Characterization of Biosynthetic Pathways for the Production of the Volatile Homoterpenes DMNT and TMTT in Zea mays

Abstract

Plant volatiles not only have multiple defense functions against herbivores, fungi, and bacteria, but also have been implicated in signaling within the plant and toward other organisms. Elucidating the function of individual plant volatiles will require more knowledge of their biosynthesis and regulation in response to external stimuli. By exploiting the variation of herbivore-induced volatiles among 26 maize (Zea mays) inbred lines, we conducted a nested association mapping and genome-wide association study (GWAS) to identify a set of quantitative trait loci (QTLs) for investigating the pathways of volatile terpene production. The most significant identified QTL affects the emission of (E)-nerolidol, linalool, and the two homoterpenes (E)-3,8-dimethyl-1,4,7-nonatriene (DMNT) and (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT). GWAS associated a single nucleotide polymorphism in the promoter of the gene encoding the terpene synthase TPS2 with this QTL Biochemical characterization of TPS2 verified that this plastid-localized enzyme forms linalool, (E)-nerolidol, and (E,E)-geranyllinalool. The subsequent conversion of (E)-nerolidol into DMNT maps to a P450 monooxygenase, CYP92C5, which is capable of converting nerolidol into DMNT by oxidative degradation. A QTL influencing TMTT accumulation corresponds to a similar monooxygenase, CYP92C6, which is specific for the conversion of (E,E)-geranyllinalool to TMTT The DMNT biosynthetic pathway and both monooxygenases are distinct from those previously characterized for DMNT and TMTT synthesis in Arabidopsis thaliana, suggesting independent evolution of these enzymatic activities.

Figure 1.

Diversity of Volatile Terpene Production among the 26 Parent Lines of the NAM Population after Elicitor Induction. Mono-, sesqui-, and diterpenes were extracted with hexane from each of 8 to 10 individual plants and were then analyzed by GC-MS as described in Methods. The diversity of the most common volatile terpenes is shown.

Figure 2.

Mapping of QTLs Involved in Plant Volatile Production. (A) The production of linalool (diamonds), (E)-nerolidol (squares), DMNT (triangles), TMTT (circles), and the ratio of DMNT to (E)-nerolidol (rectangle) were determined across a set of 5982 RILs of the NAM population and mapped against 1106 common parent-specific markers. The cumulative chromosome positions of the major QTLs are indicated on the x axis. Prominent QTLs are highlighted with boxes and are described in Results. (B) Suggested functions of the QTLs in the proposed biosynthetic pathways of these compounds. QTL₆₀₆ is associated with a terpene synthase activity able to convert GDP, FDP, and GGDP to the respective tertiary alcohols. QTL₂₉₈ and QTL₇₆₄ are associated with an oxidase activity that forms DMNT and TMTT, respectively, depending on the substrate.

Figure 3.

Mapping of QTL₆₀₆ for Nerolidol Production in a GWAS Using 1.6 Million SNP Markers. (A) The bar graph presents the counts of iterations by GWAS for each of the SNPs located near QTL₆₀₆. The counts of iterations are shown above the most prominent bars. The SNP with the most significant correlation to QTL₆₀₆ (indicated by a black bar) is positioned in the promoter region of tps2 (arrow). Shown above is the pattern of exons (boxes) and introns (lines) of tps2 with the number of nucleotides from the translation start. (B) Location of tps2, tps3, and the most relevant QTLs in a section of chromosome 5 (wide gray line). A close-up of this region (thin gray line) shows the position of SNP70571650 in relation to tps2 and tps3. For both tps2 and tps3, the respective pattern of exons (black boxes) and introns (black lines) is pictured below.

Figure 4.

The Terpene Synthase TPS2 Encodes an Active Enzyme in the Inbred Line B73. TPS2 and TPS3 were produced in E. coli, extracted, partially purified, and incubated with the substrates. The resulting terpene products were collected with a polydimethylsiloxane-coated SPME fiber and analyzed by GC-MS. The terpene synthase TPS2 produces the tertiary alcohols linalool from GDP (A), nerolidol from FDP (B), and geranyllinalool from GGDP (C). TPS3 formed no detectable products with any tested substrate. Products were identified by GC-MS. The products were identified by comparison of their retention times and mass spectra with those of authentic standards.

Figure 5.

Expression of the Terpene Synthases TPS2 and TPS3 Is Induced by Simulated Herbivory. Leaves of 14-d-old plants of the inbred line B73 were wounded and treated with indanone derivative (elicitor) for 24 h or left undamaged (control). Levels of transcripts encoding TPS2 and TPS3 were determined relative to the APT1 reference gene. One-way ANOVA was used to calculate the significance based on three plants per treatment. Error bars represent se. Triple asterisks represent significant difference from the control at P < 0.01.

Figure 6.

Terpene Synthase TPS2 Is Localized in the Chloroplasts of Maize Mesophyll Protoplasts. Images of maize mesophyll protoplasts transiently expressing 35S-YFP ([A] to [C]) or 35S-TPS2-YFP ([D] to [F]) showing YFP fluorescence ([A] and [D]), chlorophyll autofluorescence ([B] and [E]), and merged images ([C] and [F]). Bars = 20 µm.

Figure 7.

TPS2 Is Inactive in the Inbred Line Tzi8. (A) Volatiles found in leaves of the inbred lines B73 and Tzi8 after induction with indanone elicitor. The volatiles were analyzed by GC-MS. The major terpene compounds were identified as 1-linalool, 2-DMNT, 3-(E)-nerolidol, 4-TMTT, 5-(E)-α-bergamotene, and 6-(E)-β-farnesene. (B) TPS2 alleles from B73 and Tzi8 were expressed in E. coli, and protein was extracted, partially purified, and incubated with GDP/FDP/GGDP substrates. The resulting terpene products were collected with a polydimethylsiloxane-coated SPME fiber and analyzed by GC-MS. (C) Levels of transcripts encoding TPS2 in leaves of the lines Tzi8 and B73. Leaves of 14-d-old plants were wounded and treated with indanone derivative (Elicitor) for 24 h or left undamaged (Control). Transcript abundance was determined relative to the reference gene encoding APT1. One-way ANOVA was used to calculate the significance of three plants per treatment. Error bars represent se. Different letters denote significant differences at P < 0.01.

Figure 8.

TPS2 Products Were Not Detected in a Maize tps2/3::DS Transposon Insertion Line. Volatiles found in of leaves after wounding and treatment with indanone derivative (elicitor) of the inbred line W22 and a line with a tps2/3::DS transposon insertion in the W22 background. The volatiles were collected with a polydimethylsiloxane-coated SPME fiber and analyzed by GC-MS. Several terpene compounds were identified as 1-linalool, 2-DMNT, 3-(E)-nerolidol, and 4-TMTT.

Figure 9.

The P450 Monooxygenases CYP92C5 and CYP92C6 Convert Sesquiterpene Alcohols into Their Respective Homoterpenes. CYP92C5 and CYP92C6 of B73 were expressed in yeast, extracted, partially purified, and incubated in the presence and absence of the substrates nerolidol (A), geranyllinalool (B), and the cosubstrate NADPH. The resulting terpene products were collected with a polydimethylsiloxane-coated SPME fiber and analyzed by GC-MS. The DMNT and TMTT products were identified by comparison of retention time and mass spectrum with those of authentic standards.

Figure 10.

Levels of Transcripts Encoding the P450 Monooxygenases CYP92C5 and CYP92C6 Are Induced by Simulated Herbivory. Leaves of the inbred line B73 were wounded and treated with an indanone derivative elicitor (Elicitor) for 24 h or left undamaged (Control). Levels of transcripts encoding CYP92C5 (A) and CYP92C6 (B) were determined relative to the APT1 reference gene. One-way ANOVA was used to calculate the significance of three plants per treatment. Error bars represent se. Asterisks represent significant difference from the control at P < 0.01.

Figure 11.

CYP92C5 Is Inactive in the Transposon Insertion Mutant MuIII. (A) Volatiles found in of leaves of the lines B73 and transposon insertion mutant MuIII after induction with the indanone elicitor. The volatiles were collected with a polydimethylsiloxane-coated SPME fiber and analyzed by GC-MS. The major terpene compounds that were identified are 1-DMNT, 2-(E)-nerolidol, and 3-TMTT. (B) Levels of transcripts encoding CYP92C5 in leaves of the lines B73 and MuIII. Leaves of 14-d-old plants were wounded and treated with indanone derivate for 24 h. Transcript abundance was determined relative to the APT1 reference gene. One-way ANOVA was used to calculate the significance of three plants per treatment. Error bars represent se.

Figure 12.

Dendrogram Analysis of Maize TPS2 and TPS3 with Closely Related Linalool/Nerolidol/Geranyllinalool Synthases from Other Plant Species. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis includes TPS2 (NP_001105854.1) and TPS3 (NP_001105855.1), shown in bold; PtTPS3 (XM_006377207.1); MtTPS3 (AY766249); VvRiLinNer (JQ062931); FaNES1 (CAD57083); PtTPS15 (KF776502); PlTPS2 (KC012520); AtLIS (AAO85533); AmNES/LIS-1 (EF433761); AmNES/LIS-2 (ABR24418); Os02g02930 (Q6ZH94); ZmTPS23 (EU259634.1); ZmTPS10 (NM_001112380.2); AtTPS04 (AA085540); NaGLS (KJ755868); AtKS (NP_178064); and ZmTPS1 (NM_001111627.1). The FPP synthase ZmFPPS3 (BT085025) was used as an outgroup. Products of each terpene synthase are indicated by letters for linalool (L), (E)-nerolidol (N), (E,E)-geranyllinalool (GL), others (X), or inactivity (–).

Figure 13.

Overview of DMNT and TMTT Synthesis in Maize. The first steps of the pathways for the synthesis of the maize homoterpenes DMNT and TMTT are catalyzed by the terpene synthase TPS2, which converts GDP, FDP, and GGDP to the corresponding tertiary alcohols. The second steps, which are the oxidative degradations, are catalyzed by two P450 monooxygenases, CYP92C5 and CYP92C6. OPP, diphosphate.

Figure 14.

Dendrogram Analysis of Maize CYP92C5 and CYP92C6 with Closely Related P450 Monooxygenases from Other Plant Species. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The P450 monooxygenase CYP710A1 was used as an outgroup. Underlined genes were shown to encode proteins producing homoterpenes upon in vitro expression. The analysis includes CYP92C5 (ACG28049), CYP92C6 (GRMZM2G139467), and CYP82G1 (AT3G25180.2), which are underlined. It also includes CYP92A2v4 (LOC107803700), CYP92A (ABG74350.1), CYP75B1 (AT5G07990), CYP81D1 (AT3G28740.1), CYP82C2 (AT4G31970.1), CYP82A1 (AAG09208.1), CYP705A1 (AT4G15330), CYP79D6 (AHF20913.1), and CYP710A1 (AT2G34500.1).