Cellular and subcellular localization of S-adenosyl-L-methionine:benzoic acid carboxyl methyltransferase, the enzyme responsible for biosynthesis of the volatile ester methylbenzoate in snapdragon flowers

Abstract

The benzenoid ester, methylbenzoate is one of the most abundant scent compounds detected in the majority of snapdragon (Antirrhinum majus) varieties. It is produced in upper and lower lobes of petals by enzymatic methylation of benzoic acid in the reaction catalyzed by S-adenosyl-L-methionine:benzoic acid carboxyl methyltransferase (BAMT). To identify the location of methylbenzoate biosynthesis, we conducted an extensive immunolocalization study by light and electron microscopy at cellular and subcellular levels using antibodies against BAMT protein. BAMT was immunolocalized predominantly in the conical cells of the inner epidermal layer and, to a much lesser extent, in the cells of the outer epidermis of snapdragon flower petal lobes. It was also located in the inner epidermis of the corolla tube with little BAMT protein detected in the outer epidermis and in the yellow hairs within the tube on the bee's way to the nectar. These results strongly suggest that scent biosynthetic genes are expressed almost exclusively in the epidermal cells of floral organs. Immunogold labeling studies reveal that BAMT is a cytosolic enzyme, suggesting cytosolic location of methylbenzoate biosynthesis. The concentration of scent production on flower surfaces that face the pollinators during landing may increase pollination efficiency and also help to minimize the biosynthetic cost of advertising for pollinators.

Figure 1

Immunofluorescence localization of BAMT in snapdragon flowers. A, Antirrhinum flower. B, Environmental scanning electron micrograph of conical cells from the inner epidermis of the lower petal lobe. C, Transverse section of 7-d-old snapdragon lower petal lobe. D, Transverse section of 7-d-old snapdragon lower petal lobe treated with anti-BAMT antibodies and visualized by fluorescent FITC-conjugated secondary antibodies. E and F, Control sections corresponding to D. E, Transverse section of 7-d-old snapdragon lower petal lobe treated with preimmune serum and visualized by fluorescent FITC-conjugated secondary antibodies. F, Transverse section of 1-d-old snapdragon lower petal lobe treated with anti-BAMT antibodies and visualized by fluorescent FITC-conjugated secondary antibodies. Nonspecific signal is due to starch granules. G, Transverse section of corolla tube of 7-d-old snapdragon flower. H, Transverse section of corolla tube of 7-d-old snapdragon flower treated with anti-BAMT antibodies and visualized by fluorescent FITC-conjugated secondary antibodies. I, Control section corresponding to H. Transverse section of corolla tube of 7-d-old snapdragon flower treated with preimmune serum and visualized by fluorescent FITC-conjugated secondary antibodies. Vascular tissues in H and I show nonspecific labeling. vt, Vascular tissue. Scale bars = 20 μm (B) and 50 μm (C–I).

Figure 2

Immunofluorescence localization of BAMT in snapdragon flower glands. A, Environmental scanning electron micrograph of glands within corolla tube of 7-d-old snapdragon flower. B, Environmental scanning electron micrograph showing the gland's head. C, Light microscopy photograph of glands within corolla tube. Fresh samples of snapdragon corolla tube were hand cut. D, Transverse section of glands within corolla tube of 7-d-old snapdragon flower. E, Cross-section through gland head within corolla tube of 7-d-old snapdragon flower. F, Cross-section through gland head within corolla tube of 7-d-old snapdragon flower treated with anti-BAMT antibodies and visualized by fluorescent FITC-conjugated secondary antibodies. G, Control section corresponding to F. Cross-section through gland head within corolla tube of 7-d-old snapdragon flower treated with preimmune serum and visualized by fluorescent FITC-conjugated secondary antibodies. H, Cross-section through gland stalk within corolla tube of 7-d-old snapdragon flower. I, Cross-section through gland stalk within corolla tube of 7-d-old snapdragon flower treated with anti-BAMT antibodies and visualized by fluorescent FITC-conjugated secondary antibodies. J, Control section corresponding to I. Cross-section through gland stalk within corolla tube of 7-d-old snapdragon flower treated with preimmune serum and visualized by fluorescent FITC-conjugated secondary antibodies. Scale bars = 100 μm (A and D), 20 μm (B, E–G, I, and J), 300 μm (C), and 10 μm (H).

Figure 3

Immunogold localization of BAMT in snapdragon flower. A, Light microscopy photograph of transverse section of 7-d-old snapdragon lower petal lobe. Boxes with letters indicate positions where the following corresponding pictures were taken. B, Fine structure of conical cell of 7-d-old snapdragon lower petal lobe. C through F, Transmission electron microscopy (TEM) of conical cells of 7-d-old snapdragon lower petal lobe labeled with anti-BAMT antibodies and gold-conjugated goat anti-rabbit antibodies. D, The region marked with an arrow was magnified three times to show gold particles (see insert). G and H, Control sections corresponding to C through F. G, TEM of conical cells of 7-d-old snapdragon lower petal lobe treated with preimmune serum and gold-conjugated goat anti-rabbit antibodies. H, TEM of conical cells of 1-d-old snapdragon lower petal lobe labeled with anti-BAMT antibodies and gold-conjugated goat anti-rabbit antibodies. I and J, TEM of outer epidermal cell of 7-d-old snapdragon lower petal lobe labeled with anti-BAMT antibodies and gold-conjugated goat anti-rabbit antibodies. K and L, Control sections corresponding to I and J. K, TEM of outer epidermal cell of 7-d-old snapdragon lower petal lobe treated with preimmune serum and gold-conjugated goat anti-rabbit antibodies. L, TEM of outer epidermal cell of 1-d-old snapdragon lower petal lobe labeled with anti-BAMT antibodies and gold-conjugated goat anti-rabbit antibodies. M, TEM of inner epidermal cell of corolla tube of 7-d-old snapdragon flower labeled with anti-BAMT antibodies and gold-conjugated goat anti-rabbit antibodies. N, Control section corresponding to M. TEM of inner epidermal cell of corolla tube of 7-d-old snapdragon flower treated with preimmune serum and gold-conjugated goat anti-rabbit antibodies. O, TEM of gland within corolla tube of 7-d-old snapdragon flower labeled with anti-BAMT antibodies and gold-conjugated goat anti-rabbit antibodies. P, Control section corresponding to O. TEM of gland within corolla tube of 7-d-old snapdragon flower treated with preimmune serum and gold-conjugated goat anti-rabbit antibodies. c, Cytosol; cw, cell wall; v, vacuole; vt, vascular tissue. Scale bars = 50 μm (A) and 1 μm (B–P). Arrows point to regions of BAMT localization.