Terminal flower2, an Arabidopsis homolog of heterochromatin protein1, counteracts the activation of flowering locus T by constans in the vascular tissues of leaves to regulate flowering time

The flowering time of plants is tightly regulated by both promotive and repressive factors. Molecular genetic studies using Arabidopsis have identified several epigenetic repressors that regulate flowering time. Terminal flower2, (TFL2), which encodes a homolog of heterochromatin protein1 represses flowering locus T (FT) expression, which is induced by the activator constans (CO) in response to the long-day signal. Here, we show that TFL2, CO, and FT are expressed together in leaf vascular tissues and that TFL2 represses FT expression continuously throughout development. Mutations in TFL2 derepress FT expression within the vascular tissues of leaves, resulting in daylength-independent early flowering. TFL2 can reduce FT expression even when CO is overexpressed. However, FT expression reaches a level sufficient for floral induction even in the presence of TFL2, suggesting that TFL2 does not maintain FT in a silent state or inhibit it completely; rather, it counteracts the effect of CO on FT activation.     

Analysis of flowering pathway integrators in Arabidopsis

Flowering is regulated by an integrated network of several genetic pathways in Arabidopsis. The key genes integrating multiple flowering pathways are FT, SOC1 and LFY. To elucidate the interactions among these integrators, genetic analyses were performed. FT and SOC1 share the common upstream regulators CO, a key component in the long day pathway, and FLC, a flowering repressor integrating autonomous and vernalization pathways. However, the soc1 mutation further delayed the flowering time of long day pathway mutants including ft, demonstrating that SOC1 acts partially independently of FT. Although soc1 did not show an obvious defect in flower meristem determination on its own, it dramatically increased the number of coflorescences in a lfy mutant, which is indicative of a defect in floral initiation. Therefore, double mutant analysis shows that the three integrators have both overlapping and independent functions in the determination of flowering time and floral initiation. The expression analysis showed that FT regulates SOC1 expression, and SOC1 regulates LFY expression, but not vice versa, which is consistent with the fact that FT and LFY have the least overlapping functions among the three integrators. The triple mutation ft soc1 lfy did not block flowering completely under long days, indicating the presence of other integrators. Finally, vernalization accelerated flowering of flc ft soc1 and ft soc1 lfy triple mutants, which shows that the vernalization pathway also has targets other than FLC, FT, SOC1 and LFY. Our genetic analysis reveals the intricate nature of genetic networks for flowering.     

The gentian orthologs of the FT/TFL1 gene family control floral initiation in Gentiana

Gentians are herbaceous perennials blooming in summer through autumn. Although they are popular ornamental flowers in Japan, the regulation of their timing of flowering has not been studied. We identified and characterized gentian orthologs of the Arabidopsis FT/TFL1 gene family to elucidate the mechanisms of flowering initiation. We isolated three gentian orthologs of FT and TFL1, denoted GtFT1, GtFT2 and GtTFL1. Since up-regulation of GtFT1 and GtFT2 as well as down-regulation of GtTFL1 promoted floral initiation in gentian plantlets, these genes affected floral initiation in a similar way to Arabidopsis FT and TFL1. The expression levels of GtFT1 and GtFT2 in leaves of late-flowering gentian increased prior to floral initiation, whereas GtTFL1 was highly expressed in shoot apical meristem at the vegetative stage and decreased drastically just before flowering initiation. Comparison of gene expression patterns showed that GtFT1 expression increased earlier in early-flowering than in late-flowering gentian, whereas the timing of the increase in GtFT2 expression was similar in early- and late-flowering plants. The GtTFL1 expression in early-flowering gentian was extremely low throughout the vegetative and reproductive stages. These results indicated that either the up-regulation of GtFT1 or the down-regulation of GtTFL1 may determine flowering time. Furthermore, we found that early-flowering but not late-flowering gentians have a 320 bp insertion in the promoter region of GtTFL1. Thus, the negligible expression of GtTFL1 in early-flowering lines may be due to this insertion, resulting in a shortened vegetative stage.     

植物FLOWERING LOCUS T/TERMINAL FLOWER1基因家族的研究进展

植物FLOWERING LOCUS T/TERMINAL FLOWER1(FT/TFL1)基因家族是一个进化上高度保守的基因家族,它在植物的花发育过程中具有重要作用:其成员FT基因编码的蛋白产物是可以长距离转运的成花激素,在花形成过程中起关键作用;另一成员TFL1基因则在花序的形成和维持过程中起重要作用.本文就近年来国内外对植物FT/TFL1基因家族的结构、成员,以及各个成员在花发育转换过程中的功能等研究现状进行综述,并对该基因家族的研究前景提出展望.     

Arabidopsis TERMINAL FLOWER 2 gene encodes a heterochromatin protein 1 homolog and represses both FLOWERING LOCUS T to regulate flowering time and several floral homeotic genes

Floral transition should be strictly regulated because it is one of the most critical developmental processes in plants. Arabidopsis terminal flower 2 (tfl2) mutants show an early-flowering phenotype that is relatively insensitive to photoperiod, as well as several other pleiotropic phenotypes. We found that the early flowering of tfl2 is caused mainly by ectopic expression of the FLOWERING LOCUS T (FT) gene, a floral pathway integrator. Molecular cloning of TFL2 showed that it encodes a protein with homology to heterochromatin protein 1 (HP1) of animals and Swi6 of fission yeast. TFL2 protein localizes in subnuclear foci and expression of the TFL2 gene complemented yeast swi6(-) mutants. These results suggested that TFL2 might function as an HP1 in Arabidopsis: Gene expression analyses using DNA microarrays, however, did not show an increase in the expression of heterochromatin genes in tfl2 mutants but instead showed the upregulation of the floral homeotic genes APETALA3, PISTILLATA, AGAMOUS and SEPALLATA3. The pleiotropic phenotype of the tfl2 mutant could reflect the fact that TFL2 represses the expression of multiple genes. Our results demonstrate that despite its homology to HP1, TFL2 is involved in the repression of specific euchromatin genes and not heterochromatin genes in Arabidopsis.     

The <Emphasis Type="Italic">FT/TFL1</Emphasis> gene family in grapevine

The FT/TFL1 gene family encodes proteins with similarity to phosphatidylethanolamine binding proteins which function as flowering promoters and repressors. We show here that the FT/TFL1 gene family in Vitis vinifera is composed of at least five genes. Sequence comparisons with homologous genes identified in other dicot species group them in three major clades, the FT, MFT and TFL1 subfamilies, the latter including three of the Vitis sequences. Gene expression patterns are in agreement with a role of VvFT and VvMFT as flowering promoters; while VvTFL1A, VvTFL1B and VvTFL1C could be associated with vegetative development and maintenance of meristem indetermination. Overexpression of VvFT in transgenic Arabidopsis plants generates early flowering phenotypes similar to those produced by FT supporting a role for this gene in flowering promotion. Overexpression of VvTFL1A does not affect flowering time but the determination of flower meristems, strongly altering inflorescence structure, which is consistent with the biological roles assigned to similar genes in other species.     

Evolution of the PEBP gene family in plants: functional diversification in seed plant evolution

The phosphatidyl ethanolamine-binding protein (PEBP) gene family is present in all eukaryote kingdoms, with three subfamilies identified in angiosperms (FLOWERING LOCUS T [FT], MOTHER OF FT AND TFL1 [MFT], and TERMINAL FLOWER1 [TFL1] like). In angiosperms, PEBP genes have been shown to function both as promoters and suppressors of flowering and to control plant architecture. In this study, we focus on previously uncharacterized PEBP genes from gymnosperms. Extensive database searches suggest that gymnosperms possess only two types of PEBP genes, MFT-like and a group that occupies an intermediate phylogenetic position between the FT-like and TFL1-like (FT/TFL1-like). Overexpression of Picea abies PEBP genes in Arabidopsis (Arabidopsis thaliana) suggests that the FT/TFL1-like genes (PaFTL1 and PaFTL2) code for proteins with a TFL1-like function. However, PaFTL1 and PaFTL2 also show highly divergent expression patterns. While the expression of PaFTL2 is correlated with annual growth rhythm and mainly confined to needles and vegetative and reproductive buds, the expression of PaFTL1 is largely restricted to microsporophylls of male cones. The P. abies MFT-like genes (PaMFT1 and PaMFT2) show a predominant expression during embryo development, a pattern that is also found for many MFT-like genes from angiosperms. P. abies PEBP gene expression is primarily detected in tissues undergoing physiological changes related to growth arrest and dormancy. A first duplication event resulting in two families of plant PEBP genes (MFT-like and FT/TFL1-like) seems to coincide with the evolution of seed plants, in which independent control of bud and seed dormancy was required, and the second duplication resulting in the FT-like and TFL1-like clades probably coincided with the evolution of angiosperms.     

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.     

BROTHER OF FT AND TFL1 (BFT), a member of the FT/TFL1 family,shows distinct pattern of expression during the vegetative growth of Arabidopsis

Transition to the flowering stage is precisely controlled by a few classes of regulatory molecules. BROTHER OF FT AND TFL1 (BFT) is a member of FLOWERING LOCUS T (FT)/TERMINAL FLOWER 1 (TFL1) family, an important class of flower development regulators with unidentified biochemical function. BFT has a TFL1-like activity and plays a role in axillary inflorescence development. To elucidate the expression pattern of BFT, we analyzed the subcellular localization and conditional expression of BFT in this study. We generated 35S::BFT:GFP plants to investigate the subcellular localization of BFT protein. 35S::BFT:GFP plants showed late flowering, similarly as did 35S::BFT plants. BFT:GFP fusion protein was localized in the nucleus and the plasma membrane, which was different from the localization pattern of FT and TFL1. BFT expression was induced by abiotic stress conditions. ABA, drought, and osmotic stress treatments induced BFT expression, whereas cold, salt, and heat stress conditions did not, suggesting that BFT plays a role in regulating flowering time and inflorescence structure under drought conditions. The induction pattern of BFT was different from those of other FT/TFL1 family genes. Our studies indicated that BFT showed a distinct expression pattern from its homologous genes during the vegetative growth in Arabidopsis.Key words: flowering time, flowering locus T, terminal flower 1, brother of FT and TFL1, abiotic stress, subcellullar localizationThe FLOWERING LOCUS T (FT)/TERMINAL FLOWER 1 (TFL1) family is a small gene family whose members play a pivotal role in flower development in Arabidopsis. The family includes FT, TFL1, TWIN SISTER OF FT (TSF), Arabidopsis thaliana CENTRORADIALIS homologue (ATC), MOTHER OF FT AND TFL1 (MFT) and BROTHER OF FT AND TFL1 (BFT).^{3,5,6,9,15,17} FT is a floral promoter that integrates signal inputs from various pathways that regulate flowering time in Arabidopsis.^5,6 TFL1 plays an antagonistic role to that of FT, functioning as a floral inhibitor. Unlike FT, TFL1 also plays an important role in controlling plant architecture by regulating the expression of LEAFY (LFY) and APETALA1 (AP1), two important floral meristem identity genes in the shoot apical meristem (SAM).^3,7 TSF regulates flowering by a mechanism similar to that of FT, although a lesion in TSF does not have an apparent effect on the determination of flowering time. MFT has a weak FT-like activity.¹⁷ ATC acts as a floral repressor, and its role is similar to that of TFL1.⁹ Finally, BFT has a TFL1-like activity, in spite of its amino acid homology to FT,^2,4,16 and functions redundantly with TFL1 in inflorescence meristem development in Arabidopsis.¹⁶ Although genetic studies elucidated an intricate role of the FT/TFL1 family genes, not much is known about the expression pattern of the remaining members except FT and TFL1. Here, we report that BFT expression showed a distinct pattern from its homologous genes during the vegetative phase. BFT:GFP fusion protein was detected in the nucleus and the plasma membrane. BFT expression was induced by abiotic stress conditions, distinct from other FT/TFL1 family genes, raising the possibility that BFT plays a role in regulating flowering time and inflorescence structure under drought conditions.     

The Arabidopsis heterochromatin protein1 homolog (TERMINAL FLOWER2) silences genes within the euchromatic region but not genes positioned in heterochromatin

TERMINAL FLOWER2 (TFL2) is the only homolog of heterochromatin protein1 (HP1) in the Arabidopsis genome. Because proteins of the HP1 family in fission yeast and animals act as key components of gene silencing in heterochromatin by binding to histone H3 methylated on lysine 9 (K9), here we examined whether TFL2 has a similar role in Arabidopsis. Unexpectedly, genes positioned in heterochromatin were not activated in tfl2 mutants. Moreover, the TFL2 protein localized preferentially to euchromatic regions and not to heterochromatic chromocenters, where K9-methylated histone H3 is clustered. Instead, TFL2 acts as a repressor of genes related to plant development, i.e. flowering, floral organ identity, meiosis and seed maturation. Up-regulation of the floral homeotic genes PISTILLATA, APETALA3, AGAMOUS and SEPALLATA3 in tfl2 mutants was independent of LEAFY or APETALA3, known activators of the above genes. In addition, transduced APETALA3 promoter fragments as short as 500 bp were sufficient for TFL2-mediated gene repression. Taken together, TFL2 silences specific genes within euchromatin but not genes positioned in heterochromatin of Arabidopsis.     

Functional Evolution of Phosphatidylethanolamine Binding Proteins in Soybean and Arabidopsis

Gene duplication provides resources for novel gene functions. Identification of the amino acids responsible for functional conservation and divergence of duplicated genes will strengthen our understanding of their evolutionary course. Here, we conducted a systemic functional investigation of phosphatidylethanolamine binding proteins (PEBPs) in soybean (Glycine max) and Arabidopsis thaliana. Our results demonstrated that after the ancestral duplication, the lineage of the common ancestor of the FLOWERING LOCUS T (FT) and TERMINAL FLOWER1 (TFL1) subfamilies functionally diverged from the MOTHER OF FT AND TFL1 (MFT) subfamily to activate flowering and repress flowering, respectively. They also underwent further specialization after subsequent duplications. Although the functional divergence increased with duplication age, we observed rapid functional divergence for a few pairs of young duplicates in soybean. Association analysis between amino acids and functional variations identified critical amino acid residues that led to functional differences in PEBP members. Using transgenic analysis, we validated a subset of these differences. We report clear experimental evidence for the functional evolution of the PEBPs in the MFT, FT, and TFL1 subfamilies, which predate the origin of angiosperms. Our results highlight the role of amino acid divergence in driving evolutionary novelty after duplication.     

The pea GIGAS gene is a FLOWERING LOCUS T homolog necessary for graft-transmissible specification of flowering but not for responsiveness to photoperiod

Garden pea (Pisum sativum) was prominent in early studies investigating the genetic control of flowering and the role of mobile flowering signals. In view of recent evidence that genes in the FLOWERING LOCUS T (FT) family play an important role in generating mobile flowering signals, we isolated the FT gene family in pea and examined the regulation and function of its members. Comparison with Medicago truncatula and soybean (Glycine max) provides evidence of three ancient subclades (FTa, FTb, and FTc) likely to be common to most crop and model legumes. Pea FT genes show distinctly different expression patterns with respect to developmental timing, tissue specificity, and response to photoperiod and differ in their activity in transgenic Arabidopsis thaliana, suggesting they may have different functions. We show that the pea FTa1 gene corresponds to the GIGAS locus, which is essential for flowering under long-day conditions and promotes flowering under short-day conditions but is not required for photoperiod responsiveness. Grafting, expression, and double mutant analyses show that GIGAS/FTa1 regulates a mobile flowering stimulus but also provide clear evidence for a second mobile flowering stimulus that is correlated with expression of FTb2 in leaf tissue. These results suggest that induction of flowering by photoperiod in pea results from interactions among several members of a diversified FT family.     

The flowering-time geneFT and regulation of flowering inArabidopsis

Transition from vegetative to reproductive development (flowering) is one of the most important decisions during the post-embryonic development of flowering plants. More than twenty loci are known to regulate this process inArabidopsis. Some of these flowering-time genes may act at the shoot apical meristem to regulate its competence to respond to floral inductive signals and floral evocation. Genetic and phenotypic analyses of mutants suggest that the late-flowering geneFT may be a good candidate for such genes. To test this, we have cloned theFT gene using aFT-deficiency line associated with a T-DNA insertion. Cloned genes and loss-of-function mutants in hand, it is now possible to analyse the role ofFT and other genes in flowering at the biochemical and cellular levels as well as at the genetic level. The deduced FT protein has homology with TFL1 and CEN proteins believed to be involved in regulation of inflorescence meristem identity. Phylogenetic analysis suggests that theFT group and theTFL1/CEN group of genes diverged before the diversification of major angiosperm clades. This raises the interesting question of the evolutionary relationship between the regulation of vegetative/reproductive switching in the shoot apical meristem and the regulation of inflorescence architecture in angiosperms. The extended abstract of a paper presented at the 13th International Symposium in Conjugation with Award of the International Prize for Biology “Fronitier of Plant Biology”