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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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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.
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pgen-1001303-g002: 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.

Mentions: 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).


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040660&req=5

pgen-1001303-g002: 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.
Mentions: 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).

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