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

The stringency of spatial expression of phaseolin, the major storage p rotein of bean (Phaseolus vulgaris) seeds, has been rigorously evaluat ed using stable and transient transformation techniques. Transgenic to bacco plants known to be homozygous for the beta-glucuronidase (gus) r eporter sequence under the regulation of various lengths of the beta-p haseolin gene (phas) promoter were shown to express gus only in develo ping seed tissues. No expression was detected in calli initiated from stems, leaves and immature seeds, showing that expression was not leak y in undifferentiated tissues. Control plants and cultures containing gus fused to the CaMV 35S promoter actively expressed gus under identi cal conditions. It was not possible to induce expression in phas/gus c alli with ABA, GA or jasmonic acid. Treatment of the cultures with 5-a zacytidine did not result in expression, excluding methylation as the major factor regulating the phas promoter. However, strong gus express ion was detected in seed of plants regenerated from these callus cultu res, confirming that neither gene rearrangements nor deletion were res ponsible for the lack of activity seen in tissues other than the devel oping seed. In contrast to the above observations, strong transient ex pression of gus was detected in tobacco, bean and soybean leaves follo wing introduction of the phas/gus fusion constructs via biolistic appr oaches and in electroporated bean leaf and hypocotyl protoplasts. Thes e experiments show unequivocally that the phas promoter is under rigor ous 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.