Early herbivore-elicited events in terpenoid biosynthesis

Abstract

Volatile terpenoids, the major products among the herbivore-induced plant volatiles in the legume, mediate interactions that attract herbivores' natural enemies and serve as signals to neighboring plants. We recently demonstrated cross-talk among the signaling components involving Ca²⁺, jasmonic acid and ethylene, which are altogether responsible for volatile terpenoid formation in Medicago truncatula. Herbivore-stimulated Ca²⁺ transients are an additional element that has an impact on the composition of the blend of terpenoids, whose biosynthesis depends on the jasmonic acid/ethylene pathway. The molecular diversity of the blend is expanded and modulated by the transcriptional regulation of terpene synthases, some of which are multi-functional enzymes producing a large set of sesqui- and monotepenes or precursors of C₁₁ and C16 homoterpenes from different prenyl diphosphates. In this addendum, we discuss a new perspective on early events leading to terpenoid biosynthesis.

The major volatiles emitted by the legume Medicago truncatula in response to herbivore feeding consist of monoterpenes (C₁₀), sesquiterpenes (C₁₅) and homoterpenes (C₁₁ or C₁₆).¹ Jasmonic acid (JA) has been suggested to act antagonistically or synergistically with salicylic acid and ethylene to regulate a characteristic blend of terpenoids.^2–5 However, little is known about the interaction between intracellular calcium signatures and the JA/ethylene signaling pathway.

Signaling Pathways for Herbivore-Induced Terpenoid Biosynthesis

In genetic studies using M. truncatula, the ethylene-insensitive mutant skl displayed a supernodulation phenotype,^6,7 demonstrating that ethylene is an essential element in the interaction between root and rhizobia. We used skl to study whether and how ethylene is involved in the signaling cascades leading to characteristic blends of HIPVs. Compared to wild-type (WT) plants, the skl mutant produced lower amounts of sequiterpenes when challenged with feeding Spodoptera exigua (beet armyworm: BAW) larvae.⁸ As shown in Figure 1A, ethylene and JA synergistically induce the biosynthesis of certain sesquiterpenes (e.g., α-copaene, (E)-nerolidol and germacrene D) in M. truncatula. Similar effects have been shown previously for Zea mays.⁹ The production of the sesquiterpenes coincides with enhanced transcript levels of a putative terpene synthase gene, MtTPS5 and the gene encoding 1-deoxy-d-xylulose-5-phosphate synthase, MtDXS2, involved in the 2-C-methyl-d-erythritol 4-phosphate pathway.⁸ In contrast, MtTPS1 and MtTPS3 transcripts were induced only by JA treatment.

Figure 1.

Schematic representation of the signaling pathways (A) and MtTPS functions (B) required for herbivore-induced terpenoid biosynthesis in M. truncatula. The green fluorescence in the demonstrated false-color image refers to the binding of Fluo-3 AM with Ca²⁺ (see online edition).

In addition, we also demonstrated that ethylene is able to modulate the extent and mode of Ca²⁺ influx resulting from herbivore attack, either directly or through feedback from ethylene effects on downstream events. Thus, ethylene contributes to the herbivory-induced terpenoid biosynthesis at least twice: by modulating both early signaling events such as cytoplasmic Ca²⁺-influx and the downstream JA-dependent biosynthesis of terpenoids (Fig. 1A). The first event after leaf feeding is an influx of cytosolic Ca²⁺ in the damaged area.¹⁰ Ca²⁺-binding proteins decode information contained in the temporal and spatial patterns of these Ca²⁺ signals and bring about changes in metabolites and gene expression.¹¹ In tobacco a link between NtCDPK2, MAP kinases and JA and ethylene cross-talk has been suggested.¹²

Product Spectrum of Multi-Functional MtTPS

To explore the regulatory mechanisms for herbivore-induced terpenoid emissions, three TPS cDNAs were cloned from M. truncatula. Most of the plant TPS are believed to generate terpenoid(s) from a single prenyl diphosphate such as geranyl diphosphate (GDP), farnesyl diphosphate (FDP) or geranylgeranyl diphosphate (GGDP). In M. truncatula, we found that the MtTPS3 encodes a multi-functional enzyme which produces (E)-nerolidol [sesquiterpene], geranyllinalool [diterpene] and linalool [monoterpene] from different prenyl diphosphates serving as substrates (see Fig. 1B). Feeding BAW raised the expression level of MtTPS3 in WT and skl plants in the same way, but levels of (E)-nerolidol and (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT, an oxidative degradation product of (E)-nerolidol), were higher in WT plants, suggesting that ethylene might have a post-transcriptional impact on the MtTPS3 protein.⁸ Another interesting aspect is that MtTPS3 has been claimed to be a plastidial-targeted protein,¹³ which is also consistent with its ability to produce the diterpene geranyllinalool from GGDP.

MtTPS5 is a multiproduct sesquiterpene synthase which produces about eleven sesquiterpene hydrocarbons and four sesquiterpene alcohols. Multi-product TPSs contribute significantly to the plasticity of blends and are increasingly found in plants, especially in connection with herbivory, as shown previously for other plants.^14,15 In addition to olefinic sequiterpenes, the heterologously expressed MtTPS5 protein produced a number of hydroxylated products which were not detected in the gas phase of M. truncatula plants.⁸ Whether these compounds are sufficiently produced and subsequently modified in planta remains to be clarified. We assume that the function of MtTPS5 in vivo is modulated by different cytoplasmic conditions, such as the presence of divalent metal ion cofactors or the pH. Those factors might display a pronounced effect on the product spectrum of heterologously expressed MtTPS5.^15,16 Likewise, in vitro assays of amorpha-4,11-diene synthase from Artemisia annua L. revealed that the presence of Mn²⁺-or Co²⁺-cofactors reduces the number of by-products, especially the production of sesquiterpene alcohols, when compared to the presence of Mg²⁺ as cofactor.¹⁷ However, up to now, such effects have been only demonstrated using in vitro system. Hence, genetic and transgenic plant approaches, in addition to the kinetic analysis of MtTPS formations, are needed to understand the probably complex in vivo function of those enzymes.

Addendum to: Arimura G, Garms S, Maffei M, Bossi S, Schulze B, Leitner M, Mithöfer A, Boland W. Herbivore-induced terpenoid emission in Medicago truncatula: Concerted action of jasmonate, ethylene and calcium signaling. Planta. 2008;227:453–464. doi: 10.1007/s00425-007-0631-y.