Genome-Wide Identification and Characterisation of the 4-Coumarate-CoA Ligase (4CL) Gene Family in Gastrodia elata and Their Transcriptional Response to Fungal Infection

Abstract

Gastrodia elata Blume is an important medicinal orchid, yet its large-scale cultivation is increasingly threatened by fungal diseases. The 4-coumarate-CoA ligase (4CL) gene family directs a key step in phenylpropanoid metabolism and plant defence, but its composition and function in G. elata have not been investigated. We mined the G. elata genome for 4CL homologues, mapped their chromosomal locations, and analysed their gene structures, conserved motifs, phylogenetic relationships, promoter cis-elements and codon usage bias. Publicly available transcriptomes were used to examine tissue-specific expression and responses to fungal infection. Subcellular localisation of selected proteins was verified by transient expression in Arabidopsis protoplasts. Fourteen Ge4CL genes were identified and grouped into three clades. Two members, Ge4CL2 and Ge4CL5, were strongly upregulated in tubers challenged with fungal pathogens. Ge4CL2 localised to the nucleus, whereas Ge4CL5 localised to both the nucleus and the cytoplasm. Codon usage analysis suggested that Escherichia coli and Oryza sativa are suitable heterologous hosts for Ge4CL expression. This study provides the first genome-wide catalogue of 4CL genes in G. elata and suggests that Ge4CL2 and Ge4CL5 may participate in antifungal defence, although functional confirmation is still required. The dataset furnishes a foundation for functional characterisation and the molecular breeding of disease-resistant G. elata cultivars.

Keywords: 4CL gene family; Gastrodia elata Blume; fungal infection; molecular characterisation; molecular defence; transcriptome analysis.

Figure 1

Physical locations of Ge4CLs in G. elata: Chromosome numbers are marked on the left side of the chromosomes in green font, and chromosome lengths are indicated in megabases (Mb). The red colours on the chromosomes represent the highest density, and blue indicates the lowest density.

Figure 2

Collinearity analysis of Ge4CLs in G. elata: (a) Intra-species collinearity analysis of Ge4CLs. (b) Collinearity analysis of 4CLs between G. elata and five other representative plants. The grey lines represent collinear blocks, and the red lines represent collinear 4CL gene pairs.

Figure 3

Maximum-likelihood (ML) phylogenetic tree of 4-coumarate–CoA ligase (4CL) proteins from G. elata (Ge) and 43 reference plant species. The tree was reconstructed in TBtools v1.138 using the JTT + G4 model and 5000 bootstrap replicates; bootstrap support values (%) are shown at the nodes. Subfamilies I–III are colour-shaded for clarity. Protein IDs of reference sequences are given in parentheses (e.g., At4CL1 = A. thaliana At1g51680; Os4CL1 = O. sativa AK069932.1; full list provided in Materials and Methods). Scale bar indicates the number of substitutions per site.

Figure 4

Conserved motifs and gene structure analysis: (a) Conserved motifs of Ge4CLs. (b) Gene structures of Ge4CLs. (c) Motif identifications of Ge4CLs.

Figure 5

Distribution of different types of cis-acting elements in Ge4CLs: The numbers in the heatmap represent the quantities of different types of cis-acting elements in the promoter regions.

Figure 6

Analysis of codon usage bias of the Ge4CLs: (a) ENC plot analysis. GC3s: GC content at the third position of the codon; (b) PR2 plot analysis. A3s: content of base A at the third position of the codon; T3s: content of base T at the third position of the codon; G3s: content of base G at the third position of the codon; C3s: content of base C at the third position of the codon. In the figure, x = 0.5 and y = 0.5 are used as reference lines for analysing codon preference. (c) Neutral plot analysis. GC3: GC content at the third position of the codon; GC12: average GC content at the first and second positions of the codon.

Figure 7

Expression profiles of Ge4CL genes based on public RNA-Seq data: (a) Tissue specificity across four developmental tissues: 1 = mature tuber (MT), 2 = juvenile tuber (JT), 3 = mother tuber of G. elata (MT-Ge), 4 = mother tuber of juvenile (MT-JT). (b) Comparison between fungal-diseased (FD) and healthy (HT) mature tubers of G. elata f. glauca. Heatmaps were generated from log2-transformed TPM values; colour scale ranges from red (high expression) to blue (low expression). Gene IDs are listed on the left.

Figure 8

Subcellular localisation analysis of Ge4CL2 and Ge4CL5 proteins: Bright Light: The field of view under the bright-light channel, showing the cell morphology. NLS-RFP: The field of view under the red fluorescence channel; NLS-RFP was localised in the nucleus. GFP: The field of view under the green fluorescence channel, showing the localisation of the green fluorescent protein (GFP). Merged: The overlapping effect of the images under the three channels. The scale bar is 25 nm.