Cytoplasmic controls of storage protein synthesis in pea (Pisum sativum L.)

Abstract

The in vitro translation of pea storage protein subunits was investigated using four polysome driven cell-free systems, derived from wheat-germ, reticulocyte lysate, pea axes and pea cotyledons. Re-initiation of protein synthesis occurred in each of the systems and accounted for about 25%, 34%, 44% and up to 45% of protein synthesis in the polysomes driven wheat-germ, cotyledon, axes and reticulocyte lysate systems respectively. The polysome translation products of all the systems were very similar and vicilin 50,000 and 47,000 mol.wt. polypeptides, convicilin 70,000 mol.wt. polypeptide, and legumin precursor of 60,000 mol.wt. were shown to be synthesised using immuno precipitation techniques and by comparing the QNBr cleavage products of in vitro and in vivo synthesised polypeptides. Modification of the vicilin 50,000 and 47,000 mol.wt. polypeptides occurred by the removal of short amino acid sequences, when microsomal membranes were present in the cell-free system, although sequestering of the synthesised polypeptides into membrane-bound vesicles could not be demonstrated. Hhe plant derived cell-free systems were capable of a limited amount of polypeptide modification, which was absent from the reticulocyte lysate system. cDNAs mRNA hybridisation techniques were employed in a study of the abundance and complexity of the mRNA population of pea cotyledons at three seed developmental stages, (9, 14, and 19 daf). An increase in very abundant mRNAs was noted between 9 and 14 day stages (up to 5 mRNAs), during which time rapid storage protein accumulation commences, while a progressive decrease in the number of rare RNAs was apparent during development. Preliminary investigations into the proportion of single-copy DNA, and of nuclear poly (A) (^+)-RNA, present in the polyscmal poly(A)(^+)-RNA were carried out; approximately 5% of single-copy DNA was transcribed into the polysomal poly (A)(^+)-RNA of cotyledons, (at 14 daf), and approximately 50% of nuclear poly (A)(^+)-RNA may be present in polysomal poly(A) (^+)-DNA of cotyledons at 9 daf.