Effects of Protein Synthesis Inhibitors on ent - Kaurene Biosynthesis during Photomorphogenesis of Etiolated Pea

Excised shoot tips from 10-day-old etiolated pea (Pisum sadvum L. cv. Alaska) seedlings were incubated in solutions of chloramphenicol, cycloheximide, and lincomycin at different concentrations during periods of 0, 4, 8, and 12 hours of irradiation with high intensity white light. Enzyme extracts were prepared from the whole shoot tips and compared with extracts from nontreated shoot tips for their capacity to synthesize entkaurene from mevalonate. In control samples, kaurene synthesis increased during the first 8 hours of irradiation and decreased after 12 hours. Chlorophyll content increased steadily up to 12 hours of irradiation. Chloramphenicol and cycloheximide reduced both kaurene synthesis and chlorophyll formation to a similar extent during all periods of irradiation, the reduction being greatest after 8 hours of irradiation. Lincomycin, a specific inhibitor of the formation of chloroplast ribosomes in detached pea shoot tips, did not significantly affect kaurene synthesis activity but strongly inhibited chlorophyll formation. It is tentatively concluded that the increase in kaurene synthesis activity during normal photomorphogenesis in pea seedlings is due to photoinduction of de novo synthesis of one or more proteins involved in the biosynthetic pathway from mevalonate to kaurene. It was reported from our laboratory (6) that cell-free extracts from pea shoot tips of light-grown pea seedlings had a 5-fold greater capacity for synthesizing kaurene, and potentially therefore GA, from mevalonate than extracts from shoot tips of etiolated seedlings of the same age. Upon continuous irradiation of 10-day-old etiolated seedlings with high intensity white light, an exponential increase in kaurene synthesis activity was observed over a period of approximately 12 hr, attaining a level equal to that of light-grown plants of the same age. Although the lightstimulated increase in kaurene synthesis and chloroplast development occuffed concurrently, there was no evidence at that time as to whether the two processes are causally related. The possibility that a direct cause and effect relationship between chloroplast ' This work constitutes a portion of a thesis presented by G. G.-N. to the Graduate School of Oregon State University in partial fulfillment of the requirements for the M.S. degree. The investigations were supported in part by a scholarship awarded by the Latin American Scholarship Program of American Universities to the senior author and by Grant PCM75-22119 from the National Science Foundation. 2 Present address: Departamento de Ciencias Biologicas, Universidad de Chile, La Serena, Chile. 'Author to whom inquiries should be made. 4 Abbreviations: ent-kaurene, also known as (-)-kaurene, is referred to elsewhere in the text simply as kaurene; GA: gibberellin; MVA: mevalonic acid. development and light-stimulation of kaurene biosynthesis does exist is indicated by numerous reports that: (a) GAs occur in chloroplasts and other plastids (3, 4, 22); (b) at least some GA biosynthesis occurs in those organelles (18, 21, 23); and (c) light affects GA metabolism in a complex way, including apparent effects on biosynthesis ( 19), interconversions (20), apparent release from membrane-bound forms (3), and phytochrome-mediated efflux from plastids (4, 11, 13, 25). These investigations were undertaken to determine whether the observed increase in kaurene synthesis activity caused by irradiation with white light is associated with de novo enzyme synthesis and, if so, whether that essential protein synthesis is localized in chloroplasts. Use was made of selective inhibitors of protein synthesis, namely, the D-threo isomer of chloramphenicol, cycloheximide, and lincomycin. On the basis of all available data, it appears that photoinduction of de novo synthesis of one or more proteins essential for kaurene, hence GA, biosynthesis does occur during de-etiolation of pea seedling shoots. MATERIALS AND METHODS Culture and Sampling of Plants. Peas (Pisum sativum L. cv. Alaska), from W. Atlee Burpee Company, Riverside, Calif., were grown in darkness in growth chambers under a regimen consisting of 16 hr at 22 ± 1 C alternating with 8 hr at 17 ± 1 C for 10 days. The Vermiculite rooting medium was irrigated with distilled H20 every other day. Plants used for standardizing conditions for assaying kaurene synthesis in cell-free extracts of shoot tips were exposed to continuous high intensity light (1,000-1,300 ft-c; 1.8 x 104 to 2.4 x 104 erg cm-2 sec) for periods of 4, 8, or 12 hr. Shoot tips were harvested by excising them below the fourth node of the seedling axes (numbering from the cotyledonary node). All tissues above the fourth node were included in the samples, which routinely were frozen and stored in liquid N2. Plants used for investigating the effects of chloramphenicol, cycloheximide, and lincomycin were grown in darkness for 10 days. Then, etiolated shoot tips were excised as before and placed in sterile Petri dishes (approximately 1.2 g, 40 shoot tips/Petri dish), each of which contained a disc of filter paper and 10 ml of one of the following solutions: 0.1 mg/ml of water, I mg/ml, 10 mg/ml, and 33 mg/ml of chloramphenicol or cycloheximide or 0.01 utg/ml, 0.10 jMg/ml, 1 ,tg/ml, 10 ,ug/ml, and 100 jig/ml of lincomycin. After incubation for I hr in darkness (17), the Petri dishes were transferred to the irradiation chamber and illuminated for 4, 8, or 12 hr as described before. Shoot tips in Petri dishes were in contact with the test solution throughout the irradiation period. Except during irradiation with white light, live plant material always was handled under a green safelight (General Electric 15-w green fluorescent lamp covered with eight layers of amber and three layers of green cellophane). After the irradiation period, shoot tips were removed from the Petri dishes, blotted, www.plantphysiol.org on October 23, 2017 Published by Downloaded from Copyright © 1978 American Society of Plant Biologists. All rights reserved. GOMEZ-NAVARRETE AND MOORE frozen, and stored in liquid N2. Preparation of Enzyme Extracts and Reaction Conditions. General procedures were as described previously (5, 6, 18). Excised frozen shoot tips were ground in a chilled mortar with pestle to a fine powder. As the tissue thawed, insoluble PVP (0.5 g wet PVP g' fresh wt of tissue) and 0.1 M KH2PO4-K2HPO4 buffer (pH 7.1; ml g-' fresh wt of tissue) containing 20 mm DTT and 133 uM chloramphenicol were quickly added, and the mixture was homogenized immediately. The homogenate was filtered once through four layers of cheesecloth, and thefiltrate was centrifuged at10,000g for 10 min at 0 to 4 C. The10,000g supernatant was centrifuged at 100,000g for 90 min at 0 to 4 C, and the resulting supernatant was the enzyme extract. Average protein concentrations (mg/ml) in the enzyme extracts were as follows: etiolated shoot tips, 20.3; shoot tips excised from whole seedlings after irradiation for 4, 8, and 12 hr, 22.6, 18.6, and 16, respectively; and shoot tips excised prior to irradiation for 4, 8, and 12 hr, 16.1, 14.6, and 13.6, respectively. Following optimization of the reaction conditions, reaction mixtures routinely contained 19.2 /LM [2-'4C]MVA (13 mCi/mmol), 2 mm MgCl2, 2 mm MnCl2, 12 mm ATP, 0.70 ml of enzyme extract, 75 mM KH2PO4-K2HPO4, 15 mm DTT, and 100 tLM chloramphenicol in a total volume ofI ml (pH 6.1). Reaction mixtures were incubated at 30 C for 2 hr in an aerobic atmosphere. Each reaction was stopped by the addition of 2 ml of acetone containing 2.5ug of nonradioactive kaurene. The yield of radioactive kaurene then was measured as described previously (5, 6, 18). Radioactivity was measured in a Packard Tri-Carb liquid scintillation spectrometer, model 2425. Counting rates were converted to dpm by internal standardization. All vials were counted prior to addition of radioactive samples to obtain a background value for each sample. Routinely, all assays were run in duplicate or triplicate, and all experiments were repeated at least twice. The Chl content of greening shoot tips was estimated by the procedure of Arnon (2). The protein content of 10%1o trichloroacetic acid-insoluble material in the enzyme extracts was estimated by the method of Lowry et al. (16), using BSA as a standard. Reagents. [2-"4C]MVA (13 mCi/mmol) in benzene solution was purchased from Amersham/Searle. The benzene was evaporated under nitrogen, and the lactone was hydrolyzed in 100%o excess NaOH for 8 hr. PVP (insoluble Polyclar-AT) was obtained from GAF Corporation, Graselli, N.J. The PVP was purified according to the procedure of Loomis (15), washed once with 0.1 M KH2PO4K2HPO4 buffer (pH 7.1), and then suspended in 0.1 M KH2PO4K2HPO4 (pH 7.1), containing 100 uM chloramphenicol. After sedimentation, the excess buffer was removed and the wet PVP was stored at 4 C. Chloramphenicol and cycloheximide were purchased from Sigma Chemical Co. Lincomycin HCI (potency 860,ug/mg) was a gift of the Upjohn Company, Kalamazoo, Mich. Omnifluor was purchased from New England Nuclear. Kaurene was a gift from Abbott Laboratories, North Chicago, Ill.