JXB Advance Access originally published online on October 10, 2007
Journal of Experimental Botany 2007 58(12):3263-3272; doi:10.1093/jxb/erm173
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© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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RESEARCH PAPER |
Biosynthesis of lipid resorcinols and benzoquinones in isolated secretory plant root hairs
1United States Department of Agriculture, Agricultural Research Service, Natural Products Utilization Research Unit, PO Box 8048, University, MS 38677, USA
2National Center for Natural Products Research, School of Pharmacy, University of Mississippi, University, MS 38677, USA
* To whom correspondence should be addressed. E-mail: fdayan{at}olemiss.edu
Received 16 May 2007; Revised 25 May 2007 Accepted 26 June 2007
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Abstract |
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The primary functions of root hairs are to increase the root surface area and to aid plants in water and nutrient uptake. However, some root hairs also have secretory functions and exude bioactive secondary metabolites. Sorghum (Sorghum bicolor) root hairs release a substantial amount of phenolic lipids including sorgoleone, a 3-pentadecatriene benzoquinone. The activity of the key enzymes involved in the biosynthesis of lipid resorcinols and benzoquinones was measured directly in isolated root hair preparations obtained from 6-d-old roots. The purified root hair preparation readily converted long-chain acyl-CoA starter units to their corresponding lipid resorcinols and decanoyl-CoA was the best substrate, yielding a 5-n-nonyl-resorcinol. The isolated root hair preparation also had high S-adenosyl-L-methionine-dependent O-methyltransferase activity, which catalyses the methylation of several 5-n-alkyl-resorcinols. Optimum activity was with 5-n-pentyl-resorcinol. Isolated root hairs also exhibited hydroxylase activity (putatively a P450 mono-oxygenase) that reacted with the lipophilic 5-pentadecyl-resorcinol substrate. The in situ hydroxylase activity was low relative to the other enzymes studied, but was still detectable in isolated root hairs. Thus, sorghum root hairs possess the entire metabolic machinery necessary for the biosynthesis of lipid resorcinols and benzoquinones. This will have implications for the genetic engineering of bioactive lipid resorcinols and benzoquinones in sorghum and in other plant species. It also demonstrates that some root hairs can function as specialized cells for the production of bioactive secondary metabolites.
Key words: Alkyl-resorcinols, allelopathy, essential oil, hydroxylase, O-methyltransferase, P450 mono-oxygenase, polyketide synthase, root hair isolation, sorgoleone
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Introduction |
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Plants, in their continual interactions with potential pathogens and competitors, have enhanced their survival potential by producing a wide array of natural defence compounds (Duke et al., 2002; Maor and Shirasu, 2005; Dudareva et al., 2006). The synthesis of these bioactive secondary metabolites is often compartmentalized in specialized epidermal cells called trichomes (Amme et al., 2005; Boughton et al., 2005). These anatomical structures are at the interface of the plant with its biotic environment and serve as first line of defence against biotic attacks (Dayan and Duke, 2003).
Trichomes can be small single-celled or large multicellular structures and their morphology is extremely diverse (Werker, 2000). While a variety of exhaustive classification systems exist to characterize the different types, trichomes can simply be described as glandular or non-glandular (hair-like). Glandular trichomes are normally physiologically active and secrete secondary metabolites into the subcuticular space at their tips.
Root hairs are classified as trichomes present on roots (Werker, 2000), and their development is under the same genetic controls as aerial trichomes (Kellogg, 2001). Root hairs are protrusions of single epidermal cells and are structurally less diverse than other aerial trichomes (Grierson and Schiefelbein, 2002). While the functions of root hairs are typically associated with increasing the root surface area and enhancing water and nutrient uptake (Gilroy and Jones, 2000), some plants have developed specialized root hairs that produce and release bioactive secondary products to the environment. Root hairs of Sorghum spp. secrete large amounts of an oily exudate (Fig. 1A) (Hess et al., 1992; Nimbal et al., 1996a; Czarnota et al., 2003b). The oily substance exuding from the tips of root hairs (Fig. 1B) contains between 80–95% sorgoleone, a lipid benzoquinone and several structural congeners which differ in their substitution pattern on the ring and in the length and level of unsaturation in the tail (Netzly et al., 1988; Fate and Lynn, 1996; Rimando et al., 1998; Kagan et al., 2003).
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This lipid benzoquinone suppresses the growth of a large number of plant species, but it is most active on small-seeded plants (Netzly and Butler, 1986; Einhellig and Souza, 1992; Nimbal et al., 1996a; Rimando et al., 1998; de Souza et al., 1999; de Almeida Barbosa et al., 2001). The primary mechanism of action of this secondary metabolite is not fully established, but this compound is known to inhibit several physiological processes in plants. It is a structural analogue of both plastoquinone and ubiquinone and, as a result, it is a potent inhibitor of photosynthetic and mitochondrial electron transport (Rasmussen et al., 1992; Einhellig et al., 1993; Nimbal et al., 1996b; Gonzalez et al., 1997; Rimando et al., 1998; Kagan et al., 2003). More recently, lipid benzoquinones were also shown to inhibit the enzyme p-hydroxyphenylpyruvate dioxygenase and to interfere with root H+-ATPase and water uptake (Meazza et al., 2002; Hejl and Koster, 2004).
Sorghum accumulates sorgoleone and its analogues in matured root hairs (Yang et al., 2004; Dayan, 2006) and the production (approximately 18 mg g–1 root dry weight) is optimum at temperatures ranging from 25 °C to 35 °C. The biosynthesis of these molecules appears to be stimulated by the presence of root exudates of other plants, suggesting that the allelopathic potential of sorghum may be enhanced in the presence of competitors (Dayan, 2006).
It has been postulated that the biosynthesis of sorgoleone is compartmentalized in root hairs (Czarnota et al., 2003a) and the biosynthetic steps have been elucidated using retrobiosynthetic NMR analysis (Fate and Lynn, 1996; Dayan et al., 2003). Synthesis involves the convergence of the fatty acid and polyketide synthase (PKS) pathways to produce a 5-pentadecatrienyl-resorcinol metabolic intermediate. This intermediate is subsequently methylated by a type I S-adenosyl-L-methionine-dependent O-methyltransferase (OMT), and hydroxylated by a putative P450 mono-oxygenase to produce sorgoleone in its hydroquinone form. However, no biochemical study on the native enzymes present in sorghum root hairs has been carried out to date, partly because of the difficulties associated with isolating these delicate cells in the absence of large amounts of sorgoleone that interferes with enzyme assays. Therefore, it is postulated that root hairs would possess enzymes required for the synthesis of lipid resorcinols and benzoquinones (Fig. 2).
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Sorghum was used as a model system to develop a method suitable for obtaining purified root hairs that retain high enzymatic activity. The substrate specificity of native PKS and OMT participating in the biosynthesis and methylation of lipid resorcinols was tested on a variety of acyl-CoA starter units and lipid resorcinols, respectively. In situ hydroxylase activity associated with the biosynthesis of lipid benzoquinones was also observed at low levels. Therefore, the root hairs of sorghum possess all of the enzymes required to catalyse the entire biosynthesis of lipid resorcinols and benzoquinones.
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Materials and methods |
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Plant material and growth conditions
Seeds of the sorghum cultivar SX17 (S. bicolorxS. sudanense) (Dekalb Genetics, Dekalb, IL) were surface-sterilized by soaking for 10 min in 10% bleach and rinsing with deionized water. For root hair production, seeds were grown in the dark on a capillary mat system (dimensions 63x35 cm), as described by Czarnota et al. (2001), except that the heating element was omitted, and seeds were placed directly onto the screen. Roots extending below the screen were harvested 6 days after planting. Roots were stored at –80 °C until root hair isolation.
Isolation of sorghum root hairs
Small chips of dry ice (about 5 mm in diameter) were placed in a 50 ml Falcon tube maintained on ice as adapted from Yerger et al. (1992). The tube was filled with liquid nitrogen and 4 g of frozen sorghum roots were added. A few more chips of dry ice were added and most of the liquid nitrogen was allowed to evaporate. The tube was sealed and vortexed vigorously for 2 min. The root fragments were transferred to a new Falcon tube containing more dry ice fragments and vortexed again. The procedure was repeated two or three more times. Root hairs were collected by washing the inside walls of the tubes with a minimal volume of buffer. The enzyme assays were performed directly in the resuspended root hair preparations. The buffer used to recover the root hairs varied according to which enzymatic assay was to be performed (i.e. 100 mM phosphate buffer, pH 7.0 for PKS assays, 100 mM TRIS-HCl, pH 7.5 for OMT assays, and 50 mM Tricine/KOH buffer, pH 7.9 for hydroxylase). Protein concentration in the isolated root hair extracts was determined according to Bradford (1976).
Type III polyketide synthase assay
The PKS assay reaction mixture consisted of 300 µl of purified root hairs (30 µg total protein per assay) in 100 mM phosphate buffer, pH 7.0. The reaction was started by adding 25 µM acyl-CoA starter units and 45 µM 14C-malonyl-CoA (55 mCi mmol–1, American Radiolabelled Chemicals, St Louis, MO). The tubes were maintained at 30 °C on a thermomixer (Brinkman Instruments, Westbury, NY) rotating at 1400 rev. min–1 for 15 min. The reaction was stopped with the addition of 5 µl 20% HCl and the reaction products were extracted with 1 ml of ethyl acetate. The tube was vortexed, cooled, and centrifuged at 13 000 g for 5 min to separate the layers. An 800 µl aliquot of the organic layer was removed and dried in a glass test tube under N2 for analysis (see below). The acyl-CoA starter units used in the purified root hairs assays are shown in Table 1. A PKS activity versus substrate concentration curve was obtained in the purified root hair preparation by testing decanoyl-CoA as a starter unit from 5–100 µM.
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Type I S-adenosyl-L-methionine-dependent O-methyltransferase assay
OMT activity was tested in the purified root hair preparation according to a protocol modified from Wang and Pichersky (1999). The reactions assays consisted of 300 µl of purified root hairs (50–60 µg protein), 100 µM resorcinol substrates (dissolved in ethanol), and 10 µM of S-[methyl-14C] adenosyl-L-methionine (50 mCi mmol–1 in 10 mM acidified ethanol, American Radiolabelled Chemicals, St Louis, MO) in 100 mM TRIS-HCl, pH 7.5. Each assay was incubated for 1 h at 30 °C on a thermomixer (Brinkman Instrument, Westbury, NY) rotating at 1400 rev. min–1. The reaction was stopped by adding 25 µl of 6 N HCl. The radioactively labelled methylated products were extracted by adding 1 ml of hexane:ethyl acetate (1:1 v/v). A 800 µl aliquot of the organic layer was removed and dried in a glass test tube under N2 for analysis (see below). The 5-alkyl-resorcinols used in the purified root hairs assays are shown in Table 1. An OMT activity versus substrate concentration curve was obtained in the purified root hair preparation by testing n-pentyl-resorcinol between 5 µM and 167 µM.
Hydroxylase assay
To perform the hydroxylase assay, radiolabelled pentadecyl-resorcinol was prepared in a large scale PKS assay (equivalent to 50 assays as described above). Each PKS assay were done with 25 µM C16:0-CoA starter unit and 45 µM 14C-malonyl-CoA, and were run for 30 min instead of the normal 15 min to maximize the conversion of the C16-CoA substrate into the resorcinol product.
Following extraction with ethyl acetate, the samples were dried under N2, redissolved in ethyl acetate, and applied manually to an aluminium-backed silica gel 60 F254 TLC plate (EMD Chemicals Inc., Gibbstown, NJ) as described below. Pure radiolabelled pentadecyl-resorcinol was recovered in chloroform from the TLC. The extract was dried in vacuo at 30 °C (Büchi Rotovapor, Brinkmann Instrument, Westbury, NY), dissolved in acetonitrile, and passed through a 0.2 µm filter. A total of 4 µCi of purified radiolabelled pentadecyl-resorcinol was obtained.
The hydroxylase assay consisted of 1.3 µCi of radiolabelled pentadecyl-resorcinol in 200 µl of root hair extract in 50 mM Tricine/KOH buffer, pH 7.9 (Latunde-Dada et al., 2001). The assay was initiated by adding 0.5 mM reduced glutathione, 100 µM NADPH, 160 µM glucose-6-phosphate, and 0.12 unit of glucose-6-phosphate dehydrogenase. The reaction was incubated for 5 h at 30 °C on a Thermomixer (Brinkman Instrument, Westbury, NY) rotating at 1400 rev. min–1. The reaction was stopped by the addition of 25 µl of 6 N HCl and 1 ml of hexane:ethyl acetate (1:1 v/v). The tubes were vortexed and then centrifuged at 13 000 g. The organic layer was collected and dried under N2 for analysis (see below).
Analysis of the reaction products
For all samples, a 200 µl aliquot of the organic phase (top layer) was analysed by scintillation counting on a Tricarb 1600 TR (Packard Instrument, Wellesley, MA 02481). An additional 200 µl aliquot was applied to a aluminium-backed silica gel 60 F254 TLC plate (EMD Chemicals Inc., Gibbstown, NJ) using an autospotter (Analtech, Inc. Newark, DE). The plates were developed with either chloroform:ethyl acetate (7:3 v/v) for the PKS assays, or hexane:isopropanol (9:1 v/v) for the OMT and hydroxylase assays. The plates were run for approximately 5 cm. The TLC plates were air-dried and exposed to a multisensitive phosphoscreen (Perkin Elmer Life Sciences, Shelton, CT). The luminescence of the phosphoscreens was analysed using a Cyclone Storage Phosphor System (Perkin Elmer Life Sciences, Shelton, CT). Data were converted from counts min–1 to pmol mg–1 protein h–1 based on the specific activity of the substrate and using correction factors for counting efficiency, assay volume, and time. The reaction products were identified by comparing their RF values to that of authentic standards.
Tissue localization of PKS and OMT activities
A total root extract was obtained by homogenizing sorghum roots using a Brinkman polytron homogenizer (Brinkman Instrument, Westbury, NY) at full speed in water for 30 s. The homogenate was centrifuged at 11 000 g for 20 min at 4 °C and the supernatant was kept on ice. Similarly, root hairs isolated as described above and the remaining dehaired roots were also homogenized and centrifuged. The protein concentration of all three samples was quantified by the method of Bradford (1976) and adjusted to 135 µg ml–1. The extracts were buffered with 100 mM potassium phosphate, pH 7.0 or 100 mM TRIS-Cl, pH 7.5 before assaying for PKS and OMT activities, respectively. These enzymatic activities were measured in the total roots, dehaired roots, and root hair extracts as described above.
Quantitation of sorgoleone in enzyme preparations and its effect of on PKS and OMT activities
Sorgoleone was extracted from 1 ml of total root, dehaired root, and root hair preparations by phase partitioning with chloroform. The organic fractions were dried under N2 and reconstituted in ethanol. The amount of sorgoleone was quantified spectrophotometrically at 287 nm with a molar extinction coefficient of 16 000 (Kagan et al., 2003).
The effect of sorgoleone on PKS and OMT activities was tested by performing the enzyme assays as describe above in the presence of 0, 1, 3, 10, 33, and 100 µM purified sorgoleone. The reactions were carried out for 60 min at 30 °C and the products were analysed as described above.
Effect of pH and temperature on PKS and OMT activities
The effect of pH on PKS and OMT activities was tested by performing assays in 100 mM potassium phosphate adjusted to pH ranging from 4.0 to 9.0. For this experiment, the root hairs were collected in water rather than buffer, and the different pH levels were achieved by adding 30 µl of 1 M potassium phosphate buffer to 260 µl of root hair extract. Stock solutions of 1 M potassium phosphate buffers with different pH were obtained by mixing various ratios of 1 M solutions of monobasic and dibasic potassium phosphate stocks. The assays were conducted as described above for the two enzymes.
The effect of temperature on PKS and OMT activities was measured by conducting the enzyme assay over a temperature range of 10–80 °C. Prior to initiating the reaction, the enzyme preparations were incubated at the selected temperature for 10 min on a Thermomixer (Brinkman Instrument, Westbury, NY).
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Results |
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Sorghum root hairs were purified by adapting a method developed for trichome isolation using dry ice chips (Yerger et al., 1992; Fig. 1C). During the isolation process, 4 g FW of sorghum roots yielded purified root hairs sufficient for 20–25 assays. The preparations contained between 0.1 and 0.2 mg protein ml–1.
The isolated root hair preparations contained highly active type III PKS activity that utilized a long chain acyl-CoA starter unit (Table 2). While the highest specific activity was with decanoyl-CoA, the activity in the extract was able to catalyse the formation of 5-heptadecyl-resorcinol using palmitoyl-CoA as a starter unit. Furthermore, there appeared to be fewer derailment products with the longer starter units than with substrates with shorter side chains (Fig. 3A). Finally, no lipid resorcinol was produced with starter units shorter than octanoyl-CoA. Preliminary saturation kinetics indicated that the PKS activity in purified root hairs had a specific activity of 0.32±0.01 nmol mg–1 h–1 using decanoyl-CoA as a starter unit (Fig. 4A).
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Similarly, the root hair preparations contained highly active OMT catalysing the methylation of lipid resorcinols (Table 3; Fig. 2B). The highest level of activity was with 5-pentyl-resorcinol, but methylation of longer substrates was also observed. The activity of the enzyme on 5-pentadecyl-resorcinol was 33% of that observed with 5-pentyl-resorcinol (Table 3). Few other methylated products were detected by these crude preparations, suggesting that the OMT catalysing the methylation of resorcinols is highly specific in sorghum root hairs (Fig. 3B). Preliminary saturation kinetics indicated that the OMT activity in purified root hairs had a specific activity of 0.057±0.003 nmol mg–1 h–1 using 5-n-pentyl-resorcinol as a substrate (Fig. 4B).
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The tissue localization of these enzymatic reactions was determined by comparing the activity of PKS and OMT in total root, dehaired root, and isolated root hair preparations (Fig. 5A). Most of the PKS activity was in isolated root hairs. On the other hand, the OMT activity was much greater in the total root extract than in the isolated root hair or the dehaired root. This discrepancy may be due to the high concentration of sorgoleone present in the isolated root hair extract (Fig. 5B), which causes an inhibition of OMT activity (Fig. 5C).
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The pH optima for PKS and OMT were similar, ranging from 7.0–8.0 and 7.0–7.5, respectively (Fig. 6A). The PKS was quite tolerant to heat, with maximum activity during the length of the assay being 60 °C (Fig. 6B). The OMT activity was not as tolerant to heat as PKS, with optimum activity at 30 °C, followed by a rapid decline at higher temperatures (Fig. 6B).
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Hydroxylase activity was difficult to observe in the isolated sorghum root hairs. Preliminary attempts to conduct coupled assays to generate radiolabelled resorcinol by utilizing the in situ PKS activity which, in turn, would serve as a substrate for the hydroxylase present in root hairs were not successful. Therefore, large amount of radiolabelled pentadecyl-resorcinol was generated in a separate large-scale PKS assay. Incubating isolated root hairs with 1.33 µCi of purified radiolabelled pentadecyl-resorcinol for 5 h provided ample substrate and time to detect the hydroxylase activity involved in sorgoleone biosynthesis. Enzymatic activity was low under the assay conditions that converted only 3% of the pentadecyl-resorcinol into a more polar oxidized product (Fig. 7).
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Discussion |
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Glandular trichomes are recognized as sites of concentrated production of bioactive secondary products (Gershenzon et al., 1992; Tellez et al., 1999; Duke et al., 1994, 2000). While root hairs are protrusions of single root epidermal cells (trichomes) under the same genetic regulation as their aerial counterparts (Kellogg, 2001), relatively little is known about the biosynthetic capabilities of these specialized cells. An efficient method to purify biosynthetically active root hair preparations was obtained by adapting a method developed for the isolation of glandular trichomes using liquid nitrogen and dry ice chips (Fig. 1C; Yerger et al., 1992). This method proved to be more effective and easier than the traditional method of trichome isolation using a bead homogenizer developed by Gershenzon et al. (1992) where porous XAD-4 polystyrene resin, polyvinylpyrrolidone, and methyl cellulose are added to a buffer along with glass beads.
The PKS activity present in sorghum root hairs produces 5-alkyl-resorcinols (Table 2; Fig. 3A) via C2
C7 aldol condensation of a linear tetraketide intermediate to yield orsellinic acid-type rings similar to that catalysed by stilbene synthase (Tropf et al., 1995). The substrate specificity of this activity differs from most other PKSs by favouring substrates with long and lipophilic chains. While a plant Type III PKS involved in cannabinoid synthesis had specificity for mid-range length fatty acyl-CoA (Raharjo et al., 2004), most plant PKSs do not react properly with long-chain starter units, resulting in the formation of their pyrone derailment products (Abe et al., 2000, 2004; Morita et al., 2001). The PKS activity in sorghum root hairs is somewhat promiscuous, accepting starter units with carbon chains ranging from 10 to 16 carbons (Table 2). The highest specific activity was for decanoyl-CoA (Table 2), but several derailment products were also observed. Conversely, the enzyme produced fewer derailment products with starter units having longer acyl chains (palmitoyl- and stearoyl-CoA) (Fig. 3A), suggesting that these more lipophilic substrates bind more properly in the catalytic domain. Since sorgoleone, the primary component of the oil exudate, has a 5-n-pentadecyl tail, in vivo substrate availability, channelling and/or compartmentalization may favour the formation of a 5-pentadecyl-resorcinol intermediate previously identified in root hair extracts (Dayan et al., 2003). This enzymatic activity was optimum at pH ranging from 7.0 to 8.0 (Fig. 6A), which is similar to many other plant PKSs (Borejsza-Wysocki and Hrazdina, 1996; Morita et al., 2000; Abe et al., 2004). The enzyme activity was quite heat stable, with activity increasing up to 60 °C. The activity dropped sharply at higher temperatures (Fig. 6B).
Unlike other plant OMTs (Lavid et al., 2002), the enzyme activity in sorghum root hairs favours resorcinolic substrates with longer side chains (Table 3). Overall substrate specificity was greatest with alkyl side chains of five or six carbons in length (5-n-pentyl- and 5-n-hexyl-resorcinols), but activity was observed on 5-n-pentadecyl-resorcinol (Table 3; Fig. 2B). A similar 3-methyl-5-pentadecatrienyl-resorcinol product has been detected in sorghum root hairs previously (Dayan et al., 2003). The relatively low activity observed on the longer resorcinol substrates may be due to the fact that these molecules have strong amphiphilic properties that enables them to assemble in lipid monolayers or liposomes (Kozubek and Tyman, 1999; Przeworska et al., 2001), possibly making them less available to the OMT. Other biochemical mechanisms such as channelling of substrate between multi-enzyme complexes (Achnine et al., 2004; Facchini and St-Pierre, 2005) and/or compartmentalization and association with membrane systems may also be involved in the de novo biosynthesis of lipid resorcinols. Indeed, an ultrastructural investigation of sorghum root hairs has revealed highly physiologically active cells containing a dense network of membranes and vesicles that appear to participate in the synthesis and transport of sorgoleone (Czarnota et al., 2003a). Both of these physical and biochemical processes may also protect the cells from the toxic effect of sorgoleone and its precursors.
The PKS activity was much higher in the root hair extract, than in the root (Fig. 5A), which is consistent with the hypothesis that the biosynthesis of lipid resorcinols occurs exclusively in these specialized cells. Interestingly, the OMT activity was very high in the total root extract but very low in the dehaired roots, indicating that the enzyme responsible for this activity was not localized in the root itself (Fig. 5A). Unlike PKS activity, however, the OMT activity was lower in isolated root hairs than in the total root. It was noticed that the amount of sorgoleone in root hair preparations was more than 20-fold greater than in the total root or dehaired root preparations (Fig. 5B). This is due to the fact that sorgoleone, which accumulates at the tip of root hairs (Fig. 1B), was isolated along with the root hairs. Therefore, it is postulated that the OMT activity in the root hair fraction was inhibited by this excess amount of sorgoleone (1.9 mg sorgoleone mg–1 protein). OMT was indeed sensitive to the presence of this lipid benzoquinone, with 67% of the activity inhibited at 100 µM sorgoleone (Fig. 5C). The amount of sorgoleone present in the root hair fraction is equivalent to nearly 500 µM sorgoleone, which would strongly inhibit OMT activity. Unfortunately, attempts to remove sorgoleone from the root hair preparation by gel filtration also removed OMT activity, which may be associated with the root hair membranes.
The pH optimum of the OMT activity measured in isolated root hairs ranged from 7.0 to 7.5, which is similar to most other OMTs (Busam et al., 1997; Christensen et al., 1998; Gang et al., 2002). This activity was not very heat tolerant, with optimum activity at 30 °C. The effect of temperature on this activity is similar to that observed on in vivo production of lipid benzoquinones in sorghum (Dayan, 2006).
The remaining step in the biosynthesis of lipid benzoquinones requires the hydroxylation of 3-methoxy-5-alkyl resorcinol intermediates. This step is most likely catalysed by a P450 mono-oxygenase. The hydroxylase activity was tested with radiolabelled pentadecyl-resorcinol, which was obtained beforehand by purifying the product of a scaled-up PKS assay using palmitoyl-CoA as a starter unit. The hydroxylase activity was low relative to the other enzymes studied here. Quinones are strong inhibitors of P450 mono-oxygenase activity (Petersen, 1997; Soucek, 1999). Therefore, the relatively high amount of sorgoleone (Fig. 7, lane 1) still present in the isolated root hairs most likely prevents an efficient measure of the hydroxylase activity. Furthermore, other potential factors hindering the assay of hydroxylase activity include low affinity of the enzyme for the non-methylated lipid resorcinol substrate, the participation of another enzyme class, such as the 2-oxoglutarate-dependent dioxygenases (De Carolis and de Luca, 1993; Anzellotti and Ibrahim, 2004), and poor availability of these amphiphilic substrates under the conditions of this assay (Kozubek and Tyman, 1999; Przeworska et al., 2001).
It had been postulated that the biosynthesis of sorghum lipid resorcinols and benzoquinones was compartmentalized in root hairs. Our research demonstrates that the entire metabolic machinery necessary for the biosynthesis for these molecules is indeed present in these specialized cells. The compartmentalization of these pathways in root hairs will have implications for the genetic engineering of lipid phenolic biosynthesis in sorghum and in other plants species. Furthermore, protein interactions involved in channelling of the amphiphilic intermediates of this pathway, as well as biochemical and structural transport mechanisms between subcellular compartments and for the exudation of sorgoleone into the environment remain to be determined. Thus, the unique biochemistry of secretory root hairs deserves to be studied in more detail. A functional genomic approach to study these enzymes in greater detail has been initiated (Baerson et al., 2006).
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