Ultrastructure of Avocados: Ripening, Chilling Injury, and Isolation of Idioblast Oil Cells

Ripening and chilling injury of avocado (Persea americana Mill. cv. Hass) have been studied at the ultrastructural level. Changes in the organization of cellular organelles and the breakdown of the cell walls during ripening are described. Changes in the organization of the cell membrane during chilling injury have also been detected and may be the underlying cause of the physiological and biochemical dysfunctions which lead to the injury. Additionally, a technique for the isolation of specialized oil cells is described. Preliminary results on the composition of their oil contents are given, and the effect of low temperature on the ultrastructure of this oil is shown. The ripening of fruits is a complex process, including several different physiological and biochemical changes. These changes are reflected in structural modifications at the cellular level. Ripening of avocados involves, primarily, a softening of the fruit mesocarp. Biochemical analyses of avocados have shown large increases in the activities of wall hydrolytic enzymes during ripening (Awad and Young, 1979). Structural studies using the electron microscope correlated the dissolution of the cell walls with this increased enzyme activity and concomitant softening of the fruit (Platt-Aloia et al., 1980; Platt-Aloia and Thomson, 1981). Mature avocados begin ripening when they are harvested. The process can be slowed considerably by low temperature storage (4-6C); however, extended cold storage results in chilling injury, which is manifested as discoloration of the mesocarp, improper softening, and off-flavor (Couey, 1982). A previous study on the ultra-structure of chilling-injured avocados reported apparent damage to the cell membrane, evident as phase separations of the phospholipids and proteins of the bilayer (Platt-Aloia and Thomson, 1987; Platt-Aloia, 1988). The major storage component of the avocado fruit is oil, primarily triglycerides (Biale and Young, 1971). This oil is stored as droplets in the cytoplasm of the parenchyma cells of the mesocarp (Platt-Aloia and Thomson, 1981). Additionally, approximately 2% of the cells of the mesocarp are specialized (idioblast), oil cells (Cummings and Schroeder, 1942; Platt-Aloia et al., 1983). The oil contained in these specialized cells has a different appearance at both the light (Scott et al., 1963) and electron microscope (Platt-Aloia et al., 1983) levels. The composition and possible function of this oil, however, are unknown. The objectives of this article are to review the structural changes that occur in the avocado fruit cells during ripening and with chilling injury, and to describe the effects of these processes on the ultrastructure of the specialized oil cells. We also report preliminary results on isolation of the idioblasts, and biochemical characterization of the oil they contain. Materials and Methods Avocados (Persea americana, Mill. cv. Hass) were harvested from trees grown on the Riverside campus of the University of California. Ripening was monitored by the method of Awad and Young (1979). For the chilling injury study, recently-harvested fruit were placed in individual containers at 6C, and ethylene evolution was monitored. Samples for electron microscopy were fixed in 1% glutaraldehyde in 50 mM cacodylate buffer, pH 7.0, at room temperature for 2 to 4 hours, rinsed in buffer, postfixed overnight in cacodylate-buffered 1 % OsO4, dehydrated in acetone, and embedded in Spurr's resin (Spurr, 1969). Samples for freeze fracture were not fixed. Tissue was frozen in liquid propane, and replicas were prepared in a Balzers freeze fracture apparatus according to the methods described by Platt-Aloia and Thomson (1982). The idioblast oil cells were isolated from soft, ripe fruit. Tissue was homogenized in a Ten Broeck homogenizer with distilled water. The homogenate was filtered through a 200 μm nylon mesh to remove vascular strands. The filtrate was then filtered through a 48 μm nylon mesh. The residue was thoroughly washed with water and centrifuged at 80 x g for 2 to 3 min. The pellet contained the isolated oil cells, and some larger cell debris including cell walls and small pieces of vascular strands. Lipids of the isolated oil cells were extracted by a modification of the technique of Bligh and Dyer (1959). Extraction was with chloroform:methanol (2:1), followed by centrifugation. The lower phase was washed twice with distilled water to remove water soluble impurities. The lower phase was then concentrated under N2 gas. Thin layer chromatography was performed on silica plates, using a solvent system of benzene:ether:acetic acid (80:20:1), and developed with iodine vapors.