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Leaf-produced floral signals
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Leaf-produced floral signals Jan AD Zeevaart Florigen is the hypothetical leaf-produced signal that induces floral initiation at the shoot apex. The nature of florigen has remained elusive for more than 70 years. But recent progress toward understanding the regulatory network for flowering in Arabidopsis has led to the suggestion that FLOWERING LOCUS T (FT) or its product is the mobile flower-inducing signal that moves from an induced leaf through the phloem to the shoot apex. In the past year, physical and chemical evidence has shown that it is FT protein, and not FT mRNA, that moves from induced leaves to the apical meristem. These results have established that FT is the main, if not the only, component of the universal florigen. Address MSU-DOE Plant Research Laboratory and Department of Plant Biology, Michigan State University, East Lansing, MI, USA Corresponding author: Zeevaart, Jan AD ([email protected])
Current Opinion in Plant Biology 2008, 11:541–547 This review comes from a themed issue on Cell Signalling and Gene Regulation Edited by Jason Reed and Bonnie Bartel Available online 6th August 2008 1369-5266/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. DOI 10.1016/j.pbi.2008.06.009
Introduction The concept of long-distance signaling in flowering was first introduced by Knott [1]. He observed that flowering in the long-day plant (LDP) spinach took place when the leaves were in long-day conditions (LD) and the shoot tips in short days (SD), but not when the leaves were in SD and the tips in LD. Knott [1] concluded that ‘‘the part played by the foliage of spinach in hastening the response to a photoperiod favorable to reproductive growth may be in the production of some substance, or stimulus, that is transported to the growing point’’. Chailakhyan [2] extended these observations to other species and coined the term ‘florigen’ (flower-former) for the photoperiodic stimulus, which is perceived in the leaves and transmitted to the shoot apex. The stimulus is graft transmissible from an induced partner (donor) to a non-induced shoot (receptor) and between different species and genera, as well as between different photoperiodic response types [3,4]. It appeared, therefore, that florigen is universal in flowering plants. Despite abundant physiological evidence for a transmissible stimulus, its identity remained unknown for www.sciencedirect.com
70 years, so florigen became known as the ‘Holy Grail’ of the physiology of flowering. Further progress had to await the development of a regulatory network for flowering in the quantitative LDP Arabidopsis thaliana. With the convergence of classical physiological studies and moleculargenetic approaches, the search for florigen could be pursued from a new perspective. In this review, I will summarize the recent work that led to the identification of FLOWERING LOCUS T (FT) protein as the main, if not the only, component of florigen. Reviews on integration of the different genes in a signaling network for flowering [5–7] and on the phloem-mobile floral stimulus [8,9,10] have appeared recently.
Role of the CO–FT pathway in flowering In Arabidopsis, a network of four interacting pathways regulates floral induction. In the photoperiod-dependent pathway, CONSTANS (CO) and FT are the key elements that mediate the effect of daylength on flowering. Both genes are expressed in phloem companion cells of leaves. In LD, expression of CO is upregulated and its protein stabilized, which in turn induces expression of FT. Ectopic expression of CO causes early flowering, but not when expressed from a shoot meristem-specific promoter. By contrast, overexpression of FT in the shoot apex alone induces early flowering, as does expression of CO from companion-cell-specific promoters. One explanation is that CO expressed in leaf phloem regulates synthesis of a phloem-mobile signal that induces flowering [11]. This possibility was further substantiated by results of grafting experiments with plants expressing CO under control of the GALACTINOL SYNTHASE 1 (GAS1) promoter, which is restricted to the companion cells of the minor veins. These GAS1::CO donor plants induced early flowering in co-1 receptor shoots [12]. Although produced in the phloem of leaves, FT acts in the shoot apex, where it forms a complex with the bZIP transcription factor FLOWERING LOCUS D (FD). The FD/FT heterodimer promotes the transition to flowering by activating SUPPRESSION OF OVEREXPRESSION OF CO 1 (SOC1) and the floral meristem identity gene APETALA1 (AP1) (Figure 1) [13,14]. It is clear from these results that the site of production of FT mRNA in the leaf is remote from the site of FT action in the shoot apex. Thus, FT (mRNA or protein) or its product is a prime candidate for the phloem-mobile florigen signal.
FT mRNA does not move in the phloem Many mRNAs move in the phloem over long distances [15,16], so it was logical to explore whether FT mRNA can move from an induced leaf to the shoot apex and thus function as the floral stimulus. Huang et al. [17] reported Current Opinion in Plant Biology 2008, 11:541–547
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Figure 1
considered it unlikely that Hd3a mRNA moved from leaves to the shoot apex. In addition, activity of the GUS transgene driven by the Hd3a promoter was detected in the vascular bundles, but not in the shoot apex [21]. Myc-FT mRNA expressed from a companion-cell-specific promoter could be detected in the phloem by in situ hybridization, but there was no expression in the shoot apex [22]. In a novel approach, Mathieu et al. [23] employed artificial microRNAs (amiRNAs) for tissuespecific inactivation of FT mRNA. Expression of amirFT under control of the 35S or SUC2 promoter delayed flowering to the same extent as in ft-10 mutant plants. By contrast, when expression of amir-FT was driven by the shoot apex-specific FD promoter, flowering was not delayed. These results demonstrate that FT mRNA is necessary in the companion cells in order to flower, but is not required in the regions of the shoot apex where FD is expressed [13,14]. Finally, FLOWERING LOCUS T-LIKE (FTL) mRNA was undetectable by real-time RT-PCR in the phloem sap of flowering Cucurbita [24]. Collectively, none of these results provides any evidence that FT mRNA functions as the phloem-mobile signal for floral induction.
FT protein is phloem-mobile FT protein as a transmissible signal for flowering. In long days, CO protein accumulates in the leaves and induces expression of FT in the phloem companion cells. FT protein is transported in the sieve tubes to the shoot apex, where it forms a heterodimer with FD. The FD/FT complex activates expression of SOC1 and AP1, which leads to floral initiation. FM, floral meristem; LP, leaf primordium. Adapted from [10].
that a single heated leaf of a transgenic plant expressing FT under control of a heat shock promoter induced flowering in the Arabidopsis ft-7 mutant in SD and that FT mRNA was detected in the shoot apex by RT-PCR. However, this work was later retracted [18]. Several later reports failed to provide evidence that FT mRNA is mobile in the phloem. In grafting experiments with tomato overexpressing SINGLE-FLOWER TRUSS (SFT, ortholog of FT in tomato), the floral stimulus did cross the graft union, but SFT mRNA could not be detected in the flowering receptor shoots [19]. In Arabidopsis, a gene fusion of FT and GREEN FLUORESCENT PROTEIN (GFP) was expressed under control of the SUCROSE TRANSPORTER 2 (SUC2) promoter, which is expressed in the phloem companion cells. FT-GFP mRNA was detected by in situ hybridization in mature phloem, but no mRNA was detected in the protophloem or shoot apex. FT-GFP mRNA also failed to move across a graft union to ft-7 mutant receptor shoots [20]. In the short-day plant (SDP) rice, under inductive conditions for flowering, Heading-date 3a (Hd3a, ortholog of FT) mRNA accumulated in the leaf blades, but levels remained very low in the shoot apex, so the authors Current Opinion in Plant Biology 2008, 11:541–547
FT mRNA and its protein are of low abundance, so that most attempts to demonstrate localization and movement of FT were conducted with transgenic plants expressing FT as a fusion protein with GFP or Myc from a companion-cell-specific promoter. Soluble cytoplasmic proteins fused to GFP can non-specifically move from the companion cells into sieve elements, but none of the GFP fusions moved beyond the protophloem in the root [25]. Consequently, if there is specific control over entry of FT into the sieve elements, distribution of FT-GFP may not reflect the movement of native FT. One should keep this caveat in mind when evaluating experimental data claiming to demonstrate movement of FT-GFP fusions from leaves to shoot apices. Perhaps the best evidence that the fusion proteins entered the shoot apex was that mobile FT-GFP fusion proteins induced early flowering. The first experiments on movement of FT-GFP from leaves to shoot apices were conducted with Arabidopsis and rice [20,21]. Expression of SUC2::FT-GFP in the ft-7 mutant of Arabidopsis resulted in early flowering. Whereas FT-GFP mRNA was restricted to mature phloem, FT-GFP protein was also present in the protophloem and at the base of the shoot apex. Movement of FT-GFP over longer distances was observed in grafting experiments from transgenic donors expressing SUC2::FT-GFP to ft-7 receptors. The receptors flowered earlier and FT-GFP was detected in their vascular bundles, thus establishing movement of FT-GFP from donor www.sciencedirect.com
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to receptor [20]. Movement of Hd3a-GFP was also observed in rice when expressed under control of phloem-specific promoters. The transgenic plants flowered early and fluorescence was observed in the vascular tissue ending below the apex and in the region below the apex. These observations indicate that Hd3a-GFP in rice moves from source companion cells to the shoot apex [21]. Transgenic Arabidopsis plants expressing GAS1::FT also flowered early. By contrast, GAS1::FTGFP plants flowered very late, although fluorescence was observed in the minor veins of the leaves. Corbesier et al. [20] speculated that FT-GFP was too large for movement in the minor veins, with the consequence that it could not reach the shoot apex and induce flowering. These observations further imply that FT does not produce a small, mobile molecule, but that FT itself is the mobile signal. Epitope-tagged FT expressed from the SUC2 promoter induced early flowering in Arabidopsis. As detected by immunolocalization, Myc-FT was not only present in the phloem, but had moved beyond the ends of the vascular strands into the areas of floral initiation. When FT was targeted to the nucleus and thus immobilized, flowering was not promoted [22]. Expression of FT as a large fusion protein (>112 kDa) from the SUC2 promoter did not affect flowering, presumably because the protein was too big to move from the companion cells into the sieve elements. Ectopic expression of the same construct did promote flowering, showing that FT as part of the large fusion protein did retain its activity. Release of FT protein from the complex with a specific protease again promoted flowering [23]. All of these results are consistent with the notion that FT must be phloem-mobile to induce flowering. All results implying the movement of FT from leaves to shoot apices were obtained with transgenic plants expressing FT fusion proteins under control of companion-cellspecific promoters. However, in none of the results discussed so far was the presence of native FT protein in the phloem stream demonstrated. For this purpose, dayneutral Cucurbita maxima (Cm) and the SDP C. moschata (Cmo) are suitable, because phloem exudate can be readily collected from cut stems. Conservation of the function of FT in flowering was demonstrated by using zucchini yellow mosaic virus (ZYMV) to overexpress FT from Arabidopsis in Cmo under LD conditions. The virus was restricted to mature and developing leaves, but very effectively induced Cmo plants to flower under LD, indicating movement of FT from infected leaves to the shoot apex. Conversely, the constructs SUC2::CmFTLIKE (FTL)1 and -2 induced early flowering in Arabidopsis. Thus, the function of FT as an inducer of flowering was conserved in Arabidopsis and cucurbits [24]. The CmoFTL genes were expressed in the phloem of stems and leaves regardless of photoperiod, but the www.sciencedirect.com
proteins were detected only in the phloem sap of plants in SD. It was concluded, therefore, that entry of the CmoFTL proteins into the phloem stream is regulated by the photoperiod and not by transcription [24]. In grafting experiments, dayneutral Cm stocks induced flowering in the SDP Cmo scions under LD. In these graft combinations, CmTFL2 protein was identified by mass spectrometry in phloem sap collected from the Cmo receptor scions. Thus, transmission of florigen in a classical grafting experiment was correlated with transport of the native CmFTL2 protein from donor to receptor [24]. This is the first example of identification and movement of native FT-like protein in phloem sap in connection with induction of flowering.
Is FT protein the universal signal for flowering? FT protein fulfills most of the criteria of florigen. It moves from induced leaves to the shoot apex and across graft unions from donor to receptor to induce flowering. One of the expectations – extraction of florigen from flowering plants and induction of flowering in vegetative plants with applied extracts – cannot be fulfilled owing to the proteinaceous nature of FT. A basic assumption of the florigen hypothesis is that florigen is a universal signal for flowering. Results of interspecific grafting experiments and of grafts between different photoperiodic response types support this idea. But grafting has been limited by incompatibility between unrelated species and by the fact that monocots are not amenable to grafting. However, these barriers can now be overcome by ‘transplanting’ the FT gene (and its orthologs) into unrelated species. Invariably, overexpression of FT-like genes has led to premature flowering under non-inductive conditions in a variety of species. The following donor species of FT or orthologs caused early flowering in Arabidopsis as the recipient species: Arabidopsis [13,26,27], rice [28], tomato [19], Cucurbita maxima [24], Pharbitis nil [29], Populus deltoides [30], and Vitis vinifera [31,32]. Examples of species in which premature flowering was induced by ectopic expression of FT or an FT ortholog are tomato, dayneutral tobacco, the SDP Maryland Mammoth tobacco [19], the SDP Pharbitis nil [29], as well as winter wheat [33]. In trifoliate orange [34] and Populus spp. [30,35] constitutive expression of FT drastically shortened the juvenile phase. Conversely, when the function of FT was downregulated by mutation, RNAi or miRNA, flowering was much delayed in Arabidopsis and rice [26,28,36]. These results indicate that FT and its orthologs are essential for flowering and, further, that the functions of FT and its orthologs are highly conserved among unrelated species. However, there are examples of grafting experiments in which the receptor failed to flower [4]. A case in point is wild-type tomato as donor for the sft mutant, but when transgenic donor plants were used, in which SFT was ectopically expressed, the receptor shoots did flower Current Opinion in Plant Biology 2008, 11:541–547
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[19]. This result demonstrates that an increased level of SFT transcript makes the transgenic plant an effective donor. Clearly, the argument that failure to induce flowering via a graft union is due to different floral stimuli in donor and receptor is no longer tenable. In Arabidopsis and rice, FT and Hd3a, respectively, are strongly upregulated under inductive conditions [20,21,28]. Although expression of CmoFTL2 in C. moschata is also upregulated under inductive conditions, photoperiodic regulation of the function of FTL2 is at entry from the companion cells into the phloem stream and not at the transcriptional level [24]. Clearly, the regulation of FT production and mobility may vary among species, but the end product, FT, is always the same. Thus, on the basis of the currently available evidence, a strong case can be made that FT protein is the universal florigen. But the question remains: Is FT the only component of florigen that moves from leaves to the shoot apex, where it induces transition to flowering? FT protein in the phloem stream may require a chaperone-like protein for protection against proteolysis and unloading at the shoot apex [15]. If a protein were identified that was invariably associated with FT in the phloem and that was also essential for flowering (a mutation in the gene encoding this hypothetical protein would result in a late-flowering phenotype), it would have to be considered a component of florigen. That question remains to be answered by future research. For now, it can be concluded that FT is a major, if not the only component of florigen.
FT is also the product of vernalization In Arabidopsis, FT is not only the output of the photoperiodic pathway, but also of the autonomous and vernalization pathways. These three pathways converge, therefore, through the FT integrator. In cold-requiring accessions of Arabidopsis, the MADS-box transcriptional regulator FLOWERING LOCUS C (FLC) represses expression of FT in the leaf and of FD and SOC1 in the shoot apex [37]. The effect of vernalization is downregulation of FLC, thus allowing FT to be expressed in the leaves and increasing the competence of the shoot apex to respond to FT. Overexpression of FT in nonvernalized Arabidopsis promoted early flowering without affecting FLC mRNA levels, indicating that FT acts downstream of FLC [27]. FLC has not been identified outside the Cruciferae [38]. Therefore, the question arises: Does vernalization in other species also involve downregulation of a repressor? Results of recent work on vernalization in winter varieties of barley and wheat indicate that cold acts by a mechanism similar to that in Arabidopsis. In these cereals, three genes, VERNALIZATION (VRN)1, VRN2, and VRN3 control vernalization requirements [39] (In barley and wheat, VRN1 and VRN2 are different from VRN1 and VRN2 of Current Opinion in Plant Biology 2008, 11:541–547
Arabidopsis [5]). In winter varieties, VRN2 is a repressor of flowering, which is downregulated by vernalization, with a concomitant upregulation of VRN1, an ortholog of the floral meristem identity gene AP1 in Arabidopsis [40,41]. In spring varieties that do not require vernalization, VRN1 is always strongly expressed. VRN2 is also downregulated by a prolonged period of SD, but in this case VRN1 is not upregulated until the plants are transferred to LD [42]. In vernalized plants in LD, repression of VRN2 also allows upregulation of VRN3, an ortholog of FT, which further enhances expression of VRN1. Winter wheat transformed with FT/VRN3 can bypass the block in flowering imposed by VRN2 [33], demonstrating that expression of FT is necessary and sufficient for flowering in winter varieties of barley and wheat, as it is in Arabidopsis [27]. So, in both Arabidopsis and winter cereals, vernalization involves the removal of a flower repressor, which then permits upregulation of FT/VRN3 under LD. FLC and VRN2 belong to different families of transcription factors, but they have the same function [41]. Clearly, in cold-requiring cereals, as in Arabidopsis, responses to vernalization and photoperiodism are integrated through the FT protein. In common with photoperiodic induction of flowering, vernalization also results in a floral stimulus that can be transmitted across a graft union and induce flowering in plants kept in the vegetative state [3,43]. Because the donor plants were flowering, the signal transmitted was most probably the final product of thermo-induction and photo-induction, that is, florigen, now known to be FT. There is no evidence for a separate transmissible stimulus specific for vernalization, with one exception. The SDP Maryland Mammoth tobacco as donor induced flower formation in non-vernalized biennial Hyoscyamus niger not only in SD when induced to flower, but also in LD when the donor remained vegetative. This result was interpreted as vernalization giving rise to a transmissible stimulus, called ‘vernalin’, which would be required for florigen production. Annual tobacco would provide ‘vernalin’ (always present in annual plants), so that biennial Hyoscyamus in LD could then produce florigen. Vernalization is a prerequisite for LD induction of flowering, so it was postulated that in a physiological sense vernalin is the precursor of florigen [3,43]. Although this experiment was reproducible, it was observed that the non-induced Maryland Mammoth tobacco caused considerable stem elongation in the biennial Hyoscyamus before flower buds appeared ([44]; JAD Zeevaart, unpublished results). This is reminiscent of gibberellin (GA)-treated biennial Hyoscyamus niger plants in which much stem elongation preceded appearance of flower buds [43,45]. It is likely, therefore, that the non-induced tobacco shoot supplied GA, thus bypassing the vernalization requirement. Consequently, the single experimental result in favor of ‘vernalin’ can be interpreted differently, and there is no reason to propose any longer a mobile stimulus ‘vernalin’ as the product of vernalization. Besides, in the www.sciencedirect.com
Leaf-produced floral signals Zeevaart 545
context of our current understanding of vernalization, it is hard to visualize any gene product other than FT being phloem-mobile.
FT and gibberellin act independently in flowering Arabidopsis is a quantitative LDP, so floral initiation will ultimately take place in SD. This transition to flowering in SD is predominantly mediated by the GA-dependent pathway. Applied GA in SD induces expression of SOC1 and the floral meristem identity gene LEAFY (LFY), but has no effect on FT expression [46,47]. Constitutive expression of FT in the GA-deficient ga1-3 mutant in SD induced very early flowering. These results suggest that GA and FT exert their effects on flowering in Arabidopsis independently. Analysis of GAs in Arabidopsis showed large increases in GA4 in the shoot apex shortly before floral initiation in SD. This increase in GA4 was accompanied by an increase in LFY transcript and could not be explained by in situ GA biosynthesis, so it was speculated that GAs produced in the leaves were transported to the shoot apex [48]. GAs as florigenic signals have been extensively studied in the LDP Lolium temulentum. In this plant, GA5 and GA6 are signals produced in the leaf and transported to the shoot apex, where they cause floral initiation [49,50]. Besides increasing GA biosynthesis in the leaves, LD also induced a large increase in FT transcript in Lolium, but this increase occurred independently of GA [50]. These observations in Arabidopsis and Lolium suggest that GAs and FT are functionally redundant; they are both leaf-produced mobile signals that can induce flowering [48,50]. But the question arises: How general is GA-induced flowering? Applied GA (usually GA3) induces flowering in many, but not all, rosette LDPs and does not induce flowering in SDP. LFY, the target of GA, is conserved in plants [51]. It is puzzling, therefore, that applied GA fails to cause flowering in many species. Two possible reasons come to mind: The ‘wrong’ GA was applied, or the regulatory region for the GA-response site of the LFY promoter has mutated, so that LFY cannot be activated by GA. At present, it is clear that FT as an inducer of flowering is highly conserved, but it remains to be established that this is also the case for GAs.
Conclusions and perspectives With the identification of FT as the mobile flower-inducing signal, its production, transport, and action can now be explored in more detail. FT is a 20-kDa protein, which is below the selective exclusion limit of 67 kDa of companion cell plasmodesmata [25], so that it should readily diffuse into the phloem stream. However, the results with C. moschata [24] show that, at least in this species, there is tight control over exit of FT from the companion cells. Mechanisms involved in FT protein movement and its targeted release from the protophloem and movement www.sciencedirect.com
from cell to cell in the apex require further studies at the molecular and cellular levels. The molecular mechanisms involved in induction of flowering – first analyzed in Arabidopsis – appear to be conserved in other species; at least the end product, FT protein, is the same. The effects of vernalization in Arabidopsis and winter cereals involve downregulation of a repressor, followed by upregulation of FT expression. How generally this model of vernalization is valid for cold-requiring dicotyledonous plants other than Arabidopsis remains to be established. It is of interest that SD followed by LD can substitute for vernalization in winter wheat [42]. This raises the possibility that in species with a dual daylength requirement for flowering (short-longday plants and long-short-day plants [52]) the first daylength requirement also involves downregulation of a repressor, followed by FT expression under the second daylength requirement. Indirect induction [4] is an intriguing phenomenon that remains to be explained in molecular terms. One possibility is that FT induces its own production via a positive feedback loop. In the case of irreversible induction in Perilla, does FT remain activated after inductive conditions no longer prevail? These and other classical observations in the physiology of flowering can now be explored at the molecular level. Finally, besides a pivotal role in floral induction, FT can also affect vegetative growth independently of flowering [19,35,53,54]. Obviously, much remains to be learned about the roles of FT in growth and development of plants.
Acknowledgements This review is dedicated to the memory of Anton Lang (1913–1996), who was a passionate advocate of the florigen hypothesis. My apologies to colleagues whose work has not been cited because of space constraints. Preparation of this review was supported by the US Department of Energy (grant number DE-FG02-91ER20021).
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
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This paper shows that Myc-tagged FT protein expressed from a phloemspecific promoter can function as the mobile signal for flowering, but not when it is immobilized in the nucleus. Myc-FT produced in the leaves was detected in the shoot apex by immunolocalization. 23. Mathieu J, Warthmann N, Ku¨ttner F, Schmid M: Export of FT protein from phloem companion cells is sufficient for floral induction in Arabidopsis. Curr Biol 2007, 17:1055-1060. Artificial microRNA (amiRNA) under control of the shoot-apex-specific FD promoter was used to demonstrate that FT mRNA was not required at the shoot apex. Expression of FT as part of a large, immobile protein complex in the phloem prevented flowering. 24. Lin M-K, Belanger H, Lee Y-L, Varkonyl-Gasic E, Taoka K-I, Miura E, Xoconostie-Ca´zares B, Gendler K, Jorgensen RA, Phinney B et al.: FLOWERING LOCUS T-protein may act as the long-distance florigenic signal in the cucurbits. Plant Cell 2007, 19:1488-1506. In this paper, a virus vector was used to overexpress FT from Arabidopsis in the SDP Cucurbita moschata to show conservation of FT function. Native FT protein was identified by mass spectrometry in phloem exudate of the SDP Cucurbita moschata. In an elegant experiment, a correlation was established between movement of the floral stimulus and FT protein across graft unions from dayneutral C. maxima donors to C. moschata receptors. Entry of FT into the phloem stream was shown to be under photoperiodic control in C. moschata. 25. Stadler R, Wright KM, Lauterbach C, Amon G, Gahrtz M, Feuerstein A, Oparka KJ, Sauer N: Expression of GFP-fusions in Arabidopsis companion cells reveals non-specific protein trafficking into sieve elements and identifies a novel postphloem domain in roots. Plant J 2005, 41:319-331. 26. Yamaguchi A, Kobayashi Y, Goto K, Abe M, Araki T: TWIN SISTER OF FT (TSF) acts as a floral pathway integrator redundantly with FT. Plant Cell Physiol 2005, 46:1175-1189.
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15. Lough TJ, Lucas WJ: Integrative plant biology: Role of phloem long-distance macromolecular trafficking. Annu Rev Plant Biol 2006, 57:203-232.
28. Kojima S, Takahashi Y, Kobayashi Y, Monna L, Sasaki T, Araki T, Yano M: Hd3a, a rice ortholog of the Arabidopsis FT gene, promotes transition to flowering downstream of Hd1 under short-day conditions. Plant Cell Physiol 2002, 43:1096-1105.
16. Kehr J, Buhtz A: Long distance transport and movement of RNA through the phloem. J Exp Bot 2008, 59:85-92. 17. Huang T, Bo¨hlenius H, Eriksson S, Parcy F, Nilsson O: The mRNA of the Arabidopsis gene FT moves from leaf to shoot apex and induces flowering. Science 2005, 309:1694-1696. 18. Bo¨hlenius H, Eriksson, Parcy F, Nilsson O: Retraction. Science 2007, 316:367.
29. Hayama R, Agashe B, Luley E, King R, Coupland G: A circadian rhythm set by dusk determines the expression of FT homologs and the short-day photoperiodic flowering response in Pharbitis. Plant Cell 2007, 19:2988-3000. In the SDP Pharbitis nil, the length of the night controls flowering. Accordingly, two orthologs of FT, PnFT1 and PnFT2, are expressed only in darkness. A light-sensitive clock set by the light–dark transition is the main factor regulating expression of PnFTs.
19. Lifschitz E, Eviatar T, Rozman A, Shalit A, Goldshmidt A, Amsellem Z, Alvarez JP, Eshed Y: The tomato FT ortholog triggers systemic signals that regulate growth and flowering and substitute for diverse environmental stimuli. Proc Natl Acad Sci U S A 2006, 103:6398-6403. In contrast to an earlier report [17], no evidence was reported in this paper for mobility of SFT mRNA in tomato, although the floral stimulus did cross the graft union. Induction of flowering by constitutive expression of SFT in Arabidopsis, and in dayneutral and short-day tobacco demonstrated conservation of SFT function. The authors suggest that SFT may also have a role in vegetative growth independent of its role in flowering.
32. Carmona MJ, Calonje M, Martı´nez-Zapater JM: The FT/TFL1 gene family in grapevine. Plant Mol Biol 2007, 63:637-650.
20. Corbesier L, Vincent C, Jang S, Fornara F, Fan Q, Searle I, Giakountis A, Farrona S, Gissot L, Turnbull C, Coupland G: FT protein movement contributes to long-distance signaling in floral induction of Arabidopsis. Science 2007, 316:1030-1033. Along with the companion paper on rice [21], this work provides the first direct evidence for movement of FT-GFP fusion protein from leaves to the shoot apex and also across the graft union from an induced Arabidopsis donor to ft mutant receptor shoots.
33. Yan L, Fu D, Li C, Blechl A, Tranquilli G, Bonafede M, Sanchez A, Valarik M, Yasuda S, Dubcovsky J: The wheat and barley vernalization gene VRN3 is an orthologue of FT. Proc Natl Acad Sci U S A 2006, 103:19581-19586. The vernalization gene VRN3 in winter wheat and barley was cloned and shown to be homologous to FT of Arabidopsis. Winter wheat transformed with FT/VRN3 flowered earlier than non-transgenic plants, showing that overexpression of FT can bypass vernalization.
21. Tamaki S, Matsuo S, Wong HL, Yokoi S, Shimamoto K: Hd3a protein is a mobile flowering signal in rice. Science 2007, 316:1033-1036. This paper provides evidence that Hd3a-GFP fusion protein expressed from phloem-specific promoters in rice moves from leaves to the shoot apex, where it induces flowering.
34. Endo T, Shimada T, Fujii H, Kobayashi Y, Araki T, Omura M: Ectopic expression of an FT homolog from Citrus confers an early flowering phenotype on trifoliate orange (Poncirus trifoliata L. Raf.). Transgenic Res 2005, 14:703-712.
22. Jaeger KE, Wigge PA: FT protein acts as a long-range signal in Arabidopsis. Curr Biol 2007, 17:1050-1054. Current Opinion in Plant Biology 2008, 11:541–547
30. Hsu C-Y, Liu Y, Luthe DS, Yuceer C: Poplar FT2 shortens the juvenile phase and promotes seasonal flowering. Plant Cell 2006, 18:1846-1861. 31. Sreekantan L, Thomas MR: VvFT and VvMADS8, the grapevine homologues of the floral integrators FT and SOC1, have unique expression patterns in grapevine and hasten flowering in Arabidopsis. Funct Plant Biol 2006, 33:1129-1139.
35. Bo¨hlenius H, Huang T, Charbonnel-Campaa L, Brunner AM, Jansson S, Strauss SH, Nilsson O: CO/FT regulatory module controls timing of flowering and seasonal growth cessation in trees. Science 2007, 312:1040-1043. www.sciencedirect.com
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36. Komiya R, Ikegami A, Tamaki S, Yokoi S, Shimamoto K: Hd3a and RFT1 are essential for flowering in rice. Development 2008, 135:767-774. This paper describes characterization of RICE FLOWERING LOCUS T 1 (RFT1), the closest homolog of Hd3a in rice. 37. Searle I, He Y, Turck F, Vincent C, Fornara F, Kro¨ber S, Amasino RA, Coupland G: The transcription factor FLC confers a flowering response to vernalization by repressing meristem competence and systemic signaling in Arabidopsis. Genes Dev 2006, 20:898-912. An elaborate study of the effect of FLC on flowering in Arabidopsis and expression of SOC1, FT, and FD. It is shown that FLC has a dual role in vernalization. In the leaves, FLC delays flowering by repressing FT expression; in the shoot apex, it reduces response to FT by repressing expression of FD and SOC1. 38. Sung S, Amasino RM: Remembering winter: toward a molecular understanding of vernalization. Annu Rev Plant Biol 2005, 56:491-508. 39. Trevaskis B, Hemming MN, Dennis ES, Peacock WJ: The molecular basis of vernalization-induced flowering in cereals. Trends Plant Sci 2007, 12:352-357. 40. Yan L, Loukoianov A, Tranquilli G, Helguera M, Fahima T, Dubcovsky J: Positional cloning of wheat vernalization gene VRN1. Proc Natl Acad Sci U S A 2003, 100:6263-6268. 41. Yan L, Loukoianov A, Blechl A, Tranquilli G, Ramakrishna W, SanMiguel P, Bennetzen JL, Echenique V, Dubcovsky J: The wheat VRN2 gene is a flowering repressor downregulated by vernalization. Science 2004, 303:1640-1644. 42. Dubcovsky J, Loukoianov A, Fu D, Valarik M, Sanchez A, Yan L: Effect of photoperiod on the regulation of wheat vernalization genes VRN1 and VRN2. Plant Mol Biol 2006, 60:469-480.
45. Lang A: Induction of flower formation in biennial Hyoscyamus by treatment with gibberellin. Naturwissenschaften 1956, 43:284-285. 46. Bla´zquez MA, Weigel D: Integration of floral inductive signals in Arabidopsis. Nature 2000, 404:889-892. 47. Moon J, Suh SS, Lee H, Choi KR, Hong CB, Paek NC, Kim SG, Lee I: The SOC1 MADS-box gene integrates vernalization and gibberellin signals for flowering in Arabidopsis. Plant J 2003, 35:613-623. 48. Eriksson S, Bo¨hlenius H, Moritz T, Nilsson O: GA4 is the active gibberellin in the regulation of LEAFY transcription and Arabidopsis floral initiation. Plant Cell 2006, 18:2172-2181. The GA-dependent pathway for flowering is most important under SD conditions. This paper provides evidence that GA4 is the active GA for stem growth and floral initiation in Arabidopsis in SD. 49. King RW, Evans LT: Gibberellins and flowering of grasses and cereals: Prizing open the lid of the ‘Florigen’ black box. Annu Rev Plant Biol 2003, 54:307-328. 50. King RW, Moritz T, Evans LT, Martin J, Andersen CH, Blundell C, Kardailsky I, Chandler PM: Regulation of flowering in the longday grass Lolium temulentum by gibberellins and the FLOWERING LOCUS T gene. Plant Physiol 2006, 141:498-507. In L. temulentum, in LD, GA5 has florigenic properties in that it is produced in the leaf and induces floral transition in the shoot apex. FT expression was drastically increased in LD independently of changes in GA. 51. Maizel A, Busch MA, Tanahashi T, Perkovic J, Kato M, Hasebe M, Weigel D: The floral regulator LEAFY evolves by substitutions in the DNA binding domain. Science 2005, 308:260-263. 52. Zeevaart JAD: Florigen coming of age after 70 years. Plant Cell 2006, 18:1783-1789.
43. Lang A: Hyoscyamus niger. In CRC Handbook of Flowering, vol. V. Edited by Halevy AH. CRC Press; 1985:144-186.
53. Gyllenstrand N, Clapham D, Ka¨llman, Lagercrantz U: A Norway spruce FLOWERING LOCUS T homolog is implicated in control of growth rhythm in conifers. Plant Physiol 2007, 144:248-257.
44. Chailakhyan MKh: Flowering in graft hybrids with both components in the vegetative state. English Transl Bot Sci Dokl Akad Nauk SSSR 1964, 159:226-228.
54. Igasaki T, Watanabe Y, Nishiguchi M, Kotoda N: The FLOWERING LOCUS T/TERMINAL FLOWER 1 family in Lombardy poplar. Plant Cell Physiol 2008, 49:291-300.
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Current Opinion in Plant Biology 2008, 11:541–547
Leaf-produced floral signals Jan AD Zeevaart Florigen is the hypothetical leaf-produced signal that induces floral initiation at the shoot apex. The nature of florigen has remained elusive for more than 70 years. But recent progress toward understanding the regulatory network for flowering in Arabidopsis has led to the suggestion that FLOWERING LOCUS T (FT) or its product is the mobile flower-inducing signal that moves from an induced leaf through the phloem to the shoot apex. In the past year, physical and chemical evidence has shown that it is FT protein, and not FT mRNA, that moves from induced leaves to the apical meristem. These results have established that FT is the main, if not the only, component of the universal florigen. Address MSU-DOE Plant Research Laboratory and Department of Plant Biology, Michigan State University, East Lansing, MI, USA Corresponding author: Zeevaart, Jan AD ([email protected])
Current Opinion in Plant Biology 2008, 11:541–547 This review comes from a themed issue on Cell Signalling and Gene Regulation Edited by Jason Reed and Bonnie Bartel Available online 6th August 2008 1369-5266/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. DOI 10.1016/j.pbi.2008.06.009
Introduction The concept of long-distance signaling in flowering was first introduced by Knott [1]. He observed that flowering in the long-day plant (LDP) spinach took place when the leaves were in long-day conditions (LD) and the shoot tips in short days (SD), but not when the leaves were in SD and the tips in LD. Knott [1] concluded that ‘‘the part played by the foliage of spinach in hastening the response to a photoperiod favorable to reproductive growth may be in the production of some substance, or stimulus, that is transported to the growing point’’. Chailakhyan [2] extended these observations to other species and coined the term ‘florigen’ (flower-former) for the photoperiodic stimulus, which is perceived in the leaves and transmitted to the shoot apex. The stimulus is graft transmissible from an induced partner (donor) to a non-induced shoot (receptor) and between different species and genera, as well as between different photoperiodic response types [3,4]. It appeared, therefore, that florigen is universal in flowering plants. Despite abundant physiological evidence for a transmissible stimulus, its identity remained unknown for www.sciencedirect.com
70 years, so florigen became known as the ‘Holy Grail’ of the physiology of flowering. Further progress had to await the development of a regulatory network for flowering in the quantitative LDP Arabidopsis thaliana. With the convergence of classical physiological studies and moleculargenetic approaches, the search for florigen could be pursued from a new perspective. In this review, I will summarize the recent work that led to the identification of FLOWERING LOCUS T (FT) protein as the main, if not the only, component of florigen. Reviews on integration of the different genes in a signaling network for flowering [5–7] and on the phloem-mobile floral stimulus [8,9,10] have appeared recently.
Role of the CO–FT pathway in flowering In Arabidopsis, a network of four interacting pathways regulates floral induction. In the photoperiod-dependent pathway, CONSTANS (CO) and FT are the key elements that mediate the effect of daylength on flowering. Both genes are expressed in phloem companion cells of leaves. In LD, expression of CO is upregulated and its protein stabilized, which in turn induces expression of FT. Ectopic expression of CO causes early flowering, but not when expressed from a shoot meristem-specific promoter. By contrast, overexpression of FT in the shoot apex alone induces early flowering, as does expression of CO from companion-cell-specific promoters. One explanation is that CO expressed in leaf phloem regulates synthesis of a phloem-mobile signal that induces flowering [11]. This possibility was further substantiated by results of grafting experiments with plants expressing CO under control of the GALACTINOL SYNTHASE 1 (GAS1) promoter, which is restricted to the companion cells of the minor veins. These GAS1::CO donor plants induced early flowering in co-1 receptor shoots [12]. Although produced in the phloem of leaves, FT acts in the shoot apex, where it forms a complex with the bZIP transcription factor FLOWERING LOCUS D (FD). The FD/FT heterodimer promotes the transition to flowering by activating SUPPRESSION OF OVEREXPRESSION OF CO 1 (SOC1) and the floral meristem identity gene APETALA1 (AP1) (Figure 1) [13,14]. It is clear from these results that the site of production of FT mRNA in the leaf is remote from the site of FT action in the shoot apex. Thus, FT (mRNA or protein) or its product is a prime candidate for the phloem-mobile florigen signal.
FT mRNA does not move in the phloem Many mRNAs move in the phloem over long distances [15,16], so it was logical to explore whether FT mRNA can move from an induced leaf to the shoot apex and thus function as the floral stimulus. Huang et al. [17] reported Current Opinion in Plant Biology 2008, 11:541–547
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Figure 1
considered it unlikely that Hd3a mRNA moved from leaves to the shoot apex. In addition, activity of the GUS transgene driven by the Hd3a promoter was detected in the vascular bundles, but not in the shoot apex [21]. Myc-FT mRNA expressed from a companion-cell-specific promoter could be detected in the phloem by in situ hybridization, but there was no expression in the shoot apex [22]. In a novel approach, Mathieu et al. [23] employed artificial microRNAs (amiRNAs) for tissuespecific inactivation of FT mRNA. Expression of amirFT under control of the 35S or SUC2 promoter delayed flowering to the same extent as in ft-10 mutant plants. By contrast, when expression of amir-FT was driven by the shoot apex-specific FD promoter, flowering was not delayed. These results demonstrate that FT mRNA is necessary in the companion cells in order to flower, but is not required in the regions of the shoot apex where FD is expressed [13,14]. Finally, FLOWERING LOCUS T-LIKE (FTL) mRNA was undetectable by real-time RT-PCR in the phloem sap of flowering Cucurbita [24]. Collectively, none of these results provides any evidence that FT mRNA functions as the phloem-mobile signal for floral induction.
FT protein is phloem-mobile FT protein as a transmissible signal for flowering. In long days, CO protein accumulates in the leaves and induces expression of FT in the phloem companion cells. FT protein is transported in the sieve tubes to the shoot apex, where it forms a heterodimer with FD. The FD/FT complex activates expression of SOC1 and AP1, which leads to floral initiation. FM, floral meristem; LP, leaf primordium. Adapted from [10].
that a single heated leaf of a transgenic plant expressing FT under control of a heat shock promoter induced flowering in the Arabidopsis ft-7 mutant in SD and that FT mRNA was detected in the shoot apex by RT-PCR. However, this work was later retracted [18]. Several later reports failed to provide evidence that FT mRNA is mobile in the phloem. In grafting experiments with tomato overexpressing SINGLE-FLOWER TRUSS (SFT, ortholog of FT in tomato), the floral stimulus did cross the graft union, but SFT mRNA could not be detected in the flowering receptor shoots [19]. In Arabidopsis, a gene fusion of FT and GREEN FLUORESCENT PROTEIN (GFP) was expressed under control of the SUCROSE TRANSPORTER 2 (SUC2) promoter, which is expressed in the phloem companion cells. FT-GFP mRNA was detected by in situ hybridization in mature phloem, but no mRNA was detected in the protophloem or shoot apex. FT-GFP mRNA also failed to move across a graft union to ft-7 mutant receptor shoots [20]. In the short-day plant (SDP) rice, under inductive conditions for flowering, Heading-date 3a (Hd3a, ortholog of FT) mRNA accumulated in the leaf blades, but levels remained very low in the shoot apex, so the authors Current Opinion in Plant Biology 2008, 11:541–547
FT mRNA and its protein are of low abundance, so that most attempts to demonstrate localization and movement of FT were conducted with transgenic plants expressing FT as a fusion protein with GFP or Myc from a companion-cell-specific promoter. Soluble cytoplasmic proteins fused to GFP can non-specifically move from the companion cells into sieve elements, but none of the GFP fusions moved beyond the protophloem in the root [25]. Consequently, if there is specific control over entry of FT into the sieve elements, distribution of FT-GFP may not reflect the movement of native FT. One should keep this caveat in mind when evaluating experimental data claiming to demonstrate movement of FT-GFP fusions from leaves to shoot apices. Perhaps the best evidence that the fusion proteins entered the shoot apex was that mobile FT-GFP fusion proteins induced early flowering. The first experiments on movement of FT-GFP from leaves to shoot apices were conducted with Arabidopsis and rice [20,21]. Expression of SUC2::FT-GFP in the ft-7 mutant of Arabidopsis resulted in early flowering. Whereas FT-GFP mRNA was restricted to mature phloem, FT-GFP protein was also present in the protophloem and at the base of the shoot apex. Movement of FT-GFP over longer distances was observed in grafting experiments from transgenic donors expressing SUC2::FT-GFP to ft-7 receptors. The receptors flowered earlier and FT-GFP was detected in their vascular bundles, thus establishing movement of FT-GFP from donor www.sciencedirect.com
Leaf-produced floral signals Zeevaart 543
to receptor [20]. Movement of Hd3a-GFP was also observed in rice when expressed under control of phloem-specific promoters. The transgenic plants flowered early and fluorescence was observed in the vascular tissue ending below the apex and in the region below the apex. These observations indicate that Hd3a-GFP in rice moves from source companion cells to the shoot apex [21]. Transgenic Arabidopsis plants expressing GAS1::FT also flowered early. By contrast, GAS1::FTGFP plants flowered very late, although fluorescence was observed in the minor veins of the leaves. Corbesier et al. [20] speculated that FT-GFP was too large for movement in the minor veins, with the consequence that it could not reach the shoot apex and induce flowering. These observations further imply that FT does not produce a small, mobile molecule, but that FT itself is the mobile signal. Epitope-tagged FT expressed from the SUC2 promoter induced early flowering in Arabidopsis. As detected by immunolocalization, Myc-FT was not only present in the phloem, but had moved beyond the ends of the vascular strands into the areas of floral initiation. When FT was targeted to the nucleus and thus immobilized, flowering was not promoted [22]. Expression of FT as a large fusion protein (>112 kDa) from the SUC2 promoter did not affect flowering, presumably because the protein was too big to move from the companion cells into the sieve elements. Ectopic expression of the same construct did promote flowering, showing that FT as part of the large fusion protein did retain its activity. Release of FT protein from the complex with a specific protease again promoted flowering [23]. All of these results are consistent with the notion that FT must be phloem-mobile to induce flowering. All results implying the movement of FT from leaves to shoot apices were obtained with transgenic plants expressing FT fusion proteins under control of companion-cellspecific promoters. However, in none of the results discussed so far was the presence of native FT protein in the phloem stream demonstrated. For this purpose, dayneutral Cucurbita maxima (Cm) and the SDP C. moschata (Cmo) are suitable, because phloem exudate can be readily collected from cut stems. Conservation of the function of FT in flowering was demonstrated by using zucchini yellow mosaic virus (ZYMV) to overexpress FT from Arabidopsis in Cmo under LD conditions. The virus was restricted to mature and developing leaves, but very effectively induced Cmo plants to flower under LD, indicating movement of FT from infected leaves to the shoot apex. Conversely, the constructs SUC2::CmFTLIKE (FTL)1 and -2 induced early flowering in Arabidopsis. Thus, the function of FT as an inducer of flowering was conserved in Arabidopsis and cucurbits [24]. The CmoFTL genes were expressed in the phloem of stems and leaves regardless of photoperiod, but the www.sciencedirect.com
proteins were detected only in the phloem sap of plants in SD. It was concluded, therefore, that entry of the CmoFTL proteins into the phloem stream is regulated by the photoperiod and not by transcription [24]. In grafting experiments, dayneutral Cm stocks induced flowering in the SDP Cmo scions under LD. In these graft combinations, CmTFL2 protein was identified by mass spectrometry in phloem sap collected from the Cmo receptor scions. Thus, transmission of florigen in a classical grafting experiment was correlated with transport of the native CmFTL2 protein from donor to receptor [24]. This is the first example of identification and movement of native FT-like protein in phloem sap in connection with induction of flowering.
Is FT protein the universal signal for flowering? FT protein fulfills most of the criteria of florigen. It moves from induced leaves to the shoot apex and across graft unions from donor to receptor to induce flowering. One of the expectations – extraction of florigen from flowering plants and induction of flowering in vegetative plants with applied extracts – cannot be fulfilled owing to the proteinaceous nature of FT. A basic assumption of the florigen hypothesis is that florigen is a universal signal for flowering. Results of interspecific grafting experiments and of grafts between different photoperiodic response types support this idea. But grafting has been limited by incompatibility between unrelated species and by the fact that monocots are not amenable to grafting. However, these barriers can now be overcome by ‘transplanting’ the FT gene (and its orthologs) into unrelated species. Invariably, overexpression of FT-like genes has led to premature flowering under non-inductive conditions in a variety of species. The following donor species of FT or orthologs caused early flowering in Arabidopsis as the recipient species: Arabidopsis [13,26,27], rice [28], tomato [19], Cucurbita maxima [24], Pharbitis nil [29], Populus deltoides [30], and Vitis vinifera [31,32]. Examples of species in which premature flowering was induced by ectopic expression of FT or an FT ortholog are tomato, dayneutral tobacco, the SDP Maryland Mammoth tobacco [19], the SDP Pharbitis nil [29], as well as winter wheat [33]. In trifoliate orange [34] and Populus spp. [30,35] constitutive expression of FT drastically shortened the juvenile phase. Conversely, when the function of FT was downregulated by mutation, RNAi or miRNA, flowering was much delayed in Arabidopsis and rice [26,28,36]. These results indicate that FT and its orthologs are essential for flowering and, further, that the functions of FT and its orthologs are highly conserved among unrelated species. However, there are examples of grafting experiments in which the receptor failed to flower [4]. A case in point is wild-type tomato as donor for the sft mutant, but when transgenic donor plants were used, in which SFT was ectopically expressed, the receptor shoots did flower Current Opinion in Plant Biology 2008, 11:541–547
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[19]. This result demonstrates that an increased level of SFT transcript makes the transgenic plant an effective donor. Clearly, the argument that failure to induce flowering via a graft union is due to different floral stimuli in donor and receptor is no longer tenable. In Arabidopsis and rice, FT and Hd3a, respectively, are strongly upregulated under inductive conditions [20,21,28]. Although expression of CmoFTL2 in C. moschata is also upregulated under inductive conditions, photoperiodic regulation of the function of FTL2 is at entry from the companion cells into the phloem stream and not at the transcriptional level [24]. Clearly, the regulation of FT production and mobility may vary among species, but the end product, FT, is always the same. Thus, on the basis of the currently available evidence, a strong case can be made that FT protein is the universal florigen. But the question remains: Is FT the only component of florigen that moves from leaves to the shoot apex, where it induces transition to flowering? FT protein in the phloem stream may require a chaperone-like protein for protection against proteolysis and unloading at the shoot apex [15]. If a protein were identified that was invariably associated with FT in the phloem and that was also essential for flowering (a mutation in the gene encoding this hypothetical protein would result in a late-flowering phenotype), it would have to be considered a component of florigen. That question remains to be answered by future research. For now, it can be concluded that FT is a major, if not the only component of florigen.
FT is also the product of vernalization In Arabidopsis, FT is not only the output of the photoperiodic pathway, but also of the autonomous and vernalization pathways. These three pathways converge, therefore, through the FT integrator. In cold-requiring accessions of Arabidopsis, the MADS-box transcriptional regulator FLOWERING LOCUS C (FLC) represses expression of FT in the leaf and of FD and SOC1 in the shoot apex [37]. The effect of vernalization is downregulation of FLC, thus allowing FT to be expressed in the leaves and increasing the competence of the shoot apex to respond to FT. Overexpression of FT in nonvernalized Arabidopsis promoted early flowering without affecting FLC mRNA levels, indicating that FT acts downstream of FLC [27]. FLC has not been identified outside the Cruciferae [38]. Therefore, the question arises: Does vernalization in other species also involve downregulation of a repressor? Results of recent work on vernalization in winter varieties of barley and wheat indicate that cold acts by a mechanism similar to that in Arabidopsis. In these cereals, three genes, VERNALIZATION (VRN)1, VRN2, and VRN3 control vernalization requirements [39] (In barley and wheat, VRN1 and VRN2 are different from VRN1 and VRN2 of Current Opinion in Plant Biology 2008, 11:541–547
Arabidopsis [5]). In winter varieties, VRN2 is a repressor of flowering, which is downregulated by vernalization, with a concomitant upregulation of VRN1, an ortholog of the floral meristem identity gene AP1 in Arabidopsis [40,41]. In spring varieties that do not require vernalization, VRN1 is always strongly expressed. VRN2 is also downregulated by a prolonged period of SD, but in this case VRN1 is not upregulated until the plants are transferred to LD [42]. In vernalized plants in LD, repression of VRN2 also allows upregulation of VRN3, an ortholog of FT, which further enhances expression of VRN1. Winter wheat transformed with FT/VRN3 can bypass the block in flowering imposed by VRN2 [33], demonstrating that expression of FT is necessary and sufficient for flowering in winter varieties of barley and wheat, as it is in Arabidopsis [27]. So, in both Arabidopsis and winter cereals, vernalization involves the removal of a flower repressor, which then permits upregulation of FT/VRN3 under LD. FLC and VRN2 belong to different families of transcription factors, but they have the same function [41]. Clearly, in cold-requiring cereals, as in Arabidopsis, responses to vernalization and photoperiodism are integrated through the FT protein. In common with photoperiodic induction of flowering, vernalization also results in a floral stimulus that can be transmitted across a graft union and induce flowering in plants kept in the vegetative state [3,43]. Because the donor plants were flowering, the signal transmitted was most probably the final product of thermo-induction and photo-induction, that is, florigen, now known to be FT. There is no evidence for a separate transmissible stimulus specific for vernalization, with one exception. The SDP Maryland Mammoth tobacco as donor induced flower formation in non-vernalized biennial Hyoscyamus niger not only in SD when induced to flower, but also in LD when the donor remained vegetative. This result was interpreted as vernalization giving rise to a transmissible stimulus, called ‘vernalin’, which would be required for florigen production. Annual tobacco would provide ‘vernalin’ (always present in annual plants), so that biennial Hyoscyamus in LD could then produce florigen. Vernalization is a prerequisite for LD induction of flowering, so it was postulated that in a physiological sense vernalin is the precursor of florigen [3,43]. Although this experiment was reproducible, it was observed that the non-induced Maryland Mammoth tobacco caused considerable stem elongation in the biennial Hyoscyamus before flower buds appeared ([44]; JAD Zeevaart, unpublished results). This is reminiscent of gibberellin (GA)-treated biennial Hyoscyamus niger plants in which much stem elongation preceded appearance of flower buds [43,45]. It is likely, therefore, that the non-induced tobacco shoot supplied GA, thus bypassing the vernalization requirement. Consequently, the single experimental result in favor of ‘vernalin’ can be interpreted differently, and there is no reason to propose any longer a mobile stimulus ‘vernalin’ as the product of vernalization. Besides, in the www.sciencedirect.com
Leaf-produced floral signals Zeevaart 545
context of our current understanding of vernalization, it is hard to visualize any gene product other than FT being phloem-mobile.
FT and gibberellin act independently in flowering Arabidopsis is a quantitative LDP, so floral initiation will ultimately take place in SD. This transition to flowering in SD is predominantly mediated by the GA-dependent pathway. Applied GA in SD induces expression of SOC1 and the floral meristem identity gene LEAFY (LFY), but has no effect on FT expression [46,47]. Constitutive expression of FT in the GA-deficient ga1-3 mutant in SD induced very early flowering. These results suggest that GA and FT exert their effects on flowering in Arabidopsis independently. Analysis of GAs in Arabidopsis showed large increases in GA4 in the shoot apex shortly before floral initiation in SD. This increase in GA4 was accompanied by an increase in LFY transcript and could not be explained by in situ GA biosynthesis, so it was speculated that GAs produced in the leaves were transported to the shoot apex [48]. GAs as florigenic signals have been extensively studied in the LDP Lolium temulentum. In this plant, GA5 and GA6 are signals produced in the leaf and transported to the shoot apex, where they cause floral initiation [49,50]. Besides increasing GA biosynthesis in the leaves, LD also induced a large increase in FT transcript in Lolium, but this increase occurred independently of GA [50]. These observations in Arabidopsis and Lolium suggest that GAs and FT are functionally redundant; they are both leaf-produced mobile signals that can induce flowering [48,50]. But the question arises: How general is GA-induced flowering? Applied GA (usually GA3) induces flowering in many, but not all, rosette LDPs and does not induce flowering in SDP. LFY, the target of GA, is conserved in plants [51]. It is puzzling, therefore, that applied GA fails to cause flowering in many species. Two possible reasons come to mind: The ‘wrong’ GA was applied, or the regulatory region for the GA-response site of the LFY promoter has mutated, so that LFY cannot be activated by GA. At present, it is clear that FT as an inducer of flowering is highly conserved, but it remains to be established that this is also the case for GAs.
Conclusions and perspectives With the identification of FT as the mobile flower-inducing signal, its production, transport, and action can now be explored in more detail. FT is a 20-kDa protein, which is below the selective exclusion limit of 67 kDa of companion cell plasmodesmata [25], so that it should readily diffuse into the phloem stream. However, the results with C. moschata [24] show that, at least in this species, there is tight control over exit of FT from the companion cells. Mechanisms involved in FT protein movement and its targeted release from the protophloem and movement www.sciencedirect.com
from cell to cell in the apex require further studies at the molecular and cellular levels. The molecular mechanisms involved in induction of flowering – first analyzed in Arabidopsis – appear to be conserved in other species; at least the end product, FT protein, is the same. The effects of vernalization in Arabidopsis and winter cereals involve downregulation of a repressor, followed by upregulation of FT expression. How generally this model of vernalization is valid for cold-requiring dicotyledonous plants other than Arabidopsis remains to be established. It is of interest that SD followed by LD can substitute for vernalization in winter wheat [42]. This raises the possibility that in species with a dual daylength requirement for flowering (short-longday plants and long-short-day plants [52]) the first daylength requirement also involves downregulation of a repressor, followed by FT expression under the second daylength requirement. Indirect induction [4] is an intriguing phenomenon that remains to be explained in molecular terms. One possibility is that FT induces its own production via a positive feedback loop. In the case of irreversible induction in Perilla, does FT remain activated after inductive conditions no longer prevail? These and other classical observations in the physiology of flowering can now be explored at the molecular level. Finally, besides a pivotal role in floral induction, FT can also affect vegetative growth independently of flowering [19,35,53,54]. Obviously, much remains to be learned about the roles of FT in growth and development of plants.
Acknowledgements This review is dedicated to the memory of Anton Lang (1913–1996), who was a passionate advocate of the florigen hypothesis. My apologies to colleagues whose work has not been cited because of space constraints. Preparation of this review was supported by the US Department of Energy (grant number DE-FG02-91ER20021).
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Kobayashi Y, Weigel D: Move on up, it’s time for change— mobile signals controlling photoperiod-dependent flowering. Genes Dev 2007, 21:2371-2384. Review from a historical point of view on photoperiodic induction of flowering, with emphasis on time measurement and florigen. 10. Turck F, Fornara F, Coupland G: Regulation and identity of florigen: FLOWERING LOCUS T moves center stage. Annu Rev Plant Biol 2008, 59:573-594. This review discusses recent work favoring FT protein as a major component of florigen. 11. An HL, Roussot C, Sua´rez-Lo´pez P, Corbesier L, Vincent C, Pin˜eiro M, Hepworth S, Mouradov A, Justin S, Turnbull C, Coupland G: CONSTANS acts in the phloem to regulate a systemic signal that induces photoperiodic flowering of Arabidopsis. Development 2004, 131:3615-3626. 12. Ayre K, Turgeon R: Graft transmission of a floral stimulant derived from CONSTANS. Plant Physiol 2004, 135:2271-2278. 13. Abe M, Kobayashi Y, Yamamoto S, Daimon Y, Yamaguchi A, Ikeda Y, Ichinoki H, Notaguchi M, Goto K, Araki T: FD, a bZIP protein mediating signals from the floral pathway integrator FT at the shoot apex. Science 2005, 309:1052-1056.
This paper shows that Myc-tagged FT protein expressed from a phloemspecific promoter can function as the mobile signal for flowering, but not when it is immobilized in the nucleus. Myc-FT produced in the leaves was detected in the shoot apex by immunolocalization. 23. Mathieu J, Warthmann N, Ku¨ttner F, Schmid M: Export of FT protein from phloem companion cells is sufficient for floral induction in Arabidopsis. Curr Biol 2007, 17:1055-1060. Artificial microRNA (amiRNA) under control of the shoot-apex-specific FD promoter was used to demonstrate that FT mRNA was not required at the shoot apex. Expression of FT as part of a large, immobile protein complex in the phloem prevented flowering. 24. Lin M-K, Belanger H, Lee Y-L, Varkonyl-Gasic E, Taoka K-I, Miura E, Xoconostie-Ca´zares B, Gendler K, Jorgensen RA, Phinney B et al.: FLOWERING LOCUS T-protein may act as the long-distance florigenic signal in the cucurbits. Plant Cell 2007, 19:1488-1506. In this paper, a virus vector was used to overexpress FT from Arabidopsis in the SDP Cucurbita moschata to show conservation of FT function. Native FT protein was identified by mass spectrometry in phloem exudate of the SDP Cucurbita moschata. In an elegant experiment, a correlation was established between movement of the floral stimulus and FT protein across graft unions from dayneutral C. maxima donors to C. moschata receptors. Entry of FT into the phloem stream was shown to be under photoperiodic control in C. moschata. 25. Stadler R, Wright KM, Lauterbach C, Amon G, Gahrtz M, Feuerstein A, Oparka KJ, Sauer N: Expression of GFP-fusions in Arabidopsis companion cells reveals non-specific protein trafficking into sieve elements and identifies a novel postphloem domain in roots. Plant J 2005, 41:319-331. 26. Yamaguchi A, Kobayashi Y, Goto K, Abe M, Araki T: TWIN SISTER OF FT (TSF) acts as a floral pathway integrator redundantly with FT. Plant Cell Physiol 2005, 46:1175-1189.
14. Wigge PA, Kim MC, Jaeger KE, Busch W, Schmid M, Lohmann JU, Weigel D: Integration of spatial and temporal information during floral induction in Arabidopsis. Science 2005, 309:1056-1059.
27. Michaels SD, Himelblau E, Kim SY, Schomburg FM, Amasino RM: Integration of flowering signals in winter-annual Arabidopsis. Plant Physiol 2005, 137:149-156.
15. Lough TJ, Lucas WJ: Integrative plant biology: Role of phloem long-distance macromolecular trafficking. Annu Rev Plant Biol 2006, 57:203-232.
28. Kojima S, Takahashi Y, Kobayashi Y, Monna L, Sasaki T, Araki T, Yano M: Hd3a, a rice ortholog of the Arabidopsis FT gene, promotes transition to flowering downstream of Hd1 under short-day conditions. Plant Cell Physiol 2002, 43:1096-1105.
16. Kehr J, Buhtz A: Long distance transport and movement of RNA through the phloem. J Exp Bot 2008, 59:85-92. 17. Huang T, Bo¨hlenius H, Eriksson S, Parcy F, Nilsson O: The mRNA of the Arabidopsis gene FT moves from leaf to shoot apex and induces flowering. Science 2005, 309:1694-1696. 18. Bo¨hlenius H, Eriksson, Parcy F, Nilsson O: Retraction. Science 2007, 316:367.
29. Hayama R, Agashe B, Luley E, King R, Coupland G: A circadian rhythm set by dusk determines the expression of FT homologs and the short-day photoperiodic flowering response in Pharbitis. Plant Cell 2007, 19:2988-3000. In the SDP Pharbitis nil, the length of the night controls flowering. Accordingly, two orthologs of FT, PnFT1 and PnFT2, are expressed only in darkness. A light-sensitive clock set by the light–dark transition is the main factor regulating expression of PnFTs.
19. Lifschitz E, Eviatar T, Rozman A, Shalit A, Goldshmidt A, Amsellem Z, Alvarez JP, Eshed Y: The tomato FT ortholog triggers systemic signals that regulate growth and flowering and substitute for diverse environmental stimuli. Proc Natl Acad Sci U S A 2006, 103:6398-6403. In contrast to an earlier report [17], no evidence was reported in this paper for mobility of SFT mRNA in tomato, although the floral stimulus did cross the graft union. Induction of flowering by constitutive expression of SFT in Arabidopsis, and in dayneutral and short-day tobacco demonstrated conservation of SFT function. The authors suggest that SFT may also have a role in vegetative growth independent of its role in flowering.
32. Carmona MJ, Calonje M, Martı´nez-Zapater JM: The FT/TFL1 gene family in grapevine. Plant Mol Biol 2007, 63:637-650.
20. Corbesier L, Vincent C, Jang S, Fornara F, Fan Q, Searle I, Giakountis A, Farrona S, Gissot L, Turnbull C, Coupland G: FT protein movement contributes to long-distance signaling in floral induction of Arabidopsis. Science 2007, 316:1030-1033. Along with the companion paper on rice [21], this work provides the first direct evidence for movement of FT-GFP fusion protein from leaves to the shoot apex and also across the graft union from an induced Arabidopsis donor to ft mutant receptor shoots.
33. Yan L, Fu D, Li C, Blechl A, Tranquilli G, Bonafede M, Sanchez A, Valarik M, Yasuda S, Dubcovsky J: The wheat and barley vernalization gene VRN3 is an orthologue of FT. Proc Natl Acad Sci U S A 2006, 103:19581-19586. The vernalization gene VRN3 in winter wheat and barley was cloned and shown to be homologous to FT of Arabidopsis. Winter wheat transformed with FT/VRN3 flowered earlier than non-transgenic plants, showing that overexpression of FT can bypass vernalization.
21. Tamaki S, Matsuo S, Wong HL, Yokoi S, Shimamoto K: Hd3a protein is a mobile flowering signal in rice. Science 2007, 316:1033-1036. This paper provides evidence that Hd3a-GFP fusion protein expressed from phloem-specific promoters in rice moves from leaves to the shoot apex, where it induces flowering.
34. Endo T, Shimada T, Fujii H, Kobayashi Y, Araki T, Omura M: Ectopic expression of an FT homolog from Citrus confers an early flowering phenotype on trifoliate orange (Poncirus trifoliata L. Raf.). Transgenic Res 2005, 14:703-712.
22. Jaeger KE, Wigge PA: FT protein acts as a long-range signal in Arabidopsis. Curr Biol 2007, 17:1050-1054. Current Opinion in Plant Biology 2008, 11:541–547
30. Hsu C-Y, Liu Y, Luthe DS, Yuceer C: Poplar FT2 shortens the juvenile phase and promotes seasonal flowering. Plant Cell 2006, 18:1846-1861. 31. Sreekantan L, Thomas MR: VvFT and VvMADS8, the grapevine homologues of the floral integrators FT and SOC1, have unique expression patterns in grapevine and hasten flowering in Arabidopsis. Funct Plant Biol 2006, 33:1129-1139.
35. Bo¨hlenius H, Huang T, Charbonnel-Campaa L, Brunner AM, Jansson S, Strauss SH, Nilsson O: CO/FT regulatory module controls timing of flowering and seasonal growth cessation in trees. Science 2007, 312:1040-1043. www.sciencedirect.com
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36. Komiya R, Ikegami A, Tamaki S, Yokoi S, Shimamoto K: Hd3a and RFT1 are essential for flowering in rice. Development 2008, 135:767-774. This paper describes characterization of RICE FLOWERING LOCUS T 1 (RFT1), the closest homolog of Hd3a in rice. 37. Searle I, He Y, Turck F, Vincent C, Fornara F, Kro¨ber S, Amasino RA, Coupland G: The transcription factor FLC confers a flowering response to vernalization by repressing meristem competence and systemic signaling in Arabidopsis. Genes Dev 2006, 20:898-912. An elaborate study of the effect of FLC on flowering in Arabidopsis and expression of SOC1, FT, and FD. It is shown that FLC has a dual role in vernalization. In the leaves, FLC delays flowering by repressing FT expression; in the shoot apex, it reduces response to FT by repressing expression of FD and SOC1. 38. Sung S, Amasino RM: Remembering winter: toward a molecular understanding of vernalization. Annu Rev Plant Biol 2005, 56:491-508. 39. Trevaskis B, Hemming MN, Dennis ES, Peacock WJ: The molecular basis of vernalization-induced flowering in cereals. Trends Plant Sci 2007, 12:352-357. 40. Yan L, Loukoianov A, Tranquilli G, Helguera M, Fahima T, Dubcovsky J: Positional cloning of wheat vernalization gene VRN1. Proc Natl Acad Sci U S A 2003, 100:6263-6268. 41. Yan L, Loukoianov A, Blechl A, Tranquilli G, Ramakrishna W, SanMiguel P, Bennetzen JL, Echenique V, Dubcovsky J: The wheat VRN2 gene is a flowering repressor downregulated by vernalization. Science 2004, 303:1640-1644. 42. Dubcovsky J, Loukoianov A, Fu D, Valarik M, Sanchez A, Yan L: Effect of photoperiod on the regulation of wheat vernalization genes VRN1 and VRN2. Plant Mol Biol 2006, 60:469-480.
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Current Opinion in Plant Biology 2008, 11:541–547