Chloroplastic Ribosome Formation: Inhibition by 3-Amino-1,2,4-Triazole

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The application of sublethal doses of 3-amino-1, 2,4-triazole (AT) to germinating wheat seedlings results in the formation of alibinistic leaves. These leaves grow and develop nearly as well as the control leaves for periods up to 1 week following germination (9). An tultrastrutctural examination of this tissue revealed that the chloroplasits were the only subcellular organelles altered morphologically by AT treatment. These altered chloroplasts lacked normal grana and fret membranes, but rather contained a few disorganized or concentric arranged membranes (2). In this study, we examined the effect of AT on the ribosomal composition of light-grown wheat leaves and found that the 70S chloroplastic ribosomes and 18S Fraction I protein of the chloroplast were absent. In addition, we confirmed earlier investigations (4, 5, 6, 8) which indicated that the chloroplasts contained only 70S ribosomes. Abotut 15 wheat grains (Triticum vulgare L. var. Seneca and Federation) were germinated in a petri dish containing 10 ml of 0.1 mm AT or distilled water. The plants were grown tunder 1000 ft-c of light (16 hr photoperiod, 21?) or in darkness. Following germination, distilled water was tused for the required watering and shoots were harvested on the seventh day and prepared for either ultrastructutral examination or sedimentation sttudies. For sedimentation studies, approximately 10 g of fresh leaf tissue were chilled and grouind in a mortar and pestle (2?) with an eqtual weight of stucrose-tris btuffer at pH 8.4 (7). The homogenate was strained throuigh 2 layers of cheesecloth, centrifuged for 30 mintutes at 23,000 X g (max, 1?) in a Servall SS-34 head and the suipernatant material was then centrifuged for 1.5 houirs at 226,000 X g in a Spinco 50 Ti head. The pellet was resuispended in a buiffer (pH 7.5) composed of 5 mM tris (Sigma) 7 mm magnesitum acetate and 5 mm mercaptoethanol, and clarified by centrifuging for 10 minuites at 8000 X g in an SS-34 head. The supernatant solution was removed and tused as the ribosomal suspension for the ultracentriftugal analysis on a Spinco MIodel E ultracentrifuge using a standard 12 mm, 40 sector cell in an AnD rotor. For electron microscopic stuidies, fresh tissue was fixed in 6 % glutaraldehyde (9 hr, 40) and embedded in Maraglas. Sections were cut with glass knives and double post-stained with aqueous uranyl acetate and lead citrate (3). Figure 1 shows a part of a chloroplast from an untreated, light-grown plant. The stroma (S) of this chloroplast contains many 170A particles (PR) which conform to the electron microscopical criteria for ribosomes since they were preserved by glutaraldehyde and osmium tetraoxide, strained 'with turanyl acetate and digested by ribonuclease (3). In contrast, a section of a plastid from an ATtreated plant (fig 2) showed that the stroma lacked ribosomal particles while the cytoplasm contained an abundance of ribosomes (CR). The ultracentrifugal pattern obtained with the ribosomal extracts from tissuie identical to those tused in the ultrastructural study is shown in figure 5. In this figuire, the direction of sedimentation was from left to right. Extracts from control, lighit-grown leaves (lower curve) showed 3 peaks with approximate sedimentation coefficients of 18S, 70S, and 80S and they represent Fractions I protein, chloroplastic and cytoplasmic ribosomes, respectively (4,5,8). In contrast, extracts of ATtreatedl, light-grown leaves (upper curve) yielded only an SOS peak, and the 18S and /OS peaks were completely absent in 5 separate experiments. In this sttudy, corrections were not made for viscosity effects duie to residual stucrose and protein concentration, therefore, the sedimentation valves are not exact. The comibined results of the tultrastructuiral and utltracentrifugal stuidies show that the treatment of light-grown, germinating seedlings with AT catused the complete loss of chloroplastic ribosomes and Fraction I protein but not cytoplasmic ribosomes. In contrast to the stuidies involving light-grown plants, AT treatment did not appear to alter the 1 Supported by grants from the USPHS (6F2HD-23, 340-OlAl) and the American Cancer Society.