Plant Molecular Biology 38: 633–646, 1998.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.
Identiﬁcation of preferred binding sites of a light-inducible DNA-binding
factor (MNF1) within 5
-upstream sequence of C4-type
phosphoenolpyruvate carboxylase gene in maize
Laboratory of Plant Physiology, Graduate School of Agriculture, Kyoto University, Kyoto 606–8502, Japan
Received 19 December 1995; accepted in revised form 28 April 1998
Key words: C4 photosynthesis, DNA-binding protein, gel shift, phosphoenolpyruvate carboxylase, recognition
MNF1 is a factor which speciﬁcally binds to a 318 bp fragment (−1012to −695) in the 5
-ﬂanking regionof the C4-
type phosphoenolpyruvatecarboxylase gene in Zea mays (Yanagisawa et al., Mol Gen Genet 224 (1990) 325–332).
The most preferred binding site of MNF1 determined by a 2 bp mutation-scanning assay was an octamer sequence,
GTGCCCTT, which is located within the repeated sequences (RS1; −886 to −849, −846 to −807). Furthermore,
a PCR-mediated selection-ampliﬁcation assay identiﬁed both the octamer sequence, GTGCCC(A/T)(A/T), and
an additional sequence, CC(G/A)CCC, the latter of which was similar to the Sp1 sites in vertebrates. Speciﬁc
binding of MNF1 to each of the supposed binding sites was conﬁrmed with double-stranded monomers as probes.
Considering native molecular mass of MNF1 (ca. 500 kDa), a protein complex is expected. In addition, MNF1 is
anticipated to have two distinct DNA-binding proteins since the MNF1 binding to CCGCCC element was 1,10-
phenanthroline-dependent whereas the MNF1 binding to the octamer was independent. Wide distribution of the
MNF1 binding sequences within the 1 kb promoter region accounts for broad interactions of MNF1. Moreover,
speciﬁc DNA binding due to MNF1, which was not observed in the nuclear extract derived from germinated and
cultivated plants in darkness, appeared after a white-light pulse. This ﬁnding suggests the involvement of the
protein complex in the light-dependent transcriptional control in the gene expression.
Gene transcription is a coordinated event in which
numerous protein factors participate and enhancer-
binding proteins modulate the basal transcription con-
ferred by RNA polymerase II . Protein complex
forming tendencies among these trans-acting factors
have been reported in vertebrates in detail [9, 22];
however, very few have been investigated in plants .
C4-type phosphoenolpyruvatecarboxylase (PEPC,
EC 220.127.116.11) catalyzes initial CO
ﬁxation during C4
photosynthesis and is one of the most abundant solu-
ble proteins in maize leaves . Since a single copy
of C4-type PEPC gene (Ppc1) is present per maize
haploid genome , this high protein level must be
due to the strong promoter system present in maize for
the Ppc1 gene. Therefore, analysis of the regulatory
mechanism of the Ppc1 promoter has a potential for
shedding light on several unanswered questions, i.e.
high-level expression, light inducibility, possibly via
the photoreceptor phytochrome [10, 21, 28, 31], and
mesophyll cell type-speciﬁc expression [15, 24, 27].
Gel retardation studies have suggested the exis-
tence of several nuclear factors which speciﬁcally
interact with some of the DNA probes derived from
the 1 kb upstream sequence of the Ppc1 gene .
Kano-Murakami et al. reported another DNA-binding
protein (PEP-1) interacting with this region which
for its binding . The relative im-
portance of the sequence, −148 to −73 from the
transcription start site , was shown for light induc-
tion of the Ppc1 expression, in which a transient assay