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.     

Major-effect alleles at relatively few loci underlie distinct vernalization and flowering variation in Arabidopsis accessions

We have explored the genetic basis of variation in vernalization requirement and response in Arabidopsis accessions, selected on the basis of their phenotypic distinctiveness. Phenotyping of F2 populations in different environments, plus fine mapping, indicated possible causative genes. Our data support the identification of FRI and FLC as candidates for the major-effect QTL underlying variation in vernalization response, and identify a weak FLC allele, caused by a Mutator-like transposon, contributing to flowering time variation in two N. American accessions. They also reveal a number of additional QTL that contribute to flowering time variation after saturating vernalization. One of these was the result of expression variation at the FT locus. Overall, our data suggest that distinct phenotypic variation in the vernalization and flowering response of Arabidopsis accessions is accounted for by variation that has arisen independently at relatively few major-effect loci.     

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.     

Vernalization sensitivity in Arabidopsis thaliana (Brassicaceae): the effects of latitude and FLC variation

Latitudinal variation in climate is predicted to select for latitudinal differentiation in sensitivity to the environmental cues that signal plants to flower at the appropriate time for a given climate. In Arabidopsis thaliana, flowering is promoted by exposure to cold temperatures (vernalization), and several vernalization pathway loci are known. To test whether natural variation in vernalization sensitivity could account for a previously observed latitudinal cline in flowering time in A. thaliana, we exposed 21 European accessions to 0, 10, 20, or 30 d of vernalization and observed leaf number at flowering under short days in a growth chamber. We observed a significant latitudinal cline in vernalization sensitivity: southern accessions were more sensitive to vernalization than northern accessions. In addition, accessions that were late flowering in the absence of vernalization were more sensitive to vernalization cues. Allelic variation at the flowering time regulatory gene FLC was not associated with mean vernalization sensitivity, but one allele class exhibited greater variance in vernalization sensitivity.     

Analysis of the molecular basis of flowering time variation in Arabidopsis accessions

Allelic variation at the FRI (FRIGIDA) and FLC (FLOWERING LOCUS C) loci are major determinants of flowering time in Arabidopsis accessions. Dominant alleles of FRI confer a vernalization requirement causing plants to overwinter vegetatively. Many early flowering accessions carry loss-of-function fri alleles containing one of two deletions. However, some accessions categorized as early flowering types do not carry these deletion alleles. We have analyzed the molecular basis of earliness in five of these accessions: Cvi, Shakhdara, Wil-2, Kondara, and Kz-9. The Cvi FRI allele carries a number of nucleotide differences, one of which causes an in-frame stop codon in the first exon. The other four accessions contain nucleotide differences that only result in amino acid substitutions. Preliminary genetic analysis was consistent with Cvi carrying a nonfunctional FRI allele; Wil-2 carrying either a defective FRI or a dominant suppressor of FRI function; and Shakhdara, Kondara, and Kz-9 carrying a functional FRI allele with earliness being caused by allelic variation at other loci including FLC. Allelic variation at FLC was also investigated in a range of accessions. A novel nonautonomous Mutator-like transposon was found in the weak FLC allele in Landsberg erecta, positioned in the first intron, a region required for normal FLC regulation. This transposon was not present in FLC alleles of most other accessions including Shakhdara, Kondara, or Kz-9. Thus, variation in Arabidopsis flowering time has arisen through the generation of nonfunctional or weak FRI and FLC alleles.     

Effect of Vernalization,Photoperiod, and Light Quality on the Flowering Phenotype of Arabidopsis Plants Containing the FRIGIDA Gene

We have compared the flowering response to vernalization, photoperiod, and far-red (FR) light of the Columbia (Col) and Landsberg erecta (Ler) ecotypes of Arabidopsis into which the flowering-time locus FRIGIDA (FRI) has been introgressed with that of the wild types Col, Ler, and San Feliu-2 (Sf-2). In the early-flowering parental ecotypes, Col and Ler, a large decrease in flowering time in response to vernalization was observed only under short-day conditions. However, Sf-2 and the Ler and Col genotypes containing FRI showed a strong response to vernalization when grown in either long days or short days. Although vernalization reduced the responsiveness to photoperiod, plants vernalized for more than 80 d still showed a slight photoperiod response. The effect of FRI on flowering was eliminated by 30 to 40 d of vernalization; subsequently, the response to vernalization in both long days and short days was the same in Col and Ler with or without FRI. FR-light enrichment accelerated flowering in all ecotypes and introgressed lines. However, the FR-light effect was most conspicuous in the FRI-containing plants. Saturation of the vernalization effect eliminated the effect of FR light on flowering, although vernalization did not eliminate the increase of petiole length in FR light.     

Flowering time variation in oilseed rape (Brassica napus L.) is associated with allelic variation in the FRIGIDA homologue BnaA.FRI.a

Oilseed rape (Brassica napus L.) is a major oil crop which is grown worldwide. Adaptation to different environments and regional climatic conditions involves variation in the regulation of flowering time. Winter types have a strong vernalization requirement whereas semi-winter and spring types have a low vernalization requirement or flower without exposure to cold, respectively. In Arabidopsis thaliana, FRIGIDA (FRI) is a key regulator which inhibits floral transition through activation of FLOWERING LOCUS C (FLC), a central repressor of flowering which controls vernalization requirement and response. Here, four FRI homologues in B. napus were identified by BAC library screening and PCR-based cloning. While all homologues are expressed, two genes were found to be differentially expressed in aerial plant organs. One of these, BnaA.FRI.a, was mapped to a region on chromosome A03 which co-localizes with a major flowering time quantitative trait locus in multiple environments in a doubled-haploid mapping population. Association analysis of BnaA.FRI.a revealed that six SNPs, including at least one at a putative functional site, and one haplotype block, respectively, are associated with flowering time variation in 248 accessions, with flowering times differing by 13-19 d between extreme haplotypes. The results from both linkage analysis and association mapping indicate that BnaA.FRI.a is a major determinant of flowering time in oilseed rape, and suggest further that this gene also contributes to the differentiation between growth types. The putative functional polymorphisms identified here may facilitate adaptation of this crop to specific environments through marker-assisted breeding.     

Novel natural alleles at FLC and LVR loci account for enhanced vernalization responses in Arabidopsis thaliana

Vernalization, the induction of flowering by low winter temperatures, is likely to be involved in plant climatic adaptation. However, the genetic, molecular and ecological bases underlying the quantitative variation that tunes vernalization sensitivity to natural environments are largely unknown. To address these questions, we have studied the enhanced vernalization response shown by the Ll-0 accession of Arabidopsis thaliana. Quantitative trait locus (QTL) mapping for several flowering initiation traits in relation to vernalization, in a new Ler × Ll-0 recombinant inbred line (RIL) population, identified large effect alleles at FRI, FLC and HUA2, together with two small effect loci named as Llagostera vernalization response (LVR) 1 and 2. Phenotypic analyses of near isogenic lines validated LVR1 effect on flowering vernalization responses. To further characterize the FLC allele from Ll-0, we carried out genetic association analyses using a regional collection of wild genotypes. FLC-Ll-0 appeared as a low-frequency allele that is distinguished by polymorphism Del(-57), a 50-bp-deletion in the 5'-UTR. Del(-57) was significantly associated with enhanced vernalization responses and FLC RNA expression, as well as with altitude and minimum temperatures. These results are consistent with Del(-57) acting as a novel cis-regulatory FLC polymorphism that may confer climatic adaptation by increasing vernalization sensitivity.     

Attenuation of brassinosteroid signaling enhances FLC expression and delays flowering

A main developmental switch in the life cycle of a flowering plant is the transition from vegetative to reproductive growth. In Arabidopsis thaliana, distinct genetic pathways regulate the timing of this transition. We report here that brassinosteroid (BR) signaling establishes an unexpected and previously unidentified genetic pathway in the floral-regulating network. We isolated two alleles of brassinosteroid-insensitive 1 (bri1) as enhancers of the late-flowering autonomous-pathway mutant luminidependens (ld). bri1 was found to predominantly function as a flowering-time enhancer. Further analyses of double mutants between bri1 and known flowering-time mutants revealed that bri1 also enhances the phenotype of the autonomous mutant fca and of the dominant FRI line. Moreover, all of these double mutants exhibited elevated expression of the potent floral repressor FLOWERING LOCUS C (FLC). This molecular response could be efficiently suppressed by vernalization, leading to accelerated flowering. Additionally, specific reduction of the expression of FLC via RNA interference accelerated flowering in bri1 ld double mutants. Importantly, combining the BR-deficient mutant cpd with ld also resulted in delayed flowering and led to elevated FLC expression. Finally, we found increased histone H3 acetylation at FLC chromatin in bri1 ld mutants, as compared with ld single mutants. In conclusion, we propose that BR signaling acts to repress FLC expression, particularly in genetic situations, with, for example, dominant FRI alleles or autonomous-pathway mutants, in which FLC is activated.     

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.     

Integration of flowering signals in winter-annual Arabidopsis

Photoperiod is the primary environmental factor affecting flowering time in rapid-cycling accessions of Arabidopsis (Arabidopsis thaliana). Winter-annual Arabidopsis, in contrast, have both a photoperiod and a vernalization requirement for rapid flowering. In winter annuals, high levels of the floral inhibitor FLC (FLOWERING LOCUS C) suppress flowering prior to vernalization. FLC acts to delay flowering, in part, by suppressing expression of the floral promoter SOC1 (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1). Vernalization leads to a permanent epigenetic suppression of FLC. To investigate how winter-annual accessions integrate signals from the photoperiod and vernalization pathways, we have examined activation-tagged alleles of FT and the FT homolog, TSF (TWIN SISTER OF FT), in a winter-annual background. Activation of FT or TSF strongly suppresses the FLC-mediated late-flowering phenotype of winter annuals; however, FT and TSF overexpression does not affect FLC mRNA levels. Rather, FT and TSF bypass the block to flowering created by FLC by activating SOC1 expression. We have also found that FLC acts as a dosage-dependent inhibitor of FT expression. Thus, the integration of flowering signals from the photoperiod and vernalization pathways occurs, at least in part, through the regulation of FT, TSF, and SOC1.     

Natural variation involving deletion alleles of FRIGIDA modulate temperature‐sensitive flowering responses in Arabidopsis thaliana

Ambient temperature is one of the major environmental factors that modulate plant growth and development. There is extensive natural genetic variation in thermal responses of plants exemplified by the variation exhibited by the accessions of Arabidopsis thaliana. In this work we have studied the enhanced temperature response in hypocotyl elongation and flowering shown by the Tsu‐0 accession in long days. Genetic mapping in the Col‐0 × Tsu‐0 recombinant inbred line (RIL) population identified several QTLs for thermal response including three major effect loci encompassing candidate genes FRIGIDA (FRI), FLOWERING LOCUS C (FLC) and FLOWERING LOCUS T (FT). We confirm and validate these QTLs. We show that the Tsu‐0 FRI allele, which is the same as FRI‐Ler is associated with late flowering but only at lower temperatures in long days. Using transgenic lines and accessions, we show that the FRI‐Ler allele confers temperature‐sensitive late flowering confirming a role for FRI in photoperiod‐dependent thermal response. Through quantitative complementation with heterogeneous inbred families, we further show that cis‐regulatory variation at FT contributes to the observed hypersensitivity of Tsu‐0 to ambient temperature. Overall our results suggest that multiple loci that interact epistatically govern photoperiod‐dependent thermal responses of A. thaliana.     

The synergistic activation of FLOWERING LOCUS C by FRIGIDA and a new flowering gene AERIAL ROSETTE 1 underlies a novel morphology in Arabidopsis

The genetic changes underlying the diversification of plant forms represent a key question in understanding plant macroevolution. To understand the mechanisms leading to novel plant morphologies we investigated the Sy-0 ecotype of Arabidopsis that forms an enlarged basal rosette of leaves, develops aerial rosettes in the axils of cauline leaves, and exhibits inflorescence and floral reversion. Here we show that this heterochronic shift in reproductive development of all shoot meristems requires interaction between dominant alleles at AERIAL ROSETTE 1 (ART1), FRIGIDA (FRI), and FLOWERING LOCUS C (FLC) loci. ART1 is a new flowering gene that maps 14 cM proximal to FLC on chromosome V. ART1 activates FLC expression through a novel flowering pathway that is independent of FRI and independent of the autonomous and vernalization pathways. Synergistic activation of the floral repressor FLC by ART1 and FRI is required for delayed onset of reproductive development of all shoot meristems, leading to the Sy-0 phenotype. These results demonstrate that modulation in flowering-time genes is one of the mechanisms leading to morphological novelties.     

Altitudinal and climatic adaptation is mediated by flowering traits and FRI, FLC, and PHYC genes in Arabidopsis

Extensive natural variation has been described for the timing of flowering initiation in many annual plants, including the model wild species Arabidopsis (Arabidopsis thaliana), which is presumed to be involved in adaptation to different climates. However, the environmental factors that might shape this genetic variation, as well as the molecular bases of climatic adaptation by modifications of flowering time, remain mostly unknown. To approach both goals, we characterized the flowering behavior in relation to vernalization of 182 Arabidopsis wild genotypes collected in a native region spanning a broad climatic range. Phenotype-environment association analyses identified strong altitudinal clines (0-2600 m) in seven out of nine flowering-related traits. Altitudinal clines were dissected in terms of minimum winter temperature and precipitation, indicating that these are the main climatic factors that might act as selective pressures on flowering traits. In addition, we used an association analysis approach with four candidate genes, FRIGIDA (FRI), FLOWERING LOCUS C (FLC), PHYTOCHROME C (PHYC), and CRYPTOCHROME2, to decipher the genetic bases of this variation. Eleven different loss-of-function FRI alleles of low frequency accounted for up to 16% of the variation for most traits. Furthermore, an FLC allelic series of six novel putative loss- and change-of-function alleles, with low to moderate frequency, revealed that a broader FLC functional diversification might contribute to flowering variation. Finally, environment-genotype association analyses showed that the spatial patterns of FRI, FLC, and PHYC polymorphisms are significantly associated with winter temperatures and spring and winter precipitations, respectively. These results support that allelic variation in these genes is involved in climatic adaptation.     

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.     

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.     

Differential expression of genes important for adaptation in Capsella bursa-pastoris (Brassicaceae)

Understanding the genetic basis of natural variation is of primary interest for evolutionary studies of adaptation. In Capsella bursa-pastoris, a close relative of Arabidopsis (Arabidopsis thaliana), variation in flowering time is correlated with latitude, suggestive of an adaptation to photoperiod. To identify pathways regulating natural flowering time variation in C. bursa-pastoris, we have studied gene expression differences between two pairs of early- and late-flowering C. bursa-pastoris accessions and compared their response to vernalization. Using Arabidopsis microarrays, we found a large number of significant differences in gene expression between flowering ecotypes. The key flowering time gene FLOWERING LOCUS C (FLC) was not differentially expressed prior to vernalization. This result is in contrast to those in Arabidopsis, where most natural flowering time variation acts through FLC. However, the gibberellin and photoperiodic flowering pathways were significantly enriched for gene expression differences between early- and late-flowering C. bursa-pastoris. Gibberellin biosynthesis genes were down-regulated in late-flowering accessions, whereas circadian core genes in the photoperiodic pathway were differentially expressed between early- and late-flowering accessions. Detailed time-series experiments clearly demonstrated that the diurnal rhythm of CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) and TIMING OF CAB EXPRESSION1 (TOC1) expression differed between flowering ecotypes, both under constant light and long-day conditions. Differential expression of flowering time genes was biologically validated in an independent pair of flowering ecotypes, suggesting a shared genetic basis or parallel evolution of similar regulatory differences. We conclude that genes involved in regulation of the circadian clock, such as CCA1 and TOC1, are strong candidates for the evolution of adaptive flowering time variation in C. bursa-pastoris.