THE PLANT CELL, Vol 5, Issue 4 485-496, Copyright © 1993 by American Society of Plant Biologists
Dissection of the Functional Architecture of a Plant Defense Gene Promoter Using a Homologous in Vitro Transcription Initiation System
J. A. Arias, R. A. Dixon and C. J. Lamb
Plant Biology Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037
CHS15 is one of a family of bean genes encoding chalcone synthase, which
catalyzes the first reaction in a branch pathway of phenylpropanoid
biosynthesis for the production of flavonoid pigments and UV protectants
and isoflavonoid-derived phytoalexins. The functional architecture of the
CHS15 promoter was dissected by a novel homologous plant in vitro
transcription initiation system in which whole-cell and nuclear extracts
from suspension-cultured soybean cells direct accurate and efficient RNA
polymerase II-mediated transcription from an immobilized promoter template.
Authentic transcription from the CHS15 promoter template was also observed
with whole-cell extracts from suspension-cultured cells of bean, tobacco,
and the monocot rice, and the soybean whole-cell extract transcribed
several other immobilized promoter templates. Hence, this procedure may be
of general use in the study of plant gene regulation mechanisms in vitro.
Assay of the effects of depletion of the soybean whole-cell extract by
preincubation with small regions of the CHS15 promoter or defined cis
elements showed that trans factors that bind to G-box (CACGTG, -74 to -69)
and H-box (CCTACC, -61 to -56 and -121 to -126) cis elements, respectively,
make major contributions to the transcription of the CHS15 promoter in
vitro. Both cis element/trans factor interactions in combination are
required for maximal activity. Delineation of these functional cis
element/trans factor interactions in vitro provides the basis for study of
the mechanisms underlying developmental expression of CHS15 in pigmented
petal cells established by G-box and H-box combinatorial interactions, and
for characterization of the terminal steps of the signal pathway for stress
induction of the phytoalexin defense response.
