beta-Phaseolin gene activation is a two-step process: PvALF-facilitated chromatin modification followed by abscisic acid-mediated gene activation

We have shown previously that a rotationally and translationally positioned nucleosome is responsible for the absence of transcriptional expression fr om the phaseolin (phas) gene promoter in leaf tissue and that the repressiv e chromatin structure is disrupted on transcriptional activation during emb ryogenesis. To investigate how the chromatin structure is modified, we ecto pically expressed PvALF, a putative seed-specific phas activator, in leaf t issue of a tobacco line transgenic for a chimeric phas/uidA construct. DNas e I footprinting in vivo revealed that the ectopic expression of PvALF resu lted in remodeling of the chromatin architecture over the TATA region of th e phas promoter but did not lead to transcriptional activation in the absen ce of abscisic acid (ABA), Treatment of the transgenic tobacco leaves with ABA in the absence of PvALF neither alleviated the repressive chromatin arc hitecture nor activated transcription. However, in the presence of PvALF, h igh levels of beta-glucuronidase expression were obtained on exposure of le aves to ABA, These results reveal that expression from the phas promoter in volves at least two discrete steps: chromatin potentiation by PvALF followe d by ABA-mediated transcriptional activation.