Protein deposition in developing barley endosperm

Abstract

The storage proteins of barley are both qualitatively and quantitatively important in determining the nutritional quality of the grain and its technological uses. The development of the barley endosperm was followed from anthesis to grain maturity by light and electron microscopy. The deposition of storage proteins in the sub-aleurone and starchy endosperm was studied using conventional electron microscopy. Correlated studies using thick sections of zinc iodide-osmium tetroxide impregnated tissue have illustrated the three-dimensional interrelationships of the endoplasmic reticulum, Golgi apparatus and vacuoles during protein deposition. To provide a clearer understanding of protein deposition in the wild-type barley, protein body formation was also investigated in mutant barley lines where a reduced and modified synthesis of storage protein is associated with an altered protein body structure. Immunocytochemical localization of A, B, and C hordeins and the chymotrypsin inhibitors, CI-1 and CI-2, primary antibodies raised in rabbit and subsequently labelled with protein A-goId illustrated the storage protein in accumulating reserves in protein bodies of the wild-type barley, and the high-lysine mutant lines. Storage proteins were localized only in specific regions of both cytoplasmic and vacuolar protein deposits. A comparison of specimen preparation techniques including different fixation and embedding protocols indicated that for barley endosperm ,tissue post-fixed with osmium tetroxide and embedded in Spurr resin gave superior results to those embedded in LR White or Lowicryl K4M resin.In situ hybridization was used to locate mRNA for CI-1 and CI-2 chymotrypsin inhibitors in barley endosperm using a biotinylated cDNA probe. The probe was localized at an ultra structural level by incubation with avidin-peroxidase and subsequent DAB staining of the peroxidase activity. The combined approach of thin- and thick- sectioning techniques for electron microscopy, in association with the molecular techniques of immunocytochemistry and in situ hybridization, has led to the development of a new model to illustrate the course of protein body development in barley endosperm. This new model also explains those previously published results used to support apparently contradictory earlier schemes.