Structure and expression of the maize (Zea maysL.) SUN-domain protein gene family: evidence for the existence of two divergent classes of SUN proteins in plants
© Murphy et al; licensee BioMed Central Ltd. 2010
Received: 19 July 2010
Accepted: 8 December 2010
Published: 8 December 2010
The nuclear envelope that separates the contents of the nucleus from the cytoplasm provides a surface for chromatin attachment and organization of the cortical nucleoplasm. Proteins associated with it have been well characterized in many eukaryotes but not in plants. SUN (Sad1p/Unc-84) domain proteins reside in the inner nuclear membrane and function with other proteins to form a physical link between the nucleoskeleton and the cytoskeleton. These bridges transfer forces across the nuclear envelope and are increasingly recognized to play roles in nuclear positioning, nuclear migration, cell cycle-dependent breakdown and reformation of the nuclear envelope, telomere-led nuclear reorganization during meiosis, and karyogamy.
We found and characterized a family of maize SUN-domain proteins, starting with a screen of maize genomic sequence data. We characterized five different maize ZmSUN genes (ZmSUN1-5), which fell into two classes (probably of ancient origin, as they are also found in other monocots, eudicots, and even mosses). The first (ZmSUN1, 2), here designated canonical C-terminal SUN-domain (CCSD), includes structural homologs of the animal and fungal SUN-domain protein genes. The second (ZmSUN3, 4, 5), here designated plant-prevalent mid-SUN 3 transmembrane (PM3), includes a novel but conserved structural variant SUN-domain protein gene class. Mircroarray-based expression analyses revealed an intriguing pollen-preferred expression for ZmSUN5 mRNA but low-level expression (50-200 parts per ten million) in multiple tissues for all the others. Cloning and characterization of a full-length cDNA for a PM3-type maize gene, ZmSUN4, is described. Peptide antibodies to ZmSUN3, 4 were used in western-blot and cell-staining assays to show that they are expressed and show concentrated staining at the nuclear periphery.
The maize genome encodes and expresses at least five different SUN-domain proteins, of which the PM3 subfamily may represent a novel class of proteins with possible new and intriguing roles within the plant nuclear envelope. Expression levels for ZmSUN1-4 are consistent with basic cellular functions, whereas ZmSUN5 expression levels indicate a role in pollen. Models for possible topological arrangements of the CCSD-type and PM3-type SUN-domain proteins are presented.
Organization of Chromatin and the Nuclear Envelope in Animals and Plants
Genomic DNA is packaged by proteins into chromatin that resides within the nuclear space in eukaryotic organisms. Within this three-dimensional space, interphase chromosomes are often observed to occupy discrete, nonoverlapping territories [1, 2]. The architecture of the cell nucleus as a whole, in combination with chromatin dynamics, provides a basis for cells' regulation of their gene expression, DNA replication, and DNA repair [2–4]. The eukaryotic cell nucleus is surrounded by a double membrane, the nuclear envelope (NE), which is composed of the inner and outer nuclear membranes, separated by an ~30-nm perinuclear space. The two are connected through nuclear pore complexes, and the space between them is continuous with the lumen of the endoplasmic reticulum (ER). Intrinsic membrane proteins associated with the inner and outer membranes make the NE a rather dynamic membrane system with a multitude of essential functions, including nuclear migration and positioning, cell cycle-dependent NE breakdown and reformation, cytoplasmic-nuclear shuttling, calcium signaling, gene expression, genome stability, meiotic chromosome behavior, and karyogamy [3–11]. Mutations in NE-associated proteins, such as nuclear lamins, give rise to a variety of heritable diseases in animals, collectively termed laminopathies, including muscular dystrophy, lipodystrophy, diabetes, dysplasia, leukodystrophy, and progeria [12–16].
Recent advances in yeast and animal NE research have identified SUN (Sad1p/Unc-84) domain homology proteins as key residents of the NE, and their presence in plants is just beginning to be recognized and characterized [17–19]. Despite the conservation of NE-mediated functions between plants and animals and the importance of the NE in plant biology, knowledge of the plant NE proteome remains limited [20–23].
SUN-Domain Proteins Are Highly Conserved
SUN-domain proteins have gained attention as a family of widely conserved NE-associated proteins that can transmit forces between the nucleus and cytoplasmic motility systems. SUN-domain proteins were first characterized in Schizosaccharomyces pombe and Caenorhabditis elegans as NE-associated proteins associated with spindle pole-body and nuclear-migration defects, respectively [24, 25]. Since then, their analysis in other eukaryotes has extended their functions to roles in chromosome tethering, telomere maintenance, meiotic chromosome behavior, nuclear pore distribution, mitotic chromosome decondensation, and the regulation of apoptosis [13, 26–35]. Furthermore, genetic analysis revealed that knockouts within the mouse SUN1 gene disrupted the expression of piRNAs and caused a misregulation of a large number of meiosis-specific reproductive genes .
In animals and fungi, SUN proteins interact through their C-terminal SUN domains in the perinuclear space with outer-nuclear-membrane-associated KASH (Klarsicht/ANC-1/Syne-1 homology) proteins as part of the LINC (Linker of Nucleoskeleton and Cytoskeleton) complex [13, 37–43]. The other members of the KASH-domain family are proteins with cytoplasmic domains and nuclear lamins that reside in the nucleoplasm and therefore allow forces produced within the cytoplasm to be transmitted to the nuclear periphery. Evidence for the expression and NE localization of plant SUN-domain proteins has emerged from studies looking at cytokinesis in Arabidopsis and nuclear proteomics in rice [17–19]. Additional studies with AtSUN1 and AtSUN2 firmly establish that these proteins reside in the NE like their animal and fungal counterparts [17–19].
SUN-Domain Proteins and Meiotic Chromosome Behavior
Some animal and fungal SUN-domain proteins are known to have a conserved role in meiotic chromosome behavior [9, 13, 33, 34, 44]. During meiotic prophase I, a dramatic reorganization of the nucleus occurs in which the chromosomes compact and telomeres attach themselves to the NE by an unknown active mechanism, cluster into a bouquet arrangement, and finally disperse along the surface of the inner nuclear membrane. The formation and dynamics of the bouquet configuration of meiotic chromosomes contribute to proper homologous chromosome pairing, synapsis, recombination, and segregation [45–50].
In maize, meiotic telomere clustering has been demonstrated to occur de novo on the NE during meiotic prophase I, and the temporal patterns are nearly identical to those in mammals [45, 51]. Interestingly, genetic disruption of the SUN1 gene in mouse leads to defects in meiotic telomere-NE association, pairing, synapsis, and recombination, a phenotype remarkably similar to those of two maize synapsis-deficient mutants, desynaptic (dy) and desynaptic1 (dsy1) [33, 52].
We set out to identify maize SUN genes to provide a foundation for analysis of meiosis and other nuclear processes in plants. Using bioinformatics and molecular approaches, we discovered five different SUN-domain genes (here designated ZmSUN1-5) in the maize genome. We present evidence that these fall into two subfamilies, which we call canonical C-terminal SUN domain (CCSD) and plant-prevalent mid-SUN 3 transmembrane (PM3). We also provide the first evidence for expression and localization of PM3-type proteins and discuss the possible significance of this novel structural-variant subfamily.
Results and Discussion
Identification of Maize Genes Encoding Canonical C-terminal SUN-Domain (CCSD) Proteins
A reference genome sequence was recently produced for the inbred line B73 (B73 RefGen_v1 ). The SUN genes described here refer to B73 sequences where possible, although many of the public cDNA and EST sequences in GenBank are from multiple other inbred lines of maize. We identified SUN-domain protein genes in a model plant genetic system by using a BLAST homology search of the maize genome queried with a fungal SUN-domain protein Sad1p, from S. pombe . The two different putative maize SUN-domain protein genes we initially identified, ZmSUN1 and ZmSUN2, were each predicted to encode ~ 50-kDa proteins. When the predicted protein sequences were used to query the Conserved Domain Database (version 2.21, NCBI), each revealed the presence of a single conserved domain, the SUN/Sad1_UNC superfamily (pfam07738), near the C-terminus of the proteins. These maize genes are homologous to recently characterized plant SUN-domain protein genes from Arabidopsis (AtSUN1, AtSUN2 [54, 55]) and rice (OsSad1 ). Experimental evidence from heterologous expression assays with fluorescent protein fusions indicates that these Arabidopsis and rice CCSD proteins are localized at the NE. The presence of a C-terminal SUN domain and the NE localization are among the defining features of animal and fungal SUN proteins [9, 13, 38]. Plant genomes therefore appear to encode canonical C-terminal SUN-domain (CCSD) type proteins, an observation that is not surprising given the conserved role of these proteins in basic eukaryotic processes such as meiosis, mitosis, and nuclear positioning [8, 9, 38, 39, 42].
Discovery of Maize Genes Encoding PM3-type of SUN-domain Proteins
Maize genes encoding SUN-domain proteins.
5 S, bin 5.04
3 S, bin 3.04
3L, bin 3.06
8L, bin 8.06
8L, bin 8.05
Properties and motifs of maize SUN-domain protiens.
N315-K454, (6 e-39)
P294-G425 (3 e-32
F233-D357 (2 e-38)
F257-D381 (7 e-38)
H197-D321 (9 e-35)
CC1, V414-E434 CC2, K495-K523
Conservation of Two Classes of SUN-domain Proteins in Plants
Shared Gene Structures Reflect an Early Divergence of the Two Types of Maize SUN-domain Proteins
The 2.3-Gb maize genome is partitioned among 10 structurally diverse chromosomes, which are predicted to encode over 32,000 genes . The genetic map of maize is subdivided into approximately 100 10-to 15-cM bins . The genome is complex and dynamic because of extensive and recent large segmental duplications [53, 57–59] and a major expansion of long terminal repeat sequences over the last few million years. Current breeding lines and natural accessions of maize harbor large amounts of sequence diversity and many structural polymorphisms [53, 58, 60].
An analysis of intron and exon structures within the maize SUN genes showed that the gene structures are conserved within each class. The CCSD genes had two or three exons, and the SUN domain was split between the exons. On the other hand, the PM3 genes had 4-5 exons and a SUN domain that was encoded within the largest exon. Comparative analysis of the maize ZmSUN gene structures revealed that the CCSD genes shared an ancestral intron that interrupts the SUN domain (between K364 and V365 in the ORF of ZmSUN1 and between K338 and D339 in the ORF of ZmSUN2; Figure 2A). This ancestral intron position may be a hallmark of this class of SUN genes, as it is also found in the Arabidopsis, rice, sorghum, and moss homologs. ZmSUN1 and ZmSUN2 share a large intron, greater than 3 kb in size, whereas the PM3 genes all possess small introns ranging from 19 to 483 nucleotides in size.
Properties of Maize SUN-domain Proteins
Even less is known about the PM3 proteins, and their functions are completely uncharacterized. They are significantly larger than plant CCSD proteins (Figure 3C). Their shared structural features are an N-terminal transmembrane domain, an internal SUN domain, a PAD, one or more predicted coiled-coil motifs, and two closely spaced C-terminal transmembrane domains (Table 2 Figure 3C). This collection of features defines them structurally, but the central location of the SUN domain is not unique to plants. Other, nonplant mid-SUN-domain proteins, largely uncharacterized, from various species including fungi, flies, worms, and mammals can be identified by sequence-search analyses (data not shown). Whether or not these proteins reside or function in the NE remains to be determined.
Despite the similarity of domain architecture and sequence similarity within conserved domains, the remainder of the protein regions exhibit considerable sequence divergence between the SOGG3 and SOGG4 members in any given species. Overall, these analyses show that the maize genome encodes at least two multigene families of SUN-domain proteins. Each of these two subfamilies comprises at least two genes. ZmSUN1 and ZmSUN2 are CCSD-type and are most closely related to plant SUN-domain homologs AtSUN1, AtSUN2, and OsSad1. ZmSUN3, 4, and 5 are PM3-type and probably represent a previously unknown class of SUN-related proteins in plants.
mRNA Expression Profiling of ZmSUNProtein Genes
The conservation of the SUN-domain protein genes in plants suggests that they potentially have functions similar to those of their animal counterparts, for example nuclear positioning and motility within the cell, bridging the cytoplasm to the cortical layer of the nucleoplasm, and contributing to meiotic chromosome segregation through telomere tethering before synapsis and recombination [8, 9, 44]. Maize SUN domain genes that function in basic somatic cell processes such as mitosis, nuclear architecture, and chromosome tethering might be expected to show ubiquitous expression, whereas those that function in meiosis or pollen-nuclear migration or nuclear fusion at fertilization might show a more limited expression profile, being active in reproductive organs such as flowers, egg and pollen mother cells, and gametophytic tissues such as pollen grains. To begin to examine these possibilities, we looked at gene expression at the mRNA abundance level using three different sources of information: NCBI's UniGene; microarray expression data from anthers, which contain male meiotic cells; and Solexa transcriptome profiling data derived from maize inbred line B73 tissues.
Four of the five genes (all but ZmSUN3) are represented by consensus UniGene models in NCBI (Table 1), and three of these, ZmSUN1, ZmSUN2, and ZmSUN4, are accompanied by quantitative EST profile information expressed as transcripts per million, which we converted to transcripts per ten million (TPdM). The EST data were pooled according to tissue type, and only relatively deeply sequenced libraries (10,000-15,000 or more) showed evidence of expression, as summarized in Additional file 3 Figure S3. The CCSD genes, ZmSUN1 and ZmSUN2, appeared to be expressed at relatively low levels (200-2,000 TPdM) in several tissues, including ear, endosperm, embryo, meristem, pollen, and tassel. Only one PM3-type SUN-domain gene, ZmSUN4, currently has corresponding EST profile data available from NCBI. It too shows relatively low expression levels (~400-3,000 TPdM) in a variety of tissues, such as embryo, pericarp, and shoot. These values are roughly 10% of those for UniGene EST data from two control so-called house-keeping genes, alpha tubulin 4 (tua4, Zm.87258) and cytoplasmic GAPDH (Zm.3765), which are expressed in 17 of the 19 tissues at levels from ~2,200 to 21,000 TPdM.
Ascribing the meiotic telomere clustering functions to any one of the five SUN genes may prove difficult, at least partly because the anther is made up of several different cell types that include not only cells in meiosis but also a layer of epidermal, intermediate, and tapetal cells. The expression or function of plant SUN genes could be partitioned among these cell types, whereas these methods produced only a single value over the entire anther . Another consideration is that even single cells may contain multiple SUN proteins with different, related, or even cooperative functions, such as NE rearrangements, interaction with nuclear pores, or paternal storage of gene products for postmeiotic functions such as pollen mitosis, pollen tube growth, nuclear migration, and fertilization.
Solexa Transcriptome Expression Profiling
To show more clearly the variation in expression levels among the SUN genes, we replotted the same data as semi-log2 (Figure 6B). The overall expression pattern is consistent with basic functions for SUN-domain proteins in most cell types. A notable exception to the widespread pattern of expression was that of ZmSUN5, which showed a very distinct and much more restricted pollen-related pattern of expression (Figure 6 pollen). Such an expression profile predicts that ZmSUN5 should be required for specialized processes such as nuclear migration down the pollen tube and possibly double fertilization. An interesting and related observation is that fertilization involves nuclear fusion, as does karyogamy, which in yeast involves active nuclear migration and SUN-domain proteins [9, 38, 62].
The present report represents the first description of relative mRNA expression levels of all members of a SUN gene family in any plant species and may therefore prove useful to investigators of the functions of plant SUN-domain proteins. Despite some variation in the data across different expression platforms, as summarized above, a consistent trend for most of the ZmSUN genes is that they are expressed in many different tissues at relatively low levels, a finding similar to that of Graumann et al.  for the CCSD-type AtSUN2 gene. In addition, we observed a distinct exception to this overall pattern with ZmSUN5, whose expression appears to be highly specific to pollen. Given the lack of information on PM3-type SUN proteins, we set out to characterize this group further in plants. We chose to examine a PM3-type gene that was expressed in many cell types including those expressed in meiosis-stage anthers with possible roles in meiotic telomere functions.
Isolation and Characterization of a Maize PM3-type SUN-Domain Protein Gene from a Meiotic cDNA Library
The role of SUN genes in telomere-associated recombination and crossover control has been established for animals and yeast and is likely to exist in plants as well [33, 63, 64]. In this regard, we find intriguing that two different laboratories [65, 66] recently and independently mapped a recombination control QTL in maize to bin 3.06, where ZmSUN3 resides. We screened a meiosis-enriched cDNA library for ZmSUN3 and its closely related duplicate ZmSUN4 using a 639-bp PCR product corresponding to a region of the SUN domain of ZmSUN3 at a stringency of Tm-15°C. The probe has a high degree of similarity to both ZmSUN3 and ZmSUN4 yet it is not similar enough to ZmSUN5 or either of the CCSD-type genes to detect them. From approximately 500,000 plaques, we isolated two identical full-length cDNA clones of ZmSUN4 with identical insert sequences. The detection of ZmSUN4 but not ZmSUN3 is consistent with the relative expression levels for ZmSUN3 and ZmSUN4 in meiosis-stage anthers (Figure 5).
The W23 ZmSUN4 full-length cDNA is 2,158 bp in length and has a predicted open reading frame (ORF) of 1,920 bp encoding a 639-residue protein with a predicted molecular mass of ~71 kD and an acidic isoelectric point of 5.2 This full-length ZmSUN4 cDNA predicts a protein with all of the motifs and arrangents (Table 2 Figure 7B) that are typical of the entire class of PM3 proteins.
Localization of a Maize PM3-type Protein
Our examination of proteins from isolated male flowers at meiotic stages of development detected high-molecular-weight bands that were considerably larger than the predicted protein sizes. Given the number of cysteine residues and the possibility of disulfide bridges, we examined the effect of prolonged boiling times in the presence of reducing agents (0.1 M 2-mercaptoethanol, 10% SDS) on the detectable band patterns. These high-molecular-weight bands were not detected in the protein samples examined for multiple other, different, nonanther tissues (Figure 8B). The basis for this difference is not known, but it may result from more highly cross-linked SUN3/4 protein in the extracts from anthers than in those from the other tissues. After 10 or more minutes of boiling, the antibodies detected a single band of about 70 kDa (Figure 8C), similar to those detected in the multitissue survey blot (Figure 8B). Therefore, ZmSUN3, ZmSUN4, or both appear to be present in meiosis-stage anthers.
Our examination of formaldehyde-fixed cells, shown in Figure 9 revealed the strongest staining around the nuclear periphery but also detected considerable speckled cytoplasmic staining in a postmeiotic uninucleate pollen mother cell. The cytoplasmic staining may reflect nonspecific background or true signal from ER-localized PM3-type SUN-domain protein. Interestingly, we have yet to detect staining in meiotic prophase nuclei with these antibodies, possibly because of difficulty in the preservation conditions or in detecting the epitope in prophase nuclei or possibly because of an absence of PM3-type SUN-domain proteins in meiotic cells. The results of negative control experiments, using preimmune sera and secondary antibody only, are shown in Figure 9 at image scaling comparable to that used for the anti-PM3-antibody staining (Figure 9C). The lack of staining in the controls suggests that the staining patterns noted with the anti-PM3 sera were specific.
These data provide the first direct evidence of a PM3 SUN-domain protein localized to the nuclear periphery and suggest that this SUN domain in this subfamily of plant proteins can reside in the NE like the CCSD proteins. Together, these observations suggest that plant nuclei contain multiple different SUN-domain proteins.
Models of the Topology of Plant SUN-domain Proteins
If CCSD SUN proteins adopt a configuration like that of plant, animal, or fungal SUN proteins, the most likely arrangement would be that depicted by topology model "A" in Figure 10. In this configuration, the N-terminus would be in the nucleoplasm, possibly interacting with chromatin, inner-nuclear-membrane-associated proteins, or telomeres, and the SUN domain would be positioned within the perinuclear space. Connections to the cytoplasm would require interactions with other proteins embedded in the outer nuclear membrane. The configuration depicted in topology model "B" would suggest an opposite set of interactions. Given the structure of the NE, the two models are not necessarily exclusive, as the two membranes are continuous and fused around nuclear pore complexes.
For the PM3 SUN proteins, four different models (Figure 10) are presented for consideration because three transmembrane domains are involved. The C-terminal transmembrane domains are close together and unlikely, although not necessarily unable, to traverse the entire lumenal space. Only models with the last two transmembrane domains in the same membrane are therefore presented. Of these, topology models "D" and "E" are intriguing in that they predict a single protein bridge with both nucleoplasmic and cytoplasmic segments. Topology model "C" could have two different nucleoplasmic segments and thereby serve as a scafold for multiple nuclear molecules or complexes, including chromatin and nonchromatin nuclear proteins, other NE proteins, or telomeric DNA. Similarly, topology model "F" depicts a protein with two cytoplasmic segments that might be capable of interacting with two cytoplasmic partners, while requiring additional protein interaction to form a functional nucleoplasmic-cytoplasmic bridge.
In nonplant systems, SUN proteins are linked to the cytoplasm by an interaction with KASH-domain proteins that traverse the outer nuclear membrane. The KASH domain proteins connect to various cytoskeletal components to function as cargo-specific cytoskeletal adaptor proteins [13, 42, 67]. As a family, the KASH domain proteins have limited homology over a small portion of their entire protein sequence, and no plant KASH-domain protein homologs have been identified by sequence analyses thus far. Genetic or protein interaction screens may be required to identify SUN-interacting partners and their function in plants.
The maize genome encodes a family of SUN-domain protein genes that form two distinct classes; the CCSD-type, resembling canonical SUN-domain proteins, and the PM3-type, representing a novel structural class shown here to be expressed in multiple tissues of maize and concentrated at the nuclear periphery in pollen mother cells. These two subfamilies are found in flowering plants and moss and therefore probably originated early in plant evolution, if not before that. The discovery of this gene family opens new avenues for investigation of molecular mechanisms that may link nuclear architecture to chromatin dynamics and nuclear positioning in maize. Future genetic analyses will be important for defining the biological role of these plant SUN genes in vivo.
Bioinformatics and SUNGene Models
The B73 reference maize genome http://www.maizesequence.org was queried with SUN-domain protein sequences from C. elegans Unc-84 [GenBank: NP_001024707], S. pombe Sad1p [GenBank: NP_595947], and rice Sad1 [GenBank: NP_001055057], which identified CCSD protein genes (ZmSUN1 and ZmSUN2). Further BLAST searches with these sequences led to the identification of PM3 genes (ZmSUN3, 4, 5). Genomic DNA structures for ZmSUN genes were produced with full-length cDNAs, ESTs, or EST contigs with B73 genomic DNA with SPIDEY, http://www.ncbi.nlm.nih.gov/spidey. The genomic structure for ZmSUN3 was determined from available EST assembly data at PlantGDB http://www.plantgdb.org/, as no full-length B73 cDNA clone was available at the time. Protein parameters including amino acid length, molecular weight, and isoelectric points were obtained from ExPASy . Secondary structure domains, including the locations of the SUN domain, predicted coiled coils, and predicted transmembrane regions, were obtained from the NCBI conserved-domain database (version 2.21), COILS , and TMpred  prediction software respectively. The PAD located in ZmSUN3, 4, and 5 was identified by analysis of a multiple sequence alignment of full-length proteins of maize, Arabidopsis, sorghum, rice, and a moss (P. patens) with ClustalW2 http://www.ebi.ac.uk/Tools/clustalw2/. The phylogenetic tree displayed in Figure 1 was created by ClustalW2, with the default multiple-sequence-alignment matrix (Gonnet 250) and is displayed as an unrooted maximum-likelihood tree from TreeView, version 1.6.6 .
mRNA Expression Analyses of ZmSUNGenes
Expression data for mRNA levels was extrapolated from three different sources. For the UniGene EST, expression profiles are computed relative abundance values derived from NCBI's UniGene for ZmSUN1 (Zm.94705), ZmSUN2 (Zm.6043), and ZmSUN4 (Zm.17612). For the anther microarray data, relative expression levels were extracted from microarray experiments available at NCBI (Gene Expression Omnibus, http://www.ncbi.nlm.nih.gov/geo/[72, 73]). The cDNAs were originally obtained from meiosis-stage anthers that were 1 mm, 1.5 mm, or 2 mm in length. Probe signals for ZmSUN genes were determined as previously reported . For transcriptome analysis, Poly(A+) RNA was isolated from various maize tissues with Trizol (Gibco, BRL), Qiagen, MACS (Miltenyi Biotec), and FastTrack (Invitrogen) RNA isolation kits. Two Solexa-based transcript-quantification platforms were used to measure the abundance of SUN transcripts, the Solexa shole-transcriptome and Solexa dual tag-based methods [74, 75]. Both of these technologies involve 36-nt or 21-nt sequence read lengths produced from multiple locations in the transcripts. The whole-transcriptome data were not restriction-enzyme anchored, so the multiple 36-nt sequences were spread along the transcripts. For the dual-tag-based methods two four-base cutter restriction enzymes, DpnII and NlaIII, were used as initiation sites for the 21-nt sequences, and therefore deep transcript counts were obtained from fewer sites in the transcripts. Only repetitive sequence reads found at 10 or fewer distinct locations in the B73 genomic sequence (by comparison to 17,455 publicly available B73 BAC sequences) were used in determining the relative gene expression levels. Sequences found more than 10 times in the genome were classified as repetitive sequences and were excluded from the analysis. The GAPDH cytoplasmic gene is known to have a moderate and relatively ubiquitous expression level in many maize tissues  and is included for comparison. The dual tag-based analysis was carried out with an Illumina GA2 machine and cDNAs treated with two restriction enzymes, DpnII and NlaIII. The aggregate counts of the resulting sequence reads from these sites, excluding repetitive sequences, were used to quantify the overall gene expression level, reported here in parts per ten million transcripts.
Molecular Cloning and Sequence Analysis of a Maize Full-Length SUN cDNA, ZmSUN4
A full length maize PM3-type SUN cDNA was isolated by hybridization screening from a meiosis-enriched tassel cDNA library (library 11, inbred line W23, a gift from J. M. Gardiner, University of Arizona, Tucson). The library was screened with a PCR product from maize B73 genomic DNA. Maize B73 genomic DNA from leaf tissue was isolated as previously described  with slight modifications: The 2-mercaptoethanol was replaced with 3 mM dithiothreitol (DTT), homogenized tissue samples were incubated at 65°C for 20 min, and the aqueous extraction buffer was supplemented with 1% polyvinylpyrrolidone (Sigma P-5288) and 1% W:V polyvinyl polypyrrolidone (Sigma P-6755). Genomic DNA (20 ng) was used in a 20-μL PCR reaction with forward and reverse ZmSUN3/4-specific primers (cg1pf1, 5'-GTGATTTGGAGATGCCAGGTG-3' and cg1pr1, 5'-TTTGAGCAAGTTTTGCATTCG-3', respectively) to produce a 639-bp fragment corresponding to a region within exon 2. The PCR product was resolved on 1% agarose, gel purified, and then cloned into the PCR 2.1-TOPO cloning vector (Invitrogen). The plasmid, pSPM17-2, was digested with EcoRI, and the insert was gel purified, quantified, and used in a random-primed labeling reaction with α-32p-dCTP (Amersham Rediprime™ II DNA Labeling System) for use in cDNA library screening. Approximately 5 × 105 phage at a high stringency (Tm-15°C) were screened.
Antibody Production and Immunoblotting
Amino acids 244-256 (ZmSUN3) were chosen as an epitope for the production of rabbit polyclonal antisera to be used to study PM3 proteins in maize. We selected the sequence (zms3gsp1a, LDKDKDKYLRNPC) to allow for the detection of either of the closely related ZmSUN3 and ZmSUN4 proteins. A second peptide antibody was also generated against ZmSUN3 (zms3gsp2, ENKKTEPDDKTKEP). Antibody production, including synthesis of the peptides and affinity purification, was carried out by GenScript (complete affinity-purified rabbit polyclonal antibody package, SC1031, GenScript Corporation, Piscataway, NJ).
Total maize protein extracts were obtained as previously described , with slight modifications: Briefly, one gram of tissue was harvested, ground to a powder in liquid nitrogen, and then homogenized in 3 mL of extraction buffer containing 50 mM Tris-HCl (8.0), 1 mm EDTA-NaOH (8.0), 10% w:v sucrose, 100 mM dithiothreitol, and 1× protease inhibitor complex (4-(2-aminoethyl) benzenesulfonyl fluoride, bestatin, pepstatinA, E-64, leupeptin, and 1,10-phenanthroline, Sigma Aldrich). The homogenate was centrifuged at 12,000 × g for 20 min at 4°C, and the supernatant was recovered and used immediately for immunoblotting or stored at -80°C. For western analyses, protein extracts were mixed with 5× sodium dodecyl sulfate (SDS) loading buffer (25 mM Tris-HCl [6.8], 0.1 M 2-mercaptoethanol, 10% SDS, and 50% glycerol), boiled for 5 min, and separated by electrophoresis on a 10% (w/v) SDS-polyacrylamide gel. Proteins were transferred by electroblotting (overnight, 4°C, 30 mA) to a 0.45-μm polyvinylidene fluoride transfer membrane (PALL life sciences, Port Washington, NY) in a Bio-Rad Mini-PROTEAN 3 Cell. After the membranes were blocked with 5% (w/v) nonfat milk in phosphate-buffered saline plus 0.05% [v/v] Tween-20 (PBS-T) buffer, they were incubated with α-zms3gsp1a diluted 1:2,000 with PBS-T at room temperature for 1 h. After four 15-min washes in PBS-T buffer at room temperature, the membranes were incubated with a 1:5,000 dilution (in PBS-T buffer) of anti-rabbit IgG horseradish peroxidase-linked antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature, then subjected to four 15-min washes in PBS-T buffer at room temperature. The immune complexes were visualized with a chemiluminescent reaction kit for 5 min at room temperature (Millipore, Immobilon detection kit, WBKL50100, Billerica, MA).
Protein Immunolocalization and Microscopy
Maize pollen mother cells were microdissected and fixed in meiocyte Buffer A  with 1% paraformaldehyde supplemented with 100 mM DTT for 30 min at room temperature. The anthers were then rinsed in Buffer A alone for 30 min at room temperature and stored at 4°C. Cells were prepared for immunofluorescence microscopy by embedding in polyacrylamide, followed by a 1-h room-temperature treatment in permeabilization buffer (1% Triton X-100, 1 mM EDTA-NaOH, and 1% BSA in 1× PBS). The acrylamide pads on the slides were then incubated in blocking buffer (3% BSA, 5% normal sheep serum, 1 mM EDTA-NaOH, 0.1% Tween-20, and 1 mM DTT in 1× PBS) at 30°C for 2 h and then incubated with the primary antibodies (α-zmS3gsp1a, α-zmS3gsp2, or preimmune sera at 1:50) in blocking buffer or blocking buffer alone (for secondary-only control) overnight at 30°C. After four consecutive 15-min washes at room temperature with 1× PBS, cells were incubated with a FITC-conjugated goat anti-rabbit IgG (1:1500 in blocking buffer) for 1 h at 30°C then given four 15-min washes with 1× PBS at room temperature. Cells were stained with 3 μg/mL DAPI (4',6-diamidino-2-phenylindole) in 1× PBS for 30 min at room temperature, rinsed three times with 1× PBS, treated with vectashield antifading solution, and finally sealed with a 22 × 30 × 1.5 mm coverslip. Images were collected on an Olympus IX-70 epifluorescense microscope, deconvolved, and analyzed with the SoftWorx computerized workstation.
We would like to thank AB Thistle, members of the Bass laboratory (AN Brown, DM Figueroa, DL Vera, ES Howe), H Cui, and ME Stroupe for critical reading of the manuscript and insightful comments. This work was supported by an American Heart Association predoctoral fellowship to SPM (AHA, Greater Southeast Affiliate, number 0715487B) and by a CRC-planning grant to HWB from the Florida State University Research Foundation.
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