Genome-Wide Sequence and Expression Analysis of the NAC Transcription Factor Family in Polyploid Wheat

Abstract

Many important genes in agriculture correspond to transcription factors (TFs) that regulate a wide range of pathways from flowering to responses to disease and abiotic stresses. In this study, we identified 5776 TFs in hexaploid wheat (Triticum aestivum) and classified them into gene families. We further investigated the NAC family exploring the phylogeny, C-terminal domain (CTD) conservation, and expression profiles across 308 RNA-seq samples. Phylogenetic trees of NAC domains indicated that wheat NACs divided into eight groups similar to rice (Oryza sativa) and barley (Hordeum vulgare). CTD motifs were frequently conserved between wheat, rice, and barley within phylogenetic groups; however, this conservation was not maintained across phylogenetic groups. Three homeologous copies were present for 58% of NACs, whereas evidence of single homeolog gene loss was found for 33% of NACs. We explored gene expression patterns across a wide range of developmental stages, tissues, and abiotic stresses. We found that more phylogenetically related NACs shared more similar expression patterns compared to more distant NACs. However, within each phylogenetic group there were clades with diverse expression profiles. We carried out a coexpression analysis on all wheat genes and identified 37 modules of coexpressed genes of which 23 contained NACs. Using gene ontology (GO) term enrichment, we obtained putative functions for NACs within coexpressed modules including responses to heat and abiotic stress and responses to water: these NACs may represent targets for breeding or biotechnological applications. This study provides a framework and data for hypothesis generation for future studies on NAC TFs in wheat.

Keywords: NAC; gene expression; phylogenetics; transcription factors; wheat.

Figure 1

Comparison of genes identified per transcription factor family in wheat, barley, and rice. The number of genes in each family for (A) wheat, (B) barley, and (C) rice. (D) The ratio of wheat to barley (red) and wheat to rice (blue). In (D), the expected ratio (3:1) is indicated by a black line. Barley and rice data were obtained from PlantTFDBv3.0.

Figure 2

Maximum likelihood phylogeny of 667 NAC proteins from wheat, rice, and barley. The phylogeny was constructed using only the NAC domain. NAC groups a–h were assigned according to rice and barley orthologs. In cases where the group assigned to a rice or barley gene conflicted with the overall tree topology, no group was assigned (black branches). Details of individual genes are presented in Figure S1 and Table S4.

Figure 3

Conserved domains in the CTD of NAC transcription factors arranged by phylogenetic position. Known CTD motifs are shown alongside the wheat, barley, and rice NAC TFs for each group a–h (A–H), colored in accordance with Figure 2. Branches corresponding to wheat NAC TFs are solid black; those for rice and barley NAC TFs are dashed. Motifs are shown as boxes, matching (left to right) motifs i–xiii from Ooka et al. (2003). Motifs that are present in each protein (P-value < 0.05, q-value < 0.05) are shown by a solid-colored box, while absent motifs are shown by an empty outlined box. Genes with no significant motifs are shown with empty space. Barley and rice genes are indicated by the presence of a star to the right of the CTD motifs. Details are presented in Table S5, and the full phylogenetic tree is presented in Figure S2. CTD, C-terminal domain; TF, transcription factor.

Figure 4

Relationship between phylogenetic position and NAC gene expression across 308 RNA-seq samples from diverse tissues, developmental stages, and stress conditions. The origin of each sample is indicated by the colored bar under each heatmap. Each panel (A–H) represents NAC genes belonging to that group according to the classification in Figure 2. Dendrograms indicate the maximum likelihood phylogeny of genes within each group. Genes that did not meet the minimum expression criteria (> 0.5 tpm in at least three samples) do not have expression data represented (white rows). All remaining expression data (tpm) was normalized per gene to range from 0 to 1. An extended version of the figure with the full phylogenetic trees is available as Figure S3. RNA-seq, RNA sequencing; tpm, transcripts per million.

Figure 5

Distribution of genes and transcription factors (TFs) across modules. (A) Number of genes (log10), (B) percentage of genes that are TFs, (C) percentage of TFs that are NAC TFs, and (D) number of NACs from each phylogenetic group.