Identification and Expression Analysis of the WOX Transcription Factor Family in Foxtail Millet (Setaria italica L.)

Abstract

WUSCHEL-related homeobox (WOX) transcription factors are unique to plants and play pivotal roles in plant development and stress responses. In this investigation, we acquired protein sequences of foxtail millet WOX gene family members through homologous sequence alignment and a hidden Markov model (HMM) search. Utilizing conserved domain prediction, we identified 13 foxtail millet WOX genes, which were classified into ancient, intermediate, and modern clades. Multiple sequence alignment results revealed that all WOX proteins possess a homeodomain (HD). The SiWOX genes, clustered together in the phylogenetic tree, exhibited analogous protein spatial structures, gene structures, and conserved motifs. The foxtail millet WOX genes are distributed across 7 chromosomes, featuring 3 pairs of tandem repeats: SiWOX1 and SiWOX13, SiWOX4 and SiWOX5, and SiWOX11 and SiWOX12. Collinearity analysis demonstrated that WOX genes in foxtail millet exhibit the highest collinearity with green foxtail, followed by maize. The SiWOX genes primarily harbor two categories of cis-acting regulatory elements: Stress response and plant hormone response. Notably, prominent hormones triggering responses include methyl jasmonate, abscisic acid, gibberellin, auxin, and salicylic acid. Analysis of SiWOX expression patterns and hormone responses unveiled potential functional diversity among different SiWOX genes in foxtail millet. These findings lay a solid foundation for further elucidating the functions and evolution of SiWOX genes.

Keywords: WOX transcription factor family; expression pattern; foxtail millet; plant hormones.

Figure 1

Phylogenetic analysis of WOX proteins of foxtail millet, green foxtail, tomato, maize, rice, and Arabidopsis. A phylogenetic evolutionary tree was constructed using MEGA-11, where clades of the same color represent that they belong to the same subfamily. The WOX gene IDs of the six species are supplemented in Table S2. Node numbers: bootstrap values, a put back sampling statistical method used to test the credibility of evolutionary tree branches.

Figure 2

Prediction of the spatial structure of SiWOX proteins. Modern clade: SiWOX2, SiWOX3, SiWOX7, SiWOX8, SiWOX10, SiWOX11, and SiWOX12; Intermediate clade: SiWOX1, SiWOX4, SiWOX5, SiWOX9, and SiWOX13; Ancient clade: SiWOX6. The spatial structure predictions of SiWOX proteins links: https://alphafold.ebi.ac.uk/ (accessed on 5 December 2023).

Figure 3

Analysis of SiWOX gene structure and conserved motifs. (A) Phylogenetic tree. (B) Gene structure. Coding sequences (CDS) and untranslated regions (UTRs) are represented by different colored boxes, while introns are indicated by lines. (C) Conserved motifs. Conserved motifs within the SiWOX genes are represented by boxes of different colors. The weblogo diagram of the twelve conserved motifs is provided in Figure S2.

Figure 4

Chromosome distribution and gene tandem repeats of SiWOX genes. Gray lines represent tandem repeats between foxtail millet genomes, while red lines represent tandem repeats between SiWOX genes.

Figure 5

Collinearity analysis of WOX genes in Setaria viridis, Setaria italica, Zea mays, and Oryza sativa. Gray lines indicate collinearity between genes from different species, while purple lines indicate collinearity between WOX genes from different species. Sv: Setaria viridis, Si: Setaria italica, Zm: Zea mays, Os: Oryza sativa.

Figure 6

Expression pattern analysis of SiWOX genes in different tissues. The heatmap of the expression profiles of the SiWOX gene in different developmental stages is represented by normalized values using RNA-seq data, with the color from red to blue indicating the expression levels from high to low. Tissue information for SiWOX expression analysis is provided in Table S4.

Figure 7

Expression patterns of SiWOX genes in foxtail millet panicles and roots. (A) Expression patterns of SiWOX genes in panicles at different developmental stages: Stage 1, panicle length approximately 1.0–1.5 mm; stage 2, panicle length approximately 2.5–3.0 mm. (B) Expression patterns of SiWOX genes in 9-day-old seedling roots treated with ABA (2 μM) and without ABA (CK). Bar graphs represent differences in transcriptome sequencing duplicates between spike samples (A) and root samples (B). Statistical significance between panicle (A) and root (B) stages was determined by t-test (* p < 0.05). TPM: transcripts per million.

Figure 8

Expression pattern of SiWOX genes in response to plant hormones. Six plant hormones, 6-BA (100 μM), ABA (100 μM), GA3 (100 μM), SA (100 μM), MeJA (100 μM), and IAA (100 μM), were used to treat 28-day-old seedlings, and leaf samples were taken at 0 h, 0.5 h, 2 h, 6 h, and 12 h post-treatment. The gene expression levels at different time intervals were detected using the qPCR method. Line graphs represent changes in gene expression levels.