The role of glycosylation in storage-protein synthesis in developing pea seeds.

Intact pea (Pisum sativum L.) cotyledons were incubated with [(14)C]glucosamine at several stages of seed development and the resultant radioactive proteins were analysed by gel electrophoresis combined with immunoaffinity chromatography and sucrose gradient fractionation. Glucosamine was incorporated into at least five vicilin polypeptides (approx. molecular weight 70,000; 50,000, two components; 14,000, two components). No incorporation was detected into the subunits of legumin. Tunicamycin at 50 µg/ml largely inhibited glucosamine incorporation but had little effect on the incorporation of (14)C-labelled amino acids into cotyledon proteins, including vicilin. The assembly of vicilin polypeptides into full-sized protein oligomers (7-9 S) was also unaffected by tunicamycin. Chromatography on concanavalin A confirmed that glycosylation of cotyledon proteins was inhibited by tunicamycin. It is concluded that glycosylation of most cotyledonary proteins involves lipid-linked sugar intermediates, but that glycosylation itself is not an essential step in the synthesis of vicilin polypeptides nor in their assembly into oligomers.

Immunofluorescent localization of pea storage proteins in glycol methacrylate embedded tissue.

Improved immunofluorescent techniques have been developed for the high resolution light microscopic localization of intracellular antigens in plant tissue. Thin sections of pea cotyledon tissue which had been fixed in paraformaldehyde and embedded in glycol methacrylate were reacted with mono-specific antibodies to the storage proteins legumin and vicilin. These antibodies were raised in sheep, purified by affinity chromatography and tested by immunoelectrophoresis and immunodiffusion. Using the indirect technique, rhodamine-labeled antibodies permitted specific fluorescent localization of the legumin and vicilin to small (ca. 1 micrometer) cytoplasmic organelles in near mature tissue. Subsequent histochemical staining verified the proteinaceous nature of these organelles. Parameters affecting staining specificity and background fluorescence are discussed.

Heterogeneity of sulphur content in the storage proteins of pea cotyledons.

By means of crossed immunoelectrophoresis of the cotyledonary storage proteins of Pisum sativum L. it was shown that reduced accumulation of the legumin fraction, resulting from severe sulphur deficiency during growth, is accompanied by relative suppression of a quantitatively minor storage protein (Peak 3) shown previously by subunit analysis to be related to the vicilin series of holoproteins. The pattern of isotopic labelling of the storage proteins after injection of [(35)S]methionine into the pedicel during seed development under normal nutritional conditions indicated that Peak-3 protein, like legumin, has a relatively high content of sulphur amino-acids. Like certain of the vicilin molecules carrying the determinants responsible for Peak-4, Peak-3 protein binds selectively to concanavalin A.

Deoxyribonucleic Acid and Ribonucleic Acid Synthesis during the Cell Expansion Phase of Cotyledon Development in Vicia faba L.

In Vicia faba L., the tissue specific proteins, legumin and vicilin, are synthesized during the cell expansion phase of cotyledon development. During this growth period, RNA and nuclear DNA increase 8- to 10-fold. (3)H-Uridine and (3)H-adenosine are incorporated into ribosomal RNA, both 25S and 18S, and into transfer RNA. DNA isolated from cotyledons in the cell division phase of growth has been compared with DNA isolated from cotyledons undergoing expansion growth. Results indicate that the DNA increase involves replication of the whole genome (endoreduplication).

Legumin Synthesis in Developing Cotyledons of Vicia faba L.

The synthesis of legumin in developing cotyledons of Vicia faba L. has been examined as a potential system for approaching the problem of differential gene expression. The pattern of legumin synthesis was determined during the growth of the cotyledon by microcomplement fixation which provided a sensitive and specific assay for legumin in the presence of vicilin. Legumin was detected even in young cotyledons. However, when the cotyledons were about 10 millimeters long, and cell division was essentially complete, there was a sharp increase in the rate of legumin accumulation.

Studies on a Maize Mutant Sensitive to Low Temperature II. Chloroplast Structure, Development, and Physiology.

In the Zea mays L. mutant M11 grown in the dark at 15 degrees , the ultrastructure of the etioplast is abnormal. The pigment content of the etioplasts is reduced but the in vivo absorption characteristics suggest that the normal protochlorophyll (ide)-holochrome is present. The lowered synthetic ability of the etioplasts is not primarily due to a reduced complement of plastid ribosomes. The plastids of mutant M11 grown in the light at 15 degrees contain little pigment, are markedly deficient in ribosomes and their ultrastructure is abnormal. In mutant M11 grown at 15 degrees , an extreme sensitivity of the plastid membranes to light was observed.

Studies on a maize mutant sensitive to low temperature I. Influence of temperature and light on the production of chloroplast pigments.

A mutant inbred line of Zea mays L. (M11) in which chlorophyll accumulation is particularly sensitive to low temperature is described. Under natural light conditions the chlorophyll content of seedlings is negligible below 17 degrees but is normal at high temperature. Seedlings of M11 can synthesize chloroplast pigments at 16 degrees but at a rate slower than normal. When photo-oxidation is minimized, chlorophyll accumulates, and seedlings can photosynthesize efficiently at low temperature. The primary site of low temperature sensitivity in M11 is the shoot apex where new leaves are developing and undergoing rapid cell expansion. It seems that there is impaired development and associated impaired function of chloroplasts in M11 grown at low temperatures which sensitizes them to rapid photo-oxidation in the light.