Differential Synthesis in Vitro of Barley Aleurone and Starchy Endosperm Proteins

Abstract

To widen the selection of proteins for gene expression studies in barley seeds, experiments were performed to identify proteins whose synthesis is differentially regulated in developing and germinating seed tissues. The in vitro synthesis of nine distinct barley proteins was compared using mRNAs from isolated endosperm and aleurone tissues (developing and mature grain) and from cultured (germinating) aleurone layers treated with abscisic acid (ABA) and GA3. B and C hordein polypeptides and the salt-soluble proteins O-amylase, protein Z, protein C, the chymotrypsin inhibitors (CI-I and 2), the a-amylase/subtilisin inhibitor (ASI) and the inhibitor of animal cell-free protein synthesis systems (PSI) were synthesized with mRNA from developing starchy endosperm tissue. Of these proteins, f6-amylase, protein Z, and CI-1 and 2 were also synthesized with mRNA from developing aleurone cells, but ASI, PSI, and protein C were not. CI-I and also a probable amylase/protease inhibitor (PAPI) were synthesized at high levels with mRNAs from late developing and mature aleurone. These results show that mRNAs encoding PAPI and CI-I survive seed dessication and are long-lived in aleurone cells. Thus, expression of genes encoding ASI, PSI, protein C, and PAPI is tissue and stage-specific during seed development. Only ASI, CI-I, and PAPI were synthesized in significant amounts with mRNA from cultured aleurone layers. The levels of synthesis of PAPI and CI-1 were independent of hormone treatment. In contrast, synthesis of a-amylase (included as control) and of ASI showed antagonistic hormonal control: while GA promotes and ABA reduces accumulation of mRNA for a-amylase, these hormones have the opposite effect on ASI mRNA levels.One approach to understanding the molecular mechanisms controlling seed maturation and germination is to study organspecific, developmentally characteristic, and hormonally regulated seed proteins and their mRNAs. Earlier studies have identified such polypeptides in Gossypium (7) and Brassica (6) and have formed the basis of later work at the molecular level on these crops. We report here the results of similar experiments with barley seeds, a cereal of importance as livestock feed and as malt in the brewing industry.The barley seed or caryopsis is comprised of several tissues derived from the fertilized ovule (4). The seed proteins of the grain, synthesized during seed development in the endosperm and/or aleurone layer, include the hordeins (30-50% of total N, ' Present address:

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