Cis-regulatory changes at FLOWERING LOCUS T mediate natural variation in flowering responses of Arabidopsis thaliana

Abstract

Flowering time, a critical adaptive trait, is modulated by several environmental cues. These external signals converge on a small set of genes that in turn mediate the flowering response. Mutant analysis and subsequent molecular studies have revealed that one of these integrator genes, FLOWERING LOCUS T (FT), responds to photoperiod and temperature cues, two environmental parameters that greatly influence flowering time. As the central player in the transition to flowering, the protein coding sequence of FT and its function are highly conserved across species. Using QTL mapping with a new advanced intercross-recombinant inbred line (AI-RIL) population, we show that a QTL tightly linked to FT contributes to natural variation in the flowering response to the combined effects of photoperiod and ambient temperature. Using heterogeneous inbred families (HIF) and introgression lines, we fine map the QTL to a 6.7 kb fragment in the FT promoter. We confirm by quantitative complementation that FT has differential activity in the two parental strains. Further support for FT underlying the QTL comes from a new approach, quantitative knockdown with artificial microRNAs (amiRNAs). Consistent with the causal sequence polymorphism being in the promoter, we find that the QTL affects FT expression. Taken together, these results indicate that allelic variation at pathway integrator genes such as FT can underlie phenotypic variability and that this may be achieved through cis-regulatory changes.

Figure 1.—

QTL analysis of flowering time in Est-1/Col-0 AI-RIL population. (A) Distribution of flowering time (expressed as leaf number) in the AI-RIL population grown in the greenhouse under 23° LD, including the means for the Est-1 and Col-0 parents. (B) QTL maps of flowering time (measured as days to flower) under three different growth conditions: blue, 16° LD in growth room; red, 23° LD in growth chambers; black, 23° LD in green house. The horizontal line represents the significance threshold for the LOD score. (C and D) Average flowering times of the HIFs based on the genotype of the F5I14 marker. White bars, homozygous for Col-0; gray bars, heterozygous; black bars, homozygous for Est-1. Significant differences (P < 0.0001) between the Est-1 and Col-0 genotypes are indicated by asterisks. Error bars represent standard error of mean (SEM).

Figure 2.—

Genetic interactions between flowering time QTL. (A and B) Two-dimensional genome scan in Est-1/Col-0 AI-RIL population in 23° LD (A) and 16° LD (B), with epistatic interactions on top, and additive interactions on the bottom. Interactions between markers on each chromosome are shown. Color scale indicates LOD scores for epistatic (left) and additive interactions (right). (C) QTL analyses of thermosensitivity in Est-1/Col-0 AI-RIL population, contrasting flowering at 16° and 23°, using three different 23° data sets: black, growth room 1; red, growth room 2; blue, green house. The thermosensitivity QTL colocalizes with the QTL in the F5I14/FT region (Figure 1B). (D) Distribution of flowering time in Dra-1 × Ler F₂ population at 23° and 16° LD. (E) QTL analysis of flowering time in Dra-1 × Ler F₂ population. (F) Two-dimensional genome scan in Dra-1 × Ler F₂ population (see A and B for legend).

Figure 3.—

Fine mapping of the chromosome 1 Est-1/Col QTL. (A) Four-week-old NIL-Col. (B) NIL-Est of same age and grown in parallel. (C) Distributions of flowering time for Est-1, Col-0, and the Est-NIL. (D) Fine mapping of the QTL. Transcription units are in purple. The FT gene (At1g65480) is highlighted in yellow. The three levels reflect the progressive rounds of fine mapping, with the final 6.7-kb mapping interval in the FT promoter shown on the bottom. The flanking markers used for mapping are shown. FAS1 (At1g65470) is the gene to the left.

Figure 4.—

Genetic evidence for FT being causal for the chromosome 1 QTL. (A and B) Quantitative complementation assays. (A) Flowering time of F₁ plants from crosses of Dra-1 and Ler to Col-0 (“wild type”) and the isogenic ft-10 mutant. (B) Flowering time of F₁ plants from crosses of Est-1 and Col-0 to Ler (“wild type”) and the isogenic ft-1 mutant. (C) Quantitative knockdown experiment with artificial miRNA against FT (amiR-ft-1) introduced into the two NILs and the two parents. (D) Flowering time of the different genotypes under short days.

Figure 5.—

Allelic variation affects FT expression. (A) Comparison of FT expression in Col-0 under thermocycles (12 hr 22°/12 hr 12°, continuous white light) and light cycles (16 hr light/8 hr dark, constant 23°). (B) Comparison of FT expression levels in NIL-Col and NIL-Est under thermocycles. Two different lines for each NIL are shown. The second NIL-Est line had very low FT expression, and its values are barely visible.