The Flowering Hormone Florigen Functions As a General Systemic Regulator of Growth and Termination

The flowering hormone florigen functions as a general systemic regulator of growth and termination

Akiva Shalita, Alexander Rozmana, Alexander Goldshmidtb, John P. Alvarezb, John L. Bowmanc, Yuval Eshedb, and Eliezer Lifschitza,1

aDepartment of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel; bDepartment of Plant Sciences, Weizmann Institute of Science, Rehovot 76100 Israel; and cSchool of Biological Sciences, Monash University, Melbourne, Victoria 3800, Australia

Edited by Elliot M. Meyerowitz, California Institute of Technology, Pasadena, CA, and approved March 9, 2009 (received for review October 27, 2008) The florigen paradigm implies a universal flowering-inducing hor- The tomato plant presents unique opportunities to study multiple mone that is common to all flowering plants. Recent work identified aspects of florigen. Its shoots consist of developmental modules FT orthologues as originators of florigen and their polypeptides as the with homology to monopodial annuals but also feature regular likely systemic agent. However, the developmental processes tar- vegetative/reproductive oscillations typical of woody sympodial geted by florigen remained unknown. Here we identify local balances perennials. Unlike other systems, tomato is photoperiod insensitive, between SINGLE FLOWER TRUSS (SFT), the tomato precursor of thereby eliminating the influence of day length on the functional florigen, and SELF-PRUNING (SP), a potent SFT-dependent SFT inhib- analysis of florigen. Finally, the ease of grafting and a fortunate itor as prime targets of mobile florigen. The graft-transmissible battery of gene mutations can be used to monitor the effects of impacts of florigen on organ-specific traits in perennial tomato show florigen on diverse aspects of growth. Pivotal to the system are SFT, that in addition to import by shoot apical meristems, florigen is encoding florigen (6), and SP, a homologue of TFL1 that promotes imported by organs in which SFT is already expressed. By modulating growth and represses flowering (15–19). Being members of the local SFT/SP balances, florigen confers differential flowering re- same gene family, these 2 CETS (CEN, TFL, SP) genes encode sponses of primary and secondary apical meristems, regulates the signaling factors with multiple options for protein–protein interac- reiterative growth and termination cycles typical of perennial plants, tions (20). For example, the interaction with FD proved essential for accelerates leaf maturation, and influences the complexity of com- the floral-inducing function of FT (21, 22). The interaction of the pound leaves, the growth of stems and the formation of abscission 2 genes with the same proteins (20) and the contrasting flowering PLANT BIOLOGY zones. Florigen is thus established as a plant protein functioning as a modes of primary and sympodial apices of the self-pruning plants general growth hormone. Developmental interactions and a phylo- indicated to us that SP is a major component of the flowering genetic analysis suggest that the SFT/SP regulatory hierarchy is a response mechanism (7). Here we analyze the broad developmental recent evolutionary innovation unique to flowering plants. consequences of changes in the SFT/SP balances as modified, in a context-dependent manner, by the mobile, graft-transmissible form growth hormone ͉ SFT/SP ratio ͉ perennial ͉ compound leaf ͉ of SFT, florigen. Florigen is thus established as a plant protein abscission zone shown to function as a general growth hormone. Results he florigen paradigm was conceived from the study of Tphotoperiod-sensitive plants but implies, in its general form, Context-Specific Termination of Vegetative Growth in Shoot Apical a universal graft-transmissible flowering signal that although Meristems by Florigen. WT tomato plants terminate with a primary activated in leaves by species-specific stimuli is common to all inflorescence after forming 8 to 12 leaves, and subsequent sympo- plants (1–3). Unequivocal evidence for the critical tenets of graft dial units (SUs) consist of 3 leaves and a terminal inflorescence transmissibility and universality of the systemic mechanism was (Fig. 1A). Isogenic sp plants also terminate after 8 to 12 leaves, but obtained in tomato. SFT, the FT homologue encoding florigen subsequent SUs form progressively fewer leaves until the shoot is (4, 5), triggers graft-transmissible signals that complement late terminated by 2 consecutive inflorescences (SI Text, Fig. S1, and flowering in sft plants and substitutes for light dose stimuli in Table S1). It was inferred therefore that the flowering programs for day-neutral tomato and tobacco, for short days in Maryland the primary and sympodial shoots in tomato might be different (7). Mammoth tobacco and for long days in Arabidopsis (6). On the Here we describe the role of SFT and its mobile form, florigen, in the 2 flowering programs. We show that both local SFT and florigen basis of the absence of SFT mRNA beyond the graft joints, we impact differential termination and flowering in the primary and suggested that florigenic signals are generated by cell- sympodial apices. autonomous SFT transcripts. This implicated the protein as a Unlike in sp, termination of the primary shoot in sft is delayed by likely systemic agent (7), which was supported by strong circum- 5 to 6 leaves resulting in a terminating vegetative inflorescence stantial evidence (8–14). However, the developmental mecha- shoot that arrests the sympodial branching, thereby replacing the nisms targeted by florigen to transform vegetative meristems normal sympodial shoot system (SI Text and Fig. S1). Surprisingly, into reproductive organs remain unknown, and their study, by despite the opposite effects of sp and sft on WT shoot architecture, and large, is indifferent to florigen being a protein or RNA. sft single mutants and sft sp double mutants are indistinguishable A clue as to the target of florigen was inferred from the (23). Furthermore, overexpression of SP delays primary termina- observation that overexpression of SFT induces, in addition to tion, increases the number of leaves per SU, and promotes leafy precocious flowering, an overall growth retardation (6). This seemingly trivial phenomenon associated with flowering in many plants might be the consequence of stress upon flowering, but Author contributions: A.S., Y.E., and E.L. designed research; A.S., A.R., A.G., J.P.A., Y.E., and because growth retardation and precocious flowering were trig- E.L. performed research; J.L.B., Y.E., and E.L. analyzed data; and Y.E. and E.L. wrote the gered by a single gene, we hypothesized that they represent 2 facets paper. of the same mechanism. In other words, boosting flowering is just The authors declare no conflict of interest. 1 of the pleiotropic functions of florigen (6). To identify the This article is a PNAS Direct Submission. developmental targets of florigen system-wide, we dissected its 1To whom correspondence should be addressed. E-mail: [email protected]. overall growth effects by using grafting in conjunction with mutants This article contains supporting information online at www.pnas.org/cgi/content/full/ that sensitize organ-specific responses to florigen. 0810810106/DCSupplemental.

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Fig. 1. Florigen and SP-dependent termination of apical meristems. (A) A scheme of a WT main shoot of the tomato plant composed of the primary shoot (leaves 1–10) and reiterated SUs (SL1–3). LS, lateral shoot; PL, primary leaf; SL, sympodial leaf. (B) Precocious primary termination and modes of first sympodial branching in MM 35S:SFT transgenic plants. (B1) Normal resumption of sympodial branching, with the first inflorescence (arrow) positioned correctly between 2 leaves (SI Text). (B2) A prospective sympodial bud was permanently suppressed, and growth resumed from a more distal axillary bud. All subsequent SUs consisted of 3 leaves in both modes. (C) A robust 2-leaf sympodial cycling (numbered) in a receptor VFNT plant stimulated by a grafted 35S:SFT donor (arrow). (D and E) Florigen regulates 2 flowering programs in an SP-dependent manner. (D1) sp 35S:SFT plants terminate after 3 leaves, as do 35S:SFT plants, but the terminal inflorescence consists of just 1 or 2 flowers, and subsequent sympodial branching is completely arrested. (D2) Distal lateral shoots of sp 35S:SFT plants (arrow) form 1 or 2 leaves with ‘‘blind’’ apices as shown here, or with terminal flowers as in D1 (arrow). (E) Systemic induction of flowering and termination in a uf sp receptor shoot (boxes mark grafting joint). The induced shoot is terminated after 3 leaves with 2 consecutive flowers (arrow). (F) Florigen is epistatic to late-flowering inflorescence identity genes. mc bl sp plants terminate after more than 20 leaves with 1 abnormal flower with enlarged sepals, and complete arrest of laterals. mc bl sp 35S:SFT plants terminate prematurely with a similar flower (arrow) and serve as ideal donors of florigen (Fig. S4). (G) The main shoot (few laterals removed) of an ever-vegetative, 75-day-old, 2-m-tall uf sft double-mutant plant. (H) Flowering on a 35S:SFT//uf sft graft. The flowering shoot (circle) arose as a lateral from the axil of a receptor leaf below the graft joint.

inflorescences (17), but these effects are essentially masked in sft cycles in WT plants, 2 doses are required to maintain it under excess plants (Fig. S2). Thus, the terminating effect of sp is largely relevant of SFT, as suggested by the fluctuation in leaf numbers (between only in the presence of a functional SFT. 1 and 3) per SUs in sp/ϩ35S:SFT plants (Table S1). Significantly, In cultivars such as Money Maker (MM) and VFNT (resistant to all features of sp 35S:SFT were also induced in sp receptors by verticillium wilt, fusarium wilt, nematodes, and tobacco mosaic florigenic SFT signals emanating from a grafted 35S:SFT donor virus), primary shoots of WT plants expressing the constitutive (Fig. 1E). 35S:SFT transgene terminate after only 3 leaves (6) (Table S1). In To examine the effect of other flowering genes on the differential contrast, their sympodial program, although initially delayed (Fig. response of the primary and sympodial apices to SFT and florigen, 1B), maintains a typical robust regularity: 3 leaves in MM 35S:SFT we bred additional flowering mutant lines that also expressed the and surprisingly, only 2 leaves in VFNT 35S:SFT (Fig. S2). Thus, 35S:SFT transgene. falsiflora (fals, the tomato LFY), macrocalyx although primary shoots were extremely sensitive to overexpression (mc,anAP1-like), blind (bl), and other mutant lines (Table S2) of SFT, the regularity with which growth and termination alternate expressing the 35S:SFT transgene terminated, like 35S:SFT, after 3 in SUs did not change, although the size of SUs could be shortened. to 4 leaves (Fig. 1F and Fig. S3). However, the phenotypes of their As shown in Fig. 1C, 35S:SFT donors induced a 2-leaf sympodial mutant inflorescences and their proper sympodial patterns were mode in VFNT receptors, indicating that in regulating the size of maintained. The role of SP in checking SFT is therefore unique, such that SFT confers termination but not identity, and this function SUs, SFT and its mobile form are interchangeable. does not require the activity of FALS or MC. The differential response of primary and sympodial apices to To facilitate the analysis of perception and transmission of 35S:SFT (Fig. 1 and Fig. S2) contrasted with their opposite sensi- florigen by the different genotypes, uniflora (uf), a late-flowering, tivity to the inactivation of SP. Whereas florigen, or a 200-fold light-sensitive tomato mutant used previously to examine the excess of SFT (Fig. S3), failed to disrupt the regularity of sympodial universal functions of SFT (6), was combined with sft to generate cycles, this regularity readily collapses in sp plants. To analyze the a tester line that does not flower under any growth condition unless contrasting response of primary and sympodial apices to overex- grafted with a florigen donor (Fig. 1 G and H, SI Text, and Table pression of florigen and to sp inactivation, we bred sp plants S3). Grafting experiments showed that all mutant lines expressing overexpressing SFT. Primary termination in sp 35S:SFT plants also 35S:SFT as graft donors induced efficient flowering in uf sft occurred after 3 to 4 leaves, but the terminating inflorescence receptors and that as receptors, all these mutants responded to meristem produced no or at most 2 flowers, and sympodial branch- mobile florigen (Fig. S4 and Table S3). Thus, the sensitivities of the ing was completely suppressed. Although distal axillary shoots were mutant lines to endogenous SFT and a mobile florigen are com- readily released from apical dominance in these plants, they pro- parable. Together these results indicate that the tested genes are not duced only 1 or 2 leaves before terminating with a single flower or required for the mobility or perception of florigen and reveal that a blind apex (Fig. 1D). SFT is therefore a potent terminator of the differential response of primary and sympodial meristems to primary, sympodial, and inflorescence apices, but its role in sym- florigen is maintained under a wide range of genetic backgrounds. podial and flower meristems is checked primarily by SP. Although Evidence pertinent to the 2 flowering programs in Arabidopsis and 1 dose of SP in sp/ϩ heterozygotes is sufficient to maintain the 3-leaf other plants is discussed in Fig. S2.

2of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0810810106 Shalit et al. Downloaded by guest on September 30, 2021 were almost devoid of folioles. Strikingly, leaves of sp 35S:SFT A B plants were reduced to only 1 pair of lateral leaflets, and some were simple with entire margins (Fig. 2B). The arrest of lateral leaflet meristems suggests an early function of SP during leaf primary morphogenesis. In contrast, overexpression of SP induced larger blades with undulating surfaces, indicative of unregulated growth (Fig. 2B), an effect that was not displayed by sft 35S:SP plants (Fig. C D S2). As shown in Fig. 2C, all of these features were also induced by mobile SFT, suggesting that florigen is imported by leaves, where SFT is already expressed (see below), and arrest leaflet formation in an SP-dependent manner. Thus, in sft and sp 35S:SFT (i.e., under low or high SFT/SP ratios, respectively), SFT, SP, and florigenic SFT function as leaf meristem factors. The response of stem meristems to different doses of SFT and SP is demonstrated in Fig. 2A. Although normal sympodial EF H I cycling in 35S:SFT plants resumed after primary termination, normal radial expansion of sympodial internodes was permanently suppressed, indicating a context-specific function of florigen. In general, a high SFT/SP ratio promoted growth restric- G tion at the shoot apical meristems (SAMs) and lateral leaflet meristems, leading to a faster floral transition and reduced leaf complexity respectively. But higher SFT/SP ratio as in sp 35S:SFT plants resulted in the complete suppression of both Fig. 2. Florigen regulates termination of sympodial and leaf meristems in an vegetative and inflorescence meristems (Fig. 1D). SP-dependent manner. (A) Florigen regulates radial expansion of the stems. Note the effect of sft and of over-expression of SP on stem girth. (B and C) SFT Florigen Mediates Its Own Distribution by Regulating Sink–Source and florigen regulate leaflet meristems in an SP-dependent manner. (B) Relations. The reiterated phase transitions along the tomato Compound leaves with different SFT/SP ratios. WT leaves have a terminal leaflet, 3 to 5 pairs of independently formed lateral leaflets, and late inter- shoot and its evergreen nature and day length insensitivity PLANT BIOLOGY calary folioles (24). sft leaves feature elongated rachises, an increased number require that the distribution of florigen be regulated by a of folioles, and extended leaflet petioles. 35S:SFT leaves are proportionally dynamic balance on a daily basis. To characterize the balance smaller, with some reduction in leaflet number. The seventh leaf is shown. The between the dynamic needs for endogenous local SFT and its third to fifth sp 35:SFT leaves, respectively, lost the majority of leaflets and mobile form in the shoot system, we determined the minimal folioles, and their blade margins were smooth. 35S:SP blades display in- source required to induce a flowering response in the tomato terveinal bulging, indicative of ectopic cell proliferation. (C) Long-range, bush. A single mature 35S:SFT donor leaf induced flowering in SP-dependent regulation of leaf morphology by florigen. uf sp leaf of a sft uf receptor shoots for 2 months and in apices 2 m above the control homograft, and a receptor uf sp shoot grafted with a 35S:SFT donor. graft points (Fig. 2D). Therefore, every mature leaf is capable of (D–I) The florigen balance in the tomato perennial bush. (D) Systemic induction of flowering by a single leaf. Left:auf sft receptor shoot of a single leaf exporting florigen to all parts of the tomato bush. graft (see Fig. S2) formed its first flowers (circle) after 19–21 days and 7 to 8 The developmental expression of SFT and SP at the whole-plant leaves. Secondary and tertiary branches continued to generate flowers for Ϸ2 level was studied by comparing each gene’s age-dependent expres- months. By that time, the receptor had developed 30 mature leaves (Right) sion gradients within sequentially growing leaves along primary and Ϸ100 leaves altogether. (E) Opposite age-dependent expression gradi- shoots. Initially a series of leaves was collected from WT VFNT ents of SFT and SP in tomato leaves. RNA was extracted from consecutive seedlings, having 10 leaves larger than 1 cm and a primordial leaves of seedlings with 10 leaves (numbered) and primordial inflorescences. inflorescence. Leaves of all ages were collected4hafterdawn, Leaf no. 10 is 0.5–1.0 cm long. Expression profiles of other relevant genes are corresponding to the diurnal SFT peak (Fig. S3). As shown in Fig. also shown, including SP2G and SP5G, 2 CETS genes with no known role in 2E, SFT and SP display opposing age-dependent expression gra- flowering. (F and G) SFT and SP are not involved in an intergenic regulatory loop. (F) Expression gradients of SFT or SP are maintained in mutant plants of dients in which SFT RNA was relatively high in expanded mature the opposite genotype. (G) Expression profiles of the endogenous SFT and SP leaves and SP RNA relatively high in the immature leaves. For genes are not altered in 35S:SP and 35S:SFT plants, respectively. (H) Intraleaf comparison, profiles of other relevant genes were also included gradients of SFT or SP. Top: expression gradients of SFT and SP along the rachis (Fig. 2E). Note that the age-dependent gradients of SFT and SP are of 5-cm-long primary leaves. TerL, a terminal leaflet; PI-PIII, leaflet pairs. independent of plant age, and similar series of leaves taken from Bottom: Intraleaf expression gradients of SFT persist in Ϸ15-cm-long leaves postflowering plants will display the same gradients. but level off in 25-cm long leaves. (I) Inactivation of SP sensitizes the response As shown in Fig. 2F, the expression patterns of SP were not of shoot and leaf meristems to mobile florigen. sp 35S:SFT donor shoot with altered in sft plants and vice versa, suggesting that these patterns are 4 leaves and all potential growing points removed, grafted onto a uf sft sp not interdependent. We next explored the possibility that overex- stock. Note the termination by 2 consecutive inflorescences and the reduced complexity of the top compound leaves of the receptor shoot. pression of one of the genes will affect the endogenous expression of the other (Fig. 2G). Here, the expression of 35S:SFT or 35S:SP results in high (200-fold and more) but equal expression in all leaves; the endogenous differential expression of SP and SFT, Florigen Regulates Stem and Leaf Meristems in an SP-Dependent respectively, in old and young leaves was maintained. We therefore Manner. Because of their developmental versatility, the compound inferred that the functional antagonism between SFT and SP does leaves of tomato provide a highly sensitized forum to illustrate the not primarily involve mutual transcriptional feedback loops. effects of florigen as a general growth hormone (24) (Fig. 2B). SP Age-dependent gradients were also evident in leaflets along the and sp leaves of isogenic lines were indistinguishable except for a proximal–distal axis of immature leaves up to 15 cm long. But when slightly reduced serration in the sp leaf margins. However, sft leaves leaves reached approximately three quarters of their final size displayed a distinct morphology and spacing of leaflets and carried (approximately 25 cm), all their major leaflets expressed SFT additional folioles, suggesting a role for SFT in regulating midrib equally (Fig. 2H). meristems (Fig. 2B). Leaves of 35S:SFT plants, although maintain- Removal of mature leaves delays flowering in tomato (7), and we ing a compound architecture, usually lacked 1 pair of leaflets and speculated that by the time intraleaf expression gradients have

Shalit et al. PNAS Early Edition ͉ 3of6 Downloaded by guest on September 30, 2021 flowering, failed to generate graft-transmissible florigenic signals A B C (see Fig. S5 for further discussion).

Florigen Links the Transition to Flowering with Leaf Architecture. To understand the genetic basis for phenotypic responses resulting from changing SFT/SP ratios, we examined the effects of different SFT/SP balances in mutant backgrounds with altered leaf morphology. Leaves of trifoliate (tf) plants form only 1 pair of lateral leaflets (24), but we observed that leaves of tf sp plants gradually lose their lateral leaflets, resembling the sequential D E reduction of leaflets in the compound leaves of the flowering rose shoots (Fig. 4A). A dysfunctional TF therefore sets higher sensitivity thresholds for changing SFT/SP ratios. The effect of sft on the trifoliate leaf is analogous to its mild effect on WT leaves (compare Fig. 4B with Fig. 2B), but if SFT is inactivated in tf sp background the effect of sp is completely suppressed: tf sft sp and tf sft leaves were indistinguishable from each other (Fig. 4B). Significantly, sft and sft sp leaves were also indistinguishable (Fig. S2), suggesting that the allelic status of SP is irrelevant in sft leaves as in sft shoots (Table S1). Fig. 3. Restrictions on the movement of florigen in the perennial tomato bush. However, when SFT was overexpressed in tf (i.e., tf 35S:SFT), (A) Florigenic potential of tagged-SFT proteins and of SFT driven by specific a functional SP, as expected of the sensitized tf background, was ϾϾ promoters via either direct (:) or transactivation ( ). (B and C) Expression of no longer sufficient to support the formation of lateral leaflets, pSUC2 is confined to the companion cells (B) of the phloem strands (C) of mature veins. (D and E) Expression by pBLS is limited to young blades (D) and is excluded and almost all leaves were simple (Fig. 4D and Fig. S4). As shown from mature veins (E). For the detailed analyses of these patterns, see Fig. S5. in Fig. 4E, tf did not affect the expression profiles of SFT or SP. To determine whether mobile florigen also inhibits leaflet meristems in the sensitized tf leaves, tf 35S:SFT donor shoots leveled off (Fig. 2H), leaves become better exporters of florigen. were grafted onto tf and tf sft sp receptors. Both receptors bear Because SP promotes growth, its inactivation, or an elevated trifoliolate leaves (Fig. 4B) and have a similar SFT/SP balance, SFT/SP ratio brought about by imported florigen, are likely to but with functional and dysfunctional SFT and SP genes, respec- accelerate leaf maturation, converting leaf status from sink to tively. As shown in Fig. 4 C and D, florigen induced simple leaves source and leading to early release of florigen. This conjecture is in both tf and tf sft sp receptors. In addition, shoots of the tf supported by experiments comparing 35S:SFT and sp 35S:SFT receptors shifted to 2-leaf SUs, as in tf 35S:SFT (Fig. 4C). In tf donors. Donors of both genotypes were either regular-growing sft sp receptors, florigen complemented the sft gene and induced shoots with leaves, axillary buds and growing apices, or 2-leaf stem tf sp-like shoots. Thus, trifoliate leaves monitor both SFT/SP and sections with apices and axillary buds removed. Regular sp 35S:SFT sft/sp as balanced 1:1 ratios, and both genotypes are similarly donors were significantly more effective inducers of both uf sft and modified by imported florigen, which is the ultimate manifes- uf sft sp receptors, and, as expected, sp receptors were more tation of the florigen-dependent SFT/SP regulatory hierarchy. responsive (compare Fig. 2 D and I). However, when sp and SP The tomato leaf is initiated as a terminal leaflet and an donors having mature leaves only were compared, they were equally elongated rachis. Independent pairs of lateral leaflets, each effective (Table S3). Therefore, the superiority of sp donors can be capable of duplicating the compound pattern, are then formed attributed to their early maturation: 35S:SFT donors continue to (24). In WT and tf, SFT and florigen regulate, in an SP- dependent context (Fig. 2), the arrest of lateral leaflets in a generate sink organs in the form of new branches, but branching is gradient opposite to their formation. The same is observed in tf suppressed in sp 35S:SFT donors (Fig. 1D). Florigen can therefore sp or tf leaves importing florigen or overexpressing SFT (Fig. 4). be seen as a hormone that by regulating leaf maturation in an In all these cases the terminal leaflets and their elongated rachis SP-dependent manner, adjusts its own distribution at the whole- are unaffected, suggesting distinct regulatory mechanisms for plant level. the initiation of classes of leaflets. We surmise that florigen, Restrictions on the long-range function of FT are imposed by mediated by the SFT/SP ratio, regulates the leaflet initiation the size of the protein (9, 10, 12, 13). In tomato, 35S:SFT with gradient in conjunction with auxin (see SI Text and below). C-terminal translational fusions of GFP, RFP, and GR induced precocious primary flowering and, in an sp background, a typical Florigen Links the Vegetative/Reproductive Balance in the Inflores- arrest of lateral leaflets. It was, however, impossible to visualize cence with the Generation of Abscission Zones. Abscission zones fluorescent signals in the respective transgenic plants. In agree- (AZs) mark the sites where plant organs, particularly fruits and ment with results in Arabidopsis (13), all 3 fusion constructs leaves (Fig. 5A), are eventually separated from the main body of failed to generate graft-transmissible flowering signals when the plant. The formation of AZs is regulated by day length, grafted onto uf sft sp receptors (Fig. 3A). Confirming previous auxin, and ethylene, and the deciduous habit is considered an results (9), flowering was delayed in uf tomato or Arabidopsis important innovation of angiosperms (25, 26). In tomato, for- plants expressing 35S:amiR-FT/SFT (Fig. S3), generating in both mation of floral pedicel AZs is preceded by site-specific cell species effects that were similar to conventional loss of function. divisions (27), and AZs are completely missing from jointless1 As shown in Table S3, it was possible to exploit the advantages (j1) and j2 pedicels (Table S2). of the tomato system and to restore flowering when uf 35S:amiR- mc, sft, and bl condition partial vegetative inflorescences FT/SFT receptors were grafted with 35S:SFT donors. similar to those seen upon overexpression of SP (17) (Table S2). Additional restrictions on the long-range effects of florigen were In the course of studying the interactions between mc, bl, and imposed by the cells and tissues in which SFT is expressed (Fig. 3 florigen, we noticed that AZs in floral pedicels of mc, bl, and sft A–E and Fig. S5). In contrast to the 35S and the phloem-specific were incomplete, irregular, or mislocated. We found that similar SUC2 promoters, expression of SFT, driven by 3 early leaf-specific to j1, floral pedicels of sft mc, sft bl,ormc bl double mutants promoters (BLS, FIL, and 650), although inducing precocious completely lacked AZs (Fig. 5 B and C), indicating that mc and

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Fig. 5. Florigen links the formation of AZs with inflorescence genes. (A) C D Normal AZ of mature tomato fruit. (B) mc bl floral pedicels produce no AZs. (C) SFT is required for the formation of AZs in an mc background. (D) Graft- transmissible signals donated by a 35S:SFT scion restore AZs in mc sft floral pedicels. (E) Genes involved in generating floral AZs are expressed in WT floral pedicels. YP, young pedicels before AZs can be observed; MP, mature pedicels with developed AZs; YF, young flowers with pedicels removed. (F) Summary of long-range complementation tests of AZs by florigen. PLANT BIOLOGY

corresponding proteins did not interact in any combination in yeast 2-hybrid tests (data not shown). E AZs serve as a classical example of a morphogenetic trait regulated by auxin (25, 26). Auxin is also involved in regulating radial expansion of stems, formation of leaflet meristems, and various forms of apical dominance (28, 29), all shown here to involve florigen. One possible scheme by which florigen and auxin interact to regulate the sympodial cycles and leaf architecture is discussed briefly in the SI Text. It is expected that the interactions between the 2 hormones in regulating the growth balance in shoot systems will attract much attention in coming years. Discussion Fig. 4. Leaf architecture in tf plants is determined by the florigen-dependent, SFT/SP regulatory hierarchy. (A) Left: A flowering tf plant. All leaves have 3 Florigen as a Growth Hormone and the Evolution of the SFT/SP leaflets. Middle:Atf sp flowering shoot with a gradual reduction in leaf com- Regulatory Hierarchy. The need for plant organs to respond plexity. Right: Stepwise elimination of leaflets in compound leaves of the garden effectively to changing environmental signals dictates quantita- rose toward flowering. (B) Inactivation of TF sensitizes the growth response of tive regulatory schemes with inherent potentials for reversibility. leaves to changing SFT/SP ratios (ratios are listed below the corresponding im- Such tenets are satisfied by florigen functioning as a general ages). SFT/SP ratio (tf, far left) and sft/sp ratio (tf sft sp, far right) result in similar growth hormone. SFT and SP modulate a variety of signaling trifoliolate leaves. High ratios, as in overexpression of SFT or inactivation of SP (leaves 3 and 4 from left, respectively) induced simple LANCEOLATE-like leaves pathways but are not directly involved in the fate of cells or (24, 36). Note that tf sft leaves (second from left) have low SFT/SP ratio but are organs. Rather, they regulate the balance of diverse growth indistinguishable from tf sft sp leaves (right-most), and both have extended processes (20) and thus facilitate the potential for plasticity in rachises and additional leaflets characteristic of sft leaves (Fig. 3). (Cand D) Mobile growth, not much different from the proposed role of the florigen modulates the SFT/SP balance to generate 2 leaf SUs and to arrest leaflet Notch/Wnt system that pleiotropically modulates multiple sig- meristems. (C) Florigen donor induced slender stems, simple leaves, and a reduced naling processes but is not essential for any of them (30). number of leaves per SU in a tf receptor (Inset). (D) A systemic reconstitution of FT is an integrator of all flowering pathways (4), but as shown high SFT/SP balance in tf leaves. Left: The contribution of florigen by a WT donor here (Figs. 1, 2, and 4), SFT is imported by organs in which it is is insufficient to reduce the complexity of tf receptor leaves. Middle: Systemic already produced, with the exception of the SAM. It was recently reconstitution of a high SFT/SP ratio in tf sft leaves induces simple leaves. Right: Mobile florigen elevates the level of SFT in tf sft sp leaves, thereby inducing suggested that genes of the autonomous pathway carry pleiotropic ‘‘reversion’’ to a simple architecture. (E) tf does not affect expression gradients of vegetative functions (31). Mutations in SOC1 and FUL, 2 major flowering genes. flowering genes in Arabidopsis, affect vegetative functions (32). Likewise, GA promotes flowering (33) and modifies growth. Given our finding that florigen is not only a flowering-specific agent, perhaps there are no designated flowering genes at all. One reason bl act redundantly with sft in specifying these tissues. Grafting could be that ‘‘flowering’’ is an arbitrary external description (34) experiments showed that mobile florigenic signals, which com- that does not match the internal description of the plant. By the plement sft, penetrate floral pedicels to rescue AZs in sft mc and same token, the mechanism requiring movement of florigen from sft bl pedicels (Fig. 5 D and F). Although SFT, SP, MC, BL, and leaves to apices (21, 22) may not be specific to flowering. Rather it J1 are all expressed in pedicels and floral buds (Fig. 5E), the is one aspect of a more general mechanism in which the florigen

Shalit et al. PNAS Early Edition ͉ 5of6 Downloaded by guest on September 30, 2021 hormone is exploited to change local SFT/SP balances. If correct, florigen may regulate flowering in species in which SFT orthologues are normally expressed in apices, just as it regulates leaflet meristems in the compound leaf. In shoots and leaves, meristems react to florigen in an SP- dependent manner. Here we offer an evolutionary scenario for the generation of this mechanism in flowering plants. Consider the following. (i) Overexpression phenotypes define SFT as a growth retardant (6). It terminates primary SAMs, sympodial SAMs, and inflorescence meristems and arrests stem and leaflet meristems (Figs. 1, 2, and 4). (ii) The arrest of meristematic activities by Fig. 6. Genes of the SP/TFL1/CEN clade are not found in nonflowering plants. SFT/florigen is alleviated by even the low WT level of SP. SP is CETS genes from EST databases and sequenced genomes of land plants (moss, therefore an amazingly potent inhibitor of SFT. (iii) SP is function- red; lycophytes, orange; gymnosperms, blue; angiosperms, green) were sub- ally relevant only in the presence of a functional SFT, and con- jected to Bayesian phylogenetic analyses (for the complete gene phylogeny versely, its inactivation is irrelevant without a functional SFT.(iv) and a detailed evolutionary analysis, see Fig. S6). The resulting tree was rooted Expression of FT orthologues is correlated with photoperiod bud with moss sequences. Four distinct clades were identified: MFT, a putative growth suppressor, is present in all lineages; SFT/FT, the universal precursor of setting in slow-growing conifers (35). With their highly conductive florigen, is present in all flowering plants and highly related to the gymno- vasculature, fast-growing meristems, and need for a rapid response sperms FT-like; the SP/TFL1/CEN clade is unique to flowering plants. W, Y, and to environmental signals, the ability of flowering plants to exploit H are amino acids characterizing the ligand-binding pocket of the 3 major new habitats likely required high levels of FT/florigen, but these clades (37, 38). levels were also detrimental. We speculate that SP evolved specifically to alleviate the detrimental effects of SFT. This is supported, as shown in Fig. 6, by the absence of genes of the SP/TFL1/CEN formed as previously described (6). A list of primers, fragment sizes, and clade from all known nonflowering plant genomes. number of cycles for each gene is given in Table S4. Confocal imaging and microscopy were performed as described previously (39). Methods ACKNOWLEDGMENTS. We thank Sheila McCormick, Zach Lippman, and Plants were grown and grafted as specified previously (6). Monogenic lines Benny Horowitz for their critical reading of the manuscript. This work was and WT cultivars were obtained from the Rick Center at University of Califor- supported by grants from the International Science Foundation (ISF) and nia, Davis. Combinations mentioned in the text and in Tables S1–S3 were the Human Frontier Science Program (to E.L.) and from the ISF and MINERVA specifically bred for this work. Cloning, RNA isolation, and PCR were per- (to Y.E.).

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