The making of a compound inflorescence in tomato and related nightshades

Abstract

Variation in the branching of plant inflorescences determines flower number and, consequently, reproductive success and crop yield. Nightshade (Solanaceae) species are models for a widespread, yet poorly understood, program of eudicot growth, where short side branches are initiated upon floral termination. This "sympodial" program produces the few-flowered tomato inflorescence, but the classical mutants compound inflorescence (s) and anantha (an) are highly branched, and s bears hundreds of flowers. Here we show that S and AN, which encode a homeobox transcription factor and an F-box protein, respectively, control inflorescence architecture by promoting successive stages in the progression of an inflorescence meristem to floral specification. S and AN are sequentially expressed during this gradual phase transition, and the loss of either gene delays flower formation, resulting in additional branching. Independently arisen alleles of s account for inflorescence variation among domesticated tomatoes, and an stimulates branching in pepper plants that normally have solitary flowers. Our results suggest that variation of Solanaceae inflorescences is modulated through temporal changes in the acquisition of floral fate, providing a flexible evolutionary mechanism to elaborate sympodial inflorescence shoots.

Figure 1. Solanaceae Inflorescences and Mutant Phenotypes

(A) Pepper plant showing single-flower inflorescence and mature fruit (inset). (B) Tomato plant and inflorescence (red ring) showing zigzag growth (lower inset) and maturing fruits (upper inset). (C) Branched inflorescence of the species S. crispum. (D) Mutant, highly branched inflorescence of s in a mixed genotype with the wild tomato species S. pennellii. (E) Mutant inflorescence of a second allele, s-multiflora, having flowers (blue arrows) mixed with cauliflower-like tissue. (F) Mutant, branched an-classic inflorescence with cauliflower-like tissue in place of flowers. (G) A weaker an allele with sepal and carpelloid tissue. (H) Mutant, branched fa inflorescence (dashed box) with leaves in place of flowers. Scale bars in (A, B, C, D, H), 5 cm; in insets (E, F, and G), 1 cm.

Figure 2. Early Branching Patterns of Normal and Mutant Inflorescences

Scanning electron micrographs and schematics of inflorescence development. Schematics reflect sequential inflorescence sympodial units (ISU) each composed of a SIM branch (colored line with arrow) that terminates with a flower (FM, colored oval). Colored circles in micrographs reflect corresponding structures in schematics. (A) Two stages of sympodial inflorescence development and mature zigzag inflorescence. (B) s inflorescences develop extra SIMs due to mutations in the ortholog of WOX9 (red rectangle = homeodomain; mutations marked by red arrows = s-classic and s-multiflora). Additional SIMs (colored circles) eventually form flowers. Black asteriks reflect asymmetrical development of meristem branches (black arrows in schematics). (C) Strong alleles of an, defective in the tomato ortholog of UFO, produce extra SIMs instead of flowers (blue rectangle = F-box domain; mutations marked by red arrows). Same color dots and lines reflect SIMs of a similar stage that become branches of the mature inflorescence (see Figure S1 for more details). (D) S. crispum inflorescences showing an s-like SIM branching pattern (colored dots/asteriks reflect interpretation of sequential SIM production similar to the convention in (B)). The youngest inflorescence (left) has already produced three SIMs from the leading SIM (red dot), and each of these elaborates further (middle). A later stage inflorescence (right) shows more than 50 maturing flowers. As seen in s, the number and position of lateral SIMs derived from leading meristems varies between inflorescences, as does the position of differentiating flowers (see Figure S2 for more details). L = leaf; SYM = sympodial shoot meristem. Scale bars, 100 μm.

Figure 3. Cloning of the compound inflorescence (s) and anantha (an) Genes

(A) The 15-cM region of tomato chromosome 2 where s was positioned previously showing overlap with the S. pennellii introgression line segments IL2–3/2–4. (B) Tomato markers (black font) spanning the s mapping interval and corresponding homologues from Arabidopsis showing synteny with genes on chromosome 1. Markers in red were designed according to this synteny, three of which helped to delimit s to a 0.3 cM window based on three recombination crossover events (denoted by ‘X'). Two co-segregating markers (‘0') were used to isolate a BAC (blue bar), which provided an additional marker (blue font) to the right of s. (C) Physical synteny expanded from 0.3 cM of tomato in four Eudicot species sharing at least seven genes (dashed lines) with three other genes syntenic between a subset of species (solid gray lines). Tomato unigene sequences used to design syntenic PCR markers are in parentheses. Some homologues vary in size, which is due to differences in structural predictions. The three transcription factors (red fonts), two Apetala2-like (AP2) and a Wuschel-homeobox (WOX) were the primary candidate genes for s. (D) Detailed view of the S gene structure compared to its Arabidopsis homologue WOX9/STIMPY. The highly conserved 65–amino acid homeodomain is shown in red, and the two alleles of s are indicated with the nucleotides that changed (below the red bar; red font) from wild type (above the red bar; black font). The classic allele of s (LA3094; s-classic) and s-mult (LA0560) had missense mutations altering two invariant amino acids in the homeodomain. Our fast neutron induced allele (s-n5568) seems to have suffered a promoter deletion or rearrangement (Figure S3). (E) Detailed view of tomato AN, pepper AN (Ca-AN), and their orthologs from Antirrhinum majus (FIMBRIATA; FIM) and Arabidopsis (UNUSUAL FLORAL ORGANS; UFO). The F-box domain is shown in blue, and five alleles of an are indicated with their DNA sequence changes shown below the blue bar. Three strong alleles had premature stop codons due to a 62bp duplication-induced frameshift (an-e1546, red arrows), a single base deletion (an-e3430, red dash), and a nonsense mutation (an-e0002, red font). Three weak alleles (an-e1444, an-e4365, Ca-an) had missense mutations, and an-classic (LA0536) suffered from a local rearrangement (Figure S3).

Figure 4. Inflorescence Variation in Domesticated Tomatoes Is Due To Independently Arisen Alleles of s

The s-classic allele was first described 100 years ago as a highly branched variety called “Wonder of Italy,” and garden varieties resembling s remain popular for their aesthetic value and prolific fruit production [38]. Six thousand domesticated varieties were screened for inflorescence variation and 23 lines exhibited highly compound inflorescences. Among the 23 lines, at least 15 represented distinct genetic backgrounds based on differences in fruit size, shape, color, and quantitative variation in branch number. (A) Phenotypic variation from three distinct varieties is shown. Core Collection line 2064 (CC2064) was extremely compound as a result of more than 200 branching events, whereas CC944 and CC3381 branched less often, and CC3381 also developed leaves within the inflorescence (B) Variation in fruit size, shape, and color highlighting the different genetic backgrounds of the varieties with compound inflorescences. Varieties with names are indicated. (C) Cleaved amplified polymorphic sequence (CAPS) PCR genotyping assay showing that all except one of 23 varieties with compound inflorescences carry the s-classic allele. CC5721 (white asterisk), which carries the identical lesion as s-classic, arose independently from a distinct progenitor line (see text for details). Controls were varieties with weak (5–10 branching events) or no branching. Rose Quartz Multiflora was confirmed by complementation test to be an allele of s, and arose independently as a result of a genomic rearrangement (Figure S3). Scale bar in (A), 1 cm.

Figure 5. Expression Patterns of Three Inflorescence Architecture Genes

(A) RT-PCR of S, AN, and FA transcripts in normal tissues. (B–E) Detection of S and AN by in situ hybridization. Upper right denotes probe; lower left, genotype. (B) Longitudinal section of a normal inflorescence showing S expression in an immature SIM (lower red arrow), but not in a more advanced ISU (upper blue arrow). Weak expression is observed between sepal and petal primordia in flowers (black arrows). (C) Close-up of similarly staged section from (B). (D) Longitudinal section showing AN expression in an incipient FM in the upper ISU (blue arrow). Expression is absent in the lower immature SIM (red arrow). AN is also expressed between sepal and petal primordial. (E) Close-up of a similarly staged section. (F) RT-PCR of S, AN, and FA in normal and mutant infloresences (IF). (G) Expression of S in an mutants marking SIMs that remain in a pre-floral state. (H) In situ hybridization with whole-mounted tissue from an s mutant; AN expression in an advanced SIM (blue arrow), but not in a less mature SIM below (red arrow), matching a similarly staged section (inset). (I) Sequential transient expression of S and AN. The first SIM (SIM1) expresses S (S₁) and initiates the first phase of the maturation of ISUs. This expression is transient as it turns off prior to activation of AN (AN₁) during the second phase of maturation, which occurs in the same ISU (ISU-1). A newly formed SIM (SIM2) emerges laterally marked by a new round of S expression (S₂), which begins maturation of ISU-2, and this process reiterates to produce a multi-flower inflorescence. (J) Schematic for temporal development (color gradient in bar) over time (position in bar) of a normal (WT) ISU. The SIM (yellow) is short-lived and transitions rapidly (orange) to a FM (red) via activity of S (black line above yellow) followed by a short period of expression from AN (black line above orange). Mutant ISUs of s temporarily stall as SIMs (extended yellow bar) allowing extra SIMs to develop before terminating in FMs. an mutants remain in a pre-floral state (extended yellow bar with S expression) enabling SIMs to elaborate indefinitely. Se = sepals; St = stamens.

Figure 6. Single-Flower Inflorescence Branching and the an Mutant of Pepper (Ca-an)

(A) Mutant vegetative inflorescence (red ring) of the tomato sft mutant showing an isolated flower. (B and C) Double mutant inflorescences (red rings) and flowers (insets) from sft:an double mutants with a weak an allele (B) and a strong an allele (C) showing the conversion of a flower into an inflorescence 3–4 branching events (red arrows). (D) The single flower pepper inflorescence. (E) Mutant, Ca-an inflorescence and a highly branched example (F) from a mixed genetic background (three branches are marked with red arrows)