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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 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).
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pgen-1001303-g003: 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).

Mentions: 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).


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 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).
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Related In: Results  -  Collection

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pgen-1001303-g003: 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).
Mentions: 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).

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