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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 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.
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pgen-1001303-g006: 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.

Mentions: 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.


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 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.
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Related In: Results  -  Collection

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pgen-1001303-g006: 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.
Mentions: 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.

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