Plant Molecular Biology 45: 63–73, 2001.
© 2001 Kluwer Academic Publishers. Printed in the Netherlands.
DNA-binding and dimerization preferences of Arabidopsis
homeodomain-leucine zipper transcription factors in vitro
Henrik Johannesson, Yan Wang and Peter Engström
Department of Evolutionary Biology, Physiological Botany, Villavägen 6, 752 36 Uppsala, Sweden (
correspondence; e-mail: firstname.lastname@example.org)
Received 8 March 2000; accepted in revised form 4 August 2000
Key words: Arabidopsis thaliana, homeobox, homeodomain,leucine zipper, plant
Homeodomain-leucinezipper (HDZip) proteins constitute a large family of transcription factors apparentlyunique
to plants. In this report we characterize the DNA-binding and dimerization preferences in vitro of class I HDZip
proteins. Using gel-exclusion chromatography and in vitro protein binding assays we demonstrate that the HDZip
class I protein ATHB5 forms a homodimeric complex in solution. Consistent with this ﬁnding we have demon-
strated the sequence-speciﬁc interaction of ATHB5 with a 9 bp pseudopalindromicDNA sequence, CAATNATTG,
composed of two half-sites overlapping at a central position, by use of a PCR-assisted binding-site selection assay
and competitive EMSA experiments. A majority of other known members of HDZip class I interacted with similar
DNA sequences, but differed in their preference for A/T versus G/C in the central position of the binding site.
Selective heterodimerization in vitro was demonstrated between ATHB5 and different class I HDZip proteins.
Heterodimer formation between class I HDZip proteins is of potential functional signiﬁcance for the integration of
information from different signalling pathways in the control of plant development.
Homeodomain-leucine zipper (HDZip) proteins are
encoded by a large gene family apparently speciﬁc to
plants. In Arabidopsis, more than 25 HDZip genes
have been identiﬁed (Ruberti et al., 1991; Mattson
et al., 1992; Schena and Davis, 1992; Carabelli et al.,
1993; Södermanet al., 1994; Schena and Davis, 1994;
Baima et al., 1995; Di Christina et al., 1996; Sessa
et al., 1998; Lee et al., 1998) and are designated
on sequence criteria, the HDZip proteins have been
grouped into four classes, HDZip I–IV (Sessa et al.,
Functional data on HDZip class II, III and IV
genes indicate an involvement of these genes in
the control of processes that are speciﬁc to plants.
ATHB10/GLABRA2 (class IV) has been shown by
mutational analysis to control the differentiation of
speciﬁc cell types: trichomes on leaves and stems, and
root hair cells (Rerie et al., 1994; Di Christina et al.,
1996). Expression data suggest that a class III HDZip
protein, ATHB8, may have a similar developmental
control function in procambial cell differentiation in
the vascular tissue of the embryo and developing or-
gans (Baima et al., 1995). The class II HDZip protein
ATHB2 is a regulator of cell expansion, as part of the
mechanism by which the plant adapts its form in rela-
tion to light quality (Carabelli et al., 1996; Steindler
et al., 1999).
Functional information on the class I HDZip pro-
teins is limited. Results from reverse genetics experi-
ments suggest that several of these proteins, similarly
to ATHB2, affect cell elongationas componentsof dif-
ferent signal transduction pathways (Söderman, 1996;
Hanson et al., manuscript in preparation;H. Johannes-
son, unpublished results). ATHB7 (Söderman et al.,
1996) and ATHB12 (Lee et al., 1998) are transcrip-
tionally induced to high levels by the plant hormone
abscisic acid, indicating that these genes act in sig-
nal transduction pathways that mediate the growth