Biosynthesis of uterotonic diterpenes from Montanoa tomentosa (zoapatle)

ARTICLE IN PRESS Journal of Plant Physiology 166 (2009) 1961—1967

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Biosynthesis of uterotonic diterpenes from Montanoa tomentosa (zoapatle) Nemesio Villa-Ruano, Martha Guadalupe Betancourt-Jime ´nez,  Edmundo Lozoya-Gloria Departamento de Ingenierı´a Gene´tica, Centro de Investigacio ´n y de Estudios Avanzados del IPN, Unidad Irapuato, Km 9.6 Libramiento Norte Carretera Irapuato-Leo ´n, P.O. Box 629, C.P. 36821 Irapuato, Guanajuato, Me ´xico Received 1 December 2008; received in revised form 17 June 2009; accepted 17 June 2009

KEYWORDS Asteraceae; Cytochrome P450-like; Grandiflorenic acid; Kaurenoic acid; Monoginoic acid

Summary Montanoa tomentosa (zoapatle) is a Central American plant used in Mexico in traditional herbal medicine to ease childbirth labor and to cure certain female disorders. Recently, crude extracts of M. tomentosa have been reported to have an aphrodisiacal effect on male rats. The bioactive molecules are the uterotonic diterpenes kaurenoic acid (KA), grandiflorenic acid (GF), and monoginoic acid (MO). Roots of M. tomentosa contain all three diterpenes, whereas in leaves only kaurenoic and GF are present. However, despite the pharmacological importance of these compounds, specific information about their biosynthesis and localization in the plant is not available. In this investigation, we followed the metabolic transformation of a tritium-labeled diterpene-precursor via geranylgeranyl diphosphate into each of the three diterpenes. Inhibitors of gibberellin biosynthesis were used to elucidate the sequence of conversion of the intermediates. Our results suggest the biosynthetic conversion of KA into GF by a putative cytochrome P450-like desaturase. Partial characterization of the enzyme revealed that it requires NADPH and O2 but is inhibited by 50 mM paclobutrazol, suggesting a cytochrome P450 desaturase like enzyme (EC 1.14.14.-). Optimal reaction conditions are 32 1C and a pH of 7.6, respectively. Apparent kinetics parameters for KA gave a Km,app of 36.31 mM, and a Vmax, app of 13.6 nmol KA mg1 protein h 1. Based on the data presented, a putative biosynthetic pathway is proposed for the uterotonic diterpenes of M. tomentosa. & 2009 Elsevier GmbH. All rights reserved.

Abbreviations: CCC, chlormequat chloride; FPP, farnesyl pyrophosphate; FW, fresh weight; GA4, gibberellin A4; GF, grandiflorenic acid; GGPP, geranylgeranyl pyrophosphate; GPP, geranyl pyrophosphate; 3H-GGPP, tritium-labeled geranylgeranyl pyrophosphate; KA, kaurenoic acid; MO, monoginoic acid; PB, paclobutrazol; TNE, trinexapac ethyl. Corresponding author. Tel.: +52 462 6239659; fax: +52 462 6245849. E-mail address: [email protected] (E. Lozoya-Gloria). 0176-1617/$ - see front matter & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2009.06.004

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Introduction

Materials and methods

Montanoa tomentosa belongs to the Asteraceae family and is known as zoapatle in Me´xico. The name zoapatle is derived from the Na ´huatlword cihuapathli meaning women’s medicine. Extracts from this plant have been used in Mexican traditional medicine as natural contraceptives (Hahn et al., 1981) and to facilitate labor and childbirth (Levine et al., 1981; Ponce-Monter et al., 1983; Be´jar et al., 1984). Recently, aphrodisiac effects were reported in male rats (Carro-Jua ´rez et al., 2006). At least 20 different active compounds have been isolated from zoapatle (Braca et al., 2001) including the three tetracyclic diterpenes KA ([4a]-kaur-6-en-18oic acid), GF ([4a]-kaura-9 [11]-16-dien-18-oic acid), and MO (13-methyl-[4a]-norkaur-15-en-18oic acid) (Enriquez et al., 1997). These three compounds have shown uterotonic (CamposBedolla et al., 1997), anti-inflammatory (Paiva et al., 2002), anti-spasmodic (Tirapelli et al., 2005), and anti-cancer effects (Cavalcanti et al., 2006) in mammals. Other reports that have attributed antibacterial and antifungal (Cotoras et al., 2004), gibberellin-like (Vieira et al., 2005) and insecticidal activities of these diterpenes (Topc-u and Go ¨ren, 2007) suggest that they may have important ecological functions. KA, whose biosynthetic pathway has been described in several species, is a precursor of gibberellins (Helliwell et al., 1999, 2001). The level of this phytohormone is low compared with the tetracyclic diterpenes that accumulate in zoapatle roots, leaves and glandular trichomes. Glandular trichomes of M. tomentosa leaves also accumulate volatile compounds (Robles-Zepeda et al., 2009), 26 of which have been identified, mostly monoand sesquiterpenes. This strong accumulation of mono-, sesqui-, and diterpenes suggests a high demand for farnesyl pyrophosphate (FPP), geranyl pyrophosphate (GPP) and geranylgeranyl pyrophosphate (GGPP). The structural similarity of KA, GF, MO, gibberellins, and ent-beyerene (Robinson and West, 1970) indicates a close biosynthetic relationship. Indeed, a putative desaturase enzyme activity in zoapatle is predicted to produce GF from KA. Also, the production of MO in roots may involve similar reactions as those that convert ent-kaurene into ent-kaurenoic acid (KA) (Helliwell et al., 1999). We tested this hypothesis by using 3H-GGPP incorporation and treatment with the inhibitors of gibberellin biosynthesis chlormequat chloride (CCC), paclobutrazol (PB), and trinexapac ethyl (TNE).

Plant material Zoapatle plants collected from the experimental field at CINVESTAV-IPN, Unidad Irapuato in Me´xico, were certified as authentic Montanoa tomentosa (Cerv.) by Dr. Jerzy Rzedowski from the Instituto de Ecologı´a, Michoaca ´n, Me´xico where a voucher specimen (IEB-57689) was deposited. Young shoots were sterilized with 10% commercial bleach for 60 s, rinsed several times with distilled water, and placed on MCM medium (100 g L 1 Murashige and Skoog medium, 30 g L 1 sucrose, 2 mg L 1 kinetin, 0.1 mg L 1 indolacetic acid, and 7 g L 1 agar) at 27 1C and 16 h/8 h light/dark for 3 months for adventitious shoot regeneration. Individual plants were transferred to 1 L pots with sterile soil mixture and grown in a greenhouse at 23–27 1C for 4 months. After that time, plants had four pairs of leaves, representing different levels of maturity, and the youngest 2 pairs (next to the apical meristem) and the roots were used for experiments.

Chemicals Paclobutrazol (PB), chlormequat chloride (CCC), trinexapac ethyl (TNE), abietic acid, gibberellin A4 (GA4), and N, O-Bis (trimethylsilyl) triflouroacetamide with 1% trimethylchlororsilane (BSTFA+1% TMCS) were all from Sigma–Aldrich Co. Ltd. b-NADPH disodium salt and other cofactors (NADP, NADH, NAD, FMN, and FAD) were obtained from USB Corp. Labeled trans-geranylgeranyl pyrophosphate ([1]-3H-GGPP, 15 Ci/mmol) was from Amersham Ltd. Solvents were from J.T. Baker and Silica Gel 60 G from Merck. Dr. Rau ´l Enrı´quez from Instituto de Quı´mica, UNAM Me´xico kindly donated pure samples of kaurenoic acid (KA), grandiflorenic acid (GF), and monoginoic acid (MO).

Analytical procedures A Varian C18 column (250  4.5 mm I.D.; 5 mm particle size) and an Agilent 1200 Series system coupled to a UV diode array detector at 220 nm (Agilent Technologies Inc.) was used for HPLC analysis. A 100 mL injection volume and 1 mL min 1 flow rate were used. Mobile phase was an isocratic system of 70% acetonitrile with 0.05% acetic acid (v/v). Mass spectrum determination was in a GC–MS Hewlett Packard 6890 II series with a HP-1 capillary column (30 m  0.25 mm I.D., covered with a 0.25 mm of dimethylpolysiloxane plate). Mobile

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phase was He at 1 mL min 1 flow rate. Injector temperature was maintained at 200 1C, oven temperature program was 150 1C during 3 min, followed by an increase of 4 1C min 1 and finally maintained at 300 1C for 20 min.

on a pure 3H-GGPP standard curve to measure a dose–response between 0.1 and 2 mCi. All assays were performed in triplicate.

Metabolites extraction and analysis

The inhibitors of gibberellin biosynthesis were selected based on their mode of action with CCC blocking the early cyclase involved in the formation of kaurane diterpenes, PB blocking cytochrome P450-dependent monooxygenases, and TNE blocking the 3b-hydroxylase involved in the formation of highly active GAs from inactive precursors (Rademacher, 2000). Solutions of each inhibitor (0.5–10 mM) were prepared in MES buffer (20 mM, pH 6.5) and were used for dose–response experiments. Leaves of zoapatle shoots were independently sprayed with these solutions (about 10 mL per plant). For experiments with roots, whole M. tomentosa plants were grown in the absence of soil and the roots were immersed in glass flasks containing 60 mL of each inhibitory compound. All assays were performed in triplicate.

Plant tissue was ground with liquid N2, sonicated for 20 min, and extracted three times with hexane:ethyl acetate (85:15, v/v) with 200 mM abietic acid added as internal standard. This crude extract was separated by silica gel 60G onto preparative TLC plates with hexane:ethyl acetate (90:10, v/v). Plate edges were sprayed with 1 g vanillin in 100 mL methanol and 0.2 mL sulfuric acid to develop a color reaction after air dryer heating. Their blue–violet color and respective 0.5 Rf, corresponding to the Rf of pure samples, identified the GF, KA, and MO mixture. This diterpene mixture was harvested from the silica gel and extracted five times with hexane:ethyl acetate (90:10, v/v). The diterpenes recovered in the organic solvent were concentrated to dryness and suspended in 1 mL n-hexane for treatment with BSTFA+1% TMCS for GC–MS, or in 1 mL pure MeOH for HPLC analysis. Identities of diterpenes were confirmed by their respective retention times and their mass spectra as follows. KA: m/z 374 (M+ 86% rel. int.), 359 (97), 331 (66), 257 (1 0 0), 241 (68), 143 (29), 91 (30), 73 (81). GF: m/z 372 (M+ 17% rel. int.), 357 (71), 254 (35), 239 (100), 183 (10), 143 (16), 91 (11), 73 (25). MO: m/z 374 (M+ 100% rel. int.), 359 (74), 284 (71), 257 (38), 215 (44), 134 (45), 105 (48), 73 (64).

Radiolabeling experiments The feeding experiments with 3H-GGPP were carried out according to Fleming et al. (1994) with some modifications. Tissue of roots or young leaves (500 mg), sliced into segments one mm in length, were incubated in 10 mL glass tubes with 0.75 mCi of [1]-3H-GGPP (15 Ci/mmol) in 4 mL of MES buffer (20 mM, pH 6.5) with gently shaking at 25 1C for 24 h. Samples were harvested every 2 h and immediately extracted with hexane:ethyl acetate (85:15, v/v) followed by TLC pre-purification of labeled diterpenes and HPLC separation as described above. The radioactive products, separated by HPLC, were recovered (Coleman et al., 1999), aliquots mixed with 5 mL scintillation cocktail (ScintiVerse* I Cocktail Scintanalyzed* Fisher Scientific) and counted in a Liquid Scintillation Counter Tri-Carbs2100TR (Perkin Elmer). The incorporation of 3H-GGPP into diterpenes was determined based

Inhibition experiments

Enzyme activity assays Freshly harvested leaves of M. tomentosa (5 g) were ground in mortar at 4 1C with 20 mL of 100 mM Tris–HCI (pH 8.0), 10 mM b-mercaptoethanol, 1% (w/v) polyvinylpolypirrolydone, and 1% (w/v) polyvinylpyrrolydone (MW: 40,000) buffer. The homogenate was stirred for 5 min on ice, filtered through two layers of cheesecloth and centrifuged at 10,000g for 15 min. The supernatant was separated by ultracentrifugation at 190,000g for 90 min to obtain the microsomal fraction. Each microsomal pellet was suspended in 2 mL Na-phosphate buffer (pH 8.0) containing 1 mM EDTA, 1 mM L-ascorbic acid, 1 mM Na2S2O5, 200 mM MgCl2, and 10% (v/v) glycerol. Microsomal fractions were assayed immediately or stored at 80 1C until use. Enzyme assays were performed in 5 mL screw-capped tubes containing 900 mL of microsomal protein (0.4 mg), and 400 mM NADPH. The reaction was initiated by adding 300 mM KA solution dissolved in 20% EtOH (96% HPLC pure) that had been isolated and purified from M. tomentosa (Batista et al., 2005) and after 1 h incubation at 32 1C with gentle shaking at 100 rpm in a rotary shaker, the reaction was stopped with one volume of acetone and abietic acid (100 mM) was added as an internal standard. The reaction mixture was extracted three times with 2 mL hexane:ethyl acetate (85:15, v/v), the combined organic phases were concentrated to dryness with N2 gas, the dry residue dissolved in

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1 mL MeOH and the samples were analyzed by HPLC as described in Metabolite extraction and analysis. The pH optima of this reaction was determined by mixing mono- and dibasic Na-phosphate buffers to obtain the pHs 5.8, 6.4, 7.0, 7.6, and 8.0, while Tris–HCl was used for buffers at pH 8.5 and 9.0. The temperature optima of the reaction was determined using an incubator shaker at 4, 15, 25, 32, 37, and 50 1C. Apparent kinetic parameters were determined with 10–400 mM KA and data were analyzed by non-linear regression using SOLVERs Software. All assays were performed with 1 mg mL 1 of protein in the assay mixture as determined by Bradford protein assay (Bio-Rad, 500-0203, Mississauga, Ontario, Canada). Glandular trichome protein extracts were obtained according to Yerguer et al. (1992), starting from 200 g fresh weight (FW) young leaves. All assays were performed in triplicate with four repetitions each one.

Results Incubation of leaf and root sections with 3H-GGPP Tissues incubated with 3H-GGPP accumulated transiently labeled diterpenes, reaching a maximum after 8–10 h of treatment and showing a decline over the next 24 h of incubation (Figure 1A and B). The highest overall incorporation was observed into MO of roots at 4.43  104 dpm mmol 1 while the highest incorporation found in leaves was into KA at 3.37  104 dpm mmol 1. In roots, radioactivity was also found in KA and GF, however at lower levels in comparison to MO (60% and 22%, respectively, Figure 1B). In leaves, only GF was labeled in addition to KA (70%, Figure 1A). Further experiments suggested that small amounts of 3 H-GGPP were incorporated into GA4 as detected by HPLC using a commercial GA4 standard that co-chromatographed with the radioactive peak (data not shown).

Application of inhibitors of GA biosynthesis Glandular trichomes accumulate KA and GF in zoapatle, and it is known that the application of 0.3 mM PB to Arabidopsis wild-type plants suppressed trichome initiation on the abaxial epidermis (Chien and Sussex, 1996). In order to investigate if these inhibitors affect zoapatle plants as well, dose–response experiments were carried out. We found that treatment of zoapatle plants with 1.5 mM CCC, 800 mM PB, and 900 mM TNE, did

Figure 1. Incorporation of 3H-GGPP into KA, GF, and MO after incubation of leaves (A) and roots (B) for 24 h. Radioactive KA, GF, and MO were collected after HPLC separation and counted. The maximum specific activity (100%) was 3.37  104 mmol 1 in A and 4.43  104 dpm mmol 1 in B. Bars represent standard deviation.

not have any phenotypic consequence but the inhibitory effect on GAs biosynthesis was present (data not shown). A feeding experiment showed 24 h after the addition of the inhibitors that PB and CCC interrupted the incorporation of 3H-GGPP into KA, GF, and MO (Figure 2) in leaf and root tissues. TNE, on the other hand, did not inhibit the incorporation of label into KA in leaves (Figure 2A) or MO in roots (Figure 2C), but increased 50% the incorporation into GF in leaves (Figure 2B) compared with controls.

Identification of KA desaturase enzyme activity in M. tomentosa microsomes When microsomal pellets were incubated with KA in the presence of NADPH and O2, its conversion to GF was clearly detected. However, decreased or no enzyme activity was detected when NADPH was replaced with other cofactors such as NADP, NAD, NADH, NAD, FAD, and FMN (Table 1). 2 Additional enzyme assays using crude cell extracts before ultracentrifugation or crude extracts from isolated glandular trichomes also showed greatly diminished enzyme activities (Table 1). Microsomes appeared to be enriched in KA desaturase activity since it was

ARTICLE IN PRESS Biosynthesis of uterotonic diterpenes from Montanoa tomentosa (zoapatle) Table 1. Assays M. tomentosa.

of

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KA

desaturase

Assay conditionsa

Specific activity (%)b

Me+400 mM NADPH Te+400 mM NADPH Ce+400 mM NADPH Me+400 mM NADH Me+400 mM NADPH+50 mM PB Me+400 mM NADPH+100 mM NADP+ Me+400 mM NADH+100 mM NAD+ Me+400 mM NADPH+2 mM FMN+2 mM FAD

100 12 20 48 7 93 41 56

in

All assays were carried out in the presence of O2 and 300 mM KA: (Me), mesophyll microsomal extract; (Te), trichome extract and (Ce), crude extract. All assays were performed in triplicate with four repetitions. Table data are the average of all results. a Microsomal mesophyll and trichome protein concentrations were 0.4 and 0.2 mg mL 1, respectively. b The maximum specific activity (100%) was 13.2 nmol KA mg 1 protein h 1.

Discussion

Figure 2. Effect of inhibitors of gibberellin biosynthesis CCC, PB, and TNE on 3H-GGPP incorporation in KA (A) and GF (B) in leaves, and in MO (C) in roots. Controls (Ctr) are without inhibitors. Maximum specific activity (100%) was 3.44  104 dpm mmol 1 for KA, 2.93  104 dpm mmol 1 for GF, and 4.21  104 dpm mmol 1 for MO. Bars represent the standard deviation.

absent in the soluble protein fraction (190,000g supernatant). It is well-known that PB is a plant growth retardant that specifically inhibits the conversion of ent-kaurene to ent-KA biosynthesis in the gibberellin pathway (Hedden and Graebe, 1985). Enzyme assays using PB (50–100 mM) also inhibited this putative KA desaturase enzyme activity from 13.2 nmol KA mg 1 protein h 1 (100%) down to 0.924 nmol mg 1 h 1 (7%). Partial biochemical characterization of this putative KA desaturase was carried out using microsomal pellets in the presence of different amounts of substrate and the GF reaction product was assayed by HPLC. This microsomal enzyme showed typical Michaelis–Menten kinetics (Figure 3) with an apparent Km,app of 36.31 mM and Vmax,app was 13.6 nmol mg 1 h 1. The temperature and pH optima for the reaction were 32 1C and 7.6, respectively.

Our study shows that 3H-GGPP is taken up into zoapatle leaves and roots to produce bioactive diterpenes (Figure 1A and B). In leaves, 3H-GGPP is first converted into KA, which is then oxidized to GF by an oxidase found in microsomes (Table 1 and Figure 3). After an initial accumulation of KA and GF, both were rapidly metabolized. These results support previous reports suggesting that GF and other similar tetracyclic diterpenes might be used as precursors for gibberellin biosynthesis in plants that involve uncharacterized pathways (Kim et al., 1996), alternative to the pathway described in the literature (Yamaguchi, 2006). In contrast to leaves, roots appear to convert 3H-GGPP into KA and GF but also into MO (Figure 1B), and this raises the issue of the biosynthetic relationship between MO, KA, and GF. The high recovery of radioactive MO in roots (Figures 1B and 2C) suggests that an additional, possibly independent pathway is involved that differs from KA, GF, and gibberellin biosynthesis (Figure 4). This pathway could include the oxidation of ent-beyerene by a cytochrome P450 enzyme that would lead to the formation of MO in a reaction similar to the ent-kaurene oxidation (Helliwell et al., 1999). The low incorporation of 3 H-GGPP into MO in the presence of PB supports this hypothesis (Figure 2C). The effects of inhibitors of gibberellins indicate that both KA and GF share common precursors. The unexpected increase of labeled GF in TNE-treated leaves of 1.4  104 dpm mmol 1 (50%) (Figure 2B)

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Figure 3. Putative KA desaturase kinetic for 10–400 mM KA substrate. The final mixture contained 0.4 mg of microsomal protein with 400 mM NADPH at 32 1C and pH 7.6. Data were analyzed using the SOLVERs Software. Bars represent the standard deviation.

Figure 4. Proposed biosynthetic pathway for KA, GF, and MO in leaves and roots of M. tomentosa: GGPP (1), entkaurene (2), ent-beyerene (3), KA (4), GF (5), and MO (6). Inhibition sites for CCC, PB, and TNE are indicated.

suggest a key role of this diterpene in the biosynthesis of GAs in zoapatle. In roots, TNE had no effect on MO accumulation, and cold MO was always higher than the labeled compound compared with leaves (Figure 2C). This fact together with the higher amount of MO found in roots may be explained by a faster biosynthesis of MO from GGPP.

However, it is also possible that this effect was primarily due to a better incorporation of GGPP in roots compared with leaves. Our studies with cell-free extracts showed that isolated microsomes contained a putative NADPHdependent KA desaturase that converts KA into GF (Table 1). The cofactor requirements, sub-cellular localization and inhibition of this desaturase by PB suggest it to be a cytochrome P450-like enzyme. Similar desaturation processes in sterol biosynthesis (Morikawa et al., 2006) also involve the participation of P450 enzymes. Kinetic analyses of this desaturase were performed and the Km,app (36.31 mM) was higher than that of the KA hydroxylase (Kim et al., 1996) isolated from Stevia reabaudiana leaves using the same substrate. However, it was lower in comparison to other reactions catalyzing similar desaturation processes (Taton and Rahier, 1996). No enzyme activity was found in microsomal extracts of glandular trichomes suggesting that GF biosynthesis was located in the foliar tissue followed by KA and GF accumulation.

Acknowledgements We would like to thank Yolanda Rodrı´guez, Cristina Elizarraraz, Enrique Ramı´rez, Antonio Vera, and Dr. He´ctor G. Nu ´n ˜ez-Palenius from CINVESTAV-IPN Campus Guanajuato for their support and advise on HPLC, GC–MS, and LCS. This work was supported by a grant from ICGEB at Trieste, Italy. NVR is recipient of a Ph.D. scholarship from CONACyT Me´xico.

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