Molecular characterization and promoter analysis of the maize cytosolic glyceraldehyde 3-phosphate dehydrogenase gene family and its expression during anoxia

Maize cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC) is encoded by a small multi-gene family consisting of gpc1, gpc2, gpc3 and gpc4. GAPC3/4 protein is synthesized in roots during anoxic conditions and is known to be one of the 'anaerobic polypeptides'. We further analyzed the gpc gene family by isolating full-length cDNA clones of gpc2, gpc3, gpc4 and genomic clones of gpc2 and gpc4. The deduced amino acid sequence of GAPC4 has 99.4% identity with that of GAPC3 as compared to only 81% with either GAPC1 or GAPC2 amino acid sequence. Based on the deduced amino acid sequence identity we designated GAPC1 and GAPC2 as group I (97% identical) and GAPC3 and GAPC4 as group II (99.4% identical). As previously reported for gpc3, transcript levels were also induced for gpc4 by anaerobiosis. Neither heat shock, cold nor salt stress induced the expression of gpc3 or gpc4. In contrast, the transcript accumulation of gpc1 and gpc2 either remained constitutive or decreased in response to anoxia. The upstream regions of gpc2 and gpc4 contain typical eukaryotic promoter features with transcription start points at 76 and 68 bp upstream of their respective translation initiation sites. Transient expression analysis of gpc4 promoter-beta-glucuronidase (GUS) reporter gene constructs in bombarded maize suspension culture cells was used to examine the role of 5'-flanking sequence of gpc4. The gpc4 promoter (-1997 to +39 bp) was sufficient to induce GUS activity approximately three-fold in response to anaerobiosis. 5'-unidirectional deletion analysis revealed that the critical region of gpc4 required for its induced expression lies between -290 and -157. This region has reverse-oriented putative 'anaerobic response elements', G-box like sequences, and a GC motif similar to that previously defined as a regulatory element of maize adh1 and Arabidopsis adh, as well as the sequences found in other environmentally inducible genes. The relevance of these elements in conferring anaerobic induction of gpc4 gene expression is discussed.