Genome-Wide Identification and Characterization of CLAVATA3/EMBRYO SURROUNDING REGION (CLE) Gene Family in Foxtail Millet (Setaria italica L.)

Abstract

The CLAVATA3/EMBRYO-SURROUNDING REGION (CLE) genes encode signaling peptides that play important roles in various developmental and physiological processes. However, the systematic identification and characterization of CLE genes in foxtail millet (Setaria italica L.) remain limited. In this study, we identified and characterized 41 SiCLE genes in the foxtail millet genome. These genes were distributed across nine chromosomes and classified into four groups, with five pairs resulting from gene duplication events. SiCLE genes within the same phylogenetic group shared similar gene structure and motif patterns, while 34 genes were found to be single-exon genes. All SiCLE peptides harbored the conserved C-terminal CLE domain, with highly conserved positions in the CLE core sequences shared among foxtail millet, Arabidopsis, rice, and maize. The SiCLE genes contained various cis-elements, including five plant hormone-responsive elements. Notably, 34 SiCLE genes possessed more than three types of phytohormone-responsive elements on their promoters. Comparative analysis revealed higher collinearity between CLE genes in maize and foxtail millet, which may be because they are both C₄ plants. Tissue-specific expression patterns were observed, with genes within the same group exhibiting similar and specific expression profiles. SiCLE32 and SiCLE41, classified in Group D, displayed relatively high expression levels in all tissues except panicles. Most SiCLE genes exhibited low expression levels in young panicles, while SiCLE6, SiCLE24, SiCLE25, and SiCLE34 showed higher expression in young panicles, with SiCLE24 down-regulated during later panicle development. Greater numbers of SiCLE genes exhibited higher expression in roots, with SiCLE7, SiCLE22, and SiCLE36 showing the highest levels and SiCLE36 significantly down-regulated after abscisic acid (ABA) treatment. Following treatments with ABA, 6-benzylaminopurine (6-BA), and gibberellic acid 3 (GA3), most SiCLE genes displayed down-regulation followed by subsequent recovery, while jasmonic acid (JA) and indole-3-acetic acid (IAA) treatments led to upregulation at 30 min in leaves. Moreover, identical hormone treatments elicited different expression patterns of the same genes in leaves and stems. This comprehensive study enhances our understanding of the SiCLE gene family and provides a foundation for further investigations into the functions and evolution of SiCLE genes in foxtail millet.

Keywords: CLE gene family; expression pattern; foxtail millet; plant hormone.

Figure 1

Phylogenetic tree of CLE peptides from foxtail millet, Arabidopsis, rice, and maize. The phylogenetic tree was constructed with the neighbor-joining (NJ) method in MEGA 7.0 software and was divided into four subgroups.

Figure 2

Gene structure and conserved motif analysis of SiCLE peptides. (A) Phylogeny tree. (B) Gene structure. Coding sequence (CDS) and untranslated region (UTR) are represented by different colored boxes, and introns are represented by lines. (C) Motif patterns. Conserved motifs in the SiCLE peptides are represented by different colored boxes. Weblogo plots of the five conserved motifs are shown in Figure S1.

Figure 3

Cis-regulatory elements in SiCLE gene promoters. The elements are displayed in differently colored boxes.

Figure 4

Chromosomal distribution and gene duplication of the SiCLE genes. Two SiCLE genes of the same segmental duplicated gene pair are labeled with blue lines.

Figure 5

Synteny analyses of the SiCLE genes between S. italica and three representative plant species. Gray lines on the background indicate the collinear blocks within S. italica and other plant genomes; red lines highlight the syntenic S. italica SiCLE gene pairs.

Figure 6

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

Figure 7

Expression patterns of SiCLE genes in young panicles and roots of foxtail millet. (A) Expression patterns of SiCLE genes in young panicles at different formation stages: branch meristems specified (stage 1, approximately 1.0–1.5 mm) and later stage with clearly formed branch meristems (stage 2, approximately 2.5–3.0 mm). (B) Expression patterns of SiCLE genes in the roots of 9-day-old seedlings with ABA treatment (2 μM) and without ABA as the control (CK). The bars represent the variation between replicates in the transcriptome sequencing of young panicle samples (A) and root samples (B). Statistically significant differences between the two stages of the panicle (A) and the root with and without ABA treated (B) were determined according to t-test (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 8

Expression patterns of SiCLE genes response to plant hormones in foxtail millet. Seedlings that were 28 days old were treated with plant hormones, including 6-BA (100 µM) (A), ABA (100 µM) (B), GA3 (100 µM) (C), MeJA (100 µM) (D), and IAA (100 µM) (E). Samples of leaves and stems were collected at 0 h, 0.5 h, 2 h, 6 h, and 12 h after the treatments. The gene expression levels at different time intervals were detected using the qPCR method. The lowercase letters in the heatmap represent significant differences between different time intervals after treatment. The color from red to blue indicates the expression levels from high to low in the heatmap.

Figure 9

The schematic diagram illustrates the predicted functions of SiCLE genes in foxtail millet. The diagram is based on the phylogenetic relationship and expression patterns of SiCLE genes, as well as the knowledge from reported CLE genes in other species. The font color in the diagram corresponds to the background color of different groups in the phylogenetic tree.