FLOWERING LOCUS C (FLC) regulates development pathways throughout the life cycle of Arabidopsis

Abstract

FLOWERING LOCUS C (FLC) has a key role in the timing of the initiation of flowering in Arabidopsis. FLC binds and represses two genes that promote flowering, FT and SOC1. We show that FLC binds to many other genes, indicating that it has regulatory roles other than the repression of flowering. We identified 505 FLC binding sites, mostly located in the promoter regions of genes and containing at least one CArG box, the motif known to be associated with MADS-box proteins such as FLC. We examined 40 of the target genes, and 20 showed increased transcript levels in an flc mutant compared with the wild type. Five genes showed decreased expression in the mutant, indicating that FLC binding can result in either transcriptional repression or activation. The genes we identified as FLC targets are involved in developmental pathways throughout the life history of the plant, many of which are associated with reproductive development. FLC is also involved in vegetative development, as evidenced by its binding to SPL15, delaying the progression from juvenile to adult phase. Some of the FLC target genes are also bound by two other MADS-box proteins, AP1 and SEP3, suggesting that MADS-box genes may operate in a network of control at different stages of the life cycle, many ultimately contributing to the development of the reproductive phase of the plant.

Fig. 1.

Analysis of motifs overrepresented in the FLC binding sites. Motifs overrepresented in the FLC binding sites were analyzed by MEME software. Motifs were numbered 1–4 and ordered by the number of occurrences. The percentage of the motif in the binding sites was indicated to the right of each motif.

Fig. 2.

Validation and expression analyses of selected FLC target genes. (A) Binding profiles for selected target genes. The TAIR annotation of the genomic locus is shown at the top of each box. The profile in flc-3 is shown in the middle part of each box, and the profile in Col FRI is shown at the bottom of each box. Scale bars indicate sequence lengths, and arrows indicate gene orientation. The scale of the y axis is adjusted for each trace for visual clarity. (B) ChIP-PCR validation for selected FLC target genes. ChIP was done with FLC antiserum in Col FRI and C24 and compared with the corresponding flc mutant: flc-3 and flc-20. Input DNA was used as the reference in the PCR. Error bars represent SEs. (C) Transcriptional responses of the selected genes with loss of flc activity. Actin was used as the internal reference. The expression level of each gene in flc-3 was normalized to the level in Col FRI. Error bars represent SEs. Asterisks indicate a significant change (*P < 0.05; **P < 0.01; Student's t test).

Fig. 3.

GO categories enriched in the FLC target genes were analyzed with BiNGO. GOslim categories with significant enrichment in the dataset were highlighted in color, with different colors representing different levels of significance.

Fig. 4.

Comparison of abaxial trichome numbers between Col FRI and flc-3. Abaxial trichome numbers for Col FRI and flc-3 were counted 4 wk after germination. Six plants were sampled for Col FRI, and 10 plants were sampled for flc-3. Error bars represent SEM.

Fig. 5.

Floral defects of flc-3 soc1-2 agl24-1. (A) Inflorescence apex of wild-type Col. (B) Early stage of flc-3 soc1-2 agl24-1 inflorescence with many leafy structures. (C) Flowers of flc-3 soc1-2 agl24-1 with no petals or stamens. (D) Flower of flc-3 soc1-2 agl24-1 with carpelloid sepals. (E) Inflorescence of soc1-2 agl24-1 with normal floral organs. (F) Inflorescence of soc1-2 agl24-1 svp-41 with abnormal floral organs.

Fig. 6.

FLC functions in plant development. FLC is involved in various aspects of plant development, including the juvenile-to-adult transition, flowering initiation, and floral morphogenesis. Arrows indicate gene activation, and blunted lines indicate repression. For FLC binding targets, solid lines indicate confirmed expression changes in the flc seedlings, and broken lines indicate either no expression changes or was not checked in the flc seedlings.