FRIGIDA-independent variation in flowering time of natural Arabidopsis thaliana accessions

FRIGIDA (FRI) and FLOWERING LOCUS C (FLC) are two genes that, unless plants are vernalized, greatly delay flowering time in Arabidopsis thaliana. Natural loss-of-function mutations in FRI cause the early flowering growth habits of many A. thaliana accessions. To quantify the variation among wild accessions due to FRI, and to identify additional genetic loci in wild accessions that influence flowering time, we surveyed the flowering times of 145 accessions in long-day photoperiods, with and without a 30-day vernalization treatment, and genotyped them for two common natural lesions in FRI. FRI is disrupted in at least 84 of the accessions, accounting for only approximately 40% of the flowering-time variation in long days. During efforts to dissect the causes for variation that are independent of known dysfunctional FRI alleles, we found new loss-of-function alleles in FLC, as well as late-flowering alleles that do not map to FRI or FLC. An FLC nonsense mutation was found in the early flowering Van-0 accession, which has otherwise functional FRI. In contrast, Lz-0 flowers late because of high levels of FLC expression, even though it has a deletion in FRI. Finally, eXtreme array mapping identified genomic regions linked to the vernalization-independent, late-flowering habit of Bur-0, which has an alternatively spliced FLC allele that behaves as a null allele.     

Diversity of flowering responses in wild Arabidopsis thaliana strains

Although multiple environmental cues regulate the transition to flowering in Arabidopsis thaliana, previous studies have suggested that wild A. thaliana accessions fall primarily into two classes, distinguished by their requirement for vernalization (extended winter-like temperatures), which enables rapid flowering under long days. Much of the difference in vernalization response is apparently due to variation at two epistatically acting loci, FRI and FLC. We present the response of over 150 wild accessions to three different environmental variables. In long days, FLC is among those genes whose expression is most highly correlated with flowering. In short days, FRI and FLC are less important, although their contribution is still significant. In addition, there is considerable variation not only in vernalization response, but also in the response to differences in day length or ambient growth temperature. The identification of accessions that flower relatively early or late in specific environments suggests that many of the flowering-time pathways identified by mutagenesis, such as those that respond to day length, contribute to flowering-time variation in the wild. In contrast to differences in vernalization requirement, which are mainly mediated by FRI and FLC, it seems that variation in these other pathways is due to allelic effects at several different loci.     

Comparison of Flowering Time Genes in Brassica Rapa, B. Napus and Arabidopsis Thaliana

The major difference between annual and biennial cultivars of oilseed Brassica napus and B. rapa is conferred by genes controlling vernalization-responsive flowering time. These genes were compared between the species by aligning the map positions of flowering time quantitative trait loci (QTLs) detected in a segregating population of each species. The results suggest that two major QTLs identified in B. rapa correspond to two major QTLs identified in B. napus. Since B. rapa is one of the hypothesized diploid parents of the amphidiploid B. napus, the vernalization requirement of B. napus probably originated from B. rapa. Brassica genes also were compared to flowering time genes in Arabidopsis thaliana by mapping RFLP loci with the same probes in both B. napus and Arabidopsis. The region containing one pair of Brassica QTLs was collinear with the top of chromosome 5 in A. thaliana where flowering time genes FLC, FY and CO are located. The region containing the second pair of QTLs showed fractured collinearity with several regions of the Arabidopsis genome, including the top of chromosome 4 where FRI is located. Thus, these Brassica genes may correspond to two genes (FLC and FRI) that regulate flowering time in the latest flowering ecotypes of Arabidopsis.     

Role of FRIGIDA and FLOWERING LOCUS C in determining variation in flowering time of Arabidopsis

Arabidopsis (Arabidopsis thaliana) accessions provide an excellent resource to dissect the molecular basis of adaptation. We have selected 192 Arabidopsis accessions collected to represent worldwide and local variation and analyzed two adaptively important traits, flowering time and vernalization response. There was huge variation in the flowering habit of the different accessions, with no simple relationship to latitude of collection site and considerable diversity occurring within local regions. We explored the contribution to this variation from the two genes FRIGIDA (FRI) and FLOWERING LOCUS C (FLC), previously shown to be important determinants in natural variation of flowering time. A correlation of FLC expression with flowering time and vernalization was observed, but it was not as strong as anticipated due to many late-flowering/vernalization-requiring accessions being associated with low FLC expression and early-flowering accessions with high FLC expression. Sequence analysis of FRI revealed which accessions were likely to carry functional alleles, and, from comparison of flowering time with allelic type, we estimate that approximately 70% of flowering time variation can be accounted for by allelic variation of FRI. The maintenance and propagation of 20 independent nonfunctional FRI haplotypes suggest that the loss-of-function mutations can confer a strong selective advantage. Accessions with a common FRI haplotype were, in some cases, associated with very different FLC levels and wide variation in flowering time, suggesting additional variation at FLC itself or other genes regulating FLC. These data reveal how useful these Arabidopsis accessions will be in dissecting the complex molecular variation that has led to the adaptive phenotypic variation in flowering time.     

Standing genetic variation in FRIGIDA mediates experimental evolution of flowering time in Arabidopsis

The role of standing genetic variation in adaptive evolution remains unclear. Although there has been much progress in identifying candidate genes that underlie adaptive traits, we still lack direct evidence that natural allelic variation in these genes can actually mediate adaptive evolution. In this study, we investigate the role of natural allelic variation in two candidate flowering time genes, in response to selection for early flowering in Arabidopsis thaliana : FRIGIDA ( FRI ) and FLOWERING LOCUS C ( FLC ). We performed artificial selection for early flowering under 'spring-' and 'winter-annual' growth conditions using an outbred population of A. thaliana produced by intermating 19 natural accessions. FRI and FLC are involved in A. thaliana 's response to winter conditions, and nonfunctional and weak alleles at these loci are know to reduce flowering time, particularly under spring-annual conditions. Our results provide direct evidence that natural allelic variation in FRI can provide rapid and predictable adaptive evolution in flowering time under spring-annual conditions. We observed a strong response to selection, in terms of reducing flowering time, in both growth conditions (~2 standard deviation reduction). Concomitantly, the frequency of functional FRI alleles under spring-annual conditions was reduced by 68%, in agreement with predicted changes. No significant changes in allele frequencies were observed in FRI in the winter-annual growth condition or in FLC for either growth conditions. These results indicate that changes in flowering time are mediated by different genetic factors under spring- and winter-annual growth conditions, and that other loci must also be contributing to the response to selection.     

FLOWERING LOCUS C encodes a novel MADS domain protein that acts as a repressor of flowering.

Winter-annual ecotypes of Arabidopsis are relatively late flowering, unless the flowering of these ecotypes is promoted by exposure to cold (vernalization). This vernalization-suppressible, late-flowering phenotype results from the presence of dominant, late-flowering alleles at two loci, FRIGIDA (FRI) and FLOWERING LOCUS C (FLC). In this study, we report that flc null mutations result in early flowering, demonstrating that the role of active FLC alleles is to repress flowering. FLC was isolated by positional cloning and found to encode a novel MADS domain protein. The levels of FLC mRNA are regulated positively by FRI and negatively by LUMINIDEPENDENS. FLC is also negatively regulated by vernalization. Overexpression of FLC from a heterologous promoter is sufficient to delay flowering in the absence of an active FRI allele. We propose that the level of FLC activity acts through a rheostat-like mechanism to control flowering time in Arabidopsis and that modulation of FLC expression is a component of the vernalization response.     

Arabidopsis relatives of the human lysine-specific Demethylase1 repress the expression of FWA and FLOWERING LOCUS C and thus promote the floral transition

The timing of the developmental transition to flowering is critical to reproductive success in plants. Here, we show that Arabidopsis thaliana homologs of human Lysine-Specific Demethylase1 (LSD1; a histone H3-Lys 4 demethylase) reduce the levels of histone H3-Lys 4 methylation in chromatin of the floral repressor FLOWERING LOCUS C (FLC) and the sporophytically silenced floral repressor FWA. Two of the homologs, LSD1-LIKE1 (LDL1) and LSD1-LIKE2 (LDL2), act in partial redundancy with FLOWERING LOCUS D (FLD; an additional homolog of LSD1) to repress FLC expression. However, LDL1 and LDL2 appear to act independently of FLD in the silencing of FWA, indicating that there is target gene specialization within this histone demethylase family. Loss of function of LDL1 and LDL2 affects DNA methylation on FWA, whereas FLC repression does not appear to involve DNA methylation; thus, members of the LDL family can participate in a range of silencing mechanisms.     

Haplotype structure and phenotypic associations in the chromosomal regions surrounding two Arabidopsis thaliana flowering time loci

The feasibility of using linkage disequilbrium (LD) to fine-map loci underlying natural variation in Arabidopsis thaliana was investigated by looking for associations between flowering time and marker polymorphism in the genomic regions containing two candidate genes, FRI and FLC, both of which are known to contribute to natural variation in flowering. A sample of 196 accessions was used, and polymorphism was assessed by sequencing a total of 17 roughly 500-bp fragments. Using a novel Bayesian algorithm based on haplotype similarity, we demonstrate that LD could have been used to fine-map the FRI gene to a roughly 30-kb region and to identify two common loss-of-function alleles. Interestingly, because of genetic heterogeneity, simple single-marker associations would not have been able to map FRI with nearly the same precision. No clear evidence for previously unknown alleles at either locus was found, but the effect of population structure in causing false positives was evident.     

The Arabidopsis TALE homeobox gene ATH1 controls floral competency through positive regulation of FLC

Floral induction is controlled by a plethora of genes acting in different pathways that either repress or promote floral transition at the shoot apical meristem (SAM). During vegetative development high levels of floral repressors maintain the Arabidopsis SAM as incompetent to respond to promoting factors. Among these repressors, FLOWERING LOCUS C (FLC) is the most prominent. The processes underlying downregulation of FLC in response to environmental and developmental signals have been elucidated in considerable detail. However, the basal induction of FLC and its upregulation by FRIGIDA (FRI) are still poorly understood. Here we report the functional characterization of the ARABIDOPSIS THALIANA HOMEOBOX 1 (ATH1) gene. A function of ATH1 in floral repression is suggested by a gradual downregulation of ATH1 in the SAM prior to floral transition. Further evidence for such a function of ATH1 is provided by the vernalization-sensitive late flowering of plants that constitutively express ATH1. Analysis of lines that differ in FRI and/or FLC allele strength show that this late flowering is caused by upregulation of FLC as a result of synergism between ATH1 overexpression and FRI. Lack of ATH1, however, results in attenuated FLC levels independently of FRI, suggesting that ATH1 acts as a general activator of FLC expression. This is further corroborated by a reduction of FLC-mediated late flowering in fca-1 and fve-1 autonomous pathway backgrounds when combined with ath1. Since other floral repressors of the FLC clade are not significantly affected by ATH1, we conclude that ATH1 controls floral competency as a specific activator of FLC expression.     

Functional redundancy and new roles for genes of the autonomous floral-promotion pathway

The early-flowering habit of rapid-cycling accessions of Arabidopsis (Arabidopsis thaliana) is, in part, due to the genes of the autonomous floral-promotion pathway (AP). The AP promotes flowering by repressing expression of the floral inhibitor FLOWERING LOCUS C (FLC). AP mutants are therefore late flowering due to elevated levels of FLC, and this late-flowering phenotype is eliminated by loss-of-function mutations in FLC. To further investigate the role of the AP, we created a series of double mutants. In contrast to the phenotypes of single mutants, which are largely limited to delayed flowering, a subset of AP double mutants show a range of defects in growth and development. These phenotypes include reduced size, chlorophyll content, growth rate, and fertility. Unlike the effects of the AP on flowering time, these phenotypes are FLC independent. Recent work has also shown that two AP genes, FCA and FPA, are required for the repression and, in some cases, proper DNA methylation of two transposons. We show that similar effects are seen for all AP genes tested. Microarray analysis of gene expression in AP single and double mutants, however, suggests that the AP is not likely to play a broad role in the repression of gene expression through DNA methylation: very few of the genes that have been reported to be up-regulated in DNA methylation mutants are misexpressed in AP mutants. Together, these data indicate that the genes of the AP play important and sometimes functionally redundant roles in aspects of development in addition to flowering time.