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Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 569
_____________________________ _____________________________
Functional Analysis ofHomeodomain-Leucine Zipper
Transcription Factors inArabidopsis thaliana
BY
HENRIK JOHANNESSON
ACTA UNIVERSITATIS UPSALIENSISUPPSALA 2000
Dissertation for the Degree of Doctor of Philosophy in Physiological Botany presentedat Uppsala University in 2000
Abstract
Johannesson, H., 2000. Functional Analysis of Homeodomain-Leucine ZipperTranscription Factors in Arabidopsis thaliana. Acta Universitatis Upsaliensis.Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science andTechnology 569. 44 pp. Uppsala. ISBN 91-554-4816-X
Homeodomain-leucine zipper (HDZip) proteins constitute a large family oftranscription factors apparently unique to plants. To elucidate the function of thesefactors, the biochemical properties in vitro as well as the effects on transgenic plantswhen expressed at high levels were studied. The conclusion is that HDZip proteins arevery similar with respect to DNA-binding specificity in vitro but appear to be active indifferent aspects of plant development. Thus, functional specificity of HDZip proteinsis most likely determined by other aspects of proteins function, e.g. their capacity tointeract with other proteins.
High-level expression of the HDZip gene ATHB5 in transgenic plants results inhypersensitivity to the inhibitory effect of the plant hormone abscisic acid (ABA) onseed germination and seedling root growth. Furthermore, the expression of ATHB5 ingerminating seedlings is downregulated in the Arabidopsis ABA response mutants abi3and abi5. Together, these data suggest that ATHB5 acts as a regulator of seedgermination and postgerminative growth downstream of ABI3 and ABI5 in an ABAresponse-signaling pathway.
Enhanced levels of the HDZip gene ATHB13 in transgenic Arabidopsis confer asugar-dependent reduction of cotyledon width. In addition, a subset of known sugar-dependent genes was hyperinduced by sucrose in Arabidopsis seedlings overexpressingATHB13. These data suggest that ATHB13 affects both cotyledon morphology and generegulation as a component of a sucrose-signaling pathway.
Loss-of-function mutations in ATHB5 and ATHB13 did not result in any discernablemutant phenotypes, suggesting that these genes are only required under specificphysiological conditions or that they act in a redundant fashion in the plant.
Henrik Johannesson, Department of Physiological Botany, Evolutionary Biology Centre,Uppsala University, Villavägen 6, SE-752 36 Uppsala, Sweden
© Henrik Johannesson 2000
ISSN 1104-232XISBN 91-554-4816-X
Printed in Sweden by University Printers, Uppsala, 2000
This thesis is based on the following papers, which will be referred to in the text by
their Roman numerals:
I. Johannesson, H., Wang, Y. and Engström, P. 2000. DNA-binding and
dimerisation preferences of Arabidopsis homeodomain-leucine zipper
transcription factors in vitro. Plant Mol. Biol., in press.
II. Hanson, J., Johannesson, H. and Engström, P. 2000. Sugar-dependent alterations
in cotyledon and leaf development in transgenic plants expressing the HDZip
gene ATHB13. Plant Mol. Biol., in press.
III. Johannesson, H., Wang, Y. and Engström, P. 2000. High-level expression of the
homeodomain-leucine zipper gene ATHB5 in transgenic Arabidopsis results in
hypersensitivity to abscisic acid. In manuscript.
IV. Johannesson, H. Hanson, J., Söderman, E., Wang, Y. and Engström, P. 2000.
HDZip proteins in Arabidopsis thaliana: a case of functional conservation and
redundancy within a family of transcription factors. In manuscript.
Reprints of papers I and II were made with permissions from Kluwer Academic
Publishers.
CONTENTS
Page
ABBREVIATIONS 6
INTRODUCTION
Arabidopsis thaliana: the model plant 7
Transcriptional regulation 10
Homeodomain proteins 11
Homeodomain proteins in plants: KNOTTED-1 proteins 12
PHD finger proteins 12
HDZip proteins 12
DNA-binding properties of HDZip proteins 14
Dimerization properties of HDZip proteins 16
Function of HDZip proteins 17
RESULTS AND DISCUSSION
Identification of new HDZip genes 19
Identification of HDZip I target sequences in vitro 23
ATHB7 and 12 have alternative binding-site specificities 24
Heterodimer formation between HDZip I proteins 26
Expression analysis of ATHB5 27
A reverse genetics approach to understand the function of HDZip proteins 28
Overexpression of ATHB5 in transgenic Arabidopsis 30
High-level expression of ATHB13 in transgenic Arabidopsis 31
Interpretation of phenotypes resulting from constitutive expression 32
Isolation of T-DNA insertion lines in HDZip I genes 33
SUMMARY 35
ACKNOWLEDGEMENTS 36
REFERENCES 37
ABBREVIATIONS
ABA abscisic acid
abi ABA insensitive mutant
ACC 1-aminocyclopropane-1-carboxylic acid
ATHB Arabidopsis thaliana homeobox
BAC bacterial artificial chromosome
bZip basic leucine zipper
cDNA complementary DNA
HD homeodomain
HDZip homeodomain leucine zipper
HTH helix-turn-helix
NMR nuclear magnetic resonance spectroscopy
SAM shoot apical meristem
T-DNA transferred DNA
7
INTRODUCTION
Arabidopsis thaliana: the model plant
Arabidopsis thaliana, thale cress, belongs to the crucifer family (Brassicaceae). This
plant has a broad natural distribution throughout Europe, Asia and North America. In
the wild, Arabidopsis thaliana grows as a winter annual, that is, the seed germinates in
the autumn, pass the winter as a rosette and then flower early during the spring when
temperature and light conditions are favourable.
Historically, agriculturally important species such as maize, tomato, potato or
barley were the plants of choice for experimental studies. As the efforts of the research
community was spread out on several plant species, each considered as the ideal model
organism by the different researchers, the progress in the understanding of fundamental
plant processes was rather slow (Meinke et al., 1998).
Already in 1943, Friedrich Laibach, Professor at the University of Frankfurt am
Main, published an article describing the advantages of Arabidopsis thaliana as a
research tool. In his article, a number of favourable features of Arabidopsis were pointed
out, and since then, only a few additional advantages are acknowledged. The features of
this plant that make it suitable for experimental studies include several traits. One is the
short generation time of Arabidopsis; when grown under optimal conditions in the green
house it has a generation time of about six weeks. Another advantage is the small size of
the plant, which make it possible to grow a large number of plants in limited growth
facilities. The diameter of the rosette ranges from 2 to 10 cm in diameter depending on
growth conditions, the final height of the inflorescence stem reaches 20-40 and the
flowers are about 2 mm long. Furthermore, Arabidopsis flowers self-pollinate as the
buds open and each plant can produce hundreds of siliques, containing more than 5000
seeds. It is easy to perform genetic crosses of Arabidopsis and recover a fully fertile
progeny, and both induced and spontaneous mutations can be isolated with ease.
Although all these advantages of using Arabidopsis for experimental studies were
pointed out already in 1943, it would last another forty years before it really became
favoured as a model organism (Redei, 1992; Meinke et al., 1998).
8
Figure 1. Arabidopsis thaliana, thale cress, approximately four weeks old and
grown under long day conditions. (Photograph by Eva Söderman).
9
In 1983, Maarten Koorneef presented a detailed genetic map of Arabidopsis.
Furthermore, it was demonstrated that the genome of Arabidopsis is one of the smallest
genomes known among angiosperm species (Meyerowitz and Pruitt, 1985); a
characteristic that facilitates the isolation of genes through screening of genomic libraries,
which can be a time-consuming task when working with large genomes. Another
groundbreaking discovery was that it is possible to transform Arabidopsis with
Agrobacterium, making it possible to generate stable transgenic plants with ease. The
methods for Agrobacterium-mediated transformation of Arabidopsis have developed
from tissue culture techniques (Lloyd et al., 1986), through seed transformation
(Feldmann and Marks, 1987), to the whole-plant transformation methods (Bechtold et
al., 1993). With the latter method, it is possible to generate large number of
transformants with a reasonable effort.
Over the past 20 years, several thousand mutants of Arabidopsis, defective in
almost every aspect of plant growth and development, have been identified. The
development of efficient transformation methods for Arabidopsis has facilitated the
establishment of large mutant collections of T-DNA insertion lines, carrying randomly
inserted T-DNAs. Seed stocks and DNA-pools from these collections of lines are
available from public stock centers. The use of these lines for mutant screenings is
particularly advantageous as the genes that are disrupted in these lines are tagged by the
inserted T-DNA, which facilitate the isolation of the mutated gene.
Research using Arabidopsis as a model plant has provided insight into almost
every aspect of plant biology. For example, the understanding of phytohormone action
has improved drastically through genetic and molecular analysis of Arabidopsis. The
classical approach to understand the function of plant hormones has largely been based
on application of the hormone and recording the observed effect. However, this
approach has numerous limitations related to the uptake, transport and sequestration of
the exogenously applied hormone. Usually, an indirect, rather than a direct, link between
the hormone and its effect on a particular developmental process of the plant will be the
result. In contrast, the use of mutants, defective in either their ability to produce the
hormone, or in their ability to perceive and respond to the hormone, has been
particularly informative (Klee and Estelle, 1991). For example, mutant analysis of the
10
response to the gaseous plant hormone ethylene lead to the identification of the first
hormone receptor in plants (Chang et al., 1993). In addition, increasing knowledge about
the signal transduction and physiological responses to a range of different plant
hormones has emerged through the genetic analysis of hormone mutants in Arabidopsis
(reviewed in McCourt, 1999). The use of Arabidopsis for this purpose makes it
possible to isolate and characterize the mutated genes with a reasonable effort due to the
extensive amount of genetic information available for Arabidopsis.
The perhaps most important progress of Arabidopsis research is the sequencing
of the entire genome, which is performed as a collaborative effort from several
participants all over the world. This project was initiated in 1996 and estimated to be
completed in year 2004. However, this project has proceeded at a much higher speed
than expected; the completion of the genome is now scheduled to the fall, 2000. With
this information available, the field of Arabidopsis research will definitively change,
making it possible to analyze plants from the perspective of the whole genome.
Transcriptional regulation
The process of gene expression links the genetic information that resides in the DNA of
the nucleus with the activity of proteins that give the individual cell its characteristics.
The conversion of the genetic information into a protein product is to a large extent
dependent on the intermediate step, gene transcription. This process is the key-point
for the regulation of gene expression, controlling the cell type or timing-specific
expression of a certain gene, as well as expression in response to a certain signal
(Latchman, 1998). Transcription is regulated by the activity of transcription factors,
which interact with target genes and either activate or repress transcription of the gene.
In most cases, transcription factors are sequence-specific DNA-binding proteins that
bind to specific DNA target sequences and interact with the basal transcription
machinery.
11
Homeodomain proteins
Among transcription factors, several different types of DNA-binding protein motifs
have been identified and characterized. Homeodomain proteins, encoded by the
homeobox gene family, represent one class of transcription factors. Homeobox genes
have been shown to be important for the genetic control of development in range of
different eucaryotes, i.e. specification of the body plan, determination of cell fate and a
number of other developmental processes (for reviews, see e.g. Gehring, 1992; Lawrence
and Morata, 1994).
The first homeodomain protein to undergo a structural analysis was the
Drosophila homeodomain protein Antennapedia (AntP), whose three-dimensional
structure in solution was determined by nuclear magnetic resonance (NMR)
spectroscopy (Qian et al., 1989; Billeter et al., 1990). The analysis revealed that the
homeodomain of AntP is composed of three α-helices that are folded in a tight globular
structure. Helix 1 is separated from helix 2 by a loop whereas helix 2 and 3 are separated
by a turn. Sequence similarity between the homeodomain and the helix-turn-helix (HTH)
motif of prokaryotic regulatory proteins (Pabo and Sauer, 1984), in which the second
helix of the HTH motif makes specific contacts with DNA, suggested that
homeodomain proteins act as DNA-binding regulatory proteins. Subsequently, X-ray
crystallographic studies of homeodomain proteins complexed with DNA have revealed
how homeodomain proteins recognize DNA (reviewed in Gehring et al., 1994). Helix 3
are positioned in the major groove of the DNA and make sequence specific interactions
with the bases of the DNA. Amino acid residues of the N-terminal extension of the
homeodomain perform additional base-contacts in the minor groove of DNA, and amino
acid residues positioned in the loop between helix 1 and 2 and at the start of helix 2
make contacts with the DNA backbone. Structural studies show that various
homeodomain proteins from a range of different organisms interact with DNA in
essentially identical ways, demonstrating that the mode of DNA binding of
homeodomain proteins has been highly conserved during evolution.
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Homeodomain proteins in plants: KNOTTED-1 proteins
The first homeodomain-encoding gene to be isolated from plants was KNOTTED-1 from
maize (Vollbrecht et al., 1991) corresponding to the Kn1 locus, which is defined by
several dominant gain-of-function mutations that disrupt leaf development, resulting in
the formation of knots on the leaf blade. In the Kn1 mutants, KNOTTED-1 is expressed
ectopically in vascular bundles within developing leaves, thus correlating with the
phenotypic alterations evident in the mutant (Smith et al., 1992). The wild-type
KNOTTED-1 gene is expressed in the apical meristem of vegetative and floral shoots but
expression is excluded from determinate organs such as leaves (Smith et al., 1992),
suggesting that this gene is important for shoot meristem function. Mutations in the
Arabidopsis KNOTTED-like gene SHOOTMERISTEMLESS (STM) result in seedlings
that are incapable of developing shoot apical meristems during embryogenesis (Barton
and Poethig, 1993; Long et al., 1996). These observations suggest that KNOTTED-like
genes have important roles in shoot apical meristem maintenance (Long et al., 1996).
PHD finger proteins
A different class of plant homeodomain proteins are defined by virtue of a second
conserved protein motif denoted PHD-finger. An example of a PHD-finger
homeodomain protein is HAT3.1 from Arabidopsis thaliana. This protein was isolated
based on its capability to interact with the light-induced cab-E promoter of tobacco
(Schindler et al., 1993). The PHD-finger class of homeodomain proteins also includes
the maize protein Zmhox1a, which binds to the 26 bp feedback control element of the of
the Shrunken gene promoter (Bellman and Werr, 1992), and PRHA/PRHP from
Arabidopsis and parsley, respectively, that both interact with a region of the pr2
promoter required for elicitor-mediated expression of this gene (Korfhage et al., 1994).
HDZip proteins
In an effort to isolate homeobox containing genes from Arabidopsis thaliana, a strategy
previously used to isolate homeobox genes from the nematode Caenorhabditis elegans
13
(Bürglin et al., 1989) was applied. Arabidopsis cDNA libraries were screened with
degenerate oligonucleotide probes, designed to match all possible codon combinations
corresponding to eight conserved amino acids in the helix 3 region of the homeodomain
(Ruberti et al., 1991; Mattsson et al., 1992; Schena and Davies, 1992). A number of
clones were recovered and showed extensive sequence similarity to known homeobox
genes and were designated Arabidopsis thaliana homeobox genes (ATHB). In addition,
these clones contained a second sequence element positioned 3´and closely linked to the
homeobox, encoding an α-helical stretch of aminoacids, with a periodic repetition of
leucine residues at every seventh position. This configuration of aminoacid residues
resembled that of the transcription factor C/EBP, in which the leucine residues are
arranged along one side of the helix and interact with the leucine residues projecting from
a second helix, forming a dimeric “leucine zipper” complex (Landschulz et al., 1988). As
the positions of putative DNA-binding units relative the leucine zipper domain are
similar in ATHB and bZIP proteins, respectively, it was proposed that proteins with a
contiguous homeodomain-leucine zipper architecture should be referred to as HDZip
proteins (Ruberti et al., 1991).
Additional members of the HDZip gene family have subsequently been isolated
from Arabidopsis (Carabelli et al., 1993; Söderman et al., 1994; Schena and Davis, 1994;
Baima et al., 1995; Lu et al., 1996; Di Cristina et al., 1996; Sessa et al., 1998; Lee and
Chun, 1998; Kubo et al., 1999; Zhong and Ye, 1999; Hanson (II); Johannesson et al.,
(IV). HDZip genes have also been identified from a range of other plant species; tomato
(Tornero et al., 1996; Meissner and Theres, 1995; Mayda et al., 1999, sunflower (Chan
and Gonzalez, 1994; Gonzalez et al., 1997), carrot (Kawahara et al., 1995; Mattsson et
al., 1995), soybean (Moon et al., 1996), Pimpinella brachycarpa (Moon et al., 1996),
resurrection plant (Frank et al., 1998) and ferns (Aso et al., 1999). HDZip genes have
been isolated from plants, but not from any other eukaryotes, suggesting that the
HDZip class of homeodomain proteins is specific to plants.
14
DNA-binding properties of HDZip proteins
Based on sequence criteria and supported by intron positions, HDZip proteins have
been grouped in four different families, HDZip I – IV (Sessa et al., 1994). Proteins that
belong to HDZip I and II are very similar to each other with respect to the architecture
of the HDZip domain. DNA-binding studies of ATHB1 (HDZip I) and 2 (HDZip II)
Figure 2. A hypothetical model of a HDZip protein binding to DNA, based on the
three-dimensional structures of the Drosophila engrailed homeodomain and the yeast
GCN4 leucine zipper motif, respectively (K. Johansson, H. Johannesson and E.
Söderaman, unpublished).
15
revealed that these two proteins bind as homodimers to pseudopalindromic binding
sites, CAAT(A/T)ATTG and CAAT(G/C)ATTG, respectively, consisting of two 5 bp
half-sites that overlap at the central position (Sessa et al., 1993). Furthermore, it was
obvious that the spacing between the homeodomain and the leucine zipper is critical for
the function of HDZip proteins as an insertion of two amino acids in this region of the
HDZip protein abolished its DNA-binding activity. Two HDZip II proteins from other
species other than Arabidopsis thaliana, Oshox1 from rice and CPHB-1 from the
resurrection plant Craterostigma plantagineum, bind DNA with specificities similar to
that of ATHB2 (Meijer et al., 1997; Frank et al., 1998).
The class III proteins ATHB8, 9, 14 have a different architecture of their HDZip
domains as compared with HDZip I and II proteins, with insertions of four amino acids
both between helix 2 and 3 of the homeodomain and between helix 3 and the leucine
zipper domain (Baima et al., 1995; Sessa et al., 1998). Thus, the spacing
between the homeodomain and leucine zipper domain is different in these proteins as
compared to that of HDZip I and II proteins, which suggests that class III proteins
might exhibit a different mode of DNA-binding. However, DNA binding studies of the
ATHB9 protein revealed that this protein interacts specifically with the DNA sequence
GTAAT(G/C)ATTAC, thus demonstrating that the HDZip domain of class III proteins
interacts with DNA in a similar fashion as that of class I and II protein.
ATHB10/GLABRA2, a class IV protein, is identical to class I and II proteins in
the architecture of the homeodomain and spacing between the homeodomain and leucine
zipper domain (Di Christina et al., 1996). However, the leucine zipper motif of
ATHB10/GLABRA2 is separated in two subdomains by a loop of 10 amino acid
residues. Replacement of the ATHB2 leucine zipper domain with that of
ATHB10/GLABRA2 resulted in a chimeric protein that was capable of interacting with
the ATHB2 recognition site. Hence, despite the different organization of the leucine
zipper domain of ATHB10/GLABRA2, this protein is active in dimerization and most
likely exhibits a similar mode of DNA-binding as class I and II proteins.
The DNA-binding activities in vivo have been analyzed for three HDZip
proteins: ATHB1 and 2 from Arabidopsis (Aoyama et al., 1995; Steindler et al., 1999)
and Oshox1 from rice (Meijer et al., 1997). ATHB1 was able to activate transcription
16
from a promoter containing the ATHB1 target sequence CAAT(A/T)ATTG upstream
of a reporter gene in tobacco cells. On the other hand, Oshox1 and ATHB2 were shown
to repress reporter gene activity in rice suspension cells and Arabidopsis leaves,
respectively. The transcriptional repression property of Oshox1 was lost upon deletion
of the N-terminal part of the protein, indicating that the repression function of Oshox1
resides in this part of the protein (Meijer et al., 1997). Thus, based on these
observations it is obvious that the HDZip family of transcription factors include both
activators and repressors of transcription.
In another study, it was shown that the N-terminal part of the protein, which
repress transcription in rice cells, functions as a transcriptional activator in yeast cells,
suggesting that Oshox1 might function as either a transcriptional activator or repressor
depending on the promoter context of the target gene (Meijer et al., 2000). To what
extent this dual function applies to other HDZip proteins awaits further testing.
Dimerization properties of HDZip proteins
Protein-protein interaction studies in vitro have shown the ATHB1 and ATHB2
proteins to exclusively form homodimers (Sessa et al., 1993). Similarly, studies on the
interaction between the sunflower HDZip proteins Hahb-1 and -10, which belong to the
class I and II, respectively, demonstrated that these proteins were only capable of
homodimerization (Gonzalez et al., 1997). Heterodimerization has been demonstrated
between two HDZip II proteins from different species; Oshox1 and ATHB2 (Meijer et
al., 1997) and between the two HDZip II proteins CPHB-1 and -2 from the resurrection
plant Craterostigma plantagineum (Frank et al., 1998). Together, these data suggest that
heterodimerisation is possible between members of the same class, but not between
proteins of different classes. The mechanism behind this apparent specificity in
dimerization is not clear. However, the organization of amino acid residues in the N-
terminal part of the leucine zipper is different between HDZip I and II proteins and is
probably of great importance (Gonzalez et al., 1997).
17
Function of HDZip proteins
The function of three Arabidopsis HDZip genes has been resolved through mutant
analyses. The glabra2 mutation results in defective trichome and root hair development
(Rerie et al., 1994; Masucci et al., 1996). The GLABRA2 gene was later shown to be
identical to the HDZip IV gene ATHB10 ( Di Christina et al., 1996). Mutations in the
ANL2 gene (HDZip IV) result in abnormal anthocyanin accumulation in subepidermal
tissues of rosette leaves and disturbed organization of primary root cells (Kubo et al.,
1999). Disruption of the IFL1 gene (HDZip III) abolishes the formation of normal
interfascicular fibers in Arabidopsis inflorescence stems (Zhong et al., 1999).
Functional information on HDZip class II genes is based on results from
expression analyses and analyses of transgenic plants with altered expression levels of
the genes. Expression of the ATHB2 (HDZip II) gene is induced by changes in the ratio
of red to far-red light (Carabelli et al., 1996) and the phenotypic analysis of transgenic
Arabidopsis plants with altered levels of ATHB2 suggests this gene to be a mediator of
shade avoidance responses (Steindler et al., 1999). Two dehydration-stress inducible
HDZip II genes, CPHB-1 and -2, were isolated from the resurrection plant,
Craterostigma plantagineum, using a differential display technique. Expression of both
genes are induced at early stages of dehydration suggesting that these genes may be
involved in the regulation of gene expression during dehydration (Frank et al., 1998).
Overexpression of the rice Oshox1 protein (HDZip II) protein in transgenic Arabidopsis
resulted in small and lancet-shaped leaves (Meijer et al., 1997). Oshox1 is predominately
expressed in leaves and together, these data are suggestive of a role for this gene in the
control of leaf morphogenesis.
Functional information on HDZip class I proteins is limited. ATHB6, -7 and -12
are induced by exogenous ABA, water deficit and osmotic stress. The induction of
ATHB6 and -7 by drought was strictly dependent on ABA as no increase in ATHB6 and
-7 transcripts was detected in the drought-treated seedlings of the ABA-deficient mutant
aba-3. Furthermore, the induction by ABA of ATHB7 was impaired exclusively in the
ABA-response mutant abi1 whereas the induction of ATHB6 was impaired in both abi1
and abi2. These data suggest that ATHB6, 7 and 12 probably act in an ABA-dependent
signal transduction pathway, mediating the growth response to drought in the plant
18
(Söderman et al., 1996; Lee and Chun, 1998; Söderman et al., 1999). Transgenic plants
with enhanced levels of ATHB1 are defective in the palisade parenchyma as cells of this
tissue in the transgenic plants are replaced by other cells, similar to spongy mesophyll
cells. In addition, the transgenic plants exhibited deetiolated phenotypes in the dark as
well as abnormal expansion of cotyledons. Together, these observations suggest that
ATHB1 is involved in the activation of target genes that are closely linked to plant
development (Aoyama et al., 1995). Antisense suppression of the tomato HDZip I gene
H52 in transgenic tomato plants results in necrosis and abscission of leaves (Mayda et
al., 1999). The accumulation of ethylene and salicylic acid is increased and several
defense-related genes are constitutively expressed in the transgenic plants. H52 gene
expression is induced upon infection with virulent pathogens. Thus, these data are
suggestive of a role of H52 as a transcriptional regulator of genes involved in
programmed cell death (PCD), acting to protect the cells against PCD (Mayda et al.,
1999).
19
RESULTS AND DISCUSSION
Identification of new HDZip genes (II, IV)
By searching available databases, we were able to identify 26 Arabidopsis sequences
that show similarity to HDZip class I and II genes. Of these, 17 corresponded to
previously characterized HDZip genes (ATHB1, 2, Ruberti et al., 1991; ATHB3,
Mattson et al., 1992; ATHB5, 6, 7, Söderman et al., 1994; ATHB12, Lee and Chun,
1998; ATHB13, Hanson et al., paper III; ATHB16, Y. Wang, in preparation; ATHB4,
Carabelli et al., 1993; HAT22, Schena and Davies, 1992; HAT1, 2, 3, 9, 14, Schena and
Davies, 1994, ATHB17, Ruberti et al., unpublished), whereas nine represented novel
sequences encoding putative HDZip proteins. These nine genes were designated
ATHB20, 21, 22, 23, 40, 51, 52, 53 and 54, respectively.
Biochemical characterization of HD-DNA complexes have shown a majority of
HD proteins to recognize TAAT-containing DNA-sequences (reviewed in Treisman et
al., 1992). The inherent similarities in DNA-binding specificity of all these HD proteins
implicate a structural conservation at the protein-DNA interface, which has been
confirmed by structural studies of several HD-DNA complexes (reviewed in e.g.
Gehring et al., 1994). A homeodomain consensus sequence has been defined based on a
compilation of 346 homeodomain sequences (Bürglin, 1994). The amino acids conserved
in this consensus sequence can be assigned to three different functions: contributing to
the hydrophobic core, to sugar-phosphate backbone recognition or specific interactions
with the bases in the TAAT core site. The HDs of all HDZip proteins, including
ATHB20, 21, 22, 23, 40, 51, 52, 53 and 54, contain the five invariant amino acid (L16,
W48, F49, N51, R53) and six out of seven of the highly conserved residues (F20, L26, L40,
V45, I47, L57) of this homeodomain consensus sequence. In addition to the conserved
homeodomain, these protein, including ATHB20, 21, 22, 23, 40, 51, 52, 53 and 54,
contain leucine zipper motifs in identical positions C-terminal to the homeodomain.
An initial alignment of the HDZip region of the deduced aminoacid sequences
revealed that the ATHB22 sequence did not align to the other HDZip sequences in the
region upstream of helix 2 (Figure 3). However, if 8 aminoacids corresponding to the
region between helix 1 and 2 were looped out, a perfect alignment of ATHB22 along the
20
21
entire HDZip domain was possible. A similar atypical architecture of the homeodomain
is evident for the yeast homeodomain MATα2, which has an insertion of three
aminoacids in the turn between helices 1 and 2 (Hall and Johnson, 1987). The structure
of MATα2 (Wolberger et al., 1991) is very similar to the Drosophila homeodomain
proteins engrailed (en) and AntP structures, indicating that the extra aminoacids in the
MATα2 homeodomain do not influence the overall structure of this domain. Thus,
despite the atypical architecture of the ATHB22 homeodomain, it is likely that this
protein adopts a similar structure and exhibits a similar DNA-binding property as those
of other HDZip proteins.
Based on sequence criteria, HDZip proteins have been grouped in four distinct
subfamilies, HDZip I – IV (Sessa et al., 1994) and the members of each subfamily are
also related with respect to intron positions. A number of positions separate HDZip I
and II proteins. More specifically, position 46 of helix 3 is invariant within each of the
classes but distinct between HDZip I and II. The same is true for position 58 in the
region between the homeodomain and the leucine zipper domain. The alignment shows
that, according to these criteria, the nine newly identified proteins belong to HDZip I,
since the amino acid residues in positions 46 and 58 are identical to those of all the
previously identified HDZip I proteins.
The evolutionary relationship between the entire set of HDZip I and II
aminoacid sequences was analyzed by parsimony, using PAUP (Swofford, 2000). The
phylogenetic analysis resulted in four equally parsimonious trees. Within the HDZip
domain, introns can be found in five different positions. The intron positions are
conserved within the different subclasses defined by the tree and thus support the major
branching pattern of the tree. The tree revealed two distinct classes, corresponding to
the HDZip I and II classes previously identified by Sessa et al., (1994), respectively.
All of the nine newly identified sequences were included in HDZip class I, confirming
that ATHB20, 21, 22, 23, 40, 51, 52, 53 and 54 belong to HDZip I.
The chromosomal locations of each of the HDZip genes were determined based
on the positions of corresponding BAC-clones on the physical map of Arabidopsis
thaliana. As shown in Figure 4, HDZip I and II genes are evenly distributed over the
entire length of the five chromosomes of Arabidopsis.
22
Figure 4. Locations of HDZip I and II genes on the five chromosomes of Arabidopsis
thaliana. The position of each gene is according to the physical map of Arabidopsis and
their locations are indicated relative a subset of mi-RFLP markers (Liu et al., 1996)
23
Identification of HDZip target sequences in vitro (I)
HDZip proteins contain leucine zipper domain located immediately C-terminal to the
homeodomain. As the leucine zipper motifs in other classes of transcription factors
mediate dimerization it was suggested that HDZip proteins are active as protein dimers
in the binding of DNA, such that the leucine zipper juxtaposes a pair of DNA
interacting units onto the DNA (Ruberti et al., 1991). This notion was confirmed when
analyzing the DNA-binding properties of ATHB1 and ATHB2, which interacted with
the distinct pseudopalindromic sequences CAAT(A/T)ATTG and CAAT(G/C)ATTG,
respectively, as homodimers (Sessa et al., 1993) . Thus, ATHB1 and ATHB2 interact
with nearly identical DNA sequences and the principal difference is their specificity for
the central position in the recognition site. Considering the high degree of sequence
similarity within the homeodomain of the HDZip proteins, it is possible that these
proteins interact with very similar DNA binding sites. Moreover, it is likely that
proteins that belong to the same subclass exhibit identical binding site preferences, such
that all HDZip I and II proteins have the same specificity as ATHB1 and ATHB2,
respectively. To test this hypothesis, the binding specificity for a number HDZip class
I proteins was analyzed, starting with ATHB5. A PCR assisted binding-site selection
procedure was used to select for high-affinity binding sites for ATHB5 from a pool of
DNA-fragments containing a 12 bp random core sequence. Strikingly, the majority of
the clones contained the pseudopalindromic sequences CAAT(A/T)ATTG or
CAAT(G/C)ATTG, confirming that the HDZip I protein ATHB5 binds DNA in a
similar fashion as ATHB1. In contrast to ATHB1, ATHB5 did not discriminate
between binding sites in which the identity of the central positions was different as the
frequency of recovered clones containing either an A/T or a G/C pair in the central
position was essentially the same. In electrophoretic mobility shift assays, ATHB6 and
16 interacted with similar affinity to either A/T or G/C containing sequences. This result
demonstrates that ATHB6 and 16, similar to ATHB5, are independent on the identity
of the central position in the binding site. In contrast, ATHB1 showed a distinct and
ATHB3 and 13 exhibited a slight preference for an A/T pair in the central position
specificity. Thus, it is evident that HDZip proteins interact with very similar binding
sites, but central position specificity appear to vary among HDZip class I proteins.
24
What is the determinant of central position specificity in the HDZip proteins? A
DNA binding model of HDZip proteins presented by Sessa et al., (1993) predicts that
HDZip and bZip proteins interact similarly with DNA and that Arg55 of the HDZip
domain is functionally equivalent to Arg243 of the yeast bZip protein GCN4. When
complexed with the GCN4 recognition site, TGA(C/G)TCA, Arg243 of one GCN4
monomer donates hydrogen bonds to the guanidine of the central pair, whereas Arg243
of the other monomer makes phosphate backbone interactions (Ellenberger et al., 1992).
The importance of Arg55 for central position specificity of HDZip proteins was tested
experimentally by a mutational analysis of the ATHB2 HDZip domain (Sessa et al.,
1997). The results indicated that Arg55 of ATHB2 is important for central position
specificity, but residues outside helix 3 also contribute to binding-specificity. Thus,
specificity for the central position may be attributable to the conformation of the Arg55
side chains in the HDZip domain. This finding is consistent with the fact that Arg55 is
invariant among all HDZip proteins and therefore, this position alone can thus not
explain the apparent diversity of central position preference for HDZip proteins.
ATHB7 and 12 have alternative binding site specificities (I)
In contrast to ATHB1, 3, 5, 6, 13 and 16, neither ATHB7 nor ATHB12 interacted with
the HDZip consensus site CAATNATTG. This was a surprising result considering the
fact that all HDZip I and II proteins, including ATHB7 and 12, are highly similar in
sequence within the homeodomain and it is rather unlikely that these proteins would
differ dramatically in their DNA binding specificities. One possible explanation for the
result is that the ability of the ATHB7 and 12 proteins to bind DNA is dependent on
correct post-translational modifications, e.g. protein phosphorylation. This possibility
is consistent with the presence of a potential phosphorylation site at a position in the
homeodomains of ATHB7 and 12, which is absent in homeodomains of other class 1
HDZip proteins. Furthermore, the transcription of both genes are induced by ABA,
osmotic stress and drought conditions (Söderman et al., 1996; Lee and Chun, 1998), and
in the case of ATHB7 by a signaling mechanism that involves protein phosphorylation/
25
dephosphorylation (Söderman et al., 1996), suggesting that this signaling pathway might
also act on ATHB7 directly on the protein level by a similar mechanism.
Another possibility is that ATHB7 and 12 interact with different binding-sites
than those of other HDZip I proteins. In support of this hypothesis, ATHB12 has
recently been demonstrated to specifically interact with the palindromic sequence
TCAATTAATTGA (Chun and Lee, 1998, US Patent No 5,981,729). A closer
inspection of the ATHB12 recognition site reveals that it consists of two non-
overlapping TCAATT half-sites, as opposed to the HDZip consensus-binding site
CAATNATTG, which consists of two CAATN half-sites overlapping in the central
position. Thus, the principal difference between the two sites is the spacing between the
half-sites, whereas the half-sites as such are essentially identical. This finding suggests
that the interaction of ATHB12 at the protein-DNA interface is the same as for other
HDZip proteins, but that ATHB12 adopts a slightly different structure that affects the
spacing between half-sites. Furthermore, considering the high degree of sequence
similarity between ATHB7 and 12, it is likely that ATHB7 exhibits an identical DNA-
binding specificity as that of ATHB12.
The mechanism by which half-site spacing in HDZip proteins is determined is
not clear. In bZip proteins, it has been shown that the short surface spanning the fork
region, which connect the leucine zipper and the DNA-binding basic region, is the major
determinant of half-site spacing (Kim and Struhl, 1995). However, the region of HDZip
proteins that corresponds to the fork region in bZip proteins is conserved among all
HDZip proteins, including ATHB7 and 12, suggesting that this mechanism of half-site
spacing determination is not applicable to HDZip proteins.
Another possibility is that HDZip proteins, in analogy with e.g. the Drosophila
homeodomain proteins AntP and en, perform DNA contacts in the minor groove of
DNA through arginine residues at positions 3 and 5 in their N-terminal arms of the
homeodomain (reviewed in Gehring et al., 1994). A closer inspection of the region of the
homeodomain preceding helix 1, all HDZip class proteins, except ATHB7 and 12, have
a basic stretch of amino acids, which shows resemblance to the N-terminal extention of
classical homeodomain proteins. Although not tested experimentally, it is possible that
the N-terminal region of HDZip proteins is functionally equivalent to that of AntP and
26
en and determines DNA-binding specificity through minor groove interactions. If that
were the case, the alternative spacing preference of ATHB7 and 12 could be a
consequence of the alternative architecture of the N-terminal region.
In summary, HDZip proteins are similar in sequence within their HDZip
domain, which suggest that they should interact with very similar DNA sequences.
Nevertheless, the diversity in central position specificity among HDZip proteins,
manifested either as preference for a certain pair in this position or as preference for an
alternative spacing between half-sites, appear to be larger than initially expected. Thus,
the functional differences between different HDZip proteins may in part be due to this
diversity in their DNA-binding.
Heterodimer formation between HDZip class I proteins (I).
The fact that some of the HDZip proteins have very similar DNA-binding specificities
and yet appear to have distinct biological functions raise the hypothesis that the
targeting of HDZip proteins in vivo to the correct promoters is dependent on
interactions with other proteins. A special case of such protein-protein interaction is the
formation of heterodimers between different members of HDZip proteins.
The interaction between a recombinant bacterial ATHB5 protein and in vitro
translated 35S-labeled HDZip proteins was analyzed and the result showed that ATHB5
interacted with ATHB6, 7, 12 and 16 with different apparent affinity, but not with
ATHB1, suggesting that HDZip proteins show selectivity in dimerformation, specified
by the dimerization domain.
In bZip proteins, such as the protein products of the proto-oncogenes c-fos and
c-jun, the leucine zipper domain itself is capable of dictating specificity (O´Shea et al.,
1992). Specificity depends on electrostatic interactions between amino acids of e and g
positions of the heptad repeats (abcdefg)n that form the hydrophobic interface in the
two monomers, which is consistent with the known crystal structures of GCN4
(Ellenberger et al., 1992) and a Fos/Jun heterodimer (Glover and Harrison, 1995). An
interhelical salt bridge hypothesis (Vinson et al., 1993), based on functional analyses of
leucine zippers suggests that the identity of amino acid in the g and e position of each
27
unit determine which proteins that have the potential to dimerize. Applied to the class 1
HDZip proteins, this hypothesis predicts the proteins to have the capacity to form
both homo- and heterodimers. The in vitro data on ATHB5 indicate that the protein in
fact does show selectivity in dimer formation, suggesting that HDZip proteins rely on a
mechanism different from that described for bZip proteins to dictate dimerization
specificity.
The selective heterodimer formation between HDZip class I proteins might
provide the plant with transcription factors that, even when interacting with the same
target sequence as the original homodimer, have distinct activation characteristics. Thus,
it is possible that HDZip proteins constitute a functional complex of homo- and
heterodimeric transcription factors in the plant, providing a mechanism by which the
plant can increase the level of complexity in transcriptional regulation on the protein
level.
Expression analysis of ATHB5 (II)
A first step to understand the function of HDZip proteins is to determine the tissue and
timing specificity of HDZip gene expression. In the case of ATHB5, this gene was
previously shown to be expressed at moderate levels in adult leaf, stem and root tissues
(Söderman et al., 1994); however, the expression of ATHB5 at other developmental
stages was not analyzed. Analysis of the expression of ATHB5 during early
development showed that ATHB5 gene transcription was turned on early after the onset
of germination and then gradually declined with increasing age of the seedlings.
Additional experiments revealed that ATHB5 mRNA was detectable as early as 12 h
post-imbibition, but not in the dry seed (data not shown). Histochemical staining of
transgenic Arabidopsis seedlings harboring an ATHB5::GUS fusion revealed strong GUS
activity specifically in the hypocotyl of germinating seedlings, concentrated around the
transition zone, which marks the boundary between the hypocotyl and the root.
Staining was retained at the transition zone as the seedlings grew older, but was also
visible with low intensity in the petioles of cotyledons and developing true leaves. Weak
GUS staining associated to vascular tissue was found in root, stem and cauline leaf
28
tissues and no visible staining was found in flower organs. Thus, the expression analysis
of ATHB5 showed that the activity of this gene appears to be under strict
developmental control and activated by the initiation of germination.
The staining intensity at the transition zone of the ATHB5::GUS transgene
decreased markedly when seedlings were treated with 10 µM ABA for 12 hrs. This
effect was specific for ABA as the staining intensity was unaffected by the treatment
with auxin, gibberellic acid, cytokinin or the ethylene precursor ACC. These data
indicate that ATHB5 gene expression is dependent on ABA signaling. This notion was
supported by results from experiments assessing whether ATHB5 expression during
postgerminative growth was dependent on the activity of known ABA response loci.
The expression level of ATHB5 was markedly lower in abi3-1 and abi5-1 mutant
background as compared to the wild type, demonstrating that ATHB5 represents a target
gene for transcriptional regulation by ABI3 and ABI5 during postgerminative growth,
either directly or indirectly.
In summary, the expression analysis allows the distinction of cell types and
developmental stages where the gene primarily exerts its function. In the case of ATHB5,
we can conclude that this protein is functional primarily in cells of the hypocotyl during
seed germination. Moreover, the fact that the ATHB5 expression level is affected by
ABA and lower in the ABA response mutants abi3 and abi5 indicate that the function
of ATHB5 is related to ABA signaling.
A reverse genetics approach to understand the function of HDZip proteins
Approaches such as gene expression analysis to understand gene function are only
correlative and do not necessarily provide a direct link between a gene and its function.
In contrast, by the analysis of null mutations in the genes of interest it is possible to
establish their function simply by assessing the phenotypic effects resulting from the
mutation. This approach has had an enormous impact on the understanding of
developmental processes in a range of different organisms (e.g. Nüsslein-Volhard, 1994;
Burns et al., 1994; Spradling et al., 1995; Brandon et al., 1995).
29
Mutations created by insertions of either T-DNA or transposable elements are
particularly advantageous as they provide direct means by which the mutated gene can
be isolated. Individual lines bearing insertions in the gene of interest are easily identified
by amplification of flanking regions of the inserted element using polymerase chain
reaction (PCR) with a combination of gene-specific primers and primers complementary
to border sequences of the inserted element. This method was originally developed for
Drosophila where PCR was used to screen for P-element insertions (Ballinger and
Benzer, 1989; Kaiser and Goodwin, 1990) and has also been utilized in other organisms
such as Caenorhabditis elegans (Zwaal et al., 1993). In Arabidopsis, this approach has
been used to isolate plants carrying insertions in a range of different genes (e.g.
McKinney et al., 1995; Krysan et al., 1996; Winkler et al., 1998). Considering the
relatively small genome size of Arabidopsis, it has been estimated that it is sufficient to
screen around 100 000 lines in order to find a plant carrying an insert in any gene of
interest (Azpiroz-Leehan and Feldmann, 1997).
A limitation to screens for loss-of-function mutations is that the recovered
mutants often lack discernable phenotypes, due to genetic redundancy. Redundancy is
evident among members of the ethylene receptor gene family (Hua and Meyerowitz,
1998) as well as among homeotic genes that control flower development in Arabidopsis
thaliana (reviewed in Martienssen and Irish, 1999). An alternative strategy to reveal
gene function is to assess the phenotypic effect of enhanced or reduced levels of gene
activity by expressing the gene in either sense- or antisense orientation from the strong
constitutive promoter of the cauliflower mosaic virus (CaMV) 35S gene. The anti-sense
approach will suffer from the same problem as that of insertion lines, namely gene
redundancy, unless the expression of the anti-sense construct has the capacity to
inactivate several genes simultaneously that are related in sequence and act in a
redundant fashion. In contrast, overexpression of the gene can provide functional
information of a gene, which will be manifested through a visible phenotype. One
complication of high-level expression from the 35S-promoter is that it results in gene
activity in tissues or developmental stages where the gene is not normally expressed and
hence, resulting in phenotypic deviations from wild type that are not related to the
30
function of the gene. However, a careful expression analysis of the gene should facilitate
the distinction of phenotypes that reflect the actual function of the gene.
Overexpression of ATHB5 in transgenic Arabidopsis (III)
Phenotypic analysis of ATHB5 overexpressor lines (35S::ATHB5) revealed that these
plants did not differ from wild type in their overall growth and development. However,
the 35S::ATHB5 plants differed from wild type in their seedling morphology.
Hypocotyls of young seedlings were shorter and radially expanded at the base. In
addition, the primary roots as well as petioles of cotyledons of 35S::ATHB5 seedlings
were shorter than those of the wild type. Thus, the phenotypic deviations from wild
type seen in the 35S::ATHB5 plants are consistent with the results from the expression
analysis of ATHB5, which suggest that it is likely that the ATHB5 gene exerts its
function primarily in hypocotyls of germinating seedlings and not in the adult plant. The
effect of the plant hormone ABA on the intensity of GUS staining in the ATHB5
promoter GUS-fusion plants, as well as the downregulation of ATHB5 in the ABA
response mutants abi3 and abi5, suggested that overexpression of ATHB5 in transgenic
plants might affect the response of the seedlings to growth inhibition by ABA. This
notion was confirmed by experiments assessing the sensitivity of wild type and
35S::ATHB5 plants to the inhibitory effects of exogenously applied ABA on seed
germination and seedling root growth. The results from these assays showed that the
35S::ATHB5 line was more sensitive to ABA at all concentrations tested as compared
with the wild type. The ABA concentration needed to achieve 50% inhibition of either
seed germination or root growth was two-fold lower for the 35S::ATHB5 line than for
the wild type. These results suggest that overexpression of ATHB5 interferes with ABA
signaling, resulting in seedlings hypersensitive to ABA. The finding that the
35S::ATHB5 seedlings were hypersensitive to ABA was consistent with the results
from experiments assessing ABA regulated gene expression, demonstrating that the
ABA responsive gene RAB18 was hyperinduced by ABA in 35S::ATHB5 seedlings as
compared to wild-type.
31
During seed development the level of endogenous ABA peaks during mid to late
embryogenesis before returning to low levels in the dry seed (Rock and Quatrano, 1995).
ABA has been demonstrated to be important for the establishment of seed dormancy
since Arabidopsis mutants either defective in ABA biosynthesis or in the ability to
sense ABA fail to become dormant (Koorneef et al., 1982; 1984). Furthermore,
mutations that enhance the sensitivity to ABA confer hyperdormancy (Cutler et al.,
1996; Gosti et al., 1999). Consequently, seeds from 35S::ATHB5 plants that are
hypersensitive to exogenous ABA should therefore also be hyperdormant. However,
overexpression of ATHB5 does not affect seed dormancy, indicating that ATHB5 does
not regulate ABA sensitivity during seed development and consequently does not take
part in the regulation of seed dormancy. This hypothesis is consistent with the fact that
ATHB5 is not expressed in wild-type plants during seed maturation when dormancy is
established.
In summary, the functional analysis of the HDZip gene ATHB5 provides
evidence for that this gene is active in the regulation of germination and postgerminative
growth, possibly as an ABA dependent negative regulator of cell expansion. Clearly,
germination and postgerminative growth is highly dependent on ABA signaling and it
has been suggested that the inhibitory effect of ABA on these processes is related to the
inhibition of storage reserve mobilization, since this hormone alters the pools of
available nitrogen and energy as well as prevents the degradation of seed storage proteins
(Garciarrubio et al., 1997). It is possible that the mechanism by which ATHB5 regulate
germination and postgerminative growth is through restriction of energy and metabolites
as a component of an ABA response pathway in Arabidopsis thaliana.
High-level expression of ATHB13 in transgenic Arabidopsis (II)
Increased expression levels of ATHB13 in transgenic plants (35S::ATHB13) did not
result in any major phenotypic deviations in overall growth and development as
compared to the wild type. However, when grown in vitro on standard medium, the
35S::ATHB13 seedlings differed from wild type by decreased cotyledon width. It was
evident that this effect was mediated by sucrose as the phenotypic deviation
32
disappeared when metabolizable sugars were omitted from the growth medium. The
effect of sucrose on the cotyledon width of the 35S::ATHB13 seedlings was dose-
dependent, since cotyledon width decreased with increasing sucrose concentrations.
Measurements of the size of individual epidermal cells demonstrated that the sucrose-
dependent decrease in cotyledon width of 35S::ATHB13 seedlings was a consequence of
inhibited epidermal cell expansion. The effect of ATHB13 overexpression on cotyledon
cell size and shape is most likely dependent on sucrose signaling, since neither non-
metabolizable sugars nor genetic crossings with transgenic plants with altered hexokinase
activities affected the morphology of 35S::ATHB13 seedlings. This hypothesis is
consistent with results from experiments analyzing sugar regulated gene expression,
which demonstrated that the expression of a subset of known sugar-responsive genes
were induced by sucrose to a higher level in 35S::ATHB13 seedlings than in the wild-
type. Taken together, these data show that ATHB13 affect both morphology and gene
regulation in response to sucrose, thus representing the first transcription factor shown
to directly take part in a sucrose signaling pathway.
Interpretation of phenotypes resulting from constitutive expression (II, III)
As stated above, high-level expression from the 35S-promoter can result in ectopic gene
activity, i.e. the gene is active in tissues or stages where it is not normally active. In the
case of the 35S::ATHB5 plants however, the expression analysis show this gene to be
active in the hypocotyl early after the onset of germination and dependent on the ABA
response genes ABI3 and ABI5, consistent with the ABA hypersensitivity phenotype
during seed germination conferred by the gene when expressed at high levels. In addition,
the fact that the phenotypic deviations seen in the ATHB5 overexpressors are specific
for the germination stage suggests that the target genes, which ATHB5 activates, are
only accessible during this developmental stage. Thus, it is possible that ATHB5 needs
to interact with other factors that are active during postgerminative growth in order to be
functional. In contrast, the ATHB13 gene is not expressed in cotyledons where it exerts
its effect when overexpressed (J. Hanson, in preparation). Thus, the effect of ATHB13
on cotyledon cell size and shape is most likely a consequence of ectopic expression and
33
does not directly reflect the function of the gene in the wild-type plant. This hypothesis
is supported by the observation that ATHB23, a close relative to ATHB13, is expressed
in cotyledons and has a similar effect as ATHB13 on cotyledon cell shape and size when
overexpressed (J. Hanson, in preparation).
Isolation of T-DNA insertion lines in HDZip class I genes (II, III, IV)
In order to obtain loss- or reduction-of-function plants of HDZip genes, the anti-sense
approach has been used for a number of HDZip genes. However, this method has
proved to be inefficient for a majority of the genes tested. Instead, PCR based screens
for insertion mutants in HDZip I genes were performed. A population of 6000 T-DNA
insertion lines with an average of 1,5 insertion events per line has been deposited at the
Arabidopsis Biological Resource Center (ABRC) as DNA "superpools" of 1000 lines
each. A primary screen of this population identified insertions in five different HDZip
genes, ATHB5, 6, 7, 40 and 51. The insertions in ATHB5, 7, 40 and 51 most likely result
in null mutations (Azpiroz-Leehan and Feldmann, 1997) as these mutation are either
located within intron sequences or, in the case of the ATHB7 insertion, in the
5´untranslated leader sequence. In contrast, a T-DNA inserted upstream of the
transcription start site of the ATHB6 gene might affect gene expression of ATHB6,
resulting in a "knock-down" mutation (Krysan et al., 1999) of the ATHB6 gene.
In the case of ATHB13, a putative null mutant line was isolated using the same
strategy as described above but from a collection of lines harboring En-1 transposons
(Baumann et al., 1998). This insertion was located in the 5´ splice site of the first intron
of ATHB13 and most likely results in a null-mutation of the ATHB13 gene.
The mRNA and protein levels of the gene have to be assessed in order to confirm
that the insertion has created a null-mutation. This was done for the ATHB5 insertion
line and the result showed that there was no detectable ATHB5 mRNA or proteins in
extracts of this line, confirming that this insertion in the first intron of the ATHB5 gene
results in a null-mutation. To what extent the insertions in ATHB6, 7, 40 and 51 disrupt
gene transcription and translation awaits further testing.
34
When analyzing the phenotype of the insertion lines it became evident that none
of these lines exhibited any visible phenotypic deviations from wild type, neither during
seedling growth nor as adult plants. The insertion lines of ATHB5 and 13 were subjected
to more thorough phenotypic analyses with respect to ABA and sucrose sensitivity,
respectively. In neither case did the loss-of-function mutations confer any phenotypic
deviations as compared to the wild type. One explanation to the lack of distinguishable
phenotypes in the insertion lines might be that HDZip genes are only functional under
specific physiological conditions, i.e. unless the mutant plant is subjected to conditions
in which the gene is essential, no phenotypic deviations from wild-type will be visible.
Another explanation is gene redundancy, i.e. the loss of gene activity in these insertion
lines might be compensated by other HDZip genes. Considering the analysis of DNA-
binding preferences of HDZip proteins it is likely that some of these proteins interact
with identical binding sites and activate the same target genes in vivo.
In summary, the use of a PCR-based systematic approach for mutant isolation in
Arabidopsis HDZip I genes proved to be successful; from a population of 6000
insertion lines, T-DNA insertions in five different genes were identified. The fact that
these lines lack distinguishable phenotypes emphasizes the importance of establishing
careful screens for growth conditions in which the activity of the target genes are
required. Alternatively, for those genes that act redundantly, genetic crosses between
plants that bear mutations in these genes have to be performed
35
SUMMARY
HDZip proteins constitute a large family of transcription factors in plants and with the
close completion of the Arabidopsis genome sequencing, we estimate that the total
number of HDZip I and II genes will not exceed 26. HDZip proteins interact
specifically with pseudopalindromic binding sites, in which each half-site is occupied by
one HDZip monomer. The difference in binding specificity between HDZip proteins
resides primarily in their preference for the central position of the binding site, whereas
the interaction with each half-site appears to be conserved. The finding that HDZip I
proteins are capable of forming both homo- and heterodimers in vitro suggests that
HDZip proteins might be involved in a complex network of interacting transcription
factors in the plant.
The HDZip I protein ATHB5 was shown to be active in the regulation of seed
germination and postgerminative growth, possibly as an ABA-dependent regulator of
cell expansion. ATHB13 affects leaf morphology as well as gene regulation in response
to sucrose, suggesting that ATHB13 takes part in a sucrose signaling pathway. The
functional analysis of these two proteins was primarily based on the interpretation of
overexpression phenotypes. Establishment of HDZip I gene function based on
phenotypic analyses of mutant plants identified by reverse genetics screens from
collections of T-DNA insertions and transposable elements has as yet not been
successful. The reason for this is not clear; however, it is possible that HDZip genes are
redundant or that the conditions in which HDZip genes are required are not known.
36
ACKNOWLEDGEMENTS
I wish express to express my sincere thanks to all those who have contributed to this
thesis:
My supervisor, Professor Peter Engström, for accepting me as PhD-student and
teaching me scientific thinking and writing.
Johannes, for interesting discussions about the deeper secrets of HDZip science;
invaluable help with computer-problems, and for giving me the opportunity to eat fast
food every now and then.
Thanks are due to Eva Söderman for being the perfect room-mate.
Eva Söderman, Eva Sundberg and Johannes Hanson, for critical reviewing parts of this
thesis.
The members of the Prof lab, Yan, Mats and Jens, for providing interesting discussions
and good friendship during labwork; Mats and Jens for making the Prof lab the most
funky lab in the building. Thanks to Peter, again, for letting Prof lab stay funky!
I wish to thank all people that have contributed to this thesis by technical assistance:
Marie Lindersson, Marie Englund, Eva Burén, Kajsa Pöntinen, and Agneta Ottoson.
Birgitta, thank you for helping me with all imaginable administrational problems.
Thank you Ingela for nostalgic conversations about good old times! Thank you Anneli,
Sandra, Katarina, and the rest of cellskapet for making fysbot a nice place to work in!
Last, but not least, I would like to thank:
Hanna for love and support, but also for invaluable comments on all manuscripts of this
thesis.
Svante, for being the sunshine of my life!
My parents and Hannas parents for continuos support and for taking care of Svante
during the work with this thesis.
37
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