Light and Abscisic Acid Coordinately Regulate Greening of Seedlings

Abstract

The greening of etiolated seedlings is crucial for the growth and survival of plants. After reaching the soil surface and sunlight, etiolated seedlings integrate numerous environmental signals and internal cues to control the initiation and rate of greening thus to improve their survival and adaption. However, the underlying regulatory mechanisms by which light and phytohormones, such as abscisic acid (ABA), coordinately regulate greening of the etiolated seedlings is still unknown. In this study, we showed that Arabidopsis (Arabidopsis thaliana) DE-ETIOLATED1 (DET1), a key negative regulator of photomorphogenesis, positively regulated light-induced greening by repressing ABA responses. Upon irradiating etiolated seedlings with light, DET1 physically interacts with FAR-RED ELONGATED HYPOCOTYL3 (FHY3) and subsequently associates to the promoter region of the FHY3 direct downstream target ABA INSENSITIVE5 (ABI5). Further, DET1 recruits HISTONE DEACETYLASE6 to the locus of the ABI5 promoter and reduces the enrichments of H3K27ac and H3K4me3 modification, thus subsequently repressing ABI5 expression and promoting the greening of etiolated seedlings. This study reveals the physiological and molecular function of DET1 and FHY3 in the greening of seedlings and provides insights into the regulatory mechanism by which plants integrate light and ABA signals to fine-tune early seedling establishment.

Figure 1.

ABA inhibits light-induced greening of etiolated seedlings through DET1. A, Effect of ABA on the greening of etiolated Col-0 and det1-1 seedlings. Four-day-old etiolated seedlings were treated without or with 1, 10, or 30 μm of ABA for 4 h under light conditions. Scale bar = 1 mm. B, The greening phenotype of 4-d-old etiolated Col-0 and det1-1 seedlings that were irradiated with light for 0, 4, 12, or 24 h. C, Total chlorophyll contents of the seedlings shown in A and B. Values are means ± sd. FW, Fresh weight.

Figure 2.

DET1 physically interacts with FHY3 in vitro and in vivo. A and B, Yeast two-hybrid assays showing DET1 interacts with FHY3 in vitro. The positions of various fragments that used in the yeast two-hybrid assays are shown in A. The various fragments of DET1 and FHY3 were fused to DNA binding domain (BD) or activation domain (AD) of GAL4, respectively. 1:10 and 1:50 indicate the dilutions of yeast cells that were spotted on plates for X-α-gal assays. DDO, Yeast synthetic medium without Trp/Leu; QDO, yeast synthetic medium without Trp/Leu/His/Ade, but with 20 μg mL⁻¹ of X-α-gal and 125 ng mL⁻¹ of AbA; QDO+, QDO medium-plus with 40 μg mL⁻¹ of X-α-gal and 250 ng mL⁻¹ of AbA. C, BiFC assays showing DET1 interacts with FHY3 in Arabidopsis protoplasts. YFP^N and YFP^C indicate the N- or C-terminal parts of YFP, respectively. Scale bars = 5 μm. D, Co-IP assays showing DET1 interacts with FHY3 in Arabidopsis. Four-day-old various etiolated seedlings were irradiated with light for 30 min and then used to perform Co-IP assays. The fhy3-11 was used as a negative control showing the positions of endogenous FHY3 protein (indicates by asterisk) that detected by anti-FHY3 polyclonal antibodies. Anti-MYC monoclonal antibodies were used to perform the IP and detect the abundance of the MYC-DET1 fusion protein.

Figure 3.

FHY3 acts downstream of DET1 in mediating ABA responses. A and B, The greening phenotype (A) and chlorophyll contents (B) of 4-d-old various etiolated seedlings treated with 0, 1, 10, or 30 μm of ABA for 4 h under light conditions. FW, Fresh weight. Scale bar = 1 mm. Values are means ± sd. C, RT-qPCR analyses showing the relative expression of ABI5 in various seedlings. Four-day-old various etiolated seedlings were treated without (Mock) or with 30 μm of ABA for the indicated times under DD or after being transferred to light conditions (DD-L). A typical experiment out of three repeated experiments is shown, and means are values ± sd of three technical replicates are presented. D, Phenotypes of various plants germinated and continuously grown on GM without or with 0.1 μm of ABA under DD for 5 d, with 1 μm of ABA under LD for 4 d, or with 3 μm of ABA under LD for 21 d. E, The ABA inhibition response curves of seed germination for various plants under DD (upper) or LD (lower) conditions. ABA inhibition curves were calculated based on the inhibition effect of ABA on germination at the time point that ∼50% wild-type seeds germinated under 1 μm of ABA treatment (at 60 h after imbibition under LD; at 72 h after imbibition under DD, respectively) using the EC50 shift. Values are means ± sd.

Figure 4.

DET1 inhibit the transcriptional activation of ABI5 by FHY3. A and B, Co-IP analyses showing the protein interaction between DET1 and FHY3 dependent on light (A) and enhanced by ABA (B). In A, 4-d-old etiolated MYC-DET1 seedlings (DD) being irradiated with light (DD-L) for 1 h were used to perform Co-IP assays. In B, 4-d-old etiolated MYC-DET1 seedlings treated without (Mock) or with ABA (30 μm) for the indicated times under DD or DD-L conditions were used to perform Co-IP assays. In A and B, MYC-DET1 fhy3 was used as a negative control to indicate the positions (asterisk) of FHY3 protein. C and D, ChIP-qPCR assays showing the association of DET1 with the ABI5 promoter is dependent on FHY3 (C) and enhanced by ABA under light (D). In C, 4-d-old etiolated seedlings (DD) after being irradiated with light (DD-L) for 3 h were used to perform ChIP assays. In D, 4-d-old etiolated seedlings were treated without (Mock) or with ABA (30 μm) for 3 h under DD or DD-L conditions, and then used to perform a ChIP assay. E and F, Transient expression assays showing DET1 represses the transcriptional activation of ABI5 by FHY3. In E, the transformed Arabidopsis protoplasts were first incubated without (Mock) or with 0.3 μm of ABA under darkness for 16 h (DD), then irradiated with light (DD-L) or not for 3 h and used to measure the expression of LUC reporter. In F, the abundances of FHY3-3FLAG and DET1-3FLAG were detected by anti-FLAG antibodies. Data are means of five biological replicates, and error bars represent sd. In C, D, and F, asterisks indicate the statistical significance by Student's t test (*P <0.05, **P <0.01, and ***P <0.001).

Figure 5.

DET1 mediates histone modification during the dark-to-light transition and ABA response. A, Immunoblot assays showing the dynamic changes of H3K27ac and H3K4me3 abundance during dark (DD) to light (L) transition and ABA treatment. Four-day-old various etiolated seedlings were treated without (Mock) or with ABA (30 μm) under DD or DD-L for 3 h, and then used to perform immunoblot assays. The abundance of histone H3 was used as a loading control. B, ChIP-qPCR assays showing the association of H3K27ac and H3K4me3 at the promoter and exon regions of ABI5. The exon region of eIF4 (eIF4A1) was used as a negative control for ChIP-qPCR. Asterisks indicate the statistical significance by Student's t test (*P <0.05 and **P <0.01). C, RT-qPCR analysis showing the effect of ABA and TSA treatment on the expression of ABI5. Four-day-old etiolated seedlings were treated with ABA (30 μm) or ABA+TSA (10 μm) for 3 h under light (DD-L), and then used to perform RT-qPCR assays.

Figure 6.

DET1 recruits HDA6 to inhibit the transcriptional activity of FHY3. A, Co-IP assays in N. benthamiana leaves showing DET1 interacts with HDA6. B, Co-IP assays in protoplasts of wild type (Col-0) and det1-1 showing FHY3 interacts with HDA6 via DET1. C and D, Seed germination phenotypes (C) and ratio (D) of wild-type (Ws) and hda6 plants under 1-μm ABA treatment conditions. Germination ratios were determined from three biological replicates of ∼40 seeds each. E, Immunoblot assays showing the dynamic changes of H3K4me3 and H3K27ac abundance in Ws and hda6 plants during the dark-to-light transition. The abundance of histone H3 was used as a loading control. F, ChIP-qPCR assays showing the effect of HDA6 on the association of H3K4me3 and H3K27ac at ABI5 promoter during the dark-to-light transition. The exon of eIF4 was used as a negative control for ChIP assay. G, ChIP-qPCR assays showing HDA6 associates with the ABI5 promoter after etiolated seedlings irradiated with light (DD-L). The promoter of ACT was used as a negative control. H, RT-qPCR analysis showing the effect of HDA6 on the expression of ABI5. Four-day-old etiolated seedlings were treated with 30 μm of ABA for 3 h under light and then used to perform RT-qPCR assays. I, Transient expression assays in Arabidopsis protoplasts showing HDA6 and DET1 repress the transcriptional activation of ABI5 by FHY3. Data are means of five biological replicates, and error bars represent sd. In F to I,  asterisks indicate the statistical significance by Student's t test  (*P <0.05 and **P <0.01). J, Working model showing DET1-HDA6 and FHY3 coordinately regulate the effects of light and ABA in the greening of seedling during the dark-to-light transition.