Scanning Electron Microscopic Study of Modified Chloroplasts in Senescing

Structural changes in chloroplasts of broccoli ( Brassica oleracea L., Italica group) florets during senescence were examined using light microscopy, scanning electron microscopy (SEM) with freeze-fracture technique, and transmission electron microscopy (TEM) to better understand the process of chloroplast degradation, particularly at the advanced stage of senescence. Light microscopy revealed that chloroplasts, which initially were intact and green, became obscure in shape, and their color faded during senescence. Small, colored particles appeared in cells as the florets approached the final stage of senescence and became fullto dark-yellow in color. Scanning electron microscopy showed that stroma thylakoids in the chloroplast initially were parallel to each other and grana thylakoids were tightly stacked. As senescence advanced, the grana thylakoids degenerated and formed globules. The globules became larger by aggregation as senescence progressed, and the large globules, called “thylakoid plexus,” formed numerous vesicles. The vesicles ultimately were expelled into the cytosol, and the light microscope revealed many colored particles in the senescent cells. These results indicate that the degradation of chloroplasts in broccoli florets progresses systematically, with the final product being colored particles, which are visible in yellow broccoli sepal cells. Structural changes in chloroplasts have been studied with both scanning electron microscopy (SEM) (Blackmore et al., 1984; Kulandaivelu and Gnanam, 1985; Larsson et al., 1977; Mohapatra and Johnson, 1976; Tuquet and Newman, 1980) and transmission electron microscopy (TEM). Plant materials studied with TEM have included senescing leaves of wheat ( Triticum aestivum L.) (Hurkman, 1979), Phaseolus (Barton, 1966; Ronning et al., 1991), spinach ( Spinacia oleracea L.) (Mine et al., 1992), and Betula (Dodge, 1970), ripening fruit of Capsicum (Spurr and Harris, 1968) and mango (Mangifera indica L.) (Parikh et al., 1990), both developing and senescing fruit of normal and mutant tomato ( Lycopersicum esculentum Mill.) (Simpson et al., 1976), and ethylenetreated fruit of Satsuma mandarin ( Citrus reticulata Blanco) (Shimokawa et al., 1978) In this study, we examine and discuss the ultrastructural changes in chloroplasts of broccoli florets during senescence, as observed with TEM, SEM with freeze fracture, and light microscopy. Materials and Methods Fresh, green broccoli heads were obtained from the Wholesale Distribution Center, Jessup, Md. A series of florets was excised from the head and allowed to senesce to the following stages for analysis: non-yellow = no yellow florets; slight-yellow = lighter green and slight yellow florets visible; half-yellow = green and yellow florets present in about equal amounts; full-yellow = most of the florets light yellow; dark-yellow = all florets dark yellow. For analysis by light microscopy, the heads were placed in a perforated polyethylene bag at 20 °C. Florets were detached daily from a single head to obtain successive stages of senescence, and immersed in a 2% warm agar solution in a petri dish. The agar containing the florets was allowed to gel in a refrigerator and sliced into 5μm-thick sections. For SEM and TEM observations, short-stemmed branchlets weighing ≈30 g were excised from a head, placed individually in 100-mL beakers containing sufficient water to cover the base of the stem, and stored at 1 °C. One beaker containing the branchlets was transferred daily from 1 to 20 °C for 6 d to obtain florets of different stages of senescence. The sepals were removed from the florets and prepared for examination by electron microscopy. For observation of freeze-fractured specimens, sepals of florets were fixed in 2% (w/v) OsO4 in phosphate buffer (0.1 M, pH 7.4) for 2 h. Following fixation, one set of specimens was rinsed with the phosphate buffer and dehydrated through a gradient series of ethanol (50% to 70%). Next, the specimens were frozen in liquid nitrogen and fractured with a scalpel. The fractured specimens were rehydrated back to phosphate buffer through a gradient series of ethanol from 70% to 50%. A second set of specimens fixed with OsO 4 was dehydrated with a gradient series of solutions of dimethyl sulfoxide (DMSO) (25% to 50%) and fractured with DMSO instead of ethanol to minimize the formation of ice crystals, which would cause mechanical disruption of the tissues. However, DMSO residue interferes with extraction of the proteinaceous matrix of plastids, which restricts accurate observation and evaluation of the fine structural features of the thylakoid membranes. Thus, micrographs from both procedures were used for analysis and interpretation of data. The specimens fractured with ethanol or DMSO series were transferred to 0.1% OsO 4 for 8 d. The OsO4 solution was replaced daily with a fresh supply. Next, specimens were treated in sequence with 1% OsO 4 for 1 h, 1% tannic acid for 2 h, and with a second solution of 1% OsO4 for 1 h and dehydrated through a gradient series of ethanol (50% to 100%). To minimize tissue distortion that is associated with air drying, the samples were placed in an Autosamdri-810 critical point drying apparaand leaves of morning glory [ Ipomoea nil (L.) Roth] (Toyama, 1980). Gepstein (1988) postulated the following sequential changes in senescing chloroplasts: 1) stroma thylakoids lost integrity; 2) chloroplasts increased in volume, and in some plants, the number of osmiophilic granules increased; 3) grana thylakoids swelled, then gradually disintegrated; and 4) the chloroplast envelope retained its integrity as the tissue entered near the final stage of senescence. These postulations were based on observations made by Gepstein and others on chloroplasts at various stages of senescence, but not at the final stage. A better and complete understanding of ultrastructural changes in chloroplasts is obtained by two-dimensional analysis with TEM and by three-dimensional analysis with SEM using a freeze-fracture technique. Barnes and Blackmore (1984) used SEM with freezefracture technique to examine the contents of chloroplasts but concentrated on specific stage rather than following sequential changes during senescence. We were interested in the effects of various postharvest treatments on maintenance of green color in broccoli florets, but needed to know the ultrastructural changes of chloroplasts during senescence to seek effective treatments. To our knowledge, no one has reported on the ultrastructural changes of chloroplasts in these tissues. Fruit or leaf blades were used in the studies cited above.