# Plant Volatile Organic Compounds Evolution: Transcriptional Regulation, Epigenetics and Polylploidy
## 2 Biosynthesis and Epigenetic Regulation of VOCs
### Genetic Roadmap to Terpenoid Biosynthesis and Regulation  
Terpenoids are the largest family of VOCs and encompass over 550 compounds [42]. These are derived from two common five-carbon (C\({}_{5}\)) precursors, isopentenyl diphosphate (IPP) and its allylic isomer, dimethylallyl diphosphate (DMAPP) [43]. In plants, these C\({}_{5}\)-isoprene precursors are synthesized from two independent and compartmentsally separated pathways: the mevalonic acid (MVA) pathway and the methylerythritol phosphate (MEP) pathway. The MEP pathway occurs exclusively in plastids [44], providing precursors for volatile hemiterpenes (C\({}_{5}\)), monoterpenes (C\({}_{10}\)) and diterpenes (C\({}_{20}\)), whereas the MVA pathway is distributed among the cytosol, endoplasmic reticulum and peroxisomes [45; 46], producing precursors for volatile sesquiterpenes (C\({}_{15}\)). Despite being compartmentally separated, MVA and MEP pathways are connected by metabolic crosstalk [47], allowing the MEP pathway, often with a higher carbon flux, to support the biosynthesis of cytosolically formed terpenoids [47; 48; 49]. The MVA and MEP pathways are well characterized and genes are conserved across plant species, producing prenyl diphosphate precursors (farnesyl diphosphate (PP) and geranyl diphosphate (GPP)), which are substrates of terpene synthases/cyclases (TPSs) [4; 42; 50]. At the final steps of the pathways (Figure 1), TPSs are responsible for the tremendous diversity of volatile terpenoids in plants [51]. Almost half of the known TPSs can synthesize multiple products from a single substrate [51], and many of them accept more than one prenyl diphosphate precursor [52; 53], which increases the diversity of the produced terpenoids by directing bifunctional enzymes to different compartments with a wide range of available substrates [49; 54]. The _TPS_ gene family is a mid-size family that includes approximately 20 to 150 genes, identified in a variety of plant species. TPSs are present in all plant genomes and they can be phylogenetically classified into seven subfamilies (TPS-a-TPS-h) [55]. Most of them have been isolated from vegetative tissues and fruits to investigate their presumably defensive function [56], but their role in floral attraction might be equally important from the perspective of plant evolution and speciation [57]. In contrast to the vast information about terpenoid biosynthesis-related enzyme-encoding genes, the regulatory network controlling the expression of TPS genes at the transcriptional level remains unclear. However, at least six transcription factor (TF) families are implied in the regulation of terpenoid biosynthesis (Figure 1): AP2/ERF, bHLH, bZIP, ARF, MYB and WRKY [58; 59; 60]. In _Arabidopsis_, the TFs AtMYB21 and AtMYC2 regulate the expression of two sesquiterpene synthase genes, _TPS11_ and _TPS21_, via the gibberell and jasmonic acid (JA) signaling pathways [58; 61]. The role of these MYB TFs have been also shown in _Freesia \(\times\)hybrid_, which has eight TPS genes responsible for the spatiotemporal release of VOCs [56]. Alternatively, in gymnosperms (i.e., _Pinus taeda_), _PtMYB14_ regulates the terpenoid MVA pathway [62]. It was recently shown that MYB TFs interact with other bHLH TFs to form a MYB-bHLH complex, participating in the activation of sesquiterpene biosynthesis [63; 64]. In addition, AtARF6 and AtARF also regulate the synthesis of sesquiterpenes by binding to _TPS11_ and _TPS21_ promoters [58]. Finally, the defensive terpene emission in _Nicotiana attenuata_ seems to be controlled by the TFs NaWRKY3 and NaWRKY6 [65]. Furthermore, the temporal emission of TPSs is controlled by the circadian clock [66], which is under epigenetic control, as explained in further sections. In addition, microRNAs (miRNAs) and long noncoding RNAs (IncRNAs) play a key role in the terpenoid regulatory network (Figure 1).