Significance
Plants receive volatile compounds emitted by neighboring plants that are infested by herbivores, and consequently the receiver plants begin to defend against forthcoming herbivory. To date, how plants receive volatiles and, consequently, how they fortify their defenses, is largely unknown. We found that tomato plants absorbed the airborne green leaf alcohol (Z)-3-hexenol emitted by neighboring conspecific plants under attack by herbivores and subsequently converted the alcohol to a glycoside. The glycoside suppressed growth and survival rates of cutworms. The accumulation of glycoside in the receiver plants explained the defense acquired via “smelling” their neighbors. This study showed that the processing of a volatile compound is a mechanism of volatile reception in tomato plants.
Keywords: plant–plant signaling, herbivore-infested plant volatiles, green leaf volatiles, defense induction, glycosylation
Abstract
Plants receive volatile compounds emitted by neighboring plants that are infested by herbivores, and consequently the receiver plants begin to defend against forthcoming herbivory. However, to date, how plants receive volatiles and, consequently, how they fortify their defenses, is largely unknown. In this study, we found that undamaged tomato plants exposed to volatiles emitted by conspecifics infested with common cutworms (exposed plants) became more defensive against the larvae than those exposed to volatiles from uninfested conspecifics (control plants) in a constant airflow system under laboratory conditions. Comprehensive metabolite analyses showed that only the amount of (Z)-3-hexenylvicianoside (HexVic) was higher in exposed than control plants. This compound negatively affected the performance of common cutworms when added to an artificial diet. The aglycon of HexVic, (Z)-3-hexenol, was obtained from neighboring infested plants via the air. The amount of jasmonates (JAs) was not higher in exposed plants, and HexVic biosynthesis was independent of JA signaling. The use of (Z)-3-hexenol from neighboring damaged conspecifics for HexVic biosynthesis in exposed plants was also observed in an experimental field, indicating that (Z)-3-hexenol intake occurred even under fluctuating environmental conditions. Specific use of airborne (Z)-3-hexenol to form HexVic in undamaged tomato plants reveals a previously unidentified mechanism of plant defense.
In response to herbivory, plants emit specific blends of volatiles (1). When undamaged plants are exposed to volatiles from neighboring herbivore-infested plants, they begin to defend against the impending infestation of herbivores (2, 3). This so-called “plant–plant signaling” has been reported in several plant species (4). For example, a study on the expression profiles of defense-related genes when Arabidopsis was exposed to several volatiles, including green leaf volatiles and a monoterpene, showed that the manner of induction varied with the gene monitored or the volatile used, suggesting that the plant responses were specific to the individual volatile compound (5). Kost and Heil (6) reported that the secretion of extrafloral nectar (an alternative food for carnivores) in undamaged lima bean plants was enhanced by volatiles from infested conspecific plants; this reaction was specific to (Z)-3-hexenyl acetate. Recently, Kikuta et al. (7) showed that wound-induced volatile organic compounds from Chrysanthemum cinerariaefolium induced the biosynthesis of pyrethrins in volatile-exposed neighboring plants. In this plant–plant signaling system, a blend of five compounds at specific concentrations was essential for the pyrethrin biosynthesis in receiver plants.
These previous studies on plant–plant signaling raise questions about how different airborne volatiles are received by undamaged neighboring plants. Tamogami et al. (8) reported that airborne (E)-nerolidol was metabolized by Achyranthes bidentata plants into (E)-4,8-dimethyl-1,3,7-nonatriene. However, the mechanisms involved in the reception of airborne (E)-nerolidol in plants remained unclear. To date, only the receptor for ethylene, ETR1, a typical histidine kinase involved in a two-component regulatory system, has been identified (9, 10); no information exists on receptors for other volatile compounds in plants. In this study, we conducted comprehensive analyses of metabolic changes in intact tomato plants (Solanum lycopersicum) exposed to volatiles emitted from conspecifics infested with common cutworm (CCW; Spodoptera litura) and also conducted bioassays and biochemical analyses. We report that (Z)-3-hexenol emitted from herbivore-infested tomato plants is used by undamaged plants to form a glycoside with defensive function against CCW.
Results and Discussion
Response of Uninfested Tomato Plants to Volatiles from CCW-Infested Conspecifics.
In an experimental airflow system (Fig. S1), we placed an uninfested potted tomato plant in each of two chambers and then inoculated the upwind plants with five third-stadium CCWs per plant. As previously reported, these infested plants emitted several compounds that were barely emitted from uninfested conspecifics (Fig. S2) (11–14). The intact exposed plants in the downwind chamber were exposed to volatiles from the infested tomato plants for 3 d. For a control experiment, uninfested plants were placed in the upwind chamber. After exposure, both exposed and control plants were separately challenged by CCWs. We used first-stadium CCWs to measure survival rates and second-stadium CCWs to measure growth rates (weight gain). Survival rates of CCWs on exposed plants were significantly lower—by 16.7% (Fig. 1A, n = 21, P = 0.048, t test after angular transformation)—than those on control plants. Weight gains of CCWs on exposed plants were also significantly lower (Fig. 1B, n = 8, P = 0.0078, Wilcoxon paired-signed rank-sum test) than on control plants. These data showed that tomato plants became more defensive against CCWs when exposed to volatiles from CCW-infested conspecifics, indicating plant–plant signaling between tomato plants.
Fig. 1.
Performance of CCWs on exposed leaves. (A) Survival rates of CCWs. The survival rates on a leaf of an exposed plant (black bar) were significantly lower than those on a leaf of a control plant (white bar) (P = 0.048, n = 21, t test after angular transformation). (B) Weight gains of CCWs. The weight gains on detached exposed leaves (black bars) were significantly smaller than those on control leaves (white bars) (P = 0.0078, n = 8, Wilcoxon paired signed-rank test).
Comprehensive Metabolite Analyses of Leaf Extracts.
Targeted GC–time of flight mass spectroscopy (GC–TOF/MS) analyses of the extracts of exposed and control plants showed no significant differences in the profiles of major metabolites such as organic acids, amino acids, sugars, and fatty acids (Dataset S1). Only one compound among 8,226 ion peaks detected with nontargeted liquid chromatography (LC)–TOF/MS analysis accumulated significantly more in exposed plants than in control plants. The compound was purified from ∼2 kg of volatile exposed tomato leaves and identified as (Z)-3-hexenyl-O-α-l-arabinopyranosyl-(1,6)-β-d-glucopyranoside [(Z)-3-hexenylvicianoside (HexVic)] by 1H- and 13C-NMR spectroscopy (Fig. 2 and Table S1). The amount of HexVic in exposed plants [2.39 ± 0.78 µg⋅g−1 fresh weight (FW), n = 11] was about 24 times higher than in control plants (0.10 ± 0.02 µg⋅g−1 FW, n = 12, P < 0.0001, Mann–Whitney–Wilcoxon U test).
Fig. 2.
Chemical structure of (Z)-3-hexenylvicianoside, which specifically accumulated in volatile-exposed tomato plants. Represented chromatograms are shown from control (dotted lines) and exposed (solid lines) leaf extracts at m/z = 412.2. The asterisk indicates a significantly accumulated compound, and its structure as determined by NMR spectroscopy is shown in the Inset. The analysis was performed more than 11 times with four independent experiments.
Response of CCWs to HexVic.
To evaluate whether HexVic was involved in the defensive response of receiver tomato plants described above, we reared first-stadium CCWs on an artificial diet supplemented with HexVic at a concentration equivalent to that found in tomato leaves exposed to infested-plant volatiles (3 µg⋅g−1 FW). This diet suppressed the survival rate of first-stadium CCWs by 17% relative to a diet without HexVic (Fig. 3A, n = 10, P = 0.039, t test after angular transformation). Weight gain in second-stadium CCWs reared on the HexVic diet was also significantly depressed compared with those on the control diet (Fig. 3B, n = 10, P = 0.037, Wilcoxon paired-signed rank-sum test). Thus, the increased defensive response in the receiver plants was at least partly explained by the negative impact of HexVic on CCWs.
Fig. 3.
Performance of CCWs on HexVic-embedded artificial diet. (A) Survival rates of CCWs. The survival rates on the embedded diets (black bar) were significantly lower than those on control diets (white bar) (P = 0.039, n = 10, t test after angular transformation). (B) Weight gains of CCWs. The weight gains on the embedded diets (black bars) were significantly smaller than those on control diets (white bars) (P = 0.037, n = 10, Wilcoxon paired signed-rank test).
Effects of Airborne (Z)-3-Hexenol on HexVic Biosynthesis.
The aglycon of HexVic, (Z)-3-hexenol, was a major component of volatiles from CCW-infested tomato plants (Fig. S2). Thus, we hypothesized that the airborne (Z)-3-hexenol produced by infested plants was involved in HexVic synthesis. To test this idea, we used both (Z)-3-hexenol and deuterium-labeled (Z)-3-hexenol [d2-(Z)-3-hexenol] (Fig. S3). When exposed to authentic (Z)-3-hexenol at 1 µM (22.4 ppmV) for 6 h in an enclosed glass vessel (2,000 cm3), the plants accumulated HexVic (Fig. 4A) [n = 3, control vs. (Z)-3-hexenol: P = 0.0041; control vs. d2-(Z)-3-hexenol: P = 0.9974; (Z)-3-hexenol vs. d2-(Z)-3-hexenol: P = 0.0038, Tukey’s multiple comparison]. When d2-(Z)-3-hexenol was used for exposure, the plants exclusively accumulated deuterium-labeled HexVic, but not nonlabeled HexVic (Fig. 4B) [n = 3, mock vs. (Z)-3-hexenol: P = 0.9999; mock vs. d2-(Z)-3-hexenol: P = 0.00004; (Z)-3-hexenol vs. d2-(Z)-3-hexenol: P = 0.00004, Tukey’s multiple comparison], indicating that the plants took in the airborne (Z)-3-hexenol and converted it into HexVic.
Fig. 4.
Conversion of airborne (Z)-3-hexenol into glycoside. Accumulation of (Z)-3-hexenylvicianoside (HexVic) (A) and d2-HexVic (B) was analyzed by LC–MS in undamaged plants exposed to chemically synthesized (Z)-3-hexenol and d2-(Z)-3-hexenol for 6 h. HexVic exclusively accumulated in (Z)-3-hexenol–exposed plants and d2-HexVic in d2-(Z)-3-hexenol exposed plants. P < 0.05, Tukey’s multiple comparisons. Data are mean ± SE of more than three independent experiments.
In addition to the use of airborne (Z)-3-hexenol, tomato plants exposed to the mixture of volatile compounds from CCW-infested plants might also activate the biosynthesis of (Z)-3-hexenol and use the endogenously formed compound to make HexVic as well. To test this possibility, we measured endogenous (Z)-3-hexenol levels in the exposed plants. We failed to find a significant difference in endogenous (Z)-3-hexenol levels between exposed and control plants (control: 5.1 ± 2.4 ng⋅g−1 FW; exposed: 9.3 ± 2.1 ng⋅g−1 FW; n = 4, P = 0.08, Mann–Whitney–Wilcoxon U test), suggesting that exposing uninfested plants to a volatile mixture from herbivore-infested plants had little effect on the production of (Z)-3-hexenol. Taken together, we concluded that the exposed plants exclusively used airborne (Z)-3-hexenol for the biosynthesis of HexVic. Under still-air conditions, the amounts of HexVic in tomato plants that had been infested by CCWs for 1 d were significantly higher than those in uninfested tomato plants (control: 0.59 ± 0.10 µg⋅g−1 FW, n = 15; infested: 5.67 ± 0.82 µg⋅g−1 FW, n = 14, P < 0.0001, Mann–Whitney–Wilcoxon U test), indicating that the infested tomato plants used their own (Z)-3-hexenol to produce HexVic.
Effects of Jasmonates on HexVic Biosynthesis.
Defensive hydroxyl geranyllinalool diterpene glycosides accumulated in wild tobacco plants (Nicotiana attenuata) in response to feeding by the hornworm Manduca sexta and to jasmonic acid (JA) treatments (15, 16). Therefore, we examined the contribution of JA signaling to HexVic synthesis in uninfested plant tissue. We treated uninfested tomato plants with methyl jasmonate (MeJA) at 1 µM (22.4 ppmV) in a 2,000-cm3 glass vessel for 2 h and then measured the amounts of HexVic at 2, 6, and 26 h after the onset of exposure. We found that the amount of HexVic was not significantly higher in JA-treated plants than in untreated ones (Fig. 5A) (n = 3, treatment: P = 0.7179; time: P = 0.5963; interaction: P = 0.8551, two-way ANOVA). The conversion rates of (Z)-3-hexenol into HexVic in MeJA-pretreated plants did not differ from those in untreated plants (Fig. 5B) (n = 3, treatment: P = 0.7423; time: P = 0.0000; interaction: P = 0.9936, two-way ANOVA). The accumulation of HexVic resulting from (Z)-3-hexenol exposure in a mutant tomato plant insensitive to JA (jai1-1, 11.79 ± 2.35 µg⋅g−1 FW, n = 3) was not significantly different from that in the wild type (15.00 ± 4.11 µg⋅g−1 FW, n = 3, P = 0.4541, t test). Furthermore, the amounts of JA, JA-isoleucine (JA-Ile), and 12-oxophytodienoic acid (OPDA) in exposed and control plants were not significantly different (Fig. S4 A–C) [treatment: P = 0.0705; time: P = 0.7997; interaction: P = 0.9621 (JA); treatment: P = 0.8414; time: P = 0.0572; interaction: P = 0.8181 (JA-Ile); treatment: P = 0.9125; time: P = 0.5864; interaction: P = 0.1028 (OPDA), two-way ANOVA, n = 5–6). These lines of evidence indicated that JA was not involved in HexVic biosynthesis in exposed tomato plants, in sharp contrast to the increases in JA levels in corn (Zea mays) seedlings and hybrid poplar (Populus deltoids × nigra) saplings after exposure to green leaf volatiles (17, 18).
Fig. 5.
Accumulation of (Z)-3-hexenylvicianoside (HexVic) in jasmonate-treated tomato plants. (A) After treating plants with jasmonate for 2 h, the plants were placed in a container without jasmonate, and their leaves were harvested 2, 6, and 26 h after the onset of jasmonate treatment. Accumulation of HexVic was hardly observed in untreated (white bars) and MeJA-treated (black bars) tomato plants (two-way ANOVA: n = 3). (B) After treating plants with jasmonate for 2 h, the plants were placed in a container with (Z)-3-hexenol, and their leaves were harvested 0, 0.5, 1, 2, 3, and 6 h after the onset of (Z)-3-hexenol treatment. Identical rates of HexVic accumulation occurred in untreated (solid squares) and MeJA-treated (open circles) tomato plants (two-way ANOVA). Data are mean ± SE of three independent experiments.
Field Study.
To test whether HexVic accumulated under natural conditions, we conducted experiments in an experimental field (5 × 20 m) at Yamaguchi University (34°15′N, 131°48′E). In each plot, 16 plants were arranged in a 4 × 4 matrix (Fig. S5A) in which the 12 outer plants were exposed to volatiles from the four inner plants, which were either infested or intact. Any outer plants infested by herbivores were eliminated. The weather conditions were not controlled during the experimental period (Fig. S5 B and C). The amounts of HexVic in tomato plants sharing a plot with infested tomato plants (1.62 ± 0.15 µg⋅g−1 FW, n = 66) were significantly higher than in control plants sharing a plot with intact plants (1.15 ± 0.17 µg⋅g−1 FW, P = 0.0351, t test, n = 49). These data indicated that airborne (Z)-3-hexenol from the damaged tomato plants was incorporated into and processed by the exposed plants even under fluctuating environmental conditions in which exposure to (Z)-3-hexenol was intermittent.
Animal olfactory systems consist of specific receptors connected to nerve systems. Although plants have been repeatedly reported to respond to volatiles, only the receptor for ethylene, a volatile phytohormone, has been identified to date (9, 10). Our study shows a previously unidentified mechanism that receives the volatile chemical (Z)-3-hexenol in plant–plant communication, i.e., selective incorporation of the compound from neighboring plants to form HexVic. In this process, the sugar moiety of HexVic, namely, vicianose, can be called a volatile acceptor for airborne (Z)-3-hexenol. That animals consume organisms/tissues containing toxic compounds and use those compounds for their own defense is well known. For example, puffer fish and some shellfish assimilate ingredients in their food to protect themselves from predators (19). The Chinese windmill butterfly, Atrophaneura alcinous, and arctiid moth, Utetheisa ornatrix, also use toxic chemicals in their food (20, 21). In contrast, in this study, a relatively inert compound, (Z)-3-hexenol (22, 23), emitted from neighboring conspecifics into the air was taken in and converted into a defensive molecule, HexVic, by plants. This “defense in trans” is a previously unknown mode of plant defense.
Because (Z)-3-hexenol is a common volatile compound emitted by most herbivore-damaged plants, and because we found that a wide range of plant species could accumulate (Z)-3-hexenyl glycosides after exposure to volatiles (Table 1), absorption and glycosylation of exogenous airborne (Z)-3-hexenol might be a general response in plants. If so, the defensive use of HexVic against CCWs would be a function of (Z)-3-hexenyl glycosides. Because (Z)-3-hexenol itself could induce defensive responses in uninfested plants (5, 24), other physiological roles of HexVic in plants should be tested. The effects of as little as 3 µg⋅g−1 FW HexVic in an artificial diet on the performances of CCWs were significant, albeit modest (Fig. 3). The negative impact of capsaicins in pepper on several noctuid larvae and that of hydroxygeranyllinalool glycosides on tobacco budworms (Heliothis virecens) were apparent at ∼20 µg⋅mL−1 and 1 mM (922 µg⋅mL−1), respectively (25, 26). Further studies on the physiological mechanisms involved in the effects of HexVic on CCWs at such low concentrations are needed. Notably, we could not determine whether HexVic biosynthesis has been shaped by selection for defense against CCWs. Thus, the comprehensive effects of HexVic on herbivore communities in nature should be tested. To clarify these issues, a study of mutants that do not produce HexVic would be an effective approach. We are currently identifying the enzyme(s) involved in HexVic formation to prepare a plant deficient in HexVic synthesis to clarify the ecological functions of HexVic.
Table 1.
Glycoside accumulation in various plant species when exposed to green leaf volatiles
| (Z)-3-Hexenyl glucoside | (Z)-3-Hexenyl vicianoside | (Z)-3-Hexenyl primeveroside | ||||
| Species | Control | Exposed | Control | Exposed | Control | Exposed |
| Oryza sativa | 0.001 ± 0.000 | 0.313 ± 0.071* | 0.001 ± 0.001 | 0.011 ± 0.003* | 0.000 ± 0.000 | 0.055 ± 0.019* |
| Sorghum sp. | 0.002 ± 0.001 | 0.096 ± 0.011* | 0.004 ± 0.001 | 0.020 ± 0.002* | ||
| Triticum aestivum | 0.003 ± 0.001 | 0.034 ± 0.011* | ||||
| Citrulus lanatu | 0.001 ± 0.001 | 0.559 ± 0.087* | 0.007 ± 0.001 | 0.310 ± 0.064* | ||
| Cucumis melo | 0.003 ± 0.001 | 0.049 ± 0.007* | 0.004 ± 0.003 | 0.038 ± 0.009* | ||
| Cucumis sativus | 0.004 ± 0.003 | 0.251 ± 0.048* | 0.001 ± 0.000 | 0.264 ± 0.038* | 0.003 ± 0.001 | 0.029 ± 0.002* |
| Monordica charantia | 0.002 ± 0.001 | 0.157 ± 0.061* | 0.087 ± 0.037 | 0.484 ± 0.247* | 0.015 ± 0.007 | 0.080 ± 0.044* |
| Lotus japonicas | 0.002 ± 0.001 | 0.680 ± 0.22* | ||||
| Phaseolus lunatus | 0.004 ± 0.003 | 0.662 ± 0.111* | 0.004 ± 0.002 | 0.376 ± 0.011* | ||
| Phaseolus vrugaris | 0.002 ± 0.001 | 0.358 ± 0.051* | 0.002 ± 0.001 | 0.060 ± 0.006* | 0.001 ± 0.001 | 0.058 ± 0.017* |
| Trifolium repens | 0.002 ± 0.001 | 0.056 ± 0.012* | 0.003 ± 0.002 | 0.016 ± 0.003* | ||
| Abelmoschus esculentus | 0.003 ± 0.001 | 0.374 ± 0.100* | 0.005 ± 0.001 | 0.089 ± 0.009* | ||
| Arabidopsis thaliana | 0.002 ± 0.000 | 0.132 ± 0.035* | ||||
| Brassica rapa | 0.000 ± 0.000 | 0.028 ± 0.011* | ||||
| Eruca vesicaria | 0.001 ± 0.000 | 0.010 ± 0.004* | ||||
| Raphanus sativus | 0.001 ± 0.000 | 0.013 ± 0.001* | ||||
| Antirrhium majus | 0.005 ± 0.002 | 0.366 ± 0.014* | ||||
| Plantago asiatica | 0.011 ± 0.005 | 0.127 ± 0.036* | ||||
| Melissa officinalis | 0.000 ± 0.000 | 0.013 ± 0.001* | ||||
| Solanum lycopersicum | 0.001 ± 0.000 | 0.096 ± 0.007* | 0.002 ± 0.001 | 0.251 ± 0.026* | 0.007 ± 0.003 | 0.066 ± 0.017* |
| Solanum melongena | 0.004 ± 0.002 | 0.024 ± 0.006* | 0.073 ± 0.018 | 0.359 ± 0.031* | ||
| Nicotiana tabacum | 0.002 ± 0.001 | 0.236 ± 0.025* | 0.005 ± 0.001 | 0.029 ± 0.001* | ||
| Arctium lappa | 0.002 ± 0.001 | 0.075 ± 0.016* | 0.008 ± 0.001 | 0.167 ± 0.023* | ||
| Petroselium crispum | 0.000 ± 0.000 | 0.016 ± 0.004* | 0.007 ± 0.001 | 0.102 ± 0.016* | ||
Accumulated glycosides in control and green leaf volatile-exposed plants were extracted and analyzed by UFLC-Q/MS. Quantities are presented as relative peak areas normalized by formononetin on the basis of gram of fresh weight (g−1 FW) of the plant tissues. Mean values (± SE) from three to four experiments are shown. *P < 0.05 (Mann–Whitney–Wilcoxon U test).
Materials and Methods
Plants and Insects.
Tomato plants (Solanum lycopersicum cv. Micro-Tom) were grown in plastic pots (6 cm diameter, 7 cm high) with a 1:1 mixture of Metro-mix (Sun Gro Horticulture) and vermiculite in a growth room at 25 °C ± 3 °C under a 14 h light (100 µmol photons m−2⋅s−1) and 10 h dark photoperiod. We used 4- to 5-wk-old plants with five to seven leaves each for the experiments. The homozygote of the JA-insensitive mutant, jai1-1, was detected using genomic PCR as described in Li et al. (27). Spodoptera litura were maintained in the laboratory on an artificial diet (Insecta LFS, Nosan Co.) under the same temperature and light conditions as the plants.
Insect Performance Experiments.
To analyze the survival rates of first-stadium CCWs on exposed and control plants, we placed 10 first-stadium CCWs from the same egg clutch on a fully expanded leaf of a tomato plant that had been exposed to volatiles from either CCW-infested or uninfested plants for 3 d. The leaf was gently covered with a pair of plastic petri dish lids (5 cm diameter, 2 cm high) to prevent the inoculated larvae from dispersing. We bored a hole (4 cm diameter) at the bottom of the lids and covered the hole with fine nylon mesh for ventilation. Two doughnuts of plastic sponge (5.5 cm external diameter; 4.5 cm internal diameter; 5 mm thickness) were attached around the rims of the two lids to sandwich the inoculated leaf. The two lids were gently held together with a clamp. The sandwiched leaf was kept horizontal by placing the two lids on a small table. The plants were kept in a growth chamber [120 L, 25 °C ± 3 °C under a 14 h light (100 µmol photons m−2⋅s−1) and 10 h dark photoperiod]. After 5 d, we counted the number of surviving CCWs. We repeated each experiment 21 times on 7 different experimental days.
To examine the effects of HexVic on the survival rates of first-stadium CCWs, purified HexVic dissolved in 80% methanol was embedded in a piece (∼1 g) of artificial diet (28) at a concentration of 3 µg⋅g−1; this concentration was roughly equivalent to that in the tomato leaf exposed to volatiles from infested tomato plants (Comprehensive Metabolite Analyses of Leaf Extracts). As a control, 80% methanol was embedded in the diet. We placed 10 first-stadium CCWs on an artificial diet in a lidded plastic petri dish (5 cm diameter, 2 cm high) in the growth chamber. After 5 d, we counted the number of surviving CCWs. We repeated each experiment 10 times on 2 different experimental days.
To observe weight gain of CCWs on tomato leaves, we detached one of the fully expanded leaves of a tomato plant that had been exposed to volatiles from either CCW-infested or uninfested plants for 3 d. Ten second-stadium CCWs from the same egg clutch were inoculated into two types of exposed leaves for 1 d. The weight of 10 CCWs was measured before and after inoculation. We repeated both the exposure experiment and control experiment 43 times on 8 experimental days. The data of each experimental day were pooled and averaged.
To examine the effects of HexVic on weight gain of CCWs, 10 second-stadium CCWs from the same egg clutch were placed on a piece (∼1 g) of artificial diet (either HexVic-embedded or control). The artificial diets were prepared as in the measurements of survival rates. The weight of 10 CCWs was measured before and after inoculation. We repeated both the embedded experiment and control experiment 95 times on 10 experimental days. The data for each experimental day were pooled and averaged.
Metabolite Analyses.
Metabolite analyses were performed essentially as previously described (29). For the targeted analysis, metabolites were extracted from about 100 mg of leaf tissues with methanol–chloroform solvent containing 200 µg⋅mL−1 ribitol and 2 mg⋅mL−1 methyl nonadecanoate as internal standards for polar (methanol–water layer) and apolar (chloroform layer) compounds, respectively. The metabolites were subjected to GC–quadrupole (Q)–TOF/MS analysis (6890, Agilent) after methyl esterification and trimethylsilylation. For the nontargeted analysis, metabolites were extracted from about 100 mg of leaf tissues using 3 vol of methanol containing 10 µg⋅mL−1 [for ultraperformance liquid chromatography (UPLC)–TOF/MS and HPLC–Fourier transform/ion cyclotron resonance–linear ion trap/MS analyses] or 1 µg⋅mL−1 (for UPLC–Q/MS analysis) formononetin as an internal standard. The methanol extract was analyzed with an Agilent 1100 system (Agilent Technologies) and Finnigan linear ion trap quadrupole (LTQ)-FT (Thermo Fisher Scientific) and with a Shimadzu UPLC system (Shimadzu) and 3200 Q TRAP (AB SCIEX). The amount of HexVic was calculated using a calibration curve constructed with (Z)-3-hexenyl-O-β-d-xylopyranosyl-(1,6)-β-d-glucopyranoside [(Z)-3-hexenylprimeveroside]. To see the direct effect of herbivore infestation on the amount of HexVic, tomato plants were infested with five third-stadium CCWs under still-air conditions in the growth room for 1 d, and HexVic was quantified with LC–MS. (Z)-3-Hexenyl-β-d-glucopyranoside [(Z)-3-hexenylglucoside] was assigned using a standard compound prepared by partial acid hydrolysis of HexVic (Table S2). To analyze (Z)-3-hexenol accumulation in the leaf tissues, ∼100 mg of tissue was finely powdered in liquid nitrogen with 1 mL of saturated calcium chloride solution using a mortar and pestle. The powder was transferred into a glass vial (22 mL) with 100 ng of nonanyl acetate (internal standard). The vial was sealed with a butyl rubber stopper and aluminum cap. The samples were stored at –80 °C until use and thawed at 25 °C for 10 min before extraction. (Z)-3-Hexenol was extracted with a SPME fiber (50/30 µm DVB/Carboxen/PDMS StableFlex, Supelco) for 30 min and analyzed with a QP-5050A GC–MS equipped with a Stabiliwax column (Shimadzu GLC). The initial oven temperature was 40 °C, held for 5 min, ramped at 5 °C min−1 to 230 °C, and held for 1 min at a flow rate of 2 mL min−1 (helium). The MS was operated in the electron ionization mode at 70 eV and a source temperature of 230 °C with a continuous scan from m/z 40–350.
HexVic Accumulation and Synthesis in MeJA-Exposed Plants.
To examine MeJA treatment, a tomato plant in a pot was placed in a closed glass jar (2,000 cm−3) with a cotton swab impregnated with 20 µL of 100 mM MeJA (in CH2Cl2) for 2 h under the growth conditions described above. For the control, we used a swab with 20 µL of CH2Cl2. To analyze the HexVic content, plants exposed to MeJA for 2 h were removed from the jar, and their leaves were harvested immediately or 4 and 24 h after removal from the jar. The contents of HexVic were analyzed with LC–MS. Quantification was carried out in four independent experiments. To analyze the rate of HexVic synthesis, the exposed plants were transferred to new containers with (Z)-3-hexenol. The plants were collected at 0, 0.5, 1, 2, 3, and 6 h after transfer. The contents of HexVic were analyzed by LC–MS. The difference in synthetic rate of HexVic between MeJA-treated and untreated plants was analyzed by interaction of exposure time and HexVic content. Quantification was carried out in three independent experiments.
Analysis of Volatile Compounds.
Plants were put into two chambers in an airflow system (Fig. S1) with or without CCWs and with a piece of filter paper that contained 100 ng of tridecane in n-hexane as an internal standard. After 24 h, a portion of the headspace was collected into a tube with 100 mg of Tenax-TA adsorbent (20/35 mesh, GL Sciences) for 1 h at a flow rate of 100 mL⋅min−1. The collected volatile compounds were analyzed with GC–MS (HP 6890 with an HP-5MS capillary column and an HP 5973 mass selective detector, Hewlett-Packard) equipped with a thermal desorption cold trap injector (CP5010, Chrompack).
Field Experiments.
We conducted field experiments in a garden (5 × 20 m) at Yamaguchi University (34°15′N, 131°48′E) from October 13 to October 17 and from November 13 to November 17, 2011 (Fig. S5). We arranged two infested and two uninfested plots in October and four plots of each type in November for a total of 12 plots. Each plot contained 16 potted tomato plants in a 4 × 4 matrix (Fig. S5A). For treated plots, we inoculated three to five third-stadium CCWs per plant onto the four plants in the center of the plot. For the control plots, no CCWs were used. The 12 potted plants surrounding the central plants were receiver plants that were exposed to volatiles from either four infested plants or four intact ones. After the exposure period (5 d), the amount of HexVic in each receiver plant was measured.
Statistical Analyses.
Statistical analyses indicated in the text and legends were performed using R 2.11.1 (www.r-project.org/) and JMP 9.0.2 (SAS Inc.) software.
Supplementary Material
Acknowledgments
We thank G. A. Howe (Michigan State University), who kindly provided the JA-insensitive mutant jai1-1; K. Kubota (Ochanomizu University), who provided hexenyl primeveroside; and R. Karban (University of California, Davis) for critically reviewing the manuscript and providing helpful comments. This study was supported in part by grants of the priority area (S) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT); the Core-to-Core project (20004) from the Japan Society for the Promotion of Science (JSPS); the Scientific Research for Plant Graduate Students from the Nara Institute of Science and Technology supported by MEXT; Research Fellowships for Young Scientists (24·841) from JSPS; a Grant-in-Aid for Scientific Research (C) (23580151) from JSPS; a Joint Research Program implemented at the Institute of Plant Science and Resources, Okayama University, supported by MEXT; and the Japan Advanced Plant Science Network.
Footnotes
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1320660111/-/DCSupplemental.
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