In planta variation of volatile biosynthesis: an alternative biosynthetic route to the formation of the pathogen-induced volatile homoterpene DMNT via triterpene degradation in Arabidopsis roots

Abstract

Plant-derived volatile compounds such as terpenes exhibit substantial structural variation and serve multiple ecological functions. Despite their structural diversity, volatile terpenes are generally produced from a small number of core 5- to 20-carbon intermediates. Here, we present unexpected plasticity in volatile terpene biosynthesis by showing that irregular homo/norterpenes can arise from different biosynthetic routes in a tissue specific manner. While Arabidopsis thaliana and other angiosperms are known to produce the homoterpene (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) or its C16-analog (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene by the breakdown of sesquiterpene and diterpene tertiary alcohols in aboveground tissues, we demonstrate that Arabidopsis roots biosynthesize DMNT by the degradation of the C30 triterpene diol, arabidiol. The reaction is catalyzed by the Brassicaceae-specific cytochrome P450 monooxygenase CYP705A1 and is transiently induced in a jasmonate-dependent manner by infection with the root-rot pathogen Pythium irregulare. CYP705A1 clusters with the arabidiol synthase gene ABDS, and both genes are coexpressed constitutively in the root stele and meristematic tissue. We further provide in vitro and in vivo evidence for the role of the DMNT biosynthetic pathway in resistance against P. irregulare. Our results show biosynthetic plasticity in DMNT biosynthesis in land plants via the assembly of triterpene gene clusters and present biochemical and genetic evidence for volatile compound formation via triterpene degradation in plants.

Figure 1.

DMNT Is Emitted from Arabidopsis Roots upon Infection with P. irregulare or Treatment with Jasmonic Acid. (A) Axenically grown Arabidopsis roots were infected with a suspension of P. irregulare mycelium and oospores, a P. irregulare filtrate, or with a suspension of E. coli. The structure of DMNT is shown. (B) Treatment of axenically grown roots with 100 μM JA. Root volatiles were analyzed from detached roots at different time points by SPME-GC/MS. Normalized peak areas are shown. Values represent the mean ± se of three biological replicates. Different letters show significant differences based on two-way ANOVA and Tukey-Kramer HSD test; P < 0.001. The experiment was repeated at least two times with similar results.

Figure 2.

Selection of DMNT Synthase Candidate Genes. (A) GC/MS chromatograms of induced emissions of DMNT from roots of the Arabidopsis accessions Ler, Cvi, and Col-0 in response to 24 h of JA treatment. No DMNT was detected from the Cvi accession, while the volatile was produced by roots of Col-0 and Ler. (B) Identification of the QTL region for DMNT formation in the Ler × Cvi RILs on chromosome 4. The y axis is in log of the odds (LOD) units, and the x axis is in centimorgans (cM); the horizontal lines represent the 0.05 significance threshold determined by 1000 permutations. (C) A screening was conducted of publically available microarray data sets using Genevestigator for all Arabidopsis P450 genes expressed upon treatment with methyl jasmonate (MeJA) or wounding assuming that the expression of the target P450 gene would be induced in roots under these conditions. Expression of selected P450 candidate genes upon wounding and methyl jasmonate treatments is shown (At4g13290 and At4g13310 share the same probe number). (D) Volatile analysis of roots of coi1-1, its corresponding background genotype Col-6 (gl1), and wild-type Col-0. No DMNT was detected in hydroponically grown coi1-1 plants upon JA treatment. Values represent the mean ± se of three replicates. (E) RT-PCR analysis of candidate gene expression in coi1-1 and wild-type Col-0 plants under JA and mock (ethanol) treatment. JA-inducible and coi1-1-regulated genes are marked with asterisks. Candidate genes on the selected QTL region on chromosome 4 are underlined. Only the CYP705A1 locus overlaps with the DMNT QTL and shows JA-inducible and coi1-1-dependent expression.

Figure 3.

Identification of CYP705A1 as a DMNT Synthase. (A) Schematic showing the genomic locus of Arabidopsis CYP705A1 (At4g15330) in tandem with the ABDS gene. Exons are represented by gray boxes. Introns and intergenic regions are represented by the black line. Insertion sites of the T-DNA mutants used in this study are marked with inverted triangles. (B) DMNT emission in roots of wild-type and cyp705a1 mutants after 24 h of JA treatment. The retention time for the DMNT authentic standard (indicated by the arrow) is marked with a dashed line. (C) DMNT emission in mock- and JA-treated plants in wild-type background compared with representative transgenic lines. Volatiles were collected from roots after 24 h of JA treatment and analyzed by SPME-GC/MS. Mock controls were treated with ethanol. Normalized peak areas are shown and the values represent the mean ± se of three biological replicates. Different letters show significant differences based on two-way ANOVA and Tukey-Kramer HSD test, P < 0.001. N.D. indicates that no volatile was detected.

Figure 4.

Arabidiol Is the Substrate for DMNT Biosynthesis. (A) Structure of arabidiol. (B) DMNT emission in yeast and Arabidopsis plants. DMNT was detected from WAT11 yeast cells coexpressing ABDS with a wild-type CYP705A1 cDNA. In yeast cells expressing ABDS alone or coexpressing ABDS with a mutated version of CYP705A1 (mut-CYP705A1), no DMNT was observed. The yeast control line was transformed with only the empty vector used for expression of CYP705A1. Volatile analysis of Col-0 roots and abds mutants after 24 h of 100 μM JA treatment is shown. No DMNT was detected from abds mutants. Volatile products were analyzed in the yeast culture or plant tissue headspace by SPME-GC/MS. The retention time for DMNT is marked with a dashed line. (C) Arabidiol detection from Arabidopsis roots. GC/MS chromatograms of liquid extracts from 1 g JA-treated roots of wild-type Col-0, abds-1, and cyp705a1-1 are depicted. Arabidiol (indicated by arrows) was only detected in the cyp705a1-1 mutant after JA treatment as shown in TIC (total ion chromatogram) and in single ion monitoring (SIM) mode for m/z 247. (D) DMNT emission from roots of wild-type Col-0 and three Pro35S-ABDS overexpression lines in two different abds mutant backgrounds treated with JA for 24 h. Normalized peak areas are shown. Values represent the mean ± se of three biological replicates. Different letters show significant differences based on two-way ANOVA and Tukey-Kramer HSD test, P < 0.001. N.D. indicates that no volatile was detected. (E) Microsomal preparations expressing CYP705A1 converted arabidiol to DMNT (indicated by the arrow) in the presence of 2.4 mM of the P450 cofactor NADPH.

Figure 5.

Detection of 14-Apo-Arabidiol in the Yeast Coexpression System. (A) The C₁₉ degradation product 14-apo-arabidiol was detected (indicated by arrows) in WAT11 yeast cells expressing ABDS and CYP705A1 but not mut-CYP705A1. The GC chromatogram of the purified degradation product is depicted. TIC, total ion chromatogram; SIM, single ion monitoring. (B) MS spectrum of 14-apo-arabidiol with a m/z 292 molecular ion. (C) The pathway for arabidiol degradation to DMNT and 14-apo-arabidiol. The molecular structure of 14-apo-arabidiol was determined by NMR analysis.

Figure 6.

Expression of CYP705A1 Is Localized to the Root Stele and Meristematic Zone and Responds to Treatment with JA. (A) to (J) GUS activity in 12-d-old mock and JA-treated ProCYP705A1-GUS transgenic lines. (A) Whole seedling. Bar = 5 mm. (B) and (G) Cotyledon and true leaves. Bars = 1 mm. (C) and (H) Main root tip. Bars = 200 μm. (D) and (I) Lateral root tip. Bars = 200 μm. (E), (F), and (J) Lateral, main root attachment site. Bars = 0.5 mm. (K) to (N) Confocal microscopy analysis of roots of 12-d-old mock and JA-treated ProCYP705A1:CYP705A1-eYFP plants. Mock-treated roots show localization of the CYP705A1-eYFP protein in the quiescent center in the root meristematic zone (K) and in the pericycle in the root hair zone (L). A localized induction of protein after JA treatment is observed ([M] and [N]). Results are representative for at least three independent transgenic lines. Bars = 20 μm.

Figure 7.

DMNT Negatively Effects P. irregulare Oospore Germination and Formation. (A) Effect of DMNT on oospore germination of P. irregulare. DMNT was applied at different concentrations in 10 mL of maize meal agar containing streptomycin. Oospore suspensions were added into each plate and incubated at 27°C in the dark. Germination rates were determined 24 h after inoculation. Thirty percent of the oospores germinated in the control treatment. The results were plotted relative to distilled water (DW), and oospore germination rate for distilled water was arbitrary set to 1. (B) Root infection assay of P. irregulare in the wild type, DMNT biosynthetic mutants (abds-1, abds-2, and cyp705a1-1), and the control line cyp705a1-2. Representative root segments were taken 3 weeks after infection of soil-grown plants with the pathogen to measure oospore formation. Oospores were counted ∼10 mm behind the root tips. Statistical analysis was done using one-way ANOVA and Tukey-Kramer HSD test. The values represent the mean ± se of five replicates (P < 0.01) for (A) and at least six root segments from three different plants (P < 0.05) for (B). Different letters above the bars show significant differences. The experiments were repeated at least twice with similar results.

Figure 8.

Docked Conformation of Arabidiol in the Active Site of Modeled CYP705A1. Docking was performed using AutoDock-Vina. The structure of the arabidiol molecule to be docked into the active site of modeled CYP705A1 was taken form ZINC database (ZINC 59211647). The most energetically favorable orientation of arabidiol with −8.9 Kcal/mol binding affinity is shown. The side chains of residues in 5 Å of substrate are illustrated. The main chain of Thr-213 forms a H-bond to C₃-OH of arabidiol. The figure was prepared with PyMol.

Figure 9.

The Proposed Mechanism for Oxidative Degradation of Arabidiol by CYP705A1. The coordination of arabidiol in the active site based on docking experiments is shown. Radical attack by [Fe(III)-O-O]^⋅, hydrogen abstraction, and subsequent internal atomic rearrangement are proposed for the C-C bond breakage of arabidiol to form 14-apo-arabidiol and DMNT.

Figure 10.

Comparative Genome Analysis Maps of the ABDS Gene Cluster. Synteny map for ABDS cluster regions between C. rubella, A. lyrata, and A. thaliana is shown. No triterpene (oxidosqualene) cyclase (OSC) gene ortholog was found on a highly syntenic region in C. rubella. One OSC and two CYP705 P450 members were present in A. lyrata. In A. thaliana, two OSC and four CYP705 members were found. A gene duplication event followed by inversion of the CYP705 gene is likely to be the source of DMNT biosynthetic gene evolution. Different gene families are color coded. Syntenic regions flanking the ABDS gene cluster are connected with gray lines. The syntenic regions for the DMNT biosynthetic genes are shown with cyan lines. ACT, acyltransferase; CSL, cellulose synthase-like; syntenic neighboring genes are colored green.