The occurrence and formation of monoterpenes in herbivore-damaged poplar roots

Abstract

Volatiles are often released upon herbivory as plant defense compounds. While the formation of volatiles above-ground has been intensively studied, little is known about herbivore-induced root volatiles. Here, we show that cockchafer larvae-damaged roots of Populus trichocarpa and P. nigra release a mixture of monoterpenes, including (-)-α-pinene, (-)-camphene, (-)-β-pinene, p-cymene, and 1,8-cineole. Three terpene synthases, PtTPS16 and PtTPS21 from P. trichocarpa and PnTPS4 from P. nigra, could be identified and characterized in vitro. PnTPS4 was found to produce 1,8-cineole as sole product. PtTPS16 and PtTPS21, although highly similar to each other, showed different product specificities and produced γ-terpinene and a mixture of (-)-camphene, (-)-α-pinene, (-)-β-pinene, and (-)-limonene, respectively. Four active site residues were found to determine the different product specificities of the two enzymes. The expression profiles of PtTPS16, PtTPS21, and PnTPS4 in undamaged and herbivore-damaged poplar roots generally matched the emission pattern of monoterpenes, indicating that monoterpene emission in roots is mainly determined at the gene transcript level. Bioassays with Phytophtora cactorum (Oomycetes) revealed inhibitory effects of vapor-phase 1,8-cineole and (-)-β-pinene on the growth of this important plant pathogen. Thus herbivore-induced volatile monoterpenes may have a role in defense against pathogens that cause secondary infections after root wounding.

Figure 1

Dendrogram (maximum likelihood tree) of putative monoterpene synthases of Populus trichocarpa and P. nigra cloned in this study (red), their previously identified orthologues (green), other previously characterized P. trichocarpa TPS-b and TPS-g enzymes (blue), and further putative monoterpene synthases found in the P. trichocarpa genome version 3.0 (black). Bootstrap values (n = 1000) are shown next to each node. ISPS, isoprene synthase; MTS, monoterpene synthase; TPS-b and -g represent TPS subfamilies. Scale bar under the tree indicates number of substitutions per site.

Figure 2

Biochemical characterization of the newly identified poplar root terpene synthases PnTPS4 (A), PtTPS16 (B), and PtTPS21 (C). The genes were heterologously expressed in E. coli and partially purified proteins were incubated with GPP as substrate. Enzyme products were analyzed using GC-MS. 1, α-thujene; 2, α-pinene*; 3, camphene*; 4, sabinene; 5, β-pinene*; 6, myrcene; 7, α-phellandrene; 8, α-terpinene*; 9, β-phellandrene; 10, limonene*; 11, ocimene; 12, γ-terpinene*; 13, terpinolene; 14, 1.8-cineole; 15, fenchyl alcohol. Compounds marked with * were identified by comparison of retention time and mass spectrum to those of authentic standards. Others were identified by database comparisons.

Figure 3

Stereochemical analysis of PtTPS21 enzyme products (A–E) and proposed reaction mechanism of PtTPS16 and PtTPS21 (F). 2, (−)-α-pinene; 3, (−)-camphene; 5, (−)-β-pinene; 10, (−)-limonene, 2*, (+)-α-pinene; 3*, (+)-camphene; 5*, (+)-β-pinene; 10*, (+)-limonene.

Figure 4

Structure model of PtTPS16. Models of N-terminal truncated PtTPS16 (A) and the active site of PtTPS16 (B) are shown. The conserved DDxxD motif is displayed in gray and the four amino acid residues that differ between the active sites of PtTPS16 and PtTPS21 are depicted in red.

Figure 5

Biochemical characterization of PtTPS16 mutants generated using in vitro mutagenesis. GC-MS chromatograms representing the product spectra of wild type PtTPS16 (A), wild type PtTPS21 (L), and the different PtTPS16 mutants (B–K) are shown. Amino acid changes (one letter code) and their positions relative to the PtTPS16 sequence are indicated in the name of the mutants. The genes were heterologously expressed in E. coli and partially purified proteins were incubated with GPP as substrate. Enzyme products were analyzed using GC-MS. 1, α-thujene; 2, α-pinene*; 3, camphene*; 4, sabinene; 5, β-pinene*; 6, myrcene; 7, α-phellandrene; 8, α-terpinene*; 9, β-phellandrene; 10, limonene*; 11, ocimene; 12, γ-terpinene*; 13, terpinolene. Compounds marked with * were identified by comparison of retention time and mass spectrum to those of authentic standards. Others were identified by database comparisons.

Figure 6

Expression of poplar root TPS genes and emission of major TPS products. Expression of TPS genes was analyzed using qRT-PCR. TPS expression and emission of monoterpenes are displayed for M. melolontha-damaged (herb) and undamaged (ctr) roots from P. trichocarpa and P. nigra. Means ± SE are shown (n = 8). Asterisks indicate statistical significance in Student’s t-tests or from Mann-Whitney Rank Sum Tests. PtTPS13 (P ≤ 0.001, T = 36.00); PtTPS21 (P = 0.209, T = 42.00); PtTPS16 (P = 0.195, T = 55.00); PnTPS4 (P ≤ 0.001, T = 36.00); PnTPS1 (P ≤ 0.001, T = 36.00); P. trichocarpa: 1.8-cineole (P = 0.048, t = 2.168); camphene (P = 0.061, t = −2.042); p-cymene (P = 0.458, t = −0.764); P. nigra: 1.8-cineole (P = 0.578, t = −0.570); camphene (P ≤ 0.001, T = 37.00).

Figure 7

The effect of volatile monoterpenes on the growth of Phytophthora cactorum (Oomycetes). Mycelial growth of P. cactorum was measured in the presence of vapor-phase 2-phenylethanol, 1,8-cineole, (−)-limonene, and (−)-β-pinene. Pathogen growth in response to the individual compounds was compared to pathogen growth when exposed to pure mineral oil. The area of P. cactorum mycelium for the control treatment was set at 100% and growth under the influence of the different volatile compounds is shown relative to the mineral oil control. Means ± SE are shown (n = 5). Asterisks indicate significant differences (Student’s t-tests. 2-phenylethanol (P = 0.452, t = −0.797); 1,8-cineole (P = 0.001, t = −6.6); (−)-limonene (P = 0.166, t = 1.524); (−)-β-pinene (P = 0.023, t = −2.792)).