Euphorbia (Euphorbiaceae) is a genus of flowering plants with about 2000 species that is subdivided into many subgenera and sections. Distributed worldwide from desert to temperate zones, Euphorbia species range from tiny annuals to large and long-lived trees (https://www.finegardening.com/genus/euphorbia; accessed on 1 December 2021). Many Euphorbia species are used in traditional Chinese, Japanese, and Korean medicine. Shi et al. (2008) surveyed biomolecules in Euphorbia and identified 535 molecules among the terpenoids, steroids, phenolic compounds, and flavonoids. Their biological activities include cytotoxicity, effects on cell division, DNA damage, tumor promotion, and antimicrobial activity. E. maculata L., commonly called spotted spurge, is an annual herb native to North America but grows worldwide. Although the sap from E. maculata may cause skin irritation and rash in some people, extracts have been used to treat diarrhea, hemolysis, and hematuria. There are numerous reports on the bioactive phytochemicals in E. maculata, such as polyphenols, tannins, flavonol glycosides, and triterpenoids.
  Also known as isoprenoids, terpenoids are a large class of plant secondary metabolites with more than 50,000 naturally occurring members. Terpenoids are organic compounds derived from a 5-carbon isoprene (C5) called isopentyl diphosphate (IPP). Terpenoids are synthesized by the head-to-tail addition of IPP (C5) units, resulting in hemiterpenoids (C5H8), monoterpenoids (C10H16), diterpenoids (C20H32), and triterpenoids (C30H48). There are two IPP biosynthesis pathways: the cytosolic mevalonic acid (MVA) pathway, resulting in IPP; and the plastidial methylerythritol phosphate (MEP) pathway, resulting in dimethylallyl diphosphate (DMAPP), an IPP isomer [10]. The cytosolic MVA pathway begins with 2-Acetyl-CoA, which is converted to IPP by stepwise enzyme-mediated reactions; the plastidial MEP pathway starts with the condensation of pyruvate and glyceraldehyde-3-phosphate by 1-deoxy-D-xylulose-5-phosphate (DOXP) synthase. Then, DOXP is converted to DMAPP by stepwise enzymatic reactions. The IPP and DMAPP isomers are interconverted by isopentyl pyrophosphate isomerase (IDI). While triterpenoids and sesquiterpenoids are synthesized via the MVA pathway, monoterpenoids, diterpenoids, and tetraterpenoids are synthesized via the MEP pathway.
  Numerous reports document the terpenoids in Euphorbia species. Tsopmo and Kamnaing (2011) isolated 18 terpenoid molecules from whole plant parts of E. sapinii by simple acetone extraction and deciphered their molecular structures. Terpenoids were extracted from E. pedroi, and an isolated tetracyclic triterpenoid was demonstrated to be a multidrug resistance reverser. Many Euphorbia species produce a milky latex that is irritating to humans and animals. The triterpene alcohols derived from the milky latex of E. azorica have potential as chemopreventive and chemotherapeutic agents in cancer treatment. Sun et al. (2018) isolated 17 triterpenoid derivatives including two lanostane-type triterpenoids from E. maculata. The isolated triterpenes exhibited potent anti-inflammatory activities, and the authors proposed these triterpenes as candidate cancer chemopreventive agents. Terpenoids have pharmacological benefits, including antitumor, anti-inflammatory, antibacterial, antioxidation, and immunoregulation activities, and can be used in the prevention of cardiovascular diseases.
  A transcriptome is the complete set of transcripts at a defined spatial and temporal stage of an organism’s life cycle, and it provides comprehensive information on gene expression and regulation. Next-generation sequencing (NGS) technologies, such as Illumina paired-end transcriptome analysis and single-molecule real-time sequencing (PacBio SMRT) technology, have been used to isolate numerous key genes in metabolite biosynthesis pathways. The PacBio SMRT system is especially useful for plants lacking reference genome sequence data because it reads full-length transcripts. Plant metabolites are often biosynthesized in specific tissues; thus, tissue-specific transcriptomes can be compared to identify key genes involved in various complex metabolite biosynthesis pathways in plants.
  In this study, we characterized the terpenoid biosynthesis genes in E. maculata. We sequenced the leaf, root, and stem transcriptomes using Illumina short-read sequencing and PacBio SMRT techniques. The former technique allowed us to identify differentially expressed genes (DEGs) in the metabolite biosynthesis pathways, and the latter allowed us to obtain the complete sequences and isoform copy number information of transcripts involved in terpenoid biosynthesis.