Functional Analysis of the 5′-Regulatory Region of the Maize ALKALINE ALPHA-GALACTOSIDASE1 Gene

Maize ALKALINE ALPHA GALACTOSIDASE (ZmAGA) is a key enzyme in the raffinose metabolism pathway. We have previously characterized the ZmAGA1 gene as heat shock induced in germinating maize seeds. Here, we report that ZmAGA1 is induced in maize leaves by heat shock, dehydration, NaCl, abscisic acid (ABA), and salicylic acid. The 1622 bp of the 5′-regulatory region of the ZmAGA1 gene was isolated and characterized for promoter activity. The 5′-deletion assay of the regulatory region indicated that the region from −629 to −1 (relative to the start codon AUG) showed the greatest promoter activity, and the sequences upstream of this fragment act to inhibit transcription. The cis-acting element ABRE (ABA - responsive element) was identified to be functional. Increasingly, more refined 3′-deletion constructs of the regulatory region demonstrated that a 6-nucleotide (nt) RNA motif in the 5′-untranslated region (UTR) of the ZmAGA1 gene stimulates the translation of the downstream open reading frame (ORF). Deletion or triplication of this motif decreased or enhanced downstream ORF translation, respectively, while the mRNA abundance remained the same. RNA-EMSA (electrophoretic mobility shift assay) confirmed that some cytosolic protein(s) was (were) able to bind to this 6-nt RNA motif. This work contributes to our understanding of the ZmAGA1 gene regulatory mechanism and the metabolism of raffinose in maize cells.