Lotuslactone, a non-canonical strigolactone from Lotus japonicus

Phytochemistry 157 (2019) 200–205

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Lotuslactone, a non-canonical strigolactone from Lotus japonicus a

b

Xiaonan Xie , Narumi Mori , Kaori Yoneyama Koichi Yoneyamaa, Kohki Akiyamab,g,∗

a,c,d

a

e,f

, Takahito Nomura , Kenichi Uchida ,

T

a

Center for Bioscience Research and Education, Utsunomiya University, 350 Mine-machi, Utsunomiya, 321-8505, Japan Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Nakaku, Sakai, Osaka, 599-8531, Japan Graduate School of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime, 790-8566, Japan d PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012, Japan e Department of Biosciences, Teikyo University, 1-1 Toyosatodai, Utsunomiya, 320–8551, Japan f Advanced Instrumental Analysis Center of Teikyo University, 1-1 Toyosatodai, Utsunomiya, 320-8551, Japan g CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama, 332-0112, Japan b c

ARTICLE INFO

ABSTRACT

Keywords: Lotus japonicus Fabaceae Lotuslactone Strigolactone Gigaspora margarita Glomeromycotina Phelipanche ramosa Orobanche minor Striga hermonthica Orobanchaceae

Root exudates from Lotus japonicus were found to contain at least three different hyphal branching-inducing compounds for the arbuscular mycorrhizal (AM) fungus Gigaspora margarita, one of which had been previously identified as (+)-5-deoxystrigol (5DS), a canonical strigolactone (SL). One of the two remaining unknown hyphal branching inducers was purified and named lotuslactone. Its structure was determined as methyl (E)-2-(3acetoxy-2-hydroxy-7-methyl-1-oxo-1,2,3,4,5,6-hexahydroazulen-2-yl)-3-(((R)-4-methyl-5-oxo-2,5-dihydrofuran2-yl)oxy)acrylate, by 1D and 2D NMR spectroscopy, and HR-ESI- and EI-MS. Although lotuslactone, a noncanonical SL, contains the AB-ring and the enol ether-bridged D-ring, it lacks the C-ring and has a sevenmembered cycloheptadiene in the A-ring part as in medicaol, a major SL of Medicago truncatula. Lotuslactone was much less active than 5DS, but showed comparable activity to methyl carlactonoate (MeCLA) in inducing hyphal branching of G. margarita. Other natural non-canonical SLs including avenaol, heliolactone, and zealactone (methyl zealactonoate) were also found to be moderate to weak inducers of hyphal branching in the AM fungus. Lotuslactone strongly elicited seed germination in Phelipanche ramosa and Orobanche minor, but Striga hermonthica seeds were 100-fold less sensitive to this stimulant.

1. Introduction Arbuscular mycorrhizal (AM) symbiosis is a mutually beneficial association of most terrestrial plants with members of the Glomeromycotina fungi (Smith and Read, 2008). The plant–AM-fungus interaction is initiated by presymbiotic communication via diffusible signals released from the two partners (Choi et al., 2018). Host roots exude signal molecules that stimulate fungal metabolism and hyphal fine branching. 5-Deoxystrigol (5DS) (1) (Fig. 1) was isolated as the first hyphal branching factor from root exudates of the model legume Lotus japonicus (Akiyama et al., 2005). 5DS (1) is the sixth member of naturally occurring strigolactones (SLs). SLs were originally isolated as seed germination stimulants for the root parasitic plants Striga and Orobanche (Akiyama and Hayashi, 2006; Cook et al., 1966; Screpanti

et al., 2016), and are now known to be a class of phytohormones controlling different aspects of plant development, including shoot branching inhibition (Gomez-Roldan et al., 2008; Umehara et al., 2008; Waters et al., 2017). To date, approximately 30 SLs have been characterized from root exudates of various plant species (Yoneyama et al., 2018b). Natural SLs are carotenoid-derived compounds characterized by the presence of an enol ether-linked (R)-configurated methylbutenolide ring (D-ring) which is connected to a structurally variable second moiety (Al-Babili and Bouwmeester, 2015; Jia et al., 2018; Yoneyama et al., 2018b; Wang and Bouwmeester, 2018). SLs are classified into canonical and noncanonical SLs according to their variable second moiety. Strigol (2) (Fig. 1) and related compounds that contain a tricyclic lactone (ABCring) and the enol-ether–D-ring are called canonical SLs. Canonical SLs

∗ Corresponding author. Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Nakaku, Sakai, Osaka, 599-8531, Japan. E-mail addresses: [email protected] (X. Xie), [email protected] (N. Mori), [email protected] (K. Yoneyama), [email protected] (T. Nomura), [email protected] (K. Uchida), [email protected] (K. Yoneyama), [email protected] (K. Akiyama).

https://doi.org/10.1016/j.phytochem.2018.10.034 Received 11 July 2018; Received in revised form 7 October 2018; Accepted 28 October 2018 0031-9422/ © 2018 Elsevier Ltd. All rights reserved.

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Fig. 1. Structures of canonical and non-canonical strigolactones. 5-Deoxystrigol (1); strigol (2); orobanchol (3); carlactone (4); carlactonoic acid (5); methyl carlactonoate (6); 4-deoxyorobanchol (7); 18-hydroxycarlactonoic acid (8); avenaol (9); heliolactone (10); zealactone (methyl zealactonoate) (11); 3-hydroxycarlactone (12); lotuslactone (13); medicaol (14).

are divided into strigol (2)- and orobanchol (3)-type SLs according to the stereochemistry of the C-ring. In contrast, there are also SL-like compounds that contain the enol-ether–D-ring moiety but lack the canonical A-, B-, and/or C-rings are termed non-canonical SLs. The simplest non-canonical SL is carlactone (CL) (4) that contains a β-iononetype A-ring and the conserved enol-ether–D-ring (Alder et al., 2012). CL itself is an endogenous biosynthetic precursor of the downstream canonical and non-canonical SLs (Seto et al., 2014). This C19 intermediate is oxidized to carlactonoic acid (CLA) (5) by a conserved function of cytochrome P450 monooxygenase MORE AXILLARY GROWTH 1 (MAX1) (Abe et al., 2014; Yoneyama et al., 2018a; Zhang et al., 2018). The MAX1 product CLA (5) is subsequently either methyl-esterified to non-canonical C20 methyl carlactonoate (MeCLA) (6) (Abe et al., 2014) or cyclized to canonical C19 SLs by unknown enzymes (Abe et al., 2014; Iseki et al., 2018). Among MAX1 homologs, rice Os900 and Selaginella SmMAX1a and SmMAX1b can consecutively catalyze CL oxidation to CLA and its cyclization to a canonical SL, 4-deoxyorobanchol (4DO) (7), probably via 18-hydroxyCLA (8) (Yoneyama et al., 2018a; Zhang et al., 2014). A sulfotransferase, LOW GERMINATION STIMULANT 1 (LGS1), is likely to be involved in the formation of a canonical SL, 5DS (1) in sorghum (Sorghum bicolor) (Gobena et al., 2017). Non-canonical C20 SLs, avenaol (9) (Kim et al., 2014; Yasui et al., 2017), heliolactone (10) (Ueno et al., 2014) and zealactone (methyl zealactonoate) (11) (Charnikhova et al., 2017; Xie et al., 2017) were recently isolated as the major germination stimulants from root exudates of AM host plants, black oat (Avena strigosa), sunflower (Helianthus annuus), and maize

(Zea mays), respectively. All these C20 compounds are likely to be derived from MeCLA (6) or its isomers and their oxygenated derivatives (Yoneyama et al., 2018b). 3-Hydroxycarlactone (12) was most recently identified as an endogenous rice compound, which likely acts as an intermediate of SL biosynthesis (Baz et al., 2018). SLs induce an array of AM fungal presymbiotic responses including spore germination, hyphal growth, hyphal branching, respiratory activity, mitosis, expression of effector genes, and enhanced exudation of chitin tetramer and pentamer, which in turn trigger symbiotic responses in the host plant (Akiyama et al., 2005; Besserer et al., 2008, 2006; Genre et al., 2013; Tisserant et al., 2012; Tsuzuki et al., 2016). Structure-activity relationship studies of canonical (Akiyama et al., 2010) and some non-canonical (Mori et al., 2016) SLs on hyphal branching induction in the AM fungus Gigaspora margarita indicated that, although CLA (5) and MeCLA (6) are moderately active on the AM fungus, canonical SLs are, in general, more active than non-canonical SLs. CLA (5) and MeCLA (6) have been detected at trace levels in root exudates of some but not all tested plants, suggesting their role as a species-specific symbiotic signal (Yoneyama et al., 2018a; Xie, 2016). Although C20 non-canonical SLs, avenaol (9), heliolactone (10), and zealactone (methyl zealactonoate) (11), fulfill the structural requirements for induction of hyphal branching in AM fungi (Mori et al., 2016), their activities have not yet been evaluated. In our continuing studies on AM symbiotic signals produced by host plants, in addition to 5DS (1), two compounds with hyphal branchinginducing activity in G. margarita were found in root exudates of Lotus 201

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Fig. 2. HPLC profile of EtOAc-soluble neutral fraction from root exudates of L. japonicus.

japonicus. One of the two hyphal branching inducers was purified and named lotuslactone (13). The results we report herein are the isolation and structure determination of this novel non-canonical SL and its biological activity in AM fungi and root parasitic plants.

determine its structure due to the scarcity and instability of the isolated material. The molecular formula of lotuslacotne (13) was established to be C22H24O9 on the basis of the proton adduct ion at m/z 433.1487 [M +H]+ obtained by HR-ESI-TOF-MS. Table 1 lists 1H and 13C NMR spectroscopic data aided with 2D NMR experiments (1H-1H COSY, HMQC, HMBC, and NOESY). The 13C NMR spectroscopic data showed 22 carbon signals including two α,β-unsaturated ester carbonyls, one α,β-unsaturated carbonyl, one acetoxy carbonyl (overlapped with one of the two ester carbonyls), three trisubstituted olefins, one tetrasubstituted olefin, three sp3 methylenes, two sp3 oxymethines, one oxygenated quaternary sp3 carbon, one methoxy, one acetyl methyl, and two allylic methyls. The 1H-1H COSY and HMBC correlations from H-2′, H-3′, H-6′, and H-7' (Fig. 3) together with the fragment ion at m/z 97 in EI-MS as above indicated the presence of the conserved enol ether-bridged methylbutenolide (the D-ring). The HMBC correlations of the enol methine (H-6′) and methoxy protons with the ester carbonyl at C-1 revealed that the molecule lacks the C-ring lactone, but has a methyl ester at the C-1 position. The 1H-1H COSY correlations of the olefinic methine (Η−9) with the allylic methyl (Η−11), and of the methylene (Η−6) with the two allylic methylenes (H-5 and H-7) indicated that lotuslacotne (13) has a seven-membered 1-methylcyclohepta-1,3diene ring as in medicaol (14), a major SL of Medicago truncatula (Tokunaga et al., 2015). The HMBC correlations of Η−9 to the two quaternary sp2 carbons at C-4a and C-9a, and the carbonyl carbon at C10, and of the oxygenated allylic methine proton (H-4) to C-4a and C-

2. Results and discussion 2.1. Isolation and structure determination of lotuslactone (13) Lotus japonicus was grown hydroponically using tap water, and root exudates were collected by the method described previously (Akiyama et al., 2005). The root exudates were partitioned between EtOAc and 0.2 M K2HPO4 solution to obtain an EtOAc-soluble neutral fraction. Fig. 2 shows the reverse-phase HPLC chromatogram of the crude extract monitored at 236 nm, in which 5DS (1) was detected as a single peak at 25.7 min. A preliminary purification of a portion of the crude extract showed that two compounds eluted as a single peak at 12.9 and 15.9 min, respectively, induced hyphal branching in G. margarita (Supplementary Fig. 1). Intense, characteristic fragment peaks at m/z 97 in the EI-MS suggested these to be putative SL derivatives (Supplementary Fig. 2). In silica gel CC, the two branching factors were eluted in the 60–80% EtOAc in n-hexane fraction. The highly active 60% EtOAc eluate was further purified with reverse-phase HPLC to obtain the slow-eluting branching factor named lotuslactone (13). Although the fast-eluting one was suggested to be a putative monohydroxy-5DS isomer from the EI-MS as shown above, we could not Table 1 NMR spectroscopic data for lotuslactone (13) (CDCl3). 13

No.

δ

C

1 2 3 4 4a 5 6 7 8 9 9a 10 11 2′ 3′ 4′ 5′ 6′ 7′ 1″ 2″ OCH3

167.8 110.5 79.1 81.6 161.6 30.8 22.5 36.0 148.1 112.9 136.2 199.7 27.2 100.3 141.1 134.7 170.4 153.6 10.5 170.4 20.7 52.2

δ 1H (mult.)

5.79 (br.s) 2.45 (m) 1.81 (m), 1.85 (m) 2.32 (m) 5.96 (br.s) 1.89 (br.s) 5.92 (br.s) 6.80 (s) 7.58 (s) 1.93 (br.s) 2.04 (s) 3.75 (s)

HMQC and DEPT C C C CH C CH2 CH2 CH2 C CH C C CH3 CH CH C C CH CH3 C CH3 CH3

1

H–1H COSY

HMBC

NOESY

C-3, C-4a, C-9a, C-1″

H-5

H-6 H-5, H-7 H-6

C-4a, C-7, C-9a

H-4

H-11

C-4a, C-7, C-9a, C-10, C-11

H-11

H-9 H-3′, H-7′ H-2′, H-7′

C-7, C-8, C-9 C-4′, C-5′, C-6′ C-2′, C-4′, C-5′

H-9 H-3′, H-6′ H-2′, H-7′

H-2′, H-3′

C-1, C-2, C-3, C-2′ C-3′, C-4′, C-5′

H-2′ H-3′

C-5, C-6, C-8, C-9

C-1″ C-1

202

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Fig. 3. Key HMBC correlations of lotuslactone (13).

Table 2 Minimum effective concentrations (MECs) of lotuslactone (13), other natural non-canonical strigolactones (9–11, 4–6), and canonical strigolactones (1, 14) for hyphal branching-inducing activity in Gigaspora margarita. Tested compound

MEC (pg/disc)a

Lotuslactone (13) Avenaol (9) Heliolactone (10) Zealactone (11) Carlactone (4) Carlactonoic acid (5) Methyl carlactonoate (6) 5-Deoxystrigol (1) Medicaol (14)

1000 1,000,000 10,000 10,000 100,000b 100b 1,000b 3c 10d

a b c d

Fig. 4. Germination stimulating activities of lotuslactone (13) toward O. minor, P. ramosa. and S. hermonthica seeds. Data are presented as means ± SE (n = 3).

The other three natural non-canonical strigolactones (9, 10, 11) were also found to be moderate to weak inducers of hyphal branching in the AM fungus. The all tested non-canonical SLs induced the formation of low order branches, mainly consisting of long tertiary hyphae, as observed for non-canonical CL (4), CLA (5), and MeCLA (6) and canonical 5DS (1) (Mori et al., 2016). 2.3. Germination stimulating activities of lotuslactone (13) in O. minor, P. ramosa, and S. hermonthica

Determined by serial 10-fold dilutions. Mori et al. (2016). Akiyama et al. (2010). Tokunaga et al. (2015).

Germination stimulating activities of lotuslactone (13) in O. minor, P. ramosa, and S. hermonthica seeds are shown in Fig. 4. Lotuslactone (13) strongly elicited P. ramosa and O. minor seed germination even at 0.1 nM concentration, while S. hermonthica seeds required 10 nM of lotuslactone (13) for appreciable germination.

9a, and the oxygenated quaternary sp3 carbon at C-3 indicated that the 1-methylcyclohepta-1,3-diene and the cyclopentanone are fused to form 7-methyl-3,4,5,6-tetrahydroazulen-1(2H)-one structure in the ABring. As evidenced by the HMBC correlations of H-4 and the acetyl methyl (H-2″) proton to the acetyl carboxyl carbon at C-1″, this AB-ring is substituted with an acetyloxy and a hydroxy group at C-4 and C-3, respectively. The two substructures, the tetrahydroazulenone AB-ring and the D-ring-substituted methyl 3-oxyacrylate, were connected together at C-3 and C-2 as indicated by the HMBC correlation of H-6′ to C3. Thus, the structure of lotuslactone (12) was established to be methyl (E)-2-(3-acetoxy-2-hydroxy-7-methyl-1-oxo-1,2,3,4,5,6-hexahydroazulen-2-yl)-3-((4-methyl-5-oxo-2,5-dihydrofuran-2-yl)oxy)acrylate. The CD spectrum of lotuslactone (13) had a positive and negative Cotton effect around 228 nm and 252 nm, respectively, suggesting that it also has a 2′(R) configuration as previously identified natural SLs (Welzel et al., 1999). In an attempt to determine the stereochemistry at C-3 and C-4 by X-ray crystallography, this compound was decomposed due to its instability during recrystallization. Therefore, the stereochemistry at C-3 and C-4 could not be resolved and can only be unambiguously determined by stereoselective total synthesis as achieved for a non-canonical SL, avenaol (Yasui et al., 2017).

3. Concluding remarks In this study, lotuslactone (13) was identified as the second branching factor in L. japonicus. It is a non-canonical SL that contains the tetrahydroazulenone AB-ring and the conserved enol-ether bridged D-ring, but lacks the C-ring. Lotuslactone (13) is the second member of SLs that has a seven-membered ring in the A ring part as in medicaol (14) (Tokunaga et al., 2015). Consistent with the previous report (Mori et al., 2016), the all tested natural non-canonical SLs in this study are commonly active on AM fungi. Although AM symbiosis has been considered to lack absolute specificity, some degree of specific and preferential interactions between AM fungi and their plant hosts exists under field conditions (Sanders, 2003). It was observed that despite the distinctive difference in SL composition with respect to canonical SLs such as 5DS (1) and sorgomol, the levels of AM colonization and the community compositions did not differ between Striga-susceptible and -resistant maize cultivars (Yoneyama et al., 2015). In contrast to canonical SLs composed of a common ABCD ring structure, non-canonical SLs isolated so far from root exudates of various AM host plants differ each other in their skeleton. This feature could confer these non-canonical SLs to act as a species-specific signal in host discrimination by AM fungi at the presymbiotic stage. Further study is needed to clarify the biosynthetic pathway of lotuslactone (13) and 5DS (1) and their roles in the host recognition of AM fungi. These studies would provide new insight into the molecular evolution and function of canonical and non-canonical SLs as rhizosphere signals for AM fungi and root parasitic plants.

2.2. Hyphal branching-inducing activity of lotuslactone (13) in G. margarita Lotuslactone (13) and three natural non-canonical SLs, avenaol (9), heliolactone (10), and zealactone (methyl zealactonoate) (11) were tested for hyphal branching-inducing activity in germinating spores of G. margarita (Table 2). Lotuslactone (13) was much less active than 5DS (1) and medicaol (13), but showed comparable activity to MeCLA (6). 203

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4. Experimental

4.4. Hyphal branching assay

4.1. General procedures

Hyphal branching activity on a germinating spores of Gigaspora marigarita Becker & Hall (MAFF 520054) was conducted as reported previously (Akiyama et al., 2010).

1

H and 13C NMR spectra were recorded in CDCl3 on a JEOL JMNECA-500 spectrometer. Standard pulse sequence and phase cycling were used for HMQC, HMBC, 1H-1H COSY and NOESY spectral analyses. CD spectra were obtained with a JASCO J-720W spectropolarimeter in MeCN. EI-MS spectra were recorded with a JEOL JMS700 instrument. High-resolution mass spectra were obtained with an SCIEX Triple TOF 5600 mass spectrometer equipped with an ESI source.

4.5. Seed germination assay Germination assays of O. minor, P. ramosa, and S. hermonthica seeds were conducted as reported previously (Yoneyama et al., 2007). Acknowledgements

4.2. Hydroponic culture of Lotus japonicus and collection of root exudate

This work was supported by the fund of Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), the Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries and Food Industry, and JSPS KAKENHI Grant Number JP18K05452.

The seeds of Lotus japonicus B-129 Gifu (Fabaceae) were sown in plastic pot and filled with autoclaved sand. The plants were grown in a growth room maintained at 25–29 °C under natural daylight conditions for 7 days. The plants were watered with tap water as required. The 2000 seedlings were transferred to a plastic container (53.5 × 33.5 × 14 cm, W × L × H) containing 20 L of tap water. Ten containers each containing 200 seedlings was 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 4 weeks and the culture medium and activated charcoal were replaced every 2 days. The root exudates absorbed on charcoal (80 g) were eluted with acetone (1500 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 Na2SO4, and concentrated in vacuo. The concentrated samples were kept at 4 °C until use. HPLC analysis of the EtOAc-soluble neutral fraction was carried out by an Inertsil ODS-3 (4.6 × 250 mm, 5 μm; GL Sciences, Japan) with an MeCN/H2O gradient system (40:60 to 100:00 over 30 min) as the eluent at a flow rate of 0.8 mL min−1, and the column temperature was set to 40 °C. Compounds eluted from the column were monitored at 236 nm.

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4.3. Isolation of lotuslactonoate (13) The crude EtOAc extract (210.7 mg) collected during 4 weeks from Lotus japonicus seedlings grown hydroponically was subjected to silica gel 60 CC (100 g, 230–400 mesh; Merck) with stepwise elution of nhexane–EtOAc (100:0–0:100, v/v, 10% step). The 60% EtOAc eluate containing novel SL (100.9 mg) was subjected to silica gel 60 CC (30 g) using n-hexane–EtOAc (45:55, v/v) as eluting solvent system. Fractions were collected every 5 mL. Fractions 13–21 containing 13 were combined (17.60 mg) and was purified by HPLC on a Mightysil RP-18 column (10 × 250 mm, 10 μm; Kanto Chemicals, Japan) with an MeCN/H2O gradient system (20:80 to 100:00 over 60 min) as the eluent at a flow rate of 3 mL min−1, and the column temperature was set to 30 °C. The fraction eluted as a single peak at 17 min (detection at 250 nm) was collected. This fraction was further purified by isocratic (60% MeCN/H2O) HPLC on a Develosil ODS-CN column (4.6 × 250 mm, 5 μm; Nomura Chemicals, Japan) at a flow rate of 1 mL min−1 to give lotuslactone (13, 4.37 mg, Rt 21.3 min, detection at 250 nm). Lotuslactone (13); EI-MS m/z (rel. int.): 431 [M-H]+ (2), 414 [MH2O]+ (4), 372 (14), 275 (100), 243 (44), 215 (25), 165 (28), 129 (28), 97 (56), 69 (32). HR-TOF-MS m/z: 433.1487 [M+H]+ (calcd. for C22H25O9, m/z: 433.1498). CD (CH3CN) λmax (Δε) nm: 281 (12.7), 252 (−0.67), 228 (32.4). For 1H and 13C NMR spectral assignments, see Table 1. 204

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