THE REGULATION OF GENE-EXPRESSION IN TRANSFORMED MAIZE ALEURONE AND ENDOSPERM PROTOPLASTS - ANALYSIS OF PROMOTER ACTIVITY, INTRON ENHANCEMENT, AND MESSENGER-RNA UNTRANSLATED REGIONS ON EXPRESSION

Gene expression in the aleurone and endosperm is highly regulated duri ng both seed development and germination. Studies of alpha-amylase exp ression in the aleurone of barley (Hordeum vulgare) have generated the current paradigm for hormonal control of gene expression in germinati ng cereal grain. Gene expression studies in both the aleurone and endo sperm tissues of maize (Zea mays) seed have been hampered because of a lack of an efficient transformation system. We report here the rapid isolation of protoplasts from maize aleurone and endosperm tissue, the ir transformation using polyethylene glycol or electroporation, and th e regulation of gene expression in these cells. Adh1 promoter activity was reduced relative to the 35S promoter in aleurone and endosperm pr otoplasts compared to Black Mexican Sweet suspension cells in which it was nearly as strong as the 35S promoter. Intron-mediated stimulation of expression was substantially higher in transformed aleurone or end osperm protoplasts than in cell-suspension culture protoplasts, and th e data suggest that the effect of an intron may be affected by cell ty pe. To examine cytoplasmic regulation, the 5' and 3' untranslated regi ons from a barley alpha-amylase gene were fused to the firefly lucifer ase-coding region, and their effect on translation and mRNA stability was examined following the delivery of in vitro synthesized mRNA to al eurone and endosperm protoplasts. The alpha-amylase untranslated regio ns regulated translational efficiency in a tissue-specific manner, inc reasing translation in aleurone or endosperm protoplasts but not in ma ize or carrot cell-suspension protoplasts, in animal cells, or in in v itro translation lysates.