ULTRASTRUCTURE OF GRAPEFRUIT SECRETORY CAVITIES AND IMMUNOCYTOCHEMICAL LOCALIZATION OF (+)-LIMONENE SYNTHASE

Premise of research. The genus Citrus includes species that are among the most important tree fruit crops. Although fruit juice is the primary product, monoterpenoid essential oil obtained from the peel is an important value-added commodity. Peel monoterpenes are synthesized by subepidermal secretory cavities that consist of glandular cells surrounding an extracellular oil storage space. Several previous studies have focused on early secretory cavity development, ending with the initiation of secretion. In order to better understand the process of monoterpene formation, transport, and storage, it is important to obtain detailed information concerning plant oil glands during all phases of development. Methodology. TEM was performed on secretory cavities isolated from the exocarp of grapefruit (Citrus x paradisi Macfad. 'Duncan') preserved by microwave-assisted fixation or high-pressure freezing and freeze substitution. Immunocytochemistry was employed to localize (+)-limonene synthase. Tomography was used to examine leucoplast ultrastructure. Pivotal results. Glandular epithelial cells of secretory cavities persisted for several months after their formation. Lipid accumulation occurred within plastoglobule-like lipid bodies (PGs), vacuoles, and cytoplasmic lipid bodies of epithelial cells. (+)-Limonene synthase, the enzyme that catalyzes the primary flux-carrying reaction in Citrus monoterpene biosynthesis, was localized to tubules within oil gland leucoplasts and was not associated with PGs. No bulk movement of lipids from PGs to vacuoles or to the extracellular oil storage cavity was observed. Conclusions. Oil glands remain biosynthetically active throughout fruit development in grapefruit. The high degree of oil gland metabolic specialization is reflected in unique cellular features, such as leucoplasts, abundant plastid-endoplasmic reticulum membrane contact sites, and the accumulation of fibrillar material in vacuoles. The mechanism of the movement of monoterpenes from plastids to the central cavity is unknown, but our observations suggest that it does not involve vesicles or disruption of the large PGs.