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

Abstract

The florigen paradigm implies a universal flowering-inducing hormone that is common to all flowering plants. Recent work identified FT orthologues as originators of florigen and their polypeptides as the likely systemic agent. However, the developmental processes targeted by florigen remained unknown. Here we identify local balances between SINGLE FLOWER TRUSS (SFT), the tomato precursor of florigen, and SELF-PRUNING (SP), a potent SFT-dependent SFT inhibitor as prime targets of mobile florigen. The graft-transmissible impacts of florigen on organ-specific traits in perennial tomato show that in addition to import by shoot apical meristems, florigen is imported by organs in which SFT is already expressed. By modulating local SFT/SP balances, florigen confers differential flowering responses of primary and secondary apical meristems, regulates the reiterative growth and termination cycles typical of perennial plants, accelerates leaf maturation, and influences the complexity of compound leaves, the growth of stems and the formation of abscission zones. Florigen is thus established as a plant protein functioning as a general growth hormone. Developmental interactions and a phylogenetic analysis suggest that the SFT/SP regulatory hierarchy is a recent evolutionary innovation unique to flowering plants.

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.

Fig. 2.

Florigen regulates termination of sympodial and leaf meristems in an 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 and florigen regulate leaflet meristems in an SP-dependent manner. (B) Compound leaves with different SFT/SP ratios. WT leaves have a terminal leaflet, 3 to 5 pairs of independently formed lateral leaflets, and late intercalary folioles (24). sft leaves feature elongated rachises, an increased number of folioles, and extended leaflet petioles. 35S:SFT leaves are proportionally smaller, with some reduction in leaflet number. The seventh leaf is shown. The third to fifth sp 35:SFT leaves, respectively, lost the majority of leaflets and folioles, and their blade margins were smooth. 35S:SP blades display interveinal bulging, indicative of ectopic cell proliferation. (C) Long-range, SP-dependent regulation of leaf morphology by florigen. uf sp leaf of a control homograft, and a receptor uf sp shoot grafted with a 35S:SFT donor. (D–I) The florigen balance in the tomato perennial bush. (D) Systemic induction of flowering by a single leaf. Left: a uf sft receptor shoot of a single leaf graft (see Fig. S2) formed its first flowers (circle) after 19–21 days and 7 to 8 leaves. Secondary and tertiary branches continued to generate flowers for ≈2 months. By that time, the receptor had developed 30 mature leaves (Right) and ≈100 leaves altogether. (E) Opposite age-dependent expression gradients of SFT and SP in tomato leaves. RNA was extracted from consecutive leaves of seedlings with 10 leaves (numbered) and primordial inflorescences. Leaf no. 10 is 0.5–1.0 cm long. Expression profiles of other relevant genes are also shown, including SP2G and SP5G, 2 CETS genes with no known role in 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 the opposite genotype. (G) Expression profiles of the endogenous SFT and SP genes are not altered in 35S:SP and 35S:SFT plants, respectively. (H) Intraleaf gradients of SFT or SP. Top: expression gradients of SFT and SP along the rachis of 5-cm-long primary leaves. TerL, a terminal leaflet; PI-PIII, leaflet pairs. Bottom: Intraleaf expression gradients of SFT persist in ≈15-cm-long leaves but level off in 25-cm long leaves. (I) Inactivation of SP sensitizes the response of shoot and leaf meristems to mobile florigen. sp 35S:SFT donor shoot with 4 leaves and all potential growing points removed, grafted onto a uf sft sp stock. Note the termination by 2 consecutive inflorescences and the reduced complexity of the top compound leaves of the receptor shoot.

Fig. 3.

Restrictions on the movement of florigen in the perennial tomato bush. (A) Florigenic potential of tagged-SFT proteins and of SFT driven by specific promoters via either direct (:) or transactivation (≫). (B and C) Expression of 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 from mature veins (E). For the detailed analyses of these patterns, see Fig. S5.

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 leaflets. Middle: A tf sp flowering shoot with a gradual reduction in leaf complexity. Right: Stepwise elimination of leaflets in compound leaves of the garden rose toward flowering. (B) Inactivation of TF sensitizes the growth response of leaves to changing SFT/SP ratios (ratios are listed below the corresponding images). SFT/SP ratio (tf, far left) and sft/sp ratio (tf sft sp, far right) result in similar 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 (24, 36). Note that tf sft leaves (second from left) have low SFT/SP ratio but are indistinguishable from tf sft sp leaves (right-most), and both have extended rachises and additional leaflets characteristic of sft leaves (Fig. 3). (C and D) Mobile florigen modulates the SFT/SP balance to generate 2 leaf SUs and to arrest leaflet meristems. (C) Florigen donor induced slender stems, simple leaves, and a reduced number of leaves per SU in a tf receptor (Inset). (D) A systemic reconstitution of high SFT/SP balance in tf leaves. Left: The contribution of florigen by a WT donor is insufficient to reduce the complexity of tf receptor leaves. Middle: Systemic 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 “reversion” to a simple architecture. (E) tf does not affect expression gradients of flowering genes.

Fig. 5.

Florigen links the formation of AZs with inflorescence genes. (A) 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.

Fig. 6.

Genes of the SP/TFL1/CEN clade are not found in nonflowering plants. CETS genes from EST databases and sequenced genomes of land plants (moss, red; lycophytes, orange; gymnosperms, blue; angiosperms, green) were subjected to Bayesian phylogenetic analyses (for the complete gene phylogeny and a detailed evolutionary analysis, see Fig. S6). The resulting tree was rooted with moss sequences. Four distinct clades were identified: MFT, a putative growth suppressor, is present in all lineages; SFT/FT, the universal precursor of florigen, is present in all flowering plants and highly related to the gymnosperms FT-like; the SP/TFL1/CEN clade is unique to flowering plants. W, Y, and H are amino acids characterizing the ligand-binding pocket of the 3 major clades (37, 38).