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

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.

苯甲酸甲酯（methylbenzoate）作为一种苯类酯类化合物，是多数金鱼草（Antirrhinum majus）品种中检测到的丰度最高的气味成分之一。该物质通过苯甲酸的酶促甲基化反应生成，催化该反应的酶为S-腺苷-L-甲硫氨酸：苯甲酸羧基甲基转移酶（BAMT），合成部位位于花瓣的上下裂片。为明确苯甲酸甲酯的生物合成位点，本研究利用针对BAMT蛋白的抗体，通过光学显微镜与电子显微镜在细胞及亚细胞层面开展了大规模免疫定位实验。结果显示，BAMT主要分布于金鱼草花瓣裂片的内表皮层锥形细胞中，仅在外表皮层细胞中少量存在；同时BAMT也存在于花冠筒的内表皮，而在外表皮以及通往花蜜路径上的花冠筒内黄色绒毛中几乎未检测到BAMT蛋白。上述结果有力表明，气味生物合成相关基因几乎仅在花器官的表皮细胞中表达。免疫金标记实验进一步证实，BAMT为胞质酶，提示苯甲酸甲酯的生物合成发生于细胞质中。将气味合成集中于传粉者降落时朝向的花表面，既可提升传粉效率，也能最大限度降低用于吸引传粉者的生物合成成本。