Ent for the plant expression vector pMDC162 so as to create the final construct of ProCYP709B3:GUS. All constructs were sequence confirmed. All constructs had been introduced into Agrobacterium tumefaciens strain GV3101 then transformed into wild-type or cyp709b3 mutant plants by the floral dipping system [43].GUS staining assayAbbreviations PCR: Polymerase chain reaction; LC/MS: Liquid chromatography-mass spectrometry; GC/MS: Gas chromatography ass spectrometry. Competing interests The authors have declared no conflict of interests. Authors’ contributions GM and OY conceived the study, made the experiments and drafted the manuscript. GM, TS and DS performed the experiments. All authors read and approved the final manuscript. Acknowledgements This research is mainly supported by grant from NSF (MCB-0923779), but in addition by grants from DOE (DE-SC0001295) and USDA (2010-65116-20514) to O. Y. Author details 1 Donald Danforth Plant Science Center, 975 North Warson Road, St. Louis, MO 63132, USA. 2Present address: Conagen Inc., 1005 North Warson Road, St., Louis, MO 63132, USA. 3Present address: The Pennsylvania State University, 115 Agricultural Sciences and Industries Building, University Park, PA 16802, USA. Received: 9 December 2012 Accepted: 28 August 2013 Published: 28 October 2013 References 1. Werck-Reichhart D, Feyereisen R: Cytochromes P450: a good results story. Genome Biol 2000, 1(6):REVIEWS3003. two. Werck-Reichhart D, Bak S, Paquette S: Cytochromes p450. Arabidopsis Book 2002, 1:e0028. three. Bak S, Beisson F, Bishop G, Hamberger B, Hofer R, Paquette S, WerckReichhart D: Cytochromes p450. Arabidopsis Book 2012, 9:e0144.GUS staining was performed as outlined by a published strategy [44].Metabolite profiling analysisWild kind (W) and cyp709b3 (M) seedlings have been collected after two days (2D) and 4 days (4D) of growth on normal or 150 mM NaCl MS plates. Sample extraction, LC/MS and GC/MS evaluation have been performed as described [34] by METABOLON, Inc. In short, 100 mg of each sample had been extracted making use of the automated Microlab STAR method. Recovery requirements had been added prior to the first step within the extraction course of action. Sample preparation was performed applying a proprietary series of organic and aqueous extractions to get rid of the protein fraction although enabling maximum recovery of compact molecules.2820536-71-6 In stock The resulting extract was divided into two fractions; one for evaluation by LC/MS and one particular for evaluation by GC/MS. Samples had been placed briefly on a TurboVap?(Zymark) to remove the organic solvent. Every sample was then frozen and dried beneath vacuum. Samples were then prepared for the appropriate their instrument, either LC/ MS or GC/MS.AN-12-H5 intermediate-1 manufacturer Mao et al.PMID:23310954 BMC Plant Biology 2013, 13:169 http://biomedcentral/1471-2229/13/Page 13 of4. five.6. 7.eight. 9.ten.11.12.13.14.15.16.17.18.19.20.21.22.23.24. 25.Durst F, Nelson DR: Diversity and evolution of plant P450 and P450reductases. Drug Metabol Drug Interact 1995, 12(three?):189?06. Paquette SM, Bak S, Feyereisen R: Intron-exon organization and phylogeny inside a huge superfamily, the paralogous cytochrome P450 genes of Arabidopsis thaliana. DNA Cell Biol 2000, 19(five):307?17. Ralston L, Yu O: Metabolons involving plant cytochrome P450s. Phytochem Rev 2006, 5:459?72. Schuler MA, Duan H, Bilgin M, Ali S: Arabidopsis cytochrome P450s by way of the looking plass a window on plant biochemistry. Phytochem Rev 2006, 5:205?37. Schuler MA, Werck-Reichhart D: Functional genomics of P450s. Annu Rev Plant Biol 2003, 54:629?67. Narusaka Y, Narusaka M, Seki M,.
