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Regulated Expression of Three Alcohol Dehydrogenase Genes in Barley Aleurone Layers ¹

Commonwealth Scientific and Industrial Research Organization, Division of Plant Industry, Canberra, A.C.T. 2601, Australia

Three genes specify alcohol dehydrogenase (EC 1.1.1.1.; ADH) enzymes in barley (Hordeum vulgare L.) (Adh 1, Adh 2, and Adh 3). Their polypeptide products (ADH 1, ADH 2, ADH 3) dimerize to give a total of six ADH isozymes which can be resolved by native gel electrophoresis and stained for enzyme activity.

Under fully aerobic conditions, aleurone layers of cv Himalaya had a high titer of a single isozyme, the homodimer containing ADH 1 monomers. This isozyme was accumulated by the aleurone tissue during the later part of seed development, and survived seed drying and rehydration. The five other possible ADH isozymes were induced by O₂ deficit. The staining of these five isozymes on electrophoretic gels increased progressively in intensity as O₂ levels were reduced below 5%, and were most intense at 0% O₂.

In vivo³⁵S labeling and specific immunoprecipitation of ADH peptides, followed by isoelectric focusing of the ADH peptides in the presence of 8 molar urea (urea-IEF) demonstrated the following. (a) Aleurone layers incubated in air synthesized ADH 1 and a trace of ADH 2; immature layers from developing seeds behaved similarly. (b) At 5% O₂, synthesis of ADH 2 increased and ADH 3 appeared. (c) At 2% and 0% O₂, the synthesis of all three ADH peptides increased markedly.

Cell-free translation of RNA isolated from aleurone layers, followed by immunoprecipitation and urea-IEF of in vitro synthesized ADH peptides, showed that levels of mRNA for all three ADH peptides rose sharply during 1 day of O₂ deprivation. Northern hybridizations with a maize Adh 2 cDNA clone established that the clone hybridized with barley mRNA comparable in size to maize Adh 2 mRNA, and that the level of this barley mRNA increased 15- to 20-fold after 1 day at 5% or 2% O₂, and about 100-fold after 1 day at 0% O₂.

We conclude that in aleurone layers, expression of the three barley Adh genes is maximal in the absence of O₂, that regulation of mRNA level is likely to be a major controlling factor, and that whereas the ADH system of barley has strong similarities to that of maize, it also has some distinctive features.

¹ Supported in part by a Guggenheim Fellowship to A. D. H.

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