FLOWERING LOCUS C Isolation and Characterization: Two Papers that Opened Many Doors

These two classic papers from The Plant Cell archives (Michaels and Amasino, 1999; Sheldon et al., 1999) were tremendously satisfying in providing a first glimpse of the mechanism by which seasonal flowering of Arabidopsis thaliana is induced by winter cold (vernalization) through FLOWERING LOCUS C (FLC). However, their significance has been much broader than could have been imagined when they were published, as FLC became a paradigm for defining the effects of histone modifications on plant gene expression and allelic variation at FLC explained many phenotypic differences observed among A. thaliana accessions and Brassicaceae species. Here, I briefly summarize the origins, content, and influence of these landmark papers. In 1999, our genetic understanding of how plants control flowering was still in its infancy. Many A. thaliana mutants showing late flowering had been identified and placed in parallel pathways based on epistasis and physiological analysis. Mutants affecting the photoperiodic pathway were only late flowering under inductive long days. By contrast, mutants in the autonomous pathway flowered later under long and short days, and therefore impaired a pathway required for flowering under both conditions. Strikingly, the flowering delay of these autonomous pathway mutants could be corrected by exposure to vernalization, whereas that of photoperiodic pathway mutants could not. Independent work on natural genetic variation among accessions also identified flowering-time loci. In work that predated the mutant analysis, the dominant FRIGIDA (FRI) locus was shown to delay flowering of late-flowering accessions, but its effect could be overcome by vernalization (Napp-Zinn, 1979), indicating some similarity between FRI action and the autonomous pathway. Furthermore, elegant genetic analysis demonstrated that a second dominant locus was required for FRI to cause late flowering (Koornneef et al., 1994; Lee et al., 1994), and the authors gave this locus the rather enigmatic name FLC. They located FLC to a region on chromosome 5, creating the opportunity to isolate it by map-based cloning. The papers in The Plant Cell described the isolation of FLC by independent routes. Sheldon et al. (1999) identified a dominant mutation causing late flowering due to a T-DNA insertion, showed that this caused increased expression of an adjacent gene, and demonstrated that a transposon insertion in the adjacent gene suppressed the late-flowering phenotype. They concluded that this gene encodes a repressor of flowering, which they initially called FLOWERING LOCUS F (FLF), but also mentioned that its chromosomal location was similar to FLC and they might therefore be allelic. By contrast, Michaels and Amasino (1999) isolated FLC by map-based cloning, and then confirmed its isolation by molecular complementation and molecular analysis of an FLC allelic series. Their paper confirmed that FLC and FLF were indeed the same gene based on sequence comparisons. Both papers show that FLC encodes a MADS box transcription factor, demonstrating what was at the time a novel function for this class of protein in repressing floral transition rather than contributing to floral development. In addition, they demonstrate that FLC is hardly expressed in early-flowering accessions such as Columbia, but is highly expressed in the presence of active FRI or in autonomous pathway mutants, and this high expression correlates with late flowering. Exposure of these genotypes to vernalization caused them to flower early and reduced FLC transcript levels. Therefore, FRI and the autonomous pathway are linked as they both act upstream of FLC to regulate flowering, placing FLC in a central position in the flowering network. Michaels and Amasino also discuss that when plants are exposed to vernalization and returned to normal temperatures, FLC expression remains repressed through mitosis, while after meiosis in the following generation its expression is reset to levels observed before vernalization. Thus, regulation of FLC transcription mimics many of the classical physiological characteristics of vernalization. In the decades since the publication of these papers, FLC regulation has proven to be a powerful system to define general mechanisms of plant gene regulation (Whittaker and Dean, 2017). Deciphering the mechanism by which FLC is repressed during vernalization revealed the importance of histone modifications in plant gene regulation (Bastow et al., 2004; Sung and Amasino, 2004). FRI activates FLC transcription before vernalization by recruiting histone methyl transferases and histone acetyl transferases to the FLC promoter. This causes the trimethylation of lysine 4 of histone 3 (H3K4me3) and the trimethylation of lysine 36 of histone 3 (H3K36me3) that are associated with active chromatin. By contrast during vernalization, polycomb repressive complex 2 is recruited to FLC, leading to trimethylation of lysine 27 of histone 3 (H3K27me3) that is correlated with repression of transcription. Such studies revealed the key roles of histone modification in the mitotic stability of the vernalized state and its resetting at meiosis. However, more generally, slight increases in FLC mRNA levels are sufficient to confer late flowering on earlyflowering accessions such as Columbia. Therefore, in these accessions FLC acts as a canary in the coal mine, so that many mutations with general effects on histone modification or gene regulation were identified as late-flowering plants due to slightly elevated FLC transcript levels. This is exemplified by the autonomous pathway mutants, which proved not to define a dedicated flowering pathway but a set of genes with important roles in general gene regulation such as 3’-end processing, RNA-binding proteins, spliceosome subunits, and additional proteins with important roles in chromatin regulation. Plant Cell Advance Publication. Published on May 2, 2019, doi:10.1105/tpc.19. 00325