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First published online November 9, 2007; 10.1104/pp.107.109538 Plant Physiology 146:250-264 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
A Genomic and Expression Compendium of the Expanded PEBP Gene Family from Maize[W],[OA]Pioneer Hi-Bred International Inc., a DuPont business, Johnston, Iowa 50131
The phosphatidylethanolamine-binding proteins (PEBPs) represent an ancient protein family found across the biosphere. In animals they are known to act as kinase and serine protease inhibitors controlling cell growth and differentiation. In plants the most extensively studied PEBP genes, the Arabidopsis (Arabidopsis thaliana) FLOWERING LOCUS T (FT) and TERMINAL FLOWER1 (TFL1) genes, function, respectively, as a promoter and a repressor of the floral transition. Twenty-five maize (Zea mays) genes that encode PEBP-like proteins, likely the entire gene family, were identified and named Zea mays CENTRORADIALIS (ZCN), after the first described plant PEBP gene from Antirrhinum. The maize family is expanded relative to eudicots (typically six to eight genes) and rice (Oryza sativa; 19 genes). Genomic structures, map locations, and syntenous relationships with rice were determined for 24 of the maize ZCN genes. Phylogenetic analysis assigned the maize ZCN proteins to three major subfamilies: TFL1-like (six members), MOTHER OF FT AND TFL1-like (three), and FT-like (15). Expression analysis demonstrated transcription for at least 21 ZCN genes, many with developmentally specific patterns and some having alternatively spliced transcripts. Expression patterns and protein structural analysis identified maize candidates likely having conserved gene function of TFL1. Expression patterns and interaction of the ZCN8 protein with the floral activator DLF1 in the yeast (Saccharomyces cerevisiae) two-hybrid assay strongly supports that ZCN8 plays an orthologous FT function in maize. The expression of other ZCN genes in roots, kernels, and flowers implies their involvement in diverse developmental processes.
The phosphatidylethanolamine-binding protein (PEBP) genes are found in all three major phylogenetic divisions of prokaryotes, archaea, and eukaryotes (Banfield et al., 1998  ; Hengst et al., 2001; Chautard et al., 2004). Their conserved sequences indicate an ancient common origin of a basic protein functional unit. The seminal PEBP described was isolated from bovine brain as a soluble 23-kD basic cytosolic protein (Bernier and Jolles, 1984). Animal PEBP proteins were subsequently shown to act as Raf kinase inhibitors (Yeung et al., 1999; Lorenz et al., 2003; Odabaei et al., 2004) in signaling cascades that control cell growth (Keller et al., 2004). Mouse PEBP exerts activity as a Ser protease inhibitor (Hengst et al., 2001). Overall, however, mammalian PEBPs are multifunctional proteins whose physiological roles are just beginning to be understood.
Plant PEBP-related genes were originally cloned from mutants with altered inflorescence architecture. These include Antirrhinum CENTRORADIALIS (CEN; Bradley et al., 1996
The Arabidopsis FLOWERING LOCUS T (FT) gene is a member of the PEBP gene family, but it has an opposing function to TFL1: It promotes the transition from the vegetative to the reproductive phase (Kardailsky et al., 1999
The function of FT is highly conserved in plants. The rice (Oryza sativa) FT homolog encoded by Heading date3a (Hd3a; also known as OsFTL2) migrates from leaves to the shoot apical meristem to induce the floral transition (Tamaki et al., 2007
Four additional PEBP genes were found in the Arabidopsis genome by homology: TWIN SISTER OF FT (TSF), BROTHER OF FT AND TFL1 (BFT), ARABIDOPSIS THALIANA CENTRORADIALIS (ACT), and MOTHER OF FT AND TFL1 (MFT). TSF and MFT seem to act as floral integrators redundantly with FT (Yoo et al., 2004
Identification and Characterization of the ZCN Gene Family
Extensive searches of public and proprietary transcript and genomic databases with six Arabidopsis PEBP proteins as BLAST queries (TFL1, FT, ATC, BFT, TSF, and MFT) identified 25 ZCN genes that were completed sequences from the appropriate bacterial artificial chromosome (BAC) clones (Table I
). One of these, ZCN23, appears to be a pseudogene because only exon 4 was found in the 200-kb sequence of the corresponding BAC. All complete 25 maize genes included DNA fragments previously identified by Chardon and Damerval (2005)
The nearest genetic markers for each ZCN gene were determined from BAC contigs and used to position the ZCN genes on the maize genetic map (Table I; Fig. 1 ). In addition to in silico mapping, the chromosome location of each ZCN gene was confirmed by PCR using the oat (Avena sativa)-maize addition lines (Supplemental Fig. S1; Supplemental Table S1), which are a set of 10 oat lines each carrying a single maize chromosome addition (Kynast et al., 2001 ). ZCN2, ZNC13, ZCN20, ZCN21, and ZCN26 mapped near centromeres (Fig. 1).
All the maize ZCN gene family members appeared to have generally low expression judging by the small number of ESTs in all the databases queried. cDNAs were obtained by reverse transcription (RT)-PCR using pairs of gene-specific primers designed close to the start and stop codons (Supplemental Table S2). No cDNAs were obtained for ZCN13, ZCN21, and ZCN24, evidently because no expressing tissues were found (Table I). The intron/exon structures of the ZCN genes were determined from the alignments of the cDNA and genomic sequences (Fig. 2 ). If a cDNA was not obtained, the genomic sequence was used to determine the probable gene structure by alignment to the conserved coding regions and intron positions exhibited by other gene family members. Of the ZCN genes found, 22 of 24 have a conserved genomic structure consisting of four exons. The exceptions, ZCN2 and ZCN20, have three exons that apparently resulted from fusion of exons 1 and 2. Most ZCN genes have a compact gene structure in the range of 1 to 3 kb. The sizes of exon 2 and exon 3 are generally 63 and 42 bp, respectively, which are also the conserved exon sizes among other plant PEBP-related genes (Carmel-Goren et al., 2003 ; Zhang et al., 2005; Carmona et al., 2007). ZCN14, ZCN18, ZCN21, and ZCN24 are the largest genes, with sizes between 5 to 9 kb, as a result of expanded introns. The complete amino acid sequence for each gene was deduced from its cDNA sequence and in a few cases its genomic sequence (Table I).
Protein Phylogenetic Analysis and Rice Synteny
Phylogenetic analysis was used to infer the evolutionary relationship between the 24 maize and 19 rice PEBP proteins. The six Arabidopsis PEBP proteins and the wheat and barley FT functionally homologous proteins corresponding to VRN3 (Yan et al., 2006
The rice proteins, with the exception of OsTFL8, formed monophyletic subgroups with one or two maize proteins. The previously reported rice-specific OsFTL5 and OsFTL11 (Chardon and Damerval, 2005 ) are now associated with ZCN16 and ZCN17, respectively. Among the 24 maize ZCN proteins, 14 ZCN proteins appeared as seven paired monophyletic subgroups: ZCN1-ZCN3, ZCN4-ZCN5, ZCN7-ZCN8, ZCN9-ZCN10, ZCN13-ZCN21, ZCN18-ZCN24, and ZCN19-ZCN25. These gene pairs likely represent paralogs, a product of the tetraploid ancestry of maize (Gaut, 2001; Bruggmann et al., 2006).
The TFL1-like subfamily is composed of six maize and four rice proteins. Maize ZCN1-ZCN3-ZCN6 proteins group with rice proteins RCN1 and RCN3. ZCN1 and ZCN3 genes seem to be a recent duplication, because they share 89.3% nucleotide identity within exons and 79.4% nucleotide identity within introns (Supplemental Fig. S2). The other monophyletic group is composed of maize proteins ZCN2-ZCN4-ZCN5 and rice RCN2-RCN4, the latter thought to represent a duplicated rice chromosomal segment (Chardon and Damerval, 2005
The FT-like subfamily is the largest, composed of 15 maize and 13 rice genes. This subfamily forms two large monophyletic groups that we named FT-like I and FT-like II. The FT-like I group is of particular interest because it includes the key floral activators from Arabidopsis, FT, and TSF (Huang et al., 2005 Synteny analysis was performed to assess the correspondence between the phylogenetic groupings of the PEBP proteins and the prevailing rice-maize syntenic context for their gene locations, recognizing that synteny or departures therefrom can exist between these complex cereal genomes. The maize gene map positions and their syntenic rice counterpart(s) are summarized in Figure 1. In cases of a single maize-to-rice gene phylogenetic correspondence, the synteny was present and unambiguous. These examples are ZCN11-OsMFT1, ZCN2-RCN2, ZCN16-OsFTL5, ZCN17-OsFTL11, ZCN20-OsFTL7, ZCN14-OsFTL1, ZCN12-OsFTL9, and ZCN26-OsFTL12. In cases where duplicated maize genes corresponded to a single rice gene, both maize duplicated segments might be considered syntenic to a single rice locus. These examples are ZCN9/ZCN10-OsMFT2, ZCN13/ZCN21-OsFTL13, ZCN18/ZCN24-OsFTL4, and ZCN19/ZCN25-OsFTL6. In one case, a single maize gene (ZCN15) corresponded to tandemly duplicated rice genes (Hd3a/Hd3b), and the syntenic context was apparent. More challenging for syntenic interpretation are genes involved in apparently ancient segmental duplications prior to the rice-maize divergence. The large duplicated segments of rice chromosomes 2 and 4, carrying RCN2-OsFTL5 and RCN4-OsFTL6 genes, respectively, appeared to be syntenic to maize genes ZCN4-ZCN21 and ZCN5-ZCN19 on chromosomes 2 and 10, but this synteny remains somewhat ambiguous. A similar situation exists for the rice RCN1-RCN3 genes that reside in duplicated segments of rice chromosomes 11 and 12. The closely related maize genes ZCN1, ZCN3, and ZCN6 mapped to duplicated segments of chromosomes 3, 10, and 4, and synteny was difficult to assign. We were also not able to determine clear rice synteny with the maize pair ZCN7/ZCN8. The complete maize genome sequence may help clarify these and other remaining inconsistencies.
All PEBP proteins share a common structure, with a dominant feature being a central antiparallel β-sheet flanked by a small β-sheet on one side and two
To build 3-D models for the maize ZCN proteins, we used homology modeling with the Arabidopsis TFL1 (Protein Data Bank PDB:1wko A chain) and FT (PDB:1wkp A chain) proteins as structural templates. Out of 25 maize ZCN proteins, we focused on two representative candidates (ZCN2 and ZCN14) based on the highest sequence similarity to their Arabidopsis counterparts. ZCN2 has 58% amino acid sequence identity to TFL1, and ZCN14 has 72% sequence identity to FT. The models were constructed with MODELER, and refined further by guidance from stereochemistry violation penalty rules. The overall refined models abided to their Arabidopsis templates with C
Conservation of ZCN protein primary structures was evaluated from the alignment of the ligand-binding motif and the external loop (Fig. 4C). All maize proteins possess invariable residues in the critical positions forming the ligand-binding pocket corresponding to Arabidopsis TFL1/FT residues Asp-74/71, Asp-75/73, Pro-78/75, His-90/87, and His-121/118 (Fig. 4C). The critical His-88 in TFL1 that defines the opposing repressor/activator activities of TFL1 and FT, respectively, is conserved in all six maize TFL1-like proteins. All other maize ZCN proteins have in this position Tyr, corresponding to Tyr-85 in Arabidopsis FT, with the exception of ZCN11 (MFT-like) and ZCN17 (FT-like I), that have Leu and Asn, respectively. The distinct feature of MFT-like proteins is substitution of conserved Glu-112/109 for Met in ZCN9 and ZCN10 or for Val in ZCN11. All the other maize proteins have Glu in this position.
The external loop encoded by exon 4 is important for the opposing activities of Arabidopsis TFL1/FT proteins (Ahn et al., 2006
As a step toward elucidating maize ZCN gene function, we surveyed ZCN transcript accumulation across a wide range of organs and tissues using the massively parallel signature sequence (MPSS) RNA profiling database (Fig. 5
). MPSS technology is an open-ended platform that quantifies the level of transcript accumulation of virtually all genes in a sample by counting the number of individual mRNA molecules produced from each gene in the form of a 17-mer tag signature sequence (Brenner et al., 2000
The maize TFL1-like subfamiliy showed similar tissue expression patterns with expression in roots, young stems, immature ear, and tassel (Fig. 6A ). Because the Arabidopsis TFL1 gene maintains shoot apical and inflorescence meristem indeterminacy, we focused on the expression of TFL1-like genes in developing shoot apices, tassels, and ears (Fig. 6B). The closely related ZCN1 and ZCN3 genes are expressed in shoot apices during vegetative development and in both tassel and ear primordia during reproductive development (Fig. 6B). ZCN1 and ZCN3 accumulated significant amounts of unspliced pre-mRNA at early stages. ZCN3-spliced mRNA appeared in significant proportions after the floral transition in the V9 stage tassel and ear primordia, whereas ZCN1 mRNA remained mostly unspliced. None of the other TFL1-like genes accumulated unspliced transcript.
ZCN6 mRNA was detected at lower levels in shoot apices and tassel primordia, but its expression is up-regulated in developing ears. ZCN2 and ZCN6 were expressed before and after the floral transition like ZCN1 and ZCN3, but they show a distinct pattern of accumulation (Fig. 6B). ZCN2 mRNA accumulation gradually increased in shoot apices around the floral transition, peaking in V7 and V8 tassels and decreasing in later stages. ZCN6 had a complementary pattern, showing low transcript accumulation in V3 to V8 stage apices and a higher accumulation at the V9 stage. In developing ears, ZCN2 was active at all stages tested whereas ZCN6 was expressed at the V7 to V9 stages and decreased later. ZCN4 and ZCN5 transcripts accumulated after the floral transition in both tassel and ear primordia (Fig. 6B), suggesting they act specifically in the developing inflorescence after the floral transition, similar to snapdragon (Antirrhinum majus) CEN (Bradley et al., 1996 ) and pea DET (Foucher et al., 2003). Five out of the six TFL1-like maize genes were expressed in roots (Fig. 6A). MFT-like ZCN transcripts accumulated predominantly in kernels with the exception of ZCN11, which accumulated in both kernel and seedling tissues (Fig. 6C). Developmental profiling of the MFT-like genes was performed on whole kernels starting from 2 to 8 d after pollination (DAP) and on dissected embryo and endosperm tissues from kernels at 10 to 26 DAP. ZCN9 mRNA was detected in the embryo after 10 DAP and faint expression was found in the endosperm after 14 DAP. ZCN10 mRNA was detected only in the embryo after 14 DAP with a significant proportion of unspliced transcript. ZCN11 transcript accumulated in ovules before pollination and continued to accumulate in both embryo and endosperm at all postpollination stages (Fig. 6D).
The FT-like subfamily is a complex group composed of 15 maize ZCN genes in two groups named FT-like I and FT-like II. The FT-like I group is composed of seven maize genes that are further divided into two subgroups, one with Arabidopsis FT-TSF, and the other formed by the FT functionally conserved monocot homologs (Fig. 3). The duplicated ZCN19-ZCN25 pair of genes was expressed in roots. ZCN16 and ZCN20 produced unspliced mRNA in many tissues. ZCN17 is actively transcribed in roots and stems. ZCN18 is expressed in the stem and leaves producing a mixture of spliced and unspliced transcript. ZCN24 mRNA was not detected in this set of tissues (Fig. 6E). ZCN14 and ZCN15 are grouped tightly with FT homologous monocot floral activators (Fig. 3), thus being the most favorable candidates for possessing FT function. However, their expression patterns do not support their function as floral activators. In the broad set of tissues surveyed, ZCN14 mRNA was detected in the tassel and ear primordia and the endosperm, whereas ZCN15 expression was not detected in these samples (Fig. 6E). The original ZCN15 EST was found in a pedicel cDNA library. In developing kernels ZCN15 mRNA was detected in whole kernels between 4 and 8 DAP and in the dissected pedicels from later stage kernels (Fig. 6F). Dissected pedicels often contain the basal endosperm. Therefore, ZCN15 transcript accumulates in kernels after fertilization, most likely in the basal layer of the endosperm. This expression pattern is inconsistent with ZNC15 as a floral activator. ZCN14 transcript was detected in ovules and early developing kernels, but its level gradually decreased after 12 DAP (Fig. 6F). Because ZCN14 is the only FT-like gene whose transcript accumulates in the ear and tassel primordia, we investigated its expression in these tissues as well (Fig. 6F). In shoot apices, weak transcript accumulation is detected at the V5 stage, coinciding with the floral transition. ZCN14 mRNA levels increased in developing tassels at the V7 stage and remained constant up to the V9 stage. ZCN14 transcript accumulated in V7 stage ear buds and later developing ear stages. The ZCN14 expression pattern is quite different from Arabidopsis FT and rice Hd3a, which are transcribed exclusively in leaf blades (Corbesier et al., 2007 The FT-like II group is composed of six maize genes. Three maize genes of this group, ZCN8, ZCN12, and ZCN26, are expressed predominantly in leaf blades (Fig. 6G). Three other genes seem to be nonfunctional or with a limited function because they produce either unspliced mRNA (ZCN7) or their expression was not detected in any tissues tested (ZCN13 and ZCN21). We investigated further the developmental expression pattern of ZCN8, ZCN12, and ZCN26 from FT-like II and ZCN14 and ZCN18 from FT-like I in stems, immature leaves, and leaf blades at the V2 to V9 stages (Fig. 6H). We also included ZCN7 to test mRNA splicing during development; however, only unspliced ZCN7 RNA was detected in stems and immature leaves and no RNA was found in leaf blades at various developmental stages. Its paralog ZCN8 also produced unspliced transcript in stems and immature leaves, but in expanded leaf blades, ZCN8 mRNA was completely spliced (Fig. 6H). This finding indicates that ZCN8 activity may be regulated by mRNA splicing. Importantly, the spliced ZCN8 transcript was detected in leaf blades before the floral transition at the V3 stage. Its level increased at the V4 stages just before the floral transition and stayed high afterward. This expression pattern makes ZCN8 an attractive candidate as a floral activator. Another leaf-specific gene, ZCN12, was found to be activated in leaf blades at the V7 stage after the floral transition and continued to be expressed at later stages (Fig. 6H). ZCN26 is expressed predominantly in leaf blades and produced a significant amount of unspliced pre-mRNA. ZCN18 was expressed mainly in the stem with weaker expression in leaf blades. ZCN14 mRNA was barely detected by RT-PCR in immature leaves and in leaf blades at vegetative V4 and V5 stages, but after the floral transition, the ZCN14 mRNA level increased and was reliably detected in leaf blades (Fig. 6H).
In Arabidopsis the floral activator FT interacts with the bZIP transcription factor FD to activate the floral identity genes inducing flower development (Abe et al., 2005
Maize Contains an Expanded PEBP Gene Family
Growing evidence indicates that PEBP-related proteins are potent and sometimes mobile regulators of plant architecture, development, and seasonal growth adaptation (Bohlenius et al., 2006
We identified 25 ZCN genes, although one is likely a pseudogene. Eudicot genomes such as Arabidopsis and poplar have about a half-dozen PEBP genes (Mimida et al., 2001
The maize PEBP gene family expansion (to 25 members) compared to rice (19 members) could be explained by the ancient tetraploid ancestry of maize, in which a genome duplication occurred after divergence from the rice and maize common ancestor, followed by subsequent diploidization en route to modern maize (Paterson et al., 2004
Most of the ZCN genes appear to be functional, because all but one gene predicts a credible complete open reading frame, and all but three genes (ZCN13, ZCN21, and ZCN24) have transcripts detected in at least one tissue of 13 tested by RT-PCR. Although the three poorly expressed genes may be heading toward evolutionary degeneracy, their open reading frames are complete, and important cryptic expression cannot be ruled out. Further, the duplicated ZCN13 and ZCN21 genes are located near centromeres 5 and 2, where expression may be suppressed due to epigenetic silencing spreading from the heterochromatin. ZCN20 is also centromeric and shows very low expression.
The presence of duplicated genes raises the question about their functional redundancy. According to evolutionary models, duplicated genes may undergo different selection processes: nonfunctionalization where one copy loses function, hypofunctionalization where one copy decreases in expression or function, neofunctionalization where one copy gains a novel function, or subfunctionalization where the two copies partition or specialize into distinct functions (Lynch and Conery, 2000 Possible subfunctionalization trends are inferred by expression patterns for gene pairs ZCN1/ZCN3, ZCN4/ZCN5, and ZCN9/ZCN10, which showed clear expression pattern shifts. ZCN1 and ZCN3 are both expressed in the shoot apices before the floral transition and in the ear and tassel primordia after the floral transition, but they produce different levels of fully spliced mRNA. ZCN1 transcripts are mostly unspliced, whereas ZCN3 produces mostly spliced mRNA, and accumulates this fully spliced mRNA in ears and tassels at later developmental stages. ZCN4 and ZCN5 are expressed in developing ears and tassels after the floral transition, but their expression is activated at somewhat different stages of development. The duplicated genes ZCN9 and ZCN10 are expressed in kernels, but ZCN9 is expressed in the embryo and endosperm, whereas ZCN10 is embryo specific. The other duplicated pair ZCN13/ZCN21 may have undergone bilateral nonfunctionalization, as apparently neither gene is expressed.
In plants regulated RNA processing and alternative splicing are common mechanisms of the posttranscriptional regulation of gene expression (Reddy, 2007
The Arabidopsis FT protein and its orthologs in other plant species serve as long-range developmental signals in activation of the floral transition (Lifschitz and Eshed, 2006
However, the yeast two-hybrid assay provides strong evidence that ZCN8 likely functions as the FT floral activator because only the ZCN8 protein interacts with the DLF1 bZIP transcription factor similar to interaction of the FT and the bZIP transcription factor FD in Arabidopsis (Abe et al., 2005
The Arabidopsis TFL1 gene plays an opposing role to the FT gene, being a repressor of the floral transition (Kardailsky et al., 1999
We identified six maize genes whose proteins formed a well-supported monophyletic subfamily with Arabidopsis TFL1. The important TFL1 protein structures are all preserved in the six maize homologous proteins including His-88, which is critical for repressing flowering (Hanzawa et al., 2005
Despite their significant protein similarity, each ZCN family member may have partially overlapping or even distinct biological functions based on their developmental expression differences. Among the maize ZCN genes, we have ascribed eight with putative functions relating to floral transition and meristem determinacy, guided by the examples of the Arabidopsis FT/TSF and TFL1 families, respectively (Kardailsky et al., 1999 The expression patterns of maize ZCN genes hint at roles in root development. The TFL1-like maize genes ZCN1, ZCN3, ZCN2, and ZCN5 are expressed in the root tips as fully spliced mRNA, suggesting a possible function in the root apical meristem. In addition, among the FT-like subfamily, ZCN19, ZCN25, and ZCN17 are expressed mainly in the roots. These could also be involved in root development perhaps as a mobile signal akin to FT.
Another likely role for some of the ZCN genes is in kernel development. The seed-specific expression of the maize MFT-like genes ZCN9 and ZCN10, and broader kernel plus other tissues expression of ZCN11, suggests these MFT-like genes function during seed development. Indeed, the Arabidopsis MFT gene is highly expressed in early developing seeds (Supplemental Table S4). This finding supports the hypothesis that Arabidopsis MFT may act during seed development in addition to its role as a redundant floral inducer (Yoo et al., 2004
ZCN14 expression in various tissues suggests its involvement in other developmental processes beyond flowering time. ZCN14 is activated after the floral transition in the tassel and ear primordia and expressed during flower development and later in ovules and kernels. To date, ZCN14 is the only FT-like gene with apparent expression in the inflorescence meristem. Rice OsFTL1 syntenic to ZCN14 has a similar pattern of expression in spikes and kernels (Izawa et al., 2002 In conclusion, this study of the maize PEBP gene family affirms the conserved protein structures and function in flowering time, but the gene expression results, including alternative splicing, point to likely adaptation and specialization of physiological roles, further investigation of which this study may help guide.
Maize PEBP Gene Identification A local implementation of the National Center for Biotechnology Information BLASTX version 2.0 was used for sequence searching. The initial protein queries used the six publicly known Arabidopsis (Arabidopsis thaliana) founders of this gene family, AtTFL1 (At5g03840), AtCEN (At2g27550), AtBFT (At5g62040), AtFT (At1g65480), AtTSF (At4g20370), and AtMFT (At1g18100). Maize (Zea mays) sequence subject sources were proprietary ESTs and their assemblies, publicly available ESTs, CDS, GSS, BACs, and The Institute for Genomic Research genomic GSS assemblies AZM_4, AZM_5 (http://maize.tigr.org/), and MAGI_4 (http://www.plantgenomics.iastate.edu/maize/). All potential hits to conserved regions of the PEBP gene family were assembled and curated, and additional rounds of searching were performed to extend the genomic and/or transcript sequences across the most complete possible transcript region. The 33 unique sequences obtained originally by this survey were used to design overgo probes for screening genomic BAC libraries. Selected BACs were sequenced and assembled. The exon/intron structures of the ZCN genes were deduced from alignments of cDNA and BAC genomic sequences using the program Sequencher 4.7 (Gene Codes Corporation).
The BAC physical map as fingerprinting contigs (http://www.genome.arizona.edu/fpc/maize/WebAGCoL/WebFPC/) was used to find the nearest available markers to position ZCN genes on the genetic IBM2 map (http://www.maizegdb.org). For synteny comparisons, The Institute for Genomic Research Rice Annotation Release 5 gene calls, rice (Oryza sativa)-maize syntenic blocks (http://www.tigr.org/tdb/synteny/maize_IBMn/figureview_desc.shtml), and a locally developed gene-centric synteny analysis between rice and maize were used.
Maize plants (genotype B73) were grown in the field. Vegetative growth stages (V1–V9) were defined according to the appearance of the leaf collar of the uppermost leaf (Muszynski et al., 2006
Small tissues such as shoot apices and ear buds were homogenized in 300 µL of TRIzol Reagent (Roche Diagnostics Corporation) using a 1.5 pestle (VWR KT479521-1590). Immature leaves and leaf blades were ground with a mortar and pestle in liquid nitrogen. Ground tissue (50 mg) was treated with 300 µL of TRIzol. Total RNA was isolated with TRIzol Reagent in combination with Phase Lock Gel (Brinkmann Instruments Inc.) according to the manufacturer's instructions. Complementary DNA synthesis was performed with Superscript First-Strand Synthesis system (Invitrogen). RT-PCR amplification was performed using Expand Long Template DNA polymerase (Roche). Primers were designed according to the predicted gene structure deduced from BAC sequences. Primers are shown in Supplemental Table S1. Two microliters of the cDNA reaction was used for PCR amplification in a 50 µL volume. The PCR conditions were 95°C for 2 min followed by 35 cycles at 94°C for 45 s, 58°C for 45 s, 72°C for 1 min, and a final extension of 72°C for 10 min. PCR products were cloned in pCR4-TOPO (Invitrogen) and sequenced.
The commercial kit MATCHMAKER GAL4 Two-Hybrid system 3 (Clontech) was used according the manufacturer's protocol. cDNAs of interacting genes were cloned into pGBKT7 (the Bait vector) and pGADT7 (the Prey vector) in reciprocal combinations. Both plasmids were transformed into the AH109 yeast (Saccharomyces cerevisiae) strain and plated on the dropout media SD/-Leu/-Trp. Individual colonies were then replated in parallel on two plates with dropout media SD/-Leu/-Trp and SD/-Leu/-Trp/-His. Colony growth on the His-minus media was indicative of protein interactions.
The DuPont MPSS (Solexa) 17-mer expression libraries isolated from over 200 diverse maize tissues and developmental stages was queried with ZCN gene sequences. The MPSS samples were curated and grouped by tissue-developmental criteria, and from these groupings the mean parts per million for each MPSS 17-mer tag was calculated for 13 tissues, and then used to represent that ZCN gene's transcript abundance. Arabidopsis MPSS can be found at http://mpss.udel.edu/at/?/.
The starting coordinates for molecular dynamics simulations and homologous modeling were taken from the x-ray structures PDB:1wko A chain at 1.80 Å resolution and PDB:1wpb A chain at 2.60 Å resolution. The initial structural coordinates of ZCN1 and ZCN14 were constructed using InsightII's MODELER module with its autoenergy minimization procedure (Accelrys). Before further analysis and molecular dynamics, all the structures were energy minimized under various constraints to relax the structure gradually, first in virtual vacuum with the crystal waters if applicable, and then subsequently in solvent water boxes. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers EU241892 to EU241916 and EU241917 to EU241937.
The following materials are available in the online version of this article.
The authors thank Jeff Habben for general support, Mike Muszynski and Nic Bate for valuable comments, Laura Appenzeller for proofreading of the manuscript, Sergei Svitashev and Stephane Deschamps for BAC library screening and sequence assembly, Norbert Brugière for pedicel RNA samples, and Pooja Patel for help with yeast two-hybrid vector construction. Received September 21, 2007; accepted November 3, 2007; published November 9, 2007.
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: Olga Danilevskaya (olga.danilevskaya{at}pioneer.com).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.109538 * Corresponding author; e-mail olga.danilevskaya{at}pioneer.com.
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-helices on the other. All family members also possess a highly conserved putative anion-binding site located at one end of the central β-sheet, supporting the idea that the proteins are functioning in part through forming complexes with a phosphorylated ligand (Banfield et al., 1998
