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The DAG1 transcription factor negatively regulates the seed-to-seedling transition in Arabidopsis acting on ABA and GA levels

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ABSTRACT

Background: In seeds, the transition from dormancy to germination is regulated by abscisic acid (ABA) and gibberellins (GAs), and involves chromatin remodelling. Particularly, the repressive mark H3K27 trimethylation (H3K27me3) has been shown to target many master regulators of this transition. DAG1 (DOF AFFECTING GERMINATION1), is a negative regulator of seed germination in Arabidopsis, and directly represses the GA biosynthetic gene GA3ox1 (gibberellin 3-β-dioxygenase 1). We set to investigate the role of DAG1 in seed dormancy and maturation with respect to epigenetic and hormonal control.

Results: We show that DAG1 expression is controlled at the epigenetic level through the H3K27me3 mark during the seed-to-seedling transition, and that DAG1 directly represses also the ABA catabolic gene CYP707A2; consistently, the ABA level is lower while the GA level is higher in dag1 mutant seeds. Furthermore, both DAG1 expression and protein stability are controlled by GAs.

Conclusions: Our results point to DAG1 as a key player in the control of the developmental switch between seed dormancy and germination.

Electronic supplementary material: The online version of this article (doi:10.1186/s12870-016-0890-5) contains supplementary material, which is available to authorized users.

No MeSH data available.


DAG1 binds the promoter of CYP707A2. Top: graphic representation of the CYP707A2 promoter. Underlying thick lines marked by letters (A, B, C) are referred to different promoter fragments used for qPCR, containing 0, 2 and 10 Dof sites respectively. Bottom, chromatin from dag1DAG1-HA (right) seeds and from dag1 (left) seeds, as a negative control, was immunoprecipitated with anti-HA antibodies, and the amount of DNA was measured by qPCR. The values of fold enrichment were normalized to internal controls (relative to input and to PP2A), and are the average of two biological replicates presented with SD values. Significant fold enrichments were analyzed by t-test (*P ≤ 0,05)
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Fig5: DAG1 binds the promoter of CYP707A2. Top: graphic representation of the CYP707A2 promoter. Underlying thick lines marked by letters (A, B, C) are referred to different promoter fragments used for qPCR, containing 0, 2 and 10 Dof sites respectively. Bottom, chromatin from dag1DAG1-HA (right) seeds and from dag1 (left) seeds, as a negative control, was immunoprecipitated with anti-HA antibodies, and the amount of DNA was measured by qPCR. The values of fold enrichment were normalized to internal controls (relative to input and to PP2A), and are the average of two biological replicates presented with SD values. Significant fold enrichments were analyzed by t-test (*P ≤ 0,05)

Mentions: To assess whether DAG1 regulates also the CYP707A2 ABA catabolic gene by directly binding to its promoter in vivo, we performed ChIP assays, using the dag1DAG1-HA line overexpressing the DAG1-HA chimeric protein in a dag1 mutant background [15, 16, 21]. Cross-linked and sonicated protein–DNA complexes were precipitated with anti-HA antibodies, or without antibodies as a negative control. As additional negative control, we performed the same assays on dag1 mutant seeds (Fig. 5, bottom left). Three regions of the CYP707A2 promoter, one with no DOF binding sites (fragment A), one with two (fragment B), and one with ten (fragment C) were amplified by qPCR (Fig. 5, top).Fig. 5


The DAG1 transcription factor negatively regulates the seed-to-seedling transition in Arabidopsis acting on ABA and GA levels
DAG1 binds the promoter of CYP707A2. Top: graphic representation of the CYP707A2 promoter. Underlying thick lines marked by letters (A, B, C) are referred to different promoter fragments used for qPCR, containing 0, 2 and 10 Dof sites respectively. Bottom, chromatin from dag1DAG1-HA (right) seeds and from dag1 (left) seeds, as a negative control, was immunoprecipitated with anti-HA antibodies, and the amount of DNA was measured by qPCR. The values of fold enrichment were normalized to internal controls (relative to input and to PP2A), and are the average of two biological replicates presented with SD values. Significant fold enrichments were analyzed by t-test (*P ≤ 0,05)
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Related In: Results  -  Collection

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Fig5: DAG1 binds the promoter of CYP707A2. Top: graphic representation of the CYP707A2 promoter. Underlying thick lines marked by letters (A, B, C) are referred to different promoter fragments used for qPCR, containing 0, 2 and 10 Dof sites respectively. Bottom, chromatin from dag1DAG1-HA (right) seeds and from dag1 (left) seeds, as a negative control, was immunoprecipitated with anti-HA antibodies, and the amount of DNA was measured by qPCR. The values of fold enrichment were normalized to internal controls (relative to input and to PP2A), and are the average of two biological replicates presented with SD values. Significant fold enrichments were analyzed by t-test (*P ≤ 0,05)
Mentions: To assess whether DAG1 regulates also the CYP707A2 ABA catabolic gene by directly binding to its promoter in vivo, we performed ChIP assays, using the dag1DAG1-HA line overexpressing the DAG1-HA chimeric protein in a dag1 mutant background [15, 16, 21]. Cross-linked and sonicated protein–DNA complexes were precipitated with anti-HA antibodies, or without antibodies as a negative control. As additional negative control, we performed the same assays on dag1 mutant seeds (Fig. 5, bottom left). Three regions of the CYP707A2 promoter, one with no DOF binding sites (fragment A), one with two (fragment B), and one with ten (fragment C) were amplified by qPCR (Fig. 5, top).Fig. 5

View Article: PubMed Central - PubMed

ABSTRACT

Background: In seeds, the transition from dormancy to germination is regulated by abscisic acid (ABA) and gibberellins (GAs), and involves chromatin remodelling. Particularly, the repressive mark H3K27 trimethylation (H3K27me3) has been shown to target many master regulators of this transition. DAG1 (DOF AFFECTING GERMINATION1), is a negative regulator of seed germination in Arabidopsis, and directly represses the GA biosynthetic gene GA3ox1 (gibberellin 3-β-dioxygenase 1). We set to investigate the role of DAG1 in seed dormancy and maturation with respect to epigenetic and hormonal control.

Results: We show that DAG1 expression is controlled at the epigenetic level through the H3K27me3 mark during the seed-to-seedling transition, and that DAG1 directly represses also the ABA catabolic gene CYP707A2; consistently, the ABA level is lower while the GA level is higher in dag1 mutant seeds. Furthermore, both DAG1 expression and protein stability are controlled by GAs.

Conclusions: Our results point to DAG1 as a key player in the control of the developmental switch between seed dormancy and germination.

Electronic supplementary material: The online version of this article (doi:10.1186/s12870-016-0890-5) contains supplementary material, which is available to authorized users.

No MeSH data available.