Anaerobiosis-specific interaction of tobacco nuclear factors with cis-regulatory sequences in the maize GapC4 promoter

The promoter of the maize glyceraldehyde-3-phosphate dehydrogenase 4 gene ( GapC4) confers strong, specific and ubiquitous anaerobic reporter gene expr ession in tobacco. To identify factors required for heterologous anaerobic gene expression, 19 progressive 5' and 3' promoter deletions were linked to a chimeric GapC4 TATA box-beta-glucuronidase (GUS) reporter gene construct and transformed into tobacco. In all transgenic lines aerobic expression v alues were in the range obtained for negative controls while histochemical GUS assays reveal some weak expression in roots only. Anaerobic induction o f about 100-fold to more than 1000-fold above unspecific background is medi ated by a region of about 190 bp of the GapC4 promoter. Anaerobic reporter gene induction strongly decreases upon deletion of a 20 bp fragment from -2 86 to -266 relative to the transcription start point. This fragment harbour s putative cis-acting sequences. Electrophoretic mobility shift assays with a 50 bp fragment harbouring these cis sequences reveal a high-mobility com plex that is formed with nuclear extracts from aerobic and anaerobic leaf t issue while an additional low-mobility complex is anaerobiosis-specific. Th e formation of the high-mobility complex requires the sequence GTGGGCCCG. T he 50 bp fragment alone confers weak and orientation-dependent anaerobic in duction to a GapC4 TATA box-beta-glucuronidase (GUS) reporter gene.