Article Figures & Data
Figures
- Figure 1.
Proposed pathways for the biosynthesis of 2-phenylethanol in poplar. CYP79, cytochrome P450 family 79 enzyme; PPDC, phenylpyruvic acid decarboxylase; MAO, monoamine oxidase; TOX, transoximase; UGT, UDP-glycosyltransferase; β-Glu, β-glucosidase. Solid lines indicate well-established reactions/enzymes; dashed lines indicate reactions hypothesized in the literature.
- Figure 2.
Herbivore-damaged leaves of P. trichocarpa accumulate and release Phe-derived metabolites. A–E, The accumulation of l-Phe (A), phenylpyruvic acid (B), 2-phenylethylamine (C), and 2-phenylethyl-β-d-glucopyranoside (D), and the emission of 2-phenylethanol (E) were analyzed in L. dispar damaged (herb) and undamaged control (ctr) leaves. Plant material was extracted with methanol and analyzed via LC-MS/MS (A–D). Volatiles were collected for 8 h and analyzed via GC-FID (E). Means ± se are shown (A–D, n = 10; E, n = 9). Asterisks indicate statistical significance as assessed by Student’s t test or Mann-Whitney Rank Sum tests. l-Phe (P = 0.530, t = −0.64); phenylpyruvic acid (P = 0.772, t = −0.294); 2-phenylethylamine (P < 0.001, T = 55); 2-phenylethyl-β-d-glucopyranoside (P = 0.011, T = 71); 2-phenylethanol (P = 0.005, T = 58.5). DW, dry weight; FW, fresh weight; n.s., not significant; n.d., not detected.
- Figure 3.
Dendrogram analysis of AAAD family genes from P. trichocarpa and characterized AADC, AAS, and Glu/Ser/His-decarboxylase genes from other plants. Substrates of each clade are indicated in parentheses. The tree was inferred by using the maximum likelihood method and n = 1,000 replicates for bootstrapping. Bootstrap values are shown next to each node. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Genes described in this study are shown in red and bold with corresponding gene identifiers (italics). Accession numbers are given in Supplemental Table S1.
- Figure 4.
PtAAS1 and PtAADC1 are interconvertible by the mutation of a single amino acid and are likely derived by tandem gene duplication. A, The recombinant proteins PtAAS1, PtAADC1, and the corresponding mutant enzymes PtAAS1 (F338Y) and PtAADC1 (Y338F) were incubated with l-Phe in the presence of PLP. Enzyme products were analyzed using GC-MS (2-phenylacetaldehyde) and LC-MS/MS (2-phenylethylamine). B, PtAADC1 and PtAAS1 form a gene cluster on chromosome 13. Both genes are oriented in the same direction and there are no coding regions between them. Sequence data were retrieved from the P. trichocarpa Genome Assembly (version 3.0; www.phytozome.net; Tuskan et al., 2006).
- Figure 5.
A–E, Overexpression of PtAAS1 and PtAADC1 in N. benthamiana alters the emission of 2-phenylethanol (A) and the accumulation of 2-phenylethylamine (B), tyramine (C), tryptamine (D), and 2-phenylethanol-β-d-glucopyranoside (E). N. benthamiana plants were infiltrated with A. tumefaciens carrying either PtAAS1 or PtAADC1. At 5 d after infiltration, the headspaces of plants were collected and analyzed via GC-MS and GC-FID. Additionally, plants were harvested, extracted with methanol, and metabolite accumulation was analyzed via LC-MS/MS. Different letters above each bar indicate statistically significant differences as assessed by Kruskal-Wallis one-way analysis of variance and Tukey tests. 2-phenylethanol (H = 19.867, P ≤ 0.001); 2-phenylethylamine (H = 16.94, P ≤ 0.001); tyramine (H = 18.267, P ≤ 0.001); tryptamine (H = 18.645, P ≤ 0.001); 2-phenylethyl-β-d-glucopyranoside (H = 21.6, P ≤ 0.001). Means ± se are shown (n = 6). WT, wild type; FW, fresh weight.
- Figure 6.
Transcript accumulation of AAAD and SDC genes in L. dispar-damaged and undamaged P. trichocarpa leaves. Gene expression in herbivore-damaged (herb) and undamaged (ctr) leaves was analyzed by Illumina sequencing and mapping the reads to the transcripts of the P. trichocarpa Genome Assembly (version 3.0; www.phytozome.net; Tuskan et al., 2006). Expression was normalized to reads per kilo base per million mapped reads (RPKM). Significant differences in EDGE tests are visualized by asterisks. Means ± se are shown (n = 4). PtAAS1 (P = 8.23213E-12, weighted difference [WD] = 4.86618E-05); PtAAS2 (P = 0.039452261, WD = −1.11092E-05); PtAADC1 (P = 8.99025E-09, WD = 1.56932E-05); PtAADC2 (P = 0.031269756, WD = 2.02404E-06); PtAADC3 (P = 1, WD = −4.35895E-08); PtSDC1 (P = 0.576261262, WD = 1.61294E-06); PtSDC2 (P = 0.015936012, WD = 1.08813E-05).
- Figure 7.
RNAi-mediated knockdown of AADC1 in P. × canescens. Sterile P. × canescens calli were transformed via A. tumefaciens. Nontransgenic trees (WT, wild type), empty vector lines (EV-2-4), and RNAi lines (RNAi-1-4) were subjected to L. dispar feeding. Volatile 2-phenylethanol was collected from the headspace and analyzed using GC-MS/FID. Phenylethylamine and 2-phenylethyl-β-d-glucopyranoside were extracted with methanol from ground plant material and analyzed via LC-MS/MS. Biological replicates (nb) and technical replicates (nt) of EV lines and RNAi lines were used to test for statistical differences. Wild type, nb = 4; EV, nb = 3, nt = 5; RNAi, nb = 4, nt = 5. Asterisks indicate statistical significance as assessed by Student’s t test or Mann-Whitney Rank Sum tests. Phenylethylamine (P < 0.001, t = 7.940); 2-phenylethyl-β-d-glucopyranoside (P = 0.011, T = 547); 2-phenylethanol (P = 0.509, T = 404). Medians ± quartiles and outliers are shown. Each data point is represented by a circle. FW, fresh weight; n.s., not significant.
Tables
- Table 1. Kinetic parameters of P. trichocarpa PtAAS1, PtAAS2, PtAADC1, PtAADC2, and PtAADC3
Enzymes were heterologously expressed in E. coli, purified, and incubated with Phe, Tyr, or Trp. Kinetic parameters are shown as means ± se (n = 3). Volatile phenylacetaldehyde was analyzed via TDU-GC-MS; all other compounds were measured using LC-MS/MS. PHA, phenylacetaldehyde; PEA, 2-phenylethylamine; 4-OH-PHA, 4-hydroxy phenylacetaldehyde; IAAld, indole-3-acetaldehyde; TyrA, tyramine; TrpA, tryptamine.
Enzyme Substrate Product Km (mm) Vmax (nmola µg−1a min−1) kcat (s−1) kcat /Km (s−1 mm−1) PtAAS1 Phe PHA 0.46 ± 0.02 — — — Phe PEA — — — — Tyr 4-OH-PHA — — — — Trp IAAld — — — — PtAAS2 Phe PHA 0.69 ± 0.39 — — — Phe PEA — — — — Tyr 4-OH-PHAa detected — — — Trp IAAlda detected — — — PtAADC1 Phe PHA — — — — Phe PEA 0.17 ± 0.03 430 ± 12 0.39 2.3 Tyr TyrA 2.0 ± 0.2 85.4 ± 3.9 0.08 0.04 Trp TrpA 3.0 ± 0.4 7.7 ± 0.5 0.007 0.002 PtAADC2 Phe PHA — — — — Phe PEA 0.19 ± 0.01 0.89 ± 0.01 0.81 4.2 Tyr TyrA 1.2 ± 0.2 148 ± 7 0.14 0.11 Trp TrpA — — — — PtAADC3 Phe PHA — — — — Phe PEA 2.3 ± 0.2 6.7 ± 0.2 0.14 0.003 Tyr TyrA 0.043 ± 0.004 3.61 ± 0.06 0.003 0.08 Trp TrpA — — — — ↵a Compounds were chemically converted into the corresponding aldoximes before LC-MS/MS analysis (see “Materials and Methods”).
- Table 2. Enzymatic activity of poplar aldehyde reductases
Enzymes were heterologously expressed in E. coli, purified, and tested with different aldehydes as potential substrates in the presence of NADPH. Enzyme products were extracted with hexane and analyzed using GC-MS. Symbols: −, no activity detected; + activity detected.
Substrate Product PtPAR1 PtPAR2 PtAAR1 PtAAR2 PtAAR3 Citronellal Citronellol + + + + + Geranial Geraniol + + − − − Nonanal Nonanol + + + − − Decanal Decanol + + + − + Hexanal Hexanol + + − − − (E)-2-hexenal (E)-2-hexenol + + − − − Benzaldehyde Benzyl alcohol + + − − − 2-phenylacetaldehyde 2-phenylethanol + + − − −
Supplemental Data
Supplemental Figures and Tables
Files in this Data Supplement:
- Supplemental Data - Supplemental Figures 1-14 and Supplemental Tables 1-6
