THE PLANT CELL, Vol 1, Issue 6 609-621, Copyright © 1989 by American Society of Plant Biologists
Cotyledon Nuclear Proteins Bind to DNA Fragments Harboring Regulatory Elements of Phytohemagglutinin Genes
C. D. Riggs, T. A. Voelker and M. J. Chrispeels
Department of Biology and Center for Molecular Genetics, University of California, San Diego, La Jolla, California 92093-0116
The effects of deleting DNA sequences upstream from the
phytohemagglutinin-L gene of Phaseolus vulgaris have been examined with
respect to the level of gene product produced in the seeds of transgenic
tobacco. Our studies indicate that several upstream regions quantitatively
modulate expression. Between -1000 and -675, a negative regulatory element
reduces expression approximately threefold relative to shorter deletion
mutants that do not contain this region. Positive regulatory elements lie
between -550 and -125 and, compared with constructs containing only 125
base pairs of upstream sequences (-125), the presence of these two regions
can be correlated with a 25-fold and a 200-fold enhancement of
phytohemagglutinin-L levels. These experiments were complemented by gel
retardation assays, which demonstrated that two of the three regions bind
cotyledon nuclear proteins from mid-mature seeds. One of the binding sites
maps near a DNA sequence that is highly homologous to protein binding
domains located upstream from the soybean seed lectin and Kunitz trypsin
inhibitor genes. Competition experiments demonstrated that the upstream
regions of a bean [beta]-phaseolin gene, the soybean seed lectin gene, and
an oligonucleotide from the upstream region of the trypsin inhibitor gene
can compete differentially for factor binding. We suggest that these legume
genes may be regulated in part by evolutionarily conserved protein/DNA
interactions.
