Plant Molecular Biology 49: 601–610, 2002.
© 2002 Kluwer Academic Publishers. Printed in the Netherlands. 601
Characterization of five novel dehydration-responsive homeodomain
leucine zipper genes from the resurrection plant Craterostigma
plantagineum
Xin Deng1, Jonathan Phillips1,3, Annemarie H. Meijer2, Francesco Salamini1 and Dorothea
Bartels4
1Max-Planck-Institut für Züchtungsforschung, Carl-von-Linne´-Weg 10, 50829 Köln, Germany; 2Institute of Mole-
cular Plant Sciences, Leiden University, Clusius Laboratory, P.O.Box 9505, 2300 RA Leiden, Netherlands;
present addresses: 3Genesis Research and Development Corporation Ltd., 1 Fox Street, Auckland, New Zealand;
4Institute of Botany, University of Bonn, Kirschallee 1, 53115 Bonn, Germany (*author for correspondence; e-mail
dbartels@uni-bonn.de or dbartels@mpiz.koeln.mpg.de)
Received 3 July 2001; accepted in revised form 7 December 2001
Key words: Craterostigma plantagineum, homeodomain leucine zipper (HDZip), resurrection plant, transcription
factor, yeast one-hybrid system, yeast two-hybrid interaction
Abstract
Homeodomain leucine zipper (HDZip) genes encode putative transcription factors that are unique to plants. A
function in regulating processes that are specific for plants is postulated, such as responses to environmental cues
and developmental signals. This is supported by a growing body of evidence resulting from studies of HDZip
genes in a variety of species. In addition to the previously isolated CPHB-1 and -2 genes, this paper reports the
isolation of members of five families of Craterostigma plantagineum homeobox leucine zipper genes (CPHB) via
a yeast one-hybrid screening approach. Based on the sequence homology and protein interactions the encoded
proteins (CPHB-3/4/5/6/7) were classified into HDZip class II and I genes. Homo- and heterodimerization of
CPHB proteins within the same structurally related class has been demonstrated and the DNA-binding activity of
CPHB proteins to two homeodomain recognition elements (HDE1 and HDE2) has been compared in yeast. All
families of CPHB genes were modulated in their expression in response to dehydration in leaves and roots. CPHB-
6 and CPHB-7 transcripts accumulated in leaves during early stages of dehydration and decreased after prolonged
dehydration. Both transcripts were also induced in ABA-treated callus. CPHB-3/4/5 were down-regulated by
dehydration in both leaves and roots. The results support the role of HDZips in regulating programs of gene
expression in C. plantagineum that lead to desiccation tolerance.
Abbreviations: ABA, abscisic acid; AD, activation domain; BD, binding domain; HDZip, homeodomain leucine
zipper; HDE, homeodomain recognition element
Introduction
In plants dehydration is a widespread environmental
condition that damages cellular structures and inhibits
normal physiological activities, consequently reduc-
The nucleotide sequence data will appear in the EMBL, GenBank
and DDBJ Nucleotide Sequence Databases under the accession
numbers AF443619 (CPHB-3), AF443620 (CPHB-4), AF443621
(CPHB-5), AF443622 (CPHB-6) and AF44223623 (CPHB-7).
ing crop yield and restricting the geographical distrib-
ution of plants. Plant cells undergo protoplasmic dehy-
dration not only under dehydration stress, but also as a
secondary effect under cold and salt stress (Ingram and
Bartels, 1996; Shinozaki and Yamaguchi-Shinozaki,
1997; Jaglo-Ottosen et al., 1998). Significant efforts
have been made to understand the mechanisms that
plants have evolved to acquire tolerance. One of the

602
strategies to investigate dehydration responses and
protective mechanisms is the study of resurrection
plants because such plants possess extreme dehydra-
tion tolerance (Ingram and Bartels, 1996). Studies
of the resurrection plant Craterostigma plantagineum
and of other plant systems have revealed many de-
hydration up-regulated genes involved in a variety of
pathways such as osmolyte biosynthesis, regulation of
water movement through membranes (iron and wa-
ter channel proteins), macromolecules and membrane
protection and detoxification (reviewed in Ingram and
Bartels, 1996; Bray, 1997; Shinozaki and Yamaguchi-
Shinozaki, 1997).
Studies have also resulted in the identification
of potential regulatory genes encoding protein fac-
tors involved in signal transduction and dehydration-
responsive gene expression. These factors are likely
to control multiple end-product genes that contribute
to the stress response. Homeodomain leucine zip-
per (HDZip) proteins have been found exclusively
in plants, which suggests a function in events that
are specific to plants (Ruberti et al., 1991; Lee and
Chun, 1998; Söderman et al., 1999). HDZip proteins
are characterized by the presence of a DNA-binding
homeodomain with a closely linked leucine zipper
motif functioning in dimer formation. HDZips have
been described for several plants such as Arabidopsis,
carrot, tomato, rice and C. plantagineum. They are
grouped into four classes (I–IV) based on sequence
homology (Sessa et al., 1993). Some members of
HDZip proteins have been proposed to control de-
velopment of specific plant structures, such as the
vascular system (ATHB8, class III, Baima et al., 1995;
Oshox1, class II, Scarpella et al., 2000; Vahox1, class
I, Tornero et al., 1996), and root hairs and trichomes
(ATHB10, class IV, Di Cristina et al., 1996). Other
HDZip proteins are more directly responsive to en-
vironmental cues: the Arabidopsis genes Athb2 and
Athb4 (both class II) are highly induced by far-red
light, indicating a role in the shade avoidance response
(Carabelli et al., 1993); Athb6, Athb7 and Athb12
are inducible by drought as well as ABA, implying
their putative function in dehydration responses (Sö-
derman et al., 1996, 1999; Lee and Chun, 1998).
From C. plantagineum, two HDZip genes (CPHB-1
and CPHB-2, class II) are dehydration-inducible, and
one of them is ABA-inducible (CPHB-2) (Frank et al.,
1998). Therefore, they are thought to be involved
in regulation of dehydration responses through dif-
ferent branches of the dehydration-induced signalling
network, ABA-independent or ABA-dependent. In
vitro binding studies showed that CPHB-1 prefer-
entially bound to the pseudopalindromic sequence
CAAT(C/G)ATTG (namely, HDE2), and yeast two-
hybrid assays revealed that CPHB-1 was capable
of homodimerization and heterodimerization with
CPHB-2.
In this paper, we report the identification and
characterization of five novel families of dehydration-
responsive HDZip genes belonging to classes II and
I from C. plantagineum. A yeast one-hybrid screen-
ing approach was employed to isolate HDZip pro-
teins from a cDNA library prepared from dehydrated
leaves based on the conservative DNA-binding char-
acteristics of HDZip I and II proteins. DNA-binding
specificity and dimerization abilities of the HDZip
proteins were characterized in yeast. Their responses
to dehydration and ABA were studied at the mRNA
level.
Materials and methods
Plant material
C. plantagineum plants were grown in controlled-
environment conditions, and stress and ABA treat-
ments were performed as described by Bartels et al.
(1990). Plants were treated with 10 µM ABA
(cis/trans, Sigma).
cDNA library construction and screening
A pAD-GAL4 plasmid library prepared from poly(A)+
RNA from C. plantagineum leaves dried for 2 h as
described by Frank et al. (1998) was used for yeast
one-hybrid screenings. Yeast reporter strains were
constructed as described by Meijer et al. (1998). Yeast
cells were transformed according to a lithium acetate
method (Gietz and Schiestl, 1995), and screened on
selective medium lacking histidine and leucine. Plas-
mid DNAs were isolated from positive yeast colonies,
and re-transformed into the Escherichia coli XL-1
blue MRF′ strain. Plasmid DNAs were extracted from
E. coli and the DNA sequences of the inserts were
determined.
To isolate full-length CPHB-5/6/7 cDNA clones, a
cDNA library constructed in pBluescript II SK+ was
prepared from poly(A)+ RNA isolated from leaves
dried for 2 h and screened with gene-specific 32P-
labelled cDNA probes.