Regulation of Legumin Levels in Developing Pea Seeds under Conditions of Sulfur Deficiency

Abstract

It was shown previously that when peas (Pisum sativum L.) are grown with suboptimal sulfur supply the level of legumin (the more S-rich of the two major seed storage proteins) in the mature seed is selectively reduced (Randall, Thomson, Schroeder, 1979 Aust J Plant Physiol 6: 11-24). This paper reports a study of the cellular mechanisms involved in regulating legumin synthesis under these conditions. Pulse and pulse-chase labeling experiments were carried out with excised, immature cotyledons from normal and S-deficient plants. Legumin was isolated from cotyledon extracts by immunochromatography, and the proportion of legumin synthesis relative to total protein synthesis was determined. Results showed that reduced legumin accumulation could largely be accounted for by a greatly reduced level of legumin synthesis (80-88% reduction) rather than by a major increase in leumin breakdown. Legumin mRNA levels were assayed by two methods. In vitro translation of polysomal RNA from cotyledons of normal and S-deficient plants indicated a reduction of 60 to 70% in synthesis of legumin-related products by preparations from S-deficient plants. A legumin cDNA clone was constructed, characterized, and used to measure the levels of legumin mRNA in polysomal and total RNA preparations from developing cotyle-dons. Legumin mRNA levels were reduced by 90% in preparations from S-deficient plants. When restored to an adequate S supply, S-deflcient plants (or pods taken from such plants) recovered normal levels of legumin synthesis (in vivo and in vitro) and of legumin mRNA. These results indicate that reduced legumin accumulation under conditions of S deficiency is primarily a consequence of reduced levels of legumin mRNA. Legumin and vicilin are the two storage proteins accumulated in large quantities during the formation of pea (Pisum sativum L.) seeds. In the cultivar used in our experiments, legumin makes up approximately 15% of the total protein in the seed at maturity (25). Legumin has a higher content of cysteine and methionine than vicilin and could therefore be considered to be the more desirable protein from the point of view of animal nutrition. Its physicochemical properties are typical of one of the storage proteins found in a range of legume seeds including broad bean (Vicia faba), lupin (Lupinus angustifolius), and soybean (Glycine max) (8). It was shown earlier (19) that when peas are grown with suboptimal sulfur (S) nutrition there is a marked, selective reduction in the level of accumulated legumin relative to the total protein level. Legumin was virtually undetectable under severe S deficiency. Similar changes were found in the more S-rich com-47 ponent of the storage proteins of lupin under S deficiency conditions (1). This paper describes experiments that were designed to study the cellular mechanisms responsible for the reduction in the level of legumin in pea seeds developing under conditions of S deficiency. The following three mechanisms were tested: (a) a normal rate of legumin synthesis but an increased rate of degradation; (b) a normal level of legumin mRNA but reduced translation; and (c) reduced levels of legumin mRNA. The results indicate that a reduced level of legumin mRNA is the main factor responsible for the reduced accumulation of legumin. MATERIALS AND METHODS Plant Material. Peas (Pisum sativum L.) line PI/G 086, selected from cv Greenfeast, were grown in artificially lit cabinets at 20°C with a 16-h photoperiod as described (16) and pods of a known age, specified as DAF 1, were used. Plants were grown in sand with nutrient solution containing combined nitrogen (19). Sulfur was supplied as MgSO4 at I mm in the control nutrient throughout growth and 0.05 mm in the S-deficient nutrient until first flower and omitted thereafter. The level of Mg2+ was kept constant by varying MgCl2. Under these conditions, S-deficient plants showed characteristic symptoms and were smaller with fewer pods. Mean seed weight at maturity was little affected in pods from lower nodes but considerably reduced in later formed pods. For uniformity , only pods from the third to the sixth flowering node were used. Mature seeds from these pods had a mean weight of 280 mg in controls compared with 265 mg in S-deficient plants. S contents of these seeds differed considerably between the two treatments being, respectively, 0.20%1o and 0.05%. From these analyses, the degree of deficiency and the relative severity of the effects on aspects ofseed composition can be gauged by reference to previous work (19). Culture of Detached Pods. Pods were cut from the plant at the base of the peduncle, quickly re-cut under water, and the peduncle immersed in nutrient solution. The nutrient with S contained (in mM): sucrose, 292; L-asparagine, 117; L-glutamine, 39; CaCl2, 4; KH2PO4, 6; K2SO4, 2; MgSO4, 4; and iron/EDTA and micronu-trients as for whole plant culture. For treatments without S, sulfates were replaced by chlorides to give the same cation concentrations. NaOH and HCI were used to give a final solution pH of 5.0. Precautions to minimize microbial growth included treating peduncles with 0.5% NaOCl for 10 min before detachment from the plant, filter-sterilizing nutrient media before use, and immers-' Abbreviations: DAF, days after flowering; MMH, methylmercuric hydroxide; PAGE, polyacrylamide gel electrophoresis; IgG, immunoglob-ulin G, AMV, avian myeloblastosis virus.