To identify non-coding regions important for FLM expression, we performed multiple sequence alignments of Arabidopsis FLM with its closest sequence homologue MAF3 and FLM homologues from five other Brassicaceae species (Figure 2—figure supplement 1A,B) (Martinez-Castilla and Alvarez-Buylla, 2003; Van de Velde et al., 2014). Within the non-coding sequences, we detected one promoter region of 250 bp and two intron 1 regions of 373 and 101 bp with increased sequence conservation (>60%) (Figure 2A and Figure 2—figure supplement 1). We then generated PROΔ225bp, INT1Δ373bp, and INT1Δ101bp transgenic lines expressing pFLM::gFLM variants (FLMCol-0) with deletions of the three regions in the FLM deletion accession Nd-1 to measure the effects on FLM-ß and FLM-δ expression at 15°C and 23°C (Figure 2B and Figure 2—figure supplement 2) (Werner et al., 2005). To normalize for variability between the transgenic lines, we examined pools of independent T2 segregating lines (n = 21–34). We validated this pooling strategy by demonstrating that the established behaviour of FLM in the Col-0 and Kil-0 accessions could be faithfully recapitulated when performing equivalent analyses with T2 lines expressing gFLMCol-0 and gFLMCol-0 bearing the Kil-0 LINE insertion (Lutz et al., 2015) (Figure 2—figure supplement 2C,D). While we detected a strong downregulation of FLM at 15°C and 23°C in PROΔ225bp and INT1Δ373bp lines, the deletion in INT1Δ101bp had comparably minor effects on FLM expression (Figure 2C,D). This indicated that the proximal 225 bp promoter and the 337 bp intron 1 regions contained important sequences for basal FLM expression. Subsequent experiments showed that the 254 bp promoter fragment alone was, however, not sufficient to confer FLM expression of a genomic FLM fragment (PRO254bp; Figure 2B–D).10.7554/eLife.22114.003Figure 2.Phylogenetic footprinting identifies promoter and intron 1 regions required for FLM expression.(A) Phylogenetic footprinting of promoter and genomic regions of FLM and putative FLM orthologs from six Brassicaceae species. Coverage is shown in blue, identities are shown in ocre (≥30%) and red (<30%). Exons are displayed in green, regions with high non-coding sequence conservation are displayed in orange. (B) Schematic illustration of the FLM genomic region and FLMCol-0 transgenic variants used for expression analysis. (C) and (D) Mean and SD (four replicate pools with five to ten independent T2 transgenic lines) of qRT-PCR analysis of FLM-β (C) and FLM-δ (D) expression at 15°C and 23°C in ten day-old seedlings of 21–40 bulked T2 transgenic lines. Student’s t-tests: *, p≤0.05; **, p≤0.01; ***, p≤0.001; n.s., not significant.DOI: http://dx.doi.org/10.7554/eLife.22114.00310.7554/eLife.22114.004Figure 2—figure supplement 1.Phylogenetic footprinting of promoter and genomic regions of FLM and putative FLM orthologs from six Brassicaceae species.(A) and (B) Sequence alignments of promoter (A) and gene sequences (B) of FLM and MAF3 as well as putative FLM orthologs from six Brassicaceae species. Coverage is shown in blue, identities are based on a sliding window word size = 30 and displayed in ocre (≥30%) and red (<30%). FLM exonic regions are shown in green (B). Numbering is indicated according to the position in the alignment.DOI: http://dx.doi.org/10.7554/eLife.22114.00410.7554/eLife.22114.005Figure 2—figure supplement 2.The gFLMCol-0 and gFLMCol-0+LINE transgenes faithfully recapitulate FLM expression in the Col-0 and Kil-0 accessions.(A) Representative photographs of 42 day-old plants of Col-0, flm-3, and Nd-1 grown at 15°C in long day photoperiod. The plant images were spliced together but originate from the same photograph as indicated by a white vertical line. (B) Quantitative flowering time analysis of Col-0, flm-3, and Nd-1 grown at 15°C and 23°C in long day photoperiod. (C) and (D) qRT-PCR analyses of FLM-β (C) and FLM-δ (D) expression at 15°C and 23°C in ten day-old flm-3 mutants complemented with a gFLMCol-0 or gFLMCol-0+LINE allele (Lutz et al., 2015). Pools of 35–40 independent T2 transgenic lines were used in comparison to Col-0 and Kil-0 homozygous lines. Shown is the mean and SD of three (Col-0, Kil-0) and four replicate pools comprising each 8 to 10 transgenic lines. Percentages refer to the reduction in FLM expression between the gFLMCol-0+LINE transgenic line in comparison to the gFLMCol-0 line or between Kil-0 and Col-0, respectively. Similar letters indicate no significant difference of total leaf number (Tukey HSD, p<0.05).DOI: http://dx.doi.org/10.7554/eLife.22114.005