HORTSCIENCE 35(1):99–103. 2000. In this study, we examine and discuss the ultrastructural changes in chloroplasts of broc- coli florets during senescence, as observed Scanning Electron Microscopic Study with TEM, SEM with freeze fracture, and light of Modified Chloroplasts in Senescing microscopy. Broccoli Florets Materials and Methods Fresh, green broccoli heads were obtained Hirofumi Terai1 and Alley E. Watada from the Wholesale Distribution Center, Jessup, Md. A series of florets was excised Horticultural Crops Quality Laboratory, Beltsville Agricultural Research from the head and allowed to senesce to the Center, Agricultural Research Service, U.S. Department of Agriculture, following stages for analysis: non-yellow = no Beltsville, MD 20705-2350 yellow florets; slight-yellow = lighter green and slight yellow florets visible; half-yellow = Charles A. Murphy and William P. Wergin green and yellow florets present in about equal Nematology Laboratory, Beltsville Agricultural Research Center, Agricultural amounts; full-yellow = most of the florets light Research Service, U.S. Department of Agriculture, Beltsville, MD 20705-2350 yellow; dark-yellow = all florets dark yellow. For analysis by light microscopy, the heads Additional index words. broccoli, ultrastructure, chloroplast, SEM, TEM, freeze-fracture were placed in a perforated polyethylene bag at 20 °C. Florets were detached daily from a Abstract. Structural changes in chloroplasts of broccoli (Brassica oleracea L., Italica single head to obtain successive stages of group) florets during senescence were examined using light microscopy, scanning electron senescence, and immersed in a 2% warm agar microscopy (SEM) with freeze-fracture technique, and transmission electron microscopy solution in a petri dish. The agar containing the (TEM) to better understand the process of chloroplast degradation, particularly at the florets was allowed to gel in a refrigerator and advanced stage of senescence. Light microscopy revealed that chloroplasts, which initially sliced into 5-µm-thick sections. For SEM and were intact and green, became obscure in shape, and their color faded during senescence. TEM observations, short-stemmed branchlets Small, colored particles appeared in cells as the florets approached the final stage of weighing ≈30 g were excised from a head, senescence and became full- to dark-yellow in color. Scanning electron microscopy showed placed individually in 100-mL beakers con- that stroma thylakoids in the chloroplast initially were parallel to each other and grana taining sufficient water to cover the base of the thylakoids were tightly stacked. As senescence advanced, the grana thylakoids degener- stem, and stored at 1 °C. One beaker contain- ated and formed globules. The globules became larger by aggregation as senescence ing the branchlets was transferred daily from 1 progressed, and the large globules, called “thylakoid plexus,” formed numerous vesicles. to 20 °C for 6 d to obtain florets of different The vesicles ultimately were expelled into the cytosol, and the light microscope revealed stages of senescence. The sepals were re- many colored particles in the senescent cells. These results indicate that the degradation moved from the florets and prepared for ex- of chloroplasts in broccoli florets progresses systematically, with the final product being amination by electron microscopy. colored particles, which are visible in yellow broccoli sepal cells. For observation of freeze-fractured speci- mens, sepals of florets were fixed in 2% (w/v) Structural changes in chloroplasts have and leaves of morning glory [Ipomoea nil (L.) OsO4 in phosphate buffer (0.1 M, pH 7.4) for 2 been studied with both scanning electron mi- Roth] (Toyama, 1980). h. Following fixation, one set of specimens croscopy (SEM) (Blackmore et al., 1984; Gepstein (1988) postulated the following was rinsed with the phosphate buffer and de- Kulandaivelu and Gnanam, 1985; Larsson et sequential changes in senescing chloroplasts: hydrated through a gradient series of ethanol al., 1977; Mohapatra and Johnson, 1976; 1) stroma thylakoids lost integrity; 2) chloro- (50% to 70%). Next, the specimens were fro- Tuquet and Newman, 1980) and transmission plasts increased in volume, and in some plants, zen in liquid nitrogen and fractured with a electron microscopy (TEM). Plant materials the number of osmiophilic granules increased; scalpel. The fractured specimens were rehy- studied with TEM have included senescing 3) grana thylakoids swelled, then gradually drated back to phosphate buffer through a leaves of wheat (Triticum aestivum L.) disintegrated; and 4) the chloroplast envelope gradient series of ethanol from 70% to 50%. A (Hurkman, 1979), Phaseolus (Barton, 1966; retained its integrity as the tissue entered near second set of specimens fixed with OsO4 was Ronning et al., 1991), spinach (Spinacia the final stage of senescence. These postula- dehydrated with a gradient series of solutions oleracea L.) (Mine et al., 1992), and Betula tions were based on observations made by of dimethyl sulfoxide (DMSO) (25% to 50%) (Dodge, 1970), ripening fruit of Capsicum Gepstein and others on chloroplasts at various and fractured with DMSO instead of ethanol (Spurr and Harris, 1968) and mango stages of senescence, but not at the final stage. to minimize the formation of ice crystals, (Mangifera indica L.) (Parikh et al., 1990), A better and complete understanding of which would cause mechanical disruption of both developing and senescing fruit of normal ultrastructural changes in chloroplasts is ob- the tissues. However, DMSO residue inter- and mutant tomato (Lycopersicum esculentum tained by two-dimensional analysis with TEM feres with extraction of the proteinaceous Mill.) (Simpson et al., 1976), and ethylene- and by three-dimensional analysis with SEM matrix of plastids, which restricts accurate treated fruit of Satsuma mandarin (Citrus using a freeze-fracture technique. Barnes and observation and evaluation of the fine struc- reticulata Blanco) (Shimokawa et al., 1978) Blackmore (1984) used SEM with freeze- tural features of the thylakoid membranes. fracture technique to examine the contents of Thus, micrographs from both procedures were Received for publication 24 Sept. 1998. Accepted for chloroplasts but concentrated on specific stage used for analysis and interpretation of data. publication 5 May 1999. Use of company or product rather than following sequential changes dur- The specimens fractured with ethanol or name by the U.S. Dept. of Agriculture does not imply ing senescence. DMSO series were transferred to 0.1% OsO4 approval or recommendation of the product to the We were interested in the effects of various for 8 d. The OsO4 solution was replaced daily exclusion of others which also may be suitable. The postharvest treatments on maintenance of green with a fresh supply. Next, specimens were cost of publishing this paper was defrayed in part by color in broccoli florets, but needed to know treated in sequence with 1% OsO4 for 1 h, 1% the payment of page charges. Under postal regula- the ultrastructural changes of chloroplasts dur- tannic acid for 2 h, and with a second solution tions, this paper therefore must be hereby marked advertisement solely to indicate this fact. ing senescence to seek effective treatments. of 1% OsO4 for 1 h and dehydrated through a 1To whom reprint requests should be addressed. To our knowledge, no one has reported on the gradient series of ethanol (50% to 100%). To Present address: Faculty of Agriculture, Kobe Uni- ultrastructural changes of chloroplasts in these minimize tissue distortion that is associated versity, Rokkodai-cho Nada-ku, Kobe 657-8501, tissues. Fruit or leaf blades were used in the with air drying, the samples were placed in an Japan. E-mail address: [email protected] studies cited above. Autosamdri-810 critical point drying appara- HORTSCIENCE, VOL. 35(1), FEBRUARY 2000 99 POSTHARVEST BIOLOGY & TECHNOLOGY tus (Tousimis Research Corp., Rockville, Md.). the 6 d at 20 °C. Examination by light micros- in the stroma and many of the globules fused In this dryer, the ethanol was replaced with copy indicated that the chloroplasts were green together. Globules are products of the disorga- liquid carbon dioxide that was subsequently and intact at the non-yellow stage (Fig. 1a), nized thylakoid membranes, which contain raised to its critical point (31 °C and 7.3 × 106 less visible at the slight-yellow stage (Fig. 1b), lipids and carotenoids (Barton, 1966; Matile, Pa) where the fluid and vapor phases are and indistinct at the half-yellow stage because 1992). Although Thomson (1966) found that indistinguishable. Next, the dryer was vented they were masked with pigments (Fig. 1c). At there was an increase in size and number of to room pressure, and the samples were re- the full- to dark-yellow stage, small green or large globules in flavedo of ‘Valencia’ or- moved and mounted on aluminum stubs that yellow-to-brown particles were seen dispersed anges [Citrus sinensis (L.) Osbeck], Dodge were sputter-coated with Au/Pd and then in- in the cells (Fig. 1d). With SEM, chloroplasts (1970) found that size, but not the number, serted into a Hitachi S-570 SEM (Hitachi were recognizable at the non-yellow stage increased during the autumnal senescence of Scientific Instruments, Mountain View, Ca- (Fig. 2a), the slight-yellow stage (Fig. 2b), and Betula leaves. We found that the globules lif.) for observation. the half-yellow stage (Fig. 2c–f), when con- gathered and then fused together