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Essay 25.2

FT Protein, not mRNA, is the Phloem-Mobile Signal for Flowering

Jan A. D. Zeevaart, MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI

September, 2007

Introduction

Florigen as a Physiological Concept

Specific flower-inducing substances were first postulated by Julius Sachs (1865), but more convincing evidence had to await the discovery of photoperiodism (Garner and Allard 1920). The seminal finding was that in photoperiodically sensitive plants the day length is perceived by the leaves, whereas flower formation takes place in the shoot apical meristem (Knott 1934). This finding demonstrates that a long-distance signal, called the floral stimulus or florigen (Chailakhyan 1936), moves from an induced leaf to the shoot apex. The floral stimulus can be transmitted from a flowering partner (donor) via a graft union to a non-flowering partner (receptor), as illustrated in Figure 25.29 of the textbook. Furthermore, it was shown that florigen is exchangeable between related species and genera, as well as among different photoperiodic response types (Lang 1965; Zeevaart 1976; Zeevaart 2006). These observations led to the hypothesis that florigen is wide-spread, if not universal, in flowering plants, and that only the conditions that regulate its production vary among the different response types. Although certain physiological characteristics of florigen, such as its movement in the phloem (e.g., King and Zeevaart 1973), could be investigated, its identity remained unknown. Thus, florigen remained a physiological concept rather than a chemical entity.

With the isolation of auxin as the first-identified plant growth hormone in the 1930s and the discoveries of cytokinins and gibberellins in the 1950s, many attempts were made to extract florigen. In the early approaches, it was assumed that like the classical plant hormones, florigen would be a small organic molecule. Extracts prepared from flowering material were tested for flower-promoting activity in vegetative plants. Positive results were reported occasionally, but none of them was reproducible (reviewed in Zeevaart 1976). As a result, skeptics challenged the adequacy of florigen as a single substance causing flowering. Instead, it was proposed that florigen consists of multiple factors and that flowering is induced by a specific ratio of known hormones and metabolites (Bernier 1988; Bernier et al. 1993).

Molecular-Genetic Research on Flowering

With the advent of Arabidopsis as a model plant for molecular-genetic studies, genetic and molecular analyses became popular approaches in studies on flowering. Through mutagenesis, many mutants were isolated in the quantitative long-day plant (LDP) Arabidopsis thaliana. Of interest here are those mutants that exhibit changes in flowering time in comparison with wild-type (WT) plants. Mutants flowering later than WT plants represent a loss-of-function that must involve positive regulators of flowering. Conversely, early-flowering mutants have lost an inhibitor of flowering. These molecular-genetic studies have led to identification of four pathways that regulate flowering in Arabidopsis: the photoperiod, vernalization, autonomous, and GA pathways (e.g., Komeda 2004; Corbesier and Coupland 2005, 2006; Imaizumi and Kay 2006). In the present essay, we will deal mainly with the photoperiod pathway.

The genes CONSTANS (CO) and FLOWERING LOCUS T (FT) are central to LD-induced flowering in Arabidopsis. CO encodes a nuclear zinc-finger protein, which in response to LD induces transcription of FT in the phloem of leaves. Neither CO nor FT is expressed in the shoot apex. Expression of CO from a meristem-specific promoter does not enhance flowering, but early flowering is induced in short days (SD) when FT is overexpressed in the shoot apex. Expression of CO from a phloem-specific promoter is sufficient to generate a phloem-mobile stimulus that induces flowering, as shown by grafting experiments between Arabidopsis donor plants overexpressing CO and co mutant shoots as receptor (An et al. 2004; Ayre and Turgeon 2004). Because FT must act in the shoot apex in order to elicit flowering, this result gives a strong indication that FT or its product is the signal that moves from an induced leaf to the shoot apex and induces flowering.

FT acts in the shoot apex by forming a complex with the bZIP transcription factor FD. The essential role of FD in flowering is demonstrated by the finding that fd mutants flower late and that FT overexpression is partially suppressed by fd (Abe et al. 2005; Wigge et al. 2005). The FT/FD complex activates the downstream genes APETALA1 (AP1) and SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1); the latter, in turn, activates LEAFY (LFY). So, although FT and FD are produced at different sites, they act together in the shoot apex (Figure 1). This finding suggests that the FT gene product has to move from the leaf to the shoot apex and strongly implicates the FT gene product as part of florigen, if not florigen itself.

Figure 1 Multiple developmental pathways for flowering in Arabidopsis: photoperiodism, the autonomous (leaf number) and vernalization (low temperature) pathways, the energy (sucrose) pathway, and the gibberellin pathway. The photoperiodic pathway is located in the leaves and involves the production of a transmissible floral stimulus, FT protein. In LDPs such as Arabidopsis, FT protein is produced in the phloem in response to CO protein accumulation under long days. It is then translocated via sieve tubes to the apical meristem. In SDPs such as rice, the transmissible floral stimulus Hd3a protein accumulates when the repressor protein, Hd1, is not produced under short days, and the Hd3a protein is translocated via the phloem to the apical meristem. In Arabidopsis, FT binds to FD, and the FT/FD protein complex activates the AP1 and SOC1 genes, which trigger LFY gene expression. LFY and AP1 then trigger the expression of the floral homeotic genes. The autonomous (leaf number) and vernalization (low temperature) pathways act in the apical meristem to negatively regulate FLC, a negative regulator of SOC1. The sucrose and gibberellin pathways, also localized to the meristem, promote SOC1 expression. (After Blázquez 2005.) (Click image to enlarge.)

Is the Phloem-Mobile Floral Stimulus FT mRNA or FT Protein?

A substance functioning as florigen must fulfill at least the following criteria:

It must be produced in the leaf, presumably only under inductive conditions for flowering. At least, it would be expected to be more abundant in an induced than in a non-induced leaf.
It must be phloem-mobile, moving from an induced leaf to the shoot apex.
It must be required for flowering in all plants.

From the hierarchy of regulatory proteins that controls flowering, it follows that FT is the terminal gene expressed in the leaf, whereas its effect is in the shoot apex (see above; Corbesier and Coupland 2006). Consequently, three possibilities can be envisaged:

FT mRNA is phloem-mobile and moves from an induced leaf to the shoot apex.
FT protein moves from an induced leaf to the shoot apex.
FT in the leaf controls the synthesis of a small compound in the leaf that then moves to the shoot apex, where it induces FT expression to produce FT protein, which complexes with FD. Results relevant to all three possibilities will be discussed below.

Huang et al. (2005) investigated the possibility that FT mRNA is the mobile stimulus. It was shown in Arabidopsis that FT under control of a heat shock promoter was transiently expressed in a single heated leaf and FT mRNA was detected in the shoot apex by RT-PCR 6 hours later. In addition, the heat-treated leaf did induce flowering. Thus, in this work FT mRNA would seem to fulfill some of the criteria of florigen. However, it was later reported that the real-time RT-PCR data were analyzed incorrectly and that in new experiments the movement of FT mRNA from leaf to shoot apex was not detected. Consequently, “…the conclusion that FT mRNA is part of the floral inductive signal moving from leaf to shoot apex” was retracted (Böhlenius et al. 2007). Other authors also failed to obtain evidence favoring FT mRNA as a mobile floral stimulus. In the case of Arabidopsis and rice, FT mRNA as measured by real-time RT-PCR was lower in shoot apices than in leaves (Corbesier et al. 2007; Tamaki et al. 2007). Also, no FT mRNA could be detected in the shoot apex of Arabidopsis by in situ hybridization (Jaeger and Wigge 2007). In Cucurbita, FT mRNA was not detected either in the phloem sap or in the shoot apex (Lin et al. 2007). Finally, in grafting experiments with tomato SFT transcript of the donors did not move across the graft union to the receptor shoots (Lifschitz et al. 2006; Lin et al. 2007).Thus, a rather extensive body of evidence argues against FT mRNA functioning as florigen, although considering the presence of mRNAs in phloem as components of a long-distance signaling network, the possibility cannot be completely ruled out (Lough and Lucas 2006).

Simultaneously with the retraction of the publication on FT mRNA as a phloem-mobile signal, two new publications reported results that FT protein rather than the mRNA is the mobile signal in the phloem that induces floral initiation in Arabidopsis as well as in the SDP rice (Figure 1). To determine the distribution and movement of FT protein, George Coupland’s team at the Max Planck Institute for Plant Breeding Research, fused FT with the gene encoding GREEN FLUORESCENT PROTEIN (GFP) and expressed this construct in ft mutant plants. Expression from phloem-specific promoters demonstrated the presence of FT:GFP not only in leaf phloem, but also in the shoot apex. FT:GFP transgenic plants also flowered earlier than ft plants. Assuming that the promoters employed in these experiments are specific for expression in phloem, these results demonstrate that the FT protein expressed in the leaf phloem moves to the shoot apex to induce floral initiation (Corbesier et al. 2007). FT and FT:GFP were also expressed from the GALACTINOL SYNTHASE (GAS1) promoter, which acts specifically in the phloem companion cells of the minor veins of leaves. In transgenic plants expressing GAS1:FT:GFP, GFP fluorescence was observed only in the minor veins of leaves. Transgenic GAS1:FT plants flowered early, but GAS1:FT:GFP plants flowered as late as ft plants, although the transgene was active in the leaves, as shown by activation of FRUITFULL (FUL). The authors speculated that the fusion protein was not mobile in the minor veins and as a result did not move to the shoot apex and induce flowering. This result confirms that FT protein is the floral stimulus in Arabidopsis and that no secondary product of FT is involved (Corbesier et al. 2007).

In the SDP rice, Heading date 3a (Hd3a) is the ortholog of FT in Arabidopsis. Under SD conditions, Hd3a shows highest expression in leaf blades of rice. Ko Shimamoto’s group at the Nara Institute of Science and Technology, transformed rice with the Hd3a:GFP construct under control of phloem-specific promoters. The transgenic plants flowered early and GFP fluorescence was observed in the vascular tissues of the leaf blade, stem, and shoot apex. Because the Hd3a:GFP construct was expressed only in the phloem of leaf blades, whereas the protein was detected in the shoot apex, it must be concluded that Hd3a protein is moving in the phloem and that Hd3a functions as florigen in rice (Tamaki et al. 2007).

Additional evidence obtained with Arabidopsis further supports the notion that FT protein moves from an induced leaf to the shoot apex. When expressed from a phloem-specific promoter, an epitope-tagged version of FT induced early flowering; the protein was detected by immunolocalization in the shoot apex. By contrast, when MycFT was targeted to the nucleus (immobilized), it had no effect on flowering and remained localized in the phloem of the leaf, whereas the constitutive 35S promoter did induce early flowering (Jaeger and Wigge 2007). Furthermore, FT expressed ectopically as a large fusion protein promoted flowering. But in the case of a phloem-specific promoter, flowering was promoted only if the FT protein was released from the complex by a specific protease (Mathieu et al. 2007). Thus, export of FT from companion cells was required and correlated with flowering.

In the experimental approaches used in Arabidopsis to demonstrate the movement of FT protein, FT was expressed as a fusion protein in transgenic plants for ready detection by either confocal microscopy or immunolocalization. It should be realized that expression of FT transgenes is usually much higher than that of the native FT gene. Although all the results are consistent with movement of FT protein from induced leaves to the shoot apex, the presence of native FT protein in the phloem translocation stream was not demonstrated in these experiments. In a close relative of Arabidopsis, Brassica napus, FT protein has been identified in phloem exudate of inflorescence stems (Giavalisco et al. 2006), so that it is reasonable to assume that native FT protein is also present in the phloem sap of Arabidopsis.

Because of its small size, Arabidopsis is not a suitable plant for phloem translocation studies. For this purpose, Cucurbita has been a favorite, especially because phloem exudate can be readily collected from cut stems. However, cucurbits are day-neutral and had not been used for flowering studies until Bill Lucas’s laboratory at the University of California, Davis, surveyed 97 cucurbit accessions. They found one, C. moschata (Cmo), that flowered only under SD conditions. The group tested this species, along with day-neutral C. maxima (Cm), to determine whether long-distance movement of FT is required for flowering. They used the Zucchini yellow mosaic virus (ZYMV) as vector for introducing the FT gene of Arabidopsis (AtFT) into Cmo. Infection with this vector caused flowering of Cmo in LD. The virus was present in developing leaves, but not in apical tissues (Lin et al. 2007). This result demonstrates that the function of AtFT as an inducer of flowering is conserved when expressed in Cmo, and further, that flowering was most likely induced by movement of FT protein from virus-infected leaves to apical regions.

To investigate the role of FT-like (FTL) genes in Cucurbita, two orthologs of FT were isolated from both Cm (Cm-FTL1 and Cm-FTL2) and Cmo (Cmo-FTL1 and Cmo-FTL2). Transcripts of FTL genes were restricted to phloem of stems and leaves, but were not detected in phloem sap. By contrast, the FTL proteins were detected in phloem sap by a combination of liquid chromatography-tandem mass spectrometry. Cmo-FTL2 was approximately 10 times more abundant in phloem exudate than was Cmo-FTL1. Both proteins were present only in phloem sap obtained from SD-grown plants. Although transcript and protein levels of Cmo-FTL2 were much up-regulated in the phloem of stems in SD, an important regulatory mechanism by the photoperiod appeared to be entry of FTL proteins into the phloem translocation stream, in addition to transcriptional control (Lin et al. 2007). Finally, Cmo (receptor) was grafted onto Cm (donor) in long days. All receptor shoots were induced to flower and the CmFTL2 protein was identified in phloem sap collected from Cmo scions. These results show convincingly that FTL proteins are present in the phloem translocation stream of Cucurbita and that they can move across a graft union in a heterograft to induce flowering in a vegetative receptor shoot (Lin et al. 2007). This experiment elegantly demonstrates that transmission of florigen from a donor to a receptor plant is associated with transfer of FT protein from donor to receptor.

FT Protein Is the Universal Signal for Flowering

The basic tenet of the florigen hypothesis is that florigen is common to all flowering plants. Physiological evidence for the universality of florigen is based on results of grafting experiments between closely related species in which one response type (e.g., a LDP) induces flowering in a related species of another response type (e.g., a SDP). However, this approach has been limited by graft-incompatibility between unrelated species. With the advent of molecular genetics and plant transformation, this barrier can now be readily overcome. Instead of grafting, a specific gene from one species can be “transplanted” into an unrelated species and its role in flowering demonstrated. For example, the SFT gene of day-neutral tomato can substitute for the LD requirement in Arabidopsis (Lifschitz et al. 2006). Likewise, FT expressed in the SDP C. moschata induces flowering under LD conditions (Lin et al. 2007).

One of the criticisms of the universality of florigen was that there were many examples of grafting experiments in which receptor shoots failed to flower (reviewed in Zeevaart 1976). Does this mean non-identity of florigen in the two grafting partners? Work with tomato provides an answer to this question. Transgenic tomato plants overexpressing SINGLE-FLOWER TRUSS (SFT), an ortholog of FT, under control of the 35S promoter, were excellent donors, but wild-type tomato could not complement sft mutant plants in grafting experiments (Lifschitz et al. 2006). This result suggests that the failure to induce flowering in receptors is not due to non-identity of florigen, but most likely due to a low level of florigen in the donor and/or rapid decay of florigen in the receptor.

The functions of FT orthologs appear highly conserved in flowering plants regardless of response type, and in monocots as well as in dicots. Although the control of expression of FT varies across response types, the end product, FT protein, appears to be always the same. The evidence obtained so far provides strong support for the universality of FT protein as florigen not only in herbaceous plants, but also in trees (Böhlenius et al. 2006; Hsu et al. 2006). In Lolium temulentum, GAs, specifically GA₅ and GA₆, have been assigned a role as florigen (Web Essay 25.1). But it is of interest that also in this species LtFT was strongly up-regulated in the leaves after plants had been shifted from SD to LD (King et al. 2006).

The question is often asked: Why did it take so long to elucidate the molecular nature of florigen? There are several aspects to a complete answer. For many years it was assumed that florigen, like the classical plant hormones, would be a small molecule that could be extracted and re-introduced into test plants. Some physiological evidence indicated that in some species florigen has virus-like properties, but techniques to extract nucleic acid or proteins and apply them to assay plants were not available. So, further progress had to await the application of molecular-genetic techniques to studies on physiology of flowering. It then took considerable time before the various flowering pathways had been worked out in Arabidopsis and the pivotal role of FT in flowering became apparent (e.g., An et al. 2004; Ayre and Turgeon 2004; Abe et al. 2005; Wigge et al. 2005; Imaizumi and Kay 2006). Rather than applying extracts to test plants, transgenes could now be expressed and their products, mRNA and protein, could be visualized by techniques of cell biology, or identified by mass spectrometry. It took 70 years, but finally florigen has been identified as FT, a mobile protein of approximately 20 kDa.

Perspective

With the identification of FT protein as florigen, questions regarding its production, transport, and persistence can be studied at the molecular level. For example, is FT permanently activated in plants with localized induction? Does FT induce its own production via a positive feedback loop in species that exhibit the phenomenon of indirect induction of flowering? (see textbook pp. 661–662). These intriguing phenomena, as well as other classical observations on the physiology of flowering, can now be studied from a molecular-genetic perspective. It is expected that results of further studies will provide a solid underpinning for the florigen theory.

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