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• W2093069957 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. 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. Allelopathy, the chemical inhibition of one plant species by another, represents a form of chemical warfare between neighboring plants competing for limited light, water, and nutrient resources (1Inderjit Duke S.O. Planta. 2003; 217: 529-539Crossref PubMed Scopus (478) Google Scholar, 2Bais H.P. Park S.W. Weir T.L. Callaway R.M. Vivanco J.M. Trends Plant Sci. 2004; 9: 26-32Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar). Allelopathic interactions have been proposed to have profound effects on the evolution of plant communities through the loss of susceptible species via chemical interference, and by imposing selective pressure favoring individuals resistant to inhibition from a given allelochemical (2Bais H.P. Park S.W. Weir T.L. Callaway R.M. Vivanco J.M. Trends Plant Sci. 2004; 9: 26-32Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar, 3Schulz M. Wieland I. Chemoecology. 1999; 9: 133-141Crossref Scopus (53) Google Scholar). In addition, allelochemicals released by grain crop species such as barley, rye, and sorghum are thought to play a significant role in their efficacy as weed suppressants when used as cover crops or within intercropping systems (4Duke S.O. Rimando A.M. Baerson S.R. Scheffler B.E. Ota E. Belz R.G. J. Pestic. Sci. 2002; 27: 298-306Crossref Scopus (30) Google Scholar, 5Weston L.A. Duke S.O. Crit. Rev. Plant Sci. 2003; 22: 367-389Crossref Scopus (336) Google Scholar). Sorghum bicolor (L.) Moench is one of the most important cereal crops worldwide (6Dogget H. 2nd Ed. Sorghum. John Wiley & Sons, Inc., New York1988: 1-13Google Scholar), surpassed only by wheat, rice, corn, and barley in total acreage, with the United States currently accounting for a major portion of total world production and exports (FAOSTAT data). The allelopathic properties of sorghum were first suggested from observations of reduced growth of other crop species when grown in rotation; moreover, certain sorghum species such as Sudan grass (Sorghum sudanense) can produce largely weed-free monocultures without the use of synthetic herbicides (reviewed in Ref. 7Duke S.O. Belz R.G. Baerson S.R. Pan Z. Cook D.D. Dayan F.E. Outlooks on Pest Management. 2005; 16: 64-68Crossref Scopus (12) Google Scholar). Current evidence suggests that a family of allelochemicals active at micromolar concentrations, referred to as sorgoleones, may account for much of the allelopathic properties of Sorghum spp. (8Netzly D.M. Butler L.G. Crop Sci. 1986; 26: 775-778Crossref Google Scholar, 9Einhellig F.A. Souza I.F. J. Chem. Ecol. 1992; 18: 1-11Crossref PubMed Scopus (171) Google Scholar, 10Czarnota M.A. Paul R.N. Dayan F.E. Nimbal C.I. Weston L.A. Weed Technol. 2001; 15: 813-825Crossref Google Scholar). The term sorgoleone is most frequently used to describe the compound corresponding to the predominant congener identified in sorghum root exudates (11Kagan I.A. Rimando A.M. Dayan F.E. J. Agric. Food Chem. 2003; 51: 7589-7595Crossref PubMed Scopus (56) Google Scholar), 2-hydroxy-5-methoxy-3-[(Z,Z)-8′,11′,14′-pentadecatriene]-p-benzoquinone (Fig. 1), which has been estimated to account for as much as 85% of the exudate material (w/w) in some varieties (10Czarnota M.A. Paul R.N. Dayan F.E. Nimbal C.I. Weston L.A. Weed Technol. 2001; 15: 813-825Crossref Google Scholar). The remaining exudate consists largely of sorgoleone congeners differing in the length or degree of saturation of the aliphatic side chain and in the substitution pattern of the quinone ring (11Kagan I.A. Rimando A.M. Dayan F.E. J. Agric. Food Chem. 2003; 51: 7589-7595Crossref PubMed Scopus (56) Google Scholar, 12Rimando A.M. Dayan F.E. Streibig J.C. J. Nat. Prod. (Lloydia). 2003; 66: 42-45Crossref PubMed Scopus (32) Google Scholar). The fact that sorgoleone acts as a potent broad-spectrum inhibitor active against many agronomically important monocot and dicot weed species, exhibits a long half-life in soil, and appears to affect multiple cellular targets (e.g. 8–10, 13–17) may make it promising for development as a natural product alternative to synthetic herbicides (18Bertin C. Yang X. Weston L.A. Plant Soil. 2003; 256: 67-83Crossref Scopus (960) Google Scholar, 19Duke S.O. Trends Biotechnol. 2003; 21: 192-195Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Sorgoleone biosynthesis likely occurs exclusively in root hairs, which appear as cytoplasmically dense cells in sorghum, containing large osmiophilic globules deposited between the plasmalemma and cell wall, presumably associated with sorgoleone rhizosecretion (10Czarnota M.A. Paul R.N. Dayan F.E. Nimbal C.I. Weston L.A. Weed Technol. 2001; 15: 813-825Crossref Google Scholar, 20Czarnota M.A. Paul R.N. Weston L.A. Duke S.O. Int. J. Plant Sci. 2003; 164: 861-866Crossref Scopus (78) Google Scholar). Labeling studies have demonstrated that the biosynthesis of sorgoleone involves the convergence of the fatty acid and polyketide pathways (21Fate G.D. Lynn D.G. J. Am. Chem. Soc. 1996; 118: 11369-11376Crossref Scopus (53) Google Scholar, 22Dayan F.E. Kagan I.A. Rimando A.M. J. Biol. Chem. 2003; 278: 28607-28611Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) through the action of a polyketide synthase activity utilizing fatty acyl-CoA starter units, resulting in the addition of a quinone head via iterative condensations of acetate extender units (Fig. 1). 3D. Cook, A. M. Rimando, T. E. Clemente, J. Schröder, N. P. Nanayakkara, M. Fishbein, Z. Pan, I. Abe, F. E. Dayan, and S. R. Baerson, unpublished results. 3D. Cook, A. M. Rimando, T. E. Clemente, J. Schröder, N. P. Nanayakkara, M. Fishbein, Z. Pan, I. Abe, F. E. Dayan, and S. R. Baerson, unpublished results. Subsequent modifications of the alkylresorcinol intermediate are likely to be mediated by AdoMet 4The abbreviations used are: AdoMetS-adenosylmethionineGC-MSgas chromatography-mass spectrometryRTreverse transcriptionESTexpressed sequence tagHPLChigh pressure liquid chromatographyDESdesaturaseOMTO-methyltransferasePKSpolyketide synthaseUTRuntranslated regionGOgene ontologyI-7-OMTisoflavone 7-O-methyltransferaseEOMTeugenol OMTCABchlorophyll a/b-binding protein. 4The abbreviations used are: AdoMetS-adenosylmethionineGC-MSgas chromatography-mass spectrometryRTreverse transcriptionESTexpressed sequence tagHPLChigh pressure liquid chromatographyDESdesaturaseOMTO-methyltransferasePKSpolyketide synthaseUTRuntranslated regionGOgene ontologyI-7-OMTisoflavone 7-O-methyltransferaseEOMTeugenol OMTCABchlorophyll a/b-binding protein.-dependent O-methyltransferases and by hydroxylases (possibly P450 monooxygenases), yielding the reduced form of sorgoleone (dihydrosorgoleone). Upon exudation, the less stable hydroquinone rapidly oxidizes to the highly phytotoxic benzoquinone form, which can persist in soil for extended periods (9Einhellig F.A. Souza I.F. J. Chem. Ecol. 1992; 18: 1-11Crossref PubMed Scopus (171) Google Scholar, 10Czarnota M.A. Paul R.N. Dayan F.E. Nimbal C.I. Weston L.A. Weed Technol. 2001; 15: 813-825Crossref Google Scholar, 23Netzly D.M. Riopel J.L. Ejeta G. Butler L.G. Weed Sci. 1988; 36: 441-446Crossref Google Scholar). In addition, recent studies have shown that sorgoleone biosynthesis occurs constitutively during early seedling establishment, and its accumulation is apparently distinct from that of phytoalexins as levels were not observed to increase following various elicitor treatments (24Dayan F.E. Planta. 2006; 224: 339-346Crossref PubMed Scopus (97) Google Scholar). S-adenosylmethionine gas chromatography-mass spectrometry reverse transcription expressed sequence tag high pressure liquid chromatography desaturase O-methyltransferase polyketide synthase untranslated region gene ontology isoflavone 7-O-methyltransferase eugenol OMT chlorophyll a/b-binding protein. S-adenosylmethionine gas chromatography-mass spectrometry reverse transcription expressed sequence tag high pressure liquid chromatography desaturase O-methyltransferase polyketide synthase untranslated region gene ontology isoflavone 7-O-methyltransferase eugenol OMT chlorophyll a/b-binding protein. The specific sequence of biosynthetic reactions leading to the formation of dihydrosorgoleone, starting from the proposed 5-pentadecatrienyl resorcinol intermediate (Fig. 1), has not been determined previously. Moreover, despite the ecological and agronomic importance of this family of allelochemicals, a paucity of information exists concerning the genes and corresponding enzymes participating in their biosynthesis. In this study, we have identified a 3-methyl ether derivative of the previously characterized 5-pentadecatrienyl resorcinol intermediate by GC-MS analysis of sorghum root extracts, 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. To identify candidate O-methyltransferase sequences, as well as candidates representing other steps in the biosynthetic pathway, an annotated EST data set consisting of 5,468 quality 5′-sequences was generated from an S. bicolor root hair-specific cDNA library. Follow-up real time RT-PCR and recombinant enzyme studies with putative O-methyltransferase sequences obtained from this library have led to the identification of a root hair-specific O-methyltransferase (designated SbOMT3) utilizing alkylresorcinolic substrates, proposed to be involved in the biosynthesis of sorgoleone. Furthermore, the annotated root hair-specific data set we have generated directly complements the existing public sorghum EST sequences, and expands our understanding of the transcriptome of a highly specialized and unique cell type. Plant Material and Growth ConditionsSeeds of S. bicolor genotype BTx623 were purchased from Crosbyton Seed Co. (Crosbyton, TX), and SX-17 sorghum-Sudangrass hybrid seeds (S. bicolor × sudanense) were purchased from Dekalb Genetics (Dekalb, IL). SX-17 was used for sorgoleone content comparisons with BTx623; all other experiments described in this work involved only BTx623. Root tissues used for sorgoleone content determinations, analysis of C15:3 resorcinols, root hair preparations, and whole root systems used for real time RT-PCR experiments were obtained from 5- or 8-day-old dark-grown seedlings grown under soil-free conditions using a capillary mat system devised by Czarnota and co-workers (10Czarnota M.A. Paul R.N. Dayan F.E. Nimbal C.I. Weston L.A. Weed Technol. 2001; 15: 813-825Crossref Google Scholar). Immature leaves and shoot apices used for real time RT-PCR experiments were isolated from seedlings maintained in a growth chamber at 28 °C for 8 days in standard (∼20 × 40 cm) nursery flats using Premier Pro Mix PGX potting media (Hummert International, Earth City, MO) under a combination of cool-white fluorescent and incandescent lighting at an intensity of ∼400 μmol m-2 s-1 and a 16-h photoperiod. Developing panicles, mature leaves, and culm (stem) tissues used for real time RT-PCR experiments were isolated from 10-week-old greenhouse-grown plants. At the time of harvest, panicles were partially exerted from flag leaf sheaths, just prior to anthesis. All harvested plant material was directly flash-frozen in liquid nitrogen and stored at -80 °C prior to analysis, with the exception of material used for sorgoleone content determinations, which involved fresh tissue extractions. Sorgoleone Content DeterminationsRoot systems from 5-day-old seedlings were weighed, immersed in chloroform, and agitated for 30 s. Extracts were filtered through Whatman 110-mm number 1 filter disks (Whatman Inc., Florham Park, NJ) to remove debris, concentrated in vacuo at 30 °C using a rotary evaporator (Büchi Rotovapor, Brinkmann Instruments), and dried to completion under nitrogen gas, then weighed on an analytical balance. Dried extracts were then re-dissolved in acetonitrile (1.0 mg sample per ml of acetonitrile) and analyzed by high performance liquid chromatography (HPLC) using a Hewlett-Packard 1050 HPLC System (Agilent Technologies, Palo Alto, CA) equipped with an Alltech EPS C18 column (100 Å, 3 μm, 150-mm length, 4.5-mm internal diameter; Alltech Associates Inc., Deerfield, IL). The sample was eluted as follows (solvent A is 2.5% acetic acid in water; solvent B is acetonitrile): 0–15-min 45% A, 55% B isocratic; 15–22-min linear gradient from 55 to 100% B; 22–25-min 100% B; 25–26-min 100 to 55% B; 26–30-min 45% A/55% B isocratic. A flow rate of 2 ml/min was used, and the sample injection volume was 20 μl. The peak corresponding to sorgoleone was monitored at 280 nm. Quantitation was based on a calibration curve using purified sorgoleone as an external standard. GC-MS Analysis of C15:3 ResorcinolsRoot systems from 8-day-old seedlings were first immersed in chloroform with agitation for 30 s to remove sorgoleone and then lyophilized. Lyophilized material was pulverized using a mortar and pestle, followed by homogenization in methanol (∼10 g per 50 ml) for 1 min at 25,000 rpm. Homogenates were then filtered through Whatman number 1 filter disks, then evaporated using a rotary evaporator (Büchi Rotovapor, Brinkmann Instruments) at 30 °C. Residues were then re-dissolved in methanol and transferred to GC vials. GC-MS analysis was performed with a JEOL GCMate II System (JEOL USA Inc., Peabody, MA) using a J & W DB-5 capillary column (0.25-mm internal diameter, 0.25-μm film thickness, 30-m length; Agilent Technologies, Foster City, CA). The GC temperature program was initially set to 110 °C, raised to 300 °C at a rate of 6 °C/min, and then held at this temperature for 2.3 min. Ultra-high purity helium was used as carrier at a flow rate of 1.0 ml/min. The inlet (splitless), GC interface, and ion chamber temperatures were 250, 250, and 230 °C, respectively. The sample injection volume used was 2.0 μl. The mass spectrum of the peak at 21.8 min (Fig. 2, B and C) showed fragment ions m/z 314 [M+], 313 [M+ - H], 269 [313+ - CH=CH2, -OH], 255 [269+ - CH2], 241 [255+ - CH2], 227 [241+ - CH2], 213 [227+ - CH2], 199 [213+ - CH2], 187 [313+ - CH(CH2CH=CH)2H, - 2O], 171 [185+ - CH2], 159 [314+ - (CH2CH=CH)3H, -2OH], 143 [314+ - (CH2)2(CH2CH = CH)3H, -2H2O], 131 [159+ - 2CH2], 129 [143+ - CH2], and 117 [131+ - CH2] supporting the identification of the 5-(8′,11′,14′)-pentadecatrienyl resorcinol intermediate. Similarly, the mass spectrum of the peak at 18.3 min (Fig. 2C) showed fragment ions m/z 328 [M+], 313 [M+ - CH3], 285 [M+ - H - CH=CH2, -OH], 269 [M+ - CH2CH=CH2, -H2O], 243 [269+ - CH=CH], 229 [243+ - CH2], 201 [M+ - 2H - (CH2CH=CH)2H, -OH, -OCH3], 187 [201+ - CH2], 171 [M+ - H, -(CH=CHCH2)2CH=CH2, -H2O, -OCH3], 159 [M+ - (CH2CH=CH)3H, -OH, -OCH3], 145 [159+ - CH2], 132 [145+ - CH], 129 [171+ - (CH2)3], and 117 [159+ - (CH2)3], supporting the identification of the 5-(8′,11′,14′)-pentadecatrienyl resorcinol-3-methyl ether intermediate. cDNA Library ConstructionRoot hairs were isolated from dark-grown 8-day-old BTX623 seedling root systems using the method devised by Bucher et al. (25Bucher M. Schroeer B. Willmitzer L. Riesmeier J.W. Plant Mol. Biol. 1997; 35: 497-508Crossref PubMed Scopus (58) Google Scholar), involving immersion in liquid nitrogen with gentle stirring, followed by filtration through a 250-μm aluminum mesh to remove root system debris. Purity of the root hair preparations was assessed by bright field microscopy, and only highly enriched preparations were retained for subsequent cDNA library construction. Root hair preparations were stored at -80 °C prior to RNA extraction. Total RNAs were isolated from root hairs using the TRIzol reagent (Invitrogen) per the manufacturer's instructions, with an additional homogenization step of 30 s at 25,000 rpm using a hand-held homogenizer. RNA purity was determined spectrophotometrically, and integrity was assessed by agarose gel electrophoresis. Poly(A)+ mRNA was prepared from root hair total RNA using an Oligotex mRNA Midi Kit (Qiagen, Valencia, CA), and ∼1.5 μg was used for construction of a directional cDNA library with the Uni-Zap XR cDNA library construction kit (Stratagene, La Jolla, CA), per the manufacturer's instructions. A primary library of ∼3 × 106 plaque-forming units was obtained. To obtain an estimate of average insert size, 36 randomly selected plaques from a primary library plating were sampled from NZY plates using a sterile 1.0-ml pipette tip, then transferred into culture tubes containing 1.0 ml of SM buffer, and allowed to elute overnight at 4 °C with shaking. Phage eluates (2.5 μl) were then used as templates in 50-μl PCRs containing T3- and T7-specific PCR primers (Stratagene) using an Expand High Fidelity PCR kit (Roche Diagnostics) per the manufacturer's instructions. After an initial denaturation step of 94 °C for 5 min, a thermal profile of 94 °C for 30 s, then 48 °C for 1 min 30 s, followed by 72 °C for 2 min for 35 cycles was used, and aliquots of the reactions were subsequently analyzed by agarose gel electrophoresis. By this analysis, the average insert size was estimated to be ∼0.93 kb, ranging between 2.4 and 0.2 kb. EST Sequencing and Data AnalysisRecombinant plasmid-bearing colonies were obtained from the nonamplified S. bicolor root hair phagemid library by mass excision, and then plasmid mini-preparations were performed for 6,624 randomly selected isolates arrayed into 69 96-well plates. 5′ DNA sequencing reactions were performed using ABI BigDye Terminator Cycle Sequence Ready Reaction kits (versions 2 and 3; Applied Biosystems, Foster City, CA) as described previously (26Pratt L.H. Liang C. Shah M. Sun F. Wang H. Reid S.P. Gingle A.R. Paterson A.H. Wing R. Dean R. Klein R. Nguyen H.T. Ma H.M. Zhao X. Morishige D.T. Mullet J.E. Cordonnier-Pratt M-M. Plant Physiol. 2005; 139: 869-884Crossref PubMed Scopus (57) Google Scholar). Base calling on raw sequence trace data were performed using PHRED software (27Ewing B. Hillier L. Wendl M.C. Green P. Genome Res. 1998; 8: 175-185Crossref PubMed Scopus (4858) Google Scholar), and vector, adapter, and low quality sequence ends were identified using an in-house processing script (28Liang C. Sun F. Wang H. Qu J. Freeman Jr., R.M. Pratt L.H. Cordonnier-Pratt M.-M. BMC Bioinformatics. 2006; 7: 115Crossref PubMed Scopus (15) Google Scholar), resulting in 5,468 high quality sequences, or an 82.6% success rate. The average trimmed EST length, determined using a moving window with a PHRED quality score of 16, was 451 bp, of which on average 432 bp were called with a quality score equal to or greater than 20. The resulting 5,468 root hair ESTs were assembled using TGICL (29Pertea G. Huang X. Liang F. Antonescu V. Sultana R. Karamycheva S. Lee Y. White J. Cheung F. Parvizi B. Tsai J. Quackenbush J. Bioinformatics (Oxf.). 2003; 19: 651-652Crossref PubMed Scopus (1499) Google Scholar). Provisional annotation of all EST and contig consensus sequences was performed by BLASTX analysis against all full-coding length entries from the PIR-NREF data base (30Altschul S.F. Madden T.L. Schäffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59448) Google Scholar, 31Wu C.H. Huang H. Arminski L. Castro-Alvear J. Chen Y. Hu Z.Z. Ledley R.S. Lewis K.C. Mewes H.W. Orcutt B.C. Suzek B.E. Tsugita A. Vinayaka C.R. Yeh L.S. Zhang J. Barker W.C. Nucleic Acids Res. 2002; 30: 35-37Crossref PubMed Scopus (179) Google Scholar). EST data mining was performed using the MAGIC Gene Discovery software (32Cordonnier-Pratt M-M. Liang C. Wang H. Kolychev D.S. Sun F. Freeman R. Sullivan R. Pratt L.H. Comp. Funct. Genom. 2004; 5: 268-275Crossref PubMed Scopus (18) Google Scholar), and by BLASTN and TBLASTN analysis (30Altschul S.F. Madden T.L. Schäffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59448) Google Scholar). Provisional gene ontology (GO) categorization of the assembled data set was performed by BLASTX analysis against all UniProt peptide sequences downloaded from the European Bioinformatics Institute web site (EBI UniProt release 9.0). An E value cutoff of E < 10-10 was applied to the BLASTX returns, and the corresponding full GO terms from significant matches were retrieved using the association table provided by the Gene Ontology Consortium web site. Full GO terms retrieved were finally mapped to their Plant GOSlim counterparts (33Berardini T.Z. Mundodi S. Reiser L. Huala E. Garcia-Hernandez M. Zhang P. Mueller L.A. Yoon J. Doyle A. Lander G. Moseyko N. Yoo D. Xu I. Zoeckler B. Montoya M. Miller N. Weems D. Rhee S.Y. Plant Physiol. 2004; 135: 1-11Crossref Scopus (358) Google Scholar) using the “map2slim.pl” tool also available on line. Quantitative Real Time RT-PCR AnalysisQuantitative real time PCRs were performed in triplicate using the GenAmp® 5700 sequence detection system (Applied Biosystems, Foster City, CA) as described previously (34Baerson S.R. Sánchez-Moreiras A. Pedrol-Bonjoch N. Schulz M. Kagan I.A. Agarwal A.K. Reigosa M.J. Duke S.O. J. Biol. Chem. 2005; 280: 21867-21881Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). First strand cDNAs were synthesized from 2 μg of total RNA in a 100-μl reaction volume using the TaqMan reverse transcription reagents kit (Applied Biosystems) per the manufacturer's instructions. Independent PCRs were performed using the same cDNA for both the gene of interest and 18 S rRNA, using the SYBR® Green PCR Master Mix (Applied Biosystems) with the following gene-specific primer pairs: SbOMT1 forward, 5′-GCATCTTCGTTCATGTACTTGTTACAC-3′, and reverse, 5′-CGACGAAGCACATCCTTACTATGAG-3′; SbOMT2 forward, 5′-GCGCCTCGTTTTCGTATGC-3′, and reverse, 5′-GAACATACAGCTCACCTTCTCTGC-3′; SbOMT3 forward, 5′-CAATTTCCCTTTTATGTTTAGCCTGATAG-3′, and reverse, 5′-TGCCAGGGTGTGATATGTGC-3′; polyubiquitin forward, 5′-CTTCCTCTGTCCCTCTGATGGAG-3′, and reverse, 5′-AAGACACGACCACGACATGC-3′; chlorophyll a/b-binding protein forward, 5′-TGGATTGATTGATGCTGCAAG-3′, and reverse, 5′-CGTGAAACAAGAGACACACATGC-3′; 18 S rRNA forward, 5′-GGCTCGAAGACGATCAGATACC-3′, and reverse, 5′-TCGGCATCGTTTATGGTT-3′. Primers were designed using Primer Express® software (Applied Biosystems) and the Amplify program (35Engels W.R. Trends Biochem. Sci. 1993; 18: 448-450Abstract Full Text PDF PubMed Scopus (160) Google Scholar). A dissociation curve was generated at the end of each PCR cycle to verify that a single product was amplified using software provided with the GenAmp® 5700 sequence detection system. A negative control reaction in the absence of template (no template control) was also routinely performed in triplicate for each primer pair. The change in fluorescence of SYBR® Green I dye in every cycle was monitored by the GenAmp® 5700 system software, and the threshold cycle (CT) above background for each reaction was calculated. The CT value of 18 S rRNA was subtracted from that of the gene of interest to obtain a ΔCT value. The CT value of an arbitrary calibrator (e.g. the tissue sample from which the largest ΔCT values were obtained) was subtracted from the ΔCT value to obtain a ΔΔCT value. The fold changes in expression level relative to the calibrator were expressed as 2-ΔΔCT. Southern Blot AnalysisGenomic DNA from S. bicolor genotype BTx623 was prepared from young leaf tissue using the Plant DNAzol Reagent (Invitrogen). Approximately 1 g of powdered tissue was mixed with 3.0 ml of plant DNAzol reagent supplemented with RNase A at a final concentration of 1.0 mg/ml and then incubated at room temperature for 10 min with gentle shaking. The remainder of the extraction procedure was carried out per the manufacturer's instructions, with an additional chloroform:isoamyl alcohol (24:1, v/v) extraction step performed prior to ethanol precipitation. Restriction endonuclease digestions and Southern blotting procedures were performed according to standard protocols (36Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 9.31-9.57Google Scholar). Probe sequences containing partial 3′-UTR and 3′ coding sequences were generated by PCR amplification from cloned SbOMT1, SbOMT2, and SbOMT3 cDNA templates with an Expand High Fidelity PCR kit (Roche Diagnostics) using a thermal profile of 94 °C for 30 s, then 55 °C for 1 min 30 s, followed by 72 °C for 1 min for 25 cycles. The following PCR primer pairs were used to generate probe sequences for the three S. bicolor OMTs: SbOMT1 forward, 5′-CACCAGAAGAAACACTAGCATCG-3′, and reverse, 5′-TTAAGGACCAATAAGCAAGCTAGTACA-3′; SbOMT2 forward, 5′-GAAGCACAGCTGCTGATGG-3′, and reverse, 5′-CAGCAAGCAACACACATCAAGTATG-3′; and SbOMT3 forward, 5′-ATGACTGGAGCAATGATGAGTG-3′, and reverse 5′-CAGGGTGCCAGGGTGTG-3′. For SbOMT1, the amplified regions correspond to nucleotides 966–1410 (445 bp in length; GenBank™ accession number EF189707), containing 191 bp of 3′-coding sequence and 254 bp of contiguous 3′-UTR sequence; for SbOMT2, amplified regions correspond to nucleotides 961–1424 (464 bp in length; GenBank™ accession number EF189706), containing 159 bp of 3′-coding sequence and 305 bp of contiguous 3′-UTR sequence; and for SbOMT3, amplified regions correspond to nucleotides 893–1376 (484 bp in length; GenBank™ accession number EF189708), containing 290 bp of 3′-coding sequence and 194 bp of contiguous 3′-UTR sequence. The resulting PCR products were cloned using a Zero Blunt TOPO PCR cloning kit (Invitrogen) and confirmed by DNA sequence analysis. Prior to use in labeling reactions, probe sequences were excised from the cloning vectors by restriction endonuclease digestion and then gel-purified. Heterologous Expression and Purification of Recombinant OMTsDNA manipulations and Escher" @default.
• W2093069957 created "2016-06-24" @default.
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• W2093069957 date "2008-02-01" @default.
• W2093069957 modified "2023-10-16" @default.
• W2093069957 title "A Functional Genomics Investigation of Allelochemical Biosynthesis in Sorghum bicolor Root Hairs" @default.
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• W2093069957 doi "https://doi.org/10.1074/jbc.m706587200" @default.
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