Within-day Changes in the Polyribosome Content and in Synthesis of Proteins in Leaves of Capsicum annuum L

Abstract

Capsicum annuum cv. California Wonder was grown in controlled environment with a 12-hour photoperiod. The polyribosome content of leaves varied from 60 to 72% of total ribosomes with the highest level occurring in the middle of the photoperiod and the lowest in the middle of the dark period. The variation was accounted for by changes in the content of large polyribosomes (hexamers and larger). There was no indication of an immediate effect on polyribosome content of light-on or light-off.The synthesis of proteins at two times in the 24-hour cycle was compared using a dual isotope technique. Statistically significant results were obtained that suggested that protein(s) with molecular weights of 26,000 daltons were preferentially synthesized in the photoperiod compared to the dark period. No evidence was found for the differential synthesis of proteins within the photoperiod.The reduction of nitrate exhibits a marked periodicity with three peaks of activity during the photoperiod, in leaves of Capsicum annuum (18,19). The consequent variability in the supply of reduced nitrogen is reflected in the amino acid pool size in the leaves (13,16) and there is an indication of an integrated control of carbon supply and reduced nitrogen supply for amino acid synthesis (17).We report here information on the synthesis of proteins in Capsicum leaves in the 24-hr cycle. The capacity for protein synthesis has been assessed by measurement of the polyribosome content of ribosome preparations, and by using a dual label technique we have identified proteins that are synthesized preferentially at particular times in the 24-hr cycle. We were able to determine whether or not amino acids synthesized at different times in the photoperiod were used for the synthesis of different proteins in the leaf. To our knowledge, the only published information on the timing of protein synthesis in green tissue during the 24-hr cycle is from work on Lemna minor (4).Clark et al. (5) Expanding leaves were used as experimental material. They were selected using the same criteria as previously reported (16).Polysome Analysis. Each sample in experiment I consisted of leaves 4, 5, and 6 from four plants, and in experiment 2, of leaves 6, 7, and 8 from three plants. At the sampling times leaf laminae were excised, the midvein removed, and the sample rapidly weighed (about 4.5 and 7.7 g fresh weight for experiments 1 and 2, respectively). The samples were stored in liquid N2 for 2 to 23 hr.For polysome preparation the high ionic strength, and high pH systems of Davies et al. (7) and Jackson and Larkins (9) were used. The frozen samples were homogenized, using a Polytron at setting 8 for 5 sec, in a solution of 200 mm sucrose, 200 mm Tris-HCI (pH 9.0), 400 mm KCl, 35 mM MgCl2, 25 mm EGTA.2 For each g fresh weight of sample 3.6 ml of solution were used. The slurry was filtered through three layers of well washed muslin and Triton X-100 was added to the filtrate to a final concentration of 2%. The filtrate was centrifuged at 10,000g for 10 min. The su...