LOCALIZATION OF LIGHT-INDUCIBLE AND TISSUE-SPECIFIC REGIONS OF THE SPINACH RIBULOSE-BISPHOSPHATE CARBOXYLASE OXYGENASE (RUBISCO) ACTIVASE PROMOTER IN TRANSGENIC TOBACCO PLANTS

Deletions in the spinach rubisco activase (Rca) promoter in transgenic tobacco were analyzed to define the regions necessary for conferring light-inducible and tissue-specific expression. Transgenic plants were constructed with Bal 31 deletions of the Rca promoter fused to the coding region of the bacterial reporter gene beta-glucuronidase (GUS). Analysis of the Rca deletion mutants localized the region conferring normal expression downstream from - 294 relative to the Rca transcription start site. A second set of transgenic plants containing the cauliflower mosaic virus (CaMV) 35S enhancer fused to the 3' end of the Rca/GUS constructs demonstrated the presence of a light-responsive element between - 150 and - 78 active in leaves. Regions 10 bp long within the light-responsive region, which included putative G box and GT elements, were removed by recombinant polymerase chain reaction. Deletion of the G box element resulted in a loss of gene expression in the leaves of transgenic tobacco, while deletion of the GT motif caused a 10-100-fold increase in expression in roots. However, site-directed mutagenesis of the GT motif resulted in expression patterns identical to the normal promoter. These experiments demonstrated that light-inducible and tissue-specific expression of the Rca promoter involves multiple cis elements proximal to the transcription start site, and that interactions between these elements are essential for regulating expression.