Roles of the APETALA3-3 ortholog in the petal identity specification and morphological differentiation in Delphinium anthriscifolium flowers

Abstract

The genus Delphinium (Ranunculaceae) with its unique and highly complex floral structure is an ideal system to address some key questions in terms of morphological and evolutionary studies in flowers. In D. anthriscifolium, for example, the original eight petal primordia differentiate into three types at maturity (i.e., two dorsal spurred, two lateral flat, and four ventral reduced petals). The mechanisms underlying their identity determination and morphological differentiation remain unclear. Here, through a comprehensive approach combining digital gene expression (DGE) profiles, in situ hybridization, and virus-induced gene silencing (VIGS), we explore the role of the APETALLATA3-3 (AP3-3) ortholog in D. anthriscifolium. Our findings reveal that the DeanAP3-3 not only functions as a traditionally known petal identity gene but also plays a critical role in petal morphological differentiation. The DeanAP3-3 gene is expressed in all the petal primordia before their morphological differentiation at earlier stages, but shows a gradient expression level difference along the dorsventral floral axis, with higher expression level in the dorsal spurred petals, intermediate level in the lateral flat petals and lower level in the ventral reduced petals. VIGS experiments revealed that flowers with strong phenotypic changes showed a complete transformation of all the three types of petals into non-spurred sepals. However, in the flowers with moderate phenotypic changes, the transformation of spurred petals into flat petals is associated with moderate silencing of the DeanAP3-3 gene, suggesting a significant impact of expression level on petal morphological differentiation. This research also shed some insights into the role of changes in gene expression levels on morphological differentiation in plants.

Figure 1

Floral structures of the Delphinieae and the petal differentiation in Delphinium anthriscifolium. (A–C) Front views (first column) and side views (second column) of mature representative Delphinieae species flowers, along with disassembled floral organs: sepals (third column), petals (fourth column), and stamens with carpels (fifth column). Perianth diagrams in the last column illustrate flower structure. (D) Petal differentiation in Delphinium anthriscifolium floral buds from stages S9 to S14, showing side views of floral buds (top), and front views of dorsal spurred petals (DPe), lateral petals (LPe), and reduced petals (RPe, indicated by white five-pointed stars). Scale bars: 1.0 mm.

Figure 2

Phylogenetic analysis and spatiotemporal expression patterns of putative A and B function genes in Delphinium anthriscifolium. (A) Phylogenetic trees for AGL6, AP3, and PI gene lineages. Genes from Delphinium anthriscifolium are highlighted. Branch lengths represent the number of nucleotide substitutions per site. (B) Spatiotemporal expression patterns of AGL6, AP3, and PI lineage genes as determined by DGE analysis. The bottom right inset depicts sepals, petals, stamens, and carpels across developmental stages S9–S16, with color intensity indicating the stage. Ca, Carpel; DPe, dorsal spurred petal; Dse, dorsal spurred sepal; LPe, lateral petal; Lse, lateral sepal; St, Stamen; Vse, ventral sepal.

Figure 3

Spatiotemporal expression of DeanAP3–3 in Delphinium anthriscifolium flowers. Serial sections of the same floral bud or petal are labeled as serial numbers. The images F, G, I, and J shows the transverse section of the floral bud, while the remaining images display longitudinal sections. Expression models of DeanAP3–3 gene are showed in the upper right corner of each set of pictures, with the color illustrating the expression domain. The final images (K) show the results using sense probes. DPe, dorsal spurred petal; LPe, lateral petal; RPe, reduced petal. Scale bars: 200 μm.

Figure 4

Function of DeanAP3–3 as revealed by virus-induced gene silencing. (A–F) Flowers in the mock (A), TRV2-DeanPDS-DeanAP3–3 treated plants with strong (B), moderate (C and D), and weak (E and F) phenotypic changes. Each panel, from left to right, presents the front and side views of the whole flower, followed by dissections revealing the anatomical structure of sepals, petals (with reduced petals marked by a white five-pointed star), and stamens and carpels. The floral diagram in the last column illustrates the altered structure of flowers. (G) Relative expression levels of DeanAP3–3 in S12 floral buds of WT, mock and TRV2-DeanPDS-DeanAP3–3 treated plants. AP3–3_1–3 are flowers with strong phenotypic changes, and AP3–3_4 are flowers with moderate phenotypic changes. (H) Relative expression levels of DeanAP3–3 in dorsal (DPe) and lateral petals (LPe) of mock plants, and in lateral petals (D-LPe, dorsal petals transformed) of TRV2-DeanPDS-DeanAP3–3 treated plants. Scale bars: 1000 μm.

Figure 5

Morphological characteristics in Delphinium anthriscifolium petals. (A) Dorsal petal of a mock-treated plant with close-ups of the epidermal cells in the distal (A1) and proximal (A2) regions, as indicated by square regions 1 and 2, respectively. (B) Lateral petal of a mock-treated plant with close-ups of the epidermal cells in the distal (B1) and proximal (B2) regions, as indicated by square regions 1 and 2, respectively. (C) Dorsal petal of a plant exhibiting weak phenotypic changes, with detailed views of the distal (C1) and proximal (C2) epidermal cells, as indicated by square regions 1 and 2, respectively. Scale bars: 100 μm