Storage Protein Synthesis in Maize

Abstract

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...

Transfer of in vitro synthesized barley endosperm proteins into the lumen of the endoplasmic reticulum

“…Techniques for the in vitro synthesis of proteins have recently been exploited in the synthesis of various plant proteins and in particular the storage proteins, zein (13), hordein (4) and oat globulin (14). Bearing in mind the hydrophobic nature of many plant storage proteins and their confinement to membrane-bound vacuoles, it is significant that the main polysome fractions active in storage protein synthesis are found among the membrane-bound polysomes (4,6,13).…”

Storage Protein Synthesis in Maize

“…In vitro translation of the polyribosome-associated mRNAs at this stage demonstrated that zein biosynthesis is primarily associated with the membranebound polyribosome fraction (6, 7 Polyribosome Extraction. Free and membrane-bound polyribosomes were prepared according to the procedure of Larkins et al (7,9). Whole kernels were hand-ground with a chilled mortar and pestle in buffer A (0.2 M tris-HCI at pH 8.5, 0.2 M sucrose, 60 mm KCI, 50 mM MgCI2, and 5 mm dithiothreitol).…”

“…Free and membrane-bound polyribosomes were prepared according to the procedure of Larkins et al (7,9). Whole kernels were hand-ground with a chilled mortar and pestle in buffer A (0.2 M tris-HCI at pH 8.5, 0.2 M sucrose, 60 mm KCI, 50 mM MgCI2, and 5 mm dithiothreitol).…”

“…Plants were self-pollinated under controlled conditions. Ears were harvested at different stages of kernel development and frozen in liquid N2 to retain polyribosome integrity (9). Intact kernels, removed from the cob and stored at -80 C, were used for the subsequent isolation of polyribosomes.…”

“…Since the opaque-2 mutant has higher endogenous levels of ribonuclease than normal maize (21), an assay for endolytic messenger ribonuclease activity (4) was conducted to determine if polysome degradation occurred during the extraction procedure. RNase activity is determined by the conversion of large polysomes to small polysomes (4,9). Purified membrane-bound polysomes from normal maize were incubated at 0 C in postribosomal supernatant from opaque-2 kernels.…”