Polyribosomes from Peas

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Undegraded free and membrane-bound polysomes were isolated from developing kernels of Zea mays L. frozen in liquid nitrogen. Freezing in liquid nitrogen was a prerequisite for preserving polysome structure in stored kernels. Membrane-bound polysomes from 22-day post-pollination kernels ground in high pH buffers containing 50 mM Mg2+ contained unique classes of large polysomes. These large polysomes were sensitive to ribonudcease, and electron micrographs verified that they were not formed by aggregation. The membrane-bound polysomes were the prindpal site of zein synthesis, since the major protein synthesized in vitro was similar to purified zein in its ethanol solubility and mobility on sodium dodecyl sulfate polyacrylamide gels.The major class of storage protein synthesized in the developing endosperm of cereal grains is a hydrophobic protein, prolamine. This protein is characterized by its solubility in 70% ethanol and its unique amino acid composition. In maize this protein, zein, is found mainly in structures called protein bodies (26).Zein, which can account for 50% of the total endosperm protein in normal maize varieties (19), is particularly rich in glutamic acid (glutamine), alanine, and leucine, and nearly devoid of the essential amino acids lysine and tryptophan (20). The discovery of opaque-2 and floury-2 endosperm mutants, which have reduced zein but increased non-zein protein (hence increased lysine and tryptophan), has greatly improved the nutritional quality of maize (17,21). Although the reduced size of protein bodies in the mutant endosperm has been noted (26), the biochemistry of zein synthesis and the mechanisms by which the mutations reduce zein synthesis are unknown.In this communication we report optimal conditions for freezing and storing post-pollination kernels, and procedures for isolating polysomes which synthesize zein in vitro. A preliminary report of these results appeared elsewhere (12). MATERIALS AND METHODSHarvesting and Storage of Kernels. Maize kernels were harvested by two procedures. In the first, kernels were cut off the cob 22 days after pollination, placed in plastic bags, frozen on dry ice, and stored at -20 C. In the second, whole ears harvested at 22 days after pollination were frozen in liquid N2. The kernels were then removed and stored at -20 and -80 C. Only whole kernels were used for polysome isolation.Polyribosome Isolation. Free and membrane-bound polysomes were isolated by a procedure adapted from Larkins and I This research was supported in part by a grant from the Lilly Endowment to C. Y. T. Journal paper no. 6074 of the Purdue University Agricultural Experiment Station. Davies (14). Kernels were ground in buffer A (0.2 M tris-HCl, pH 8.5, 0.2 M sucrose, 60 mM KCl, 50 mM MgCl2, and 5 mM dithiothreitol), strained through four layers of cheesecloth, and centrifuged at 5OOg for 5 min. The supernatant fraction was centrifuged at 37,000g for 10 min to separate free and membrane-bound polysomes. The supernatant containing free polysomes was decanted and lay...

Section: Resultsmentioning

confidence: 99%

Storage Protein Synthesis in Maize

Larkins¹,

Bracker²,

Tsai³

1976

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“…The methods described by Davies et al (9) for pea stem internodes, Lin et al (22) for soybean roots, and Bewley (3) for moss also resulted in poor yields of the polysome fraction with a high yield of free ribosomal material. The unmodified technique of Jachymczyk and Cherry (18) produced yields only slightly lower than the present method.…”

Section: Methodsmentioning

confidence: 99%

Polyribosome Formation and Protein Synthesis in Imbibed but Dormant Lettuce Seeds

Fountain¹,

Bewley²

1973

Dormancy is maintained in Grand Rapids lettuce (Lactuca sativa) seeds imbibed on water in darkness at 25 C. Poly. ribosome formation and protein synthesis occur early in the imbibition phase and considerable polysomal material is also present after 24 and 48 hours, even though the seeds have failed to germinate. Incorporation of labeled leucine into protein following a 24-hour preincubation period shows that these polysomes are active in protein synthesis.Grand Rapids lettuce seeds imbibed in darkness at 25 C fail to germinate unless supplied with a particular germination stimulus. Thus, light (11,12,21) or gibberellic acid (20) stimulates almost 100% germination within 24 hr, while few untreated seeds germinate within the same time period. Such water-imbibed seeds maintain their dormant condition for long periods, up to 12 weeks (27), but retain their light sensitivity for only a matter of hours (16) or a few days (7, 13), before becoming skotodormant.Germination in darkness of these seeds is promoted by both D-and L-threo chloramphenicol and by actinomycin D (6, 7). Furthermore, both isomers, at low concentrations where germination is not promoted, prevent the onset of skotodormancy. Black and Richardson (7) suggest these inhibitors exert their effects by reducing a protein synthesis-dependent inhibitory process occurring in dark-imbibed dormant seeds, but the possibility also exists that they are acting indirectly by initially decreasing respiration (15).Although dark-imbibed dormant Grand Rapids lettuce seeds show these responses to external stimuli, to date the status of protein synthesis in the seed tissues remains obscure. Bewley and Black (4) showed protein synthesis occurring in darkimbibed dormant seeds from hours 5 to 15. Similarly, a ribosomal fraction extracted from seeds imbibed in darkness on water at times prior to, and including the time of germination of light-treated seeds, was shown to support in vitro protein synthesis without the addition of artificial mRNA, although this declined to a low level by 24 hr (10). In hazel seeds, GA. treatment substitutes for a chilling requirement, and a dormant condition, seemingly similar to that in dark imbibed seeds of

“…Isolation of RNA. The extraction procedure was modified from Davies et al (13), with heparin addition based on Palmiter (37). In a typical extraction, frozen kernels were cooled in liquid N2, 2 Reference to a company and/or product named by the department is only for purposes of information and does not imply approval or recommendation of the product to the exclusion of others which may also be suitable.…”

Section: Materials and Methods2mentioning

confidence: 99%

In Vitro Synthesis of Wheat (Triticum aestivum L.) Storage Proteins

Greene

1981

Free and membrane-associated polysomes were isolated in approximately equal amounts from endosperm of wheat kernels harvested 20 days after anthesis. The presence of heparin in the homogenizing buffer minimized polysome degradation. Ribonucleic acid from the isolated polysomes, when translated in vitro in a wheat germ system, yielded products ranging in size from about 12,000 to about 80,000 daltons, including at least two polypeptides that co-migrated with seed extract proteins in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The nature of the translation products of free and membrane-associated RNA are distinctly different, with membrane-associated RNA yielding a higher proportion of polypeptides in the size range of 30,000 to 37,000 daltons. Analysis of membraneassociated 3'-terminal polyadenylyl-containing RNA in vitro translation products, by solubility in 70% ethanol and by immunoprecipitation, indicates that the 33,000-to 37,000-dalton polypeptides contain gliadins, and the analysis provides evidence that these proteins are synthesized in association with membranous cell organelles. Gliadin polypeptides synthesized in vitro are larger than authentic gliadins and probably are precursors which, in vivo, undergo modification to yield the smaller final products.