High-quality Lindera megaphylla genome analysis provides insights into genome evolution and allows for the exploration of genes involved in terpenoid biosynthesis

Abstract

Lindera megaphylla, a Lauraceae species, is valued for timber, horticulture, landscape architecture, and traditional medicine. Here, a high-quality genome of L. megaphylla was obtained at the chromosome level. A total of 96.77% of genomic sequences were mapped onto 12 chromosomes, with a total length of 1309.2 megabase (Mb) and an N50 scaffold of 107.75 Mb. Approximately, 75.91% of genome consists of repetitive sequences and 7004 ncRNAs were predicted. We identified 29 482 genes, and 28 657 genes were annotated. Gene family analysis showed expanded gene families were mainly involved in energy metabolism and cellular growth, while contracted ones were associated with carbohydrate metabolism and signal transduction. Our analysis revealed that L. megaphylla has undergone two rounds of whole-genome duplication (WGD). Our results revealed that volatile compounds in L. megaphylla leaves inhibited the growth of several fungi and bacteria. Fifty-two terpene synthase (TPS) genes were identified and classified into six subfamilies, with significant expansion observed in the TPS-b, TPS-f, and TPS-g subfamilies in L. megaphylla. Transcriptomic and metabolomic co-analysis revealed that 43 DEGs were correlated with 117 terpenoids. Further analysis revealed that LmTPS1 was significantly correlated with caryophyllene oxide content. The overexpression of LmTPS1 in transgenic tomato lines significantly increased the contents of β-caryophyllene and humulene, which further improved the resistance of transgenic tomato plants to common fungal and bacterial diseases. The integrated analysis of genome, metabolome, and transcriptome provides comprehensive insights into the evolution of L. megaphylla and clarifies the molecular mechanisms underlying the protective effects of caryophyllene against biotic stress.

Figure 1

Phenotype, Hi-C contact map, and gene family analysis of the L. megaphylla genome. (a-d) Phenotypes of the whole plant, leaves, flowers, and fruits of L. megaphylla. (e) All-by-all interactions across the 12 chromosomes obtained by Hi-C sequencing within the L. megaphylla genome. (f) The number of genes in each gene family in L. megaphylla and 15 representative species. (g) Gene family expansion and contraction in L. megaphylla and 15 representative species.

Figure 2

Schematic representation of the phylogenetic tree with the Ks density curve. The circles denote whole-genome triplication (WGT) events, whereas the squares indicate WGD events.

Figure 3

A detailed visualization of the global collinearity alignment, complemented by a partial homologous gene dot matrix. Distinct color-coded rectangles are employed to delineate and highlight the collinear regions, facilitating an intuitive understanding of the genomic correspondences among the species.

Figure 4

Antimicrobial activity of volatile compounds from L. megaphylla leaves. (a) Volatile compounds from L. megaphylla leaves against E. coli, S. aureus, S. typhimurium, and B. subtilis. (b) Volatile compounds from L. megaphylla leaves against Penicillium, A. flavus, and Rhizopus.

Figure 5

TPS family gene phylogenetic tree and conserved motif distribution. TPS-a, TPS-b, TPS-c, TPS-e, TPS-f, and TPS-g represent the 6 subfamily classifications of the TPS family. Ten different colors represent motifs 1 to 10.

Figure 6

Identification of DEGs in different comparison groups. (a-c) KEGG enrichment analysis of DEGs in the comparisons of LY vs. SY, SZ vs. LY, and SZ vs. SY. (d) Module analysis of DEGs. The Y- and X-axes indicate the P values and tissues, respectively. (e) A heatmap was constructed to display the expression of DEGs associated with terpenoid synthesis based on RNA-seq data. (f) RT-qPCR verification of 12 candidate genes associated with terpenoid synthesis. The error bars reflect the standard error of three replicates, indicating the variability in the expression data. Note: SY, LY, and SZ represent leaves, leaf buds, and annual shoots, respectively.

Figure 7

Transcriptomic and metabolomic co-analysis of DEGs and DEMs involved in the terpenoid biosynthesis pathway. (a-c) KEGG enrichment analysis of DEMs in the comparisons of LY vs. SY, SZ vs. LY, and SZ vs. SY. (d) Correlation analysis was conducted between a subset of DEGs and DEMs related to terpenoid synthesis in L. megaphylla. (e) Tissue-specific relative expression profiles of DEGs implicated in terpenoid biosynthesis. The red and blue boxes indicate the up- and down-regulated DEGs, respectively, in different tissues of L. megaphylla. Cytosol represents the MVA pathway, and plastid represents the MEP pathway.

Figure 8

Protein sequence alignment, phylogenetic analysis, and subcellular localization of LmTPS1. (a) Multiple sequence alignment of LmTPS1 and homologous proteins from other plants. Conserved motifs are highlighted in dashed rectangle. (b) Phylogenetic analysis showing the evolutionary relationships between LmTPS1 and homologous proteins from other plants. (c) Subcellular localization of the LmTPS1 protein in tobacco leaves. GFP alone or the LmTPS1 gene fused with GFP driven by the CaMV35S promoter was transiently expressed in tobacco epidermis cells. Transgenic plants expressing candidate genes fused to GFP were detected by laser scanning confocal microscopy. Scale bar: 100 μm.

Figure 9

Overexpression of LmTPS1 improved the resistance of transgenic tomato to biotic stress. (a) Structural diagram of LmTPS1-pCAMBIA2300. (b) Tissue-specific correlation analysis between LmTPS1 expression and β-Caryophyllene accumulation in L. megaphylla. (c) RT-qPCR verification of LmTPS1 in transgenic tomato lines (OE1-OE3). (d) Measurement of terpenoids in transgenic tomato lines and WT plants using GC–MS/MS. (e) The contents of three main terpenoids in the transgenic tomato lines. (f) The inhibitory effects of β-caryophyllene on S. aureus, E. coli, S. typhimurium, and B. subtilis. (i-h) Tomato fruits injected with β-caryophyllene presented increased resistance to biotic stress. (j) Overexpression of LmTPS1 improved the resistance of transgenic tomato to biotic stress. The leaf lesion areas were captured under 100x and 180x magnification using a super depth-of-field microscope, with arrows indicating the diseased regions on the leaf. Scale bar: 500 μm.