DMRs as well as the involvement of small RNAs in AGL36 imprinting [72].

The PRC2 FIS-complex regulates maternal expression of AGL36

Distinct from the expression pattern of AGL36 that subsides at the time of cellularization in wild-type seeds, AGL36 maternal expression in mea mutant seeds was highly elevated and sustained throughout seed development. Recently, Walia et al. also reported AGL36 upregulation obtained in five days old seeds from selfed fis2^+/− plants [65]. Our results show that FIS-complex mediated repression acts exclusively on the expression of the maternal allele of AGL36. The paternal allele was efficiently silenced throughout endosperm development.

Surprisingly, weak paternal pAGL36::GUS expression could be observed in 6 DAP early heart stage embryos when the mother was homozygous for mea. MEA has been shown to have biallelic expression in the embryo [28], and thus the observed paternal expression in hemizygous mea embryos is not caused by the lack of functional MEA. This could hint to dosage-dependent regulation of paternal AGL36 expression by MEA, directly or indirectly, but in lack of further experiments this remains speculation.

Different PRC2 complexes can regulate common genes [30]. However, in mutants of CLF and SWN, the paralogues of MEA, no significant effect on AGL36 expression levels was found, indicating that AGL36 regulation is specific to PRC2^FIS. H3K27 trimethylation mediates PRC2s repressive function, and in a whole-genome assay for H3K27 methylation more than 4400 target genes were detected [73] (Daniel Bouyer, personal communication). AGL36 was however not part of this set of genes. Since this material was obtained from seedlings and may not reflect the situation in the seed, it is not known whether AGL36 is a direct target of H3K27 trimethylation.

AGL36 identifies a dual regulation mechanism by DME and the FIS PRC2-complex

Repression of the maternal AGL36 allele identifies a novel means of dual epigenetic regulation of imprinted genes. In this scenario, the expressed maternal AGL36 allele is antagonistically activated by DME and repressed by PRC2^FIS. To our knowledge, this is the first report of an imprinted gene where the expressed allele is concurrently regulated by a repressive epigenetic mark.

We asked whether this type of regulation was specific for AGL36 by investigating the fis mutant for expression of three other imprinted genes that are activated by DME. We found that these genes fall into two distinct groups; FWA and FIS2 which were largely unaffected by the lack of FIS, and MPC along with AGL36 which showed strong upregulation. This suggests that additional PRC2 regulation of DME-activated alleles defines a common mechanism that applies to a subset of imprinted genes.

In Arabidopsis, three imprinted genes, MEA, PHE1 and AtFH5 are known to have their silenced allele repressed by PRC2^FIS, and two of these genes, MEA and PHE1 are additionally regulated by DNA methylation [55]. In these cases however, the repressed allele is silenced by PRC2 whereas the active allele is regulated by DNA methylation [74]. Here, we show that AGL36 defines a novel type of regulation where the same allele is activated by DME and repressed by PRC2^FIS in a sequential fashion. This suggests that maternal AGL36 expression after DME activation needs to be dampened and developmentally regulated by PRC2^FIS, in accordance with the strong AGL36 expression observed in hypomethylated met1^−/− plants. Interestingly, DME is required to activate both PRC2^MEA and AGL36, and is thus a key player in developmental tuning of parent-of-origin specific AGL36 expression.

The role of AGL36 in seed development

AGL36 was identified in our transcript profiling as a downregulated gene when the paternal contribution to the endosperm was absent. A simple hypothesis to account for this regulation would be that AGL36 is under the control of one or more paternally expressed factor(s) that activate the maternal allele of AGL36. The identity of such factor(s) remain unknown, and was not approached in this work, but a simple prediction from this hypothesis is that AGL36 would be upregulated in paternal excess interploidy crosses. In a recent report, AGL36 is indeed upregulated in such crosses, as well as in crosses with unreduced diploid jas pollen [66]. Such parental cross-talk is however likely to involve complex genetic and epigenetic regulatory mechanisms, and the mechanism that cause the observed transcriptional response of AGL36 and other previously described imprinted genes in cdka;1^p seeds remains to be clarified.

In our study, we have shown that AGL36 is only maternally expressed. Our current model suggests that the paternal allele is silenced by the action of MET1 and the maternal allele activated by DME (Figure 8). In addition, we have also shown that PRC2^FIS regulates the expression of the maternal AGL36 allele at the transition between proliferation and cellularization (Figure 8).

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Figure 8. A model for the imprinted expression of AGL36.

Paternal silencing of AGL36 is maintained by the action of MET1 in the male germline prior to fertilization (1). The maternal copy of AGL36 is repressed after fertilization, either directly or indirectly, by the maternally expressed FIS-complex (2) (only MEA is indicated in the figure). DME is expressed only in the central cell and activates the maternal allele of MEA and AGL36 (3 & 4). The autoregulatory repression of maternal MEA upon paternal MEA is indicated (5). Solid lines indicate direct regulation. Dashed lines indicate possible indirect regulation.

https://doi.org/10.1371/journal.pgen.1001303.g008

Although AGL36 is identified as a novel target of the imprinting machinery in Arabidopsis, we have limited knowledge about its function during plant and seed development. Since expression of AGL36 and its interacting partners coincide with the transition of endosperm from proliferation to differentiation, we speculate that it plays an important role in this process. This is in agreement with recent findings [65], showing that suppression of an AGL cluster including AGL36 is critical for successful transition of endosperm from syncytial to cellularized stage.

In this work we have identified a novel imprinted gene that is controlled by a novel type of dual epigenetic regulation in the seed. This underscores the importance of further investigations to identify imprinted genes in order to unravel the complex network of epigenetic regulation of parent-of-origin effects in seed development.

Plant strains and growth conditions

All plant lines used in these experiments were obtained from the Nottingham Arabidopsis Stock Centre (NASC) unless otherwise stated. The mutant lines cdka;1-1 (SALK_106809; [40], [41]), ddm1-2 (a kind gift from E. Richards; [53]), dme-6 (GK-252E03-014577; Figure S9), mea-8 (SAIL_55_C04; [75]), mea-9 (SAIL_724_E07; Figure S9), met1-4; (SAIL_809_E03; [76] and swn-4 (SALK_109121; [77]) were in the Col accession. The mutant lines ago4-1 (N6364; [78]), clf-2 (N290; [79]), cmt3-7 (N6365; [80]), fis1 (a kind gift from A. Chaudhury; [14]) and kyp-2 (N6367; [51] were in the Ler accession. The drm1;drm2 (N6366; [81]) line was in the Ws-2 accession. Mutants used in this study were genotyped using gene-specific and T-DNA specific primers as described in Table S2. The ddm1-2 mutant line was genotyped by an allele-specific PCR test using dCAPS primers DDM1f and ddm1-2Rsa, as described by [68], allowing digestion of the PCR fragment of the ddme1-2 allele with RsaI restriction endonuclease, generating a ∼130 bp band.

We obtained the agl36-1 allele from the Koncz collection [48]. Allele-specific PCR, using the primers HOOK1 (left border T-DNA primer) and AGL36-AS2-KONCZ (genomic AGL36 primer), was carried out to verify the T-DNA insertion, followed by sequencing analysis of the PCR product using the HOOK1 primer. The left border of the insertion was verified to be 16 bp upstream of the ATG start codon of AGL36. In addition, there is an 11 bp long DNA filler located between the genomic sequence and the T-DNA sequence.

Arabidopsis seeds were surface-sterilized using EtOH, bleach and Tween20 prior to plating out on MS-2 plates [82] supplemented with 2% Sucrose, containing the correct selection when necessary. Seeds on the MS-2 plates were stratified at 4°C O.N before they were incubated for 14 days at 18°C to germinate. The seedlings were then put on soil and grown in long day conditions (16 hr light) at 18°C.

Seed isolation, RNA extraction, and cDNA synthesis

To increase tissue specificity, siliques were cut open and seeds were isolated directly in tubes containing pre-chilled ceramic beads (Roche MagNA Lyser Green Beads). Isolated tissues were stored at −80°C. Homogenization was performed by the addition of Lysis buffer containing β-ME (Sigma Spectrum Plant Total RNA Kit) directly to the samples, followed by 3×15 second intervals of homogenization using a MagNA Lyser Instrument (Roche). To prevent RNA degradation, samples were chilled on ice two minutes between each homogenization interval. After the last homogenization step, the samples were centrifuged at 4°C for one minute prior to the transfer of the lysate to a new 1.5 ml tube. RNA extraction was performed according to the Sigma Plant Total RNA Kit protocol, except that all centrifugation steps were done at 4°C and not at room temperature as indicated in the protocol. RNA was eluted in 50 µl volume. cDNA was synthesized by first preparing the RNA for real-time PCR by treatment with DNase I (Sigma) followed by Reverse Transcription using Oligo(dT) and SuperScript III Reverse Transcriptase (Invitrogen) according to the manufacturer's protocols. The synthesized cDNA was purified utilizing QIAquick PCR Purification kit (Qiagen) and eluted in 30 µl volume prior to measurement of cDNA concentration using a NanoDrop 1000 Spectrophotometer.

Microarray analysis

Plants were grown and seeds isolated as described above. Total RNA was isolated as described above. For microarray analysis, three biological replicas were generated, each consisting of approximately 35 hand-pollinated siliques from ten different plants. The microarray experiment was conducted by the NARC Microarray Service in Trondheim. Microarray slides were printed by the Norwegian Microarray Consortium (Trondheim, Norway). A custom made Arabidopsis chip with 32567 unique 70-mer oligo probes was used in the experiments. Total RNA (15 mg) and Super-Script III reverse transcriptase (Invitrogen) were used in a reverse transcription reaction. A 3DNA Array 350 kit with Cy3- and Cy5-labelled dendrimers (Genisphere Inc.) was used for labeling. Hybridizations were performed in a Slide Booster Hybridization Station (Advalytix), and the slides were washed according to the manufacturers' descriptions (Genisphere and Advalytix). The slides were scanned at 10 mm resolution on a G2505B Agilent DNA microarray scanner (Agilent Technologies). The resulting image files were processed using GenePix 5.1 software (Axon Instruments). Spots identified as not found or manually flagged out as bad were filtered out. Spots with more than 50% saturated pixels were also excluded. The data sets were log-transformed and normalized using the print-tip Loess approach [83]. Within-array replicated measurements for the same gene were merged by taking the average between the replicates. The data were then scaled so that all array data sets had the same median absolute deviation. The differentially expressed genes were identified using the Limma software package [84]. The resulting set of p-values were used to compute the q-values as described [85].

The microarray data generated in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE24809 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE24809).

Bioinformatics analyses

We defined the following sub-sets for our microarray data (see Table S3): All expressed  =  all genes having a present call (17223 genes); Down 0.8 =  in Ler x cdka;1 downregulated genes with q≤0.35 and arithmetic ratio (ar) ≤0.8 (602 genes); Up 1.5 =  in Ler x cdka;1 upregulated genes with q≤0.35 and ar ≥1.5 (323 genes); Up 1.2 =  in Ler x cdka;1 upregulated genes with q≤0.35 and ar ≥1.2 (1030 genes). The q-value is the false discovery rate (FDR) of the p-value, and was adjusted with Storey's q-procedure [85]. The threshold for analysis was set to q≤0.35 since this value detected paternally expressed genes at an arithmetic ratio (ar) ≤0.8. A functional classification was done at http://www.arabidopsis.org/tools/bulk/go/ using the GO-Slim Molecular Function classification system. For the detailed transcription factor analysis we used the Transcription Factor (TF) classification from the Arabidopsis transcription factor database (AtTFDB) hosted on the Arabidopsis Gene Regulatory Information Server (AGRIS, http://arabidopsis.med.ohio-state.edu/AtTFDB). The MADS TFs were sub-grouped as in de Folter et al [45]. We compared our microarray data with seed expression data generated by the Goldberg & Harada laboratories, available at http://seedgenenetwork.net/analyze?project=Arabidopsis.

For data comparison a reference set of genes was used that contained all genes covered by the Operon chip used in our study (Arabidopsis thaliana 34K NARC serie 8; GEO Platform GPL11051GPL) and the Affimetrix chip used by Goldberg & Harada (Ath1, GEO Platform GPL198). For the Ath1 chip we used the annotation provided by Goldberg & Harada available at http://seedgenenetwork.net/media/Arab_Final_Annotations_09-07-07_completed.txt. For the operon chip we used the current TAIR 9.0 annotation. From these annotations all AGIs for nuclear genes were extracted and the overlap was calculated. This reference set contained 22130 genes.

We used the reference set overlap of the following Goldberg/Harada datasets for comparison: GH seed  =  call all present and experiment in Arabidopsis ATH1 Array/Arabidopsis/Globular Stage/Seed; GH seed coat  =  call all present and experiment in Arabidopsis ATH1 Array/Arabidopsis/Globular Stage/Chalazal Seed Coat or Arabidopsis ATH1 Array/Arabidopsis/Globular Stage/General Seed Coat; GH endosperm  =  call all present and experiment in Arabidopsis ATH1 Array/Arabidopsis/Globular Stage/Chalazal Endosperm or Arabidopsis ATH1 Array/Arabidopsis/Globular Stage/Micropylar Endosperm or Arabidopsis ATH1 Array/Arabidopsis/Globular Stage/Peripheral Endosperm; GH embryo  =  call all present and experiment in Arabidopsis ATH1 Array/Arabidopsis/Globular Stage/Embryo Proper.

Venn diagrams were generated using the VENN diagram generator designed by Tim Hulsen at http://www.cmbi.ru.nl/cdd/biovenn/ [86]. The test for statistical significance of the overlap between two groups of genes was calculated by using software provided by Jim Lund accessible at http://elegans.uky.edu/MA/progs/overlap_stats.html.

Plasmid construction

To generate the pAGL36::GUS construct we utilized the Gateway cloning technology (Gateway; Invitrogen). The promoter region (÷1740–12) spanning the ATG start codon was amplified using the attB sequence containing primers attB1-pAGL36-AS7 and attB2-pAGL36-S4 (Table S2), and cloned into the pMDC162 GUS-vector [87]. The resulting construct, after checking the DNA sequence, was introduced to Col ecotype by Agrobacterium tumefaciens mediated transformation using the floral-dip method [88].

β-Glucuronidase expression analysis and histology

Histochemical assays were performed after a modified protocol from Grini et al. (2002) by incubating the tissues in staining buffer (2 mM X-Gluc; 50 mM NaPO₄, pH 7.2; 2 mM K₄Fe(CN)₆ x 3H₂O; 2 mM K₃Fe(CN)₆; 0.1% Triton) overnight at 37°C before the reaction was terminated using 50% EtOH. The tissues were cleared and mounted on slides according to Grini et al. (2002), and inspected using an Axioplan 2 Carl Zeiss Microscope. Images were acquired with an AxioCam HRc Carl Zeiss camera and processed with AxioVs40 V 4.5.0.0 software.

Real-time quantitative PCR

Real-time PCR was performed using a Light-cycler LC480 instrument (Roche) according to the manufacturer's protocol. To ensure high PCR efficiency and to avoid undesired primer dimers, all oligonucleotide pairs were initially tested by melting point analysis using SYBR Premix Ex Taq (TaKaRa). To obtain higher level of gene specificity, probe-based real-time PCR with confirmed primers were performed using Universal Probe Library (UPL) hydrolysis probes (Roche) in combination with Premix Ex Taq (TaKaRa).

For AGL36 real-time PCR, we used primers AGL36-160-LP and AGL36-160-RP, which gave a 60 bp amplicon (Table S2). Comparison of the sequences of the coding region and the 3′UTR of AGL36 with AGL34 and AGL90, revealed more than 85% and 84% sequence similarity respectively between these genes (Figure S8). To ensure that the abovementioned primers are only amplifying AGL36, we cloned the obtained amplicon from four independent reactions into the pCR2.1 vector (Invitrogen), and subsequently sequenced two clones of each construct with M13-Forward and M13-Reverse primers. Sequence results revealed exclusive and specific AGL36 amplification.

ACTIN11 (ACT11), a housekeeping gene that is strongly expressed in the developing ovules [89], was shown in a preliminary analysis not to be affected by our experimental conditions (data not shown), and was therefore selected as a reference gene. GLYCERALDEHYDE-3-P DEHYDROGENASE A-SUBUNIT (GAPA) was used as an additional reference gene. The oligo sequences, their amplicons and appropriate UPL probes are shown in Table S2.

Real-time PCR of all samples and reference controls were performed in two independent biological replicates and repeated at least two times (technical replicas) unless otherwise stated. The PCR efficiency was determined independently for all replicates (biological and technical) by series of dilutions (100 ng, 50 ng, 20 ng, 5 ng template/rxn) for each experiment. This allowed us to obtain the efficiency for each single reaction. Calculations of relative expression ratios were performed according to a model described by Pfaffl [90] with minor exceptions. Since we had efficiency for all reactions (four values for each calculation corresponding to E^{target-sample}, E^{target-standard}, E^{reference-sample} and E^{reference-standard}), we calculated the average E^target and E^reference values from the standards and the samples, ending up with two E-values that we could use in the formula described by Pfaffl.

Single Nucleotide Polymorphism analysis

RNA was isolated and cDNA synthesized and purified as described above. Polymorphisms between various ecotypes were identified using TAIR Genome Browser (www.arabidopsis.org) and/or the Arabidopsis SNP Sequence Viewer tool provided by the Salk Institute Genomic Analysis Laboratory (http://natural.salk.edu/cgi-bin/snp.cgi). A selected region spanning the SNP of interest was amplified by PCR using TaKaRa Ex Taq DNA polymerase applying 100 ng template per reaction, and the following PCR parameters in a 50 µl reaction: 94°C-3 min, 35×(94°C-1 min, 58°C-30 sec, 72°C-1 min/kb), 72°C-5 min, 4°C-∞. Parental-specific expression based on SNP was determined by setting up an appropriate restriction digest. For AGL36 SNP analysis, 20 µl of the SNP PCR reaction was digested with 15 U of AlwNI at 37°C for a duration of 2.5 hrs, followed by a 20 min inactivation at 65°C. For the FWA control SNP, due to the absence of a restriction site in the SNP region in both Col and Ler ecotypes, dCAPS primers were used, generating a NheI restriction site in the Col ecotype. The obtained amplicons for both ecotypes were digested with NheI [11].

In cases where the detected SNP did not result in digestion in neither ecotype, a primer sequence was designed to introduce a base exchange adjacent to the SNP, leading to restriction digestion of one of the ecotypes. The obtained amplicon for both ecotypes were then treated with the appropriate restriction enzyme. In all experiments either genomic DNA or cDNA from wild-type plants from both ecotypes used in the study was used as controls for the presence or absence of digestion. The digested samples were analyzed using DNA-1000-LabOnChip and 2100 Bioanalyzer (Agilent Technologies).

To rule out that the primers used for AGL36 SNP PCR (AGL36-SP7-SNP and AGL36-ASP6-SNP) (Table S2) would amplify the highly similar AGL90, we oriented the AGL36-SP7-SNP primer such that it was located in a region that was annotated as intron in AGL90 but not in AGL36 (Figure S8). First, the presence of the intron in AGL90 was confirmed by amplifying the intron-flanking region (AGL90-SP1-subcloning and AGL90-ASP2-subcloning primers (Table S2)), and comparing the size differences obtained between the genomic PCR and cDNA PCR. Due to high sequence similarity, we suspected to amplify both AGL36 and AGL90 in these PCR reactions. To distinguish between these two amplicons, we took advantage of the presence of two unique restriction sites (MslI and BspBI) in the amplified region of AGL36 that are absent in AGL90.

Sequence comparison between the abovementioned AGL36-SNP primers and AGL34 showed that there was approximately 70% and 91% sequence similarity between the primers and the AGL34 gene. However, if these primers were functional in amplifying AGL34, they would result in a smaller amplicon than AGL36 amplicon (373 bp versus 399 bp respectively). This difference could easily be detected using a DNA-1000-LabOnChip. Our SNP data only showed the expected 399 bp band, verifying that AGL34 was not amplified using the above primers. The paternally imprinted FWA gene was used as a positive control by utilizing primers FWA-RTf and FWA-dNheI (Table S2) for PCR amplification followed by NheI restriction digest.