Genome organization of more than 300 defensin-like genes in Arabidopsis

Abstract

Defensins represent an ancient and diverse set of small, cysteine-rich, antimicrobial peptides in mammals, insects, and plants. According to published accounts, most species' genomes contain 15 to 50 defensins. Starting with a set of largely nodule-specific defensin-like sequences (DEFLs) from the model legume Medicago truncatula, we built motif models to search the near-complete Arabidopsis (Arabidopsis thaliana) genome. We identified 317 DEFLs, yet 80% were unannotated at The Arabidopsis Information Resource and had no prior evidence of expression. We demonstrate that many of these DEFL genes are clustered in the Arabidopsis genome and that individual clusters have evolved from successive rounds of gene duplication and divergent or purifying selection. Sequencing reverse transcription-PCR products from five DEFL clusters confirmed our gene predictions and verified expression. For four of the largest clusters of DEFLs, we present the first evidence of expression, most frequently in floral tissues. To determine the abundance of DEFLs in other plant families, we used our motif models to search The Institute for Genomic Research's gene indices and identified approximately 1,100 DEFLs. These expressed DEFLs were found mostly in reproductive tissues, consistent with our reverse transcription-PCR results. Sequence-based clustering of all identified DEFLs revealed separate tissue- or taxon-specific subgroups. Previously, we and others showed that more than 300 DEFL genes were expressed in M. truncatula nodules, organs not present in most plants. We have used this information to annotate the Arabidopsis genome and now provide evidence of a large DEFL superfamily present in expressed tissues of all sequenced plants.

Figure 1.

Clustering of related DEFLs in Arabidopsis. Each filled circle represents a single DEFL gene. DEFLs that fall within a 100,000-bp window are stacked vertically. The largest clusters are numbered for reference elsewhere. Sequences within labeled clusters are color-coded to reflect membership in sequence-related subgroups. Sequences are colored only if they match at least one other sequence of the same subgroup in a labeled cluster. Overlapping subgroups (subgroups whose members score against a neighboring subgroup's HMM with E value < 10⁻⁴) are assigned a single color.

Figure 2.

Representative alignment of DEFLs from four distinct clusters in Arabidopsis. Clusters correspond to those identified in Figures 1 and 3 and Table I. Not all sequences from each cluster are included. Identical residues are shaded black, while similar residues are shaded gray. A box labeled Signal Peptide designates the size and position of the signal peptide. Boxes labeled C or G designate conserved Cys or Gly residues within each group. Locus identifiers, issued by TAIR, are shown to the left of the alignment. Below the figure, key residues are shown that correspond to the CSαβ motif common to defensins (Cornet et al., 1995; Lay et al., 2003b) and the γ-core motif common to all of classes Cys containing AMPs (Yount and Yeaman, 2004).

Figure 3.

Expression of DEFLs in Arabidopsis flowers, shoots, and roots. Plus and minus reverse-transcriptase cDNA libraries were constructed from Arabidopsis flower (F, F−), shoot (S, S−), and root (R, R−) RNA. SEC and ACT7 primers were controls for cDNA synthesis and genomic DNA contamination. Genomic DNA (G) and water (H) were positive and negative controls for PCR amplification. Primer names are located to the left of gel images and correspond to clusters in Figure 1. The sizes of the amplified cDNAs appear to the right of the image. Primer pair 14.1 was not expected to amplify genomic DNA since one of the primers spanned the boundaries of exon 1 and 2. Multiple bands within a lane reflect the ability of primers to amplify multiple DEFLs.

Figure 4.

Plot of nonsynonymous (K_a) versus synonymous (K_s) substitution rates for Arabidopsis DEFL sequences. Nucleotide substitution ratios were determined for the coding sequences of gene pairs clearly arising from a local duplication event. The Arabidopsis clusters used in this analysis are numbered (1–16) in Figure 1. The DEFL sequences were divided into two parts: the signal peptide and the trimmed mature peptide minus conserved Cys codons. Only sequences for which a K_a and K_s value could be determined for both signal and mature peptides were included. The line represents neutral selection (K_a/K_s = 1). Points above this line are likely under divergent selection, while points below are conserved. Statistical analyses of these results are provided in Supplemental Table IV.