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First published online November 30, 2007; 10.1105/tpc.107.055772 The Plant Cell 19:3379-3390 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
A Feedback Regulatory Module Formed by LITTLE ZIPPER and HD-ZIPIII Genes[W],[OA]Department of Plant Biology, Carnegie Institution, Stanford, California 94305 1 Address correspondence to kbarton{at}stanford.edu.
The Arabidopsis thaliana REVOLUTA (REV) protein is a member of the class III homeodomain-leucine zipper (HD-ZIPIII) proteins. REV is a potent regulator of leaf polarity and vascular development. Here, we report the identification of a gene family that encodes small leucine zipper–containing proteins (LITTLE ZIPPER [ZPR] proteins) where the leucine zipper is similar to that found in REV, PHABULOSA, and PHAVOLUTA proteins. The transcript levels of the ZPR genes increase in response to activation of a steroid-inducible REV protein. We show that the ZPR proteins interact with REV in vitro and that ZPR3 prevents DNA binding by REV in vitro. Overexpression of ZPR proteins in Arabidopsis results in phenotypes similar to those seen when HD-ZIPIII function is reduced. We propose a negative feedback model in which REV promotes transcription of the ZPR genes. The ZPR proteins in turn form heterodimers with the REV protein, preventing it from binding DNA. The HD-ZIPIII/ZPR regulatory module would serve not only to dampen the effect of fluctuations in HD-ZIPIII protein levels but more importantly would provide a potential point of regulation (control over the ratio of inactive heterodimers to active homodimers) that could be influenced by other components of the pathway governing leaf polarity.
Early in their development, leaf primordia become polarized along their adaxial/abaxial axes. The adaxial domain of the leaf primordium is closest to the center of the meristem and develops into the upper half of the leaf. The abaxial domain of the leaf primordium is furthest from the center of the meristem and develops into the lower half of the leaf (for reviews, see Canales et al., 2005  ; Carraro et al., 2006)
The establishment of ad/abaxial polarity is fundamental to several aspects of shoot development. First, it is critical for establishing the cellular specializations important for leaf function. The upper portion of the leaf, with its tightly packed, chloroplast-rich palisade cells, is optimized for light capture, while the lower portion of the leaf, with its loosely packed spongy mesophyll cells, is optimized for gas exchange. Second, the establishment of ad/abaxial polarity in the leaf is critical for blade outgrowth. The absence of a properly specified ad/abaxial boundary in the leaf primordium results in rod-shaped leaves that lack blade (Waites and Hudson, 1995
Class III homeodomain-leucine zipper (HD-ZIPIII) proteins are conserved plant proteins that act as potent regulators of ad/abaxial polarity in Arabidosis (Talbert et al., 1995
There are five HD-ZIPIII genes encoded in the Arabidopsis genome: PHABULOSA (PHB)/ATHB14, REVOLUTA (REV), PHAVOLUTA (PHV)/ATHB9, INCURVATA4/CORONA/ATHB15, and ATHB8 (Sessa et al., 1994
Consistent with their redundant action in the plant, the HD-ZIPIII genes are expressed in overlapping patterns. Expression is found principally in the adaxial domains of primordia and in the immature vasculature (Baima et al., 1995
In several cases, mutants in the HD-ZIPIII genes cause alterations in vascular development. For instance, ectopic and overexpression of ATHB8 results in increased expression of xylem (Baima et al., 2001
The HD-ZIPIII proteins contain four recognizable domains. From N terminus to C terminus, these are a DNA binding homeodomain followed immediately by a leucine zipper domain (Ruberti et al., 1991
Unlike animal homeodomain proteins, such as ANTENNAPEDIA, which can bind DNA as monomers, HD-ZIP DNA binding requires dimerization through the leucine zipper domain (Sessa et al., 1993
The HD-ZIPIII proteins are part of a larger network of regulatory factors that establish ad/abaxial leaf fates. The ASYMMETRIC LEAVES1 (AS1) protein, a MYB-domain containing protein, and the AS2 protein, a LOB domain–containing plant-specific protein, act to promote adaxial fates in this network (Byrne et al., 2000
A subset of the transcription factors involved in the establishment of ad/abaxial polarity is controlled by small RNAs. mRNAs encoding the HD-ZIPIII factors are targeted by microRNAs 165 and 166 (Reinhart et al., 2002 Here, we report the discovery of a new family of plant genes, the LITTLE ZIPPER (ZPR) genes, which are transcriptionally upregulated by HD-ZIPIII activity. The ZPR proteins consist principally of a stretch of leucine zipper similar to the leucine zipper in HD-ZIPIII proteins. Furthermore, we show that the ZPR proteins interact with and repress HD-ZIPIII activity, thus forming a negative feedback loop. The balance between HD-ZIPIII:HD-ZIPIII homodimers and HD-ZIPIII:ZPR heterodimers provides a potential point of regulation. We speculate that the START and PAS domains of the HD-ZIPIII protein may affect this balance through interaction with small molecule ligands or protein–protein interactions.
A System for Inducible HD-ZIPIII Function To identify genes whose transcript levels are affected by HD-ZIPIII transcription factor activity, we generated an HD-ZIPIII protein with inducible activity. One of the five Arabidopsis HD-ZIPIII proteins, REV, was placed under glucocorticoid control by fusing the glucocorticoid receptor (GR) domain to its N terminus (Figure 1A ). The GR domain prevents the GR-REV protein from acting in the absence of exogenously added glucocorticoid. The GR-REV gene fusion was placed under the control of the highly and constitutively expressed viral cauliflower mosaic virus 35S promoter (CaMV35S) promoter. Since the HD-ZIPIII genes are negatively controlled by microRNAs 165/166, it was also necessary to introduce mutations that disrupted the microRNA target sequence. Transgenic Arabidopsis seedlings carrying this construct, called 35S:GR-REVd, developed normal leaves in the absence of the glucocorticoid dexamethasone. However, when plate-grown seedlings were flushed with 50 micromolar dexamethasone for 5 min and then transplanted to soil, they developed adaxialized, trumpet-shaped leaves (Figure 1B). Thus, the 35S:GR-REVd fusion construct is capable of directing adaxial leaf development in response to dexamethasone treatment. Subsequent leaves developed normally (Figure 1C), indicating that HD-ZIPIII activity does not persist after this short dexamethasone treatment. (Similar constructs with the GR domain fused to the C terminus of the REV protein, or with a wild-type microRNA complementary site, did not cause adaxialization in this assay; data not shown).
Identification of ZPR Genes as Targets of REV Control Using genome-wide transcriptional profiling, we screened for genes whose transcripts were rapidly upregulated following dexamethasone treatment of 35S:GR-REVd plants. RNA was harvested from 35S:GR-REVd and wild-type Landsberg erecta (Ler) plants after 1 h of dexamethasone treatment or after 1 h of mock treatment. The RNA was labeled and hybridized to Affymetrix microarrays. In this screen, At2g45450 (subsequently named ZPR1) was the most highly induced transcript in 35S:GR-REVd plants in the presence of dexamethasone (see Supplemental Table 1 online for levels of ZPR1 and ZPR3 relative to actin. Data for the entire experiment can be retrieved from the Gene Expression Omnibus repository (series record number GSE7003). Although ZPR1 was annotated in the Arabidopsis database as encoding a chloroplast protein, more intensive comparison of the protein sequence showed that it encodes a leucine zipper–containing protein where the leucine zipper shows similarity to the leucine zipper in the REV protein (see below). ZPR1 is one of four members of a family of small genes, only two of which are represented on the Affymetrix microarray (ZPR1 and ZPR3). To verify that ZPR1 is upregulated in response to increased HD-ZIPIII activity and to test the other three family members for induction, we performed quantitative RT-PCR experiments comparing RNA harvested from treated and untreated seedlings. We found that all four ZPR genes were upregulated in 35S:GR-REVd plants within 2 h of dexamethasone treatment (Figure 1D).
Expression of ZPR Genes in Planta Correlates with HD-ZIP Gene Activity In situ hybridization experiments show that ZPR3 is expressed within the domain known to express REV and are therefore consistent with HD-ZIPIII control of ZPR transcription. ZPR1 and ZPR3 mRNAs are expressed within the domain of HD-ZIPIII–expressing cells; both are expressed in the adaxial epidermis of the cotyledons and in the vascular cylinder of wild-type torpedo stage embryos (Figures 2A to 2D and 2G ). In these experiments, we did not observe ZPR1 or ZPR3 expression in earlier stages (late globular to early torpedo) of cotyledon development.
Because ZPR3 is expressed at very low levels, we constructed a reporter to analyze the spatial distribution of the ZPR3 protein. Therefore, we fused 2.3 kb of 5' regulatory sequences and the ZPR3 coding sequence (including introns) in frame to the bacterial uidA gene (β-glucuronidase [GUS]). Similar to the in situ hybridization signal, the expression of this reporter was limited to the adaxial cotyledon domain and the vascular cylinder (Figures 2E and 2F; note that the dark outline surrounding the torpedo stage embryo in Figure 2E is a shadow around the whole-mount embryo and does not reflect GUS expression). We also examined expression of the GUS transgene in vegetatively growing plants (Figures 2H and 2I). Similar to our findings in cotyledons, expression was seen principally in the adaxial epidermis of leaves. Expression in subepidermal layers occurred in the area over the midvein. In the petiole, expression was seen more extensively in the adaxial domain.
Structure of ZPR Proteins Suggests a Model for Their Action
Leucine zippers are coiled-coil domains consisting of heptad repeats with a Leu residue in the "d" position (Landschulz et al., 1988 ). Each heptad is two turns of the  -helix. In plants, the related amino acids Ile and Met are often found in place of Leu residue at the "d" position (Deppmann et al., 2004). Amino acids at both the "a" and "d" positions are at the interhelical interface (Figure 3C). The "a" position is occupied by Asn in heptads two, three, four, and six in the leucine zippers of both ZPR and HD-ZIPIII proteins. Asn at the "a" position interacts favorably with Asn and unfavorably with aliphatic amino acids at the "a" position in the partner helix (Acharya et al., 2002). Based on the similar pattern of Asn residues at "a" positions in both the ZPR and HD-ZIPIII leucine zippers, the ZPR and HD-ZIPIII proteins have the potential to form parallel heterodimers with one another (Figure 3C).
Unlike many animal homeodomain proteins that bind DNA as monomers, HD-ZIP proteins bind DNA as dimers (Sessa et al., 1993 The structural features of the ZPR proteins, and their induction by HD-ZIPIII activity, suggest a model for the action of the ZPR genes as part of a negative feedback loop on HD-ZIPIII function (Figure 3E). In the proposed regulatory module, HD-ZIPIII proteins activate transcription of the ZPR genes. The ZPR proteins then form heterodimers with the HD-ZIPIII proteins, preventing or altering their DNA binding.
In Vitro Studies of the Interaction between REV and ZPR Proteins
As a further test of the model, we asked whether ZPR3 could prevent DNA binding by REV (Figure 4B). REV protein in the presence of GAD protein bound to a radiolabeled oligonucleotide carrying one HD-ZIPIII binding site causing this oligonucleotide to migrate slower in an acrylamide gel. REV protein did not bind to a similar oligonucleotide in which the sequence was scrambled. In the presence of ZPR3, the ability of REV to bind DNA was abolished.
Overexpression of ZPR1 and ZPR3 Genes
Additional phenotypic similarities between mutants overexpressing ZPR3 and lacking HD-ZIPIII activity are seen in the leaf blade. 35S:ZPR3 plants make leaves with a roughened blade texture and ridges along the midvein and blade (Figure 5F). The roughened leaf with midvein ridges has been seen in mutants with reduced activity of three of the five HD-ZIPIII genes (e.g., phv; phb/+; rev mutants; Prigge et al., 2005 ). 35S:ZPR3 plants with rough blades showed an increased density of veins (Figure 5H). In addition, the veins were surrounded by ridges of small cells, indicating ectopic cellular proliferation (Figure 5J). These ridges were seen on the adaxial but not the abaxial surface. This extra proliferation on the adaxial surface may be, at least in part, responsible for the downward curling of the mutant leaf. In sum, overexpressing ZPR1 and ZPR3 causes leaf abnormalities similar to those seen when HD-ZIPIII function is reduced. Mutants showing dramatically reduced levels of ZPR2 and ZPR3 mRNAs were isolated from publicly available T-DNA insertion collections (see Supplemental Figure 3 online). These mutants were indistinguishable from the wild type. Given the high degree of similarity between the ZPR proteins, this is likely due to redundancy between members of the ZPR gene family. Determination of the exact role these genes play in leaf development will therefore likely required the generation of appropriate double and perhaps higher-order mutant combinations.
Discovery of a Family of ZPR Genes In this article, we have described the discovery of a small family of plant-specific ZPR genes that encode small proteins (67 to 105 amino acids long) that include a stretch of leucine zipper motifs. This stretch of leucine zipper is approximately six heptads in length and is similar to the leucine zipper found in the class III HD-ZIP proteins. In particular, the pattern of Asn residues at the "a" positions of the leucine zippers is similar to that of the HD-ZIPIII proteins, and this pattern predicts that the ZPR proteins and HD-ZIPIII proteins should heterodimerize. Both the ZPR1/ZPR2 (central leucine zipper) and ZPR3/ZPR4 (N-terminal leucine zipper) types of proteins are present in rice and maize as well as Arabidopsis, indicating that the two types of ZPR genes diverged prior to the split between monocots and eudicots. Our searches of other plant databases have yet to turn up homologs of the ZPRs, indicating that they may not be present outside of angiosperm plants. However, we cannot rule out that their small size (making them difficult to annotate) and low abundance may have prevented us from detecting them. It is currently unclear whether the ZPR genes and the HD-ZIPIII genes share a common ancestor or whether their similar leucine zipper regions are examples of convergent evolution. There are three additional classes of HD-ZIP proteins in addition to the class III type studied here. These are the class I, II, and IV types. While these all have leucine zippers, the leucine zipper motif is distinct in all four classes, and the ZPR proteins described here are not predicted to interact with them. However, it remains possible that these families are regulated by other families of as yet undiscovered leucine zipper–containing proteins. Our work on this gene family was prompted by the discovery that ZPR1 transcript levels are upregulated by the REV protein, a potent regulator of adaxial leaf development. Increases in ZPR transcript levels were also seen in gain-of-function mutants for two other HD-ZIPIII genes (PHB and PHV), indicating that upregulation of ZPR transcript levels may be a more general feature of HD-ZIPIII function. Given the relatively rapid upregulation of ZPR transcript levels, it is possible that HD-ZIPIII transcription factors bind to DNA at the ZPR loci and act as direct transcriptional activators of these loci. However, evidence for a role of HD-ZIPIII transcription factors as direct activators of ZPR transcription awaits further experimentation.
Interestingly, for the two ZPR genes examined, expression begins well after HD-ZIPIII expression is first established in the embryo. Whereas expression of REV, PHB, and PHV is seen in the early globular embryo (McConnell et al., 2001
A Negative Feedback Loop What then is the role of this feedback loop in the wild-type plant? The wild-type phenotype of the single mutants isolated thus far indicates that these genes are likely to act redundantly to one another. It will likely be necessary to maker higher-order mutant combinations to see a phenotype. The relatively late onset of ZPR1 and ZPR3 expression indicates that at least these two ZPR genes play a role in adaxial leaf development that occurs after the initial establishment of polarity in the primordium. Likely roles include promoting vascular development and growth in the adaxial leaf domain by preventing the action of HD-ZIPIII proteins. Such late events in leaf development would likely be important for the fine-tuning of leaf development to prevailing environmental conditions.
The discovery of the ZPRs and the finding that HD-ZIPIII proteins have the potential to exist in two types of dimers, inactive heterodimers and active homodimers, adds a new point of potential regulation in which the ratio of inactive heterodimers to active homodimers could be controlled. Note that we use homodimers to refer to dimers made of two HD-ZIPIII proteins, whether these are dimers of one HD-ZIPIII (e.g., REV-REV dimer) protein or dimers of two different HD-ZIPIII proteins (e.g., REV-PHB dimer). The presence of a START domain in the HD-ZIPIII proteins has puzzled researchers because, although indicating that HD-ZIPIII activity is modulated by a small hydrophobic ligand, the role of such a ligand has eluded understanding. An attractive hypothesis that follows from the results presented here is that the ligand could control HD-ZIPIII activity by influencing the type of dimer that is formed, for instance by promoting or inhibiting the formation of either active HD-ZIPIII/HD-ZIPIII homodimers or inactive HD-ZIPIII/ZPR heterodimers. Chandler et al. (2007) The role of this feedback loop in leaf development adds a second layer of negative regulation of the HD-ZIPIIIs to that mediated by microRNAs 165 and 166. Whether the two negative regulatory loops function independently of one another or are in some way interconnected remains to be determined.
Generation of 35S:GR-REVd Transgenic Plants The N-terminal coding sequences of REV were amplified using PCR from the wild-type cDNA using primers REV 5' fusion and HB8L2 with a combination of Pfu and Taq polymerases to produce a product with 3' A overhangs. This gave the first 251 codons of the REV coding sequence and incorporated the ATG start codon into an NcoI site. This PCR product was cloned into pGEM T-EASY and cut out again with an EcoRI site in the vector polylinker and a SalI site in the REV coding sequence to isolate the 51 N-terminal-most codons. This fragment was then fused to each of three REV cDNAs; wild-type, G189D, and  miRNA, the latter two of which contain a gain-of-function mutation in the miR165/166 binding site as described by Emery et al. (2003). These cDNAs were then subcloned into the shuttle vector pART7 carrying a CaMV35S promoter followed by modified GR coding sequences (Wagner et al., 1999) ending in an NcoI site for in-frame fusion of the NcoI-REV cDNAs and followed by 3' untranslated region sequences from ocs to provide a poly(A) signal. The entire 35S:GR:REV:ocs fusion was transferred into the pMLBART binary vector using two flanking NotI sites. Constructs were introduced into Agrobacterium tumefaciens strain GV3101 by electroporation and then into Arabidopsis thaliana Ler plants by floral dipping. Plasmids pART7 and pMLBART were obtained by J.E. from Bart Janssen and Kim Richardson (Horticultural and Food Research Institute of New Zealand, Auckland).
Microarray Experiment
Histology
Real-Time RT-PCR
In Situ Hybridization
In Vitro Studies For in vitro DNA binding assays, proteins were individually synthesized (for 45 min) using the Promega TNT T7 quick coupled transcription/translation system in the presence of nonradioactive Met. Proteins (REV + GAD; REV + ZPR3) were then mixed and incubated for another 45 min. Single-stranded oligonucleotides were denatured at 95°C for 10 min and then cooled to room temperature allowing the formation of a double strand. The DNA sequences of the duplexes were 5'-CAGATCTGTAATGATTACGAGAAT-3' for the HB-9 oligonucleotide harboring the HD-ZIPIII binding site and 5'-CAGATCTGTATAATATACGAGAAT-3' for the HB-9mut oligonucleotide in which the core nucleotides of the HD-ZIPIII binding site were altered.
Duplex DNA was end-labeled with T4 polynucleotide kinase and [
Accession Numbers
Supplemental Data
We thank Doris Wagner (University of Pennsylvania) for the gift of the plasmid containing the glucocorticoid response sequences and Michael Prigge (University of Indiana) for the gift of the rev/+ phv phb line. Khar-Wai Lye provided excellent technical assistance. The work was supported by a grant from the National Institutes of Health (to M.K.B.) and a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft (to S.W.; DFG WE4281/1-1).
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: M.K. Barton (kbarton{at}stanford.edu).
[W] Online version contains Web-only data.
[OA] Open access articles can be viewed online without a subscription. www.plantcell.org/cgi/doi/10.1105/tpc.107.055772 Received September 20, 2007; Revision received October 23, 2007. accepted November 8, 2007.
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ABSTRACT
INTRODUCTION
20% in the direction of the y axes. The photo was cropped at the bottom; no bands below those shown were seen. The top of the gel photo was not cropped.
-32P]ATP. DNA binding reactions were performed using the Promega gel shift assay system. TNT-produced protein mixes were incubated in gel-shift binding buffer for 1 h on ice. After adding the radiolabeled nucleotides, binding reactions were incubated at room temperature for 20 min and subsequently separated on 6% DNA retardation gels (Invitrogen), dried, and exposed to a phosphor imager screen. 