Genetic control of floral zygomorphy in pea (Pisum sativum L.)

Abstract

Floral zygomorphy (flowers with bilateral symmetry) has multiple origins and typically manifests two kinds of asymmetries, dorsoventral (DV) and organ internal (IN) asymmetries in floral and organ planes, respectively, revealing the underlying key regulators in plant genomes that generate and superimpose various mechanisms to build up complexity and different floral forms during plant development. In this study, we investigate the loci affecting these asymmetries during the development of floral zygomorphy in pea (Pisum sativum L.). Two genes, LOBED STANDARD 1 (LST1) and KEELED WINGS (K), were cloned that encode TCP transcription factors and have divergent functions to constitute the DV asymmetry. A previously undescribed regulator, SYMMETRIC PETALS 1 (SYP1), has been isolated as controlling IN asymmetry. Genetic analysis demonstrates that DV and IN asymmetries could be controlled independently by the two kinds of regulators in pea, and their interactions help to specify the type of zygomorphy. Based on the genetic analysis in pea, we suggest that variation in both the functions and interactions of these regulators could give rise to the wide spectrum of floral symmetries among legume species and other flowering plants.

Fig. 1.

Mutants affecting floral dorsoventral asymmetry in papilionoid legumes. Front and side views of flowers and flattened petals from wild type and mutants are shown. (a) Wild-type pea flower. Yellow arrow: two ventral petals form a keel. Red line with arrow: the DV asymmetry in floral plane. Red lines: the IN asymmetry of lateral and ventral petals. (b) k-1 flowers with keeled wings (white arrow) in pea. (c) lst1-1 flowers with lobed standards (white arrow) in pea. (d) k-1 lst1-1 flowers in pea. Most the dorsal petals are small and not fully expanded, possessing a keel structure (white arrow). Dashed lines: where the petal has been cut to make it flat. Red triangle: dorsal petals with prominent IN asymmetry. Red pentacle: abnormal lateral petal occasionally found. (e) Wild-type flower of Lotus. (f) kew1 flower in Lotus with keeled wings (white arrow). (g) kew1 flower in Lotus with repetitive morph forms at lateral region. Red star in d and g: abnormal lateral petal occasionally found. (Scale bar, 5 mm.)

Fig. 2.

Petal development in wild type and mutants. (a) Floral organ development stages 2, 4 and 5 (21, 22). At stage 4, two instead of one ventral sepals are initiated in syp1-1 floral meristem (yellow star). At stage 5, one dorsal petal and two stamens are initiated in wild type (blue ellipse), whereas the dorsal primordia fail to differentiate in k-1 lst1-1 (blue ellipse). At the same stage, extra primordia can be found in syp1-1 (yellow ellipse) in comparison with the wild type (yellow ellipse). Blue and yellow arrows indicate the dorsal and ventral region of the floral meristem, respectively. (b) Percentage of flowers with variable organ number. Blue: in the dorsal region. Yellow: in the ventral region. wt: JI992; k: k-1; lst1: lst1-1; k lst1: k-1 lst-1; syp1: syp1-1; k-1 lst1-1 syp1: k-1 lst1-1 syp1-1. Eighty flowers were scored in each genetic background. (c) Epidermal cell on the adaxial side of mature petals. In wild type, a representative cell type of dorsal petals is a long strip with wavy margins, one of the lateral petals is jigsaw puzzle-like, and the one of the ventral petals is ripple-like. The petal cells of the lateral petals in k are identical to that of the ventral petal, and the dorsal petal cells of lst1 resemble that of the lateral petal in wild type. Cells of all petals in k lst1 bear those of the ventral petals in wild type, whereas no differences between wild type and syp1 were observed. (d) Petal development, stage 6. Red arrow: the asymmetric shape of both lateral and ventral petals is obvious in the wild type. Yellow arrow: the shape of the lateral petal in k mimics that of the ventral petal. Blue arrow: the retarded dorsal petals in lst1. Dashed lines: IN asymmetry of lateral and ventral petals is lost in syp1-1. The blue, orange, and yellow color marks the different cell types in dorsal, lateral, and ventral petals, respectively. DP, dorsal petal; LP, lateral petal; VP, ventral petal. (Scale bar, 50 μm.)

Fig. 3.

Cloning of K and LST1 genes. (a) Physical map of the 295-kb region in Chr. 5 of Lotus, where KEW1 was located (upper lane), and the gene structure of LjCYC3 is shown in the lower lane. (b) Phylogenetic tree of TCP family members. CYC (CAA76176) and DICH (AAF12817) are from Antirrhium; PCF1 from rice (BAA23142); LjCYCs from L. japonicus (LjCYC1, ABB36471; LjCYC2, ABB36472; LjCYC3, ABB36473; LjCYC5, ABB36474),and PsCYCs from pea. (c) Genomic Southern blot analysis reveals the deletion of the K gene in k-1 and k-3. (d–f) Expression patterns of PsCYCs in wild type (d) and mutants (e and f). R, root; ST, stems; YL, young leaves; VA, vegetative apex; IM, inflorescence meristem. VIGS phenotype (g–m) side or front view of flowers and lateral or dorsal petals after inoculation are shown. (g) Control, VIGS-GFP. (h and i) VIGS-PsCYC3 in wild-type. (h) VIGS-PsCYC3 flowers with weak k phenotype (arrow) and (i) strong k phenotype (arrow). (j) VIGS-GFP control. (k) VIGS-PsCYC2 in wild-type, dorsal petal with lobes (arrow). (l) VIGS-GFP control in k-1. (m) VIGS-PsCYC1 in k-1, dorsal petal is retarded (arrow).

Fig. 4.

Interaction between PsCYCs and SYP1, floral diagrams, and the establishment of floral zygomorphy. (a–f) syp1 mutants and syp1-1 in different genetic backgrounds. Front and side views of flowers and flattened petals from wild type and mutants are shown. (a) Wild-type pea flower. (b) syp1-1 flower bears symmetrical lateral and ventral petals (white arrow). The ventral petals possess a keel structure. Yellow arrow: the additional ventral petal, which is cut along the edge of the keel. (c) syp1-2 flower with symmetrical lateral petals (white arrow). (d) lst1-1 syp1-1 flower displays an additive phenotype. (e) k-1 syp1-1 flower has abnormal dorsal petals (white arrow). (f) k-1 lst1-1 syp1-1 flower has radial symmetry with five ventralized symmetrical petals. In d–f, all ventralized petals were cut to make them flat. (Scale bar, 10 mm.) (g) Floral diagrams of wild type and various default states in Antirrhinum and pea. In wild type, both flowers have three distinct petals and possess DV (red lines with arrow) and IN asymmetry (broken lines). In the ventralized (−DV) default forms, all petals of pea flower manifest IN asymmetry, whereas those of Antirrhinum are symmetric. In syp1, the (−IN) default form still possesses DV asymmetry. In k lst1 syp1, the (−DV−IN) default form displays radial symmetry. Based on work on Lotus, another radial symmetry form is expected when PsCYCs are ectopically expressed, and all petals mimic the dorsal. Furthermore, flowers with left-right asymmetry could be found as the variation of (−DV) default form. (h) Functions and interactions of DV and IN regulators in pea. DV and IN asymmetries are separately controlled by PsCYCs and SYP1. PsCYCs can suppress the manifestation of IN asymmetry and couple IN with DV asymmetry. SYP1 can interact with PsCYCs to regulate organ primordium initiation, presumably modifying DV orientation. The interaction of SYP1 and PsCYCs is important for zygomorphic development in legumes.