Structure and expression of the maize (Zea mays L.) SUN-domain protein gene family: evidence for the existence of two divergent classes of SUN proteins in plants

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Figure 1

Phylogenetic relationships among selected SUN-Domain proteins in the plant kingdom. An unrooted phylogenetic tree of SUN-domain proteins is shown, deduced from full-length cDNAs from maize (Zea mays, Zm), Arabidopsis (At), rice (Os), Sorghum bicolor (Sb), and moss (Physcomitrella patens, Pp). GenBank accession numbers are given in the figure, except for those of maize, which are from sequences listed in Table 1. The protein maximum-likelihood tree was created with TreeView, version 1.6.6 [71]. Proteins belonging to the canonical (CCSD, green shaded area) and mid-SUN (PM3, yellow shaded area) classes are indicated. Four SUN orthologous grass groups (SOGG1-4) are also indicated. A partial EST from sorghum (Sb03g010590/PUT-157a-Sorghum_bicolor-11155) aligns with the SOGG2 group but was excluded from the analysis because it lacked a full-length ORF. Scale bar (0.1) represents 10 expected amino-acid changes for every 100 residues.

Figure 2

Genomic structures for the two subfamilies of maize SUN-domain protein genes. The locations of exons, start (ATG), and stop (TGA, TAA) codons are shown for each gene. The diagrams were drawn from predictions made by the SPIDEY program http://www.ncbi.nlm.nih.gov/spidey/ on the basis of alignments of cDNA to genomic DNA sequences (from Table 1). The mRNA coordinates for the exon bases are listed above the diagrams. Exons are numbered, and the intron lengths (bp) appear below the diagrams. (A) The canonical C-terminal SUN domain genes show a large intron at a conserved location interrupting the SUN domain region (yellow box) within the ORF. (B) The plant-prevalent mid-SUN 3 transmembrane genes all share a large exon that contains the entire SUN domain plus a domain of unknown function (black box) associated with these genes, as well as two small introns before the last exon.

Figure 3

Conservation of functional domains in plant and animal SUN-domain proteins. Comparative diagrams of SUN-domain proteins depicting protein sizes and domain locations (see Table 2). The positions of transmembrane (red), coiled-coil (blue), SUN (yellow), and PM3-associated (PAD) domains (black) are indicated for each protein. (A) Known nonplant SUN-domain proteins (human, Hs; mouse, Mm; nematode; Ce; fission yeast, Sp) of various sizes, but all with a single C-terminal SUN domain are shown (UniProt accession numbers: HsSUN1, O94901; HsSUN2, Q9UH99; MmSUN1, Q9D666; MmSUN2, Q8BJS4; CeSUN1, Q20924; CeUNC84, Q20745; SpSAD1, Q09825). (B) CCSD and (C) PM3 plant proteins grouped by their orthologous groups (see Figure 1).

Figure 4

Multiple sequence alignment of PM3 SUN domains and PAD regions. Multiple sequence alignments from ClustalW2 for isolated domains of PM3 proteins from five plant species. Box shade alignment displays show conserved residues (identical black, similar grey) and an alignment consensus sequence at the bottom. (A) Alignment of the SUN domains with amino-acid numbers indicated. (B) Alignment of PAD regions composed of a ~38-amino acid segment.

Figure 5

Expression of ZmSUN genes in meiosis-stage anthers. Relative expression levels shown by maize SUN-domain protein genes obtained from published microarray experiments (Gene Expression Omnibus [73,79]). The cDNAs were from meiosis-stage anthers 1 mm, 1.5 mm, and 2 mm in length. The histogram depicts signals relative to the whole-chip mean. Dye-normalized values for each channel generated by Feature Extraction software were divided by the median intensity for that channel on each array, and then the log base 2 was taken, as previously described [61]. The table at the bottom tabulates the gene name (Gene), Probe ID (the gene model/contig being targeted), and feature number (chip oligo 60-mer).

Figure 6

Expression profiling of ZmSUN genes by Solexa tag-based and whole-transcriptome sequencing. mRNA from various B73 tissues was subjected to two Solexa sequencing platforms, Solexa whole-transcriptome (SWT) and Solexa dual-tag based (STB). The vertical axis represents the number of 36-nt (SWT) or 21-nt (STB) sequence tag matches per ten million transcripts. (A) Expression levels of ZmSUN genes and the control gene, cytoplasmic GAPDH, are graphed for comparison. (B) The same data are plotted as semi-log₂ for easier comparisons among the low-expression ZmSUN genes.

Figure 7

ZmSUN4 cDNA and protein features. (A) ZmSUN4 (genotype W23) full-length cDNA, showing the 5' and 3' UTRs, open reading frame (ORF), and poly-A tail. A diagram of the protein indicates domain locations as described in Figure 3. (B) Annotated protein sequence predicted from full-length cDNA ORF (GenBank GU453173). Color scheme is the same as in Figure 3. Amino acid residues below the ZmSUN4 sequence show divergent residues of the duplicated locus on chromosome 3L, ZmSUN3, genotype B73.

Figure 8

Western blot of proteins ZmSUN3 and ZmSUN4. (A) Two peptide antibodies were made against synthetic peptides within (zms3gsp1a) and just after (zms3gsp2) the SUN-domain of the maize ZmSUN3 protein. The corresponding regions in ZmSUN3 and ZmSUN4 are aligned, and asterisks indicate divergent residues in ZmSUN4. (B) Western-blot detection (top panel) of ZmSUN3 and ZmSUN4 in various plant tissues. Protein was loaded on an equal-fresh-weight basis for leaf, root, silk, husk, earshoot, embryo, meiosis-stage tassel, and postmeiotic tassel, resulting in the detection of a single band of ~72 kDa. (C) Immunoblot showing the effect of increased sample boiling time on bands detected. Protein from meiosis-stage anthers appeared as a single band at ~70 kDa (arrow) after the protein was boiled in SDS for 10 min or more.

Figure 9

Immunolocalization of PM3 SOGG3 Proteins at the nuclear periphery. Combined antisera (zms3gsp1a and zms3gsp2) or preimmune control sera were used to stain formaldehyde-fixed uninucleate pollen mother cells. The immune complex was visualized by deconvolution microscopy in the FITC channel with A488-goat-anti-rabbit sera. Images from a single cell are shown. (A-C) Projection of the central 2/3 of the three-dimensional set of data shows DAPI image (A), FITC image (B), and pseudocolor overlay (C). Zoom up of a region of the nucleus-cytoplasm boundary is shown for the FITC (D) and overlay (E) images. Control staining with preimmune sera (F) or secondary only (G) are shown with a color scheme (red DAPI, green FITC) and scaling parameters that match those of panel C.

Figure 10

Maize SUN topology models relative to the membranes of the nuclear envelope. Possible protein arrangement models with the SUN (yellow) domain in the perinuclear space are shown for the CCSD (A-B) and PM3 (C-F) proteins. Models do not attempt to depict multimer interactions that may occur with the SUN or coiled-coil (not shown) domains.