Identification of biologically relevant compounds in aboveground and belowground induced volatile blends

Abstract

Plants under attack by aboveground herbivores emit complex blends of volatile organic compounds (VOCs). Specific compounds in these blends are used by parasitic wasps to find their hosts. Belowground induction causes shifts in the composition of aboveground induced VOC blends, which affect the preference of parasitic wasps. To identify which of the many volatiles in the complex VOC blends may explain parasitoid preference poses a challenge to ecologists. Here, we present a case study in which we use a novel bioinformatics approach to identify biologically relevant differences between VOC blends of feral cabbage (Brassica oleracea L.). The plants were induced aboveground or belowground with jasmonic acid (JA) and shoot feeding caterpillars (Pieris brassicae or P. rapae). We used Partial Least Squares--Discriminant Analysis (PLSDA) to integrate and visualize the relation between plant-emitted VOCs and the preference of female Cotesia glomerata. Overall, female wasps preferred JA-induced plants over controls, but they strongly preferred aboveground JA-induced plants over belowground JA-induced plants. PLSDA revealed that the emission of several monoterpenes was enhanced similarly in all JA-treated plants, whereas homoterpenes and sesquiterpenes increased exclusively in aboveground JA-induced plants. Wasps may use the ratio between these two classes of terpenes to discriminate between aboveground and belowground induced plants. Additionally, it shows that aboveground applied JA induces different VOC biosynthetic pathways than JA applied to the root. Our bioinformatic approach, thus, successfully identified which VOCs matched the preferences of the wasps in the various choice tests. Additionally, the analysis generated novel hypotheses about the role of JA as a signaling compound in aboveground and belowground induced responses in plants.

Fig. 1

Preference of Cotesia glomerata wasps expressed as the percentage of wasps preferring the plant (±s.e.m. calculated over 8–18 replicate test runs of 10–15 females per run). a and c Dual-choice tests between plants treated with jasmonic acid (JA) to the roots (RJA), to the shoots (SJA) or with acidic water (CON) 3 d before 10 Pieris brassicae (a) or 10 Pieris rapae (c) larvae were added. Asterisks denote a significant difference in preference between the two plants (* P Gp < 0.01, ** P Gp < 0.001, see Table 1. b and d) Triple choice tests between CON, RJA and SJA plants. Asterisks denote an overall significance in preference between the three plants (** P Gp < 0.001)

Fig. 2

Two-dimensional PLS-Discriminant fingerprints for VOCs emitted by Brassica oleracea plants with a. 10 Pieris brassicae larvae or b 10 P. rapae larvae. The separation of individual plants on each discriminant function are given on the left vertical (RJA vs. SJA) and lower horizontal (CON vs. (RJA + SJA)) axes in each figure. Open circles = control plants, grey squares = root-induced (RJA) plants; black riangles = shoot-induced (SJA) plants. The two-dimensional plots in the middle show the contribution of each VOC to the separation. The position of each point is determined by its importance for the contrasts. The numbered VOCs (see Table 1) contributed significantly to the separation of treatment groups (encircled: (CON vs. (RJA + SJA)), crosses: for (RJA vs. SJA). Statistical significance was determined by testing the discriminant function value of a VOC against a null distribution of 1000 models on randomly permuted treatment group assignments (P < 0.05)

Fig. 3

Box plots showing the median value, the 25th percentile, and 75th percentile of the peak area per ml sampled air for a limonene, and b sabinene. Grey bars: values in Pieris brassicae experiment, white bars: values in P. rapae experiment. CON = control plants, RJA = root jasmonic acid (JA) induced plants, SJA = shoot JA induced plants. The error bars above and below the box indicate the 90th and 10th percentile

Fig. 4

Box plots showing the median value, the 25th percentile, and 75th percentile of the peak area per ml sampled air for homo-and sesquiterpenes. a DMNT.’ < 3E > -4,8-dimethyl-1,3,7-nonatriene; b Zingiberene plus sesquiterpene 2 (zingiberene alike); Legend (DMNT) Grey bars: values in Pieris brassicae experiment, white bars: values in P. rapae experiment. CON = control plants, RJA = root jasmonic acid (JA)-induced plants, SJA = shoot JA-induced plants. The error bars above and below the box indicate the 90th and 10th percentile