Phytochrome B in the mesophyll delays flowering by suppressing FLOWERING LOCUS T expression in Arabidopsis vascular bundles

Light is one of the most important environmental factors that determine the timing of a plant's transition from the vegetative to reproductive, or flowering, phase. Not only daylength but also the spectrum of light greatly affect flowering. The shade of nearby vegetation reduces the ratio of red to far-red light and can trigger shade avoidance responses, including stem elongation and the acceleration of flowering. Phytochrome B (phyB) acts as a photoreceptor for this response. Physiological studies have suggested that leaves can perceive and respond to shade. However, little is known about the mechanisms involved in the processing of light signals within leaves. In this study, we used an enhancer-trap system to establish Arabidopsis thaliana transgenic lines that express phyB-green fluorescent protein (GFP) fusion protein in tissue-specific manners. The analysis of these lines demonstrated that phyB-GFP in mesophyll cells affected flowering, whereas phyB-GFP in vascular bundles did not. Furthermore, mesophyll phyB-GFP suppressed the expression of a key flowering regulator, FLOWERING LOCUS T, in the vascular bundles of cotyledons. Hence, a novel intertissue signaling from mesophyll to vascular bundles is revealed as a critical step for the regulation of flowering by phyB.     

CONSTANS activates SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 through FLOWERING LOCUS T to promote flowering in Arabidopsis

CONSTANS (CO) regulates flowering time by positively regulating expression of two floral integrators, FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1), in Arabidopsis (Arabidopsis thaliana). FT and SOC1 have been proposed to act in parallel pathways downstream of CO based on genetic analysis using weak ft alleles, since ft soc1 double mutants showed an additive effect in suppressing the early flowering of CO overexpressor plants. However, this genetic analysis was inconsistent with the sequential induction pattern of FT and SOC1 found in inducible CO overexpressor plants. Hence, to identify genetic interactions of CO, FT, and SOC1, we carried out genetic and expression analyses with a newly isolated T-DNA allele of FT, ft-10. We found that ft-10 almost completely suppressed the early flowering phenotype of CO overexpressor plants, whereas soc1-2 partially suppressed the phenotype, suggesting that FT is the major output of CO. Expression of SOC1 was altered in gain- or loss-of-function mutants of FT, whereas expression of FT remained unchanged in gain- or loss-of-function mutants of SOC1, suggesting that FT positively regulates SOC1 to promote flowering. In addition, inactivation of FT caused down-regulation of SOC1 even in plants overexpressing CO, indicating that FT is required for SOC1 induction by CO. Taken together, these data suggest that CO activates SOC1 through FT to promote flowering in Arabidopsis.     

Synchronized expression of FLOWERING LOCUS T between branches underlies mass flowering in Fagus crenata

Masting is the intermittent and synchronized production of a large amount of flower and seed in plant populations. This population-level phenomenon is caused by individual-level variability in reproduction and its synchrony between individuals. The variability at the individual level is induced by synchronized reproduction between branches within an individual because a tree is an assemblage of branches that are considered as semiautonomous units. However, there have been no empirical studies that quantify the degree of reproductive synchrony at the branch level within the same tree in masting species. Here, we evaluated the reproductive synchrony within individuals by monitoring flowering dynamics and expression level of a flowering-time gene at the branch-level in a typical masting species, Fagus crenata Blume. The 4-year census showed that the branch-level gene expression was highly variable between years and was strongly synchronized between branches. The branch-level synchrony in flowering-time gene expression was followed by coherent flowering cycle at the whole individual. To examine the causal relationship between gene expression and climatic factors, we performed a nonlinear statistical analysis called convergent cross-mapping using the time course data of gene expression and environmental variables. Our results indicated that the observed gene expression pattern was well cross-mapped by temperature or precipitation. However, this cross-mapping skill was lower than that of randomly generated seasonal dynamics, implying a combination of internal and external environmental signals is more likely to regulate gene expression dynamics in F. crenata. Our results provide the first empirical evidence that synchronized expression of a flowering-time gene between branches underlies integrated flowering behavior at the individual level.     

Repression of FLOWERING LOCUS C and FLOWERING LOCUS T by the Arabidopsis Polycomb repressive complex 2 components

Polycomb group (PcG) proteins are evolutionarily conserved in animals and plants, and play critical roles in the regulation of developmental gene expression. Here we show that the Arabidopsis Polycomb repressive complex 2 (PRC2) subunits CURLY LEAF (CLF), EMBRYONIC FLOWER 2 (EMF2) and FERTILIZATION INDEPENDENT ENDOSPERM (FIE) repress the expression of FLOWERING LOCUS C (FLC), a central repressor of the floral transition in Arabidopsis and FLC relatives. In addition, CLF directly interacts with and mediates the deposition of repressive histone H3 lysine 27 trimethylation (H3K27me3) into FLC and FLC relatives, which suppresses active histone H3 lysine 4 trimethylation (H3K4me3) in these loci. Furthermore, we show that during vegetative development CLF and FIE strongly repress the expression of FLOWERING LOCUS T (FT), a key flowering-time integrator, and that CLF also directly interacts with and mediates the deposition of H3K27me3 into FT chromatin. Our results suggest that PRC2-like complexes containing CLF, EMF2 and FIE, directly interact with and deposit into FT, FLC and FLC relatives repressive trimethyl H3K27 leading to the suppression of active H3K4me3 in these loci, and thus repress the expression of these flowering genes. Given the central roles of FLC and FT in flowering-time regulation in Arabidopsis, these findings suggest that the CLF-containing PRC2-like complexes play a significant role in control of flowering in Arabidopsis.     

HISTONE DEACETYLASE6 interacts with FLOWERING LOCUS D and regulates flowering in Arabidopsis

Histone acetylation and deacetylation play an important role in epigenetic controls of gene expression. HISTONE DEACETYLASE6 (HDA6) is a REDUCED POTASSIUM DEPENDENCY3-type histone deacetylase, and the Arabidopsis (Arabidopsis thaliana) hda6 mutant axe1-5 displayed a late-flowering phenotype. axe1-5/flc-3 double mutants flowered earlier than axe1-5 plants, indicating that the late-flowering phenotype of axe1-5 was FLOWERING LOCUS C (FLC) dependent. Bimolecular fluorescence complementation, in vitro pull-down, and coimmunoprecipitation assays revealed the protein-protein interaction between HDA6 and the histone demethylase FLD. It was found that the SWIRM domain in the amino-terminal region of FLD and the carboxyl-terminal region of HDA6 are responsible for the interaction between these two proteins. Increased levels of histone H3 acetylation and H3K4 trimethylation at FLC, MAF4, and MAF5 were found in both axe1-5 and fld-6 plants, suggesting functional interplay between histone deacetylase and demethylase in flowering control. These results support a scenario in which histone deacetylation and demethylation cross talk are mediated by physical association between HDA6 and FLD. Chromatin immunoprecipitation analysis indicated that HDA6 bound to the chromatin of several potential target genes, including FLC and MAF4. Genome-wide gene expression analysis revealed that, in addition to genes related to flowering, genes involved in gene silencing and stress response were also affected in hda6 mutants, revealing multiple functions of HDA6. Furthermore, a subset of transposons was up-regulated and displayed increased histone hyperacetylation, suggesting that HDA6 can also regulate transposons through deacetylating histone.     

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.     

The microRNA156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE3 module regulates ambient temperature-responsive flowering via FLOWERING LOCUS T in Arabidopsis

The flowering time of plants is affected by modest changes in ambient temperature. However, little is known about the regulation of ambient temperature-responsive flowering by small RNAs. In this study, we show that the microRNA156 (miR156)-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE3 (SPL3) module directly regulates FLOWERING LOCUS T (FT) expression in the leaf to control ambient temperature-responsive flowering. Overexpression of miR156 led to more delayed flowering at a lower ambient temperature (16°C), which was associated with down-regulation of FT and FRUITFULL expression. Among miR156 target genes, SPL3 mRNA levels were mainly reduced, probably because miR156-mediated cleavage of SPL3 mRNA was higher at 16°C. Overexpression of miR156-resistant SPL3 [SPL3(-)] caused early flowering, regardless of the ambient temperature, which was associated with up-regulation of FT and FRUITFULL expression. Reduction of miR156 activity by target mimicry led to a phenotype similar to that of SUC2::rSPL3 plants. FT up-regulation was observed after dexamethasone treatment in GVG-rSPL3 plants. Misexpression and artificial microRNA-mediated suppression of FT in the leaf dramatically altered the ambient temperature-responsive flowering of plants overexpressing miR156 and SPL3(-). Chromatin immunoprecipitation assay showed that the SPL3 protein directly binds to GTAC motifs within the FT promoter. Lesions in TERMINAL FLOWER1, SHORT VEGETATIVE PHASE, and EARLY FLOWERING3 did not alter the expression of miR156 and SPL3. Taken together, our data suggest that the interaction between the miR156-SPL3 module and FT is part of the regulatory mechanism controlling flowering time in response to ambient temperature.     

DICER-LIKE 1 and DICER-LIKE 3 redundantly act to promote flowering via repression of FLOWERING LOCUS C in Arabidopsis thaliana

In Arabidopsis thaliana, DICER-LIKE 1 and DICER-LIKE 3 are involved in the generation of small RNAs. Double mutants between dicer-like 1 and dicer-like 3 exhibit a delay in flowering that is caused by increased expression of the floral repressor FLOWERING LOCUS C. This delayed-flowering phenotype is similar to that of autonomous-pathway mutants, and the flowering delay can be overcome by vernalization.     

The Medicago FLOWERING LOCUS T homolog, MtFTa1, is a key regulator of flowering time

FLOWERING LOCUS T (FT) genes encode proteins that function as the mobile floral signal, florigen. In this study, we characterized five FT-like genes from the model legume, Medicago (Medicago truncatula). The different FT genes showed distinct patterns of expression and responses to environmental cues. Three of the FT genes (MtFTa1, MtFTb1, and MtFTc) were able to complement the Arabidopsis (Arabidopsis thaliana) ft-1 mutant, suggesting that they are capable of functioning as florigen. MtFTa1 is the only one of the FT genes that is up-regulated by both long days (LDs) and vernalization, conditions that promote Medicago flowering, and transgenic Medicago plants overexpressing the MtFTa1 gene flowered very rapidly. The key role MtFTa1 plays in regulating flowering was demonstrated by the identification of fta1 mutants that flowered significantly later in all conditions examined. fta1 mutants do not respond to vernalization but are still responsive to LDs, indicating that the induction of flowering by prolonged cold acts solely through MtFTa1, whereas photoperiodic induction of flowering involves other genes, possibly MtFTb1, which is only expressed in leaves under LD conditions and therefore might contribute to the photoperiodic regulation of flowering. The role of the MtFTc gene is unclear, as the ftc mutants did not have any obvious flowering-time or other phenotypes. Overall, this work reveals the diversity of the regulation and function of the Medicago FT family.     

Polycomb-Group Proteins and FLOWERING LOCUS T Maintain Commitment to Flowering in Arabidopsis thaliana

The switch from vegetative to reproductive growth is extremely stable even if plants are only transiently exposed to environmental stimuli that trigger flowering. In the photoperiodic pathway, a mobile signal, florigen, encoded by FLOWERING LOCUS T (FT) in Arabidopsis thaliana, induces flowering. Because FT activity in leaves is not maintained after transient photoperiodic induction, the molecular basis for stable floral commitment is unclear. Here, we show that Polycomb-group (Pc-G) proteins, which mediate epigenetic gene regulation, maintain the identity of inflorescence and floral meristems after floral induction. Thus, plants with reduced Pc-G activity show a remarkable increase of cauline leaves under noninductive conditions and floral reversion when shifted from inductive to noninductive conditions. These phenotypes are almost completely suppressed by loss of FLOWERING LOCUS C (FLC) and SHORT VEGETATIVE PHASE, which both delay flowering and promote vegetative shoot identity. Upregulation of FLC in Pc-G mutants leads to a strong decrease of FT expression in inflorescences. We find that this activity of FT is needed to prevent floral reversion. Collectively, our results reveal that floral meristem identity is at least partially maintained by a daylength-independent role of FT whose expression is indirectly sustained by Pc-G activity.