Mycorrhiza-Triggered Transcriptomic and Metabolomic Networks Impinge on Herbivore Fitness

Abstract

Symbioses between plants and mycorrhizal fungi are ubiquitous in ecosystems and strengthen the plants' defense against aboveground herbivores. Here, we studied the underlying regulatory networks and biochemical mechanisms in leaves induced by ectomycorrhizae that modify herbivore interactions. Feeding damage and oviposition by the widespread poplar leaf beetle Chrysomela populi were reduced on the ectomycorrhizal hybrid poplar Populus × canescens Integration of transcriptomics, metabolomics, and volatile emission patterns via mass difference networks demonstrated changes in nitrogen allocation in the leaves of mycorrhizal poplars, down-regulation of phenolic pathways, and up-regulation of defensive systems, including protease inhibitors, chitinases, and aldoxime biosynthesis. Ectomycorrhizae had a systemic influence on jasmonate-related signaling transcripts. Our results suggest that ectomycorrhizae prime wounding responses and shift resources from constitutive phenol-based to specialized protective compounds. Consequently, symbiosis with ectomycorrhizal fungi enabled poplars to respond to leaf beetle feeding with a more effective arsenal of defense mechanisms compared with nonmycorrhizal poplars, thus demonstrating the importance of belowground plant-microbe associations in mitigating aboveground biotic stress.

Figure 1.

Visits of C. populi, feeding damage, and oviposition on poplar leaves of mycorrhizal or nonmycorrhizal plants. Data show cumulative visits of poplar leaf beetles and feeding damage to leaf biomass (A and inset) and cumulative number of eggs deposited on poplar leaves (B). Data indicate means ± se (n = 4). Count data (beetle visits and eggs) for the whole time course were analyzed by Poisson GLM and biomass at harvest by ANOVA, with different letters denoting significantly different values.

Figure 2.

PCA score plot of volatile organic profiles emitted by poplar leaves. Data for the emitted volatiles are shown in Supplemental Table S3. Data were log₁₀ transformed and Pareto scaled prior to analysis.

Figure 3.

PCA score plot of metabolite analyses in poplar leaves for (−)LC-MS data (A) and (+)LC-MS data (B). The measurement data are shown in Supplemental Table S4.

Figure 4.

Numbers of compounds affected by mycorrhiza or by feeding beetles in poplar leaves. Data show numbers of annotated discriminant molecular formulas for EMF inoculation (A) and for beetle exposure (B). Bars indicate the numbers of unique or overlapping metabolites that were increased (up arrows) or decreased (doe arrows) in response to the treatment.

Figure 5.

Flavonoid biosynthesis exemplifies transcriptomic-metabolomic data matching via MDBs. A, Log₂ fold changes of metabolite (black) and transcript (red) levels of the proanthocyanidin pathway. B and C, (−)UPLC-qToF-MS MDiN with transcripts matched on the MDBs (B; for color code of up- and down-regulated mass features, see Fig. 4) and with an expanded view of flavonoid/proanthocyanidin biosynthesis (C). Abbreviations are as follows: dihydrokaempferol (DHK), dihydroquercetin (DHQ), leucoanthocyanidin reductase (LAR), leucoanthocyanidin dioxygenase (LDOX), leucocyanidin (leucocy), quercetin (Que). The color code of the MDiN in B reflects the modularity of the nodes.

Figure 6.

Z-scores obtained by MDEA of the EMF effect in the (−)LC-MS samples. A and B, Overrepresented MDBs in the MC/NC comparison (A) and the MB/NB comparison (B). C and D, MDBs highlighted with dashed lines in A and B pertain to the cyanoamino acid metabolism KEGG pathway map starting from Phe (C), and MDBs highlighted in gray start from either Cys or Ile (D). cond., Condensation; DiOH, dihydroxy; FMN, flavin mononucleotide; MDA, malondialdehyde; OH, hydroxyl. Red background, detected with GC-MS; purple background, detected via transcriptomics.

Figure 7.

Cross-platform MDiN. Discriminant molecular formulas were colored according to the EMF effect (A) and according to the beetle effect (B).

Figure 8.

Illustration of systemic changes resulting from EMF inoculation within a poplar cell. Transcriptional changes of enzymes are represented by colored boxes, and those of transmembrane transporters are represented by colored circles; biosynthetic routes are illustrated in rounded rectangles that are colored according to the enzymatic and metabolic regulation patterns found within them. Metabolites and compound classes also are colored according to their respective regulation patterns. Metabolites that are colored in red were detected by GC-MS measurements. ACC, Acetyl-CoA carboxylase; Ac-CoA, acetyl-CoA; biosyn, biosynthesis; CHS, chalcone synthase; Cyto, cytosol; DHAP, dihydroxyacetone phosphate; E4P, erythrose 4-phosphate; FA, fatty acid; G3P, glyceraldehyde 3-phosphate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GDH2, Glu dehydrogenase2; GOGAT, Gln oxoglutarate aminotransferase; GS, Glu synthase; GST, glutathione S-transferase; ISPS, isoprene synthase; KAT, 3-ketoacyl-CoA thiolase; KPI, Kunitz protease inhibitor; NAS3, nicotianamine synthase; NIA, nitrite reductase; NRT1, nitrate transporter; OA, oxaloacetate; 2OG, 2-oxogluterate; PAL, Phe ammonia lyase; PEP, phosphoenolpyruvate; PPP, pentose phosphate pathway; PPT, phosphoenolpyruvate/phosphate translocator; Phpyr, phenylpyruvate; PolyOxid, polyamine oxidase; Pyr, pyruvate; SAM, S-adenoysl-l-Met; TPI, triosephosphate isomerase; trans, transporter; XGE, xyloglucan endotransglucosylase.