Herbivore-induced terpenoid emission in Medicago truncatula: concerted action of jasmonate, ethylene and calcium signaling

Abstract

Plant volatiles emitted by Medicago truncatula in response to feeding larvae of Spodoptera exigua are composed of a complex blend of terpenoids. The cDNAs of three terpene synthases (TPSs), which contribute to the blend of terpenoids, were cloned from M. truncatula. Their functional characterization proved MtTPS1 to be a beta-caryophyllene synthase and MtTPS5 to be a multi-product sesquiterpene synthase. MtTPS3 encodes a bifunctional enzyme producing (E)-nerolidol and geranyllinalool (precursors of C11 and C16 homoterpenes) from different prenyl diphosphates serving as substrates. The addition of jasmonic acid (JA) induced expression of the TPS genes, but terpenoid emission was higher from plants treated with JA and the ethylene precursor 1-amino-cyclopropyl-1-carboxylic acid. Compared to infested wild-type M. truncatula plants, lower amounts of various sesquiterpenes and a C11-homoterpene were released from an ethylene-insensitive mutant skl. This difference coincided with lower transcript levels of MtTPS5 and of 1-deoxy-D: -xylulose-5-phosphate synthase (MtDXS2) in the damaged skl leaves. Moreover, ethephon, an ethylene-releasing compound, modified the extent and mode of the herbivore-stimulated Ca2+ variations in the cytoplasm that is necessary for both JA and terpene biosynthesis. 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. 1

Volatile profiles and mRNA levels in wild-type (WT) and in skl plants. a Volatiles emitted from WT and from skl plants damaged by beet armyworms (BAW); (+) with larvae, (−) without larvae for 48 h. Emission is presented as relative peak area (g shoot tissue)⁻¹ collected over a 48-h period + SE (n = 4). Means followed by small letters for each set of volatiles that are significantly different according to Scheffe’s test (P < 0.05). 1 1-octene-3-ol, 2 (Z)-3-hexenyl acetate, 3 limonene, 4n-nonanal, 5 DMNT, 6n-decanal, 7 (+)-cyclosativene, 8 α-ylangene, 9 α-copaene, 10 β-caryophyllene, 12 α-himalachene, 14allo-aromadendrene, 15 γ-muurolene, 16 germacrene D, 17 unidentified sesquiterpene (I), 18 α-muurolene, 20 (E)-nerolidol, 21 TMTT, 22 β-himachalol. Relative peak areas from integration of the reconstructed ion chromatogram were used without additional calibration. Peak areas of germacrene D and unidentified sesquiterpene (16 + 17) were determined as the sum due to insufficient separation. b Relative mRNA levels for genes involved in terpenoid biosynthesis in BAW-infested leaves. Data represent the mean + SE (n = 4). *P < 0.05 (ANOVA). DXS DXP synthase, HMGR HMG-CoA reductase, TPS terpene synthase

Fig. 2

a Sesquiterpenes formed by the assay of the recombinant MtTPS enzymes with FDP as substrate. An assay of the extract prepared from the BL21-CodonPlus(DE3) strain transformed with a plasmid without the TPS cDNA insert serves as control. b Stereochemistry of (E)-nerolidol was identified by comparing the retention time with that of authentic standards of (3R)-(E)-nerolidol and (3S)-(E)-nerolidol using GC–MS. Also shown is the sesquiterpene product formed by recombinant MtTPS3 enzyme in vitro

Fig. 3

Mono- and diterpenes formed by the assay of the recombinant MtTPS enzymes with GDP (a) and GGDP (b) as substrates, respectively. The GDP- and GGDP-derived products trapped by SPME and pentene, respectively, are illustrated. Assays of the extract prepared from the BL21-CodonPlus(DE3) strain transformed with a plasmid without the TPS cDNA insert serve as control

Fig. 4

Effect of exogenous application of JA and ACC on relative mRNA levels for genes involved in the biosynthesis of terpenoids in WT leaves. Treatments: JA (J), the ethylene precursor ACC (A), or both (+) for 2 and 24 h (mean + SE, n = 3–4). Means followed by small letters for each set of volatiles that are significantly different according to Scheffe’s test (P < 0.05)

Fig. 5

Effect of exogenous application of JA and ACC: volatiles released from WT plants treated with water (control), JA, ACC, JA + ACC, or JA + ACC + STS for 24 h. Emission is presented as relative peak area (g shoot tissue)⁻¹ collected over a 24-h period + SE (n = 4−5). Relative peak areas from integration of the reconstructed ion chromatogram were used without additional calibration. Peak areas of germacrene D and unidentified sesquiterpene (16 + 17) were determined as the sum due to insufficient separation. Means followed by small letters for each set of volatiles are significantly different according to Scheffe’s test (P < 0.05). 2 (Z)-3-hexenyl acetate, 3 limonene, 4n-nonanal, 5 DMNT, 6n-decanal, 7 (+)-cyclosativene, 8 α-ylangene, 9 α-copaene, 10 β-caryophyllene, 11 β-copaene, 12 α-himalachene, 13 α-humulene, 14allo-aromadendrene, 15 γ-muurolene, 16 germacrene D, 17 unidentified sesquiterpene (I), 18 α-muurolene, 19 δ-cadinene, 20 (E)-nerolidol, 22 β-himachalol

Fig. 6

Pathway allocation in terpenoid biosynthesis in JA + ACC induced M. truncatula. a Degree of labelling of terpenoids emitted from JA + ACC-induced plants pre-treated with [²H₂]-DOX. b Volatiles from JA + ACC-induced plants after pre-treatment with lovastatin (L), fosmidomycin (F), or water (C). Emission is presented as relative peak area (g shoot tissue)⁻¹ collected over a 24-h period + SE (n = 3−4). Relative peak areas from integration of the reconstructed ion chromatogram were used without additional calibration. Means followed by small letters for each set of volatiles are significantly different according to Scheffe’s test (P < 0.05)

Fig. 7

Effect of ethylene on cytoplasmic Ca²⁺ concentrations. a A WT leaf was treated with Fluo-3 AM for 1 h and damaged with a BAW larva. The confocal laser scanning microscope analysis showed false-colour subcellular localization of the dyes, proving that the dyes are loaded mainly into the cytoplasm. The green fluorescence refers to the binding of Fluo-3 AM with Ca²⁺, whereas the chloroplasts are evidenced by a bright red colour caused by chlorophyll fluorescence. b The dye-loaded WT or skl leaf was damaged by a BAW larva. After 10 min, the leaf was treated with the ethephon solution (10 mM) or buffer (Control), indicated by a blue arrow. c Alternatively, the dye-loaded WT or skl leaf was pre-treated with ethephon (10 mM) for 20 min and exposed to a BAW larva for 10 min. Demonstrated false-colour images show differences between cellular Ca²⁺ concentrations in WT and skl leaves 10 min after ethephon treatment. Data represent the ratio of Fluo-3 AM fluorescence ± standard deviation (SD) (n = 4)

Fig. 8

Biosynthesis of oxylipins (a) and volatiles (b) in WT leaves after treatment with BAPTA or water (control). Leaves to be used for oxylipin analysis were harvested 6 h after BAW exposure (mean + SE, n = 4). Volatiles emitted from the infested plants were collected for 48 h starting with BAW damage. Emission is presented as relative peak area (g shoot tissue)⁻¹ collected over a 48-h period + SE (n = 4). *P < 0.05, **P < 0.01 (ANOVA). Peak areas from integration of the reconstructed ion chromatogram were used without additional calibration