CHROMOSOMAL INTEGRATION IS REQUIRED FOR SPATIAL REGULATION OF EXPRESSION FROM THE BETA-PHASEOLIN PROMOTER

The stringency of spatial expression of phaseolin, the major storage protein of bean (Phaseolus vulgaris) seeds, has been rigorously evaluated using stable and transient transformation techniques. Transgenic tobacco plants known to be homozygous for the beta-glucuronidase (gus) reporter sequence under the regulation of various lengths of the beta-phaseolin gene (phas) promoter were shown to express gus only in developing seed tissues. No expression was detected in calli initiated from stems, leaves and immature seeds, showing that expression was not leaky in undifferentiated tissues. Control plants and cultures containing gus fused to the CaMV 35S promoter actively expressed gus under identical conditions. It was not possible to induce expression in phas/gus calli with ABA, GA or jasmonic acid. Treatment of the cultures with 5-azacytidine did not result in expression, excluding methylation as the major factor regulating the phas promoter. However, strong gus expression was detected in seed of plants regenerated from these callus cultures, confirming that neither gene rearrangements nor deletion were responsible for the lack of activity seen in tissues other than the developing seed. In contrast to the above observations, strong transient expression of gus was detected in tobacco, bean and soybean leaves following introduction of the phas/gus fusion constructs via biolistic approaches and in electroporated bean leaf and hypocotyl protoplasts. These experiments show unequivocally that the phas promoter is under rigorous spatial control when integrated into the genome, but lacks spatial control when present as extrachromosomal naked DNA. A putative model explaining these differences is presented.