Florigen Activation Research Articles

A gain-of-function mutation at the C-terminus of FT-D1 promotes heading by interacting with 14-3-3A and FDL6 in wheat.

Vernalization and photoperiod pathways converging at FT1 control the transition to flowering in wheat. Here, we identified a gain-of-function mutation in FT-D1 that results in earlier heading date (HD), and shorter plant height and spike length in the gamma ray-induced eh1 wheat mutant. Knockout of the wild-type and overexpression of the mutated FT-D1 indicate that both alleles are functional to affect HD and plant height. Protein interaction assays demonstrated that the frameshift mutation in FT-D1eh1 exon 3 led to gain-of-function interactions with 14-3-3A and FDL6, thereby enabling the formation of florigen activation complex (FAC) and consequently activating a flowering-related transcriptomic programme. This mutation did not affect FT-D1eh1 interactions with TaNaKR5 or TaFTIP7, both of which could modulate HD, potentially via mediating FT-D1 translocation to the shoot apical meristem. Furthermore, the 'Segment B' external loop is essential for FT-D1 interaction with FDL6, while residue Y85 is required for interactions with TaNaKR5 and TaFTIP7. Finally, the flowering regulatory hub gene, ELF5, was identified as the FT-D1 regulatory target. This study illustrates FT-D1 function in determining wheat HD with a suite of interaction partners and provides genetic resources for tuning HD in elite wheat lines.

The 'Candidatus phytoplasma ziziphi' effectors SJP1 and SJP2 destabilise the bifunctional regulator ZjTCP7 to modulate floral transition and shoot branching.

Phytoplasmic SAP11 effectors alter host plant architecture and flowering time. However, the exact mechanisms have yet to be elucidated. Two SAP11-like effectors, SJP1 and SJP2, from 'Candidatus Phytoplasma ziziphi' induce shoot branching proliferation. Here, the transcription factor ZjTCP7 was identified as a central target of these two effectors to regulate floral transition and shoot branching. Ectopic expression of ZjTCP7 resulted in enhanced bolting and earlier flowering than did the control. Interaction and expression assays demonstrated that ZjTCP7 interacted with the ZjFT-ZjFD module, thereby enhancing the ability of these genes to directly bind to the ZjAP1 promoter. The effectors SJP1 and SJP2 unravelled the florigen activation complex by specifically destabilising ZjTCP7 and ZjFD to delay floral initiation. Moreover, the shoot branching of the ZjTCP7-SRDX transgenic Arabidopsis lines were comparable to those of the SJP1/2 lines, suggesting the involvement of ZjTCP7 in the regulation of shoot branching. ZjTCP7 interacted with the branching repressor ZjBRC1 to enhance suppression of the auxin efflux carrier ZjPIN3 expression. ZjTCP7 also directly bound to and upregulated the auxin biosynthesis gene ZjYUCCA2, thereby promoting auxin accumulation. Our findings confirm that ZjTCP7 serves as a bifunctional regulator destabilised by the effectors SJP1 and SJP2 to modulate plant development.

The Significance of Florigen Activation Complex in Controlling Flowering in Rice

Yield potential, plant height and flowering time are three classes of traits that determine the productivity of rice. Rice heading greatly depends on the accurate measurement of environment changes, particularly in day length and temperature. We discuss all genes reported to be related to the heading time in this review. The accumulation of florigen, such as Hd3a or RFT1, is the key factor for rice heading. Florigen is found in the rice leaf and then transports into the shoot apical meristem (SAM), where floral initiation occurs. We discuss the formation of florigen activation complex (FAC) with 14-3-3 and FD1 proteins to induce the expression of flowering genes. In addition, two key pathways related to heading date, GI-Hd1-Hd3a and Ghd7-Ehd1-Hd3a/RFT1 are introduced in this review. We summarize proteins with histone modification functions, due to the modification of histone is also significant for heading date. The flowering time also extremely dependent on the environments, including nutrient availability, abiotic and biotic stresses, we argue the phenotypes of rice heading date under different extreme environments, as well. Lastly, we gather genes that are reported having a change in heading time, but their detail molecular regulation is still elusive. Thus, this review introduces heading time systematically, providing knowledge and ideas for researchers to study further the phenotype of heading time.

VERNALIZATION1 represses FLOWERING PROMOTING FACTOR1-LIKE1 in leaves for timely flowering in Brachypodium distachyon.

FLOWERING PROMOTING FACTOR1 (FPF1), a small protein without any known domains, promotes flowering in several plants; however, its functional mechanism remains unknown. Here, we characterized 2 FPF1-like proteins, FPL1 and FPL7, which, in contrast, function as flowering repressors in Brachypodium distachyon. FPL1 and FPL7 interact with the components of the florigen activation complex (FAC) and inhibit FAC activity to restrict expression of its critical target, VERNALIZATION1 (VRN1), in leaves, thereby preventing overaccumulation of FLOWERING LOCUS T1 (FT1) at the juvenile stage. Further, VRN1 can directly bind to the FPL1 promoter and repress FPL1 expression; hence, as VRN1 gradually accumulates during the late vegetative stage, FAC is released. This accurate feedback regulation of FPL1 by VRN1 allows proper FT1 expression in leaves and ensures sufficient FAC formation in shoot apical meristems to trigger timely flowering. Overall, we define a sophisticated modulatory loop for flowering initiation in a temperate grass, providing insights toward resolving the molecular basis underlying fine-tuning flowering time in plants.

Adaptation to high latitudes through a novel allele of Hd3a strongly promoting heading date in rice.

A novel Hd3a allele strongly promoting rice heading date was identified, and it functions through florigen activation complex (FAC) and was selected during the spread of rice cultivation to high-latitude areas. Heading date is a critical agronomic trait for rice that determines the utilization of light and temperature conditions and thereby affects grain yield. Rice is a short day (SD) plant, and its photoperiodic information is processed by complex pathways and integrated by florigens to control flowering. In this study, we identified a novel allele for the florigen gene Heading date 3a (Hd3a), characterized by a C435G substitution in its coding region, by a genome-wide association study (GWAS) approach in a panel of 199 high-latitude japonica rice varieties. The C435G substitution induces plants to flower 10days earlier in high-latitude area (long day condition). Then, we mutated C435 to G in Hd3a by prime editing and found the point mutation plants flowered 12days earlier. Further molecular experiments showed the novel Hd3a protein can interact with GF14b protein and increase the expression of OsMADS14, the output gene of florigen activation complex (FAC). Molecular signatures of selection indicated that the novel Hd3a allele was selected during the process of rice cultivation expansion into high-latitude areas. Collectively, these results provide new insights into heading date regulation in high-latitude areas and advance improvements to rice adaptability to enhance crop yield.

OsFTL12, a member of FT-like family, modulates the heading date and plant architecture by florigen repression complex in rice.

FLOWERING LOCUS T (FT), a florigen in Arabidopsis, plays critical roles in floral transition. Among 13 FT-like members in rice, OsFTL2 (Hd3a) and OsFTL3 (RFT1), two rice homologues of FT, have been well characterized to act as florigens to induce flowering under short-day (SD) and long-day (LD) conditions, respectively, but the functions of other rice FT-like members remain largely unclear. Here, we show that OsFTL12 plays an antagonistic function against Hd3a and RFT1 to modulate the heading date and plant architecture in rice. Unlike Hd3a and RFT1, OsFTL12 is not regulated by daylength and highly expressed in both SD and LD conditions, and delays the heading date under either SD or LD conditions. We further demonstrate that OsFTL12 interacts with GF14b and OsFD1, two key components of the florigen activation complex (FAC), to form the florigen repression complex (FRC) by competing with Hd3a for binding GF14b. Notably, OsFTL12-FRC can bind to the promoters of the floral identity genes OsMADS14 and OsMADS15 and suppress their expression. The osmads14 osmads15 double mutants could not develop panicles and showed erect leaves. Taken together, our results reveal that different FT-like members can fine-tune heading date and plant architecture by regulating the balance of FAC and FRC in rice.

A bZIP transcription factor (CiFD) regulates drought- and low-temperature-induced flowering by alternative splicing in citrus.

Drought and low temperature are two key environmental factors that induce adult citrus flowering. However, the underlying regulation mechanism is poorly understood. The bZIP transcription factor FD is a key component of the florigen activation complex (FAC) which is composed of FLOWERING LOCUS T (FT), FD, and 14-3-3 proteins. In this study, isolation and characterization of CiFD in citrus found that there was alternative splicing (AS) of CiFD, forming two different proteins (CiFDα and CiFDβ). Further investigation found that their expression patterns were similar in different tissues of citrus, but the subcellular localization and transcriptional activity were different. Overexpression of the CiFD DNA sequence (CiFD-DNA), CiFDα, or CiFDβ in tobacco and citrus showed early flowering, and CiFD-DNA transgenic plants were the earliest, followed by CiFDβ and CiFDα. Interestingly, CiFDα and CiFDβ were induced by low temperature and drought, respectively. Further analysis showed that CiFDα can form a FAC complex with CiFT, Ci14-3-3, and then bind to the citrus APETALA1 (CiAP1) promoter and promote its expression. However, CiFDβ can directly bind to the CiAP1 promoter independently of CiFT and Ci14-3-3. These results showed that CiFDβ can form a more direct and simplified pathway that is independent of the FAC complex to regulate drought-induced flowering through AS. In addition, a bHLH transcription factor (CibHLH96) binds to CiFD promoter and promotes the expression of CiFD under drought condition. Transgenic analysis found that CibHLH96 can promote flowering in transgenic tobacco. These results suggest that CiFD is involved in drought- and low-temperature-induced citrus flowering through different regulatory patterns.

Don't FRET: screening for inhibitors of the florigen activation complex

Don't <scp>FRET</scp>: screening for inhibitors of the florigen activation complex

Multifunctional chemical inhibitors of the florigen activationcomplex discovered by structure-based high-throughput screening.

Structure-based high-throughput screening of chemical compounds that target protein-protein interactions (PPIs) is a promising technology for gaining insight into how plant development is regulated, leading to many potential agricultural applications. At present, there are no examples of using high-throughput screening to identify chemicals that target plant transcriptional complexes, some of which are responsible for regulating multiple physiological functions. Florigen, a protein encoded by FLOWERING LOCUS T (FT), was initially identified as a molecule that promotes flowering and has since been shown to regulate flowering and other developmental phenomena such as tuber formation in potato (Solanum tuberosum). FT functions as a component of the florigen activation complex (FAC) with a 14-3-3 scaffold protein and FD, a bZIP transcription factor that activates downstream gene expression. Although 14-3-3 is an important component of FAC, little is known about the function of the 14-3-3 protein itself. Here, we report the results of a high-throughput invitro fluorescence resonance energy transfer (FRET) screening of chemical libraries that enabled us to identify small molecules capable of inhibiting FAC formation. These molecules abrogate the invitro interaction between the 14-3-3 protein and the OsFD1 peptide, a rice (Oryza sativa) FD, by directly binding to the 14-3-3 protein. Treatment with S4, a specific hit molecule, strongly inhibited FAC activity and flowering in duckweed, tuber formation in potato, and branching in rice in a dose-dependent manner. Our results demonstrate that the high-throughput screening approach based on the three-dimensional structure of PPIs is suitable in plants. In this study, we have proposed good candidate compounds for future modification to obtain inhibitors of florigen-dependent processes through inhibition of FAC formation.

Transcription factor StABI5-like 1 binding to the FLOWERING LOCUS T homologs promotes early maturity in potato

Potato (Solanum tuberosum L.) maturity involves several important traits, including the onset of tuberization, flowering, leaf senescence, and the length of the plant life cycle. The timing of flowering and tuberization in potato is mediated by seasonal fluctuations in photoperiod and is thought to be separately controlled by the FLOWERING LOCUS T-like (FT-like) genes SELF-PRUNING 3D (StSP3D) and SELF-PRUNING 6A (StSP6A). However, the biological relationship between these morphological transitions that occur almost synchronously remains unknown. Here, we show that StABI5-like 1 (StABL1), a transcription factor central to abscisic acid (ABA) signaling, is a binding partner of StSP3D and StSP6A, forming an alternative florigen activation complex and alternative tuberigen activation complex in a 14-3-3-dependent manner. Overexpression of StABL1 results in the early initiation of flowering and tuberization as well as a short life cycle. Using genome-wide chromatin immunoprecipitation sequencing and RNA-sequencing, we demonstrate that AGAMOUS-like and GA 2-oxidase 1 genes are regulated by StABL1. Phytohormone profiling indicates an altered gibberellic acid (GA) metabolism and that StABL1-overexpressing plants are insensitive to the inhibitory effect of GA with respect to tuberization. Collectively, our results suggest that StABL1 functions with FT-like genes to promote flowering and tuberization and consequently life cycle length in potato, providing insight into the pleiotropic functioning of the FT gene.

Characterization of Frond and Flower Development and Identification of FT and FD Genes From Duckweed Lemna aequinoctialis Nd.

Duckweeds (Araceae: Lemnoideae) are aquatic monocotyledonous plants that are characterized by their small size, rapid growth, and wide distribution. Developmental processes regulating the formation of their small leaf-like structures, called fronds, and tiny flowers are not well characterized. In many plant species, flowering is promoted by the florigen activation complex, whose major components are florigen FLOWERING LOCUS T (FT) protein and transcription factor FD protein. How this complex is regulated at the molecular level during duckweed flowering is also not well understood. In this study, we characterized the course of developmental changes during frond development and flower formation in Lemna aequinoctialis Nd, a short-day plant. Detailed observations of frond and flower development revealed that cell proliferation in the early stages of frond development is active as can be seen in the separate regions corresponding to two budding pouches in the proximal region of the mother frond. L. aequinoctialis produces two stamens of different lengths with the longer stamen growing more rapidly. Using high-throughput RNA sequencing (RNA-seq) and de novo assembly of transcripts from plants induced to flower, we identified the L. aequinoctialis FT and FD genes, whose products in other angiosperms form a transcriptional complex to promote flowering. We characterized the protein-protein interaction of duckweed FT and FD in yeast and examined the functions of the two gene products by overexpression in Arabidopsis. We found that L. aequinoctialis FTL1 promotes flowering, whereas FTL2 suppresses flowering.

Components and Functional Diversification of Florigen Activation Complexes in Cotton.

In shoot apex cells of rice, a hexameric florigen activation complex (FAC), comprising flowering locus T (FT), 14-3-3 and the basic leucine zipper transcription factor FD, activates downstream target genes and regulates several developmental transitions, including flowering. The allotetraploid cotton (Gossypium hirsutum L.) contains only one FT locus in both of the A- and D-subgenomes. However, there is limited information regarding cotton FACs. Here, we identified a 14-3-3 protein that interacts strongly with GhFT in the cytoplasm and the nuclei, and five FD homoeologous gene pairs were characterized. In vivo, all five GhFD proteins interacted with Gh14-3-3 and GhFT in the nucleus. GhFT, 14-3-3 and all the GhFDs interacted in the nucleus as well, suggesting that they formed a ternary complex. Virus-induced silencing of GhFD1, -2 and -4 in cotton delayed flowering and inhibited the expression of floral meristem identity genes. Silencing GhFD3 strongly decreased lateral root formation, suggesting a function in lateral root development. GhFD overexpression in Arabidopsis and transcriptional activation assays suggested that FACs containing GhFD1 and GhFD2 function mainly in promoting flowering with partial functional redundancy. Moreover, GhFD3 was specifically expressed in lateral root meristems and dominantly activated the transcription of auxin response factor genes, such as ARF19. Thus, the diverse functions of FACs may depend on the recruited GhFD. Creating targeted genetic mutations in the florigen system using Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) genome editing may fine-tune flowering and improve plant architecture.

Identification and functional characterization of two bamboo FD gene homologs having contrasting effects on shoot growth and flowering

Bamboos, member of the family Poaceae, represent many interesting features with respect to their fast and extended vegetative growth, unusual, yet divergent flowering time across species, and impact of sudden, large scale flowering on forest ecology. However, not many studies have been conducted at the molecular level to characterize important genes that regulate vegetative and flowering habit in bamboo. In this study, two bamboo FD genes, BtFD1 and BtFD2, which are members of the florigen activation complex (FAC) have been identified by sequence and phylogenetic analyses. Sequence comparisons identified one important amino acid, which was located in the DNA-binding basic region and was altered between BtFD1 and BtFD2 (Ala146 of BtFD1 vs. Leu100 of BtFD2). Electrophoretic mobility shift assay revealed that this alteration had resulted into ten times higher binding efficiency of BtFD1 than BtFD2 to its target ACGT motif present at the promoter of the APETALA1 gene. Expression analyses in different tissues and seasons indicated the involvement of BtFD1 in flower and vegetative development, while BtFD2 was very lowly expressed throughout all the tissues and conditions studied. Finally, a tenfold increase of the AtAP1 transcript level by p35S::BtFD1 Arabidopsis plants compared to wild type confirms a positively regulatory role of BtFD1 towards flowering. However, constitutive expression of BtFD1 had led to dwarfisms and apparent reduction in the length of flowering stalk and numbers of flowers/plant, whereas no visible phenotype was observed for BtFD2 overexpression. This signifies that timely expression of BtFD1 may be critical to perform its programmed developmental role in planta.

Phosphorylation of OsFD1 by OsCIPK3 promotes the formation of RFT1-containing florigen activation complex for long-day flowering in rice

Heading date is a critical trait that determines the regional adaptability and grain productivity of many crops. Although rice is a facultative short-day plant, its domestication led to the Ghd7-Ehd1-Hd3a/RFT1 pathway for adaptation to long-day conditions (LDs). The formation of the “florigen activation complex” (FAC) containing florigen Hd3a has been characterized. However, the molecular composition of the FAC that contains RFT1 for long-day flowering is unclear. We show here that RFT1 forms a ternary FAC with 14-3-3 proteins and OsFD1 to promote flowering under LDs. We identified a calcineurin B-like-interacting protein kinase, OsCIPK3, which directly interacts with and phosphorylates OsFD1, thereby facilitating the localization of the FAC to the nucleus. Mutation in OsCIPK3 results in a late heading date under LDs but a normal heading date under short-day conditions. Collectively, our results suggest that OsCIPK3 phosphorylates OsFD1 to promote RFT1-containing FAC formation and consequently induce flowering in rice under LDs.

Roles of the 14-3-3 gene family in cotton flowering

BackgroundIn plants, 14-3-3 proteins, also called GENERAL REGULATORY FACTORs (GRFs), encoded by a large multigene family, are involved in protein–protein interactions and play crucial roles in various physiological processes. No genome-wide analysis of the GRF gene family has been performed in cotton, and their functions in flowering are largely unknown.ResultsIn this study, 17, 17, 31, and 17 GRF genes were identified in Gossypium herbaceum, G. arboreum, G. hirsutum, and G. raimondii, respectively, by genome-wide analyses and were designated as GheGRFs, GaGRFs, GhGRFs, and GrGRFs, respectively. A phylogenetic analysis revealed that these proteins were divided into ε and non-ε groups. Gene structural, motif composition, synteny, and duplicated gene analyses of the identified GRF genes provided insights into the evolution of this family in cotton. GhGRF genes exhibited diverse expression patterns in different tissues. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that the GhGRFs interacted with the cotton FLOWERING LOCUS T homologue GhFT in the cytoplasm and nucleus, while they interacted with the basic leucine zipper transcription factor GhFD only in the nucleus. Virus-induced gene silencing in G. hirsutum and transgenic studies in Arabidopsis demonstrated that GhGRF3/6/9/15 repressed flowering and that GhGRF14 promoted flowering.ConclusionsHere, 82 GRF genes were identified in cotton, and their gene and protein features, classification, evolution, and expression patterns were comprehensively and systematically investigated. The GhGRF3/6/9/15 interacted with GhFT and GhFD to form florigen activation complexs that inhibited flowering. However, GhGRF14 interacted with GhFT and GhFD to form florigen activation complex that promoted flowering. The results provide a foundation for further studies on the regulatory mechanisms of flowering.

Characterization and identification of OsFTL8 gene in rice

The rice genome contains 13 Flowering Locus T-Like (FT-Like) genes, among which, OsFTL2/Hd3a and OsFTL3/RFT1 are characterized as florigen-encoding genes, promoting rice flowering under short-day and long-day conditions, respectively. However, the functions of the other FT-Like members are still unknown in rice. In this study, we characterized and identified a member of FT-Like family, OsFTL8 gene. The investigation of photoperiodic expression pattern showed that OsFTL8 transcription was mainly induced under short-day conditions. The qRT-PCR and GUS staining assays indicated that OsFTL8 was broadly expressed in various organs, including stems, leaves and panicles. OsFTL8 overexpression transgenic lines and its knockout mutants displayed no obvious change in their heading date, compared with the wild type. However, the pollen viability and seed setting rate were decreased in the overexpression transgenic lines. OsFTL8 localizes in both nucleus and cytoplasm. And we found that OsFTL8 does not interact with OsFD1 in vivo, suggesting that OsFTL8 may not be the component of florigen activation complex (FAC). In summary, our results indicate that OsFTL8 is associated with pollen viability and not involved in regulating rice flowering time, implying functional divergence of FT-Like members.

Foxtail mosaic virus-induced flowering assays in monocot crops.

Virus-induced flowering (VIF) exploits RNA or DNA viruses to express flowering time genes to induce flowering in plants. Such plant virus-based tools have recently attracted widespread attention for their fundamental and applied uses in flowering physiology and in accelerating breeding in dicotyledonous crops and woody fruit-trees. We now extend this technology to a monocot grass and a cereal crop. Using a Foxtail mosaic virus (FoMV)-based VIF system, dubbed FoMViF, we showed that expression of florigenic Flowering Locus T (FT) genes can promote early flowering and spikelet development in proso millet, a C4 grass species with potential as a nutritional food and biofuel resource, and in non-vernalized C3 wheat, a major food crop worldwide. Floral and spikelet/grain induction in the two monocot plants was caused by the virally expressed untagged or FLAG-tagged FT orthologs, and the florigenic activity of rice Hd3a was more pronounced than its dicotyledonous counterparts in proso millet. The FoMViF system is easy to use and its efficacy to induce flowering and early spikelet/grain production is high. In addition to proso millet and wheat, we envisage that FoMViF will be also applicable to many economically important monocotyledonous food and biofuel crops.

Hd3a and OsFD1 negatively regulate rice resistance to Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola

In rice, Hd3a, GF14 and OsFD1 proteins, forming florigen activation complex, are key components in flowering time regulation. GF14 genes in rice response to biotic and abiotic stress has also been well addressed. The role of GF14e in rice defense has been well studied. However, whether Hd3a and OsFD1 play roles in defense is unclear. In present study, we identified that Hd3a and OsFD1 expression is repressed by Xoo and JA, and validated that Hd3a and OsFD1 negatively regulate resistance to Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc). hd3a and osfd1 mutants increase resistance to Xoo and Xoc, and activate JA responsive genes expression. Our data also demonstrate that OsFD1 binds to the promoters of and activates the expression of JAZ genes; Hd3a, cooperating with GF14e, promotes OsFD1 action on JAZ gene expression. The functional confirmation of Hd3a and OsFD1 in rice defense makes them to be promising targets in molecular rice breeding.

TFL1-Like Proteins in Rice Antagonize Rice FT-Like Protein in Inflorescence Development by Competition for Complex Formation with 14-3-3 and FD.

Hd3a, a rice homolog of FLOWERING LOCUS T (FT), is a florigen that induces flowering. Hd3a forms a ternary 'florigen activation complex' (FAC) with 14-3-3 protein and OsFD1 transcription factor, a rice homolog of FD that induces transcription of OsMADS15, a rice homolog of APETALA1 (AP1), which leads to flowering. TERMINAL FLOWER 1 (TFL1) represses flowering and controls inflorescence architecture. However, the molecular basis for floral repression by TFL1 remains poorly understood. Here we show that RICE CENTRORADIALIS (RCN), rice TFL1-like proteins, compete with Hd3a for 14-3-3 binding. All four RCN genes are predominantly expressed in the vasculature, and RCN proteins are transported to the shoot apex to antagonize florigen activity and regulate inflorescence development. The antagonistic function of RCN to Hd3a is dependent on its 14-3-3 binding activity. Our results suggest a molecular basis for regulation of the balance between florigen FT and anti-florigen TFL1.

A Virus-Induced Assay for Functional Dissection and Analysis of Monocot and Dicot Flowering Time Genes.

Virus-induced flowering (VIF) uses virus vectors to express Flowering Locus T (FT) to induce flowering in plants. This approach has recently attracted wide interest for its practical applications in accelerating breeding in crops and woody fruit trees. However, the insight into VIF and its potential as a powerful tool for dissecting florigenic proteins remained to be elucidated. Here, we describe the mechanism and further applications of Potato virus X (PVX)-based VIF in the short-day Nicotiana tabacum cultivar Maryland Mammoth. Ectopic delivery of Arabidopsis (Arabidopsis thaliana) AtFT by PVX/AtFT did not induce the expression of the endogenous FT ortholog NtFT4; however, it was sufficient to trigger flowering in Maryland Mammoth plants grown under noninductive long-day conditions. Infected tobacco plants developed no systemic symptoms, and the PVX-based VIF did not cause transgenerational flowering. We showed that the PVX-based VIF is a much more rapid method to examine the impacts of single amino acid mutations on AtFT for floral induction than making individual transgenic Arabidopsis lines for each mutation. We also used the PVX-based VIF to demonstrate that adding a His- or FLAG-tag to the N or C terminus of AtFT could affect its florigenic activity and that this system can be applied to assay the function of FT genes from heterologous species, including tomato (Solanum lycopersicum) SFT and rice (Oryza sativa) Hd3a Thus, the PVX-based VIF represents a simple and efficient system to identify individual amino acids that are essential for FT-mediated floral induction and to test the ability of mono- and dicotyledonous FT genes and FT fusion proteins to induce flowering.