Both 5[prime] and 3[prime] Sequences of Maize adh1 mRNA Are Required for Enhanced Translation under Low-Oxygen Conditions

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Abstract
Alcohol dehydrogenase-1 (ADH1) synthesis in O2-deprived roots of maize (Zea mays L.) results from induced transcription and selective translation of ADH1 mRNA. The effect of ADH1 mRNA sequences on message stability and translation was studied in protoplasts of the maize cell line P3377. 5[prime] capped and 3[prime] polyadenylated mRNA constructs containing the firefly gene (luc) for luciferase (LUC) 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% O2. A LUC mRNA with a 17-nucleotide polylinker 5[prime] untranslated region (UTR) was expressed 10-fold higher under aerobic conditions than under hypoxic conditions. Expression of five chimeric ADH1-GUS mRNAs was measured relative to this LUC mRNA. An mRNA containing the 5[prime]-UTR and the first 18 codons of adh1 in a translational fusion with the GUS coding region and followed by the 3[prime]-UTR of adh1 was expressed 57-fold higher at 5% O2. Progressive deletion of adh1 5[prime]-UTR and coding sequences reduced expression of the GUS-mRNA at 5% O2, but had little impact on expression of 40% O2. Enhancement of expression in hypoxic protoplasts conferred by the adh1 5[prime]-UTR and the first 26 codons decreased more than 3-fold when the adh1 3[prime]-UTR was removed. In addition, the adh1 3[prime]-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 were largely independent of mRNA stability. Thus, both adh1 5[prime] and 3[prime] mRNA sequences are required for enhanced translation in protoplasts under O2 deprivation.