Floral patterning in Lotus japonicus

Abstract

Floral patterning in Papilionoideae plants, such as pea (Pisum sativum) and Medicago truncatula, is unique in terms of floral organ number, arrangement, and initiation timing as compared to other well-studied eudicots. To investigate the molecular mechanisms involved in the floral patterning in legumes, we have analyzed two mutants, proliferating floral meristem and proliferating floral organ-2 (pfo-2), obtained by ethyl methanesulfonate mutagenesis of Lotus japonicus. These two mutants showed similar phenotypes, with indeterminate floral structures and altered floral organ identities. We have demonstrated that loss of function of LjLFY and LjUFO/Pfo is likely to be responsible for these mutant phenotypes, respectively. To dissect the regulatory network controlling the floral patterning, we cloned homologs of the ABC function genes, which control floral organ identity in Arabidopsis (Arabidopsis thaliana). We found that some of the B and C function genes were duplicated. RNA in situ hybridization showed that the C function genes were expressed transiently in the carpel, continuously in stamens, and showed complementarity with the A function genes in the heterogeneous whorl. In proliferating floral meristem and pfo-2 mutants, all B function genes were down-regulated and the expression patterns of the A and C function genes were drastically altered. We conclude that LjLFY and LjUFO/Pfo are required for the activation of B function genes and function together in the recruitment and determination of petals and stamens. Our findings suggest that gene duplication, change in expression pattern, gain or loss of functional domains, and alteration of key gene functions all contribute to the divergence of floral patterning in L. japonicus.

Figure 1.

Ontogeny of Gifu flower. a, Gifu flower; bar = 1 mm. b, Dissection of Gifu flower; bar = 1 mm. c to k, SEM showing the floral ontogeny of wild-type L. japonicus. Sp, Sepal; Pt, petal; Sti, inner stamen; Sto, outer stamen; Ca, carpel. Bar = 50 μm. The adaxial side of all floral primordia faces up in all micrographs. c, Stage 0, Floral primordia (crosses) are initiated from the I2. d, Stage 1, A bract (arrowhead) develops from floral primordia opposite the adaxial position (cross). e, Stage 2, A sepal primordium (arrowhead) is initiated at the abaxial position of the floral meristem. f, Stage 3, One anlage (common primordium, arrowhead) forms in the axil of the abaxial sepal. The other four sepal primordia (arrow) appear at the lateral and adaxial positions of the floral meristem. g, Stage 4, A carpel primordium (arrowhead) appears in the center of the floral meristem. Two anlagen (black arrowhead) are initiated in the axils of the lateral sepals. Two abaxial petal primordia (keel, arrow) and one abaxial outer stamen primordium (cross) form from the flattened anlage. h, Stage 5, Two lateral petal primordia (wing, arrow) and two outer stamen primordia (arrowhead) form from the two lateral anlagen. i, Stage 6, One adaxial petal primordium (the standard, arrowed) and two outer stamen primordia (arrowhead) are initiated. j, Stage 7, The five inner stamen primordia are initiated. k, After Stage 7 organ primordia begin elongating.

Figure 2.

Ontogeny of mutant flowers. a, An inflorescence of pfm; bar = 1 mm. b to d, Dissection of a pfm mutant inflorescence; bar = 1 mm. e, An inflorescence of pfo-2; bar = 1 mm. f to h, Dissection of pfo-2 mutant inflorescence; bar = 1 mm. i to r, SEM showing the floral ontogeny of the pfm mutant; bar = 50 μm. i, The I2 (I₂) initiates from the I1 (I₁). j, Primary floral meristems (FM) initiate progressively from the I2 meristem. k to m, Early stages of pfm primary floral development. n, The floral meristem of pfm initiates four to five anlagen or primordia (cross) between the sepals and the carpel. Trichomes (arrowhead) appear at the boundaries of the flattened primordia. o, The anlagen contract into ball-like structures. p, Another round of floral-like meristems (cross) form, at the periphery of which another whorl of sepals (arrowhead) initiates. q and r, Repeated pattern of floral-like meristems. Broken rings indicate the successive floral-like meristems.

Figure 3.

Point mutations in the LjLFY and Pfo sequences of the pfm and pfo-2 mutants, respectively. a, Schematic representation of the LjLFY ORF. Nucleotide and predicted amino acid sequences are shown for the pfm mutant and the corresponding region in Gifu. b, Schematic representation of the Pfo ORF, with the F-box shaded black. Nucleotide and predicted amino acid sequences are shown for the pfo-2 mutant and the corresponding region in Gifu.

Figure 4.

Sequence analysis of ABC function gene homologs in L. japonicus. a, A phylogenetic tree generated using neighbor-joining method from the predicted amino acid sequences of genes in the MIKC subfamily of MADS from Arabidopsis, Antirrhinum, pea, M. sativa, soybean (for accession no., see Supplemental Table II), and L. japonicus (dot). b, Alignment of the predicted MADS domain amino acid sequences from the AP1, PI, AP3, AG, and SEP subfamilies. c, Alignment of the predicted C-terminal amino acid sequences from the AP1 and PI subfamilies.

Figure 5.

Expression patterns of floral patterning genes in Gifu. Probe: a1 to 4, LjLFY; b1 to 4, Pfo; c1 to 4, LjAP1a; d1 to 4, LjAP1b; e1 to 4, LjPIa; f1 to 4, LjPIb; g1 to 4, LjAGa; h1 to 4, LjAGb; i1 to 4, LjSEP3; and j1 to 3, LjAP3. SAM was labeled as a cross. (c1), Transverse section of a Gifu florescence; F, floral primordium; I₁, I1 meristem; I₂, I2 meristem. Bar = 50 μm.

Figure 6.

Expression patterns of floral patterning genes in pfm and pfo-2. a to i, pfm and j to r, pfo-2. a to c and j to m, LjAP1a probe; d to f, n, and o, LjAP1b probe; g and p, LjAGa probe; and h, i, q, and r, Pfo probe. Bar = 50 μm.

Figure 7.

Summary of the expression patterns of A, B, C, and E function genes during floral ontogenesis. The false colorings superimposed on SEM of the SAM indicate the expression patterns of different function genes. Bar = 50 μm.