FLOWERING LOCUS C Gene Research Articles

Adaptation to reductions in chilling availability using variation in PLANT HOMOLOGOUS TO PARAFIBROMIN in Brassica napus.

Winter annual crops are sown in late summer or autumn and require chilling to promote flowering the following spring. Floral initiation begins in autumn and winter, and in winter oilseed rape (OSR), continued chilling during flower development is necessary for high yield potential. This can be a problem in areas where chilling is not guaranteed, or as a result of changing climates. Here, we used chilling disruption and low chilling to identify loci with the potential to increase chilling efficiency in winter OSR. We report that time to flowering and yield potential under low chill conditions are affected by variation at the PLANT HOMOLOGOUS TO PARAFIBROMIN gene, a component of the plant PAF1c complex. We show that increases in winter chilling given to developing flowers can improve seed yields and that loss of function of BnaPHP.A05 leads to early flowering in B. rapa and B. napus and an increase in seed set where chilling is limited. Because PHP is known to specifically target the FLOWERING LOCUS C (FLC) gene in Arabidopsis, we propose that variation at PHP is useful for breeding modifications to chilling responses in polyploid crops with multiple copies of the FLC gene.

Integrating analog and digital modes of gene expression at Arabidopsis FLC.

Quantitative gene regulation at the cell population level can be achieved by two fundamentally different modes of regulation at individual gene copies. A 'digital' mode involves binary ON/OFF expression states, with population-level variation arising from the proportion of gene copies in each state, while an 'analog' mode involves graded expression levels at each gene copy. At the Arabidopsis floral repressor FLOWERING LOCUS C (FLC), 'digital' Polycomb silencing is known to facilitate quantitative epigenetic memory in response to cold. However, whether FLC regulation before cold involves analog or digital modes is unknown. Using quantitative fluorescent imaging of FLC mRNA and protein, together with mathematical modeling, we find that FLC expression before cold is regulated by both analog and digital modes. We observe a temporal separation between the two modes, with analog preceding digital. The analog mode can maintain intermediate expression levels at individual FLC gene copies, before subsequent digital silencing, consistent with the copies switching OFF stochastically and heritably without cold. This switch leads to a slow reduction in FLC expression at the cell population level. These data present a new paradigm for gradual repression, elucidating how analog transcriptional and digital epigenetic memory pathways can be integrated.

Epigenetics for crop adaptation to climate change

Epigenetics for crop adaptation to climate change

Polymerase II-Associated Factor 1 Complex-Regulated FLOWERING LOCUS C-Clade Genes Repress Flowering in Response to Chilling.

RNA polymerase II–associated factor 1 complex (PAF1C) regulates the transition from the vegetative to the reproductive phase primarily by modulating the expression of FLOWERING LOCUS C (FLC) and FLOWERING LOCUS M [FLM, also known as MADS AFFECTING FLOWERING1 (MAF1)] at standard growth temperatures. However, the role of PAF1C in the regulation of flowering time at chilling temperatures (i.e., cold temperatures that are above freezing) and whether PAF1C affects other FLC-clade genes (MAF2–MAF5) remains unknown. Here, we showed that Arabidopsis thaliana mutants of any of the six known genes that encode components of PAF1C [CELL DIVISION CYCLE73/PLANT HOMOLOGOUS TO PARAFIBROMIN, VERNALIZATION INDEPENDENCE2 (VIP2)/EARLY FLOWERING7 (ELF7), VIP3, VIP4, VIP5, and VIP6/ELF8] showed temperature-insensitive early flowering across a broad temperature range (10°C–27°C). Flowering of PAF1C-deficient mutants at 10°C was even earlier than that in flc, flm, and flc flm mutants, suggesting that PAF1C regulates additional factors. Indeed, RNA sequencing (RNA-Seq) of PAF1C-deficient mutants revealed downregulation of MAF2–MAF5 in addition to FLC and FLM at both 10 and 23°C. Consistent with the reduced expression of FLC and the FLC-clade members FLM/MAF1 and MAF2–MAF5, chromatin immunoprecipitation (ChIP)-quantitative PCR assays showed reduced levels of the permissive epigenetic modification H3K4me3/H3K36me3 and increased levels of the repressive modification H3K27me3 at their chromatin. Knocking down MAF2–MAF5 using artificial microRNAs (amiRNAs) in the flc flm background (35S::amiR-MAF2–5 flc flm) resulted in significantly earlier flowering than flc flm mutants and even earlier than short vegetative phase (svp) mutants at 10°C. Wild-type seedlings showed higher accumulation of FLC and FLC-clade gene transcripts at 10°C compared to 23°C. Our yeast two-hybrid assays and in vivo co-immunoprecipitation (Co-IP) analyses revealed that MAF2–MAF5 directly interact with the prominent floral repressor SVP. Late flowering caused by SVP overexpression was almost completely suppressed by the elf7 and vip4 mutations, suggesting that SVP-mediated floral repression required a functional PAF1C. Taken together, our results showed that PAF1C regulates the transcription of FLC and FLC-clade genes to modulate temperature-responsive flowering at a broad range of temperatures and that the interaction between SVP and these FLC-clade proteins is important for floral repression.

Flowering Times of Wild Arabidopsis Accessions From Across Norway Correlate With Expression Levels of FT, CO, and FLC Genes.

Temperate species often require or flower most rapidly in the long daylengths, or photoperiods, experienced in summer or after prolonged periods of cold temperatures, referred to as vernalization. Yet, even within species, plants vary in the degree of responsiveness to these cues. In Arabidopsis thaliana, CONSTANS (CO) and FLOWERING LOCUS C (FLC) genes are key to photoperiod and vernalization perception and antagonistically regulate FLOWERING LOCUS T (FT) to influence the flowering time of the plants. However, it is still an open question as to how these genes vary in their interactions among wild accessions with different flowering behaviors and adapted to different microclimates, yet this knowledge could improve our ability to predict plant responses in variable natural conditions. To assess the relationships among these genes and to flowering time, we exposed 10 winter-annual Arabidopsis accessions from throughout Norway, ranging from early to late flowering, along with two summer-annual accessions to 14 weeks of vernalization and either 8- or 19-h photoperiods to mimic Norwegian climate conditions, then assessed gene expression levels 3-, 5-, and 8-days post vernalization. CO and FLC explained both FT levels and flowering time (days) but not rosette leaf number at flowering. The correlation between FT and flowering time increased over time. Although vernalization suppresses FLC, FLC was high in the late-flowering accessions. Across accessions, FT was expressed only at low FLC levels and did not respond to CO in the late-flowering accessions. We proposed that FT may only be expressed below a threshold value of FLC and demonstrated that these three genes correlated to flowering times across genetically distinct accessions of Arabidopsis.

Identification and characterization of MADS box gene family in pigeonpea for their role during floral transition.

The online version contains supplementary material available at 10.1007/s13205-020-02605-7.

The Role of FLOWERING LOCUS C Relatives in Cereals.

FLOWERING LOCUS C (FLC) is one of the best characterized genes in plant research and is integral to vernalization-dependent flowering time regulation. Yet, despite the abundance of information on this gene and its relatives in Arabidopsis thaliana, the role FLC genes play in other species, in particular cereal crops and temperate grasses, remains elusive. This has been due in part to the comparative reduced availability of bioinformatic and mutant resources in cereals but also on the dominant effect in cereals of the VERNALIZATION (VRN) genes on the developmental process most associated with FLC in Arabidopsis. The strong effect of the VRN genes has led researchers to believe that the entire process of vernalization must have evolved separately in Arabidopsis and cereals. Yet, since the confirmation of the existence of FLC-like genes in monocots, new light has been shed on the roles these genes play in both vernalization and other mechanisms to fine tune development in response to specific environmental conditions. Comparisons of FLC gene function and their genetic and epigenetic regulation can now be made between Arabidopsis and cereals and how they overlap and diversify is coming into focus. With the advancement of genome editing techniques, further study on these genes is becoming increasingly easier, enabling us to investigate just how essential FLC-like genes are to modulating flowering time behavior in cereals.

Feedback regulation of COOLAIR expression controls seed dormancy and flowering time.

Plants integrate seasonal signals, including temperature and day length, to optimize the timing of developmental transitions. Seasonal sensing requires the activity of two proteins, FLOWERING LOCUS C (FLC) and FLOWERING LOCUS T (FT), that control certain developmental transitions in plants. During reproductive development, the mother plant uses FLC and FT to modulate progeny seed dormancy in response to temperature. We found that for regulation of seed dormancy, FLC and FT function in opposite configuration to how those same genes control time to flowering. For seed dormancy, FT regulates seed dormancy through FLC gene expression and regulates chromatin state by activating antisense FLC transcription. Thus, in Arabidopsis the same genes controlled in opposite format regulate flowering time and seed dormancy in response to the temperature changes that characterize seasons.

In silico Functional Analysis of FLC and FT-Genes Responsible for Postponingand Accelerating the Onset of Flowering

In silico functional analyses of FLOWERING LOCUS C (FLC) and FLOWERING LOCUS T (FT) genes were investigated herein. The recognition of cis-regulatory modules and their organization are a prerequisite to information on the regulation of gene expression. Accordingly, this study carried out a promoter analysis on FT and FLC genes in Arabidopsis and its orthologs in Prunus persica by PlantPan and PLANCARE servers in which the integrating transcription factor binding site (TFBs) was involved in hormonal and stress signals. Light responsive elements were also studied. Among the responsive elements identified in FT and FLC, Arabidopsis had the highest number of responsive elements. This is partly because P. persica is day-neutral, whereas Arabidopsis is a facultative long day plant. The results of motif recognition in FT and FLC proteins with the MEME server showed that FT is highly conserved, but the number of motifs suggests that the FLC protein had far more numerous motifs, which is likely due to its higher amino acid length. Some specified TFs like AGAMOUS (AG), AGAMOUS LIKE (AGL), LEAFY (LFY) and APETALA1 (AP1) were found by the PlantPan web server in FT and FLC promoters’ analysis. They were also present in the protein-protein interaction network analysis which raises the question whether or not FT or FLC can be positive or negative regulators of their own expression in a feedback loop.

Arabidopsis HIGH PLOIDY2 Sumoylates and Stabilizes Flowering Locus C through Its E3 Ligase Activity.

Flowering Locus C (FLC), a floral repressor, plays an important role in flowering. The mechanisms regulating FLC gene expression and protein function have been studied extensively; however, post-translational regulation of FLC remains unclear. Here, we identified Arabidopsis HIGH PLOIDY2 (HPY2) as an E3 SUMO ligase for FLC. In vitro and vivo pull-down assays showed that FLC physically interacts with HPY2. In vitro assays showed that the stimulation of FLC sumoylation by HPY2 was dependent on SUMO-activating enzyme E1 and -conjugating enzyme E2, indicating that HPY2 was an E3 SUMO ligase for FLC. In transgenic plants, inducible HPY2 overexpression increased the concentration of FLC, indicating that HPY2 stabilized FLC through direct sumoylation. Flowering time in hpy2-2 mutants was shorter than in wild-type plants under long- and short-day conditions, with a greater effect under short-day conditions, and FLC was downregulated in hpy2-2 mutants. These data indicate that HPY2 regulates FLC function and stability at both the transcriptional and post-translational levels through its E3 SUMO ligase activity.

Quantitative regulation of FLC via coordinated transcriptional initiation and elongation

The basis of quantitative regulation of gene expression is still poorly understood. In Arabidopsis thaliana, quantitative variation in expression of FLOWERING LOCUS C (FLC) influences the timing of flowering. In ambient temperatures, FLC expression is quantitatively modulated by a chromatin silencing mechanism involving alternative polyadenylation of antisense transcripts. Investigation of this mechanism unexpectedly showed that RNA polymerase II (Pol II) occupancy changes at FLC did not reflect RNA fold changes. Mathematical modeling of these transcriptional dynamics predicted a tight coordination of transcriptional initiation and elongation. This prediction was validated by detailed measurements of total and chromatin-bound FLC intronic RNA, a methodology appropriate for analyzing elongation rate changes in a range of organisms. Transcription initiation was found to vary ∼ 25-fold with elongation rate varying ∼ 8- to 12-fold. Premature sense transcript termination contributed very little to expression differences. This quantitative variation in transcription was coincident with variation in H3K36me3 and H3K4me2 over the FLC gene body. We propose different chromatin states coordinately influence transcriptional initiation and elongation rates and that this coordination is likely to be a general feature of quantitative gene regulation in a chromatin context.

JACALIN-LECTIN LIKE1 Regulates the Nuclear Accumulation of GLYCINE-RICH RNA-BINDING PROTEIN7, Influencing the RNA Processing of FLOWERING LOCUS C Antisense Transcripts and Flowering Time in Arabidopsis.

Lectins selectively recognize sugars or glycans for defense in living cells, but less is known about their roles in the development process and the functional network with other factors. Here, we show that Arabidopsis (Arabidopsis thaliana) JACALIN-LECTIN LIKE1 (AtJAC1) functions in flowering time control. Loss of function of AtJAC1 leads to precocious flowering, whereas overexpression of AtJAC1 causes delayed flowering. AtJAC1 influences flowering through regulation of the key flowering repressor gene FLOWERING LOCUS C (FLC). Genetic analysis revealed that AtJAC1's function is mostly dependent on GLYCINE-RICH RNA-BINDING PROTEIN7 (GRP7), an upstream regulator of FLC. Biochemical and cell biological data indicated that AtJAC1 interacted physically with GRP7 specifically in the cytoplasm. AtJAC1 influences the nucleocytoplasmic distribution of GRP7, with predominant nuclear localization of GRP7 when AtJAC1 function is lost but retention of GRP7 in the cytoplasm when AtJAC1 is overexpressed. A temporal inducible assay suggested that AtJAC1's regulation of flowering could be compromised by the nuclear accumulation of GRP7. In addition, GRP7 binds to the antisense precursor messenger RNA of FLC through a conserved RNA motif. Loss of GRP7 function leads to the elevation of total FLC antisense transcripts and reduced proximal-distal polyadenylation ratio, as well as histone methylation changes in the FLC gene body region and increased total functional sense FLC transcript. Attenuating the direct binding of GRP7 with competing artificial RNAs leads to changes of FLC antisense precursor messenger RNA processing and flowering transition. Taken together, our study indicates that AtJAC1 coordinates with GRP7 in shaping plant development through the regulation of RNA processing in Arabidopsis.

INDUCER OF CBF EXPRESSION1 integrates cold signals into FLOWERING LOCUS C-mediated flowering pathways in Arabidopsis.

Plants constantly monitor changes in photoperiod and temperature throughout the year to synchronize flowering with optimal environmental conditions. In the temperate zones, both photoperiod and temperature fluctuate in a somewhat predictable manner through the seasons, although a transient shift to low temperature is also encountered during changing seasons, such as early spring. Although low temperatures are known to delay flowering by inducing the floral repressor FLOWERING LOCUSC (FLC), it is not fully understood how temperature signals are coordinated with photoperiodic signals in the timing of seasonal flowering. Here, we show that the cold signaling activator INDUCER OF CBF EXPRESSION1 (ICE1), FLC and the floral promoter SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) constitute an elaborate signaling network that integrates cold signals into flowering pathways. The cold-activated ICE1 directly induces the gene encoding FLC, which represses SOC1 expression, resulting in delayed flowering. In contrast, under floral promotive conditions, SOC1 inhibits the binding of ICE1 to the promoters of the FLC gene, inducing flowering with a reduction of freezing tolerance. These observations indicate that the ICE1-FLC-SOC1 signaling network contributes to the fine-tuning of flowering during changing seasons.

Gene regulatory variation mediates flowering responses to vernalization along an altitudinal gradient in Arabidopsis.

Steep environmental gradients provide ideal settings for studies of potentially adaptive phenotypic and genetic variation in plants. The accurate timing of flowering is crucial for reproductive success and is regulated by several pathways, including the vernalization pathway. Among the numerous genes known to enable flowering in response to vernalization, the most prominent is FLOWERING LOCUS C (FLC). FLC and other genes of the vernalization pathway vary extensively among natural populations and are thus candidates for the adaptation of flowering time to environmental gradients such as altitude. We used 15 natural Arabidopsis (Arabidopsis thaliana) genotypes originating from an altitudinal gradient (800-2,700 m above sea level) in the Swiss Alps to test whether flowering time correlated with altitude under different vernalization scenarios. Additionally, we measured the expression of 12 genes of the vernalization pathway and its downstream targets. Flowering time correlated with altitude in a nonlinear manner for vernalized plants. Flowering time could be explained by the expression and regulation of the vernalization pathway, most notably by AGAMOUS LIKE19 (AGL19), FLOWERING LOCUS T (FT), and FLC. The expression of AGL19, FT, and VERNALIZATION INSENSITIVE3 was associated with altitude, and the regulation of MADS AFFECTING FLOWERING2 (MAF2) and MAF3 differed between low- and high-altitude genotypes. In conclusion, we found clinal variation across an altitudinal gradient both in flowering time and the expression and regulation of genes in the flowering time control network, often independent of FLC, suggesting that the timing of flowering may contribute to altitudinal adaptation.

Functional Consequences of Splicing of the Antisense Transcript COOLAIR on FLC Transcription

SummaryAntisense transcription is widespread in many genomes; however, how much is functional is hotly debated. We are investigating functionality of a set of long noncoding antisense transcripts, collectively called COOLAIR, produced at Arabidopsis FLOWERING LOCUS C (FLC). COOLAIR initiates just downstream of the major sense transcript poly(A) site and terminates either early or extends into the FLC promoter region. We now show that splicing of COOLAIR is functionally important. This was revealed through analysis of a hypomorphic mutation in the core spliceosome component PRP8. The prp8 mutation perturbs a cotranscriptional feedback mechanism linking COOLAIR processing to FLC gene body histone demethylation and reduced FLC transcription. The importance of COOLAIR splicing in this repression mechanism was confirmed by disrupting COOLAIR production and mutating the COOLAIR proximal splice acceptor site. Our findings suggest that altered splicing of a long noncoding transcript can quantitatively modulate gene expression through cotranscriptional coupling mechanisms.

The BAF60 Subunit of the SWI/SNF Chromatin-Remodeling Complex Directly Controls the Formation of a Gene Loop at FLOWERING LOCUS C in Arabidopsis

SWI/SNF complexes mediate ATP-dependent chromatin remodeling to regulate gene expression. Many components of these complexes are evolutionarily conserved, and several subunits of Arabidopsis thaliana SWI/SNF complexes are involved in the control of flowering, a process that depends on the floral repressor FLOWERING LOCUS C (FLC). BAF60 is a SWI/SNF subunit, and in this work, we show that BAF60, via a direct targeting of the floral repressor FLC, induces a change at the high-order chromatin level and represses the photoperiod flowering pathway in Arabidopsis. BAF60 accumulates in the nucleus and controls the formation of the FLC gene loop by modulation of histone density, composition, and posttranslational modification. Physiological analysis of BAF60 RNA interference mutant lines allowed us to propose that this chromatin-remodeling protein creates a repressive chromatin configuration at the FLC locus.

R2R3 MYB transcription factor PtrMYB192 regulates flowering time in Arabidopsis by activating FLOWERING LOCUS C

R2R3 MYB transcription factors regulate multiple aspects of plant growth and development. Here we report the identification of PtrMYB192, a Populus R2R3 MYB transcription factor, as a negative regulator of flowering time. By using quantitative RT-PCR, we found that PtrMYB192, but not its closely homologous gene PtrMYB028, is highly expressed in mature leaves in Populus. Heterologously expression of PtrMYB192 under control of 35S promoter in Arabidopsis resulted in late flowering phenotypes under both long and short day conditions, indicating that PtrMYB192 controls flowering time independent of the photoperiod pathway. Domain swapping experiment showed that neither PtrMYB028DB-192AD nor PtrMYB192DB-028AD affected flowering time when heterologously expressed in Arabidopsis. However, when recruit to the promoter of a GAL4-GUS reporter gene by a GAL4 DNA binding domain in Arabidopsis protoplasts, both of PtrMYB028DB-192AD and PtrMYB192DB-028AD activated the reporter gene. Quantitative RT-PCR results showed an elevated expression of the floral repressor gene FLOWERING LOCUS C (FLC), but not the flowering-promoting gene CONSTANS (CO) in PtrMYB192 transgenic plants. Taken together, these results suggest that PtrMYB192 is a transcription activator that negatively regulating flowering time in Arabidopsis by activating FLC and possible other genes, and that both R2R3 DNA binding domain and activation domain maybe required for its full function.

Coordination of the Vernalization Response through a VIN3 and FLC Gene Family Regulatory Network in Arabidopsis

Vernalization is an environmentally induced epigenetic switch in which winter cold triggers epigenetic silencing of floral repressors and thus provides competence to flower in spring. Vernalization triggers the recruitment of chromatin-modifying complexes to a clade of flowering repressors that are epigenetically silenced via chromatin modifications. In Arabidopsis thaliana, VERNALIZATION INSENSITIVE3 (VIN3) and its related plant homeodomain finger proteins act together with Polycomb Repressive Complex 2 to increase repressive histone marks at floral repressor loci, including FLOWERING LOCUS C (FLC) and its related genes, by vernalization. Here, we show that VIN3 family of proteins nonredundantly functions to repress different subsets of the FLC gene family during the course of vernalization. Each VIN3 family protein binds to modified histone peptides in vitro and directly associates with specific sets of FLC gene family chromatins in vivo to mediate epigenetic silencing. In addition, members of the FLC gene family are also differentially regulated during the course of vernalization to mediate proper vernalization response. Our results show that these two gene families cooperated during the course of evolution to ensure proper vernalization response through epigenetic changes.

Role of vernalization and of duplicated FLOWERING LOCUS C in the perennial Arabidopsis lyrata

FLOWERING LOCUS C (FLC) is one of the main genes influencing the vernalization requirement and natural flowering time variation in the annual Arabidopsis thaliana. Here we studied the effects of vernalization on flowering and its genetic basis in the perennial Arabidopsis lyrata. Two tandemly duplicated FLC genes (FLC1 and FLC2) were compared with respect to expression and DNA sequence. The effect of vernalization on flowering and on the expression of FLC1 was studied in three European populations. The genetic basis of the FLC1 expression difference between two of the populations was further studied by expression quantitative trait locus (eQTL) mapping and sequence analysis. FLC1 was shown to have a likely role in the vernalization requirement for flowering in A.lyrata. Vernalization decreased its expression and the northern study populations showed higher FLC1 expression than the southern one. eQTL mapping between two of the populations revealed one eQTL affecting FLC1 expression in the genomic region containing the FLC genes. Most FLC1 sequence differences between the study populations were found in the promoter region and in the first intron. Variation in the FLC1 sequence may cause differences in FLC1 expression between late- and early-flowering A.lyrata populations.

Vernalization-mediated chromatin changes

Proper flowering time is vital for reproductive fitness in flowering plants. In Arabidopsis, vernalization is mediated primarily through the repression of a MADS box transcription factor, FLOWERING LOCUS C (FLC). The induction of a plant homeodomain-containing protein, VERNALIZATION INSENSITIVE 3 (VIN3), by vernalizing cold is required for proper repression of FLC. One of a myriad of changes that occurs after VIN3 is induced is the establishment of FLC chromatin at a mitotically repressed state due to the enrichment of repressive histone modifications. VIN3 induction by cold is the earliest known event during the vernalization response and includes changes in histone modifications at its chromatin. Here, the current understanding of the vernalization-mediated chromatin changes in Arabidopsis is discussed, with a focus on the roles of shared chromatin-modifying machineries in regulating VIN3 and FLC gene family expression during the course of vernalization.