Plant Molecular Biology 38: 609–622, 1998.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.
The Arabidopsis Athb-8, -9 and -14 genes are members of a small gene
family coding for highly related HD-ZIP proteins
, Corinna Steindler
, Giorgio Morelli
and Ida Ruberti
Centro di studio per gli Acidi Nucleici, c/o Dipartimento di Genetica e Biologia Molecolare, Universit`adi
Roma La Sapienza, P.le Aldo Moro 5, 00185 Rome, Italy (
author for correspondence).
Unit`a di Nutrizione
Sperimentale, Istituto Nazionale della Nutrizione, Via Ardeatina 546, 00178 Rome, Italy; †The ﬁrst two authors
contributed equally to this work
Received 4 February 1998; accepted in revised form 23 April 1998
Key words: binding site selection, gene expression, homeobox-leucine zipper genes, protein-DNA interaction
We report the isolation and characterization of two Arabidopsis homeobox genes highly related to the Athb-8 gene.
The full-length cDNAs encode proteins of 841 and 852 amino acids which we have designated Athb-9 and -14,
respectively. Athb-8, −9 and -14 are members of a small family of HD-Zip proteins (HD-ZIP III) characterized by
a HD-Zip motif conﬁned to the N-terminus of the polypeptide. The spatial organization of the HD-Zip domain of
Athb-8, -9 and -14 is different from that of the Athb-1 (a member of the HD-ZIP I family) and Athb-2 (a member of
the HD-ZIP II family) HD-Zip domains. DNA binding analysis performed with random-sequence DNA templates
showed that the Athb-9 HD-Zip (HD-Zip-9) domain, but not the Athb-9 HD alone, binds to DNA. The HD-Zip-
9 domain recognizes a 11 bp pseudopalindromic sequence (GTAAT(G/C)ATTAC), as determined by selecting
high-afﬁnity binding sites from random-sequence DNA. Moreover, gel retardation assays demonstrated that the
HD-Zip-9 domain binds to DNA as a dimer. These data support the notion that the HD-ZIP III domain interacts
with DNA recognition elements in a fashion similar to the HD-ZIP I and II domains.
Homeodomain-leucine zipper (HD-Zip) proteins are
putative transcription factors identiﬁed only in plants.
A number of HD-Zip proteins identiﬁed in Arabidop-
sis are thought to constitute a large class of proteins
[2, 4, 12, 14, 17-19, 21, 23, 27]. We have tentatively
grouped these HD-Zip proteins into four differentfam-
ilies, named HD-ZIP I, II, III and IV . Expression
analysis in Arabidopsis and in transgenic plants of
Athb-1 and -7 (HD-ZIP I) [1, 28], Athb-2 (HD-ZIP
II) [4, 5, 20, 29], Athb-8 (HD-ZIP III) , Athb-
10/GLABRA2 (Athb-10/GL2)andATML1 (HD-ZIP
IV) [6, 12, 13, 17] suggests that the HD-Zip proteins
might control important aspects of plant development.
The nucleotide sequence data reported will appear in the
EMBL, GenBank and DDBJ Nucleotide Sequence Databases under
the accession numbers Y10922 (Athb-9 gene) and Y11122 (Athb-14
The presence of a putative leucine zipper closely
linked to a homeodomain suggested that the HD-Zip
proteins could, via dimerization of the leucine zip-
pers, recognize dyad-symmetric DNA sequences .
DNA binding studies on several HD-Zip proteins sup-
port this model. We have proved that the HD-Zip
domain of Athb-1, a member of the HD-ZIP I fam-
ily, and that of Athb-2, a member of the HD-ZIP II
family, bind DNA as dimers [22, 24]. We found that
the HD-Zip-1 and -2 binding sites are pseudopalin-
dromes of two 5 bp half-sites that overlap at a central
position, CAAT(A/T)ATTG and CAAT(G/C)ATTG,
respectively [22, 23]. Moreover, we showed that the
spacing between the zipper and the helix-3 is critical
for function; in fact, insertion of two amino acids at
the beginning of the Athb-1 leucine zipper resulted in
a HD-Zip complex incapable of DNA binding .
Recently, we have also demonstrated that the leucine
zipper of Athb-10/GL2, a member of the HD-ZIP IV