FKF1 and GIGANTEA complex formation is required for day-length measurement in Arabidopsis

Abstract

Precise timing of CONSTANS (CO) gene expression is necessary for day-length discrimination for photoperiodic flowering. The FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1), and GIGANTEA (GI) proteins regulate CO transcription in Arabidopsis. We demonstrate that FKF1 and GI proteins form a complex in a blue-light-dependent manner. The timing of this interaction regulates the timing of daytime CO expression. FKF1 function is dependent on GI, which interacts with a CO repressor, CYCLING DOF FACTOR 1 (CDF1), and controls CDF1 stability. GI, FKF1, and CDF1 proteins associate with CO chromatin. Thus, the FKF1-GI complex forms on the CO promoter in late afternoon to regulate CO expression, providing a mechanistic view of how the coincidence of light with circadian timing regulates photoperiodic flowering.

Figure 1. FKF1 interacts with GI in a blue-light dependent manner

(A) Interaction between FKF1 and GI proteins in yeast. LOV+F contains LOV and F-box domains. F+kelch contains F-box and kelch repeat domains (7). ASK2 is known to interact with F-box domain. SD-WL medium is a control; SD-WLH medium is for selection of protein interaction. (B to F) GI-TAP and HA-FKF1 protein profiles in coimmunoprecipitation experiments under various light conditions. The 35S‷HA-FKF1 35S‷GI-TAP line and the 35S‷HA-FKF1 line were grown for 10 days in long days (B) or short days (C). The long-day-grown 35S‷HA-FKF1 35S‷GI-TAP #18 / fkf1 line was kept in the dark on day 10 (D). The 35S‷HA-FKF1 35S‷GI-TAP #18 / fkf1 line was incubated under blue or red light (both 25 μmol/m²/sec) on day 10 (E). The 35S‷HA-FKF1 35S‷GI-TAP #18 / fkf1 line was incubated under different intensities of blue light for 1 hour (F). The bar color represents the light conditions. (G) In vitro reconstitution of the FKF1-GI interaction. Samples were incubated in the dark or under white light (80 μmol/m²/sec). DB71 staining showed GST and GST-GI-N proteins precipitated.

Figure 2. The FKF1-GI complex is formed in late afternoon and regulates daytime CO expression

(A to C) GI-TAP and HA-FKF1 protein profiles in coimmunoprecipitation experiments with a line expressing GI-TAP and HA-FKF1 under endogenous promoter regulation. The FKF1‷HA-FKF1 GI‷GI-TAP / fkf1 gi-2 transgenic plants were grown under long day (A), short day (B) or short day to long day (C) conditions. The white and black bars represent the white light and dark conditions. The hatched bar represents extended light incubation. (D) Flowering phenotypes of plants with various levels of FKF1 and GI expression in long days and short days. Data are mean ± S.E.M. for 16 plants. (E) CO expression in wild type plants, fkf1-2, gi-2, and fkf1-2 gi-2 mutants in long days. IPP2 expression (15) was used for normalization. CO expression in each panel is shown relative to the average value of wild-type plant data. (F and G) CO expression in wild type plants and the 35S‷HA-FKF1 35S‷GI-TAP #18 / fkf1 in long days (F) and short days (G).

Figure 3. GI-FKF1-CDF1 complex associates with CO promoter in vivo

(A) In vivo interaction between HA-CDF1 and GI-TAP. The 35S‷HA-CDF1 #17 (GI-TAP-) and 35S‷GI-TAP 35S‷HA-CDF1 #17 (GI-TAP+) lines were grown in long days, harvested 4 hours after light onset on day 10, and coimmunoprecipitation assays were performed (7). (B) HA-CDF1 expression in the 35S‷HA-CDF1 and gi-2 35S‷HA-CDF1 lines. Plants were harvested at day 10 in long days. ACT was used as a loading control. (C) CO chromatin regions associated with GI-TAP protein. Plants were harvested 13 hours after light onset on day 10. The ratio between the specific enrichment value in the GI‷GI-TAP sample and that in the wild type sample on each amplicon was calculated from 7 independent ChIP analyses (7). ACT2 and UBQ10 genes were used as controls. The dotted line indicates no enrichment. (D) Schematic drawing of the CO locus and the amplicon locations for ChIP analysis. The 17 amplicon locations are shown. White and light gray boxes represent exons, and 5'- and 3'-untranslated regions (UTR). (E and F) CO promoter regions associated with FKF1-TAP and HA-CDF1 proteins. Plants were harvested 13 hours [FKF1-TAP (E)] and 4 hours [HA-CDF1 (F)] after light onset on day 10. Data were calculated from 4 independent analyses.

Figure 4. A model of day-length dependent CO transcriptional regulation

In long days, the circadian-regulated coincidence of FKF1 and GI peak expression and the light-induced FKF1 interaction with GI enable the formation of the FKF1-GI complex in late afternoon. When the complex is formed on the CO promoter, CDF1 associated with GI is degraded by FKF1 to facilitate the induction of daytime CO expression. Then CO protein is stabilized and activated by light to induce FT expression (13, 17). In short days, FKF1 peaks in the dark at a different time than GI, thus only a small quantity of the complex forms.