Delayed burst in induced volatile emissions in plants exposed to volatiles of a herbivore-attacked neighbor. Emission kinetics of herbivore-induced plant volatile (HIPV)-induced terpenes in undamaged receiver plants are shown. Dark green points represent mean emission of herbivore-damaged sender plants connected to undamaged receiver plants, with the emissions from damaged sender plants only subtracted. Black points represent the same but with undamaged sender plants. Blue rectangle represents the dark phase. Abbreviations. DMNT, 4,8-dimethylnona-1,3,7-triene; TMTT, 4,8,12-trimethyltrideca-1,3,7,11-tetraene. Error bars = SE. n = 8-10.

The delayed burst in terpene emission is not explained by terpene overaccumulation during the night. Accumulation of terpenes and induction of terpene biosynthesis genes in receiver plants was measured over time. A-D) Internal foliar pools of terpenes in receiver plants. E-H) Expression of terpene biosynthesis genes in receiver plants. Dark green bars represent receiver plants exposed to herbivore-induced plant volatiles (HIPVs) and light grey bars represent receiver plants exposed to control plant volatiles. Blue rectangle represents the dark phase. Abbreviations: TMTT, 4,8,12-trimethyltrideca-1,3,7,11-tetraene; FPPS3, Farnesyl pyrophosphate synthase 3; TPS2, Terpene synthase 2; TPS10, Terpene synthase 10; CYP92C5, Dimethylnonatriene/trimethyltetradecatetraene synthase. = p < 0.1, * = p < 0.05, ** = p < 0.01, *** = p < 0.001 as determined by a Welch’s two-sample t-test. Bars = mean ± SE. n = 4-6.

Welch’s two-sample t-test results for foliar terpene pools, biosynthesis genes and phytohormone levels. Bold values: p < 0.05, underlined values: p < 0.1. Abbreviations: β-car, β-caryophyllene; β-farn, β-farnesene; α-berg, α-bergamotene; TMTT, 4,8,12-trimethyltrideca-1,3,7,11-tetraene; FPPS3, Farnesyl pyrophosphate synthase 3; TPS2, Terpene synthase 2; TPS10, Terpene synthase 10; CYP92C5, Dimethylnonatriene/trimethyltetradecatetraene synthase; OPDA = 12-oxophytodienoic acid; JA = jasmonic acid; JA-Ile = jasmonic acid-isoleucine; OPR7 = oxo-phytodienoate reductase 7.

The delayed burst in terpene emission is associated with clocked jasmonate production. Foliar jasmonate concentrations (A-C) and jasmonate biosynthesis (OPR7; D) in receiver plants over time are shown. Dark green bars represent receiver plants exposed to herbivore-induced plant volatiles (HIPVs) and light grey bars represent receiver plants exposed to control plant volatiles. Blue rectangle represents the dark phase. Abbreviations. OPDA = 12-oxophytodienoic acid, JA = jasmonic acid, JA-Ile = jasmonic acid-isoleucine, OPR7 = oxo-phytodienoate reductase 7. = p < 0.1, * = p < 0.05, ** = p < 0.01, *** = p < 0.001 as determined by a Welch’s two-sample t-test. Bars = mean ± SE. n = 3-6.

The delayed volatile burst is conserved under continuous light. Emission kinetics of herbivore-induced plant volatiles (HIPV)-induced terpenes in undamaged receiver plants under continuous light are shown. Dark green points represent the mean emission of herbivore damaged sender plants connected to undamaged receiver plants with the emissions from damaged sender plants only subtracted. Black points represent the same but with undamaged sender and receiver plants. Yellow rectangle represents when the lights would typically be turned off. Abbreviations: DMNT, 4,8-dimethylnona-1,3,7-triene; TMTT, 4,8,12-trimethyltrideca-1,3,7,11-tetraene. Error bars = SE. n = 8-10.

Green leaf volatile (GLV) and indole emissions in Spodoptera exigua-damaged plants. Light green points represent the mean emission of herbivore-damaged sender plants. Grey points represent the mean emission of undamaged sender plants. Blue rectangle represents the dark phase. Yellow rectangle represents when the lights would typically be turned off. Error bars = SE. n = 8-10.

Receiver plant terpene emissions tightly correlate with bioactive sender plant signals under normal light conditions. The left panels depict scatter plot correlation matrices of volatile emissions from both damaged sender plants and herbivore-induced plant volatile (HIPV)-exposed receiver plants, from the first measurement following the addition of herbivores to sender plants. Upper scatter plot (in blue box) shows correlations under normal light conditions and lower scatter plot (yellow box) depicts correlations under continuous light. Each black point represents the mean value of all individuals at a given time point after herbivory began. Panels on the right hand side depict heat maps based on the value of Pearson’s correlation coefficient between two given compounds. Numbers in the center of each square are Pearson’s correlation coefficient. Correlation coefficients contained in a pink rectangles indicate a significant correlation (p < 0.05). Hexenyl acetate (HAC), Hexenal (Hexa), Hexen-1-ol (Hexo) and indole (Ind) were from sender plants, and sesquiterpenes (SQT), monoterpenes (MNT), 4,8-dimethylnona-1,3,7-triene (DMNT) and 4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT) were from receiver plants.

The combination of volatile priming and sender emission kinetics can explain the delayed terpene burst in receiver plants. Sender plants were connected to receiver plants 30 min prior to herbivore exposure on sender plants and left connected for 1.25 hr following exposure (time between perforated vertical lines). After 1.25 hr, chambers were disconnected and measurements were collected from receiver plant chambers only. The following day, after light was restored, plants were treated with (Z)-3-hexenyl acetate (HAC) dispensers to simulate bioactive signals (indicated by red solid vertical line). * = p < 0.05, ** = p < 0.01, *** = p < 0.001 as determined by aligned rank transformed nonparametric factorial repeated measures ANOVA. Abbreviations: HIPV, herbivore-induced plant volatile; DMNT, 4,8-dimethylnona-1,3,7-triene; TMTT, 4,8,12-trimethyltrideca-1,3,7,11-tetraene. Colored points represent mean emissions standardized by fresh weight (fw). Error bars = SE. n = 12-16.

Aligned rank transformed nonparametric factorial repeated measures ANOVA results. Bold values: p < 0.05 and underlined values: p < 0.1. Abbreviations: HAC, (Z)-3-hexenyl acetate; SQT, sesquiterpenes; MNT, monoterpenes; DMNT, 4,8-dimethylnona-1,3,7-triene; TMTT, 4,8,12-trimethyltrideca-1,3,7,11-tetraene.

Experimental setup schemes for volatile profiling

Gene identifiers and qRT-PCR primer sequences used in this study.