Discordant longitudinal clines in flowering time and phytochrome C in Arabidopsis thaliana

Using seasonal cues to time reproduction appropriately is crucial for many organisms. Plants in particular often use photoperiod to signal the time to transition to flowering. Because seasonality varies latitudinally, adaptation to local climate is expected to result in corresponding clines in photoperiod-related traits. By experimentally manipulating photoperiod cues and measuring the flowering responses and photoperiod plasticity of 138 Eurasian accessions of Arabidopsis thaliana, we detected strong longitudinal but not latitudinal clines in flowering responses. The presence of longitudinal clines suggests that critical photoperiod cues vary among populations occurring at similar latitudes. Haplotypes at PHYC, a locus hypothesized to play a role in adaptation to light cues, were also longitudinally differentiated. Controlling for neutral population structure revealed that PHYC haplotype influenced flowering time; however, the distribution of PHYC haplotypes occurred in the opposite direction to the phenotypic cline, suggesting that loci other than PHYC are responsible for the longitudinal pattern in photoperiod response. Our results provide previously missing empirical support for the importance of PHYC in mediating photoperiod sensitivity in natural populations of A. thaliana. However, they also suggest that other loci and epistatic interactions likely play a role in the determination of flowering time and that the environmental factors influencing photoperiod in plants vary longitudinally as well as latitudinally.     

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.     

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.     

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.     

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.     

Longitudinal trends in climate drive flowering time clines in North American Arabidopsis thaliana

Introduced species frequently show geographic differentiation, and when differentiation mirrors the ancestral range, it is often taken as evidence of adaptive evolution. The mouse-ear cress (Arabidopsis thaliana) was introduced to North America from Eurasia 150-200 years ago, providing an opportunity to study parallel adaptation in a genetic model organism. Here, we test for clinal variation in flowering time using 199 North American (NA) accessions of A. thaliana, and evaluate the contributions of major flowering time genes FRI, FLC, and PHYC as well as potential ecological mechanisms underlying differentiation. We find evidence for substantial within population genetic variation in quantitative traits and flowering time, and putatively adaptive longitudinal differentiation, despite low levels of variation at FRI, FLC, and PHYC and genome-wide reductions in population structure relative to Eurasian (EA) samples. The observed longitudinal cline in flowering time in North America is parallel to an EA cline, robust to the effects of population structure, and associated with geographic variation in winter precipitation and temperature. We detected major effects of FRI on quantitative traits associated with reproductive fitness, although the haplotype associated with higher fitness remains rare in North America. Collectively, our results suggest the evolution of parallel flowering time clines through novel genetic mechanisms.     

Re-establishment of clinal variation in flowering time among introduced populations of purple loosestrife (Lythrum salicaria, Lythraceae)

Range expansion during biological invasion requires that invaders adapt to geographical variation in climate, which should yield latitudinal clines in reproductive phenology. We investigated geographic variation in life history among 25 introduced populations of Lythrum salicaria, a widespread European invader of North American wetlands. We detected a strong latitudinal cline in initiation of flowering and size at flowering, which paralleled that reported among native populations. Plants from higher latitudes flowered earlier and at a smaller size than those from lower latitudes, even when raised in a uniform glasshouse. Early flowering was associated with greatly reduced reproductive output, but this was not associated with latitudinal variation in abundance, and probably did not result from a genetic correlation between time to and size at flowering. As introduction to North America c. 200 years ago, L. salicaria has re-established latitudinal clines in life history, probably as an evolutionary response to climatic selection.     

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.     

Polymorphism of DNA sequences of cryptochrome genes is not associated with the photoperiodic flowering of wild soybean along a latitudinal cline

Both cultivated soybean and its wild relative Glycine soja exhibit strong photoperiodic sensitivity at different latitudes. Recent studies have demonstrated that the blue light-absorbing cryptochrome gene, CRY1a, is involved in the photoperiodic flowering of soybeans. However, no sequence variation was found in the cDNA among cultivars at different latitudinal clines. In the present study, we examined whether positive selection due to polymorphisms in the cryptochrome genes of G. soja occurs. Partial DNA sequences, mainly exons, of cryptochrome genes CRY1a-1d and CRY2a-2c were analyzed for 18 accessions in the Japanese archipelago. The neutral evolutionary pattern of the polymorphisms for all cryptochrome genes except for CRY1a was summarized using Tajima's D test and low nucleotide diversity was shown for all genes. Although CRY1a did not show neutral evolution, balancing selection was recognized in the intron while not in the exon. No geographical pattern of polymorphisms was observed in the cryptochrome genes. These results reject the possibility of cryptochrome genes being involved in the photoperiodic flowering of wild soybeans along a latitudinal cline.     

Evidence of genetic change in the flowering phenology of sea beets along a latitudinal cline within two decades

Sea beets grown from seeds collected in 1989 and 2009 along the coasts of France and adjacent regions were compared for flowering date under controlled conditions. Seeds from both collection years were sown simultaneously and cultivated under the same glasshouse conditions. Date of flowering onset and year of first flowering were recorded. There was an overall northward shift in flowering time of about 0.35° latitude (i.e. 39 km) over the 20‐year period. The southern portion of the latitudinal gradient – that is, from 44.7°N to 47.28°N – flowered significantly later by a mean of 1.78 days, equivalent to a 43.2‐km northward shift of phenotypes. In the northern latitudes between 48.6°N and 52°N, flowering date was significantly earlier by a mean of 4.04 days, corresponding to a mean northward shift of 104.9 km, and this shift was apparently due to a diminished requirement of exposure to cold temperatures (i.e. vernalization), for which we found direct and indirect evidence. As all plants were grown from seed under identical conditions, we conclude that genetic changes occurred in the sensitivity to environmental cues that mediate the onset of flowering in both the northern and the southern latitudes of the gradient. Microevolution and gene flow may have contributed to this change. There was no significant change in the frequency of plants that flowered without vernalization. The lack of vernalization requirement may be associated with environmental instability rather than with climate conditions.     

Quantifying latitudinal clines to light responses in natural populations of Arabidopsis thaliana (Brassicaceae)

Evidence of adaptation in Arabidopsis thaliana (Brassicaceae) phenotypic traits has rarely been shown. We demonstrate latitudinal clines in two A. thaliana traits: hypocotyl responses to red and far-red light. Natural populations of A. thaliana were sampled along a latitudinal gradient from southern to northern Norway. Seeds from maternal families within each population were subjected to 1 wk of constant red, far-red, blue, white, and dark treatments. Hypocotyl lengths were measured for each maternal family within each population. Significant variability within and among populations in hypocotyl responses for the various treatments was found. There was a significant latitudinal cline in hypocotyl responses for red and far-red treatments, with northern populations being more de-etiolated than southern populations. These results suggest that northern populations are more responsive to red and far-red light than southern populations. Thus, differentiation of seedling traits in natural populations of A. thaliana seems in part to be mediated by the phytochrome pathway. There was no correlation between hypocotyl responses and flowering time for any treatment. This suggests that flowering time variability and variability in hypocotyl responses may not be governed by genes shared between the pathways, such as those involved in photoreception or the circadian clock.     

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.     

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.     

The role of a pseudo-response regulator gene in life cycle adaptation and domestication of beet

Life cycle adaptation to latitudinal and seasonal variation in photoperiod and temperature is a major determinant of evolutionary success in flowering plants. Whereas the life cycle of the dicotyledonous model species Arabidopsis thaliana is controlled by two epistatic genes, FLOWERING LOCUS C and FRIGIDA, three unrelated loci (VERNALIZATION) determine the spring and winter habits of monocotyledonous plants such as temperate cereals. In the core eudicot species Beta vulgaris, whose lineage diverged from that leading to Arabidopsis shortly after the monocot-dicot split 140 million years ago, the bolting locus B is a master switch distinguishing annuals from biennials. Here, we isolated B and show that the pseudo-response regulator gene BOLTING TIME CONTROL 1 (BvBTC1), through regulation of the FLOWERING LOCUS T genes, is absolutely necessary for flowering and mediates the response to both long days and vernalization. Our results suggest that domestication of beets involved the selection of a rare partial loss-of-function BvBTC1 allele that imparts reduced sensitivity to photoperiod that is restored by vernalization, thus conferring bienniality, and illustrate how evolutionary plasticity at a key regulatory point can enable new life cycle strategies.     

Focus on Chromatin/Epigenetics: Flowering Locus C’s Lessons: Conserved Chromatin Switches Underpinning Developmental Timing and Adaptation

Analysis of how seasonal cues influence the timing of the floral transition has revealed many important principles for how epigenetic regulation can integrate a variety of environmental cues with developmental signals. The study of the pathways that necessitate overwintering in plants and their ability to respond to prolonged cold (the vernalization requirement and response pathways) has elaborated different chromatin regulatory pathways and the involvement of noncoding RNAs. The major target of these vernalization pathways in Arabidopsis (Arabidopsis thaliana) is Flowering Locus C (FLC). A relatively simple picture of FLC regulation is emerging of a few core complexes and mechanisms that antagonize each other’s actions. This balance provides a fine degree of control that has nevertheless permitted evolution of a wide range of natural variation in vernalization in Arabidopsis. Similar simple routes of adaptation may underlie life history variation between species.The time at which different species flower is an important marker of seasonal and climatic changes and is ecologically and economically important. The sessile nature of plants means that they experience the full range of environmental changes over the seasons. Flowering time control in many species is highly responsive to environmental cues and therefore very sensitive to local climate conditions. The impact of this on many ecosystem and agricultural processes has made understanding flowering time control an important objective.Many genetic pathways influence flowering time, either as part of seasonal (photoperiod and past and present temperature), developmental (developmental phase and age), or stress response (overcrowding and nutrient stress). Despite the variety of competing inputs, these many and various mechanisms are integrated at the action of a small number of nodes in Arabidopsis (Arabidopsis thaliana) termed floral pathway integrators (Simpson and Dean, 2002). Analyses of the genes identified by flowering time mutants have shown many have roles as chromatin modifiers (Andrés and Coupland, 2012; Pajoro et al., 2014). Timing of flowering seems particularly sensitive to chromatin regulation, potentially due to the necessity for long-term storage of seasonal information.In this review, we focus on vernalization in Arabidopsis and summarize our understanding of how chromatin modifiers interact with other proteins and noncoding RNAs to integrate developmental and temperature cues into chromatin changes at the key integrating locus Flowering Locus C (FLC). Further, we explore how changes in these mechanisms underlie different life history strategies and vernalization responses in different accessions and species. A key observation we wish to convey is that the complexity of these systems at the molecular level belies simplicity in balancing forces that enable a fine degree of control and adaptive responses at the phenotypic level.     

A latitudinal cline and response to vernalization in leaf angle and morphology in Arabidopsis thaliana (Brassicaceae)

Adaptation to latitudinal patterns of environmental variation is predicted to result in clinal variation in leaf traits. Therefore, this study tested for geographic differentiation and plastic responses to vernalization in leaf angle and leaf morphology in Arabidopsis thaliana. Twenty-one European ecotypes were grown in a common growth chamber environment. Replicates of each ecotype were exposed to one of four treatments: 0, 10, 20 or 30 d of vernalization. Ecotypes from lower latitudes had more erect leaves, as predicted from functional arguments about selection to maximize photosynthesis. Lower-latitude ecotypes also had more elongated petioles as predicted by a biomechanical constraint hypothesis. In addition, extended vernalization resulted in shorter and more erect leaves. As predicted by functional and adaptive hypotheses, our results show genetically based clinal variation as well as environmentally induced variation in leaf traits.     

Genetic and physiological analysis of biennialism in Hyoscyamus niger

A genetic and physiological study of biennialism in the diploid selfer Hyoscyamus niger (black henbane), an obligate long-day plant, is described. Three annual and two biennial accessions that were homozygous for their respective growth habits were selected. The early-flowering trait of two annual accessions was dominant over the late-flowering trait of the third annual accession. The late-flowering annual accession, but not the early-flowering ones, responded to vernalization. Two biennial accessions remained vegetative after more than 1 year in soil and thus had an obligate vernalization requirement. Crosses between annual and biennial accessions showed that biennialism was conferred through a single dominant gene. However, plants containing only one copy of this dominant gene were transformed from biennials into very late-flowering winter-annual plants that responded more rapidly to vernalization than biennials. Taken together, these results indicated that there were allelic differences in photoperiod-specific flowering time genes and that biennialism was a dose-dependent trait with incomplete dominance. Models for flowering time regulation in henbane involving photoperiod-, vernalization-, and most likely gibberellin-specific pathways are discussed.     

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.