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Chloroplasts, isolated from the primary leaves of 7-day-old seedlings, were incubated in vitro at 256C with 2-chloroethylphosphonic acid (ethephon) under light (0.16 milliwatts per square centimeter) and dark conditions. Ethephon at 1 micromolar (0.1445 ppm), 0.1 and 1 millimolar, or 5 microliters ethylene promoted the deterioration of chloroplasts, increased proteolysis, and reduced the chlorophyll content and PSI and PSII during 72 hours under,both light and dark conditions. The decline in PSI and PSII occurred prior to a measurable loss of chlorophyll. The loss of photosynthetic activity affected by ethephon was initiated prior to 12 hours of incubation. After 24 hours in light, 0.1 millimolar (1.445 ppm) epthephon significantly reduced PSI and PSII and promoted the total free amino acid liberation in isolated chlorophsts. In darkness the rate of loss of PSI activity was about 50%O of that in light. After 24 hours, in light at 1 millimolar epthephon, PSII activity was 55% of the control, yet nearly 90% of the chlorophyll remained, which indicates that the loss of thylakoid integrity was promoted by ethephon. Ethylene injected in the chloroplast medium at 5 microliters (0.22 micromolar per milliliter) reduced PSI by nearly 50% of the initial in 12 hours. In leaf sections floated in 5 microliters per milliliter suspension medium, a 36% loss of chlorophyll of the control in 36 hours was observed. Cycloheximide at 0.5 millimolar masked the effect of 1 millimolar ethephon and maintained the initial chlorophyll content during the 72 hour period.To promote maturity or color of apples, blackberries, blueberries, cranberries, pineapple, and tomatoes, foliar sprays of an ethephon solution ranging from 200 to 1000 actual ppm have been the recent field practice (24). Ethylene evolved from ethephon has been known to hasten maturity and the senescence phenomena in plant tissues. Ethylene released from ethephon promotes the loss of Chl (4, 18, 1-9), hydrolysis of polymers (12,14), climacteric rise in respiration-(1, 14), and early maturation or accelerated senescence (1,2,13 MATERIALS AND METHODSChloroplasts were isolated from oat (Avena sativa cv. 'Garry Spring') seedlings which were grown for 7 d in vermiculite at 25°C. Cool white fluorescent tubes provided light for 14 h/d at approximately 0.81 mW cm-2 at the plant level. A bundle of 10 g of apical 5-cm leaves were chilled, cut, surface sterilized, finely minced with a razor blade, and homogenized in a freshly prepared chilled isolation medium A (5). This medium A contained 30 mm Tes, 0.33 M sorbitol, 1 mM EDTA, 1 mM MgCl2, 5 mM sodium ascorbate, and 0.1% BSA fraction V and was adjusted to pH 7.4 with 1 N NaOH. Chloroplasts were aseptically isolated at 0 to 4°C from the homogenate as previously described (6)

The retention of photosystems I and II and or RuDP carboxylase activity in chloroplasts isolated from the first leaves of Victory oat (Avena sativa L.) seedlings was followed as the chloroplasts senesced in darkness. Both photosystems (PS) I and II retained their full activity after 3 days at 1°C, while even after 7 days at 1°C around 80% of the activity was still present. After 3 days at 25°C, PS I lost only 20% and PS II 50% of the initial activity. Acid pH increased the rate of decay of both systems, PS II falling almost to zero after 3 days at pH 3.5 (at 25°C). The preparations were almost bacteria-free, and addition of antibiotics not only did not improve their stability, but accelerated the rates of loss of photosynthetic activity. This is held to indicate that the enzymes are undergoing some turnover even in isolated chloroplasts. If the leaves were allowed to senesce in the dark first and the chloroplasts then isolated, their photosynthetic activities had greatly decreased, showing that senescence is more rapid in situ than in isolation. Under these conditions PS I decayed more rapidly than PS II. Ribulosediphosphate carboxylase, as measured by CO2 fixation, declined more rapidly than the photosystems, though the addition of kinetin and indole-3-acetic acid somewhat decreased the rate of loss, at least for the first 24 h. When the intact (detached) leaves were held in the dark, the rate of oxygen evolution declined rapidly, but in monochromatic blue light (450 nm) at 25°C about 30% of the initial rate was retained after 72 h.

The changes in chlorophyll and protein in senescing chloroplasts isolated from the first leaves of 7-day-old oat (Avena sativa) seedlings have been investigated. In darkness the chlorophyll in these plastids is highly stable, losing only 5 to 10% of its content after 7 days at 26 C. This result contrasts with the behavior of chlorophyll in intact leaves, in which about 80% of the pigment would have disappeared in that time. The protein is less stable than the chlorophyll, though more stable than in the leaf; probably a small amount of protease is present in the plastids. Some protein is also being synthesized in the chloroplasts along with its breakdown; gains of up to 38% in protein and 13% in chlorophyll were observed under different conditions. L-Serine, which actively promotes senescence in the leaf, has only a very slight effect on the chloroplasts, and kinetin antagonizes it. Kinetin also has a small but significant effect in preserving the protein from breakdown. Acid pH somewhat promotes the breakdown, both of chlorophyll and protein. A loss of chlorophyll and protein comparable to that occurring in the senescence of the leaf could not be induced in the chloroplasts by suspending them in malate, in cytoplasmic extract, or in any of a number of enzymes tested alone. Incubation with a mixture of four enzymes was the only treatment which approximated the senescent process in the leaf, causing 34% loss of chlorophyll at pH 5 and 40% loss of protein at pH 7.4, both in 72 hours.In white light, the chlorophyll and the carotenoids, but not the protein, disappear rapidly. This disappearance was shown to be prevented in an atmosphere of nitrogen or in air by a number of reducing agents, of which ascorbic acid was the most effective. It is, therefore, ascribed to photooxidation rather than to normal senescence.The senescence of leaves, which has been studied in a number of laboratories over the years (3, 7, 10, 11, 18, 19, 21-23, 25, 26, 29, 31, 33-39), involves loss of Chl, decrease in carotenoids, enhanced proteolysis, hydrolysis of starch and other polysaccharides, and, in some cases, increase in respiration. In detached leaves, the hydrolyzed products accumulate and may be partly converted to organic acids (21, 23, 37), whereas if the leaves remain on the plant the amino acids, and perhaps 'This work was supported in part by National Science Foundation Grant GB35238 to K. V. T. other products, are re-exported to the stem or base (34). Both proteolysis and Chl loss can be effectively prevented by cytokinins and, in a few cases, also by gibberellins (e.g. 4, 13). The mechanism of this interference with hydrolytic reactions is under active study.In our own laboratory, detailed studies of the senescent process in the detached first leaves of 7-day-old oat seedlings have shown that senescence is largely prevented not only by cytokinins but also by anaerobiosis and by certain inhibitors of protein synthesis, i.e. it is dependent on some synthetic processes, probably formation of enzymes, at the start. There ...

Because previous work indicated that senescence of oat leaves in darkness probably centers in the cytoplasm, the senescence of chloroplasts isolated from the same material and carefully purified was studied. The rate of loss of chlorophyll was about one-tenth of that which takes place in the isolated leaves at the same temperature, while the loss of protein, though slightly more rapid, was still only 36% of the rate observed in the leaf after 7 days. This unexpected stability of the chloroplasts is matched by their photosynthesis, 81% of photosystem I and 35% of photosystem II being present after 3 days at 25°. Traces of system I, but not of system II, were still detectable even after 7 days.

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Lipoxygenase (LOX), one of the main oxidative catalysts in plants, is involved in the regulation of growth and senescence. We investigated changes in LOX activity or its properties as they related to the development of oat plants at four stages (germination, growth, natural senescence, and dark-incubated senescence). LOX activity was high during early growth and at senescence. At pH 4.5, activity showed an abrupt surge compared with a normal enzyme reaction pattern at pH 6.5. The optimum reaction temperature was 25~ LOX and peroxidase exhibited similar activity patterns. Polyacrylamide gel electrophoresis revealed that the purified LOX consisted of three isoenzymes in germinating seeds, two in growing seedlings, and three during both natural and dark-induced senescence. As determined by isoelectric focusing, the isoelectric points (pl) of LOX ranged from 3.6 to 6.5 throughout the four developmental stages; for natural or dark-induced senescence, the pl was 9.0.