A 68 bp element of the beta-phaseolin promoter functions as a seed-specific enhancer

Abstract

In beans, expression of the beta-phaseolin gene (phas), encoding the major seed storage protein of bean (Phaseolus vulgaris) is confined to the cotyledons of developing embryos. Phaseolin has not been detected in the endosperm, which remains liquid and is lost early in development. However, fusion constructs between the phas promoter and the gus-coding region yield expression in both embryo and endosperm of developing seeds from transgenic tobacco (Nicotiana tabacum) plants. Although elements extending 1470 bp upstream of the transcription start site are known to modulate phas expression, the proximal 295 bp (p295) are sufficient to drive high levels of seed-specific GUS activity. This region was dissected into three elements: a 68 bp element (seed specific enhancer, SSE: -295 to -227), a middle region (-227 to -109) and a basal phas promoter (-109 to +20: p109). Different promoter constructs containing the SSE or middle region upstream of p109 or a CaMV 35S basal promoter (-64 to +6) were fused to gus. Each construct was expressed in seed, but not in vegetative tissues. Use of the various phas promoter regions yielded notable differences in relative GUS activity in embryo or endosperm. Addition of both the SSE and middle region resulted in higher activity than the sum of adding either element alone to p109, indicating synergistic interaction between these elements. Seeds from plants transformed with the proximal 227 bp of promoter (p227) showed embryo-specific GUS activity. In contrast, constructs containing two copies of the SSE element were preferentially expressed in the endosperm. These results illustrate the modular nature of the proximal phas promoter, where distinct elements contribute to high levels of expression in different parts of the seed.