# Unraveling the Biosynthesis of Carvacrol in Different Tissues of _Origanum vulgarae_
## 1 Introduction  
Throughout the plant world, numerous species generate aromatic essential oils (EOs) that include complex mixes of volatile organic compounds (VOCs), such as terpene or phenylpropanol metabolites, which are of tremendous value in a variety of sectors, ranging from food chemistry to pharmaceutics [1]. Oregano belongs to the Lamiaceae family and is a well-known herb that is widely distributed in the Asia and the Mediterranean which can be used to extract an EO mainly dominated by compositions of phenolic monoterenes (carvacrol, thymol) and their precursors- [2,3]. Despite the fact that the plant contains several chemotypes, the carvacrol chemotype (containing up to 80% carvacrol) is the most valuable due to its therapeutic activity and flavoring characteristics. Carvacrol (C\({}_{10}\)H\({}_{14}\)O) is a liquid phenolic monoterenoid with antibacterial action and some pharmacological effects that have attracted interest [4,5]. Its use as a food additive is highly desirable because of its inclusion in the European Commission's list of chemical flavorings and Food and Drug Administration (FDA) clearance as a toxicologically safe chemical (FDA, 2017). The cytosolic mevalonate pathway (MVA) or the plastid-based methylerythritol pathway (MEP) are both viable routes for terpene production. Isopentenyl diphosphate (IPP)is generated in the cytosol by the condensation of two acetyl-CoA moieties via the MVA route. Starting with glyceraldehyde 3-phosphate (G3P) and pyruvate (PYR), the plastid MEP pathway produces the isomer dimethylallyl diphosphate (DMAPP). Later, two C5 units fuse to produce geranyl diphosphate (GPP), a monoterpene precursor, and three C5 units fuse to form farnesyl diphosphate (FPP), a sesquiterpene precursor [6]. Typically, monoterpene synthases catalyze the cyclization of GPP via the \(\alpha\)-terpinyl cation intermediate, and this very unstable intermediate can subsequently be transformed into certain monoterpenes [7]. Terpene biosynthesis pathways contain the terpene synthases (TPSs) and cytochrome P450-dependent monooxygenases (CYPs) as their central enzymes [8]. An important class of CYPs is responsible for the oxidation and conjugation of the first TPS products, which are involved in the modification of monoterpenes and sesquiterpenes [9]. In addition, a recent study demonstrated that the aromatic backbones of carvacrol are produced by the CYP71D subfamily and short-chain dehydrogenase/reductase (SDR) in conjunction with dehydrogenases via unstable intermediates [10]. Glandular trichomes are often multicellular and serve as the site of biosynthesis and storage of significant amounts of VOCs in a liquid state, which are only released when trichomes are broken [11]. Many plant species have glandular trichomes, and much literature on this topic has demonstrated the enormous variation in their shape and size [12]. The number of specific metabolites generated by a plant is frequently closely connected to the density of glandular trichomes present at the epidermis's surface [13]. Increasing glandular trichome density is a novel plant breeding strategy for increasing beneficial chemical output. The advent of short-read sequencing with Illumina technology and single-molecule real-time (SMRT) sequencing with the PacBio RS system has accelerated the creation of genome and transcriptome resources for various plant species [14,15]. Correlations between the expression of paired genes may be used to determine relationships based on transcript expression data, and a gene co-expression network can be built by discovering non-random gene-gene expression connections [16]. Hence, we explored the mechanisms underlying the generation of monoterpenes and sesquiterpenes in different elaborate tissues (stems, STs; leaves, LEs; bracts, BRs; sepals, SEs; petals, PEs) of _Origanum vulgare_ 'Hot and Spicy' (_O.