Negative regulatory roles of DE-ETIOLATED1 in flowering time in Arabidopsis

Abstract

Arabidopsis flowers early under long days (LD) and late under short days (SD). The repressor of photomorphogenesis DE-ETIOLATED1 (DET1) delays flowering; det1-1 mutants flower early, especially under SD, but the molecular mechanism of DET1 regulation remains unknown. Here we examine the regulatory function of DET1 in repression of flowering. Under SD, the det1-1 mutation causes daytime expression of FKF1 and CO; however, their altered expression has only a small effect on early flowering in det1-1 mutants. Notably, DET1 interacts with GI and binding of GI to the FT promoter increases in det1-1 mutants, suggesting that DET1 mainly restricts GI function, directly promoting FT expression independent of CO expression. Moreover, DET1 interacts with MSI4/FVE, which epigenetically inhibits FLC expression, indicating that the lack of FLC expression in det1-1 mutants likely involves altered histone modifications at the FLC locus. These data demonstrate that DET1 acts in both photoperiod and autonomous pathways to inhibit expression of FT and SOC1. Consistent with this, the early flowering of det1-1 mutants disappears completely in the ft-1 soc1-2 double mutant background. Thus, we propose that DET1 is a strong repressor of flowering and has a pivotal role in maintaining photoperiod sensitivity in the regulation of flowering time.

Figure 1. Flowering-time phenotypes of det1-1 mutants.

(a) Phenotypes of wild-type (WT, Col-0 ecotype) and det1-1 mutant plants. Plants were grown at 22°C under cool-white fluorescent light (90–100 μmol m⁻²s⁻¹) in LD (16-h light:8-h dark) or SD (10-h light:14-h dark), and photographed at 2 to 4 days after bolting. Scale bars = 2 cm. (b–c) Genetic analysis to show epistasis between det1-1 and flowering mutants using double (b) and triple mutants (c). The number of rosette leaves of WT (Col-0) and flowering-time mutants grown under LD (16-h light:8-h dark) and SD (10-h light:14-h dark) in (b), and LD (16-h light:8-h dark) and SD (8-h light:16-h dark) conditions in (c) (see Table S1). Flowering time was measured as the number of rosette leaves at bolting. Means and standard deviations were obtained from more than 20 plants.

Figure 2. Effect of det1-1 mutation on GI, FKF1, CO, FT, SOC1, and FLC expression under SD.

The expression of GI (a), FKF1 (b), CO (c), FT (d), SOC1 (e), and FLC (f) was analyzed in Col-0 and det1-1 mutants by real-time PCR using 3-week-old plants. Plants were grown at 22°C under SD (8-h light:16-h dark) conditions, and plant tissues were harvested every 3 h. ACT2 expression was used for normalization. Means and standard deviations were obtained from three biological replicates.

Figure 3. Flowering time of det1-1 mutants under reduced diurnal cycles.

(a) Effect of reduced diurnal cycles on the flowering time of det1-1 mutants. Plants were entrained in SD (light [L]:dark [D] = 1:2) of 24 h (24 T = 8 L:16 D), 21 h (21 T = 7 L:14 D), and 18 h (18 T = 6 L:12 D). T represents environmental time period. Means and standard deviations were obtained from more than 20 plants. Col-0 means Columbia-0 ecotype (wild type). (b) Phenotypes of det1-1 mutants after bolting under SD of 24 T, 21 T, and 18 T. Plants were grown at 22–24°C under cool-white fluorescent light (90–100 μmol m⁻² s⁻¹). Scale bars = 2 cm.

Figure 4. DET1 directly interacts with GI.

(a) Comparison of GI protein stability between pGI:GI-HA and pGI:GI-HA det1-1 plants under SD conditions. The plant tissues were collected every 2 h during the daytime and every 4 h during the nighttime, using 3-week-old seedlings. GI protein was detected with an anti-HA antibody. RFT5 expression was used for normalization. Means and standard deviations were obtained from three biological replicates. (b) Interaction of DET1-GI was tested by yeast 2-hybrid assay. The bait was full-length DET1. For prey, GI was divided into three pieces: N-terminal (N; 1–507), middle (M; 401–907), and C-terminal (C; 801–1173). Gal4 indicates a positive control. Empty pGBKT7 (BD) and pGADT7 (AD) vectors were used as the negative control. SD medium (-LWHA; lacking tryptophan, leucine, histidine, and adenine) was used to select for the interaction between bait and prey proteins. β-galactosidase (β-Gal) activity assays were performed according to the manufacturer's protocol. Means and standard deviations were obtained from three biological replicates. (c) BiFC analysis of the interaction of between DET1 and GI in the nucleus of an onion epidermal cell. nYFP-ELF3 and cYFP-ELF4 plasmids served as a positive control. For the negative control, empty nYFP/GI-cYFP and nYFP-DET1/cYFP were used. Scale bar = 50 μm. (d) Coimmunoprecipitation of DET1 and GI. Total protein was extracted from 2-week-old seedlings of p35S:TAP-DET1 pGI:GI-HA gi-2 and p35S:TAP-GFP pGI:GI-HA gi-2. IgG beads were used for the pull-down. An anti-HA antibody was used for GI-HA protein band. p35S:TAP-GFP pGI:GI-HA gi-2 plants served as a negative control. The upper panel is a coimmunoprecipitated sample, and the middle panel is the input sample for GI-HA protein. The lower panel shows input samples of p35S:TAP-GFP and p35S:TAP-DET1.

Figure 5. DET1 affects GI binding to the FT promoter.

(a) Gene structure of FT and the amplicon regions for the ChIP assay. Six amplicon locations (I, II, III, IV, V and VI) are shown. (b) FT promoter binding affinity of GI in the det1-1 mutant, relative to the wild type. All samples were harvested at ZT8 under SD (8-h light:16-h dark) conditions. Chromatin isolated from these samples was immunoprecipitated with anti-HA. Relative enrichment in Col-0, pGI:GI-HA gi-2, and pGI:GI-HA gi-2 det1-1 are shown. Means and standard deviations were obtained from three biological replicates. This experiment was replicated at least three times with similar results. UBIQUITIN 10 (UBI10) was used as a negative control. Black, gray, and white boxes represent Col-0, pGI:GI-HA gi-2, and pGI:GI-HA gi-2 det1-1, respectively. Asterisks indicate statistically significant differences compared to pGI:GI-HA as determined by Student's t-test (*P < 0.05 and **P < 0.01, respectively).

Figure 6. DET1 interacts with MSI4 and regulates histone methylation of the FLC locus.

(a) BiFC analysis of the interaction between MSI4 and DET1 in onion epidermal cells. For negative controls, nYFP/cYFP-MSI4 and DET1-nYFP/cYFP were used. Scale bar = 50 μm. (b) Relative levels of histone modifications on the FLC locus were examined by ChIP analysis using H3K4me3 and H3K27me3 antibodies in Col-0 and det1-1 plants. The top of the panel represents the FLC gene structure and the region used for primers (I, II and III) in the ChIP-quantitative PCR analyses. Chromatin was prepared from 14-day-old seedlings grown under SD (8-h light:16-h dark). FUSCA 3 (FUS3) was used for the normalization of the quantitative PCR analysis. Means and standard deviations were obtained from three biological replicates. This experiment was replicated at least three times with similar results. Asterisks indicate statistically significant difference compared to Col-0 as determined by Student's t-test (*P < 0.05).

Figure 7. Working model of DET1 function in floral repression in Arabidopsis.

DET1 suppresses FT and SOC1 expression through the photoperiod and autonomous pathways of flowering. In the photoperiod pathway, DET1 mainly represses flowering by modulating GI-mediated floral induction at the transcriptional and post-translational levels during daytime under SD. DET1 represses the function of daytime-expressed GI by preventing GI from binding to the FT promoter in a CO-independent pathway. In the autonomous pathway, DET1 interacts with MSI4/FVE and possibly modulates trimethylation of FLC chromatin to epigenetically induce FLC expression. Genes and proteins are represented as rectangles and ovals, respectively.