# Identifying Terpenoid Biosynthesis Genes in _Euphoria maculata_ via Full-Length cDNA Sequencing
## 4 Discussion
Proteome plasticity from alternative splicing plays a major role in adaptation to environmental stresses [52]. In plants, alternative splicing occurs in about 24% of transcripts in wheat (_Triticum aestivum_) to 60% in _Arabidopsis_ in intron-containing genes [44]. In the _E. maculata_ transcriptome, about 35.8% of the unigenes had isoforms; two examples are shown in Figure 3. Different paralogs had different isoform patterns. Furthermore, the expression patterns of paralogs differed among root, stem, and leaf tissues. Thus, paralogs and their isoforms might help plants adapt to stresses, as demonstrated in cassava under cold stress [44]. Terpenoids are the major bioactive compounds in _E. maculata_. We isolated the genes, as well as their isoforms and paralogs, involved in the MVA, MEP, and terpenoid biosynthesis. The _E. maculata_ genome has a unique _E. maculata_ genome, but it is not a single _E. maculata_ genome, but it is not a single _E. maculata_ genome. thesis pathways in _E. maculata_. The MVA pathway begins with Acetoacetyl-CoA synthase (AAC thiolase), which catalyzes the condensation of two 2-Acetyl-CoA (AAC) molecules. AAC is subsequently transformed into five intermediate molecules to form IPP, which involves five enzymes: HMG-CoA synthase, HMG-CoA reductase, MVA kinase, MVAP kinase, and MVAPP decarboxylase (Figure 7) [11]. In _E. maculata_, the genes encoding these enzymes were present as single-copy up to five-copy genes, with one to three isoforms per gene (Table 4). HMG-CoA reductase is a key regulatory enzyme in the MVA pathway in plants [53] and catalyzes the conversion of HMG-CoA to MVA, which is a rate-limiting step in the MVA pathway [10; 13]. The HMG-CoA reductase gene is highly conserved among organisms, and we identified 1929 HMG-CoA reductase mRNAs among all biological kingdoms from viruses to bacteria to eukaryotes in the NCBI database (data not shown). The gene encoding HMG-CoA reductase had five copies in _E. maculata_, and each paralog was expressed differently in stem, leaf, and root tissue. Developmental and organ-specific expression of the HMG-CoA reductase gene was also reported in plants [53]. The HMG-CoA reductase gene was expressed higher in stems than in roots and leaves in lavender (_Lavandula pubescens_), which also produces terpenoids [54]. In _E. maculata_, one of the HMG-CoA reductase-paralogous genes was highly expressed in stems. The various paralogs expressed differently among the three organs, which may be highly coordinated for plant development. The MEP pathway, also known as the non-mevalonate (non-MVA) pathway [13], occurs in plastids; thus, animals do not have this pathway, which has spurred interest as a potential strategy to develop anti-bacterial or herbicide products [55; 56]. We identified all enzyme-encoding genes of the MEP pathway in _E. maculata_. Except for the gene encoding CDP-ME kinase, all other enzyme-encoding genes had two to four copies and several isoforms. IPP derived from the MVA pathway and DMAPP derived from the MEP pathway are structurally unrelated isomers that are interconverted by IDI. Because IPP is derived directly from the MVA pathway, IDI is not essential for plant survival; thus, IDI may play a role in modulating the IPP/DMAPP ratio in the cell [13]. IPP is a C\({}_{5}\) molecule that undergoes enzyme-mediated sequential head-to-tail condensation to become GPP (C\({}_{10}\)), FPP (C\({}_{15}\)), and GGPP (C\({}_{20}\)) [12]. There were two, one, and two copies of GPP synthase, FPP synthase, and GGPP synthase in _E. maculata_, respectively. GPP is converted to monoterpenes by monoterpene synthase, which was encoded by two paralogous genes, and both copies had very high expression in the three organs in our analysis. Monoterpenoids have not been reported in _E. maculata_, but several monoterpenoid compounds were reported in other _Euphoria_ species [57; 58]. FPP is converted to sesquiterpenenes (C\({}_{15}\)) by sesquiterpene synthase or squalene (C\({}_{30}\)) by squalene synthase.