THE PLANT CELL, Vol 4, Issue 4 473-483, Copyright © 1992 by American Society of Plant Biologists
Binding Sites for Two Novel Phosphoproteins, 3AF5 and 3AF3, Are Required for rbcS-3A Expression
L. P. Sarokin and N. H. Chua
Laboratory of Plant Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, New York 10021
Previous studies of boxes II (-151 to -138) and III (-125 to -114), binding
sites for the nuclear factor GT-1 within the -166 deleted promoter of the
ribulose-1,5-bisphosphate carboxylase-3A (rbcS[middot]3A) gene, suggested
that GT-1 might act in concert with an additional protein to confer
light-responsive rbcS[middot]3A expression. In this work, S1 analysis of
RNA isolated from transgenic tobacco plants carrying mutant rbcS[middot]3A
constructs led to the identification of two short sequences located at the
5[prime] and 3[prime] ends of box III that are required for expression.
These two sequences serve as binding sites for two novel proteins, 3AF5 and
3AF3. Gel shift studies using tetramerized binding sites for both 3AF5 and
3AF3 showed that complexes with faster mobilities were formed using nuclear
extracts prepared from dark-adapted plants compared with those from
light-grown tobacco plants. Phosphatase treatment of extracts from
light-grown plants resulted in the formation of complexes with faster
mobility. Although the binding of 3AF3 to its target site is dependent upon
phosphorylation, the binding of 3AF5 does not appear to be affected by its
phosphorylation state. These results suggest that the phosphorylated forms
of both 3AF5 and 3AF3 are required for -166 rbcS[middot]3A expression but
that the mechanisms differ by which phosphorylation regulates the
activities of 3AF5 and 3AF3.
