Comparative Analysis and Identification of Terpene Synthase Genes in Convallaria keiskei Leaf, Flower and Root Using RNA-Sequencing Profiling

Abstract

The 'Lilly of the Valley' species, Convallaria, is renowned for its fragrant white flowers and distinctive fresh and green floral scent, attributed to a rich composition of volatile organic compounds (VOCs). However, the molecular mechanisms underlying the biosynthesis of this floral scent remain poorly understood due to a lack of transcriptomic data. In this study, we conducted the first comparative transcriptome analysis of C. keiskei, encompassing the leaf, flower, and root tissues. Our aim was to investigate the terpene synthase (TPS) genes and differential gene expression (DEG) patterns associated with essential oil biosynthesis. Through de novo assembly, we generated a substantial number of unigenes, with the highest count in the root (146,550), followed by the flower (116,434) and the leaf (72,044). Among the identified unigenes, we focused on fifteen putative ckTPS genes, which are involved in the synthesis of mono- and sesquiterpenes, the key aromatic compounds responsible for the essential oil biosynthesis in C. keiskei. The expression of these genes was validated using quantitative PCR analysis. Both DEG and qPCR analyses revealed the presence of ckTPS genes in the flower transcriptome, responsible for the synthesis of various compounds such as geraniol, germacrene, kaurene, linalool, nerolidol, trans-ocimene and valencene. The leaf transcriptome exhibited genes related to the biosynthesis of kaurene and trans-ocimene. In the root, the identified unigenes were associated with synthesizing kaurene, trans-ocimene and valencene. Both analyses indicated that the genes involved in mono- and sesquiterpene biosynthesis are more highly expressed in the flower compared to the leaf and root. This comprehensive study provides valuable resources for future investigations aiming to unravel the essential oil-biosynthesis-related genes in the Convallaria genus.

Keywords: differential gene expression; floral volatile; monoterpenes; sesquiterpenes; transcriptome.

Figure 1

KEGG annotations of the MVA and MEP pathways and their volatile compounds in C. keiskei (KEGG pathway map reference 00900 and 00909).

Figure 2

Phylogenetic tree analysis and identification of the TPS gene family in C. keiskei.

Figure 3

Venn diagram of the overlap of DE unigenes among the leaf, flower, and root of C. keiskei. (A) Upregulated genes and (B) downregulated genes.

Figure 4

Volcano plot showing comparative differential expression of unigenes and the top four TPS genes in C. keiskei.

Figure 5

The expression profile of genes in the MVA/MEP (terpenoid backbone) pathway in C. keiskei. HMGS: Hydroxymethylglutaryl–CoA synthase; HMGR: Hydroxymethylglutaryl–CoA reductoisomerase; ACAT: Acetyl–CoA C-acetyltransferase; DXS: 1–deoxy–D–xylulose–5–phosphate synthase; DXR: 1–deoxy–D–xylulose–5–phosphate reductoisomerase; ISPD and ISPF: 2–C–methyl–D–erythritol 4–phosphate cytidylyltransferase; CDPMEK: 4–(Cytidine–5–diphospho)–2–C–methyl–D–erythritol kinase; HDS: 4–hydroxy–3-methylbut–2–enyl diphosphate synthase; HDR: 4–hydroxy–3–methylbut–2–enyl diphosphate reductase; GGPP: Geranylgeranyl pyrophosphate synthase; FFPP: farnesyl pyrophosphate.

Figure 6

Terpene synthase expression patterns in C. keiskei. (A). Comparative differential expressed C. keiskei terpene synthase genes. FPKM: fragment per kilobase of transcript per million fragments mapped, size: FPKM value. (B). A Venn diagram of the expected distribution of ckTPS genes in C. keiskei transcriptome.

Figure 7

Comparative quantitative analysis of the terpene backbone pathway and TPS genes. The error bar indicates the mean of ±SD, and * (p ≤ 0.05), ** (p ≤ 0.001) and *** (p ≤ 0.0001) indicate the significant changes based on t-test calibration.