THE PLANT CELL, Vol 6, Issue 3 317-332, Copyright © 1994 by American Society of Plant Biologists
Site-Specific Mutagenesis of the Nodule-Infected Cell Expression (NICE) Element and the AT-Rich Element ATRE-BS2* of the Sesbania rostrata Leghemoglobin glb3 Promoter
K. Szczyglowski, L. Szabados, S. Y. Fujimoto, D. Silver and F. J. de Bruijn
Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824-1312
Sesbania rostrata leghemoglobin glb3 (Srglb3) promoter sequences
responsible for expression in infected cells of transgenic Lotus
corniculatus nodules were delimited to a 78-bp Dral-Hinfl fragment. This
region, which is located between coordinates -194 to -116 relative to the
start codon of the Srglb3 gene, was named the nodule-infected cell
expression (NICE) element. Insertion of the NICE element into the truncated
nopaline synthase promoter was found to confer a nodule-specific expression
pattern on this normally root-enhanced promoter. Within the NICE element,
three distinct motifs ([A]AAAGAT, TTGTCTCTT, and CACCC[T]) were identified;
they are highly conserved in the promoter regions of a variety of plant
(leg)hemoglobin genes. The NICE element and the adjacent AT-rich element
(ATRE-BS2*)were subjected to site-directed mutagenesis. The expression
patterns of nine selected Srglb3 promoter fragments carrying mutations in
ATRE-BS2* and 19 with mutations in the NICE element were examined.
Mutations in ATRE-BS2* had varying effects on Srglb3 promoter activity,
ranging from a two- to threefold reduction to a slight stimulation of
activity. Mutations in the highly conserved (A)AAAGAT motif of the NICE
element reduced Srglb3 promoter activity two- to fourfold, whereas
mutations in the TCTT portion of the TTGTCTCTT motif virtually abolished
promoter activity, demonstrating the essential nature of these motifs for
Srglb3 gene expression. An A-to-T substitution in the CACCC(T) motif of the
NICE element also abolished Srglb3 promoter activity, while a C-to-T
mutation at position 4 resulted in a threefold reduction of promoter
strength. The latter phenotypes resemble the effect of similar mutations in
the conserved CACCC motif located in the promoter region of mammalian
[beta]-globin genes. The possible analogies between these two systems will
be discussed.
