Synthesis of amino acid esters of menthol (ment-aa)
All commercially available reagents and solvents were used without further purification. Reactions were monitored by thin-layer chromatography (TLC) carried out on silica gel-coated aluminum sheets 60F254 (Merck KGaA, Darmstadt, Germany). Spots on the TLC sheet were visualized using anisaldehyde sulfuric acid and ninhydrin reagents. Flash chromatography was performed on Wakogel C-200 (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). 1H NMR spectra were recorded on an Avance DRX-600 or Avance NEO 400 spectrometer (Bruker, Billerica, MA) at 298 K. Chemical shifts (δ) were reported in parts-per-million (ppm) with respect to tetramethylsilane as internal reference (δ = 0.00 ppm in CDCl3). Signal splitting patterns are described as singlet (s), doublet (d), triplet (t), multiplet (m), and broad signal (br). The assignment of 1H resonance was achieved by a combined employment of 1D and 2D (COSY, HSQC, and HMBC) techniques.
The amino acid esters of menthol were synthesized based on a previous report (Harada et al. 1964). One equivalent mol of amino acid (glycine, L-alanine, L-valine, D-valine, L-leucine, L-isoleucine, or L-phenylalanine; FUJIFILM Wako Pure Chemical Corporation), l-menthol (1.5 mol eq., FUJIFILM Wako Pure Chemical Corporation), and p-toluenesulfonic acid monohydrate (1.3 mol eq., FUJIFILM Wako Pure Chemical Corporation) were suspended in toluene and refluxed with a Dean-Stark apparatus for 24–72 hr. The reaction mixture was diluted with toluene, washed with 1 M NaOH aq. and brine. The organic phase was dried (Na2SO4) and concentrated in vacuo. The resulting oil was purified by flush column chromatography to obtain the amino acid ester of menthol.
Glycine menthyl ester (ment-Gly): 1H NMR (600 MHz, CDCl3): δ 4.74 (ddd, J = 4.1, 10.8, 10.8 Hz, 1H, H-1), 3.43–3.36 (m, 2H, Gly-CH2), 2.00–1.98 (m, 1H, H-6a), 1.86–1.81 (m, 1H, 2’-CH), 1.74 (br s, 2H, Gly-NH2), 1.71–1.67 (m, 2H, H-3a, 4a), 1.57–1.42 (m, 1H, H-5), 1.40–1.36 (m, 1H, H-2), 1.10–1.03 (m, 1H, H-3b), 1.01–0.95 (m, 1H, H-6b), 0.91–0.85 (m, 7H, H-4b, 2’-CH3, 5-CH3), 0.76 (d, J = 7.0 Hz, 3H, 2’-CH3).
L-Alanine menthyl ester (ment-Ala); 1H NMR (600 MHz, CDCl3): δ 4.63 (ddd, J = 3.9, 10.6, 10.6 Hz, 1H, H-1), 3.46–3.39 (m, 1H, Ala-α-CH), 1.92–1.86 (m, 1H, H-6a), 1.84–1.75 (m, 1H, 2’-CH), 1.66–1.55 (br m, 4H, Ala-NH2, H-3a, 4a), 1.47–1.38 (m, 1H, H-5), 1.35–1.30 (m, 1H, H-2), 1.26–1.21 (m, 3H, Ala-β-CH3), 1.04–0.96 (m, 1H, H-3b), 0.95–0.86 (m, 1H, H-6b), 0.86–0.77 (m, 7H, H-4b, 2’-CH3, 5-CH3), 0.69 (d, J = 7.0 Hz, 3H, 2’-CH3).
L-Valine menthyl ester (ment-Val); 1H NMR (600 MHz, CDCl3): δ 4.77–4.70 (m, 1H, H-1), 3.29–3.24 (m, 1H, Val-α-CH), 2.08–1.95 (m, 2H, Val-β-CH, H-6a), 1.92–1.83 (m, 1H, 2’-CH), 1.73–1.65 (m, 2H, H-3a, 4a), 1.55–1.45 (m, 1H, H-5), 1.45–1.36 (m, 2H, Val-NH2, H-2), 1.12–1.02 (m, 1H, H-3b), 1.02–0.96 (m, 4H, Val-γ-CH3, H-6b), 0.94–0.83 (m, 10H, Val-γ-CH3, H-4b, 2’-CH3, 5-CH3), 0.80–0.73 (m, J = 7.0 Hz, 3H, 2’-CH3).
D-Valine menthyl ester (ment-DVal); 1H NMR (400 MHz, CDCl3): δ 4.70 (ddd, J = 4.3, 10.9, 10.9 Hz, 1H, H-1), 3.27–3.26 (m, 1H, Val-α-CH), 2.13–1.98 (m, 2H, Val-β-CH, H-6a), 1.97–1.85 (m, 1H, 2’-CH), 1.74–1.65 (m, 2H, H-3a, 4a), 1.61–1.35 (m, 4H, Val-NH2, H-5, H-2), 1.13–0.94 (m, 5H, H-3b, Val-γ-CH3, H-6b), 0.94–0.81 (m, 10H, Val-γ-CH3, H-4b, 2’-CH3, 5-CH3), 0.74 (d, J = 7.0 Hz, 3H, 2’-CH3).
L-Leucine menthyl ester (ment-Leu); 1H NMR (600 MHz, CDCl3): δ 4.71 (ddd, J = 3.5, 10.6, 10.6 Hz, 1H, H-1), 3.41 (t, J = 7.0, 1H, Leu-α-CH), 1.99–1.92 (m, 1H, H-6a), 1.91–1.84 (m, 1H, 2’-CH), 1.83–1.75 (m, 1H, Leu-γ-CH), 1.74–1.61 (m, 4H, Leu-NH2, H-4a, H-3a), 1.58–1.45 (m, 2H, Leu-β-CHa, H-5), 1.44–1.34 (m, 2H, H-2, Leu-β-CHb), 1.13–1.02 (m, 1H, H-3b), 1.02–0.82 (m, 14H, H-4b, H-6b, Leu-δ-CH3 × 2, 2’-CH3, 5-CH3), 0.77 (d, J = 7.0 Hz, 3H, 2’-CH3).
L-Isoleucine menthyl ester (ment-Ile); 1H NMR (600 MHz, CDCl3): δ 4.73 (ddd, J = 4.3, 11.0, 11.0 Hz, 1H, H-1), 3.35–3.32 (m, 1H, Ile-α-CH), 2.00–1.94 (m, 1H, H-6a), 1.91–1.83 (m, 1H, 2’-CH), 1.78–1.72 (m, 1H, Ile-β-CH), 1.71–1.65 (m, 2H, H-3a, 4a), 1.55–1.36 (br m, 5H, Ile-NH2, H-5, H-2, Ile-γ-CHa), 1.24–1.15 (m, 1H, Ile-γ-CHb), 1.10–1.02 (m, 1H, H-3b), 1.01–0.82 (m, 14H, H-6b, H-4b, 2’-CH3, 5-CH3, Ile-β-CH3 Ile-δ-CH3), 0.75 (d, J = 7.0 Hz, 3H, 2’-CH3).
L-Phenylalanine menthyl ester (ment-Phe); 1H NMR (600 MHz, CDCl3): δ 7.31–7.25 (m, 2H, Phe-Ar-CH), 7.24–7.18 (m, 3H, Phe-Ar-CH), 4.77–4.655 (m, 1H, H-1), 3.72–3.65 (m, 1H, Phe-α-CH), 3.06 (dd, J = 4.8, 13.6 Hz, 1H, Phe-β-CHa), 2.83 (dd, J = 8.0, 13.6 Hz, 1H, Phe-β-CHb), 1.93–1.87 (m, 1H, H-6a), 1.86–1.77 (m, 1H, 2’-CH), 1.70–1.61 (m, 2H, H-3a, 4a), 1.52–1.41 (m, 3H, Phe-NH2, H-5), 1.41–1.33 (m, 1H, H-2), 1.09–0.97 (m, 1H, H-3b), 0.96–0.80 (m, 8H, H-4b, H-6b, 2’-CH3, 5-CH3), 0.74 (d, J = 7.0 Hz, 3H, 2’-CH3).
Plants
Soybean plants (Glycine max cv. Enrei), Pisum sativum, Brassica rapa (var. perviridis, cv. Natsurakuten), tobacco (Nicotiana tabacum cv. SR1), lettuce (Lactuca sativa var. Crispa) and Zea mays (cv. Royal Dent), were grown in soil for 14 days (except that tobacco and lettuce were grown for 4 weeks), in a climate-controlled room at 24 ± 1°C with a photoperiod of 16 h (80 µE m–2 s–1). The light period was from 07:00 to 23:00.
Chemical treatment
A single leaf branch (3 leaves) of a potted plant was evenly sprayed with 3 mL of 10 mM MES buffer (pH 6.0) containing 1% ethanol and 0.1, 1, or 10 µM menthol or ment-Val. Application of 10 mM MES buffer (pH 6.0) alone served as control. The plants were then incubated in a climate-controlled room at 24 ± 1°C with a photoperiod of 16 h (80 µE m–2 s–1) for 24 h or 5 days.
For histone acetyltransferase (HAT) inhibitor (garcinol) treatment, a single leaf branch (3 leaves) of soybean plants was evenly sprayed with 1 mL of an aqueous solution of garcinol (0.1 mM, Cayman Chemical, Ann Arbor, MI, USA) 24 h before ment-Val treatment.
RNA extraction, cDNA synthesis and quantitative polymerase chain reaction (PCR)
Approximately 100 mg of leaf tissues were homogenized in liquid nitrogen, and total RNA was isolated and purified using Sepasol®-RNA I Super G (Nacalai Tesque, Kyoto, Japan) following the manufacturer’s protocol. First-strand cDNA synthesis and qPCR were performed according to the method described previously (Uemura et al. 2020). Primers used in this study are listed in Table S1. Relative transcript abundances were determined after normalization of raw signals with the transcript abundance of a housekeeping gene (actin). We did not use samples or data when sufficient amounts or quality of RNA (> 83 ng µL-1) were not obtained from leaves or when abnormal quantification cycle (Cq) values for the actin gene were obtained.
Leaf damage and herbivore growth assays
Eggs of Spodoptera litura (Fabricius) were obtained from Sumika Technoservice Co. Ltd. (Takarazuka, Japan). They were incubated in a climate-controlled room at 24 ± 1°C with a photoperiod of 16 h, as reported previously (Uemura et al. 2020).
Five third-instar larvae of S. litura (1.8–2.0 mg) were starved overnight and released onto 3 sets of the secondary leaves of a potted soybean plant, in which each solution had been sprayed with each solution before 24 h. The leaves were covered with a mesh bag and kept for 2 h at 24 ± 1°C. The leaves were then scanned, and the total leaf area and the consumed leaf area were determined using ImageJ. Replicate analyses were conducted with 5 independent samples.
Otherwise, a third-instar larva of S. litura (1.8-2.0 mg) was incubated on 3 grams of artificial diet (Insecta LFS, Nihon Nosan Kogyo Ltd., Tokyo, Japan) supplemented with 500 µL of menthol or ment-Val in MES (1 µM) or MES alone, in a plastic petri dish (55 mm diameter, 15 mm deep). The fresh diet with each solution was supplied daily. The net body weight that S. litura larvae gained was determined during the following 96 h. When a larva died or was lost during the assay, we excluded that sample, and final replicate analyses were conducted with 10 independent samples.
Mite oviposition assays
Tetranychus urticae Koch (Acari: Tetranychidae) were reared as reported previously (Iida et al. 2019). A T. urticae adult female (10 days after oviposition) was transferred onto a leaf disc (1.8 cm2) of soybean on wet cotton in a plastic Petri dish (90 mm diameter). We prepared 1 leaf disc from each of 3 secondary soybean leaves that had been sprayed with each solution before 24 h. The means of the 3 discs were evaluated as a single independent replicate, and final replicate analyses were conducted with 5 independent samples.
Statistics and reproducibility
We performed one-way ANOVA with Holm's sequential Bonferroni post hoc test and post hoc Tukey’s HSD using the program (http://astatsa.com/OneWay_Anova_with_TukeyHSD/) for comparing multiple samples. The sample sizes and number of replicates for all of the sets of assays and analyses are indicated in the legends of the corresponding figures.