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Keywords:

  • abiotic stress;
  • HD-Zip;
  • phytohormone;
  • plant development;
  • transcription factor

Abstract

  1. Top of page
  2. Abstract
  3. I. Introduction
  4. II. The role of HD-Zip transcription factors in plant growth adaptation to environmental changes and the phytohormone network
  5. III. Dissecting the common cis element, dimerization and cell specificity of HD-Zip I and HD-Zip II transcription factors
  6. IV. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

Contents

 Summary823
I.Introduction824
II.The role of HD-Zip transcription factors in plant growth adaptation to environmental changes and the phytohormone network825
III.Dissecting the common cis element, dimerization and cell specificity of HD-Zip I and HD-Zip II transcription factors830
IV.Conclusions834
 Acknowledgements834
 References834

Summary

Plant development is adapted to changing environmental conditions for optimizing growth. This developmental adaptation is influenced by signals from the environment, which act as stimuli and may include submergence and fluctuations in water status, light conditions, nutrient status, temperature and the concentrations of toxic compounds. The homeodomain-leucine zipper (HD-Zip) I and HD-Zip II transcription factor networks regulate these plant growth adaptation responses through integration of developmental and environmental cues. Evidence is emerging that these transcription factors are integrated with phytohormone-regulated developmental networks, enabling environmental stimuli to influence the genetically preprogrammed developmental progression. Dependent on the prevailing conditions, adaptation of mature and nascent organs is controlled by HD-Zip I and HD-Zip II transcription factors through suppression or promotion of cell multiplication, differentiation and expansion to regulate targeted growth. In vitro assays have shown that, within family I or family II, homo- and/or heterodimerization between leucine zipper domains is a prerequisite for DNA binding. Further, both families bind similar 9-bp pseudopalindromic cis elements, CAATNATTG, under in vitro conditions. However, the mechanisms that regulate the transcriptional activity of HD-Zip I and HD-Zip II transcription factors in vivo are largely unknown. The in planta implications of these protein–protein associations and the similarities in cis element binding are not clear.


I. Introduction

  1. Top of page
  2. Abstract
  3. I. Introduction
  4. II. The role of HD-Zip transcription factors in plant growth adaptation to environmental changes and the phytohormone network
  5. III. Dissecting the common cis element, dimerization and cell specificity of HD-Zip I and HD-Zip II transcription factors
  6. IV. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

1. Plant development is adapted to the environment

Plants face many environmental challenges during development, and they must optimize growth to adapt to the prevailing circumstances. They may also be limited by the availability of resources in their immediate environment. Consequently, plants are sensitive to these abiotic stresses and adapt their growth to variations in water status, light conditions, nutrient status, temperature, concentrations of toxic compounds and submergence. These developmental adaptations are stimulated by signals from the environment (Sultan, 2010) and involve regulation of cell multiplication, differentiation and expansion through the action of phytohormones (Wolters & Jürgens, 2009; Perrot-Rechenmann, 2010; Skirycz & Inzé, 2010). Phytohormones, in turn, affect development through antagonistic and cooperative pathways that integrate endogenous developmental and exogenous environmental signals (Achard et al., 2006; Santner et al., 2009; Wolters & Jürgens, 2009; Perrot-Rechenmann, 2010; Stamm & Kumar, 2010). The effects of phytohormones on growth are transduced through signalling networks that induce changes in the transcriptome and proteome (Santner et al., 2009; Stamm & Kumar, 2010). The changes to the transcriptome are achieved through transcription factors (TFs), which repress or activate suites of genes to modulate plant growth (Nakashima et al., 2009). TFs of homeodomain-leucine zipper (HD-Zip) families I and II contribute to the plasticity of plant growth and are responsible for modulating plant development in response to environmental stimuli (Agalou et al., 2008). These stimuli are involved in changes that occur to the transcriptome, together with phytohormones, through the control of cell differentiation, division and expansion. The transcriptional mechanisms by which the HD-Zip I and HD-Zip II TFs manipulate growth are currently unknown, although it is known that members of these families form homo- and heterodimers exclusively with other members of their own family as a precursor to DNA binding. Also, dimers have been shown to target similar cis elements under in vitro conditions. However, the way in which the dimerization of the HD-Zip I and HD-Zip II TFs and the similarity of the target cis element sequences translate into function remains largely unexplored.

In this review, we present our account of the current knowledge that relates to the roles of the HD-Zip I and HD-Zip II TFs during plant adaptation under changing environmental conditions. We also briefly describe how these TFs integrate with phytohormone-mediated responses, and indicate the limits of the current knowledge that relate to the mechanism of transcriptional activity and address the issue of how to overcome these limitations.

2. Discovery of the homeodomain transcription factors

Homeosis is the abnormal conversion of one body part into another and is the result of aberrations in the normal developmental program of an organism (Eckardt, 2003). While homeotic abnormalities have been observed in many different organisms (Eckardt, 2003), the first characterization of a homeotic gene was made in Drosophila melanogaster. The Drosophila antennapedia allele is responsible for the development of antennae in the position of the second leg pair, when the gene is expressed (Garber et al., 1983). Further study established that a common region of DNA, named the homeobox (HB), is present in many genes involved in the developmental program of Drosophila. It is now known that the HB encodes a DNA-binding domain termed the homeodomain (HD). The first characterized HB-containing gene in plants was KNOTTED1, which was identified in maize (Zea mays) as a mutated gene (Vollbrecht et al., 1991). The gain-of-function allele kn1 induces areas of irregular cell division along secondary veins of the lamina and the displacement of the ligule (Hake et al., 1989). It was also found through mutagenesis studies that the HB-containing TENDRIL-LESS gene of Pisum sativum inhibits lamina development and is responsible for the conversion from leaflet to tendril (Hofer et al., 2009). A strategy used to isolate HB genes from the nematode Caenorhabditis elegans was developed by Burglin et al. (1989) and was used to isolate HB sequences from plant cDNA libraries. This strategy was also used to isolate the first HB genes from Arabidopsis, which encoded a protein with an HD and a closely associated leucine zipper of the HD-Zip family; this association of an HD and leucine zipper motif is unique to plants (Ruberti et al., 1991). Many HD-Zip sequences were further isolated from rice (Oryza sativa; Meijer et al., 2000), the resurrection plant Craterostigma plantagineum (Deng et al., 2002) and wheat (Triticum aestivum; Lopato et al., 2006), using the yeast one hybrid system. With the increasing abundance of sequenced plant genomes it is now possible to identify an entire complement of plant HD-Zip TF sequences (Mukherjee et al., 2009).

Higher plants contain four families of HD-Zip proteins (HD-Zip I–IV). An analysis of available plant genome sequences has identified these genes in flowering plants and mosses. These analyses showed that these genes are absent from the genomes of unicellular green and red algae (Mukherjee et al., 2009). Individual families can be distinguished by conservation within the HD-Zip domain, other motifs in the amino acid sequences, and also specific intron and exon positions (Ariel et al., 2007). When the phylogeny of each family is scrutinized in rice and Arabidopsis, it becomes evident that subgroups of genes associate within each family. These subgroups form paralogous gene pairs that have arisen through genome duplication, as they are associated with duplicated regions of chromosomes in the Arabidopsis and rice genomes (Henriksson et al., 2005; Agalou et al., 2008; Ciarbelli et al., 2008). The phylogeny and structures of the HD-Zip I–IV genes and proteins have recently been investigated by Henriksson et al. (2005), Agalou et al. (2008) and Ciarbelli et al. (2008).

3. Structure of the homeodomain helix-turn-helix, the role of the leucine zipper in HD-Zip proteins and three-dimensional structures of nonplant HD and leucine zippers

The HB is a 180-nucleotide region of DNA encoding a 60-amino-acid-long HD that consists of three characteristic α-helices (Fig. 1; Gehring et al., 1994). α-helix 3, considered the recognition helix, is the most conserved across HD proteins (Gehring et al., 1994) and is responsible for the specificity of protein–DNA interactions. α-helix 3 typically lies within the major groove of DNA next to the core sequence ATTA (or TAAT), bound by most HD proteins (Gehring et al., 1994). In contrast, a leucine zipper is an α-helix comprised of seven amino acid (heptad) repeats. The residues of the heptad are designated an, bn....gn (n being the number of the heptad), where residue d is a leucine. During dimerization through the monomeric α-helices, the structure forms a coiled coil (Fig. 1). Residues a and d form the hydrophobic interface of the coiled coil, while residues at positions e and g form complementary charge interactions that permit or inhibit dimerization between two monomers (Szilák et al., 1997). The leucine zipper of HD-Zip proteins is immediately downstream of the HD and enables dimerization of HD-Zip proteins, which is a prerequisite for DNA binding. Many HD proteins bind strongly as monomers to DNA, but HD-Zip proteins possess a very weak affinity for DNA as monomers and require dimerization for increased DNA-binding efficiency (Palena et al., 1999). If the spacing between the HD and leucine zipper components of HD-Zip I and HD-Zip II proteins is modified, DNA binding is disrupted, indicating that dimerization is essential for the correct spacing of the HDs with regard to their position on the DNA (Sessa et al., 1993). While structural information on HD-Zip proteins is currently unavailable, three-dimensional structures of HDs and leucine zippers (bZIP proteins) from various sources, mainly insect, yeast and mammalian systems, are available (Fig. 1). The advantage of the tandem binding arrangements of HDs is that they provide the opportunity for a larger and more specific DNA sequence read-out. These dimeric arrangements could create specific structural patterns that are recognized by DNAs.

image

Figure 1. Three-dimensional structures of Drosophila and yeast proteins containing homeodomains (HDs) and leucine zippers (bZIPs). (a) A tandem arrangement of two HDs (HD-A and HD-B) in the Eve HD-DNA complex from Drosophila, in which each HD represents a helix-loop-helix-turn-helix structure and where both HDs bind an AT-rich AATTAAATTC oligonucleotide (Hirsch & Aggarwal, 1995) (Protein Data Bank (PDB) accession 1JGG). α-helix 3 can be seen aligning with the major groove of the B-DNA. (b) A GCN4 (General Control Nonderepressible 4) bZIP dimer from yeast (bZIP-A and bZIP-B) illustrating the interaction of the two monomeric α-helices. The basic regions of the bZip proteins bind the pseudo-palindrome AATGACTCAT/TACTGAGTA, centered on a GC base pair (in bold) (Ellenberger et al., 1992) (PDB accession 1YSA).

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II. The role of HD-Zip transcription factors in plant growth adaptation to environmental changes and the phytohormone network

  1. Top of page
  2. Abstract
  3. I. Introduction
  4. II. The role of HD-Zip transcription factors in plant growth adaptation to environmental changes and the phytohormone network
  5. III. Dissecting the common cis element, dimerization and cell specificity of HD-Zip I and HD-Zip II transcription factors
  6. IV. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

In this section we will review studies of the functions of HD-Zip I and HD-Zip II TFs, which have suggested that these TFs play a role in the plant response to water deficit, ABA, salinity stress and changes in light conditions. Further, the integration of HD-Zip I and HD-Zip II TFs with phytohormone pathways and plant development will be summarized. It appears that HD-Zip I and HD-Zip II TFs contribute to the regulation of plant cell expansion, division and differentiation. The default body plan can thus be influenced by external parameters through HD-Zip I and HD-Zip II TFs that induce adaptation of established and nascent organs in response to prevailing conditions. The role of HD-Zip I and HD-Zip II TFs was determined largely through work with the model species Arabidopsis and rice. Making comparisons of the characteristics of homologous genes from these two model species has proved difficult. Although Agalou et al. (2008) grouped closely aligned HD-Zip I proteins from Arabidopsis, C. plantagineum and rice into clades that were established by Henriksson et al. (2005) for Arabidopsis, comparison of functional characterizations of assumed homologs reveals divergent roles and expression patterns (Henriksson et al., 2005; Agalou et al., 2008). Phylogenetic trees for Arabidopsis and rice have been produced by combining the HD-Zip I and HD-Zip II proteins with the addition of relevant sequences from other species (Fig. 2). The clades previously established by Henriksson et al. (2005) and Agalou et al. (2008) for HD-Zip I and by Ciarbelli et al. (2008) for HD-Zip II have been used to help shed light on similar or distinct characters of the HD-Zip I and HD-Zip II paralogs and homologs within and between each species, respectively.

image

Figure 2. An unrooted radial phylogenetic tree of the selected homeodomain-leucine zipper (HD-Zip) I and HD-Zip II transcription factors. The tree is based on full-length amino acid sequences. The alignment was created using ClustalX (Thompson et al., 1997) and branch lengths are drawn to scale. HD-Zip I clades are circled and identified as α, β1, β2, γ, δ, ζ, ϕ1 and ϕ2 and HD-Zip II clades as α, β, γ and δ, as previously established (Henriksson et al., 2005; Agalou et al., 2008; Ciarbelli et al., 2008). Two-letter prefixes for sequence identifiers indicate species of origin. At, Arabidopsis thaliana; Cp, Craterostigma plantagineum; Mt, Medicago truncatula; Na, Nicotiana attenuata; Os, Oryza sativa; HB, homeobox; HOX, homeobox. The tree was bootstrapped using the N-J algorithm (Thompson et al., 1997) and bootstrap values for reproducibility out of 1000 are shown at the confluence of the clusters, for which these values were > 500. The bar indicates substitutions per site.

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1. The role of HD-Zip transcription factors in water deficit, salinity stress and ABA-modulated development

Plants are dependent on their environment for access to water for heat relief, photosynthesis and growth through cell expansion (Skirycz & Inzé, 2010). When a plant senses that water is becoming limited, it will reallocate this valuable resource by restricting transpiration and growth and will frequently flower earlier (Skirycz & Inzé, 2010). As summarized in this section, the role of HD-Zip I TFs in the drought response is to mediate ABA signaling and control growth by limiting cell expansion.

HD-Zip I  Several specific HD-Zip I family members have been characterized with respect to their roles in regulating drought responses. Of the Arabidopsis γ-clade (Fig. 2), AtHB7 and AtHB12 transcripts are expressed in young expanding tissues and transcript levels are dramatically increased, especially in the vasculature, upon application of exogenous ABA or water deficit (Olsson et al., 2004). When over-expressed in Arabidopsis, AtHB7 and AtHB12 confer a reduced growth phenotype typical of water-limiting conditions (Olsson et al., 2004). Conversely, it has been observed that athb12 mutant plants have slight but reproducible increases in stem length (Son et al., 2010). Hjellstrom et al. (2003) have shown that retardation of stem growth in AtHB7 over-expressing plants is caused by decreased cell elongation. In AtHB12 over-expression lines, stem retardation is associated with down-regulation of GA20 oxidase 1 activity, which is responsible for GA-stimulated cell elongation (Son et al., 2010). It has been proposed that the role of AtHB7 and AtHB12 is to reduce growth under water deficit, and the inhibition of GA biosynthesis is consistent with this role (Hjellstrom et al., 2003; Olsson et al., 2004; Son et al., 2010).

The sunflower (Helianthus annuus) HB4 (HaHB4) gene clusters within the drought/ABA inducible γ-clade (Fig. 2) and is strongly induced by water deficit and ABA (Gago et al., 2002). Work with two forms of the HaHB4 promoter identified an ABA response element (ABRE) that responds to ABA application and also an ABRE that responds to NaCl treatment via an ABA-independent mechanism (Manavella et al., 2008a). When over-expressed in Arabidopsis, HaHB4 confers a similar reduced growth phenotype as seen when AtHB7 and AtHB12 are over-expressed (Olsson et al., 2004; Dezar et al., 2005). When tested under drought stress, Arabidopsis plants expressing HaHB4 constitutively or under a drought-inducible promoter showed increased survival through a mechanism that inhibited drought-related senescence (Dezar et al., 2005; Manavella et al., 2006). Arabidopsis plants over-expressing HaHB4 that were grown under well-watered conditions also showed a delay in the developmental senescence that occurs at the end of the life cycle (Manavella et al., 2006). Microarray and Q-PCR data suggested that the role of HaHB4 in Arabidopsis and H. annuus is to suppress the expression of ethylene-related genes and delay senescence. It was then found that the increasing levels of HaHB4 expression in sunflower correlated with the developmental age of leaves, as older leaves approached senescence (Manavella et al., 2006). It was concluded that HaHB4 was delaying the onset of senescence in Arabidopsis under drought stress and also acting as an antagonist to developmental senescence in both species. A delay in senescence has not been reported in transgenic Arabidopsis over-expressing AtHB7 and AtHB12 genes under well-watered or water-deficit conditions, making it difficult to compare the roles of these genes in the two species.

The Medicago truncatula HD-Zip I member HB1 (MtHB1), belonging to the γ clade, (Fig. 2) is induced by ABA and salinity stress in roots and plays a role in lateral root emergence (Ariel et al., 2010). When MtHB1 is over-expressed in the roots of composite plants, the primary root is longer and lateral root emergence is reduced, a phenotype typically seen in wild-type plants exposed to severe salt stress. Two TILLING-derived mthb1 mutant lines showed a reciprocal phenotype and had shorter roots with an increased number of emerged lateral roots (Ariel et al., 2010). The molecular mechanism responsible for the lateral root emergence phenotypes involves suppression of LOB-binding domain 1 (LBD1) by MtHB1. The lateral organ boundaries (LOB) domain TFs of Arabidopsis and rice play a role in auxin-regulated lateral root initiation and adventitious root formation, respectively (Liu et al., 2005; Okushima et al., 2007). It is proposed that the role of MtHB1 is to inhibit lateral root emergence, when roots are exposed to adverse conditions, which reduces the surface area exposed to soil stress (Ariel et al., 2010).

NaHD20, a Nicotiana attenuataγ-clade homolog, is also induced in roots and leaves by exogenous ABA and soil water deficit (et al., 2011). It was observed that under water stress N. attenuata plants with reduced NaHD20 transcripts, through virus-induced gene silencing, had reduced levels of ABA, N. attenuata 9-cis-epoxycarotenoid dioxygenase 1 (NaNCED1; an ABA biosynthesis gene) and N. attenuata osmotin 1 (NaOSM1) but that N. attenuata lipid transfer protein 1 (NaLTP1) induction was not affected (et al., 2011).

OsHOX6 (O. sativa Homeobox 6), OsHOX22 and OsHOX24, which are considered the rice homologs of AtHB7 and AtHB12 (Fig. 2), are also up-regulated by water deficit but there are no reports clarifying the functional roles of the corresponding proteins in the rice drought adaptation response (Agalou et al., 2008). These rice genes differ from their Arabidopsis counterparts in their basal expression patterns and there are also differences between the rice paralogs themselves. Under well-watered conditions, OsHOX6 has a relatively high basal level of expression in all tissues, while OsHOX22 transcript is expressed mainly in the blade and panicles. Further, OsHOX24 is strongly expressed in panicles and weakly in other tissues (Agalou et al., 2008). Under extended drought, OsHOX22 and OsHOX24 transcripts are increasingly up-regulated in leaf tissues in both drought-resistant and drought-sensitive cultivars, whereas OsHOX6 is only slightly up-regulated in a drought-sensitive cultivar (Agalou et al., 2008).

The Arabidopsis HD-Zip I β-clade gene AtHB6 (Fig. 2) is ubiquitously expressed in all tissues of mature plants, but in leaves it is detected predominantly in the vasculature (Söderman et al., 1999). AtHB6 is thought to play a role in the regulation of cell division or differentiation as its expression in developing leaves declines basipetally, withdrawing with the wave of epidermal cell differentiation, but AtHB6 still remains in the guard cells (Söderman et al., 1999). Expression of AtHB6 in Arabidopsis is increased by water deficit and ABA, but remains within the same cell types as under untreated conditions (Söderman et al., 1999). When AtHB6 is over-expressed in transgenic Arabidopsis, plants show reduced stomatal closure and diminished inhibition of germination by ABA (Himmelbach et al., 2002); these are also characteristics of the ABA-insensitive mutants abi1 and abi2 (Leung et al., 1997). These data imply that AtHB6 plays a role as a negative regulator in the ABA response under water deficit (Deng et al., 2006).

AtHB5 of the β clade (Fig. 2) shows a similar expression pattern to AtHB6 and is found in all major tissues (Söderman et al., 1994; Henriksson et al., 2005). The AtHB5 promoter is active in the hypocotyl of germinating seedlings, but is suppressed by ABA application. After ABA treatment its expression is restricted to a discrete band in the transition zone between the hypocotyl and root (Johannesson et al., 2003). Transgenic Arabidopsis with ectopically increased levels of AtHB5 are more sensitive to root growth inhibition by ABA at the seedling stage and to germination inhibition. The two observations, that AtHB5 is down-regulated by ABA and that increased levels of ABA enhance ABA-specific responses, have led the authors to suggest a role in seedling development under short-term water-limiting conditions that is not sustained through long-term water deficit (Johannesson et al., 2003).

The HD-Zip I TFs of C. plantagineum that group with this β clade under phylogenetic analysis (Fig. 2), CpHB4 and CpHB5, are down-regulated in C. plantagineum leaves and roots upon exposure to water deficit, whereas CpHB6 and CpHB7 are up-regulated (Deng et al., 2002). Under normal growth conditions, CpHB7 promoter activity in Arabidopsis is considered comparable to that of AtHB6. The AtHB6 over-expression phenotype is also paralleled by ectopic expression of CpHB7 in Arabidopsis (Deng et al., 2006).

OsHOX4 of the rice ζ clade (Fig. 2) is also drought responsive but the level of mRNA transcripts decreases upon exposure to water deficit (Agalou et al., 2008). There are contrasting reports regarding the characteristics of transgenic rice over-expressing OsHOX4. While transgenic plants are shorter than wild-type plants, this phenotype is reported to be attributable to a decrease in the number of cells per internode (Agalou et al., 2008) or to a reduction in cell elongation in the stem (Dai et al., 2008). The mechanism behind stem height reduction is thought to involve OsHOX4 up-regulation of YAB1 (YABBY1, encodes a protein with a YABBY domain) expression, a negative regulator of the GA response (Dai et al., 2008). These results, when interpreted in isolation, imply that under water deficit OsHOX4 is down-regulated, leading to a decrease in YAB1 expression, which enables a stronger plant response to GA. However, the implication of a drought response mechanism involving OsHOX4 in the adaptation of rice development is unclear.

The transcript levels of the Arabidopsis δ-clade genes (Fig. 2) AtHB21, AtHB40 and AtHB53 are up-regulated upon exposure to ABA and salinity stress. However, no role in environmental adaptation has been established for these HD-Zip I TFs, although a role in ovule development has been proposed for all three genes (Skinner & Gasser, 2009).

HD-Zip II  There is little functional evidence to suggest a role for HD-Zip II TFs in plant growth adaptation responses to water deficit. However, expression studies using microarrays have shown that HAT2 (Homeobox from Arabidopsis thaliana 2) and HAT22 expression is up-regulated under drought in Arabidopsis (Huang et al., 2008).

In rice, OsHOX11 transcripts are dramatically decreased upon drought exposure in a drought-resistant cultivar and OsHOX27 is up-regulated under mild drought, but transcript levels decrease as the severity of the water deficit increases in both drought-resistant and drought-sensitive cultivars (Agalou et al., 2008). OsHOX19 expression is increased by the imposition of drought stress in both drought-sensitive and drought-resistant cultivars.

Transcripts of HD-Zip II TFs from C. plantagineum are also regulated by water availability and ABA. CpHB1 and CpHB2 show tissue-specific differences in expression under water deficit and ABA treatment. CpHB1 expression is induced in leaves by water deficit but not by ABA, and the level of CpHB2 transcript is up-regulated in the roots by water deficit and ABA (Deng et al., 2002).

2. The role of HD-Zip transcription factors in plant growth adaptation to light and their expression and function

Light stimulates germinating seedlings to de-etiolate and triggers the change to a light-harvesting photoautotroph (Franklin & Quail, 2010). Once a plant is harvesting light, the red : far-red ratio (R : FR) is an indicator of shade or competition for light from neighboring plants (Franklin, 2008). During this post-germinative stage, FR-rich light acts as a stimulus that is interpreted by the phytochrome signaling system to induce the shade avoidance response (Stamm & Kumar, 2010). Blue light is perceived by cryptochromes and phototropins, which regulate stem elongation, floral induction, the circadian clock and phototropism (Lin & Shalitin, 2003; Inoue et al., 2010; Möglich et al., 2010).

HD-Zip I  Of the four α-clade HD-Zip I TFs (Fig. 2), neither AtHB13 nor AtHB20 transcripts were detected in plants grown in darkness (Henriksson et al., 2005) but they appear to have distinct roles. AtHB13 is implicated in sucrose-responsive development (Hanson et al., 2001), while AtHB20 is involved in vascular patterning and integration of light with internal ABA signals during seed germination (Mattsson et al., 2003; Barrero et al., 2010). AtHB20 is involved in vascular patterning in leaves and is associated with areas around emerging procambial strands in very early leaf primordia. Expression of AtHB20 in developing leaves mirrors that of auxin localization and, as leaves mature, transcripts are localized to the vasculature and eventually to the fascicular cambium (Mattsson et al., 2003). It has also been found that AtHB20 is involved in germination and is thought to inhibit the action of ABA near the micropyle (Barrero et al., 2010). During germination, the micropyle weakens and allows the root cap to emerge, and ABA, which inhibits germination, inhibits this weakening. AtHB20 was shown to be expressed in the endosperm at the micropylar end and also in the root cap (Barrero et al., 2010). When seeds were germinated under light, there was an increase in the transient expression of AtHB20 3 h after imbibition. Barrero et al. (2010) suggested that AtHB20 acts as an integrator of internal ABA signaling and external light signaling, which is required for germination of Arabidopsis seeds.

Like all members of the Arabidopsis β1 and β2 clades (Fig. 2), AtHB1 and AtHB16 transcripts are expressed ubiquitously, but unlike AtHB5 and AtHB6 they are up-regulated when Arabidopsis plants experience periods of darkness (Henriksson et al., 2005). When ectopically over-expressed, AtHB1 overrides the etiolation response of dark-grown seedlings and results in defects in palisade cell formation, suggesting a role in leaf cell fate determination (Aoyama et al., 1995). AtHB16 negatively affects hypocotyl elongation in response to blue light. When mRNA over-expression or antisense transgenic lines are exposed to blue light, the transcript levels are negatively correlated with the hypocotyl elongation response, when compared with wild-type plants (Wang et al., 2003). AtHB16 expression, however, is not affected by blue light or CRYPTOCHROME1 (CRY1) and CRY2; neither does it affect CRY1 or CRY2 expression. Under white light (WL) conditions, AtHB16 is highly expressed in leaves and acts as a negative regulator of leaf cell expansion. This response was confirmed by an increase in leaf size in both directions upon mRNA antisense transcript suppression (Wang et al., 2003).

The Arabidopsis HD-Zip I gene AtHB52, for which there is little functional information, also shows a strong up-regulation of transcript level in response to blue light and especially darkness (Henriksson et al., 2005). Large-scale expression analyses under different light regimes have not been reported for the rice HD-Zip I family.

HD-Zip II  Many of the HD-Zip I genes appear to be regulated by water deficit or light conditions. By contrast, five of the Arabidopsis HD-Zip II genes are known to respond to changes in light, but there is little evidence to imply a role in adaptation to water stress. Little is known about the environmental conditions regulating the remaining five HD-Zip II members other than that they are not responsive to changes in light (Ciarbelli et al., 2008). Of the three γ-clade Arabidopsis HD-Zip II genes (Fig. 2), AtHB2/HAT4 has been investigated most thoroughly. This gene is highly expressed in dark-grown etiolated seedlings (Carabelli et al., 1996) and is strongly inhibited upon exposure to either R or FR light. AtHB2/HAT4 mRNA antisense and over-expression lines have shorter and longer hypocotyls, respectively. The biological role of the corresponding protein during germination appears to be in the regulation of hypocotyl elongation, which is dependent upon seed reserves, until light is sensed, at which time elongation can cease and the plant develops autotrophic capabilities. Over-expression lines show smaller cotyledons through decreased cell expansion, which supports a role in determining developmental differences between heterotrophic and autotrophic habits (Steindler et al., 1999). Once development has proceeded past germination, AtHB2/HAT4 is expressed at all stages of leaf development and increases with leaf age (Ciarbelli et al., 2008). When plants are grown under WL, AtHB2/HAT4 is strongly, but reversibly, induced by low R : FR light conditions (Steindler et al., 1997), which are typical under canopy shade. This leads in turn to the induction of a shade avoidance response, with plant characteristics such as shoot elongation and a reduction of leaf size. When over-expressed under WL conditions, AtHB2/HAT4 decreases leaf size by repressing its cell division.

Ectopic over-expression of the two remaining Arabidopsis γ-clade HD-Zip II genes, HAT1 and HAT2 (Fig. 2), results in a phenotype similar to that seen in AtHB2/HAT4 over-expression lines (Steindler et al., 1999; Sawa et al., 2002; Ciarbelli et al., 2008). Of the three members of the γ-clade, HAT1 is more closely related to HAT2 than AtHB2/HAT4, but functionally has more similarity with AtHB2/HAT4. Both the AtHB2/HAT4 and HAT1 genes are rapidly up-regulated by FR-rich light, presumably through the direct action of the phytochrome system (Ciarbelli et al., 2008). The HAT2 gene is also up-regulated by low R : FR light but is delayed, compared with AtHB2/HAT4, as a result of regulation by an auxin-mediated pathway that is induced by FR-rich light (Ciarbelli et al., 2008). HAT1 over-expressing lines share an identical phenotype with AtHB2/HAT4 over-expressors, and HAT2 over-expressors have a similar phenotype but show additional features that are characteristic of IAA over-producing mutants such as epinastic cotyledons (Sawa et al., 2002). The reduction in leaf size seen in the over-expression phenotype of the three γ-clade genes has been attributed to a reduction in the number of cells via reduced cell division (Ciarbelli et al., 2008).

The sunflower HaHB10 gene is strongly up-regulated in dark-grown seedlings and is expressed mainly in mature photosynthetic organs of sunflower (Rueda et al., 2005). HaHB10 was considered to be closely related to AtHB2/HAT4 by Ariel et al. (2007), because of similar responses to light and their apparent roles in adaptation to changes in light conditions. However, the extreme C-terminal end of the HD-Zip II protein shares a near-identical amino acid sequence with HAT22 and HAT9 of the Arabidopsis HD-Zip II β clade (our own observation), although the expression patterns of these genes show no response to changes in light quality (Ciarbelli et al., 2008).

As with members of the γ clade, the δ-clade genes AtHB4 and HAT3 (Fig. 2) are also induced by an increase in FR light (Ciarbelli et al., 2008). The phenotype of Arabidopsis over-expressing AtHB4 under continuous WL is also similar to that of γ-clade over-expression lines (Sorin et al., 2009). However, unlike the response of plants over-expressing the γ-clade genes, it was shown that over-expression of AtHB4 can inhibit FR-induced hypocotyl elongation, when compared with the wild type.

3. Integration of endogenous and environmental signaling through phytohormones and the HD-Zip I and HD-Zip II transcription factors

Coordination of plant development involves the integration of many factors, including those involved in interactions of plant phytohormones and HD-Zip I and HD-Zip II TFs. This coordination involves regulation, at different levels, of phytohormones and HD-Zip TFs, separately or in combination. The complexity of phytohormone regulation of an HD-Zip I TF is seen in the induction process of AtHB53, which is up-regulated by NaCl and ABA in seedlings and in the root meristem and elongation zone by auxin. Up-regulation in the roots by ABA is inhibited by co-application of cytokinin (Son et al., 2005). Demonstrating the influence of HD-Zip I TFs on phytohormone signaling and integration with environmental signals, OsHOX4 is suppressed under water deficit and over-expression results in ectopic expression of YAB1, a negative regulator of the GA response (Dai et al., 2008). Through ABA, the HD-Zip I drought-inducible γ clade has been shown to influence water deficit by inhibiting the action of GA and auxin. AtHB12 is up-regulated by ABA and down-regulates GA20 oxidase 1 in Arabidopsis (Son et al., 2010). Medicago truncatula HB1 is induced by ABA and NaCl in roots and suppresses auxin-mediated LBD1 expression (Ariel et al., 2010). Integrating water deficit, through ABA, with ethylene signaling, HaHB4 suppresses senescence under drought (Manavella et al., 2006). By integrating pathways via plant hormones that are considered to be abiotic and biotic stress related, HaHB4 expression could also be induced by application of methyl jasmonate, while NaHD20 is induced by ABA, jasmonic acid, salicylic acid and ethephon (which releases ethylene; Manavella et al., 2008b; et al., 2011). Of the HD-Zip II TFs involved in de-etiolation and the shade avoidance response, many TFs were shown to require or inhibit the action of phytohormones. To fulfill its role in the shade avoidance response, the AtHB2 gene requires the auxin transport system for cell elongation in the hypocotyl (Steindler et al., 1999), and HAT2 is induced under FR-rich light conditions but via an auxin-mediated pathway (Sawa et al., 2002). AtHB4 regulates hypocotyl length in the seedling shade avoidance response and down-regulates auxin and/or brassinosteroid induction of genes that are also up-regulated by FR-rich light (Sorin et al., 2009). HaHB10 is also involved in plant growth adaptation to light conditions and expression is strongly induced in the hypocotyls of dark-grown seedlings and by GA (Rueda et al., 2005). There is also evidence that HaHB10 is up-regulated by the biotic stress-related salicylic acid and that it down-regulates salicylic acid biosynthetic genes, although levels of the phytohormone itself are not affected under control conditions (Dezar et al., 2010).

III. Dissecting the common cis element, dimerization and cell specificity of HD-Zip I and HD-Zip II transcription factors

  1. Top of page
  2. Abstract
  3. I. Introduction
  4. II. The role of HD-Zip transcription factors in plant growth adaptation to environmental changes and the phytohormone network
  5. III. Dissecting the common cis element, dimerization and cell specificity of HD-Zip I and HD-Zip II transcription factors
  6. IV. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

HD-Zip I and HD-Zip II TFs cis are involved in adapting plant growth to changes in environmental conditions. The mechanism by which they exert their control over plant development has not yet been identified. HD-Zip I and HD-Zip II TFs are perceived to bind common cis elements that were revealed using in vitro methods (Frank et al., 1998; Meijer et al., 2000; Deng et al., 2002; Lopato et al., 2006). It was also shown in vitro that dimerization is a prerequisite for DNA binding and it is now assumed that HD-Zip I and HD-Zip II TFs can heterodimerize within their own families (discussed in section III). These observations suggest that a large network exists, where dimerization partners confer different transcriptional characters and two families compete over similar cis elements. This would enable environmental and endogenous signals to regulate fluxes in the network to establish developmental programs through differential gene expression (Fig. 3). We have recently found that two members of the HD-Zip I and HD-Zip II families can antagonistically regulate a group of genes involved in the same developmental process (S. Lopato et al., unpublished data). Whether this is direct or indirect regulation through the same cis element remains to be demonstrated. However, to date, very few data have been presented that demonstrate the effects of the protein–DNA and protein–protein interactions in planta. Problems hindering validation of this network will be addressed here.

image

Figure 3. The proposed plant developmental regulatory network involving homeodomain-leucine zipper (HD-Zip) I and HD-Zip II transcription factors (TFs). Black arrows indicate an interaction. The black dashed line represents a promoter, the cross-hatched box represents cis elements downstream of other trans-acting factors, the CAATNATTG box represents the HD-Zip I and HD-Zip II cis elements, the right-angled arrow represents the transcriptional start site, and the ‘Downstream gene’ box represents genes whose transcription will be either suppressed or activated. 1The arrowed box represents post-translational modification that affects the ability of the HD-Zip TFs to dimerize and/or bind DNA (Himmelbach et al., 2002; Tron et al., 2002).

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1. Defining target cis elements within downstream genes

When the specificity of HD-Zip I and HD-Zip II cis element-binding interactions has been investigated in vitro, the results have generally yielded the sequence CAAT (A/T)ATTG (binding site 1 (BS1)) or CAAT(C/G)ATTG (BS2). It is considered that BS1 and BS2 are composed of two overlapping yet different HD-related cis elements composed of 5′-TAATTG-3′ and 3′-GTTATT-5′, or 5′-TGATTG-3′ and 3′-GTTACT-5′, respectively (the difference in the central nucleotide is highlighted in bold). The consequence of this is that each HD of a dimer is interacting with a different BS, depending on the orientation of the dimer relative to the DNA. Also, only one HD of any dimer makes specific contacts with the central nucleotide and each monomer has specific preferences for the orientation of a cis element (Tron et al., 2005). Key amino acid residues in the HD which contribute to binding at the central nucleotide are conserved in members within each family (Sessa et al., 1997). However, it is now evident that these conservations are not solely responsible for the distinction between the BSs. There are many examples of members of each family that bind to both BS1 and BS2 sequences in vitro, albeit with varying degrees of efficiency (Frank et al., 1998; Meijer et al., 2000; Deng et al., 2002; Lopato et al., 2006). Also, AtHB2 is known to regulate its own promoter and DNA footprinting assays confirmed that it can interact strongly with TAATCATTA and TAATTATTA, but also weakly with TAATCATCT (deviations from the typical BS are underlined; Ohgishi et al., 2001). Therefore, while it is considered that there are two different cis elements, clearly there are ambiguities. A more definitive study has been performed on the DNA-binding properties of MtHB1, which negatively regulates LBD1 expression (Ariel et al., 2010). Initial gel shift assays confirmed that MtHB1 interacted with both BS1 and BS2. Mutation analysis of BS1, found in the promoter of the LBD1 gene, showed that BS1 was necessary to maintain the usual spatial expression pattern regulated by MtHB1 (Ariel et al., 2010). ChIP-PCR (Chromatin immuno precipitation PCR) assays confirmed that MtHB1-histidine fusion proteins could indeed associate with the LBD1 promoter. Establishing the promoters that are directly regulated by HD-Zip I and HD-Zip II TFs will enable the precise identification of target cis elements. This can be achieved through transcriptomic approaches that identify genes that are co-regulated with HD-Zip I and HD-Zip II transcripts under specific induction conditions, or through up-regulation of potential target genes by an ectopically expressed HD-Zip I or HD-Zip II TF. Once potential downstream genes have been revealed, investigations of their promoter regions can be carried out to identify these cis elements and validate them using mutations and promoter activation studies. Performing ChIP-PCR will confirm that the HD-Zip TF of interest interacts with the suspected promoter under in vivo conditions.

Clearly, plant HD-Zip I and II proteins have similar DNA-binding selectivity but current models that define HD-Zip–DNA interactions are based on comparisons between HD proteins from insect or mammalian sources, structures of which have been determined by X-ray crystallography and NMR spectroscopy (Kissinger et al., 1990; Qian et al., 1993). The atomic structure of a fruit-fly HD provided the rationale for site-directed mutagenesis experiments in plant dimeric HD-Zip TFs. Targeted amino acid residue substitutions of the N-terminal arm led to significant changes in DNA-binding affinities of HaHB4 (Palena et al., 2001) and enabled determination of the molecular basis of DNA binding that is assigned to helix III (recognition helix) and to the loop between helix I and helix II of HaHB4 (Tron et al., 2004). Access to the atomic structure of this particular HD-Zip TF would provide more accurate alterations in DNA-binding affinities and would shed light on the structural basis of oligomerization patterns. More atomic structures, in particular those of plant HD-Zip TFs, are needed to establish the precise roles of the structural elements that guide DNA binding and dimerization.

2. Regulation of cis element binding through post-translational modification

It has been shown using in vitro assays that binding of target cis elements by HD-Zip I and HD-Zip II TFs may be regulated by post-translational modification. The redox status of a plant cell changes to an oxidative environment under stress. Implicating cellular redox status in post-translational control of HD-Zip II TF DNA binding, the conserved cysteines at a2, a3 and g2 of the leucine zipper and the conserved CPSCE motif (a highly conserved string of five amino acids consisting of Cys, Pro, Ser, Cys and Glu found in the C-terminus) inhibit DNA binding in an oxidative environment (Tron et al., 2002). The phosphorylation status of AtHB7 and AtHB12 has been suggested as a form of post-translational control that may explain the inability of these HD-Zip I TFs to bind either BS1 or BS2 in vitro (Johannesson et al., 2001). However, AtHB7 is able to homodimerize and AtHB7 and AtHB12 can activate a reporter construct with six repeats of BS1 (Johannesson et al., 2001; Henriksson et al., 2005). Although Son et al. (2010) found that they could not determine AtHB12 binding to the promoter of GA20 oxidase 1 in vitro, AtHB12 over-expression did inhibit activation of the GA20 oxidase 1 promoter in transient assays. Himmelbach et al. (2002) found that ABI1 (ABA Insensitive), a serine/threonine phosphatase, interacts with AtHB6 in yeast-2-hybrid assays. A role for dephosphorylation in activating AtHB6 transcriptional activity was revealed by the observation that phosphorylation of AtHB6 by protein kinase A inhibited DNA-binding activity. Similarly, in transient assays, AtHB6 activated a promoter with four repeats of the HD-Zip binding sequence more strongly when ABA was applied, suggesting dephosphorylation by an ABA-dependent factor (Himmelbach et al., 2002). These in vitro observations have demonstrated that post-translational regulation can influence DNA binding.

3. Dimerization and the roles of members of the HD-Zip I and HD-Zip II families in the cell- and condition-specific interactome

Currently it is assumed that each HD-Zip I and HD-Zip II TF is able to heterodimerize only with other members of its own family. However, Gonzalez et al. (1997) noted that not all leucine zippers have a theoretically favorable conformation for hetero- or homodimerization. Selected studies indicate that at least five factors could contribute to oligomerization of leucine zipper domains (Gonzalez et al., 1997; Szilák et al., 1997; Casteel et al., 2010). These factors include: (1) specific positions of leucine and threonine residues at a1 and d1 positions; (2) changes in packing interactions at oligomer interfaces; (3) identity of residues in the close vicinity of a1 and d1 positions, which are typically positively and negatively charged, respectively; (4) overall length of coiled-coil structures; and (5) protein dynamics of HD-Zip TFs, as contributing HD components would affect overall folding patterns and inherent protein motions of HD-Zip TFs. In families 1A, 1B and 1C (Fig. 4), the allowed variations in the a and d positions correspond to substitutions that would not perturb tight hydrophobic packing of coiled-coil assemblies at the dimer interface. More information is needed on the interaction profile of HD-Zip I and HD-Zip II TFs and the properties that specific interaction partners contribute to cis element binding. Such studies have been successful in defining the characteristics of the MADS box TFs (de Folter et al., 2005).

image

Figure 4. An unrooted radial phylogenetic tree of leucine zipper domains from the homeodomain-leucine zipper (HD-Zip) I and HD-Zip II complements of the rice and Arabidopsis genomes. Amino acid sequences were aligned with ClustalX (Thompson et al., 1997) and branch lengths are drawn to scale. Families, circled and designated IA, IB, IC and II, are grouped according to differences in the amino acid residues at positions a and d of the leucine zipper heptads. Drought- and light-responsive genes scattered throughout the clades are highlighted with closed or open white ovals, respectively (HAT2 is considered drought- and light-responsive). Two-letter prefixes for sequence identifiers indicate species of origin. The tree was bootstrapped using the N-J algorithm (Thompson et al., 1997), and bootstrap values for reproducibility out of 1000 are shown at the confluence of the clusters, for which these values were > 500. The letters a and d indicate the positions of the seven amino acid residues of a heptad (Gonzalez et al., 1997). At, Arabidopsis thaliana; Os, Oryza sativa; HB, homeobox; HOX, homeobox.

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The strength of the DNA–protein interaction is also dependent on the dimers that make up the unit as demonstrated in vitro with OsHOX4 and OsHOX5, which bind DNA more strongly as heterodimers than as homodimers (Meijer et al., 2000). It was also shown that the presence of stimulus-specific factors can modulate the DNA-binding properties of HD-Zip I and HD-Zip II TFs. A co-activator of transcription, which was specifically induced in response to a biotic stress, Solanum tuberosum multiprotein bridging factor 1, increased the DNA-binding affinity of two sunflower HD-Zip TFs from different families under in vitro conditions (Zanetti et al., 2004).

Some members of the HD-Zip I and HD-Zip II families are widely expressed in plant tissues throughout plant development, while others have specific spatial, temporal, developmental and/or inducible expression characteristics. Over-expression of different HD-Zip genes may result in phenotypes that are not physiologically relevant when analyzed (Supporting Information Table S1). The presence of an HD-Zip I or HD-Zip II TF, when ectopically expressed, may interfere with the network that is operating within a specific tissue or cell when dimerization occurs. The sucrose concentration-dependent small cotyledon phenotype observed in Arabidopsis ectopically expressing AtHB13 reveals this anomalous effect, as a similar response is not seen in wild-type plants (Hanson et al., 2001). In this case, AtHB13 is thought to interfere with the role of an HD-Zip TF that is involved in cotyledon expansion but is unrelated to sucrose concentration. Contrary to this idea that an HD-Zip I or HD-Zip II TF can interfere with the interactome when ectopically expressed, over-expression of a single HD-Zip I or HD-Zip II TF can result in little overall change of a plant phenotype. This suggests that other spatial, temporal or conditional factors necessary for activity of the ectopic TF are not present. This has been observed with ectopic over-expression of OsHOX1 in Arabidopsis and rice, where a distinct effect on leaf shape or no other discernible changes in phenotype were observed, respectively (Scarpella et al., 2000). It was proposed that OsHOX1 is only transcriptionally active only in a transient cell type in zones of vascular differentiation; these observations imply that, outside of this environment, the TF has little or no discernible effect on rice phenotype. A further example demonstrates that the contribution of other specific conditional or spatial factors is required for HD-Zip I or HD-Zip II TF activity. Under control conditions, root growth of Arabidopsis over-expressing AtHB7 or AtHB12 remains similar to that of wild-type plants, whereas leaves and stems are smaller. However, treating the roots with ABA results in increased inhibition of root growth compared with that seen in wild-type plants, suggesting that an ABA-inducible factor is necessary for the response in the roots (Olsson et al., 2004). An example of a developmentally specific role comes from Arabidopsis plants over-expressing AtHB5. Arabidopsis plants over-expressing AtHB5 were found to have an increased sensitivity to ABA, which was characterized by inhibited germination and seedling root growth with no observable effect seen in adult plants (Johannesson et al. 2003). Johannesson et al. (2003) argued that AtHB5 modulates growth in response to water deficit strictly at the seedling stage and that at other developmental stages additional factors required for this response are not present. With different roles in two developmentally different tissue types, AtHB2/HAT4 over-expression induces hypocotyl elongation and represses cotyledon expansion in transgenic plants. This suggests that AtHB2/HAT4 plays distinct roles in specific tissues in response to changes in light quality.

Clearly, HD-Zip I and HD-Zip II TFs are able to dimerize within their respective families and to bind a common cis element under in vitro conditions, which suggests a large network. To investigate the influence that any given HD-Zip I or HD-Zip II TF may have on this proposed network, as a first step, the presence of viable dimerization partners must be confirmed. Information on cell- and condition-specific transcriptomes accumulating in public databases will aid these efforts. The ability to dimerize with the partners that are present would then indicate involvement with the HD-Zip I and HD-Zip II network, which could be tested by analysis of downstream target genes.

4. Redundancies in the roles of HD-Zip I and HD-Zip II transcription factor paralogs

Arabidopsis and rice HD-Zip families I and II consist of many related genes that have presumably arisen through genome and gene duplications, although this phenomenon will need to be assessed in other species as complete genome sequences arise. These duplications can lead to variations in expression patterns and differences in the roles that each of the paralogous genes plays. As is evident in the HD-Zip I and HD-Zip II families of rice and Arabidopsis, this has led to functional redundancies, which make it difficult to determine a role for a single member of the families through down-regulation (Table S1). The degree of stem retardation seen in AtHB7 and AtHB12 over-expressing plants is additive, when both are over-expressed in concert. This suggests that the effect on the genes targeted by AtHB7 and AtHB12 is additive or, more likely, that they act on the same target genes (Olsson et al., 2004). Duplication of the HD-Zip TFs would have relieved pressure to adhere to a strict functional role and provided an opportunity for new functionality. Transcription of both AtHB7 and AtHB12 is strongly induced by ABA in all tissues, but the kinetics of induction differs and these genes are also regulated differently by ABI1 and ABI2 (Lee & Chun, 1998). It was also suggested that AtHB5 may not play an exclusive role, as Arabidopsis lines homozygous for an AtHB5 allele with a T-DNA insertion show no differences in phenotype from wild-type plants (Johannesson et al., 2003). In light of the phylogenetic grouping within the β2 clade, it was suggested that AtHB6 and AtHB16 may share redundancies with AtHB5 (Johannesson et al., 2003). This remains to be verified, especially as these genes play distinct roles and possess unique induction characteristics, as discussed in section II.

As noted above, the closely related genes HAT1, HAT2, HAT3, AtHB2 and AtHB4 share many similar characteristics though they have different expression patterns (Ciarbelli et al., 2008). All are transcriptionally induced by changes in light quality, although HAT2 by an auxin-mediated path, over-expression of each gene results in a similar phenotype, and no phenotype is observed in knock out lines of individual genes (Sorin et al., 2009). However, double knock-out lines have revealed that hat1/hat2 plants show reduced hypocotyl elongation in response to FR light, and in athb4/hat3 plants hypocotyl elongation in response to FR light is abolished (Sorin et al., 2009).

OsHOX4 of the rice ζ clade is thought to share redundancies in function with its paralog OsHOX20. These genes are similar in structure and, although they possess different tissue-specific expression patterns, both are down-regulated in leaf blades upon exposure to drought stress (Agalou et al., 2008). When OsHOX4 expression was reduced in transgenic rice, using RNA interference, no phenotypic differences were observed. It is evident through mRNA knock-down experiments that there are redundancies in the functions of HD-Zip I and HD-Zip II TFs that cannot always be extended to redundancies in expression. These examples of the facets of redundancy found within the HD-Zip I and HD-Zip II families make it difficult to clarify the role of genes in isolation. Where redundancies exist between related members, paralogs would need to be analyzed using single, double and even triple mutant knockout lines to tease apart both redundant and specific roles.

IV. Conclusions

  1. Top of page
  2. Abstract
  3. I. Introduction
  4. II. The role of HD-Zip transcription factors in plant growth adaptation to environmental changes and the phytohormone network
  5. III. Dissecting the common cis element, dimerization and cell specificity of HD-Zip I and HD-Zip II transcription factors
  6. IV. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

Plants cope with a variety of environmental stresses by modifying their growth pattern to minimize the impacts of stress or to escape damage. The HD-Zip I and HD-Zip II TFs play an integral role in the signaling network that is triggered by endogenous and external stimuli, which leads to the modified growth characteristic of stressed plants (Fig. 3). This growth adaptation is achieved through the regulation of cell differentiation, division and expansion by HD-Zip I and HD-Zip II TFs. However, there are still mechanistic and functional aspects of the HD-Zip I and HD-Zip II network that we need to understand before we are able to characterize the roles of each family in plant growth adaptation to environmental stresses. We know very little of the downstream genes that are ultimately regulated by the HD-Zip I and HD-Zip II TFs. Identification of their target genes is needed to build a comprehensive view of the regulatory pathways and will enable validation of the suite of promoters controlled by HD-Zip I and HD-Zip II proteins to define the cis-acting elements. More information is needed on the interaction profiles of HD-Zip I and HD-Zip II TFs, and the properties that specify interaction partners which contribute to cis element binding. The increasing amount of cell-specific microarray data available in public databases will also enable determination of potential interaction partners that are dependent upon expression in the same cell. If transcriptional regulation by the HD-Zip I and HD-Zip II TFs is conferred through a common cis element, that implies that there are factors determining the specificity of transcriptional activity, as transgenic plants over- or under-expressing HD-Zip I and HD-Zip II TFs have variant phenotypes. Elements contributing to the specificity of action remain elusive. Obtaining data on the three-dimensional structures of the HD-Zip I and HD-Zip II TFs will also decisively contribute to our understanding of the nature of the protein–protein and protein–DNA interactions.

References

  1. Top of page
  2. Abstract
  3. I. Introduction
  4. II. The role of HD-Zip transcription factors in plant growth adaptation to environmental changes and the phytohormone network
  5. III. Dissecting the common cis element, dimerization and cell specificity of HD-Zip I and HD-Zip II transcription factors
  6. IV. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. I. Introduction
  4. II. The role of HD-Zip transcription factors in plant growth adaptation to environmental changes and the phytohormone network
  5. III. Dissecting the common cis element, dimerization and cell specificity of HD-Zip I and HD-Zip II transcription factors
  6. IV. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

Table S1 Phenotypes of transgenic or mutant plants with altered transcript levels of homeodomain-leucine zipper (HD-Zip) I and HD-Zip II transcription factors as reported in the literature

Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office.

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NPH_3733_sm_TableS1.doc68KSupporting info item