Florigen trafficking integrates photoperiod and temperature signals in Arabidopsis

The transition to flowering is the most dramatic phase change in flowering plants and is crucial for reproductive success. A complex regulatory network in plants has evolved to perceive and integrate the endogenous and environmental signals. These signals perceived, including day length and temperature, converge to regulate FLOWERING LOCUS T (FT), which encodes a mobile stimulus required for floral induction in Arabidopsis. Despite the discovery of modulation of FT messenger RNA (mRNA) expression by ambient temperature, whether the trafficking of FT protein is controlled in response to changes in growth temperature is so far unknown. Here, we show that FT transport from companion cells to sieve elements is controlled in a temperature‐dependent manner. This process is mediated by multiple C2 domain and transmembrane region proteins (MCTPs) and a soluble N‐ethylmaleimide‐sensitive factor protein attachment protein receptor (SNARE). Our findings suggest that ambient temperatures regulate both FT mRNA expression and FT protein trafficking to prevent precocious flowering at low temperatures and ensure plant reproductive success under favorable environmental conditions.     

Proteins from the FLOWERING LOCUS T‐like subclade of the PEBP family act antagonistically to regulate floral initiation in tobacco

Flowering is an important agronomic trait that often depends on the integration of photoperiod, vernalization, gibberellin and/or autonomous signaling pathways by regulatory proteins such as FLOWERING LOCUS T (FT), a member of the phosphatidylethanolamine‐binding protein (PEBP) family. Six PEBP family proteins control flowering in the model plant Arabidopsis thaliana, and their regulatory functions are well established, but variation in the number and structural diversity of PEBPs in different species means their precise functions must be determined on a case‐by‐case basis. We isolated four novel FT‐like genes from Nicotiana tabacum (tobacco), and determined their expression profiles in wild‐type plants and their overexpression phenotypes in transgenic plants. We found that all four genes were expressed in leaves under short‐day conditions, and at least NtFT3 expression was restricted to phloem companion cells. We also found that the NtFT1, NtFT2 and NtFT3 proteins are floral inhibitors (atypical for FT‐like proteins), whereas only NtFT4 is a floral inducer. We were unable to detect the expression of these genes under long‐day conditions, suggesting that all four tobacco FT‐like proteins may control flowering in response to short days. Phylogenetic analysis of PEBP family proteins and their functions in different solanaceous species confirmed that gene duplication and divergence within the FT‐like clade has led to the evolution of antagonistic regulators that may help to fine‐tune floral initiation in response to environmental cues.     

Arabidopsis thaliana CENTRORADIALIS homologue (ATC) acts systemically to inhibit floral initiation in Arabidopsis

Floral initiation is orchestrated by systemic floral activators and inhibitors. This remote‐control system may integrate environmental cues to modulate floral initiation. Recently, FLOWERING LOCUS T (FT) was found to be a florigen. However, the identity of systemic floral inhibitor or anti‐florigen remains to be elucidated. Here we show that Arabidopsis thaliana CENTRORADIALIS homologue (ATC), an Arabidopsis FT homologue, may act in a non‐cell autonomous manner to inhibit floral initiation. Analysis of the ATC null mutant revealed that ATC is a short‐day‐induced floral inhibitor. Cell type‐specific expression showed that companion cells and apex that express ATC are sufficient to inhibit floral initiation. Histochemical analysis showed that the promoter activity of ATC was mainly found in vasculature but under the detection limit in apex, a finding that suggests that ATC may move from the vasculature to the apex to influence flowering. Consistent with this notion, Arabidopsis seedling grafting experiments demonstrated that ATC moved over a long distance and that floral inhibition by ATC is graft transmissible. ATC probably antagonizes FT activity, because both ATC and FT interact with FD and affect the same downstream meristem identity genes APETALA1, in an opposite manner. Thus, photoperiodic variations may trigger functionally opposite FT homologues to systemically influence floral initiation.     

Zebularine treatment is associated with deletion of FT‐B1 leading to an increase in spikelet number in bread wheat

The number of rachis nodes (spikelets) on a wheat spike is a component of grain yield that correlates with flowering time. The genetic basis regulating flowering in cereals is well understood, but there are reports that flowering time can be modified at a high frequency by selective breeding, suggesting that it may be regulated by both epigenetic and genetic mechanisms. We investigated the role of DNA methylation in regulating spikelet number and flowering time by treating a semi‐spring wheat with the demethylating agent, Zebularine. Three lines with a heritable increase in spikelet number were identified. The molecular basis for increased spikelet number was not determined in 2 lines, but the phenotype showed non‐Mendelian inheritance, suggesting that it could have an epigenetic basis. In the remaining line, the increased spikelet phenotype behaved as a Mendelian recessive trait and late flowering was associated with a deletion encompassing the floral promoter, FT‐B1. Deletion of FT‐B1 delayed the transition to reproductive growth, extended the duration of spike development, and increased spikelet number under different temperature regimes and photoperiod. Transiently disrupting DNA methylation can generate novel flowering behaviour in wheat, but these changes may not be sufficiently stable for use in breeding programs.     

Genetic interactions reveal the antagonistic roles of FT/TSF and TFL1 in the determination of inflorescence meristem identity in Arabidopsis

During the transition to the reproductive phase, the shoot apical meristem switches from the developmental program that generates vegetative organs to instead produce flowers. In this study, we examined the genetic interactions of FLOWERING LOCUS T (FT)/TWIN SISTER OF FT (TSF) and TERMINAL FLOWER 1 (TFL1) in the determination of inflorescence meristem identity in Arabidopsis thaliana. The ft‐10 tsf‐1 mutants produced a compact inflorescence surrounded by serrated leaves (hyper‐vegetative shoot) at the early bolting stage, as did plants overexpressing TFL1. Plants overexpressing FT or TSF (or both FT and TFL1) generated a terminal flower, as did tfl1‐20 mutants. The terminal flower formed in tfl1‐20 mutants converted to a hyper‐vegetative shoot in ft‐10 tsf‐1 mutants. Grafting ft‐10 tsf‐1 or ft‐10 tsf‐1 tfl1‐20 mutant scions to 35S::FT rootstock plants produced a normal inflorescence and a terminal flower in the scion plants, respectively, although both scions showed similar early flowering. Misexpression of FT in the vasculature and in the shoot apex in wild‐type plants generated a normal inflorescence and a terminal flower, respectively. By contrast, in ft‐10 tsf‐1 mutants the vasculature‐specific misexpression of FT converted the hyper‐vegetative shoot to a normal inflorescence, and in the ft‐10 tsf‐1 tfl1‐20 mutants converted the shoot to a terminal flower. TFL1 levels did not affect the inflorescence morphology caused by FT/TSF overexpression at the early bolting stage. Taking these results together, we proposed that FT/TSF and TFL1 play antagonistic roles in the determination of inflorescence meristem identity, and that FT/TSF are more important than TFL1 in this process.     

Gibberellic acid signaling is required for ambient temperature‐mediated induction of flowering in Arabidopsis thaliana

Distinct molecular mechanisms integrate changes in ambient temperature into the genetic pathways that govern flowering time in Arabidopsis thaliana. Temperature‐dependent eviction of the histone variant H2A.Z from nucleosomes has been suggested to facilitate the expression of FT by PIF4 at elevated ambient temperatures. Here we show that, in addition to PIF4, PIF3 and PIF5, but not PIF1 and PIF6, can promote flowering when expressed specifically in phloem companion cells (PCC), where they can induce FT and its close paralog, TSF. However, despite their strong potential to promote flowering, genetic analyses suggest that the PIF genes seem to have only a minor role in adjusting flowering in response to photoperiod or high ambient temperature. In addition, loss of PIF function only partially suppressed the early flowering phenotype and FT expression of the arp6 mutant, which is defective in H2A.Z deposition. In contrast, the chemical inhibition of gibberellic acid (GA) biosynthesis resulted in a strong attenuation of early flowering and FT expression in arp6. Furthermore, GA was able to induce flowering at low temperature (15°C) independently of FT, TSF, and the PIF genes, probably directly at the shoot apical meristem. Together, our results suggest that the timing of the floral transition in response to ambient temperature is more complex than previously thought and that GA signaling might play a crucial role in this process.     

Natural variation involving deletion alleles of FRIGIDA modulate temperature‐sensitive flowering responses in Arabidopsis thaliana

Ambient temperature is one of the major environmental factors that modulate plant growth and development. There is extensive natural genetic variation in thermal responses of plants exemplified by the variation exhibited by the accessions of Arabidopsis thaliana. In this work we have studied the enhanced temperature response in hypocotyl elongation and flowering shown by the Tsu‐0 accession in long days. Genetic mapping in the Col‐0 × Tsu‐0 recombinant inbred line (RIL) population identified several QTLs for thermal response including three major effect loci encompassing candidate genes FRIGIDA (FRI), FLOWERING LOCUS C (FLC) and FLOWERING LOCUS T (FT). We confirm and validate these QTLs. We show that the Tsu‐0 FRI allele, which is the same as FRI‐Ler is associated with late flowering but only at lower temperatures in long days. Using transgenic lines and accessions, we show that the FRI‐Ler allele confers temperature‐sensitive late flowering confirming a role for FRI in photoperiod‐dependent thermal response. Through quantitative complementation with heterogeneous inbred families, we further show that cis‐regulatory variation at FT contributes to the observed hypersensitivity of Tsu‐0 to ambient temperature. Overall our results suggest that multiple loci that interact epistatically govern photoperiod‐dependent thermal responses of A. thaliana.     

Molecular cloning and functional analysis of the FLOWERING LOCUS T (FT) homolog GhFT1 from Gossypium hirsutum

FLOWERING LOCUS T (FT) encodes a member of the phosphatidylethanolamine‐binding protein (PEBP) family that functions as the mobile floral signal, playing an important role in regulating the floral transition in angiosperms. We isolated an FT‐homolog (GhFT1) from Gossypium hirsutum L. cultivar, Xinluzao 33 GhFT1 was predominantly expressed in stamens and sepals, and had a relatively higher expression level during the initiation stage of fiber development. GhFT1 mRNA displayed diurnal oscillations in both long‐day and short‐day condition, suggesting that the expression of this gene may be under the control of the circadian clock. Subcellular analysis revealed that GhFT1 protein located in the cytoplasm and nucleus. Ectopic expression of GhFT1 in transgenic arabidopsis plants resulted in early flowering compared with wild‐type plants. In addition, ectopic expression of GhFT1 in arabidopsis ft‐10 mutants partially rescued the extremely late flowering phenotype. Finally, several flowering related genes functioning downstream of AtFT were highly upregulated in the 35S::GhFT1 transgenic arabidopsis plants. In summary, GhFT1 is an FT‐homologous gene in cotton that regulates flower transition similar to its orthologs in other plant species and thus it may be a candidate target for promoting early maturation in cotton breeding.     

Cytokinin promotes flowering of Arabidopsis via transcriptional activation of the FT paralogue TSF

Cytokinins are involved in many aspects of plant growth and development, and physiological evidence also indicates that they have a role in floral transition. In order to integrate these phytohormones into the current knowledge of genetically defined molecular pathways to flowering, we performed exogenous treatments of adult wild type and mutant Arabidopsis plants, and analysed the expression of candidate genes. We used a hydroponic system that enables synchronous growth and flowering of Arabidopsis, and allows the precise application of chemicals to the roots for defined periods of time. We show that the application of N⁶‐benzylaminopurine (BAP) promotes flowering of plants grown in non‐inductive short days. The response to cytokinin treatment does not require FLOWERING LOCUS T (FT), but activates its paralogue TWIN SISTER OF FT (TSF), as well as FD, which encodes a partner protein of TSF, and the downstream gene SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1). Treatment of selected mutants confirmed that TSF and SOC1 are necessary for the flowering response to BAP, whereas the activation cascade might partially act independently of FD. These experiments provide a mechanistic basis for the role of cytokinins in flowering, and demonstrate that the redundant genes FT and TSF are differently regulated by distinct floral‐inducing signals.