Abstract
Sorghum is considered to be one of the more allelopathic crop species, producing phytotoxins such as the potent benzoquinone sorgoleone (2-hydroxy-5-methoxy-3-[(Z,Z)-8',11',14'-pentadecatriene]-p-benzoquinone) and its analogs. Sorgoleone likely accounts for much of the allelopathy of Sorghum spp., typically representing the predominant constituent of Sorghum bicolor root exudates. Previous and ongoing studies suggest that the biosynthetic pathway for this plant growth inhibitor occurs in root hair cells, involving a polyketide synthase activity that utilizes an atypical 16:3 fatty acyl-CoA starter unit, resulting in the formation of a pentadecatrienyl resorcinol intermediate. Subsequent modifications of this resorcinolic intermediate are likely to be mediated by S-adenosylmethionine-dependent O-methyltransferases and dihydroxylation by cytochrome P450 monooxygenases, although the precise sequence of reactions has not been determined previously. Analyses performed by gas chromatography-mass spectrometry with sorghum root extracts identified a 3-methyl ether derivative of the likely pentadecatrienyl resorcinol intermediate, indicating that dihydroxylation of the resorcinol ring is preceded by O-methylation at the 3'-position by a novel 5-n-alk(en)ylresorcinol-utilizing O-methyltransferase activity. An expressed sequence tag data set consisting of 5,468 sequences selected at random from an S. bicolor root hair-specific cDNA library was generated to identify candidate sequences potentially encoding enzymes involved in the sorgoleone biosynthetic pathway. Quantitative real time reverse transcription-PCR and recombinant enzyme studies with putative O-methyltransferase sequences obtained from the expressed sequence tag data set have led to the identification of a novel O-methyltransferase highly and predominantly expressed in root hairs (designated SbOMT3), which preferentially utilizes alk(en)ylresorcinols among a panel of benzene-derivative substrates tested. SbOMT3 is therefore proposed to be involved in the biosynthesis of the allelochemical sorgoleone.
Highlights
Sorghum is considered to be one of the more allelopathic crop species, producing phytotoxins such as the potent benzoquinone sorgoleone (2-hydroxy-5-methoxy-3-[(Z,Z)-8,11,14pentadecatriene]-p-benzoquinone) and its analogs
Analyses performed by gas chromatography-mass spectrometry with sorghum root extracts identified a 3-methyl ether derivative of the likely pentadecatrienyl resorcinol intermediate, indicating that dihydroxylation of the resorcinol ring is preceded by O-methylation at the 3-position by a novel 5-n-alk(en)ylresorcinol-utilizing O-methyltransferase activity
Identification of Pentadecatrienyl Resorcinol and Pentadecatrienyl Resorcinol-3-methyl Ether Intermediates in S. bicolor Genotype BTx623—Given the likelihood that sorgoleone is synthesized predominantly in root hair cells [10, 20], it follows that the biosynthetic enzymes are exclusively or predominantly localized in this cell type
Summary
Identification of Pentadecatrienyl Resorcinol and Pentadecatrienyl Resorcinol-3-methyl Ether Intermediates in S. bicolor Genotype BTx623—Given the likelihood that sorgoleone is synthesized predominantly in root hair cells [10, 20], it follows that the biosynthetic enzymes are exclusively or predominantly localized in this cell type. Given the ultimate goal of identifying all of the key enzymes required for sorgoleone biosynthesis, which would include the biochemical characterization of fatty acid desaturases (DESs), polyketide synthases (PKSs), O-methyltransferases (OMTs), and possibly P450 monooxygenases (Fig. 1), the identification of additional pathway intermediates is crucial for the understanding of the in vivo substrates used by the O-methyltransferase and hydroxylase activities likely to be involved Toward this end, methanol extracts were prepared from roots of 8-day-old BTx623 and SX-17 seedlings and analyzed by GC-MS, as shown, B and C (representative chromatograms for genotype BTx623 shown). GO:0005634 nucleus GO:0005840 ribosome GO:0005737 cytoplasm GO:0005783 endoplasmic reticulum GO:0005856 cytoskeleton GO:0005575 cellular component
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