A genomic and expression compendium of the expanded PEBP gene family from maize

Abstract

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.

Figure 1.

ZCN gene positions on maize IBM2 genetic map and syntenic rice genes. ZCN positions are inferred from the nearest markers of the corresponding BAC fingerprinting contigs (Table I). Centromeric regions are shown as black rectangles. Pseudogene, ZCN23, is marked by an asterisk (*). Seven duplicated pairs of genes are marked by various colors: ZCN1/ZCN3, blue; ZCN4/ZCN5-ZCN19/ZCN25, red; ZCN9/ZNC10, burgundy; ZCN18/ZCN24, orange; ZCN7/ZCN8, green; ZCN13/ZCN21, framed red over the centromeres. Rice genes (their chromosomes in parentheses) with the highest probability of synteny are shown on the left side of the maize chromosome.

Figure 2.

ZCN exon/intron structures. Genomic structure of the ZCN genes is represented by black boxes as exons and spaces between the black boxes corresponds to introns. The sizes of the exons and introns can be estimated using the vertical lines. Genes are arranged into phylogenetic subfamilies. The major subfamilies are denoted on the right.

Figure 3.

Phylogenetic analysis of the maize, rice, and Arabidopsis PEBP proteins. The protein sequences of 51 plant PEBP gene products including 45 Poaceae monocot (black) and six Arabidopsis (red) were limited to the conserved EDL/DPL…..RRR/GGR region encompassing most of the complete proteins. The phylogenetic test used was the Bootstrap test by minimum evolution method, as implemented by MEGA version 3.1 (Kumar et al., 2004). Dendogram branches are labeled with percentage of 1,000 iterations supporting each branch. The scale bar at bottom reflects the frequency of amino acid substitutions between sequences as determined by the Poisson corrective distance model.

Figure 4.

Structural models of two ZCN proteins and alignment of maize and Arabidopsis PEBP proteins. Protein structure ribbon presentation of ZCN2 (A) and ZCN14 (B). The secondary structures are assigned by Pymol and colored as orange. The external loop is highlighted as magenta. The conserved residue side chains in the binding pocket are shown as sticks and the triad residue side chains are represented as stick and balls with atoms color coded: green for carbon, blue for nitrogen, and red for oxygen. C, Multiple sequence alignment of the conserved ligand-binding motif and the external loop of maize ZCN and the Arabidopsis TFL1, MFT, FT, and TSF proteins. The external loop is boxed. Invariant amino acids are highlighted yellow, similar amino acids are highlighted green or blue. Intron positions are marked by arrows above the alignment. Asterisks (*) indicate invariable and pound signs (#) variable, but critical amino acids forming the ligand-binding pocket. Invariable residues correspond to the following position in TFL1/FT proteins: Asp-74/71, Asp-75/73, Pro-78/75, His-90/87, His-121/118. Variable His-88/Tyr-85 residues define the opposing activity of TFL1 and FT, respectively. Ampersand (&) signs mark Arg-133/130 and Arg-143/139 in the external loop that is conserved in most proteins, with the exceptions of ZCN2, ZCN19, and ZCN25.

Figure 5.

Maize ZCN family transcript survey across various tissues using MPSS technology. Relative ZCN gene transcript abundance measured as mean frequency of 17-mer tags in part per million in varies tissues. ZCN13 was omitted from the survey due to lack of a gene-specific tag. ZCN2, ZCN20, and ZCN21 do not produce expressed 17-mer tags.

Figure 6.

ZCN gene expression patterns revealed by RT-PCR. RNA expression patterns of TFL1-like genes in a broad set of tissues (A), and in developing tassels and ears (B). Expression patterns of MFT-like genes in a broad set of tissues (C) and in developing kernels (D). Expression patterns of FT-like I genes in a broad set of tissues (E). ZCN14 and ZCN15 expression in developing kernels, pedicel (ZCN15), and in developing tassels and ears (ZCN14; F). Expression patterns of FT-like II genes in a broad set of tissues (G) and in developing stems and leaves (H). Horizontal arrows indicate spliced mRNA. White stars indicate the floral transition. Asterisks (*) mark genes with no detectable expression. V3 to V10 indicates the vegetative stages of plant development defined according to the appearance of the leaf collar of the uppermost leaf. The superscript numbers 1 and 2, mark the early and later stages of V4 and V7, respectively, as judged by the shorter internode length at the early stages. Beginning of the floral transition was determined by the elongation of the shoot apical meristem followed by the appearance of the spikelet pair meristem that typically takes 1 to 2 d. Tissues are abbreviated as: EM, Embryo; EN, endosperm; IL, immature leaves; IT, immature tassel; IE, immature ear; LB, leaf blade; SAM, shoot apical meristem; R, root; S, stem.