Integration of two herbivore-induced plant volatiles results in synergistic effects on plant defence and resistance

Abstract

Plants can use induced volatiles to detect herbivore- and pathogen-attacked neighbors and prime their defenses. Several individual volatile priming cues have been identified, but whether plants are able to integrate multiple cues from stress-related volatile blends remains poorly understood. Here, we investigated how maize plants respond to two herbivore-induced volatile priming cues with complementary information content, the green leaf volatile (Z)-3-hexenyl acetate (HAC) and the aromatic volatile indole. In the absence of herbivory, HAC directly induced defence gene expression, whereas indole had no effect. Upon induction by simulated herbivory, both volatiles increased jasmonate signalling, defence gene expression, and defensive secondary metabolite production and increased plant resistance. Plant resistance to caterpillars was more strongly induced in dual volatile-exposed plants than plants exposed to single volatiles.. Induced defence levels in dual volatile-exposed plants were significantly higher than predicted from the added effects of the individual volatiles, with the exception of induced plant volatile production, which showed no increase upon dual-exposure relative to single exposure. Thus, plants can integrate different volatile cues into strong and specific responses that promote herbivore defence induction and resistance. Integrating multiple volatiles may be beneficial, as volatile blends are more reliable indicators of future stress than single cues.

Keywords: (Z)-3-hexenyl acetate; benzoxazinoids; indole; induced resistance; insects; jasmonic acid; maize; plant defence; plant herbivore interactions; volatile communication.

Figure 1

Simultaneous pre‐exposure to (Z)‐3‐hexenyl acetate (HAC) and indole synergistically increases abscisic acid (ABA) and jasmonic acid (JA) biosynthesis in induced maize plants. (a)‐(e) Average concentrations of the stress hormones ABA (a), 12‐oxophytodienoic acid (OPDA, b), JA (c), JA‐isoleucine (JA‐Ile, d), and salicylic acid (SA, e) in plants that were pre‐exposed to HAC, indole, or both volatiles simultaneously (HAC + Indole) and induced by simulated herbivory (+SE, n = 5). (f) Average transcript levels of ZmLOX10, ZmAOS, ZmPR1, and ZmPR5 (+SE, n = 5). FW, fresh weight. n.s., not significant. Treat., treatment. Gene expression is shown relative to the expression level of the control treatment. P values of one‐way analyses of variance (ANOVAs) are shown (*P < 0.05, **P < 0.01, ***P < 0.001). Dashed lines indicate calculated additive effects of single volatile exposures. Letters indicate significant differences between different volatile exposure treatments (P < 0.05, one‐way ANOVA followed by multiple comparisons through FDR‐corrected LSMeans). Stars indicate a significant difference between the double exposure treatment and the calculated additive effect of both single treatments (*P < 0.05, Student's t tests)

Figure 2

Simultaneous pre‐exposure to (Z)‐3‐hexenyl acetate (HAC) and indole specifically and synergistically increases defence gene expression in induced maize plants. Average transcript levels of ZmMPI (a), ZmSerPIN (b), ZmRIP2 (c), and ZmCyst (d) in plants that were pre‐exposed to HAC, indole, or both volatiles simultaneously (HAC + Indole) and induced by simulated herbivory (+SE, n = 5). n.s., not significant. Treat., treatment. Gene expression is shown relative to the expression level of the control treatment. P values of one‐way analyses of variance (ANOVAs) are shown (*P < 0.05, **P < 0.01, ***P < 0.001). Dashed lines indicate calculated additive effects of single volatile exposures. Letters indicate significant differences between different volatile exposure treatments (P < 0.05, one‐way ANOVA followed by multiple comparisons through FDR‐corrected LSMeans). Stars indicate a significant difference between the double exposure treatment and the calculated additive effect of both single treatments (*P < 0.05, Student's t tests)

Figure 3

Simultaneous pre‐exposure to (Z)‐3‐hexenyl acetate (HAC) and indole synergistically regulates benzoxazinoid (BX) biosynthesis in induced maize plants. (a) Average concentrations of benzoxazinoids in plants that were pre‐exposed to HAC, indole, or both volatiles simultaneously (HAC + Indole) and induced by simulated herbivory (+SE, n = 5). (b)‐(c) Average transcript levels of ZmBx10/11 and ZmBx14 (+SE, n = 5). FW, fresh weight. L.O.D, below limit of detection. n.s., not significant. Treat., treatment. Gene expression is shown relative to the expression level of the control treatment. P values of one‐way analyses of variance (ANOVAs) are shown (*P < 0.05, **P < 0.01, ***P < 0.001). Dashed lines indicate calculated additive effects of single volatile exposures. Letters indicate significant differences between different volatile exposure treatments (P < 0.05, one‐way ANOVA followed by multiple comparisons through FDR‐corrected LSMeans). Stars indicate a significant difference between the double exposure treatment and the calculated additive effect of both single treatments (*P < 0.05, **P < 0.01, Student's t tests). DIMBOA‐Glc, 2‐(2,4‐dihydroxy‐7‐methoxy‐1,4‐benzoxazin‐3‐one)‐β‐d‐glucopyranose; DIM₂BOA‐Glc, 2‐(2,4‐dihydroxy‐6,7‐dimethoxy‐l,4‐benzoxazin‐3‐one)‐β‐d‐glucopyranose; DIBOA‐Glc, 2‐(2,4‐dihydroxy‐1,4‐benzoxazin‐3‐one)‐β‐d‐glucopyranose; HDMBOA‐Glc, 2‐(2‐hydroxy‐4,7‐dimethoxy‐1,4‐benzoxazin‐3‐one)‐β‐d‐glucopyranose; HDM₂BOA‐Glc, 2‐(2‐hydroxy‐4,7,8‐trimethoxy‐1,4‐benzoxazin‐3‐one)‐β‐d‐glucopyranose; DIMBOA: 2,4‐dihydroxy‐7‐methoxy‐1,4‐benzoxazin‐3‐one; MBOA: 6‐methoxy‐benzoxazolin‐2‐one

Figure 4

Simultaneous pre‐exposure to (Z)‐3‐hexenyl acetate (HAC) and indole does not synergistically regulate volatile production in induced maize plants. (a)‐(e) Average relative amounts (peak areas) of linalool (a), (3E)‐4,8‐dimethyl‐1,3,7‐nonatriene (DMNT, b), (E)‐α‐bergamotene (c), (E)‐α‐farnesene (d), and indole (e) in plants that were pre‐exposed to HAC, indole, or both volatiles simultaneously (HAC + Indole) and induced by simulated herbivory (+SE, n = 5). (f) Average transcript levels of ZmCYP92C5, ZmTPS2, ZmTPS3, ZmTPS10, and ZmIGL (+SE, n = 5). FW, fresh weight. n.s., not significant. Treat., treatment. Gene expression is shown relative to the expression level of the control treatment. P values of one‐way analyses of variance (ANOVAs) are shown (*P < 0.05, **P < 0.01, ***P < 0.001). Dashed lines indicate calculated additive effects of single volatile exposures. Letters indicate significant differences between different volatile exposure treatments (P < 0.05, one‐way ANOVA followed by multiple comparisons through FDR‐corrected LSMeans). Stars indicate a significant difference between the double exposure treatment and the calculated additive effect of both single treatments (*P < 0.05, Student's t tests)

Figure 5

Simultaneous pre‐exposure to (Z)‐3‐hexenyl acetate (HAC) and indole increases herbivore resistance of maize plants. (a) Average growth rate of Spodotera littoralis caterpillars feeding on plants that were pre‐exposed to HAC, indole, or both volatiles simultaneously (HAC + Indole, +SE, n = 10). (b) Average consumed leaf area (+SE, n = 10). n.s., not significant. Treat., treatment. The results of one‐way analyses of variance (ANOVAs) are shown (**P < 0.01, ***P < 0.001). Dashed lines indicate calculated additive effects of single volatile exposures. Letters indicate significant differences between different volatile exposure treatments (P < 0.05, one‐way ANOVA followed by multiple comparisons through FDR‐corrected LSMeans)

Figure 6

Pre‐exposure to (Z)‐3‐hexenyl acetate (HAC), but not indole, directly induces defence gene expression in maize plants. (a) Average transcript levels of genes involved in JA biosynthesis, SA signalling and benzoxazinoid biosynthesis in plants that were pre‐exposed to HAC, indole, or both volatiles simultaneously (HAC + Indole) without subsequent induction (+SE, n = 5). (b) Average transcript levels of putative proteinase inhibitors and a ribosome‐inactivating gene ZmRIP2 (+SE, n = 5). (c) Average transcript levels of genes involved in terpene and indole biosynthesis (+SE, n = 5). n.s., not significant. Treat., treatment. Gene expression is shown relative to the expression level of the control treatment. P values of one‐way analyses of variance (ANOVAs) are shown (*P < 0.05, **P < 0.01, ***P < 0.001). Dashed lines indicate calculated additive effects of single volatile exposures. Letters indicate significant differences between different volatile exposure treatments (P < 0.05, one‐way ANOVA followed by multiple comparisons through FDR‐corrected LSMeans). Stars indicate a significant difference between the double exposure treatment and the calculated additive effect of both single treatments (*P < 0.05, Student's t tests)

Figure 7

Simultaneous pre‐exposure to (Z)‐3‐hexenyl acetate (HAC) and indole results in specific defence signatures in maize plants. Principal component analyses of maize defence markers (a) 0 min, (b) 45 min, and (c) 5 hr after induction by simulated herbivory. Plants were pre‐exposed to HAC, indole, or both volatiles simultaneously (HAC + Indole, n = 5) prior to induction by simulated herbivory. PCAs include data on defence gene expression at 0 min, phytohormones and signalling related gene expression at 45 min, and defence gene expression and secondary metabolite production at the 5 hr time point. Data points represent individual replicate samples. Vectors of individual defence markers are shown as grey arrows. P values of permutational analyses of variance (“Adonis test”) between treatments are shown