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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.


GA induces DAG1 expression. a Relative expression level of DAG1 in 24 h-imbibed wild type (WT) seeds, in the presence of water (H2O), GA4+7 (100 μM), Paclobutrazol (PAC) (100 μM), or ABA (3 μM). The values of relative expression levels are the mean of three biological replicates, presented with SD values. Expression levels were normalized with that of the UBQ10 (At4g05320) gene. b and c Chromatin from seeds imbibed 24 h with H2O (white bars) or GA4+7 (100 μM) (black bars), was immunoprecipitated with specific antibodies against the H3K27me3 (b), or the H3K4me3 (c) epigenetic marks. The amount of DNA was measured by qPCR. The regions of the DAG1 locus used for qPCR are as in Fig. 1. 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 differences were analyzed by t-test (*P ≤ 0,05)
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Fig6: GA induces DAG1 expression. a Relative expression level of DAG1 in 24 h-imbibed wild type (WT) seeds, in the presence of water (H2O), GA4+7 (100 μM), Paclobutrazol (PAC) (100 μM), or ABA (3 μM). The values of relative expression levels are the mean of three biological replicates, presented with SD values. Expression levels were normalized with that of the UBQ10 (At4g05320) gene. b and c Chromatin from seeds imbibed 24 h with H2O (white bars) or GA4+7 (100 μM) (black bars), was immunoprecipitated with specific antibodies against the H3K27me3 (b), or the H3K4me3 (c) epigenetic marks. The amount of DNA was measured by qPCR. The regions of the DAG1 locus used for qPCR are as in Fig. 1. 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 differences were analyzed by t-test (*P ≤ 0,05)

Mentions: Since the levels of bioactive GAs increase during seed imbibition as does the level of expression of DAG1, and DAG1 controls GA and ABA levels, we wondered whether DAG1 expression in seeds might be regulated by these hormones. RT-qPCR analysis performed on seeds imbibed 24 h in the presence of GA4+7, or ABA, showed that the DAG1 transcript level was significantly induced by GAs (up to 4-fold), but not by ABA. Accordingly, in the presence of paclobutrazol (PAC), an inhibitor of GA biosynthesis, DAG1 expression level was comparable to control seeds imbibed with water (Fig. 6a).Fig. 6


The DAG1 transcription factor negatively regulates the seed-to-seedling transition in Arabidopsis acting on ABA and GA levels
GA induces DAG1 expression. a Relative expression level of DAG1 in 24 h-imbibed wild type (WT) seeds, in the presence of water (H2O), GA4+7 (100 μM), Paclobutrazol (PAC) (100 μM), or ABA (3 μM). The values of relative expression levels are the mean of three biological replicates, presented with SD values. Expression levels were normalized with that of the UBQ10 (At4g05320) gene. b and c Chromatin from seeds imbibed 24 h with H2O (white bars) or GA4+7 (100 μM) (black bars), was immunoprecipitated with specific antibodies against the H3K27me3 (b), or the H3K4me3 (c) epigenetic marks. The amount of DNA was measured by qPCR. The regions of the DAG1 locus used for qPCR are as in Fig. 1. 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 differences were analyzed by t-test (*P ≤ 0,05)
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Fig6: GA induces DAG1 expression. a Relative expression level of DAG1 in 24 h-imbibed wild type (WT) seeds, in the presence of water (H2O), GA4+7 (100 μM), Paclobutrazol (PAC) (100 μM), or ABA (3 μM). The values of relative expression levels are the mean of three biological replicates, presented with SD values. Expression levels were normalized with that of the UBQ10 (At4g05320) gene. b and c Chromatin from seeds imbibed 24 h with H2O (white bars) or GA4+7 (100 μM) (black bars), was immunoprecipitated with specific antibodies against the H3K27me3 (b), or the H3K4me3 (c) epigenetic marks. The amount of DNA was measured by qPCR. The regions of the DAG1 locus used for qPCR are as in Fig. 1. 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 differences were analyzed by t-test (*P ≤ 0,05)
Mentions: Since the levels of bioactive GAs increase during seed imbibition as does the level of expression of DAG1, and DAG1 controls GA and ABA levels, we wondered whether DAG1 expression in seeds might be regulated by these hormones. RT-qPCR analysis performed on seeds imbibed 24 h in the presence of GA4+7, or ABA, showed that the DAG1 transcript level was significantly induced by GAs (up to 4-fold), but not by ABA. Accordingly, in the presence of paclobutrazol (PAC), an inhibitor of GA biosynthesis, DAG1 expression level was comparable to control seeds imbibed with water (Fig. 6a).Fig. 6

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.