TRANSIENT EXPRESSION ACTIVITY OF RBCS PROMOTER REGIONS FROM BRASSICA-NAPUS IN MESOPHYLL PROTOPLASTS FROM NICOTIANA-TABACUM

The 5' upstream DNA of RbcS genes from Brassica napus contains sequence elements that specifically interact with DNA-binding proteins of the transcription apparatus in vitro. To clarify if these sequences are functional in vivo, we have studied them in transient expression experiments. Fragments generated by successive deletions of the 5' upstream regions of two RbcS genes were fused in frame to the beta-glucuronidase (gusA) reporter gene and GUS activity was monitored fluorometrically after polyethylene glycol (PEG)-mediated DNA uptake in mesophyll protoplasts from Nicotiana tabacum SR1. The results indicate modular organization of cis-elements that together account for full promoter activity. Whereas the TATA-box region alone resulted in basal expression levels, enhanced GUS activity was observed in the presence of additional further-upstream sequences, including the G-box and the G(s)-box. For maximal activity, the presence of a region containing the I-box was required. In addition to these putative activating DNA regions, evidence was obtained for regions that negatively affected GUS activity and hence may contain silencer element(s).