A peroxisomal β-oxidative pathway contributes to the formation of C6-C1 aromatic volatiles in poplar

Abstract

Benzenoids (C6-C1 aromatic compounds) play important roles in plant defense and are often produced upon herbivory. Black cottonwood (Populus trichocarpa) produces a variety of volatile and nonvolatile benzenoids involved in various defense responses. However, their biosynthesis in poplar is mainly unresolved. We showed feeding of the poplar leaf beetle (Chrysomela populi) on P. trichocarpa leaves led to increased emission of the benzenoid volatiles benzaldehyde, benzylalcohol, and benzyl benzoate. The accumulation of salicinoids, a group of nonvolatile phenolic defense glycosides composed in part of benzenoid units, was hardly affected by beetle herbivory. In planta labeling experiments revealed that volatile and nonvolatile poplar benzenoids are produced from cinnamic acid (C6-C3). The biosynthesis of C6-C1 aromatic compounds from cinnamic acid has been described in petunia (Petunia hybrida) flowers where the pathway includes a peroxisomal-localized chain shortening sequence, involving cinnamate-CoA ligase (CNL), cinnamoyl-CoA hydratase/dehydrogenase (CHD), and 3-ketoacyl-CoA thiolase (KAT). Sequence and phylogenetic analysis enabled the identification of small CNL, CHD, and KAT gene families in P. trichocarpa. Heterologous expression of the candidate genes in Escherichia coli and characterization of purified proteins in vitro revealed enzymatic activities similar to those described in petunia flowers. RNA interference-mediated knockdown of the CNL subfamily in gray poplar (Populus x canescens) resulted in decreased emission of C6-C1 aromatic volatiles upon herbivory, while constitutively accumulating salicinoids were not affected. This indicates the peroxisomal β-oxidative pathway participates in the formation of volatile benzenoids. The chain shortening steps for salicinoids, however, likely employ an alternative pathway.

Figure 1

Volatiles emitted from undamaged (ctr) and Chrysomela populi-damaged (herb) P. trichocarpa leaves. Volatile profiles of control and herbivore-damaged P. trichocarpa leaves (A), and enlargements of the volatile profiles (B) measured and analyzed using GC-MS, and quantification of benzenoid volatiles (C). Means and se are shown (n = 8). 1, (E)-3-methylbutyraldoxime; 2, (E)-2-methylbutyraldoxime; 3, (Z)-2-methylbutyraldoxime; 4, 1-hexanol; 5, (Z)-3-methylbutyraldoxime; 6, α-pinene^#; 7, benzaldehyde^#; 8, sabinene; 9, β-pinene^#; 10, p-cymene; 11, limonene^#; 12, 1,8 cineole; 13, benzylalcohol^#; 14, (Z)-β-ocimene; 15, salicylaldehyde^#; 16, (E)-β-ocimene^#; 17, 2-phenylethanol^#; 18, (E)-4,8-dimethylnona-1,3,7-triene; 19, benzyl cyanide; 20, α-terpineol^#; 21, methyl salicylate^#; 22, (E)-phenylacetaldoxime; 23, salicylalcohol; 24, (Z)-phenylacetaldoxime; 25, indole; 26, 2-phenylnitroethane; IS, internal standard (nonyl acetate); 27, eugenol; 28, α-copaene; 29, calarene; 30, β-cubebene; 31, isoamyl benzoate; 32, α-amorphene; 33, (Z,E)-α-farnesene; 34, (E,E)-α-farnesene; 35, γ-cadinene; 36, δ-cadinene; 37, cis-3-hexenyl benzoate^#; 38, (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene/hexenyl benzoate; 39, cadinol; 40, benzyl benzoate^#. Compounds marked with^# were identified by comparison of retention time and mass spectrum to those of authentic standards. Other compounds were identified by database comparisons. fw, fresh weight; TIC, total ion chromatogram. Asterisks indicate statistical significance as assed by Student's t test (ST) or Mann-Whitney Rank Sum Test (MW) (*P < 0.05; **P < 0.01; ***P < 0.001). benzaldehyde (P < 0.001, T = 36.000, MW); benzylalcohol (P < 0.001, T = 36.000, MW); salicylaldehyde (P = 0.007, T = 43.000, MW); methyl salicylate (P = 0.031, t = −2.398, ST); benzyl benzoate (P = 0.036, t = −2.315, ST).

Figure 2

Benzenoid volatiles emitted from Chrysomela populi-damaged P. trichocarpa leaves (herb) incubated with water (H₂O) or D₇-cinnamic acid (D₇-CA). Volatiles were measured and analyzed using GC-MS. Volatile profiles (A and C) and mass spectra (B and D) of unlabeled and deuterium-labeled benzenoid volatiles are displayed. 1, D₅-benzaldehyde; 2, benzaldehyde^#; 3, D₅-benzylalcohol; 4, benzylalcohol^#; 5, D₄-salicylaldehyde; 6, salicylaldehyde^#; 7, D₅-cis-3-hexenyl benzoate; 8, cis-3-hexenyl benzoate^#; 9, D₅-hexenyl benzoate; 10, hexenyl benzoate; 11, D₁₀-benzyl benzoate; 12, benzyl benzoate^#. Compounds marked with ^# were identified by comparison of retention time and mass spectrum to those of authentic standards. Other compounds were identified by database comparisons. TIC, total ion chromatogram.

Figure 3

Enzymatic activity of Populus trichocarpa cinnamate-CoA ligase 4 (PtCNL4), cinnamoyl-CoA hydratase/dehydrogenase 1 (PtCHD1), and 3-ketoacyl-CoA thiolase 1 (PtKAT1). PtCNL4 (A), PtCHD1 (B), and PtKAT1 (C) were heterologously expressed in E. coli as His₆-tag fusion proteins and recombinant proteins were purified using affinity chromatography. Purified proteins were incubated with the potential substrates trans-cinnamic acid (PtCNL4), cinnamoyl-CoA (PtCHD1), cinnamoyl-CoA (+ PtCHD1; PtKAT1) and the respective cosubstrates ATP, CoA (PtCNL4); NAD⁺ (PtCHD1); CoA, NAD⁺ (PtKAT1). Reaction products were analyzed using LC–MS/MS. cps, counts per second.

Figure 4

Expression of putative poplar CNL, CHD, and KAT genes in Chrysomela populi-damaged (herb) and undamaged (ctr) Populus trichocarpa leaves. Absolute normalized expression values were obtained from transcriptomes (n = 4 biological replicates). RPKM, reads per kilo base per million mapped reads. Significant differences in EDGE tests are visualized by asterisks. Means ± se are shown. PtCNL1 (P = 2.38E−07, WD = 1.71E−05); PtCNL3 (P = 1.0, WD = 1.2E−07); PtCNL4 (P = 1.53E−11, WD = 1.31E−04); PtCNL5 (P = 1.0, WD = −1.36E−08); PtCNL6 (P = 2.5E−03, WD = 6.5E−06); PtCNL7 (P = 1.0, WD = 1.53E−07); PtCHD1 (P = 1.33E−04, WD = 1.30E−04); PtCHD2 (P = 1.60E−01, WD = −1.47E−05); PtCHD3 (P = 3.04E−11, WD = 3.89E−05); PtKAT1 (P = 2.76E−11, WD = 7.94E−05); PtKAT2 (P = 1.88E−01, WD = 4.38E−05); PtKAT3 (P = 1.0, WD = −3.67E−07).

Figure 5

Effect of RNAi-mediated knockdown of the CNL1 and 4 in Populus x canescens on the emission of aromatic compounds. Transcript accumulation of P. x canescens CNL4 (A), and the relative emission of benzaldehyde (B), benzylalcohol (C), salicylaldehyde (D), benzyl benzoate (E), and methyl salicylate (F) of Chrysomela populi-damaged wild-type (WT) and transgenic P. x canescens trees are shown. Gene expression was measured using RT-qPCR, with ubiquitin (Ramírez-Carvajal et al., 2008) as housekeeping gene. Volatiles were analyzed using GC-MS. Medians ± quartiles, and outliers are shown (n = 4 biological replicates). EV, empty vector; ns, not significant. Asterisks indicate statistical significance as assed by Student's t test (ST); *P < 0.05; **P < 0.01). PcCNL4 (P = 0.045, t = −2.519, ST); benzaldehyde (P = 0.002, t = −5.491, ST); benzylalcohol (P = 0.007, t = −4.080, ST); salicylaldehyde (P = 0.492, t = −0.731, ST); benzyl benzoate (P = 0.002, t = −5.123, ST); methyl salicylate (P = 0.020, t = −3.131, ST).

Figure 6

Effect of RNAi-mediated knockdown of the CNL1 and 4 in Populus x canescens on the formation of salicinoids. Concentration of salicin (A), salicortin (B), tremulacin (C), salirepin (D), salicin-7-sulfate (E), and salirepin-7-sulfate (F) in leaves of Chrysomela populi-damaged wild-type (WT) and transgenic P. x canescens trees are shown. Compounds were extracted with methanol from freeze-dried leaf material and analyzed using LC–MS/MS and HPLC-UV. Medians ± quartiles, and outliers are shown (n = 4 biological replicates). EV, empty vector; dw, dry weight; ns, not significant. Differences between WT/EV and RNAi knockdown lines were analyzed by Student's t test (ST). Salicin (P = 0.111, t = −1.865, ST); salicortin (P = 0.385, t = −0.936, ST); tremulacin (P = 0.168, t = −1.566, ST); salirepin (P = 0.098, t = 1.961, ST); salicin-7-sulfate (P = 0.242, t = 1.298, ST); salirepin-7-sulfate (P = 0.131, t = 1.748, ST).

Figure 7

Schematic overview of benzenoid (C₆–C₁) biosynthetic pathways in plants. Displayed are the peroxisomal β-oxidative pathway investigated and two proposed nonoxidative cytosolic pathways in the biosynthesis of benzenoid compounds from the common precursor cinnamic acid. Already known enzymatic steps from plants are displayed with solid arrows, unknown enzymatic steps with dashed arrows. Enzymes investigated in this study are highlighted in red, already characterized enzymes from other plants are displayed in black. BALDH, benzaldehyde dehydrogenase; BEBT, benzoyl-CoA:benzyl alcohol O-benzoyltransferase; BSMT, benzoic acid/salicylic acid methyltransferase; 4CL, 4-coumarate CoA-ligase; CoA, Coenzyme A; 3H3PP, 3-hydroxy-3-phenylpropionic acid; 3H3PP-CoA, 3-hydroxy-3-phenylpropanoyl-CoA; 3O3PP, 3-oxo-3-phenylpropanoyl-CoA; PAL, phenylalanine ammonia lyase; TE, thioesterase.