THE PLANT CELL, Vol 5, Issue 11 1681-1692, Copyright © 1993 by American Society of Plant Biologists
Gibberellin Treatment Stimulates Nuclear Factor Binding to the Gibberellin Response Complex in a Barley [alpha]-Amylase Promoter
T. D. Sutliff, M. B. Lanahan and THD. Ho
Department of Biology, Washington University, St. Louis, Missouri 63130
The promoters of a majority of cereal [alpha]-amylase genes contain three
highly conserved sequences (gibberellin response element, box I, and
pyrimidine box). Recent studies have demonstrated the functional importance
of four regions that either coincide with or are immediately proximal to
these three conserved elements as well as an upstream Opaque-2 binding
sequence. In this study, we describe the characterization of nuclear
protein factors from barley aleurone layers whose binding activity toward
gibberellin response complex sequences from the barley low-pl
[alpha]-amylase gene (Amy32b) promoter is stimulated by gibberellin A3
(GA3) treatment. Barley proteins isolated from crude nuclear extracts
prepared from aleurone layers incubated with or without GA3 were
fractionated by anion exchange fast protein liquid chromatography and
studied using band shift assays, sequence-specific competitions, and DNase
I footprinting. A GA3-dependent binding activity eluting at 210 mM KCl was
shown to bind specifically to the gibberellin response element and the
closely associated box I. DNase I footprinting with the proteins in this
fraction indicated interactions with sequences in the gibberellin response
element and box I. A second DNA binding activity eluting at 310 mM KCl was
present constitutively in extracts prepared from tissues incubated both in
the absence and in the presence of hormone. Proteins in this fraction were
able to bind to many DNA sequences and, in general, were largely
nonspecific. DNase I footprinting with the proteins in this fraction
indicated a large area of protection with a single unoccupied region
located at the 3[prime] end of box I. The possible function of such an
activity in hormone regulation of the [alpha]-amylase genes is discussed.
