BOTH 5'-SEQUENCES AND 3'-SEQUENCES OF MAIZE ADH1 MESSENGER-RNA ARE REQUIRED FOR ENHANCED TRANSLATION UNDER LOW-OXYGEN CONDITIONS

Alcohol dehydrogenase-1 (ADH1) synthesis in O-2-deprived roots of maiz e (Zea mays L.) results from induced transcription and selective trans lation of ADH1 mRNA. The effect of ADH1 mRNA sequences on message stab ility and translation was studied in protoplasts of the maize cell lin e P3377. 5' capped and 3' polyadenylated mRNA constructs containing th e firefly gene (luc) for luciferase (LUG) or the Escherichia coli gene (uidA) for beta-glucuronidase (GUS) coding region were synthesized in vitro and electroporated into protoplasts that were cultured at 40 or 5% O-2. A LUC mRNA with a 17-nucleotide polylinker 5' untranslated re gion (UTR) was expressed 10-fold higher under aerobic conditions than under hypoxic conditions. Expression of five chimeric ADH1-GUS mRNAs w as measured relative to this LUC mRNA. An mRNA containing the 5'-UTR a nd the first 18 codons of adh 1 in a translational fusion with the GUS coding region and followed by the 3'-UTR of adh1 was expressed 57-fol d higher at 5% O-2. Progressive deletion of adh1 5'-UTR and coding seq uences reduced expression of the GUS-mRNA at 5% O-2, but had little im pact on expression of 40% O-2. Enhancement of expression in hypoxic pr otoplasts conferred by the adh1 5'-UTR and the first 26 codons decreas ed more than 3-fold when the adh1 3'-UTR was removed. In addition, the adh1 3'-UTR slightly inhibited expression in aerobic protoplasts. The physical half-lives of the GUS and LUC mRNAs were similar under both anaerobic and hypoxic conditions, indicating that expression levels we re largely independent of mRNA stability. Thus, both adh1 5' and 3' mR NA sequences are required for enhanced translation in protoplasts unde r O-2 deprivation.