From the United States Department of Agriculture/Agricultural Research Service, Natural Products Utilization Research Unit, University, Mississippi 38677
Received for publication, April 22, 2003 , and in revised form, May 21, 2003.
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ABSTRACT |
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INTRODUCTION |
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The primary mechanism of phytotoxic action of 1 is associated with inhibition of photosynthesis in higher plant systems by competing for the binding site of plastoquinone on photosystem II (46). This lipophilic p-benzoquinone is also known to hinder electron transfer reactions involved in mitochondrial respiration and to inhibit the enzyme p-hydroxyphenylpyruvate dioxygenase (7, 8).
The herbicidal and allelopathic properties of 1 make isolation of the genes responsible for its biosynthesis desirable, as manipulation of those genes in sorghum or their introduction in other plant species could provide a better understanding of the role of 1 in plant-plant interaction and natural weed control. To identify the genes and characterize the enzymes they encode, it is necessary to determine the steps involved in the biosynthesis of 1. Little has been reported on this subject.
Sorgoleone is found exclusively as a hydrophobic droplet exuding from the tips of root hairs (2), and a recent investigation of sorghum roots suggested that the production of 1 is compartmentalized in the highly physiologically active root hairs (9). The biosynthesis of secondary metabolites occurring in root hairs has not been studied in detail, although other specialized cells at the interface between a plant surface and its environment (i.e. trichomes) are known to produce unique secondary metabolites (1012).
It has been postulated that the biosynthesis of 1 and related resorcinolic lipids is the result of the convergence of two pathways, namely the fatty acid biosynthetic pathway for the synthesis of the aliphatic tail and the activity of polyketide synthase-type enzymes for the formation of the quinone head (13, 14). Although this convergence of pathways has been demonstrated for aflatoxin biosynthesis in fungi (15), examples in plants have not been completely elucidated but have been derived from incomplete experimental data complemented with biogenetic assumptions that have not necessarily been demonstrated for these compounds (14). In the case of 1, Fate and Lynn (16) demonstrated that exogenous isotopically labeled acetate was incorporated into the quinone head but not into the tail. No further isotopic labeling studies of the hydrophobic tail, nor any studies of the biogenesis of the substituents on the quinone head, have been reported. Consequently, many questions regarding the biosynthesis of this important allelochemical have been left unanswered (17). The objective of this study was to determine the entire biosynthetic pathway of 1 via retrobiosynthetic NMR analysis of the root exudate of sorghum seedlings grown in the presence of suitable isotopically labeled substrates and to identify key metabolic intermediates.
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EXPERIMENTAL PROCEDURES |
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Seed Sterilization and Growth of SeedlingsSorghum (SX17) seeds were surface sterilized in 20% Clorox bleach (final concentration of 1% sodium hypochlorite) for 10 min and rinsed with deionized water. The seeds were poured into a gravity funnel lined with cheesecloth and allowed to dry overnight in a laminar flow hood. Although not completely aseptic, this method allowed the seed to grow free of observable microbial contamination during the experiments.
Twenty seeds (approximately 500 mg) were placed in 20 x 100-mm sterile polystyrene Petri dishes (Falcon) over the surface of sterile Whatman No. 1 filter paper (90 mm diameter). Five ml of sterile water was added to the dish, and the seeds were covered with a second sterile filter paper. The dishes were sealed and incubated in the dark at 25 °C. Sorgoleone was extracted from 6-day-old seedlings in all experiments unless otherwise indicated.
Labeling StudiesStock solutions of the labeled compounds
were prepared as a 1:1 or 1:3 ratio of 13C:12C isotopes
at 10 mM concentration of the combined forms, except for acetate
and pyruvate, which were prepared in 1 mM stock solutions. The
labeled precursors were added to the Petri dishes on the 4th day. The Petri
dishes were opened, and the upper filters were discarded. Excess water in the
dishes was removed and replaced with 2 ml of either 500 µM
[4-13C]3-hydroxybutyrate, 500 µM
[1,2-13C2]palmitate, 500 µM
[13C]methyl-L-methionine, 1 mM
[1-13C]acetate, 1 mM [2-13C]acetate, or 2
mM D-[2-13C]glucose. The excess water in the
Petri dish was discarded because it contained 0.15% of the total amount
of 1 produced by the roots. The same procedure was repeated on the 5th
and 6th day. At the end of the labeling period, the roots were excised, and
1 was extracted as described below. All labeling procedures were done
under low-intensity green light to prevent the formation of anthocyanins by
sorghum roots.
Extraction and Purification of SorgoleoneSorgoleone was extracted by immersing the roots in chloroform for 1 min. Sorgoleone absorbed to the bottom filter paper (about 20% of the final weight of sorgoleone) was also extracted with chloroform. The extract was filtered through Whatman No. 1 filter paper and evaporated in vacuo (Büchi Rotovapor, Brinkmann Instruments) at 30 °C. The dried yellow residue was redissolved in chloroform, transferred to vials, and dried under nitrogen. The crude extract was applied to 20 x 20-cm aluminum-backed silica thin layer plates and separated using hexane:2-propanol (9:1 v/v). The sorgoleone band (RF 0.36) was cut out and eluted from the silica by washing twice with 200 ml of CHCl3. The pooled material was dried under nitrogen and stored at 20 °C until NMR analysis.
NMR and Mass Spectrometry Analysis13C NMR experiments were carried out in CDCl3 on a Bruker Avance DPX 300 (Bruker, Billerica, MA) instrument using standard Bruker software (XWINNMR version 1.3), with the following variables: 30° pulse, 2.17 µs; repetition time, 3 s; spectral width, 15.1 kHz; temperature, 27 °C. The decoupling pulse was 90 µs at 18 db.
13C NMR spectra of labeled and unlabeled (natural abundance) sorgoleone were obtained under the same experimental conditions. Each carbon peak was integrated and 13C enrichments were calculated using the formula: (a b)/b, where a is the integrated NMR signal of the enriched carbon in the labeled sorgoleone and b is the integrated NMR signal of the same carbon in the unlabeled sorgoleone (19).
Gas chromatography-mass spectrometry (GC-MS)1 was performed on a JEOL (JEOL USA, Inc., Peabody, MA) GCMate II system. The GC temperature program was as follows: the initial 120 °C was raised to 280 °C at a rate of 20 °C/min and held at this temperature for 1 min, then raised to 300 °C at a rate of 10 °C/min and held at this temperature for 4 min. The GC capillary column was ZB-50 (0.25 mm inner diameter, 0.25 mm film thickness, 30 m length; Phenomenex, Torrance, CA). The carrier gas was ultrahigh purity helium at a 1 ml/min flow rate. The inlet (splitless), GC interface, and ion chamber temperatures were 250, 250, and 230 °C, respectively. Samples were prepared in CHCl3 at 1 mg/ml concentrations, and the volume of samples injected was 1 µl.
Isolation and Identification of Resorcinolic Metabolic Intermediates in Sorghum bicolor Root HairsRoot hairs were isolated from unlabeled sorghum roots as described by Röhm and Werner (20), with the addition that the liquid nitrogen containing the isolated root hairs was decanted by pouring it through a stainless steel mesh sieve (No. 60, 250 µm) to remove larger root debris. Isolated root hairs were powdered in liquid nitrogen, extracted with 10 ml of 95% MeOH, and sonicated in a sonication bath (Branson 3510 Ultrasonic Cleaner) for 15 min. The extract was filtered through a Whatman No. 1 filter. The extraction procedure was repeated with the residue two more times, and the organic supernatants were pooled. The organic phase was dried with magnesium sulfate and evaporated in vacuo. The residue was dissolved in a small amount of ethyl acetate/hexane (80:20 v/v) and applied to aluminum-backed silica plates. The extract was chromatographed in CHCl3/ethyl acetate/methanol (85:8:7 v/v), and the UV active band with an RF identical to that of a 5-pentadecyl resorcinol (Chem Service, Inc. West Chester, PA) was eluted from the plate (21). The semipurified sample (12 mg) was diluted in 1 ml of CHCl3 and injected in a JEOL GCmate mass spectrometer as described previously. The injection volume was 1 µl.
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RESULTS AND DISCUSSION |
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Labeling with [2-13C]acetate and [2-13C]malonate yielded similar patterns of incorporation on carbon atoms 2, 4, and 6 (151.9, 182.1, and 102.5 ppm, respectively) (Figs. 2B and 3). The percent incorporation was similar for both precursors ranging from 3.6 to 6.2% (Table I). Labeling with [1-13C]acetate led to enrichment in carbons 1 and 5 (183.2 and 161.5 ppm, respectively), but no incorporation was detected in carbon atom 3 (Fig. 2C). These results indicate that, at the mechanistic level, the polyketide synthase (PKS) involved in the production of 2 catalyzes an orsellinic acid-type cyclization of the linear tetraketide intermediate (Fig. 3, steps III and IV), such as that catalyzed by stilbene synthase rather than a phloroacetophenone-type cyclization typically associated with chalcone synthase (23, 24). Although stilbene and chalcone synthase have similar nucleotide sequences, this difference in their reaction mechanism may provide useful information to help identify the genes encoding for the proper polyketide synthase participating in the biosynthesis of 1.
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The fact that carbon 3 (119.6 ppm) of the quinone head is not labeled in
the presence of [1-13C]acetate
(Table I), whereas carbons 1
and 5 become isotopically labeled, precludes the possibility that acetate is
the starter unit for the PKS involved in the synthesis of 1. Because
1, with a pentadecatriene lipid tail, accounts for >80% the sorghum
root exudate, the starter unit is likely to be a novel 9,12,15
C16:3-CoA. However, the exudate also contains minor components consisting of
sorgoleone derivatives with aliphatic tails of 15 or 17 carbons and various
degrees of unsaturation (3),
suggesting that the PKS involved in the synthesis of 1 can utilize
other C16 and C18 fatty acyl-CoAs as starter units.
The product of PKS activity is 2 (2-[(8'Z,11'Z)-8',11',14'-pentadecatriene]resorcinol), a cardol-like intermediate as shown in Fig. 3, which was isolated and identified by GC-MS in sorghum root hair extracts. The extracted ion chromatogram showed m/z 313.1 [M+ H] indicative of 2 (retention time 7 min 55 s) with characteristic fragment ions m/z 282 [M+ O2], 206 (the pentadecatriene fragment), 149 [206+ C4H7], 135 [149+ CH2], 123 (methyl resorcinol fragment) supporting the identity of 2. The existence of 5-pentadecatriene resorcinol and related analogues has also been reported in grass species phylogenetically related to sorghum (14, 21, 2528).
Attempts to label the lipid tail with acetate, malonate, and pyruvate were unsuccessful. Other substrates tested, such as L-acetylcarnitine (which is involved in the transport of fatty acids across the mitochondrial membranes and possibly fatty acid biosynthesis (29, 30)) and 3-hydroxybutyrate (in which the acyl carrier protein conjugate is an intermediate in the early stages of fatty acid biosynthesis (29)), also failed to be incorporated. Further labeling experiments, bypassing fatty acid synthesis by providing labeled palmitate or palmitoyl-CoA, also proved unsuccessful. However, inhibition of root growth caused by palmitate and its low water solubility (log p = 7.15) and the low root uptake of palmitoyl-CoA were limiting factors interfering with incorporation of these substrates.
Labeling of the alkyl carbons, in a pattern consistent with the catalytic mechanism of fatty-acid synthase (31), was achieved only with D-[2-13C]glucose (Fig. 2, D and F; and Fig. 4, step II). Enrichment was observed in carbons 2', 4', 6', 8', 10', 12', and 14', corresponding to increased signals at 28.4, 29.7, 30.0, 130.8, 25.9, 127.2, and 137.3 ppm, respectively (Fig. 4). The level of isotope incorporation in the tail ranged from 1.2 to 3.7% (Table I).
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Glucose is often used in retrobiosynthetic NMR analysis of pathways because it overcomes limitations associated with subcellular compartmentalization (22). In this study, the contrast between the labeling patterns obtained with acetate, malonate, and glucose is particularly striking because fatty-acid synthase uses both acetate and malonate as substrate, yet no incorporation in the tail was observed with these latter substrates.
Incorporation of biosynthetic precursors into fatty acids is known to be problematic because their synthesis is compartmentalized in plastids. In most plant cells, exogenous acetate is not usually incorporated into C18 and shorter fatty acids because acetate does not readily cross the plastidic membranes, and plastids have their own endogenous pool of acetate used for fatty acid synthesis (3234). Even when endogenous acetate has been reported to be incorporated in short fatty acids, glucose has been demonstrated to be a far better substrate, because it can either be imported in nongreen plastids directly as glucose 6-phosphate and used as the source for the plastidic pool of acetate (35, 36) or be broken down to triose phosphates in the cytosol and taken up in plastids via triose phosphate translocators (37).
An important aspect of the synthesis of 1 is the unusual
desaturation pattern of the aliphatic tail. Most of the steps leading to the
formation of 9 C16:1 fatty acids are ubiquitous in plants. However, the
next two desaturation steps required to yield
9,12 C16:2 and especially
9,12,15 C16:3 are more unusual and must be catalyzed by as-yet-unknown
fatty-acid desaturases (Fig. 4,
reaction IV). It is important to differentiate this novel
9,12,15 hexadecatrienoyl-CoA derivative (3) involved in the
biosynthesis of 1 from the well characterized
3 and
6
hexadecatrienoic acids that are important components of plastidic membranes
(
7,10,13 and
4,7,10, respectively)
(3840).
The remaining steps involved in the biosynthesis of 1 are the methylation of the hydroxyl on carbon 5 and the addition of two hydroxyl groups on carbons 2 and 4. Whereas the order of these reactions is not certain, the methylation is catalyzed by an O-methyltransferase (OMT) (41), and the addition of the hydroxyl groups is most likely catalyzed by the action of a P450 monooxygenase on the resorcinol ring (4246). As expected, isotopic labeling of this methyl group was achieved with [13C]methyl-methionine, suggesting the presence of a S-adenosyl-L-methionine-dependent OMT. Methylation with labeled methionine yielded the greatest percent incorporation (nearly 27%) relative to the other substrates used in this study (Table I).
The presence of 2 in root hair suggests that the methylation most likely occurs on 2 (Fig. 3) rather than a putative tetrahydroxy resorcinolic lipid intermediate. In any case, both intermediates are symmetrical with respect to the position of the hydroxyl groups. Therefore, the OMT would not distinguish between the hydroxyl group on carbon 1 and the one on carbon 5. The OMT found in sorghum root hairs appears to be quite specific for this substrate because a 4,6-dimethylated form of 1 is present only in small amounts in sorghum exudate (16, 47, 48). OMTs have been recognized to catalyze highly regiospecific reactions and to be quite substrate-specific (43, 4952). In light of the dual hydrophobic/hydrophilic nature of 1, the OMT involved in this pathway is also expected to be substrate-specific with a substrate-binding domain that can accommodate the very lipophilic tail of 1. Once a single methylation occurs, subsequent hydroxylation by a putative P450 monooxygenase (53) leads to the formation of 1 in its reduced hydroquinone form. The enzymatic synthesis apparently ends with the formation of the reduced form of 1 (a hydroquinone known as sorghum xenognosin for Striga germination) (54). The oxidation of the hydroquinone into the benzoquinone is most likely the result of autoxidation upon exposure to air (1, 16).
In conclusion, the biosynthetic pathway of 1 initiates in the
plastids with the synthesis of an unusual 9,12,15 hexadecatrienoic acid
through the action of fatty-acid synthase and fatty-acid desaturases. Outside
of the plastids, this polyunsaturated fatty acid serves as the starter unit
for PKS yielding a resorcinolic lipid intermediate. The action of a
S-adenosylmethionine-dependent OMT subsequently methylates the
hydroxyl group on carbon 5, and the enzymatic synthesis ends with the
hydroxylation in the aromatic ring by a P450 monooxygenase. Our research
laboratory is now focusing on the characterization of the biochemical
machinery involved in the biosynthesis of 1 and on identifying the
genes encoding these enzymes.
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FOOTNOTES |
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To whom correspondence should be addressed. Tel.: 662-915-1039; Fax:
662-915-1035; E-mail:
fdayan{at}ars.usda.gov.
1 The abbreviations used are: GC-MS, gas chromatography-mass spectrometry;
PKS, polyketide synthase; OMT, O-methyltransferase.
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ACKNOWLEDGMENTS |
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REFERENCES |
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