Introduction
Plants interact with the environment by releasing various volatiles that possess physiological and ecological functions (O’Connor, 2015). The theory of herbivore-induced plant volatiles (HIPVs), proposed by Ehrlich and Raven (1964), states that plants release various volatiles to communicate and defend against herbivore attack. Thereafter, studies have been increasingly focused on relationships between plant compounds and insects. A recent report showed that the cottons emitting increased levels of volatile compounds have fewer eggs of Helicoverpa armigera (Liu et al. 2018), suggesting that volatiles play important roles in protecting plants from pests and enhancing plant survival. Indeed, numerous plant-derived volatile compounds, particularly terpenes and terpenoids, are documented to protect plants. For example, farnesene protects plants from aphids (Myzus persicae ) because it acts as an alarm pheromone and attracts the predatory ladybugs (Harmonia axyridis ) (Francis et al.2004; Zhu et al. 2005). Another common compound in flowers and fruits is linalool, which has studied in Arabidopsis thaliana , indicating it attracts thrips; however, oxides of linalool, catalysed by CYP71D, can repel these insects (Boachon et al . 2015). Meanwhile, HIPVs, including mint-derived volatiles such as limonene, 1,8-cineole, and carvone, can also attract species that predate on herbivores, such as predatory mites that feed on two-spotted spider mites (Togashi et al. 2019). Therefore, volatile compounds may be part of a defence system used directly or indirectly by plants to protect themselves via tritrophic interactions (Meena et al . 2017). Furthermore, as they deter herbivores, these plant volatiles are likely expressed in higher concentrations during early stages of development than in mature stages and tissues (Meena et al. 2017); thereafter, expression of these compounds is maintained at a constant level or reduced( Niederbacher et al . 2015). This shows that plants dynamically regulate expression of metabolites in order to adapt to the environment and reproduce.
Most HIPVs are terpenes/terpenoids and are biosynthesized via two pathways. The first is the mevalonate (MVA) pathway, which occurs in the cytosol/endoplasmic reticulum, and leads to the formation of farnesyl diphosphate (FPP) as a precursor to sesquiterpenoids. The other is 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway, occurring in plastids; this pathway generates geranyl diphosphate (GPP) as a precursor to monoterpenoids, catalysed by the associated terpene synthases (TPSs) (McGarvey and Croteau, 1995). These TPSs are a mid-sized gene family in plants, consisting of TPS-a, -b, -c, -d, -e/f, and -g (Chen et al . 2011).
Recently, neryl diphosphate (NPP), which is an isomer of GPP, and (Z,Z)-FPP, were shown to be precursors to several terpenes in the tomato plant (Schilmilleret al. 2009; Akhtar et al . 2013). This indicates that terpene/terpenoid biosynthesis needs to be further investigated in various plant species. Moreover, the structure and function of terpenes that are post-processed by co-expressed genes have been extensively studied in the peppermint (Mentha x piperita ) (Rodneyet al . 2005), Artemisia annua L. (Teoh et al . 2006), and A. thaliana (Boachon et al , 2019). However, few studies have examined these compounds in the lavender.
Lavender (Lavandula angustifolia ) is an important aromatic plant generating as many as 70 volatile metabolites, including limonene,α- pinene, linalool. Lavender has also been proposed as a model to study the regulation of terpene biosynthesis (Guitton et al.2010a). Although lavender metabolism has been investigated for several years, enzymes, such as 3-carene synthase, fenchol synthase, α -pinene synthase, andβ -phellandrene synthase are mainly identified usingin-vitro assays (Demissie ZAet al . 2011; Benabdelkaderet al . 2014; Adal AM et al . 2017). In addition, terpenes synthesized by TPSs are potential to be transported into endoplasmic reticulum where these compounds are converted by cytochrome P450 (CYP) into their respective derivatives (Karunanithi and Zerbe 2019). This enables plants to increase the expression of metabolites that can perform multiple functions. Although the CYP enzyme family, which accounts for 1% of the genome in most plants (Nelson and Werck-Reichhart 2011), has not been functionally identified in the lavender, 30 unique CYPs genes were predicted using sequence tags (ESTs) (Lane et al. 2010).
Physiological functions of volatiles and their genes within tritrophic interactions in lavenders remain elusive. Previous studies have described volatile organic compound (VOC) content during inflorescence ontogeny, and revealed that the terpenes involved fell into three groups identifiable via three developmental phases of flowering. 3-carene, limonene, myrcene, bornyl acetate, borneol, camphor, 1,8-cineol, and trans -ocimene belonged to the first group and may play a protective role in repelling damaging insects (Guittonet al . 2010b). Previously, we found that aphids and ladybugs were dominant insects in lavender fields during early spring in Beijing. This suggests probable tritrophic interaction among predators, preys and volatiles (Li et al.2019) . Y-tube olfactometer experiments showedβ -trans-ocimene and (+)-R-limonene get rid of 74.71% and 78.41% aphids (Li et al. 2019).
In this study, we investigated the tactics used by the lavender plant to defend against herbivore attack during immature developmental stages and in young plant tissues.