PMC

Seedlings of afr1 AFR1:HA, agl15 agl18 afr1 AFR1:HA, co afr1 AFR1:HA, and WT (negative CK) were harvested at ZT16 and subjected to ChIP assays with anti-HA. The fold enrichments of AFR1:HA in FT-P (a proximal promoter region) in the AFR1:HA-expressing lines over CK, are presented. Error bars indicate SD of triplicate measurements. A biological repeat of this analysis is presented as .

(A) AFR1 and AFR2 gene structures. Exons are represented by black boxes, and arrows indicate transcription start sites (TSS); triangles for T-DNA insertion sites. (B) afr1, afr2, and afr1 afr2 mutants grown in LDs. The arrow indicates a main bolt with flowers. (C) Flowering times of the indicated genotypes grown in LDs. 19–23 plants were scored for each line. Double asterisks indicate statistically significant differences in the means between Col (WT) and the indicated mutants, as revealed by two-tailed Student's t test (**, p<0.01). Bars indicate SD (for standard deviation). (D) Flowering times of the indicated genotypes grown in short days. 11–15 plants were scored for each line. Double asterisks indicate statistically significant differences in the means between Col and the indicated mutants. (E) Leaf phenotype of the afr1 afr2 double mutant grown in LDs.

Amounts of the immunoprecipitated genomic fragments were quantified by qPCR. The fold enrichments were calculated as follows: for each examined FT region, the amount of DNA fragments from WT or afr1 afr2 at each time point (ZT8 or ZT16) was first normalized to the constitutively expressed TUBULIN2 (TUB2) in each sample, and subsequently, the TUB2-normalized values for the afr1 afr2 at ZT8, the afr1 afr2 at ZT16, or the WT at ZT16 were divided by the value for the WT at ZT8 to obtain fold enrichments. Shown are the means and SD of two ChIP experiments. An analysis of H3 acetylation on FT chromatin in Col and afr1 afr2 seedlings is presented in .

(A) Interactions of AtSAP18 with AT1G75060 and AT1G19330 in yeast. The indicated proteins of full-length were fused with the GAL4-BD or AD domain. Yeast cells harboring the fusion proteins, BD and/or AD (as indicated), were grown on selective synthetic defined media lacking of Trp, Leu, and His. (B) BiFC analysis of the interactions of AtSAP18 with AT1G75060 and AT1G19330 in onion epidermal cells. Onion epidermal cells were co-transformed transiently by a pair of plasmid, as indicated, via biolistic gene bombardment. Yellowish-green signals indicate physical associations of paired proteins in the nuclei. Blue fluorescence from a DAPI (4′,6-diamidino-2-phenylindole) staining indicates a nucleus. Bar = 20 µm. (C) Interactions of HDA19 with AT1G75060 and AT1G19330 in yeast. The indicated full-length proteins were fused with the GAL4-BD or AD domain. Yeast cells were grown on selective synthetic defined media lacking of Trp, Leu, and His. (D) BiFC analysis of the interactions of HDA19 with AT1G75060 and AT1G19330 in onion epidermal cells. Bar = 20 µm.

The coincidence of high CO mRNA expression with light exposure at the day's end leads to the CO protein accumulation towards dusk. CO directly binds to the FT proximal promoter, and CO activity at the FT locus may change the chromatin state and enables/gates AGL18 (and presumably AGL15) binding to the FT proximal promoter. AGL18 recruits AFR1/AFR2-HDAC to FT chromatin at dusk. In addition, the CO activity may also enable the recruitment of a HAT to FT chromatin. The opposing activities of HAT and AFR-HDAC on FT chromatin at the end of LDs conceivably modulate the acetylation dynamics of FT chromatin and set FT expression at an adequate level at dusk. At night, CO is rapidly degraded by proteasomes, which prevents the actions of HAT and AFR-HDAC on FT chromatin, resulting in a “silent” chromatin state. In early day, FT chromatin remains ‘silent’ due to lack of the CO protein. Day and night are indicated with white and gray shadings, respectively.

(A,B) Flowering times of the indicated genotypes grown in LDs. 11–17 plants were scored for each line. The afr1 afr2 double mutant is afr1-1 afr2-1. Double asterisks indicate statistically significant differences in the means between the indicated genotypes, as revealed by two-tailed Student's t test (**, p<0.01). Bars indicate SD. (C,D) Relative CO (C) and FT transcript levels (D) in the seedlings of indicated genotypes at the end of LDs (ZT16), quantified by qRT-PCR. The transcript levels were normalized first to the endogenous control UBQ10, and relative fold changes to WT are presented (note that the FT transcript level in co was less than 1% of that in WT). Bars indicate SD of triplicate measurements. One of two biological repeats with similar results is shown. (E) FT mRNA levels in Col and afr1 afr2 seedlings over a 24-h LD cycle, as quantified by qRT-PCR. FT transcript levels were normalized to UBQ10; bars indicate SD of triplicate measurements. A biological repeat of this analysis is included as . White and dark bars below the x-axis indicate light and dark periods, respectively.

(A) Spatial expression patterns of AFR1-GUS, AFR2-GUS, and FT-GUS. LD-grown Col seedlings or rosette leaves were stained for 6 h except for AFR1-GUS staining with 8.5 h. Arrows indicate stained veins. (B) Nuclear localization of the AFR1:GFP and AFR2:GFP fusion proteins in Arabidopsis root cells. Scale bars are 50 µm. The blue DAPI staining indicates nuclei. (C) AFR1 and AFR2 mRNA levels in Col (WT) seedlings over a 24-h LD cycle. The mRNA levels were normalized to UBQ10; bars indicate SD of triplicate measurements. A biological repeat of this analysis is included as . White and dark bars below the x-axis indicate light and dark periods, respectively. (D) AFR1:HA and AFR2:FLAG protein levels in Col seedlings over a 24-h LD cycle. Total proteins loaded in a duplicated SDS-PAGE gel were stained with Coomassie Blue, serving as loading controls. (E) ChIP analysis of AFR1:HA enrichment at the FT locus. Amounts of the immunoprecipitated genomic fragments were measured by qPCR, and normalized first to the endogenous control TUBULIN8 (TUB8). The fold enrichment of AFR1:HA in each examined region (at each time point) was calculated by dividing the TUB8-normalized amount of examined region from the AFR1:HA-expressing line, by that of WT (without AFR1:HA) at each time point. Error bars indicate SD of triplicate quantifications (technical replicates). A biological repeat of this analysis is presented as . (F) ChIP analysis of AFR2:FLAG enrichment at the FT locus. The fold enrichments of AFR2:FLAG were calculated in a way similar to those of AFR1:HA. Error bars indicate SD of triplicate quantifications. A biological repeat of this analysis is presented as .

(A) BiFC analysis of the interaction of AGL18 with AFR1 in onion epidermal cells. Yellowish-green signals indicate the physical association of AGL18 with AFR1 in the nuclei (indicated by the blue fluorescence from DAPI). Bar = 20 µm. (B) Co-immunoprecipitation of AFR1 with AGL18 in Arabidopsis seedlings. Total protein extracts from F₁ seedlings of the doubly hemizygous AGL18:FLAG and AFR1:HA, were immunoprecipitated with anti-HA agarose; subsequently, the precipitates were analyzed by western blotting with anti-FLAG (recognizing AGL18:FLAG) and anti-HA (recognizing AFR1:HA). (C) Flowering times of the indicated genotypes grown in LDs. 12–16 plants were scored for each line. Double asterisks indicate a statistically significant difference in the means between Col and agl15 agl18, as revealed by two-tailed Student's t test (**, p<0.01). Bars indicate SD. (D) Relative FT transcript levels in the seedlings of indicated genotypes at ZT16, quantified by qRT-PCR. The transcript levels were first normalized to UBQ10, and relative fold changes to Col are presented. Bars indicate SD of triplicate measurements. One of two biological repeats with similar results is shown. (E) ChIP analysis of AGL18:FLAG enrichment at the FT locus. Amounts of immunoprecipitated genomic fragments were measured by qPCR, and normalized first to the endogenous control TUB8. The fold enrichment of AGL18:FLAG in each examined region (at each time point) was calculated by dividing the TUB8-normalized amount of examined region from the AGL18:FLAG-expressing line, by that of WT (without AGL18:FLAG) at each time point. Error bars indicate SD of triplicate measurements. A biological repeat of this analysis is presented as .