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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.


The DAG1 protein is stabilized by GA. a Protein level of DAG1-HA in dag1DAG1:HA seeds imbibed for 24 or 48 h in the presence of water (H2O), GA4+7, paclobutrazol (PAC) or ABA. b Protein level of DAG1-HA in 48 h-imbibed seeds (0), then treated for 4 or 8 h with cycloheximide (CHX), or with CHX and GA. Western blot (top) and densitometric analysis (bottom). c Protein level of DAG1-HA in 5-day-old dag1DAG1:HA seedlings treated with MG132 or DMSO as control. TUB or total protein content were used as loading control. Western blot (top) and densitometric analysis (bottom). GA4+7 (100 μM), PAC (100 μM), ABA (3 μM), CHX (50 μM), MG132 (50 μM). The protein levels are the mean of three biological replicates, presented with SD values. Significant differences were analyzed by t-test (*P ≤ 0,05)
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Fig7: The DAG1 protein is stabilized by GA. a Protein level of DAG1-HA in dag1DAG1:HA seeds imbibed for 24 or 48 h in the presence of water (H2O), GA4+7, paclobutrazol (PAC) or ABA. b Protein level of DAG1-HA in 48 h-imbibed seeds (0), then treated for 4 or 8 h with cycloheximide (CHX), or with CHX and GA. Western blot (top) and densitometric analysis (bottom). c Protein level of DAG1-HA in 5-day-old dag1DAG1:HA seedlings treated with MG132 or DMSO as control. TUB or total protein content were used as loading control. Western blot (top) and densitometric analysis (bottom). GA4+7 (100 μM), PAC (100 μM), ABA (3 μM), CHX (50 μM), MG132 (50 μM). The protein levels are the mean of three biological replicates, presented with SD values. Significant differences were analyzed by t-test (*P ≤ 0,05)

Mentions: To investigate whether the stability of the DAG1 protein would be affected by ABA or GAs, we utilized the dag1 mutant line overexpressing a 35S:DAG1-HA translational fusion [15], whose transcription is not induced by either ABA or GAs (Additional file 2: Figure S2). We performed an immunoblot analysis on DAG1-HA seeds imbibed for 24 and 48 h in the presence of ABA, GA4+7 or PAC compared to water-imbibed controls. Addition of exogenous GAs increased the level of DAG1-HA, at 24 and 48 h (1.7 and 1.6-fold, respectively); consistently, the presence of PAC reduced the amount of the chimeric protein to the level of the corresponding control (Fig. 7a). Interestingly, the amount of DAG1-HA increased during imbibition up to 48 h, as a consequence of the increase of the endogenous GA levels. In contrast, the level of DAG1-HA was not affected by ABA (Fig. 7a). To gain insight on the molecular mechanism underlying this GA-mediated control of the DAG1 protein, we performed an immunoblot analysis of DAG1-HA seeds imbibed for 48 h then treated for 4/8 h with cycloheximide (CHX), to inhibit protein synthesis, or with CHX and GAs. As shown in Fig. 7b, the increase of DAG1-HA during imbibition is mainly due to new synthesis of the protein, since in the presence of CHX the amount of DAG1-HA was drastically reduced. Interestingly, addition of exogenous GAs resulted in an increase of the protein level, suggesting that GAs stabilize DAG1-HA by increasing its half-life (Fig. 7b).Fig. 7


The DAG1 transcription factor negatively regulates the seed-to-seedling transition in Arabidopsis acting on ABA and GA levels
The DAG1 protein is stabilized by GA. a Protein level of DAG1-HA in dag1DAG1:HA seeds imbibed for 24 or 48 h in the presence of water (H2O), GA4+7, paclobutrazol (PAC) or ABA. b Protein level of DAG1-HA in 48 h-imbibed seeds (0), then treated for 4 or 8 h with cycloheximide (CHX), or with CHX and GA. Western blot (top) and densitometric analysis (bottom). c Protein level of DAG1-HA in 5-day-old dag1DAG1:HA seedlings treated with MG132 or DMSO as control. TUB or total protein content were used as loading control. Western blot (top) and densitometric analysis (bottom). GA4+7 (100 μM), PAC (100 μM), ABA (3 μM), CHX (50 μM), MG132 (50 μM). The protein levels are the mean of three biological replicates, presented with SD values. Significant differences were analyzed by t-test (*P ≤ 0,05)
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Fig7: The DAG1 protein is stabilized by GA. a Protein level of DAG1-HA in dag1DAG1:HA seeds imbibed for 24 or 48 h in the presence of water (H2O), GA4+7, paclobutrazol (PAC) or ABA. b Protein level of DAG1-HA in 48 h-imbibed seeds (0), then treated for 4 or 8 h with cycloheximide (CHX), or with CHX and GA. Western blot (top) and densitometric analysis (bottom). c Protein level of DAG1-HA in 5-day-old dag1DAG1:HA seedlings treated with MG132 or DMSO as control. TUB or total protein content were used as loading control. Western blot (top) and densitometric analysis (bottom). GA4+7 (100 μM), PAC (100 μM), ABA (3 μM), CHX (50 μM), MG132 (50 μM). The protein levels are the mean of three biological replicates, presented with SD values. Significant differences were analyzed by t-test (*P ≤ 0,05)
Mentions: To investigate whether the stability of the DAG1 protein would be affected by ABA or GAs, we utilized the dag1 mutant line overexpressing a 35S:DAG1-HA translational fusion [15], whose transcription is not induced by either ABA or GAs (Additional file 2: Figure S2). We performed an immunoblot analysis on DAG1-HA seeds imbibed for 24 and 48 h in the presence of ABA, GA4+7 or PAC compared to water-imbibed controls. Addition of exogenous GAs increased the level of DAG1-HA, at 24 and 48 h (1.7 and 1.6-fold, respectively); consistently, the presence of PAC reduced the amount of the chimeric protein to the level of the corresponding control (Fig. 7a). Interestingly, the amount of DAG1-HA increased during imbibition up to 48 h, as a consequence of the increase of the endogenous GA levels. In contrast, the level of DAG1-HA was not affected by ABA (Fig. 7a). To gain insight on the molecular mechanism underlying this GA-mediated control of the DAG1 protein, we performed an immunoblot analysis of DAG1-HA seeds imbibed for 48 h then treated for 4/8 h with cycloheximide (CHX), to inhibit protein synthesis, or with CHX and GAs. As shown in Fig. 7b, the increase of DAG1-HA during imbibition is mainly due to new synthesis of the protein, since in the presence of CHX the amount of DAG1-HA was drastically reduced. Interestingly, addition of exogenous GAs resulted in an increase of the protein level, suggesting that GAs stabilize DAG1-HA by increasing its half-life (Fig. 7b).Fig. 7

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.