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First published online January 20, 2006; 10.1104/pp.105.070565 Plant Physiology 140:1095-1108 (2006) © 2006 American Society of Plant Biologists The Arabidopsis Zinc Finger-Homeodomain Genes Encode Proteins with Unique Biochemical Properties That Are Coordinately Expressed during Floral Development1Department of Molecular, Cellular, and Developmental Biology (Q.K.-G.T., V.F.I.) and Department of Ecology and Evolutionary Biology (V.F.I.), Yale University, New Haven, Connecticut 06520–8104
Arabidopsis (Arabidopsis thaliana) contains approximately 100 homeobox genes, many of which have been shown to play critical roles in various developmental processes. Here we characterize the zinc finger-homeodomain (ZF-HD) subfamily of homeobox genes, consisting of 14 members in Arabidopsis. We demonstrate that the HDs of the ZF-HD proteins share some similarities with other known HDs in Arabidopsis, but they contain distinct features that cluster them as a unique class of plant HD-containing proteins. We have carried out mutational analyses to show that the noncanonical residues present in the HDs of this family of proteins are important for function. Yeast (Saccharomyces cerevisiae) two-hybrid matrix analyses of the ZF-HD proteins reveal that these proteins both homo- and heterodimerize, which may contribute to greater selectivity in DNA binding. These assays also show that most of these proteins do not contain an intrinsic activation domain, suggesting that interactions with other factors are required for transcriptional activation. We also show that the family members are all expressed predominantly or exclusively in floral tissue, indicating a likely regulatory role during floral development. Furthermore, we have identified loss-of-function mutations for six of these genes that individually show no obvious phenotype, supporting the idea that the encoded proteins have common roles in floral development. Based on these results, we propose the ZF-HD gene family encodes a group of transcriptional regulators with unique biochemical activities that play overlapping regulatory roles in Arabidopsis floral development.
The homeodomain (HD) is a 60-amino acid DNA-binding domain (BD) found in many transcription factors. HD-containing proteins are found in diverse organisms such as humans, Drosophila, nematode worms, and plants, where they play important roles in development (for example, McGinnis et al., 1984a  , 1984b; Scott and Weiner, 1984; Chan et al., 1998; Veraksa et al., 2000). The crystal structures of different HDs bound to DNA have been isolated and the structures have been found to be remarkably similar. The HD consists of three  helices and a flexible N-terminal arm. Helix III, the recognition helix, packs against the DNA major groove and makes specific contacts with the DNA (for review, see Gehring et al., 1994; Wolberger, 1996). HD proteins have been explicitly shown to act as transcription factors because they bind to promoter sequences and act to modulate mRNA synthesis (for example, see Biggin and Tjian, 1989).
In Arabidopsis (Arabidopsis thaliana), about 100 HD-encoding genes have been identified, and they belong to a few specific classes (Chan et al., 1998
The WOX class of HD-containing genes appears to be essential for embryonic patterning, specifying different regions in the embryo (Haecker et al., 2004
Class III HD-Leu zipper (ZIP) proteins are characterized by a START domain and a HD-ZIP at the N terminus. The five Class III HD-ZIP members in Arabidopsis all appear to be regulated by mRNAs (Bowman, 2004
The zinc finger (ZF)-HD family of HDs has not been functionally characterized in Arabidopsis. In Flaveria trinervia, four ZF-HD proteins were identified and found to bind to the regulatory regions of the C4 phosphoenolpyruvate carboxylase gene, using a yeast one-hybrid screen (Windhövel et al., 2001
To identify DNA-binding proteins involved in Arabidopsis floral development, we carried out a yeast one-hybrid screen using floral-specific promoter sequences. The DNA-binding protein ATHB33, one of the members of the ZF-HD family, was identified in this screen (Q. K.-G. Tan and V.F. Irish, unpublished data). There are 14 members of this family in Arabidopsis (Fig. 1A ) and 11 members had been previously assigned ATHB names (Windhövel et al., 2001 ).
Conservation of Domains of ZF-HD Family Members
All the Arabidopsis ZF-HD family members encode small proteins, with the longest sequence containing only 334 amino acids. None of the genes contain introns in their coding sequences. Members of the ZF-HD class contain one conserved region upstream of the HD (Fig. 1A). This upstream region corresponds to the ZF domain and contains conserved His and Cys residues. The ZF domain has been shown to be sufficient in mediating dimerization between different ZF-HD proteins in Flaveria, with the Cys residues being important for interaction (Windhövel et al., 2001). The ZF (CX+NHAX3GX4DGCXEFX8–15CX2CXCHRXFH) is characterized by a CHCC3H2 motif with a variable length spacer between the first CHC residues and the more carboxy terminal C3H2 residues. It forms a distinct group compared with other ZF motifs characterized in Arabidopsis (Takatsuji, 1998
The HDs of the Arabidopsis family members are aligned in Figure 1B, together with other representative HDs. The HD in the ZF-HD family is most closely related to the HDs that are associated with LIM domains (Windhövel et al., 2001
We examined the importance of the HD in DNA binding by generating a variety of mutated versions of ATHB33. While full-length ATHB33 binds to target promoter sequences in yeast one-hybrid assays, removal of the HD from ATHB33 abolished the binding to target promoter sequences (Fig. 2
). Most HD-containing transcription factors identified contain a canonical Phe at residue 49 in the HD region (Fig. 1B; Hirsch and Aggarwal, 1995
Mapping of the ZF-HD Consensus-Binding Sequence
Yeast one-hybrid assays were used to delimit the domain to which ATHB33 binds to 30 bp of the original 202-bp promoter fragment (data not shown). Within this region, there is a binding sequence for the
To explicitly map the binding sequence of ATHB33, we carried out electrophoretic mobility shift assays. Two overlapping fragments spanning the 30-bp binding region were initially used to localize the binding of ATHB33 to a 20-bp fragment that contains the consensus NNATTA (Fig. 3A
). A series of 20-bp oligonucleotides, in which one or two bases were mutated, were then used to define sequences required for binding (Fig. 3, A and B). Both the BM1 and BM2 mutated oligonucleotides retained the ability to bind ATHB33, while BM3 and BM4 abrogated binding. In addition, competition assays were used to assess the ability of these oligonucleotides to disrupt binding of ATHB33 to the B fragment (Fig. 3C). The relative strength of competition was BM1 > BM2 > BM4 > BM3, indicating that the mutated bases in BM4 and BM3 were the least effective in disrupting binding. Together, these results show that ATHB33 binds to a core consensus sequence of ATTA, similar to that of other HD-containing proteins (Gehring et al., 1994
The ZF-HD Family Comprises a Distinct Family of HDs
To compare the HD in the 14 Arabidopsis ZF-HD proteins with the HDs found in the other major Arabidopsis HD families, phylogenetic analyses were carried out (Swofford, 2003
BLAST searches revealed that no homologs of the ZF-HD proteins are present in Homo sapiens, C. elegans, Mus musculus, Drosophila melanogaster, and Danio rerio, and no homologs were found when the ZF-HD sequence was used in BLAST searches against 16 fungal and 303 bacterial genomes. However, a hypothetical protein with a region similar to the ZF was found in the fungus Magnaporthe grisea and a Ser protease in the Bdellovibrio bacteriovorus bacterial genome. The HD was most similar to that of LHX8 found in humans and mouse. The ZF-HD sequence was searched against plant genomic databases (www.plantgdb.org), using tBLASTn against expressed sequence tags, cDNAs, or genomic sequences. Genes containing both the ZF and HD were found in a variety of plant genomes, including gymnosperms (Pinus taeda), as well as a number of angiosperm species, including monocots (e.g. Triticum aestivum, maize, and rice) and eudicots (e.g. Citrus sinensis, Glycine max, Gossypium arboreum, Medicago truncatula, and Phaseolus vulgaris). Sequences similar to the ZF were found in the fern Ceratopteris richardii, while a noncanonical similar HD sequence was found in the moss Physcomitrella patens. These data suggest that the ZF-HD gene family is specific to the plant kingdom, and implies that such genes have plant-specific functions. Furthermore, the apparent absence of the combination of the ZF and HD motifs in clades other than gymnosperms and angiosperms suggests that the ZF-HD family may have arisen during the evolution of seed plants.
To analyze the expression patterns of the ZF-HD family members, northern-hybridization analyses were carried out (Fig. 5
). Strikingly, RNA of 13 of the family members was detected predominantly or exclusively in floral tissue. The expression of the ATHB26 gene could not be detected by northerns, nor could it be detected by Massively Parallel Signature Sequencing with confidence (Meyers et al., 2004
A comparison was carried out between the expression data we obtained and the expression profiles of the ZF-HD family members as assessed by microarray analyses. Using the Gene Atlas feature of the Genevestigator Web site (Zimmermann et al., 2004 ), which collates data from different microarray experiments (Edgar et al., 2002; Brazma et al., 2003; Craigon et al., 2004), the expression profile of these genes is largely similar to our northern data. One exception is ATHB27, for which we only see high levels of expression in open flowers, but is reported to be fairly ubiquitously expressed by Gene Atlas. High levels of seed expression are observed in Gene Atlas for ATHB22 and ATHB29, which may explain the particularly high expression level we obtained for RNA extracted from open flowers corresponding to these two genes. Strong floral expression is seen for both data sets for ATHB28 and ATHB31, although there are some minor variations in expression levels seen in other tissues.
The similarity in expression domains of the ZF-HD family members suggests that they may have similar roles in regulating floral development. To explicitly test this possibility, we identified T-DNA insertional mutations in ATHB22, ATHB23, ATHB25, ATHB29, ATHB31, ATHB32, ATHB33 (two insertions), and ATHB34 (data not shown). Homozygous mutant lines were identified for each of these mutations using PCR genotyping (see "Materials and Methods"); however, none of these nine mutant lines showed any detectable developmental or morphological phenotype (data not shown). Reduced levels of transcripts for the mutations in ATHB31 and ATHB33 were confirmed using northern analyses (data not shown).
To characterize the dimerization partners of the Arabidopsis ZF-HD family members, we carried out a matrix of yeast two-hybrid assays, with each family member linked to the yeast GAL4AD or GAL4BD. We carried out these interaction studies for each family member in both combinations. As shown in Table I , the strengths of the interactions between these proteins reveal interesting trends. The Arabidopsis proteins appear to form stronger heterodimers compared to homodimers, as assessed by reporter gene expression. The only exception is ATHB22, which forms a strong interaction with itself, as well as with many other members. Some of the other members, namely ATHB25 and ATHB29, are likewise promiscuous in their interactions. ATHB29 possesses its own AD, since the construct with ATHB29 linked to BD could activate reporter gene expression when coexpressed with just the AD alone. On the other hand, when linked to the AD, ATHB29 interacts with virtually all family members. This is an interesting finding, since ATHB29 could potentially serve as the partner that confers activation of transcription, whereas the other partner could dictate DNA-binding specificity. According to the northern data, the ATHB29 gene is expressed at high levels in flowers only, with low expression in roots (Fig. 5). It is likely that ATHB29 confers floral specificity on the transcriptional complex containing ZF-HD family members.
In general, the dimerization characteristics of the proteins are consistent in both combinations tested. The few exceptions are ATHB23/ATHB29, ATHB23/ATHB31, ATHB25/ATHB34, ATHB28/ATHB33, ATHB28/ATHB34, ATHB33/ATHB34, ATHB22/ATHB34, and ATHB23/ATHB34, which differ to some degree in strength. We only identified one ZF-HD, ATHB26, which does not dimerize at all with any of the other family members. As previously described, ATHB26 does not appear to be expressed. ATHB27 and ATHB32 are two members that form weak dimers with some other members of the family. It may be possible that these weak interactions indicate that ATHB27 and ATHB32 require the presence of cofactors to stabilize the resulting complex. Another possibility is that ATHB27 and ATHB32 contain repressor domains, which overcome the activity of the yeast AD in this assay.
A Plant-Specific ZF-HD Family We have characterized the ZF-HD family of homeobox genes in Arabidopsis. To date, ZF-HD proteins have only been identified in plants, and likely play plant-specific roles. Our phylogenetic analyses support the idea that these genes arose during land plant evolution, and further show that some of the ZF-HD genes likely arose by gene duplication events. We have also shown that members of this family possess a noncanonical residue within the conserved HD, and that this variant residue is required for DNA binding to a core consensus sequence of ATTA. These data, in combination with our findings that all Arabidopsis members of this family are expressed in flowers, and that at least six of these genes have redundant roles, suggest the intriguing possibility that such genes coordinately function in regulating plant reproductive development.
We have shown that the ZF-HD family members contain a noncanonical residue at position 49 in the HD that is required for DNA binding. Mutation of the Met in this position in ATHB33 completely abrogates DNA binding (Fig. 2), demonstrating the importance of this noncanonical residue for binding in the context of these variant HD-containing proteins. Thus, the binding properties of the ZF-HD family members may differ from other HDs with a Phe at position 49. Various HD-DNA structures have been crystallized (Kissinger et al., 1990
In terms of residues that contact the major groove of DNA, the ZF-HD proteins contain a conserved Asp at position 51, as in all HDs. Position 47 in the ZF-HD family is occupied by Val or Ile in most of the family members in Arabidopsis, which is similar to HD proteins that contact DNA bases using this residue. For most of the ZF-HD family members, residue 54 is a polar group (Gln, Asp), suggesting that this residue may confer some binding similarity to the family, since this residue may mediate some DNA-binding specificity (Wolberger et al., 1991
As for the other residues in the HD involved in contacting DNA, the ZF-HD family possesses the conserved Trp at position 48. The ZF-HD proteins also contains a Lys at position 53, which has a positive side chain and thus is similar to the Arg at position 53 in other HD proteins, and likely makes the same kind of contacts to the DNA backbone. The residue at position 25 for the family is not a Tyr, as in many HDs crystallized to date, but a positive residue, in 11 of the 14 family members. It is interesting to note that a Lys residue also resides in the equivalent position in the Oct-1 HD, and this residue also makes phosphate contacts similar to the others with a Tyr in this position (Klemm et al., 1994
As also noted by Windhövel et al. (2001)
All in all, the HDs crystallized to date, although varying in the identity of some residues that contact DNA, appear to bind DNA in a very similar spatial manner. Supporting this idea, we have found that the ZF-HD family member ATHB33 binds to a core consensus sequence of ATTA, similar to that of other HD-containing proteins (Gehring et al., 1994
For all but one of the ZF-HD family members, we could detect strong expression in flowers (Fig. 5). We could not detect any expression for ATHB26 using northern hybridization (data not shown), and since the expression of ATHB26 is not identified by microarray experiments as well (Zimmermann et al., 2004
The northern data raises the interesting possibility that the ZF-HD family may share important roles in floral development. This idea is consistent with the observation that members of some classes of transcription factors perform similar functions in plants. For example, the YABBY family of transcription factors in Arabidopsis specifies abaxial identity in various organs (Bowman, 2000 It is likely that the Arabidopsis ZF-HD members have largely overlapping or redundant roles. For instance, ATHB23, ATHB34, and ATHB30 are closely related to each other (Fig. 4) and have similar patterns of expression (Fig. 5). As for the factors that are largely floral specific, ATHB21 and ATHB31 are close relatives (Fig. 4). This suggests that members that are closely related to each other may play similar roles in plant development. Because it is likely that the ZF-HD gene products play redundant roles, loss-of-function mutations in multiple family members will likely have to be generated to identify their functions. Individual loss-of-function mutations have been obtained in ATHB23, ATHB25, ATHB31, ATHB32, ATHB33, and ATHB34, but none of them display mutant phenotypes (data not shown), supporting the idea that there is a high degree of redundancy in this family.
Using yeast two-hybrid analyses, we found that the ZF-HD family members in general form strong heterodimeric interactions. This raises the intriguing possibility that heterodimerization between different ZF-HD members serves to regulate the transcription of floral genes, since all the members are expressed in flowers. Heterodimerization of HD proteins is one mechanism that can modulate the specificity of target gene activation. For instance, the HD proteins Aristaless-like4 (Alx4) and Goosecoid are capable of self dimerization, as well as forming heterodimers with each other. The homodimers and heterodimer preferentially bind to different DNA sequences (Tucker and Wisdom, 1999
Heterodimerization likely occurs through sequences in the ZF region, although dimer stability in vivo may involve sequences in the HD. Heterodimerization through the ZF domain has been found for the ZF-HD family members in Flaveria (Windhövel et al., 2001
Some of the interactions between HDs and other proteins occur through the three-amino acid extension (between helix I and II) in the TALE class of HD proteins (for review, see Burglin, 1997 In addition to the 14 family members, it is very intriguing that there are two expressed sequences in Arabidopsis that only contain the upstream ZF regions (At1g74660 and AAM63930, data not shown) and not the HD. Because these factors do not contain the HD, they presumably cannot bind DNA (this study; Windhövel et al., 2001). One possibility is that these sequences play a regulatory role in titrating the function of the ZF-HD class. It could be envisioned that these non-HD-containing factors may heterodimerize with the ZF-HD proteins, and modify their DNA-binding activity through modulating target site specificity or affinity, or through affecting interactions with cofactors.
ATHB29 is expressed predominantly in floral tissues and possesses its own AD. The insertional mutant of ATHB29 does not have obvious floral phenotypes (data not shown), so it is likely that ATHB29 is not the only protein that confers transcriptional activation to the rest of the family. Other ZF-HD members may have transcriptional activation capabilities that we could not detect in the two-hybrid assay. Moreover, HD-containing proteins are known to interact with other activators/repressors to regulate transcriptional specificity or activity. For example, the yeast
Phylogenetic Analyses
The HD sequences were downloaded from GenBank. HD sequences from two members from each homeobox class in Arabidopsis (Arabidopsis thaliana) were used for the alignment (classes were selected based on Chan et al., 1998
The HD sequences were aligned with ClustalX (1.81), and alignments refined by hand using MacClade 4.03.68K (Maddison and Maddison, 1989
Phylogenetic analyses to generate maximum parsimony trees were performed using PAUP 4.0b10 (Swofford, 2003
Bayesean analyses were carried out using MrBayes 3.1 (Ronquist and Huelsenbeck, 2003
SLAT lines: The SLAT collection of Arabidopsis insertion lines was created by Dr. Jonathan Jones, Sainsbury Laboratory, John Innes Center, Norwich, UK. The lines are of the Columbia ecotype containing dSpm (a nonautonomous Spm derivative) insertions in the genome. DNA from the iPCR products of the SLAT plants (approximately 48,000 transposants) was arranged into superpools and subpools and spotted onto a filter. The filter was probed with the gene of interest, and plants from the subpool with a hit were grown, genomic DNA was isolated from these plants, and PCR was used to isolate the plant with the insert. dSpm primers and PCR protocols were obtained from the SLAT line Web site (http://arabidopsis.info/CollectionInfo?id=51).
SAIL lines: The SAIL T-DNA lines were generated by the Torrey Mesa Research Institute/Syngenta Biotechnology, San Diego, CA (Sessions et al. 2002
SALK Lines: The SALK SiGNAL T-DNA lines (Alonso et al., 2003 For all mutant lines, plant tissues were ground in liquid nitrogen using an eppendorf tube pestle. Three-hundred microliters of extraction buffer (200 mM Tris, pH 7.5; 250 mM NaCl; 25 mM EDTA; and 0.5% SDS) was added to the ground tissue and the tube was incubated at 50°C for 20 to 30 min. An equal volume of phenol:chloroform was then added to the mixture, mixed, and then centrifuged for 5 min. Three-hundred microliters of isopropanol was added to the supernatant, mixed, and frozen at –20°C for 30 min. The DNA was obtained by a 10-min centrifugation, and it was washed with 70% ethanol, dried, dissolved in Tris-EDTA, and used in PCR reactions. Taq polymerase was obtained from Promega or NEB and Pwo polymerase was obtained from Roche. PCR reactions were performed as recommended by the respective Web sites.
Seedling tissue from Arabidopsis Landsberg erecta was collected at 6 d postgermination, grown on Murashige and Skoog plates (all plant media from Sigma) or soil. Root tissue was collected from plants grown on Murashige and Skoog plates. The other tissues were collected from soil-grown plants. All plants were grown in standard long-day conditions (16-h light/8-h dark; 22°C). RNAs from tissues were prepared using Trizol (Invitrogen) following the manufacturer's protocol. For RNA blots, 25 µg of RNA was loaded per lane; the gels were then treated with 50 mM NaOH/10 mM NaCl, and subsequently with 100 mM Tris, pH 7.5. They were blotted onto a Hybond-N membrane (GE Healthcare Amersham). The probes used for hybridization were unique regions for each gene. Hybridization was carried out at 42°C overnight in a solution containing 50% formamide, 3x SSC, 0.1 mg/mL ssDNA, 5x Denhardt's, 5% Dextran Sulphate, and 25 mM EDTA, pH 8. The filters were then washed in SSC. All chemicals were obtained from Sigma.
The –285 to –83 region of the promoter of APETALA3 (At3g54340; Hill et al., 1998
For the no-HD construct, the HD of ATHB33 was removed using BglII and ligated to the pGAD424 vector, which contains the yeast GAL4AD (BD Biosciences Clontech). This construct lacks the HD sequence and 77 bp 5' to the HD. As for the FL construct, the full-length ATHB33 fragment (coding region and 33 bp of 3' untranslated region) was first cloned into pCR2.1 using the TOPO TA cloning kit (Invitrogen). The insert was then digested with EcoRI, and cloned into the EcoRI site in pGAD424 to give FL. For the no-AD construct, the AD sequence in FL (in pGAD424) was cut out using KpnI and a partial digest of EcoRI, and the plasmid was then blunt ended with Klenow polymerase (NEB) and religated. The MtoA (Met 49 to Ala) and MtoF (Met 49 to Phe) mutations were engineered using PCR, as described in Innis et al. (1990)
LacZ liquid assays provide a quantitative measure of the amount of reporter expression. For the liquid assay, the yeast colonies were grown on synthetic dextrose (SD)/Leu liquid media for 2 d at room temperature. They were then transferred to 2 mL of yeast peptone dextrose and grown for 3 to 4 h at room temperature. One milliliter of the culture was then centrifuged and resuspended in 800 µL of Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, and 50 mM
Each construct was assayed using five independent transformants and the assays were performed at least twice for consistency.
The open reading frame sequences of all ZF-HD family members were obtained from Ler wild-type tissue using reverse transcription-PCR. Five micrograms of the total RNA was reverse transcribed in a 20 µL reaction, using 0.5 µg of oligo dT primer and Superscript II (Invitrogen), according to the manufacturer's recommendations. The cDNA was diluted 20x, and 5 µL was used in PCR reactions using the Expand HiFidelity PCR system (Roche). PCR products were cloned into pCR2.1 (Invitrogen) and sequenced. All the constructs were ligated into either the pGAD424 vector or the pGBT9 vector (containing the GAL4BD sequence). The plasmids containing the AD sequence were transformed into Y187 yeast strain (MAT
The ATHB33 coding region was cloned into pSP72A (Promega) and a transcription reaction was performed with SP6 RNA polymerase (Roche) at 37°C for 7 h. The mixture was then treated with DNAse I (Roche). Five micrograms of RNA was used for in vitro translation using the Rabbit Reticulocyte Lysate system (Promega). 35S Met (Amersham) was used to label the protein, and the reaction was carried out following the manufacturer's protocol. As a control, the Luc control RNA was also translated, and a control reaction containing no RNA was also set up. The integrity of the proteins was checked on a polyacrylamide gel.
Oligonucleotides were generated by the Keck Facility at Yale University. To anneal, complementary pairs of oligonucleotides were dissolved in 0.05 M NaCl, boiled, then slowly cooled to room temperature. A total of 1.5 units of shrimp alkaline phosphatase (USB) were then added to dephosphorylate the ends of the DNA. After the reaction, the phosphatase was inactivated and removed, and the DNA was precipitated. The DNA was labeled with gamma32P ATP (Amersham) using 10 units of T4 polynucleotide kinase (NEB). Labeled DNA was spotted onto DE81 filters to measure the specific activity of the labeling reaction. The excess probe was then removed using sephadex G50 microspin columns (Amersham). Electrophoretic mobility shift reactions were run on an 8% polyacrylamide gel. For binding reactions, 6 fmol of each probe was used in a mixture containing 0.8 µg poly dIdC, 20 mM HEPES, pH 7.8, 50 mM NaCl, 10 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, and 10% glycerol (Windhövel et al., 2001
We thank members of the Irish lab for their constructive comments during the course of this work, and thank Sang-Tae Kim and Irvin Pan for help with the phylogenetic analyses. The SLAT filter was kindly generated and donated by Drs. Stephen Rutherford and Jane Langdale. Received August 26, 2005; returned for revision December 16, 2005; accepted January 5, 2006.
1 This work was supported by a grant from the U.S. Department of Agriculture (grant no. 2001–35304–2226 to V.F.I.). 
2 Present address: Department of Pediatrics, Yale University School of Medicine, New Haven, CT 06520–8064. 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.plantphysiol.org) is: Vivian F. Irish (vivian.irish{at}yale.edu). Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.070565. * Corresponding author; e-mail vivian.irish{at}yale.edu; fax 203–432–5711.
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-galactosidase reporter gene-containing vector pLacZi (BD Biosciences Clontech). The promoter fragments were cloned in the sense or antisense orientation with respect to the reporter genes. These reporter constructs were then linearized with the appropriate enzymes and transformed into yeast (Saccharomyces cerevisiae) strain YM4271. The resultant yeast strain had the promoter-LacZ reporter integrated into its genome. 
