| 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.MOLECULAR analysis of the phenotypic variation in life history traits is key to understanding how plants evolve in diverse natural environments. Among such traits, flowering time is critical for the reproductive success of the plant and is highly variable among natural Arabidopsis thaliana strains, providing an attractive paradigm for studying adaptive evolution (Johanson et al. 2000; Hagenblad and Nordborg 2002; Stinchcombe et al. 2004; Lempe et al. 2005; Shindo et al. 2005; Werner et al. 2005a). Two major environmental parameters that modulate flowering time are light and temperature (Koornneef et al. 1998). Temperature and light conditions vary substantially within the geographical range of A. thaliana, and natural populations presumably need to adapt to the local environment to ensure reproductive success. Flowering in A. thaliana is generally accelerated by long photoperiods, vernalization (exposure to winter-like conditions), and elevated ambient temperatures (Bäurle and Dean 2006). All these cues favor flowering of A. thaliana during spring or early summer, although the contribution from each individual cue and the interactions among them vary depending on the local environmental conditions (Wilczek et al. 2009).Flowering time is controlled through several genetic cascades that converge on a set of integrator genes including FLOWERING LOCUS T (FT), which encodes a protein that is highly conserved in flowering plants (Kardailsky et al. 1999; Kobayashi et al. 1999; Ahn et al. 2006). FT and its homologs are very likely an integral part of the mobile signal (florigen) that is produced in leaves and travels to the shoot apex to induce flowering (Abe et al. 2005; Wigge et al. 2005; Lifschitz et al. 2006; Corbesier et al. 2007; Jaeger and Wigge 2007; Lin et al. 2007; Mathieu et al. 2007; Tamaki et al. 2007; Notaguchi et al. 2008). In A. thaliana, FT expression is controlled by photoperiod, vernalization, and ambient growth temperature. Photoperiod in conjunction with the circadian clock promotes daily oscillations in FT RNA levels, which are greatly elevated at the end of long days. The central role of FT in determining the timing of flowering appears to be conserved in many species, making FT an attractive target for altering flowering time in cereals and other plants of economic importance (recently reviewed by Kobayashi and Weigel 2007; Turck et al. 2008).Wild strains of A. thaliana show extensive variation in flowering time and much of this is due to variation in the activity of the floral repressor FLOWERING LOCUS C (FLC). While some of this variation maps to FLC itself, much of it is due to differential activity at the epistatically acting FRIGIDA (FRI) locus (Michaels and Amasino 1999; Sheldon et al. 1999; Johanson et al. 2000; Michaels et al. 2003; Lempe et al. 2005; Shindo et al. 2005, 2006). Flowering is typically substantially delayed when the FRI/FLC system is active, unless these plants are first vernalized. However, FRI and FLC do not explain all of the flowering time variation seen in wild strains, and functionally divergent alleles of several additional flowering regulators, including CRYPTOCHROME 2 (CRY2), HUA2, FLOWERING LOCUS M (FLM), PHYTOCHROME C (PHYC), and PHYTOCHROME D (PHYD), have been identified in different strains of A. thaliana (Aukerman et al. 1997; Alonso-Blanco et al. 1998; El-Assal et al. 2001; Werner et al. 2005b; Balasubramanian et al. 2006a; Wang et al. 2007). Finally, there are many genotype-by-environment interactions that dramatically affect the contribution of a specific locus to the overall phenotype.The study of natural variation in A. thaliana has been greatly facilitated through the use of recombinant inbred line (RIL) populations (Koornneef et al. 2004). We have recently established two advanced intercross (AI)-RIL sets, in which the genetic map is greatly expanded, allowing for high-resolution QTL mapping (Balasubramanian et al. 2009). Here we use one of the new AI-RIL populations along with an independent F2 population to identify the molecular basis of a light and temperature-sensitive flowering time QTL that mapped to the promoter of the FT gene. We show that FT is likely the causal gene for variation in light and temperature-sensitive flowering. Our results, in combination with those from other species, suggest that cis-regulatory variation rather than structural variation at FT contributes to phenotypic variation in natural populations. |
