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Genome-wide transcript profiling of endosperm without paternal contribution identifies parent-of-origin-dependent regulation of AGAMOUS-LIKE36.

Shirzadi R, Andersen ED, Bjerkan KN, Gloeckle BM, Heese M, Ungru A, Winge P, Koncz C, Aalen RB, Schnittger A, Grini PE - PLoS Genet. (2011)

Bottom Line: In Arabidopsis, analyses of mutants in the cell-cycle regulator CYCLIN DEPENDENT KINASE A;1 (CKDA;1) have revealed the importance of a paternal genome for the effective development of the endosperm and ultimately the seed.Among those, AGAMOUS-LIKE (AGL) genes encoding Type-I MADS-box transcription factors were significantly overrepresented.Interestingly, our data also show that the active maternal allele of AGL36 is regulated throughout endosperm development by components of the FIS Polycomb Repressive Complex 2 (PRC2), revealing a new type of dual epigenetic regulation in seeds.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences (IMBV), University of Oslo, Oslo, Norway.

ABSTRACT
Seed development in angiosperms is dependent on the interplay among different transcriptional programs operating in the embryo, the endosperm, and the maternally-derived seed coat. In angiosperms, the embryo and the endosperm are products of double fertilization during which the two pollen sperm cells fuse with the egg cell and the central cell of the female gametophyte. In Arabidopsis, analyses of mutants in the cell-cycle regulator CYCLIN DEPENDENT KINASE A;1 (CKDA;1) have revealed the importance of a paternal genome for the effective development of the endosperm and ultimately the seed. Here we have exploited cdka;1 fertilization as a novel tool for the identification of seed regulators and factors involved in parent-of-origin-specific regulation during seed development. We have generated genome-wide transcription profiles of cdka;1 fertilized seeds and identified approximately 600 genes that are downregulated in the absence of a paternal genome. Among those, AGAMOUS-LIKE (AGL) genes encoding Type-I MADS-box transcription factors were significantly overrepresented. Here, AGL36 was chosen for an in-depth study and shown to be imprinted. We demonstrate that AGL36 parent-of-origin-dependent expression is controlled by the activity of METHYLTRANSFERASE1 (MET1) maintenance DNA methyltransferase and DEMETER (DME) DNA glycosylase. Interestingly, our data also show that the active maternal allele of AGL36 is regulated throughout endosperm development by components of the FIS Polycomb Repressive Complex 2 (PRC2), revealing a new type of dual epigenetic regulation in seeds.

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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.
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pgen-1001303-g007: 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.

Mentions: 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).


Genome-wide transcript profiling of endosperm without paternal contribution identifies parent-of-origin-dependent regulation of AGAMOUS-LIKE36.

Shirzadi R, Andersen ED, Bjerkan KN, Gloeckle BM, Heese M, Ungru A, Winge P, Koncz C, Aalen RB, Schnittger A, Grini PE - PLoS Genet. (2011)

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.
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Related In: Results  -  Collection

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pgen-1001303-g007: 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.
Mentions: 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).

Bottom Line: In Arabidopsis, analyses of mutants in the cell-cycle regulator CYCLIN DEPENDENT KINASE A;1 (CKDA;1) have revealed the importance of a paternal genome for the effective development of the endosperm and ultimately the seed.Among those, AGAMOUS-LIKE (AGL) genes encoding Type-I MADS-box transcription factors were significantly overrepresented.Interestingly, our data also show that the active maternal allele of AGL36 is regulated throughout endosperm development by components of the FIS Polycomb Repressive Complex 2 (PRC2), revealing a new type of dual epigenetic regulation in seeds.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences (IMBV), University of Oslo, Oslo, Norway.

ABSTRACT
Seed development in angiosperms is dependent on the interplay among different transcriptional programs operating in the embryo, the endosperm, and the maternally-derived seed coat. In angiosperms, the embryo and the endosperm are products of double fertilization during which the two pollen sperm cells fuse with the egg cell and the central cell of the female gametophyte. In Arabidopsis, analyses of mutants in the cell-cycle regulator CYCLIN DEPENDENT KINASE A;1 (CKDA;1) have revealed the importance of a paternal genome for the effective development of the endosperm and ultimately the seed. Here we have exploited cdka;1 fertilization as a novel tool for the identification of seed regulators and factors involved in parent-of-origin-specific regulation during seed development. We have generated genome-wide transcription profiles of cdka;1 fertilized seeds and identified approximately 600 genes that are downregulated in the absence of a paternal genome. Among those, AGAMOUS-LIKE (AGL) genes encoding Type-I MADS-box transcription factors were significantly overrepresented. Here, AGL36 was chosen for an in-depth study and shown to be imprinted. We demonstrate that AGL36 parent-of-origin-dependent expression is controlled by the activity of METHYLTRANSFERASE1 (MET1) maintenance DNA methyltransferase and DEMETER (DME) DNA glycosylase. Interestingly, our data also show that the active maternal allele of AGL36 is regulated throughout endosperm development by components of the FIS Polycomb Repressive Complex 2 (PRC2), revealing a new type of dual epigenetic regulation in seeds.

Show MeSH
Related in: MedlinePlus