MesophyH protoplasts isolated from primary leaves of wheat seedlings were used to folow the localization of proteases and the breakdown of chloroplasts during dark-induced senescence. Protoplasts were readily obtained from leaf tissue, even after 80% of the chlorophyll and protein had been lost. Intact chloroplasts and vacuoles could be isolated from the protoplasts at al stages of senescence. Al the proteolytic activity associated with the degradation of ribulose bisphosphate carboxylase in the protoplasts could be accounted for by that localized within the vacuole. Moreover, this localization was retained late into senescence. Protoplasts isolated during leaf senescence first showed a decline in photosynthesis, then a decline in ribulose bisphosphate carboxylase activity, followed by a decline in chloroplast number. There was a close correlation between the decline in chloroplast number and the loss of chlorophyll and soluble protein per protoplast, suggesting a sequential degradation of chloroplasts during senescence. Ultrastructural studies indicated a movement of chloroplasts in toward the center of the protoplasts during senescence. Thus, within senescing protoplasts, chloroplasts appeared either to move into invaginations of the vacuole or to be taken up into the vacuole.Leaf senescence has often been referred to as a well-organized event (2, 4) because of the sequential loss of organelles with their corresponding function during senescence. Yet, evidence is lacking on how these changes might be controlled. If MATERIALS AND METHODSPlant Material. Wheat (Triticum aestivum L. var. Arthur) seeds were planted in vermiculite in 6-cm pots and held in a growth cabinet at 20°C and 75% RH. The photoperiod was 16 h, with a quantum flux density of 250 ,uE m s'. After 10 days, the second leaf had just emerged and the first leaf was fully expanded. The seedlings were then transferred to a dark room at 22°C to induce senescence. At different times, sections were taken from the primary leaves (8-cm sections cut 2 cm from the tip) and used for protoplast isolation.Protoplast, Chloroplast, and Vacuole Isolation. Protoplasts were isolated as previously described (6), except that 0.1% pectolyase Y-23 was substituted for prectinase and hemicellulase in the digestion enzyme mixture (5), which shortened the incubation time to 1 to 1.5 h. Chloroplasts were isolated according to the procedure of Robinson and Walker (10), as outlined previously (6).Vacuoles were isolated from protoplasts according to a modification of the method of Wagner (11). Twenty-eight ml of 0.5 mm DTT in 170 mm phosphate buffer (pH 8.0) were added to the centrifuge tube containing the protoplast pellet. The mixture was gently stirred for 5 min, poured through three layers of glasswool, and centrifuged at 200g for 3 min to spin down the majority of the protoplasts. The supernatant was transferred to a beaker, and 12 ml of 60o sucrose (w/v) were added to form an 18% sucrose mixture. The mixture was then divided into two centrifuge tubes, and 6 ml o...

Changes in photosynthesis,rlbulose bisphosphate carboxylase (RuBPCase), and proteolytic activity were followed in the leaves of field-grown soybeans [Giycine max (L.) Merr. cv. Kent] from flowering through senescence. These parameters were followed in relation to changes in leaf resistance, chlorophyll, protein, starch, total N levels, and seed development. In addition, changes in leaf ultrastructure were observed. The initial symptoms of senescence (evident 3 to 4 weeks after flowering) were a decline in photosynthesis, chlorophyll, and total leaf N and an increase in proteolytic activity. Preceding these changes there was a swelling of the chloroplasts and a disorientation of the chloroplast lamellae, possibly resulting from the apparent increase in starch deposition. Also, large numbers of osmiophilic granules appeared within the chloroplasts.These changes were evident prior to the time the seed entered its most rapid period of growth which was 4 to 7 weeks after flowering, The initial decline in photosynthesis did not appear to be due to an increase in leaf resistance or a decline in RuBPCase activity or level. The decline in protein levels began between 5 and 6 weeks after flowering and was paralleled by the decline in carboxylase activity and level. Associated with these changes were an increase in the size of the osmiophilic granules within the chloroplasts, a decrease in the number of chloroplasts with a corresponding increase in the apparent cellular breakdown products, and a dissolution of the vacuoles. No large increase in leaf resistance or change in specific activity of carboxylase was observed until late in senescence.

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