Methyl zealactonoate, a novel germination stimulant for root parasitic weeds produced by maize

Abstract

One of the germination stimulants for root parasitic weeds produced by maize (Zea mays) was isolated and named methyl zealactonoate (1). Its structure was determined to be methyl (2E,3E)-4-((RS)-3,3-dimethyl-2-(3-methylbut-2-en-2-yl)-5-oxotetrahydrofuran-2-yl)-2-((((R)-4-methyl-5-oxo-2,5-dihydrofran-2-yl)oxy)methylene)but-3-enoate using by 1D and 2D NMR spectroscopy and ESI and EI–MS spectrometry. Feeding experiments with ¹³C-carlactone (CL), a biosynthetic intermediate for strigolactones, confirmed that 1 is produced from CL in maize. Methyl zealactonoate strongly elicits Striga hermonthica and Phelipanche ramosa seed germination, while Orobanche minor seeds are 100-fold less sensitive to this stimulant.

Maize (Zea mays) is a major host of the root parasitic weeds known as witchweeds, Striga hermonthica and S. asiatica, and has been reported to produce several strigolactones (SLs).^1,2) Among known SLs, strigol was isolated as the major Striga seed germination stimulant in maize root exudates.³⁾ In addition to this SL, sorgomol and 5-deoxystrigol were found in maize root exudates.⁴⁾ Recently, Jamil et al. reported the detection of novel germination stimulants tentatively called SL1 and SL2 in maize root exudates based on LC–MS/MS analysis.⁵⁾ SL1 and SL2 were found to have the molecular weights of 348 and 276, respectively. Herein we report the isolation and structure determination of SL2, named methyl zealactonoate,⁶⁾ and its germination stimulation activities on S. hermonthica, Phelipanche ramosa, and Orobanche minor seeds.

The crude EtOAc extract (350.7 mg) of root exudates collected over 5 weeks from ca. 800 maize seedlings grown hydroponically was purified using silica gel column chromatography (CC) with stepwise elution using n-hexane and EtOAc. The 30% and 40% EtOAc fractions which were found via LC–MS analysis to contain SL1 and SL2 were combined and further purified by silica gel CC (25% EtOAc in n-hexane). Since SL1 was found to be a mixture of at least two isomers with very similar chromatographic behaviors, we focused on the purification of SL2. The fractions containing SL2 were combined and purified by HPLC on an ODS column followed by HPLC on an ODS-CN column to afford pure germination stimulant 1 (7.83 mg) as an amorphous solid.

The molecular formula of 1 was established to be C₂₀H₂₄O₇ on the basis of protonated and sodium adduct ions obtained by HR–ESI–TOF–MS, indicative of nine degrees of unsaturation. The molecular ion observed at m/z 376 [M]⁺ in GC–MS also supported this molecular formula.

The ¹H NMR spectrum of 1 displayed two singlet methyl protons at 0.73 and 0.99 ppm, two methyl protons with small coupling (<2 Hz) at 1.25 and 1.64 ppm; one oxygenated methyl proton at 3.35 ppm; two geminal coupled methylene protons at 1.90 and 2.25 ppm, five olefinic protons at 4.71, 4.94, 5.20, 5.53, and 7.45 ppm; and two coupled olefinic protons with 16.75 Hz, suggesting the trans olefine (Table 1, Supplemental Fig. S1). The ¹³C NMR spectrum revealed the presence of 20 carbons including three ester carbonyl carbons, four methyl carbons, one methoxy carbon, six olefinic carbons, one methylene carbon and five quaternary carbons, the multiplicities of which were determined by DEPT experiment (Table 1, Supplemental Fig. S2). Additionally, the HMQC spectrum indicated that two olefinic protons (at 4.71 and 5.20 ppm) were exo-methylene protons (Table 1, Supplemental Fig. S3). In the ¹H−¹H COSY spectrum, the cross peaks of H-4/H-18, H-15/H-11, and H-15/H-12 indicated that C-4 and C-15 were allyl methyl groups, which was also supported by their small coupling constants in the ¹H NMR spectrum (Table 1, Supplemental Fig. S4). The HMBC correlations of H-15 to C-12, C-13, and C-14 and H-12 to C-11 established a butenolide moiety with the methyl group at C-13 (Table 1, Supplemental Fig. S5). The chemical shifts of the proton and carbon at C-11 suggested that the butenolide moiety was bound with an ether bridge. The presence of ether-bridged butenolide was also supported by the fragment ion at m/z 97 in LC–MS/MS and GC–MS, which was also observed in SLs. The HMBC correlations from H-2 to C-1, C-3, C-6, C-16, and C-17; H-16 to C-6 and C17; and H-17 to C-6 and C-16 indicated the presence of a γ-lactone moiety with two methyl groups at C-1 (Table 1, Supplemental Fig. S5). A conjugated diene moiety consisted of C-7, C-8, C-9, and C-10 was established by the HMBC correlations of H-10 to C-8 and C-9. The HMBC correlations from H-8, H-10, and H-20 to C-19 indicated the presence of a methoxycarbonyl group at C-9. The configuration at C-9 was suggested to be a Z form from the chemical shift of the H-10 proton. The HMBC correlations of H-7 and H-8 to C-6 indicated the connection between C-6 and C-7. In addition, the ether bridge between C-10 and C-11 was established from the HMBC correlations of H-10 to C-11 and H-11 to C-10 (Table 1, Supplemental Fig. S5). The remaining unit was deduced to be an isopropenyl group connected to C-6, suggested from the HMBC correlations (Table 1, Supplemental Fig. S5). The key NOESY correlations of 1 are shown in Fig. 1B (Supplemental Fig. S6). Thus, the chemical structure of 1 was determined to be methyl (2E,3E)-4-((R,S)-3,3-dimethyl-2-(3-methylbut-2-en-2-yl)-5-oxotetrahydrofuran-2-yl)-2-((((R)-4-methyl-5-oxo-2,5-dihydrofran-2-yl)oxy)methylene)but-3-enoate, which was named methyl zealactonoate.⁶⁾ Although 1 contains only the D-ring moiety of canonical SLs and thus is classified as a non-canonical SL,²⁾ feeding experiments with ¹³C-CL confirmed that 1 is synthesized from CL in maize plants (Supplemental Fig. S7). This also indicates that 1 has an (11R) configuration since natural CL exists only in only an (11R) isomer.⁷⁾ However, further study is needed to confirm its absolute stereochemistry. The stimulant 1 strongly elicits seed germination in S. hermonthica and P. ramosa, but O. minor seeds are 100-fold less sensitive to the stimulant (Fig. 2).

Table 1. NMR spectral data for compound 1 (C₆D₆).

No.	δH (mult., J Hz)	HMQC and DEPT	δC	HMBC	NOESY
1		C	43.1
2a	1.90 (d, 16.8)	CH2	44.1	C-1, 3, 6, 17	H-17
2b	2.25 (d, 16.8)			C-1, 3, 16, 17	H-16
3		C	174.6
4a	4.71 (bdq, 1.4, 1.4)	CH2	112.5	C-6, 18	H-18
4b	5.20 (brs)			C-5, 6, 18
5		C	143.5
6		C	92.8
7	7.33 (d, 15.9)	CH	132.8	C-6, 9	H-16, 18
8	6.96 (d, 15.9)	CH	117.9	C-6, 19
9		C	111.9
10	7.45 (s)	CH	153.9	C-8, 9, 11, 19
11	4.94 (bdq, 1.4, 1.4)	CH	100.3	C-10
12	5.53 (bdq, 1.6, 1.6)	CH	141.2	C-11
13		C	135.4
14		C	170.2
15	1.25 (bdd, 1.5, 1.5)	CH3	10.6	C-12, 13, 14
16	0.99 (s)	CH3	23.9	C-6, 17	H-2b, 7
17	0.73 (s)	CH3	24.5	C-6, 16	H-2a
18	1.64 (brs)	CH3	21.2	C-4, 5, 6	H-4a, 7
19		C	166.5
20	3.35 (s)	CH3	51.4	C-19

Fig. 1. Chemical structure of methyl zealactonoate (1) (A) and key NOESY correlations (B).

Fig. 2. Germination stimulation activities of methyl zealactonoate (1) on Striga hermonthica, Phelipanche ramosa, and Orobanche minor seeds.

Experimental

1. Instruments

¹H and ¹³C NMR spectra were recorded in C₆D₆ (δ_H 7.26, δ_C 128.4) on a JEOL JMN-ECA-500 spectrometer. Standard pulse sequence and phase cycling were used for HMQC, HMBC, COSY and NOE spectral analyses. CD spectra were recorded with a JASCO J-720W spectropolarimeter in MeCN. EI-GC/MS spectra were obtained with a JEOL JMS-Q1000GC/K9 on a DB-5 (J&W Scientific, Agilent) capillary column (5 m×0.25 mm) using a He carrier gas (3 mL/min). High-resolution mass spectra were obtained with an Agilent 6520 Q-TOF mass spectrometer equipped with an ESI source. An LC-MS/MS analysis of the proton adduct ion was performed with a triple quadrupole/linear ion trap (LIT) instrument (QTRAP5500; AB SCIEX) with an electrospray source. The LC-MS/MS analytical conditions were the same as those described previously.⁸⁾

2. Chemicals

1-¹³C-Carlactone was prepared as reported.⁷⁾ The other analytical-grade chemicals and HPLC solvents were obtained from Kanto Chemical Co., Ltd. and Wako Pure Chemical Industries Ltd.

3. Plant material and root exudate collection

The seeds of maize (Zea mays cv. Pioneer 2817) were sown in plastic pots filled with autoclaved sand and were grown in a growth room maintained at 25–29°C under natural daylight conditions for 7 days. The pots were watered with tap water as required. The seedlings were transferred to a plastic container (53.5×33.5×14 cm, W×L× H) containing 20 L of tap water and 10 mM Ca(NO₃)₂. Ten containers each containing 80 seedlings were placed in a growth room maintained at 25–29°C under natural daylight conditions. Root exudates released into the culture medium were adsorbed on activated charcoal (4 g×2 for 20 L) using two water circulation pumps. The plants were grown for 5 weeks and the culture medium and activated charcoal were replaced every 3 days. The root exudates absorbed on charcoal (ca. 80 g in total) were eluted with acetone (1,500 mL). After evaporation of the acetone in vacuo, the aqueous residue (ca. 200 mL) was extracted with EtOAc (3×200 mL). The EtOAc extracts were combined, washed with 0.2 M K₂HPO₄ (300 mL, pH 8.3), dried over anhydrous MgSO₄, and concentrated in vacuo. The concentrated samples were kept at 4°C until use.

4. Seed germination assay

Germination assays of S. hermonthica, P. ramosa, and O. minor seeds were conducted as reported previously.⁹⁾

5. Isolation of methyl zealactonoate

The crude EtOAc extract (350.7 mg) collected during 5 weeks from maize seedlings grown hydroponically was subjected to silica gel CC (85 g) with stepwise elution of n-hexane–EtOAc (100 : 0–0 : 100, v/v, 10% step) to give fractions 1–11. Fractions 4 and 5 (30% and 40% EtOAc, respectively) containing novel SLs were combined (87.6 mg) and subjected to silica gel CC (30 g) using n-hexane–EtOAc (75 : 25, v/v) as an eluting solvent system. Fractions were collected every 10 mL. Fractions 17–25 were found to contain novel germination stimulant 1 based on LC–MS/MS and GC–MS analyses. Fractions 17–25 were combined (20.17 mg) and purified by HPLC on an ODS column (Mightysil RP-18, 10×250 mm, 10 µm; Kanto Chemicals, Japan) with an MeCN/H₂O gradient system (40 : 60 to 100 : 00 over 60 min) as the eluent at a flow rate of 3 mL/min, and the column temperature was set to 30°C. The active fraction eluted as a single peak at 23.4 min (detection at 254 nm) was collected. This fraction was further purified by isocratic (70% MeCN/H₂O) HPLC on a Develosil ODS-CN column (4.6×250 mm, 5 µm; Nomura Chemicals, Japan) at a flow rate of 0.8 mL/min to give methyl zealactonoate (1, 7.83 mg, R_t 27.7 min, detection at 254 nm).

Methyl zealactonoate (1) CD (c 0.0005, MeCN) λ_max (Δε) nm: 273 (40.96), 255 (–0.70); GC-MS, 70 eV, m/z (rel. int.): 376 (30), 279 (10), 223 (31), 153 (100), 111 (87), 97 (27). HR–TOF–MS m/z: 377.1604 [M+H]⁺ (calcd. for C₂₀H₂₅O₇, m/z: 377.1595). ¹H and ¹³C NMR spectroscopic data are shown in Table 1, Fig. 1B, and Supplemental Figs. S1–S6.

6. Feeding experiments

Two-week-old maize seedlings grown hydroponically with tap water were treated with fluridone (1 µM) for 24 hr. LC-MS analysis confirmed that this fluridone treatment completely inhibited methyl zealactonoate production (data not shown). After being washed thoroughly with tap water, three seedlings were transferred to a glass tube containing 300 mL of 1-¹³C-CL (0.15 µg/mL) tap water medium and incubated for another 24 hr. The culture medium+washing and maize roots were separately extracted with EtOAc as described previously.¹⁰⁾ The experiment was repeated three times. The EtOAc extracts were analyzed using LC–MS/MS (Supplemental Fig. S7).

Electronic supplementary material

The online version of this article contains supplementary materials (Supplemental Figures S1-S7), which is available at http://www.jstage.jst.go.jp/browse/jpestics/.