Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 618
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The Role of the Homeobox Gene ATHB16 in Development Regulation in Arabidopsis thaliana
BY
YAN WANG
ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2001 Dissertations for the Degree of Doctor of Philosophy in Physiological Botany presented at Uppsala University in 2001
ABSTRACT Wang, Y., 2001. The Role of the Homeobox Gene ATHB16 in Development Regulation in Arabidopsis thaliana. Acta Univ.Ups., Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 618. 50 pp. Uppsala. ISBN 91-554-4983-2.
There are 42 members of the homeodomain-leucine zipper (HDZip) family of transcription factors in Arabidopsis thaliana. This thesis focuses on the functional analysis of one member of this family, ATHB16, and on the biochemical properties of HDZip proteins. To assess the function of the ATHB16 gene, the expression of ATHB16 was altered in transgenic Arabidopsis plants by using sense and antisense RNA constructs under the control of the 35S promoter. The reciprocal phenotypic effects associated with elevated and reduced levels of ATHB16 expression suggested that, in wild-type plants, ATHB16 acts as a mediator of blue and red light effects on the regulation of plant growth and the timing of the floral transition. In wild-type Arabidopsis, expression of ATHB16 is high in leaves, intermediate in adult roots and inflorescences, and low in stems and siliques. The expression of ATHB16 in the root is markedly increased in response to exogenous abscisic acid (ABA) treatment, but is reduced in the ABA response mutants abi1 and abi2, suggesting that ATHB16 may be involved in ABA signal transduction. This hypothesis was corroborated by observations of alterations in sensitivity to ABA inhibition of root growth in seedlings of a T-DNA insertion mutant of ATHB16 and of transgenic plants with elevated ATHB16 levels. HDZip proteins bind DNA as dimers. DNA-binding studies showed that different HDZip proteins interact with very similar target sequences in vitro and that they selectively form heterodimers with each other. For example, it was demonstrated that ATHB16 can heterodimerize with ATHB6 and ATHB7 in yeast and with ATHB5 in vitro, suggesting that ATHB16 may interact with other HDZip proteins in Arabidopsis. This interaction may have functional significance, since it may provide a mechanism for the plant to integrate different input signals, like light of different spectral qualities and water availability in the regulation of its growth.
Yan Wang, Department of Physiological Botany, Evolutionary Biology Center, Uppsala University, Villavägen 6, SE-752 36 Uppsala, Sweden
Yan Wang 2001
ISSN 1104-232X ISBN 91-554-4983-2
Printed in Sweden by Uppsala University, Tryck & Medier, Uppsala 2001 To my family This Thesis is based on the following papers, which will be referred to in the text by their Roman numerals:
I. Wang, Y., Sundberg, E. and Engström, P. The Arabidopsis homeobox gene, ATHB16, is a potential mediator of blue and red light effects on plant growth and development. (manuscript).
II. Wang, Y., Johannesson, H., Qiu, J.L. and Engström, P. The HDZip protein ATHB16 mediates abscisic acid signaling and affects root sensitivity to abscisic acid in Arabidopsis. (manuscript).
III. Johannesson, H., Wang, Y. and Engström, P. (2001) DNA-binding and dimerization preferences of Arabidopsis homeodomain-leucine zipper transcription factors in vitro. Plant Mol. Biol., 45, 63-73.
IV. Johannesson, H., Hanson, J., Söderman, E., Wang, Y. and Engström, P. HDZip proteins in Arabidopsis thaliana: a case of functional conservation and redundancy within a family of transcription factors. (manuscript).
Reprint of paper III was made with kind permission from Kluwer Academic Publishers. TABLE OF CONTENTS
ABBREVIATIONS 7 INTRODUCTION 8 Arabidopsis: a model plant 8 Plant growth and development 8 Floral initiation 9 The shoot apical meristem and phase transition 9 Multiple pathways regulating the floral transition 11 Genes specifying the floral meristem 12 Light signaling 14 Photoreceptors 14 Phytochromes 14 Cryptochromes 16 The plant hormone abscisic acid 17 HDZip transcription factors 19 Homeobox genes 19 HDZip genes 19 DNA binding and dimerization properties of HDZip proteins 20 Functions of HDZip genes 22 RESULTS AND DISCUSSION 24 Identification of ATHB16, a new member of the HDZip I family (I and IV) 24 Expression of ATHB16 in wild-type plants (I and II) 24 Expression of ATHB16 is upregulated by abscisic acid in the root (II) 25 Alteration of ATHB16 expression levels in transgenic plants (I) 26 ATHB16 reduces the sensitivity of the flowering response to photoperiod (I) 26 ATHB16 causes pleiotropic effects on plant growth and development (I) 27 ATHB16 affects the blue light sensitivity of the plant (I) 28 ATHB16 may mediate light effects on plant growth (I) 28 ATHB16 mediates blue/red light-dependent regulation of flowering (I) 29 Isolation and identification of a T-DNA insertion line in ATHB16 (II) 30 ATHB16 affects sensitivity to ABA in a root growth assay (II) 32 Identification of HDZip I target sequences in vitro (III) 33 Heterodimer formation between HDZip I proteins (II and III) 34 CONCLUDING REMARKS 36 ACKNOWLEDGEMENTS 37 REFERENCES 39 ABBREVIATIONS
ABA abscisic acid cry cryptochrome EMSA eletrophoretic mobility shift assay EST expressed sequence tag GA gibberellins HDZip homeodomain leucine-zipper IAA indole-3-acetic acid LD long day phy phytochrome RACE rapid amplification of cDNA ends SAM shoot apical meristem SD short day
The following nomenclature conventions have been used in this thesis: Names of genes are written in italicised upper-case letters, e.g. ATHB16. Names of proteins are written in non-italicised upper-case letters, e.g. ATHB16. Names of mutants are written in italicised lower-case letters, e.g. athb16. Names of photoreceptor holoproteins are written in non-italicised lower-case letters, e.g. phyA, cry1. Names of photoreceptor apoproteins are written in non-italicised upper-case letters, e.g. PHYA, CRY1. INTRODUCTION
Arabidopsis: a model plant
Biologists rely on model organisms. Arabidopsis thaliana, a small weed related to mustard, emerged in the 1980s as the most popular model in plant biology, in large part because of its small size, short life cycle and the ease with which it could be grown and bred. This plant, also known as thale cress, is widely distributed throughout the Northern Hemisphere (Rédei, 1975). In nature, it grows as a winter annual, which means that the seed germinates and grows during the autumn, the plant survives the winter as a rosette and then flowers in the spring. The distribution of natural populations (ecotypes) of Arabidopsis reflects the adaptations of the species to different natural environments. Another feature that makes Arabidopsis an attractive model plant is its relatively small genome, which contains a complete set of genes for controlling developmental patterns, metabolism, responses to environmental cues and disease resistance. By the end of the year 2000, its complete genome had been sequenced by an international public effort (Arabidopsis Genome Initiative, 2000). The genome contains 25,498 genes, encoding proteins from 11,000 families. Fewer than 10% of the Arabidopsis genes have been studied in experiments on gene function. However, completion of the genome sequence dramatically broadens and facilitates research into gene function.
Plant growth and development
The general body plan of plants is established during embryogenesis. However, much of the plant development occurs post-embryonically and the development is highly dependent on the external conditions at the growth site. Plants have evolved many plant-specific functions, such as photomorphogenesis, gene regulation and metabolism to control their growth and development. The mechanisms underlying these biological processes and the developmental regulation of plants are, therefore, of particular interest for plant biologists. Here, I briefly summarize current knowledge concerning selected aspects of plant-specific development, including floral transition, photomorphogenesis and ABA responses, since the function of the ATHB16 gene, which is the major subject of this thesis, is related to all these processes.
Floral initiation
The shoot apical meristem and phase transition During plant development, new organs are continually generated from pools of undifferentiated cells called meristems. The meristem of higher plants is a center of continuous cell division that has two distinct functions. Proliferation at the center maintains the meristem, while generation of cells at the flanks leads to organogenesis and ultimately differentiation of tissues and organs. In many plant species, the shoot apical meristem (SAM) is formed in embryogenesis, and leaf primordia are initiated later from the flanks of the vegetative meristems. Once the floral transition occurs, floral primordia are initiated from the flanks of the SAM and floral organs develop (Steeves and Sussex, 1989). Arabidopsis belongs to a group of plants that characteristically grow as rosettes during their vegetative growth phase, because of the reiterative production of leaves without internode elongation. The initiation of reproductive development is marked by the elongation of the internode (bolting) that gives rise to an inflorescence, bearing cauline leaves with coflorescence meristems at their basal nodes, and flowers without subtending leaves at their apical nodes, as shown in Figure 1. The vegetative phase of Arabidopsis can be subdivided into juvenile vegetative (JV) and adult vegetative (AV) phases, which can be distinguished by differences in the shape of the leaves and the distribution of trichomes on the leaf surface (Chien and Sussex, 1996; Telfer et al., 1997). Juvenile leaves produce trichomes only on the upper (adaxial) surfaces, whereas adult leaves produce them on both their upper and lower (abaxial) surfaces (Figure 1). In inflorescence development, two phases can also be distinguished, the early reproductive (ER) and late reproductive (LR) phases, which differ in the lateral structures produced by the SAM. During the early reproductive phase, cauline leaf primordia that subtend axillary inflorescence meristems are produced. Determinate floral primordia that develop into a bractless flower are produced in the later reproductive phase (Schultz and Haughn, 1993).
Figure 1. Vegetative and reproductive phase changes in Arabidopsis development. JV, AV, ER and LR represent juvenile vegetative, adult vegetative, early reproductive and late reproductive phases, respectively. Following the transition to flowering, apical dominance decreases, allowing the secondary inflorescence (which originates from meristems in the axils of the rosette leaves) to develop. The figure is adopted from Simpson et al. (1999) and Araki (2001), with kind permission from the authors. Multiple pathways regulating the floral transition The transition of SAM from vegetative growth to flowering is the most dramatic developmental switch in the plant life cycle and is crucial for seed production. To ensure that seed set occurs during the appropriate season, plants have evolved complex schemes to regulate the timing of flowering. Some plants are completely dependent upon environmental signals to induce flowering, whereas others rely on internal developmental cues, perhaps correlated with plant size to signal the time of transition (Lang, 1965; Bernier, 1988; McDaniel et al., 1996). Most plants integrate both environmental and developmental signals to elicit flowering. These environmental factors include photoperiod (i.e., day length), light quality (spectral composition), light quantity (photon flux density), vernalization (exposure to a long period of cold) and various stresses (e.g., nutrient deficiency and water availability). The most significant advances in the dissection of these diverse influences and the molecular events that mediate their effects have been made through the molecular genetic analysis of Arabidopsis. Over the years, two complementary strategies have been used to identify genes or loci that affect flowering time in Arabidopsis. The first exploits the variation in naturally occurring ecotypes of Arabidopsis that differ in flowering time. A number of genes, such as FRIGIDA (FRI), FLOWERING LOCUS C (FLC), FLOWERING KIRUNA (FKR) and various quantitative trait loci (QTLs) have been identified by this approach (reviewed by Koornneef et al., 1998). The second approach is to generate mutations by diverse methods that result in either early or late flowering plants. Multiple mutants and genes that control flowering time have been identified by this approach, from three laboratory progenitor ecotypes, Landsberg erecta (Ler), Columbia (Col) and Wassilewskija (Ws), (reviewed by Martinez-Zapater et al., 1994; Haughn et al., 1995; Weigel, 1995; Amasino, 1996; Hicks et al., 1996; Coupland, 1997; Levy and Dean, 1998; Piñeiro and Coupland, 1998). Altogether, there are currently more than 80 genes and loci in Arabidopsis known to affect flowering time that have been characterized through one or other of these approaches. The study of how these mutants respond to different environmental treatments, combined with the genetic epistasis analysis, has established the existence of at least three pathways involved in the control of floral transition (reviewed by Simpson et al., 1999 and Araki, 2001). As shown in Figure 2, the first is the autonomous pathway that probably monitors endogenous cues from the developmental states. The autonomous pathway promotes the floral transition by reducing the levels of the floral repressor FLC (Michaels and Amasino, 1999). FLC is also negatively regulated by vernalization (reviewed by Reeves and Coupland, 2000). The second is the photoperiodic promotion pathway (or long day promotion pathway), which mainly integrates daylength into the flowering decision through a series of genes that sense and respond to photoperiod. CONSTANS (CO; Putterill et al., 1995) has been suggested to be the key regulator in this pathway. CRY2 (CRYPTOCHROME 2) acts as a blue light receptor within the pathway that increases CO expression under LD conditions (Guo et al., 1998). The third pathway operates primarily in SD, and requires the involvement of the gibberellin (GA) class of phytohormones. Extensive cross-talk among the pathways and integrating genes has been suggested to occur (Samach et al., 2000; Kobayashi et al., 1999; Swarup et al., 1999).
Genes specifying the floral meristem How does the identity of a meristem switch from making leaves to making flowers? Years of study have shown that the determination of floral meristem identity (including the switch of meristem fate from vegetative to floral and the following development of the floral meristem) is controlled by FLIP (floral initiation process) genes. In Arabidopsis, these include LEAFY (LFY), APETALA1 (AP1), APETALA2 (AP2), CAULIFLOWER (CAL), and UNUSUAL FLORAL ORGANS (UFO) (Schultz and Haughn, 1991 and 1993; Weigel et al., 1992; Bowman et al., 1993; Weigel and Meyerowitz, 1994; Kempin et al., 1995; Levin and Meyerowitz, 1995; Ruiz-Garcia et al., 1997). Analyses of single and double mutant phenotypes, gene sequences and expression, and transgenic plants have all provided evidence suggesting that high enough levels of FLIP genes in the floral primordium ensure a complete switch to the floral program (reviewed by Pidkowich et al., 1999). Therefore, the signals that regulate flowering in response to environmental and endogenous cues must act through FLIP genes (such as LFY, shown in Figure 2) to specify the floral fate.
Figure 2. Multiple genetic pathways involved in regulating the transition from the vegetative (dotted regions) to the reproductive (striped regions) phase in Arabidopsis. The autonomous pathway, which is active in both LD and SD, promotes the floral transition by reducing the levels of the floral repressor FLC. Vernalization can directly modulate FLC expression. The photoperiod pathway mediates signals from LD photoperiods and acts via CO. The gibberellin pathway plays an essential role in SD conditions. Regulation of LFY by the photoperiod and gibberellin pathways is mediated by different cis elements on the LFY promoter (Blázquez and Weigel, 2000). JV, AV, ER and LR represent juvenile vegetative, adult vegetative, early reproductive and late reproductive phases, respectively. The figure is adopted from Araki (2001) with kind permission from the author. Light signaling
Photoreceptors Light, as an energy resource exploited in plant photosynthesis, affects all stages of plant growth and development, from germination and de-etiolation to aspects of leaf expansion, stem growth, floral initiation, stomatal opening, phototropism and regulation of gene expression (Chory et al., 1996; Chory, 1997). Plants are sensitive to the quality, quantity, duration and direction of light, through photoreceptors that consist of a light-absorbing pigment, or chromophore, bound to a protein effector molecule, or apoprotein. Absorption of light by the chromophore induces a chemical or conformational change in the receptor apoprotein that is transmitted to photoresponsive downstream genes, which regulate appropriate growth and developmental responses (reviewed by Terzaghi and Cashmore, 1995; Fankhauser and Chory, 1997). Two different types of photoreceptors have been identified (Figure 3). The first are the phytochromes (phy), which absorb primarily in the red and far-red regions of the visible spectrum (600-800 nm). The other type are the cryptochromes (cry) and a photoreceptor named (after the gene that encodes it) 'non-phototropic hypocotyl 1' (nph1), which specifically absorbs blue/UV-A light (350-500 nm). The photoreceptors that detect UV-B light have not yet been characterized at a molecular level.
Phytochromes Five phytochrome genes, PHYA to PHYE have been characterized in Arabidopsis (Clack et al., 1994; Quail et al., 1995). They are reversibly photochromic, existing in two photointerconvertible isoforms: the biologically inactive form, Pr, and the active form, Pfr (Furuya and Song, 1994). Some phytochromes induce distinct light responses, while others stimulate similar responses to each other, but under differing conditions with respect to light quantity, quality, or timing. For example, PHYA seems to have a primary role in germination and in the regulation of seedling Figure 3. Photoreceptors and simplified schematic diagram illustrating proposed photosensory signaling pathways. Blue/UV-A light signals are perceived by the nph1, cry1 and cry2 photoreceptors; red/far-red signals are perceived by the phyA through phyE photoreceptor family. Nph1 seems to be more specifically implicated in phototropism. The remaining photoreceptors control various aspects of photomorphogenesis (germination, de- etiolation, inhibition of hypocotyl elongation, vegetative morphology and initiation of reproductive growth). Crosstalk between the signal transduction pathways linked to the different photochromes and cryptochromes results in synergistic or antagonistic light responses. morphogenesis by far-red light (Parks and Quail, 1993). In contrast, PHYB is an essential component of the shade-avoidance mechanism, and modulates the expression of genes in response to red light (Reed et al., 1993). PHYC, -D and -E appear to have similar photosensory activities to PHYB, but there are also functional differences between PHYB, C, D and E. For instance, PHYC may have a specialized role in controlling primary leaf cell expansion (Qin et al., 1997), while PHYE has a distinct function in regulating internode elongation (Devlin et al., 1998). A few recent discoveries have dramatically improved our understanding of the biochemical function and molecular targets of phytochromes. For example, PIF3, a DNA-binding basic helix-loop-helix (bHLH) protein, has been identified and shown to be mainly involved in phyB signaling (Ni et al., 1998) while HFR1 encodes an atypical bHLH protein, which acts in phyA signal transduction (Fairchild et al., 2000).
Cryptochromes Two cryptochrome genes, CRY1 and CRY2, have been characterized in Arabidopsis (Figure 3). The first Arabidopsis cryptochrome gene (CRY1) was identified by the isolation of the hy4 mutant, which has an elongated hypocotyl in blue light (Koornneef et al., 1980; Ahmad and Cashmore, 1993). CRY1 encodes a protein associated with a flavin chromophore (FAD) that absorbs blue/UV-A light (Lin et al., 1995). The second cryptochrome gene, CRY2, was cloned using CRY1 cDNA as a hybridization probe (Lin et al.,1996a). The amino acid sequences of CRY1 and CRY2 are about 50% identical, most of the sequence similarity being within the N- terminal chromophore-binding domain. CRY1 mediates many blue light responses, including the inhibition of hypocotyl elongation, the accumulation of anthocyanin, the regulation of leaf and cotyledon expansion and the expression of blue light-regulated genes (Short and Briggs, 1994; Ahmad and Cashmore, 1996; Lin et al., 1996b). Interestingly, CRY2 has been shown to affect the timing of reproductive development more strongly than hypocotyl elongation, and the cry2 mutant was recently demonstrated to be allelic to fha (Guo et al., 1998): a photoperiod insensitive late-flowering mutation previously characterized by Koornneef (1991). Studies of chimeric proteins between CRY1 and CRY2 have indicated that they are functionally redundant in blue light induced de- etiolation (Ahmad et al., 1998). However, they differ in photosensory sensitivity; CRY2 is active primarily under low fluence rates, while CRY1 is active under both low and high fluence rates (Lin et al., 1998). In the past few years, considerable progress has been made towards understanding the mechanisms of blue light signaling, but the mode of action of cryptochromes is still poorly defined. Recently, cryptochrome genes have been isolated from various animals, including the fruit fly, mouse and man (Cashmore et al., 1999). Studies of mouse and fruit fly cryptochromes have indicated that these proteins play important roles in the function and regulation of the animal circadian clock (Thresher et al., 1998).
The plant hormone abscisic acid
The phytohormone abscisic acid (ABA) is a sesquiterpenoid for which mevalonic acid is a precursor. It regulates various aspects of plant growth and development, including seed maturation and dormancy, as well as adaptation of vegetative tissues to abiotic environmental stresses such as drought and high salinity (reviewed by Zeevaart and Creelman, 1988). Significant progress towards understanding ABA action has come from the characterization of several mutants that are either defective in ABA biosynthesis or ABA responsiveness. The mutants, aba1-aba3 block the chain of ABA biosynthesis at different steps and their phenotypes can be reversed to wild-type by exogenous addition of ABA (Koornneef et al., 1982; Léon-Kloosterziel et al., 1996; Marin et al., 1996; Schwartz et al., 1997). Unlike biosynthetic mutants, ABA-insensitive (abi1-abi5) and enhanced response to ABA (era1-era3) mutants have alterations in their responsiveness to ABA. They do not have a reduced endogenous ABA content and their phenotypes cannot be reversed to wild-type by exogenous supply of ABA (Koornneef et al., 1984; Finkelstein, 1994; Cutler et al., 1996). Thus, such response mutants can be used to study components of ABA signal transduction cascades and to unravel their pathways (reviewed by Merlot and Giraudat, 1997). Phenotypically, the abi1, abi2 and abi3 mutants display marked reductions in seed dormancy and abi3, abi4 and abi5 mutants exhibit defects in various aspects of seed maturation (Koornneef et al., 1984; Finkelstein, 1994; Nambara et al., 1995 and 2000). Aside from the seed-specific defects, the abi1 and abi2 mutants also clearly affect ABA responses in vegetative tissues. The ABI1 to ABI5 genes have been cloned. The ABI1 and ABI2 genes encode similar class 2C serine/threonine protein phosphatases (Leung et al., 1994 and 1997; Meyer et al., 1994 and Rodriguez et al., 1998). ABI3, ABI4 and ABI5 encode putative transcriptional regulators. ABI3 is orthologous to the maize Viviparous 1 protein (McCarty et al., 1991 and Giraudat et al., 1992), ABI4 contains an APETALA2-like DNA binding domain (Finkelstein et al., 1998) and ABI5 is a member of the basic leucine zipper transcription factor family (Finkelstein and Lynch, 2000). Many of the actions of ABA, in both seeds and vegetative tissues, involve modifications of gene expression at the transcriptional level. A number of genes, for which the expression is regulated by ABA, have been characterized and some of them can also be regulated by various adverse environmental stimuli in either an ABA-dependent or ABA-independent manner (Chandler and Robertson, 1994; Ishitani et al., 1997). Studies of these genes give important clues to the nature of the ABA signal transduction pathway. It is becoming clear that ABA biosynthesis and signal transduction are not simple linear pathways, but rather they involve complex, ramified and redundant branches. In addition, ABA often acts in conjunction with other hormones or stimuli. HDZip transcription factors
Homeobox genes A homeobox is a 180 base pair sequence motif, which encodes a 60 amino acid domain called the homeodomain. Homeodomain-containing proteins act as transcription factors; they bind to specific sequences of DNA and regulate the expression of target genes (reviewed by Bürglin, 1994). Determination of the three- dimensional structure of the homeodomain, by nuclear magnetic resonance (NMR) spectroscopy (Qian et al., 1989; Billeter et al., 1990), has shown that it contains three α -helices, of which helix 1 is separated from helix 2 by a loop whereas helix 2 and 3 are separated by a turn. Study of the structure of the homeodomain-DNA complex by X-ray crystallography has revealed that helix 3 is positioned in the major groove of the DNA and interacts sequence specifically with the bases of the DNA. Amino acid residues of the N-terminal extension of the homeodomain make additional base- contacts in the minor groove of DNA, while 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. Homeobox containing genes were first identified from Drosophila melanogaster, and then from other animals and plants. Drosophila homeobox genes have been demonstrated to be important in homeotic transformations during embryogenesis (McGinnis et al., 1984; Scott and Weiner, 1984). The first homeobox gene identified from plants was KNOTTED-1 in maize (Vollbrecht et al., 1991). KNOTTED-1 was shown to play an important role in shoot apical meristem maintenance during plant development (Smith et al., 1992).
HDZip genes Homeobox containing genes from Arabidopsis (Arabidopsis thaliana homeobox genes, or ATHB genes) have been isolated by screening Arabidopsis cDNA libraries with a degenerate oligonucleotide probe, designed to match all possible codons of the most conserved part of helix 3 of the Drosophila Antp gene (Ruberti et al., 1991; Mattsson et al., 1992; Schena and Davies, 1992). The genes obtained from these screens were found to contain not only a sequence encoding the homeodomain, but also a sequence encoding a leucine-zipper domain (referred to as HDZip). The leucine-zipper domain is characterized by a periodic repetition of leucine residues at every seventh position and is closely linked to the C-terminal of the homeodomain. Subsequently, many more HDZip genes were isolated from Arabidopsis on the basis of sequence similarity. By the completion of the genome sequence, 42 HDZip genes had been identified in Arabidopsis (Arabidopsis Genome Initiative, 2000). They are grouped in four different families, HDZip I – IV, based on sequence criteria (Sessa et al., 1994). The genes of HDZip families I and II appear to share a common origin while genes belonging to HDZip families III and IV are more distantly related. HDZip genes have been found in many other plant species as well, such as tomato (Meissner and Theres, 1995), sunflower (Chan and Gonzalez, 1994) and rice (Meijer et al., 1997). As yet, no HDZip genes have been identified in animals, indicating that this group of genes is most likely plant specific.
DNA binding and dimerization properties of HDZip proteins Since the leucine-zipper motif is known to mediate protein dimerization in other classes of transcription factors, this structural feature of the HDZip proteins suggest that they bind DNA as protein dimers (a hypothetical model is shown in Figure 4). This hypothesis was confirmed by in vitro DNA-binding studies of ATHB1 (HDZip I) and ATHB2 (HDZip II). 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). Later, ATHB9 (HDZip III) was demonstrated to bind specifically to the DNA sequence GTAAT(G/C)ATTAC (Sessa et al., 1998). ATHB10 (HDZip IV) interacts with DNA in a similar fashion to that of the ATHB2 protein (Di Cristina et al., 1996). All four classes of HDZip proteins in Arabidopsis seem to exhibit a similar mode of DNA binding. In addition, two HDZip II proteins from other plants, Oshox1 from rice and CPHB-1 from the resurrection plant Craterostigma plantagineum, have been shown to bind DNA with specificities similar to that of the Arabidopsis HDZip II protein, ATHB2 (Meijer et al., 1997; Frank et al., 1998).
Figure 4. A hypothetical model of a HDZip protein binding to DNA. A model (Söderman, 1996) based on the three-dimensional structures of the Drosophila ENGRAILED homeodomain (Kissinger et al., 1990) and the yeast GCN4 leucine zipper motif (Ellenberger et al., 1992). Protein-protein interaction studies in vitro have shown that ATHB2 (HDZip II) could not form a heterodimer with ATHB1 (HDZip I, Sessa et al., 1993), but could heterodimerize with Oshox1 (HDZip II) from rice (Meijer et al., 1997). Similarly, two sunflower HDZip proteins, Hahb-1 and Hahb-10, which belong to HDZip families I and II, respectively, were only capable of homodimerization, and were unable to interact with each other (Gonzalez et al., 1997). These data suggest that heterodimerisation is possible between members of the same class of HDZip, but not between proteins of different classes.
Functions of HDZip genes HDZip genes have been shown to be involved in a wide range of processes in plant growth and development. The Arabidopsis HDZip I gene ATHB1 is associated with leaf development (Aoyama et al., 1995) and ATHB7, ATHB12 and ATHB6 have been reported to be transcriptionally activated by addition of exogenous ABA, water deficit and osmotic stress conditions (Söderman et al., 1996 and 1999; Lee and Chun, 1998). The expression of the HDZip II gene ATHB2 is strongly induced by far-red light (Carabelli et al., 1993 and 1996). This expression pattern, together with evidence from analysis of transgenic plants, suggests that ATHB2 plays a developmental role in the auxin-dependent shade avoidance response (Steindler et al., 1999). The HDZip III genes ATHB8 and INTERFASCICULAR FIBERLESS1 (IFI1) have roles in the differentiation of the vascular system and interfascicular fibers, respectively (Baima et al., 1995; Zhong and Ye, 1999). The HDZip IV gene ATHB10 is identical to the GLABRA2 (GL2) gene (Rerie et al., 1994; Di Cristina et al., 1996), which is required for both normal trichome differentiation and for the production of root hairs (Masucci et al., 1996). Another HDZip IV gene, ANTHOCYANINLESS2 (ANL2) affects anthocyanin accumulation and root development in Arabidopsis (Kubo et al., 1999). In other species, CPHB1 and CPHB2, two HDZip II genes from the resurrection plant Craterostigma plantagineum, are regulated by drought and may be involved in gene regulation in response to drought (Frank et al., 1998). H52, a HDZip I gene from tomato, is involved in limiting the spread of programmed cell death (Mayda et al., 1999). The rice HDZip II gene, Oshox1, has been suggested to have a role in the control of leaf morphogenesis (Meijer et al., 1997). In this thesis, the main focus of attention is the function of a novel Arabidopsis HDZip I gene, ATHB16. RESULTS AND DISCUSSION
Identification of ATHB16, a new member of the HDZip I family (I and IV) An EST clone, 72B4T7 (GenBank accession number R 86816), was found in a search for sequences with similarities to previously identified HDZip I genes. By use of RACE, the total transcript length was determined to be 1410 nucleotides, encoding a protein of 294 amino acids. The deduced amino acid sequence contains a stretch of 60 residues, which shows a distinct similarity to homeodomains from other proteins and has a putative leucine zipper motif with five leucines and 1 isoleucine in every seventh position, C-terminal to the homeodomain. It was shown to be a HDZip protein that had not been previously characterized, which we refer to as ATHB16 (Paper I).
Comparison of the amino acid sequence of ATHB16 to other Arabidopsis HDZip proteins showed that it has extensive similarity to a specific HDZip I protein, ATHB6 (GenBank accession number AF 104900). ATHB16 and ATHB6 share 93% amino acid identity over the homeodomain and 73% amino acid identity over the full length of the proteins.
In an experiment in which the ATHB16 cDNA was used as probe to screen an Arabidopsis genomic library, the ATHB16 gene was shown to exist as a single copy in the genome and it was mapped to the bottom of chromosome IV. Comparison of the ATHB16 cDNA sequence with that of the genomic clone showed that the ATHB16 open reading frame is split by two introns: one located upstream of the homeobox, and the other downstream of the homeobox. Introns at identical positions in relation to the homeobox are found in most of the HDZip I genes (Paper IV).
Expression of ATHB16 in wild-type plants (I and II)
Northern blot experiments showed that ATHB16 is expressed in all organs tested in Arabidopsis (Paper I). Expression of ATHB16 was strong in leaves, intermediate in adult roots and inflorescences, and low in the stem and siliques (Paper I). Analysis of the spatial expression pattern of ATHB16 using the reporter gene GUS (Paper II) revealed that strong expression of ATHB16 is localised in leaf primordia of seedlings, adult leaves, flower buds, the apical part of carpels, receptacles and expanding lateral roots. In addition, GUS staining was evident in the vascular strand of cotyledons, pedicels, sepals and petals, as well as in the funiculus of siliques. In contrast, GUS activity was undetectable in root tips, lateral root primordia, stamens, stigma and embryos under normal growth conditions.
Expression of ATHB16 is upregulated by abscisic acid in the root (II)
The staining intensity in roots of seedlings expressing transgenic ATHB16::GUS constructs is weak, but can be markedly increased by exogenous abscisic acid (ABA) treatment. This effect is specific to the root, since no altered staining pattern or intensity was observed in leaves, cotyledons or hypocotyls after ABA treatment (Paper II).
The ATHB16 transcript level in the root was found to be upregulated 3-4 fold by exogenous abscisic acid (ABA) in northern blot analysis, which is consistent with the ATHB16::GUS transgene expression analysis. Further analysis of ATHB16 expression levels in response to ABA in the ABA-insensitive mutants, abi1, abi2 and abi3, showed that its induction requires the ABI1 and ABI2 proteins and is partially dependent on ABI3. These results indicate that the regulation of ATHB16 expression is dependent on a functional ABA signal transduction pathway.
Like ATHB16, three other HDZip I genes, ATHB6 (Söderman et al., 1999), ATHB7 (Söderman et al., 1996), and ATHB12 (Lee and Chun, 1998) have been shown to be transcriptionally activated by exogenous ABA. Expression of ATHB6 requires the ABI1 and ABI2 gene products, but unlike ATHB16, ATHB6 is independent of ABI3 for its transcriptional activation. ATHB7 requires only the ABI1 gene product and is thus independent of ABI2 and ABI3 for its transcriptional activation. Whether ATHB12 activation is dependent on any of these ABA signaling genes is not known. Thus, the HDZip I gene family includes genes participating in partly overlapping pathways of ABA response mechanisms.
Alteration of ATHB16 expression levels in transgenic plants (I) To assess the function of the ATHB16 gene, we generated two sets of transgenic Arabidopsis plants with elevated and reduced levels of gene activity by expressing the gene in sense and antisense orientations, respectively, from the strong constitutive promoter of the cauliflower mosaic virus (CaMV) 35S gene. The phenotypic effects observed in plants with elevated and reduced levels of ATHB16 expression are reciprocal, indicating that they directly reflect the function of ATHB16 in the wild-type plants. Moreover, plants that simultaneously expressed both ATHB16 cDNA and ATHB16 antisense cDNA had further reductions in ATHB16 transcript levels, and enhanced phenotypic deviations, compared to the ATHB16 antisense plants. Phenotypically, changes in expression levels of ATHB16 were found to be associated with alterations in flowering time and defects in leaf expansion and shoot elongation. Each of these responses is discussed below.
ATHB16 reduces the sensitivity of the flowering response to photoperiod (I) Arabidopsis is a facultative long-day plant, meaning that LD promotes flowering and SD delays it. In our growth conditions, the flowering of wild-type plants was delayed by a period of three to 11 weeks when grown in SD. Transgenic plants with increased levels of ATHB16 expression (35S::ATHB16 plants) flowered later in LD, but earlier in SD, compared to wild-type, with the result that the flowering of 35S::ATHB16 plants was delayed only 5.5 to 10 weeks by SD. In contrast, transgenic plants with decreased levels of ATHB16 expression (i.e. 35S::antiATHB16 plants and the plants simultaneously expressing both the 35S::ATHB16 and 35S::antiATHB16 constructs) flowered later in SD, compared to wild-type. For instance, flowering in the 35S::antiATHB16 plants was delayed three to 12-13 weeks by SD. These reciprocal effects of elevated and reduced ATHB16 levels on flowering time strongly suggest that ATHB16 reduces the sensitivity of the flowering response to photoperiod in wild-type plants. Gibberellins (GAs) have been shown to be important factors in flower induction in Arabidopsis, especially under SD conditions (Wilson et al., 1992). To test whether ATHB16 influences flowering time by affecting GA signaling, exogenous GA was sprayed repeatedly on wild-type and ATHB16 transgenic plants in SD conditions. The response of 35S::antiATHB16 plants to exogenous GA was like that of the wild- type. This result indicates that the effects of alterations in ATHB16 levels on flowering time are not due to changes in the responsiveness of the plant’s GA signal transduction pathways. Instead, the ATHB16 protein may act as a negative regulator of photoperiodic sensitivity, as part of the photoperiodic promotion pathway (LD promotion pathway) in the regulation of the transition from vegetative to reproductive development.
ATHB16 causes pleiotropic effects on plant growth and development (I) In addition to its effects on flowering time, ATHB16 also affects leaf and stem growth. Elevated levels of ATHB16 expression (in the 35S::ATHB16 plants) resulted in a dwarf phenotype with a reduced size of almost every organ under both LD and SD conditions, e.g. smaller rosette leaves, shorter internodes, smaller flowers and siliques, and delayed senescence. Further anatomical study revealed that the smaller size of the rosette is mainly due to leaf cells being smaller than wild-type, since the relative difference in leaf epidermal cell size between the 35S::ATHB16 and wild- type plants was similar to the relative difference in leaf size between the plants. In contrast, reduced levels of ATHB16 expression (in 35S::antiATHB16 plants) led to larger rosette leaves as compared to wild-type plants in LD. The relative difference in leaf epidermal cell size between the 35S::antiATHB16 and wild-type plants was similar to the relative difference in leaf size between the plants. These data indicate that ATHB16 may affect the size of the leaves and stems by affecting cell expansion in the organs. The reciprocal character of the effects caused by elevated and reduced levels of ATHB16 expression indicates that the gene acts as a regulator of cell expansion in wild-type plants.
ATHB16 affects the blue light sensitivity of the plant (I) Under white light, red light, far-red light and darkness, ATHB16 transgenic plants (both 35S::ATHB16 and 35S::antiATHB16) showed no distinguishable phenotypic differences at the seedling stage from wild-type seedlings. However, under blue light, the seedlings with reduced levels of ATHB16 expression (35S::antiATHB16 plants) had longer hypocotyls than the wild-type seedlings. Furthermore, this effect was quantitatively dependent on the degree of the reduction in gene expression, since plants that simultaneously expressed both the 35S::ATHB16 and 35S::antiATHB16 constructs showed an even greater reduction in transcript levels and stronger phenotypic deviation from wild-type. In contrast, the seedlings with increased levels of ATHB16 expression (35S::ATHB16 plants) had a shorter hypocotyl than wild-type seedlings in blue light. This finding indicates that ATHB16 is a positive regulator of blue light-dependent inhibition of hypocotyl growth. Thus, ATHB16 may specifically affect the response of the plant to blue light.
ATHB16 may mediate light effects on plant growth (I) The defects in organ development observed in ATHB16 transgenic plants are consistent with the photoresponse affecting hypocotyl elongation, which implies that the effect of ATHB16 on plant growth might be mediated by changes in sensitivity to light. This hypothesis can be assessed by considering several additional lines of evidence. First, plants with elevated levels of ATHB16 expression (35S::ATHB16 plants) displayed a significantly higher level of anthocyanin pigments under blue light compared with wild-type plants and plants with reduced levels of ATHB16 expression (our unpublished observations). The anthocyanin accumulation process is known to be induced by blue light (Feinbaum et al., 1991), suggesting that blue light sensitivity is increased in plants with elevated levels of ATHB16 expression. Second, plants with elevated levels of ATHB16 expression (35S::ATHB16 plants) showed a general increase in sensitivity to light. For instance, seedlings germinated in darkness did not survive after transfer to white light conditions, whereas wild-type plants and plants with reduced levels of ATHB16 expression survived and developed normally (our unpublished observations). Third, like ATHB16, high levels of expression of the blue light receptor CRY1, caused a reduction in the size of the leaves and inflorescence stems (Lin et al., 1996b). Moreover, the effects of ATHB16 on leaf size were not affected by exogenous treatment with indole-3-acetic acid (IAA), GA3, kinetin or epibrassinolide, the plant hormones known to affect cell expansion. Thus, it is unlikely that the role of ATHB16 is related to the function of any of these hormones as regulators of cell expansion. Taken together, these data suggest that ATHB16 may mediate light effects on organ development.
ATHB16 mediates blue/red light-dependent regulation of flowering (I) The effect of ATHB16 on flowering time is unlikely to be an indirect consequence of the difference in leaf size, since altered levels of expression of ATHB16 have similar effects on leaf size in LD and SD, whereas the effects on flowering time differ between these conditions. To investigate whether the effect of ATHB16 on flowering time was dependent on the spectral qualities of the light, we analyzed the flowering time of 35S::ATHB16 and wild-type plants grown in continuous white, blue and red light. We found that the flowering of 35S::ATHB16 plants is delayed compared to that of corresponding wild-type plants under white light, as well as under blue and red light conditions. This suggests that ATHB16 may mediate the blue and red light signaling that regulates floral initiation.
Blue light promotes and red light inhibits flowering (Eskins, 1992), and the signaling pathways initiated by blue and red light interact with each other in controlling the timing of flowering, as demonstrated in comparative studies on wild-type Arabidopsis plants and a blue light photoreceptor mutant, cryptochrome 2 (cry2). The cry2 mutant flowers late in white light, but not in blue or red light, and the delayed flowering in white light can be completely phenocopied by growing cry2 under blue-plus-red light (Guo et al., 1998). These results indicate that the regulation of floral induction is mediated by the antagonistic actions of cry2 and the red light receptor phyB (Mockler et al., 1999). Recent studies have confirmed that genetic interaction between cry2 and phyB is involved in the control of flowering time and that cry2 and phyB physically interact in the nucleus (Más et al., 2000). Possibly, ATHB16 may act downstream to both CRY2 and PHYB in the regulation of flowering time. However, exactly how ATHB16 regulates photoperiodic flowering remains to be elucidated.
Isolation and identification of a T-DNA insertion line in ATHB16 (II) An alternative strategy used to study ATHB16 function was to identify knock-out alleles of ATHB16. Several T-DNA and transposon insertion lines were screened for insertions in the ATHB16 gene. In the T-DNA collection in the Ws-2 ecotype from the Arabidopsis Knock-out Facility (Krysan et al., 1999), we identified one insertion in the untranslated leader of the ATHB16 gene, 2 bp downstream of the transcriptional initiation site (this allele was denoted athb16-1). The athb16-1 line may represent a loss-of-function mutant of ATHB16 since a very low abundance of ATHB16 transcript was detected in this line by northern blot analysis. Interestingly, it did not confer a phenotype that was morphologically distinct from the Ws-2 wild- type at any developmental stage under normal LD growth conditions. However, when ABA was exogenously applied, athb16-1 displayed a reduced sensitivity to ABA inhibition of root growth.
Judging from the phenotypes of the ATHB16 transgenic plants, we would expect athb16-1 to generate similar or more severe phenotypic deviations than those seen in the ATHB16 antisense plants, e.g. a longer hypocotyl under blue light and larger rosettes in LD. One explanation for the lack of a loss-of-function phenotype in the athb16-1 mutant is that athb16-1 is a mutant form of the Ws-2 ecotype, whereas the transgenic lines described above were generated in the Col-0 ecotype. More than 20 ATHB16 antisense transgenic lines generated in the Ws-2 background were screened and none of them showed any distinguishable phenotype as compared to the Ws-2 wild-type (our unpublished results).
The Ws-2 ecotype has been demonstrated to contain a naturally occurring mutation within the PHYD gene (Aukerman et al., 1997). The monogenic phyD mutant created by introgression of the Ws PHYD gene into the Landsberg erecta ecotype showed increased hypocotyl elongation and decreased cotyledon expansion under white light conditions, in comparison with the wild-type. In addition, phyB/phyD double mutants displayed an enhanced reduction in leaf area relative to the phyB monogenic mutant (Aukerman et al., 1997; Devlin et al., 1999), suggesting that PHYD has a role in leaf expansion. Probably, the morphological deviation from the wild-type caused by the mutation of ATHB16 in the athb16-1 line was masked by the effect of the mutation in the PHYD gene. We therefore believe that ATHB16 and PHYD genes may act epistatically in a light-signaling pathway. However, we cannot exclude the possibility that other genomic differences between Ws-2 and Col resulted in the lack of phenotypic deviations in the athb16-1 line. This hypothesis could be tested in studies involving introgression of the T-DNA insertion in the ATHB16 gene from the athb16-1 line into the Col ecotype, or further screening of other T-DNA and transposon insertion collections in order to generate additional insertion lines of ATHB16 in different ecotypes.
Alternatively, the lack of a discernible athb16-1 phenotype could be explained by ATHB16 being functionally redundant to other genes. Considering the highly conserved homeodomain and leucine-zipper domain of the HDZip proteins, together with their similar DNA-binding properties (Paper III), it is very likely that the HDZip genes act redundantly (Paper IV). Thus, other HDZip genes in Arabidopsis might compensate for the loss of ATHB16 activity in athb16-1. It is possible that the overlapping functions of HDZip proteins provide a mechanism whereby alteration of ATHB16 levels in the plant could also affect the expression of other HDZip genes. Hence, the phenotypic changes observed in ATHB16 transgenic plants may be influenced by changes in the expression not only of ATHB16, but also of other HDZip genes. To test this possibility, we examined transcript levels of ATHB6, which has the highest sequence similarity to ATHB16, in ATHB16 transgenic plants (unpublished data). We found similar to wild-type levels of ATHB6 mRNA in transgenic plants with both elevated and reduced levels of ATHB16 expression. These results indicate that the phenotypic changes observed in ATHB16 transgenic plants are probably not caused by an alteration in ATHB6 levels. However, since northern blots have limited sensitivity to quantitative changes, the possibility that a certain percentage of variation was present, e.g., 20-30%, in ATHB6 transcript levels could not be excluded. Our data on ATHB16 show that increased and decreased levels of expression of the gene cause reciprocal phenotypic effects, both on photoperiodic flowering and on cell expansion (Paper I). These findings provide a strong indication that the observed effects directly reflect the function of the gene in the wild-type plant, rather than being indirect results of ATHB16 artificially interfering with the function of a second HDZip gene.
ATHB16 affects sensitivity to ABA in a root growth assay (II) The expression of ATHB16 in roots is upregulated by abscisic acid (ABA), suggesting that ATHB16 may be involved in the ABA responses of the plants. To test this hypothesis, we examined the root growth of 35S::ATHB16, athb16-1 and wild- type seedlings grown on media containing 0, 10 or 100 µM ABA. The results showed that the root growth of athb16-1 seedlings was less strongly inhibited by ABA than the corresponding wild-type seedlings. In contrast, the 35S::ATHB16 seedlings were hypersensitive to ABA inhibition of root growth compared to its corresponding wild-type. These results strongly suggest that ATHB16 has a role in ABA-mediated regulation of root growth. To investigate whether ATHB16 affects other ABA responses, we compared the seed germination of 35S::ATHB16, athb16-1 and wild-type plants grown on media containing different concentrations of ABA. The results showed that the germination of seeds from both athb16-1 and 35S::ATHB16 plants have the same sensitivity to ABA as their wild-type counterparts, suggesting that ATHB16 activity may not affect ABA sensitivity during seed germination. Thus, ATHB16 may have a specific function in the regulation of root growth in response to environmental stimuli, such as drought. This would be consistent with the root-specific effects of ABA on ATHB16 expression noted above.
Identification of HDZip I target sequences in vitro (III) DNA binding studies of an HDZip I protein (ATHB1) and an HDZip II protein (ATHB2) showed that they interact with nearly identical pseudopalindromic sequences, CAAT(A/T)ATTG and CAAT(G/C)ATTG, respectively (Sessa et al., 1993). The principal difference between the two sites is in the preferred bases at the central position. To extend this study, we analyzed the binding specificity for a number of HDZip I proteins. Initially, the purified recombinant ATHB5 protein (HDZip I) was used to select high- affinity binding sites from a pool of DNA-fragments containing a 12 bp random core sequence. The results showed that ATHB5 binds to the pseudopalindromic sequences CAAT(A/T)ATTG and CAAT(G/C)ATTG, in a similar fashion to ATHB1. However, in contrast to ATHB1, ATHB5’s binding is non-specific with respect to the base pair in the central position, since the frequencies of recovered subclones with A/T and G/C pairs in the central position were essentially identical. Subsequently, more HDZip I proteins were analyzed with respect to their DNA binding specificity by EMSA experiments using in vitro translated proteins and synthetic oligonucleotides containing either the CAAT(A/T)ATTG or the CAAT(G/C)ATTG core. The results showed that ATHB6 and ATHB16, like ATHB5, interacted with similar affinity to either A/T or G/C containing sequences, whereas ATHB3 and ATHB13 exhibited a slight preference for the A/T pair in the central position. Taken together, these data suggest that HDZip I proteins interact with very similar binding sites, but that central position specificity appears to vary within the HDZip I class. Interestingly, ATHB7 and ATHB12 did not interact with these binding sites containing either an A/T or a G/C pair in the central position. One possible explanation is that the ability of the ATHB7 and ATHB12 proteins to bind DNA is dependent on correct post-translational modifications, such as phosphorylation or destabilization. Another possibility is that ATHB7 and ATHB12 interact with different binding-sites from those of other HDZip I proteins.
Heterodimer formation between HDZip I proteins (II and III) The HDZip genes contain a DNA binding domain linked to a dimerization domain, which allows them to interact with each other (Sessa et al., 1993). We made use of an in vitro protein-binding assay to analyze the dimerization properties of ATHB5. The results showed that in addition to forming homodimers, ATHB5 could form heterodimers with ATHB6, -7, -12 and -16, with differing apparent affinities, but not with ATHB1. This suggests that HDZip I proteins show selectivity in dimer formation (Paper III). The ability of dimers to form between different HDZip I proteins was tested in vivo using the yeast two-hybrid interaction cloning protocol (Fields and Song, 1989). A sequence encoding a C-terminally truncated ATHB7, which lacked the ability to activate transcription (Jin-Long Qiu, unpublished results) was fused to the sequence encoding the GAL4 DNA-binding domain and used as bait. A set of target plasmids was prepared, which each contained a sequence encoding the GAL4 activation domain and a full-length copy of a gene encoding one of the following: ATHB1, ATHB3 (Mattsson et al., 1992), ATHB6, ATHB7, ATHB13 (Hanson, 2000), ATHB16 or the HDZip II protein, ATHB2. These target plasmids were then each used, separately, to co-transform yeast together with the bait plasmid. Growth of the resulting yeast transformants on media without histidine (-LTH media) and expression of the LacZ reporter gene was then tested. The results showed that ATHB7 could interact with ATHB6 and ATHB16, but not with ATHB1, ATHB3 or ATHB13, indicating that heterodimer formation between members of class I HDZip proteins in vivo is selective (Jin-Long Qiu, unpublished results). ATHB7 could not interact with the tested HDZip II-ATHB2 combination, which is consistent with the hypothesis that no heterodimerisation occurs between proteins of the HDZip I and II classes (Meijer et al., 2000). The ability of ATHB16 and ATHB6 to form heterodimers was also tested in yeast, and the results showed that these two proteins could interact each other (Paper II). The high similarity of in vitro DNA binding properties of most HDZip I and II proteins implies that their functional specificity in vivo might be dependent on dimer formation. The selective heterodimer formation between HDZip I proteins may be of great functional significance, since it might provide the plant with a means for integrating information from several different input signals, such as light of different spectral qualities and water availability, in the regulation of plant growth. This is particularly interesting since ATHB6, ATHB7 and ATHB16, which have been demonstrated to be capable of forming heterodimers with each other, are all involved in ABA transduction pathways in different fashions (Söderman et al., 1996 and 1999; Paper II). The ability to form specific heterodimers between pairs of ATHB6, ATHB7 or ATHB16 gene products could thus increase the level of complexity in transcriptional regulation at the protein level in the ABA signaling pathways. On the other hand, the functional complex of homodimeric ATHB16, ATHB6 or ATHB7 could have distinct activation characteristics without the involvement of any heterodimers. CONCLUDING REMARKS
This thesis describes the characterization and functional analysis of an Arabidopsis HDZip I gene, ATHB16, and the biochemical properties of HDZip proteins. The ATHB16 gene was shown to mediate effects of blue and red light on the timing of the floral transition and on plant growth, and also to affect the sensitivity of seedling root growth to inhibition by exogenous ABA. The information obtained on the expression pattern of ATHB16 shows there is a direct link between expression of the gene and its functions, providing evidence that ATHB16 has dual functions dependent on both light and ABA. It is becoming increasingly clear that the light and ABA signaling pathways interact in the regulation of gene expression and plant development (Weatherwax et al., 1996; Rohde et al., 2000). Therefore, the dual role of ATHB16 might be explained by ATHB16 acting on a focal point of ABA and light signal cascades. However, the underlying molecular mechanism is unclear. The biochemical studies on HDZip proteins suggested that they exert their effects as dimers. The selective heterodimer formation between HDZip proteins may be of functional significance in the integration of information from different signaling pathways in the control of plant development. Thus, we suggest that ATHB16 may interact with ATHB6 or ATHB7 in mediating the adaptive responses of plants to drought, whereas the ATHB16 homodimer may function as a mediator of light effects on plant growth and development. In summary, HDZip transcription factors constitute a large protein family that is apparently unique to plants. In Arabidopsis alone, 42 HDZip proteins have been identified, 26 of which belong to subclasses I and II of the HDZip family (Paper IV). The functional information available on HDZip I and II genes indicates that at least some of the genes mediate the effects of external factors on plant growth and development. Since HDZip I and II proteins have very similar DNA binding specificities, we believe that HDZip I and II proteins may be involved in a complex network of interacting transcription factors in the plant. ACKNOWLEDGEMENTS
I would like to express my sincere gratitude to all of the people who contributed to the completion of this work in various ways. Thank you all. I would like to express my warmest thanks especially to:
Professor Peter Engström, my supervisor, for accepting me as a PhD student and for introducing me to the field of plant molecular biology. I extend special thanks to him for reading and correcting manuscripts of my papers and this thesis.
Professor Stellan Hjertén, Department of Biochemistry, Uppsala University, for opening the Swedish scientific gates for me. What I learned in his lab has been of great benefit to my career.
Eva Sundberg, Eva Söderman and Kerstin Nordin Henriksson for valuable discussion and critical reviews of this thesis.
Henrik Johannesson, Johannes Hanson, Mattias Hjellström for struggling together with me in the HDZip project and for many helpful discussions.
All the PhD students in “Cellskapet”: Alessia, Anna, Annelie, Eva, Henrik, Ingela, Jens, Joel, Johannes, Katarina, Mats, Mattias and Sandra, for scientific discussions, collaborations and for the nice parties we had.
The members of the "Prof lab", Henrik, Jens and Mats, for providing interesting talks and creating a nice atmosphere in the lab. I won't forget the beautiful view of "botaniska trädgården" from my bench.
Johannes, Jens, Pia and Joel for invaluable help in solving computer problems.
Birgitta for excellent secretarial assistance.
John Blackwell for corrections of this manuscript. Stefan Gunnarsson for help with image processing.
Marie Englund for being a nice roommate and doing in-situ hybridization work on ATHB16, hopefully we might interpret the data later. Agneta Ottosson for excellent skill in plant transformation and assistance in caring for the plants. Marie Lindersson for sequencing work and helping in various ways. Caisa Pöntinen for making lots of growth media. Afsaneh Ahmadzadeh and Eva Büren for sequencing and Gun-Britt Berglund for technical assistance during my busiest time. Anita and her family for caring, providing opportunities for me to learn Swedish culture and providing kind help in many different ways.
My former colleagues: Kate Wilson for being a close friend and many kinds of help inside and outside the lab; Jinlong Qiu for trusting me and the nice time we had together; Anika Sundås for valuable discussion of my "strange" results.
The former and present members of Professor Lindbrad's group and of the Department of Comparative Physiology, for being nice neighbors.
The foundations of Liljewalch, Rector and Kungl.Skogs-och Lantbruksakademien for travel expenses.
Thank to my friends Ronnie Zhang/Jiali Liao, Hongbin Henricsson, Aijie Liu/Tao Weitao, Jinping Li/Xiao Zhang, Fanyi Jiang/Paul Xie, Ailing Zhang/Kui Huang, Yijia Liu/ Yimin Li, Hong Li/Yinong Rao, Zhiping Ren, Wei Shi and many others for your warm friendship and for sharing joys during my years in Uppsala.
The members of "Sernanders" group: Linhong Weng, Lumin Liu, Zhiqiang Cui, Wansheng Liu, Chengming Zeng and others for sharing the experience of life in Sweden and for the fun we had together.
Javier Martinéz for understanding and friendship. Ellen /Volker Ziemann and Magdalena Gunnarsdotter for sharing nice times together.
Thanks to my parents, for endless love, encouragement, understanding and taking care of Wei. To my sister for forgiving my absence and taking care of our parents, and for being the best of friends all the time. I miss you all. Thanks to my parents-in- law for caring and treating me as their daughter.
Finally, to Yanguo and Wei for love, support and for always being there for me. REFERENCES
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