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Current Trends in Biotechnology and PharmacyVol. 5 (1) 1064-1072 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1065been characterized using an activation taggingapproach. For example, a HIS kinase involvedin cytokinin signalling isolated fromArabidopsis has been characterized (2) andgenes conferring disease resistance have alsobeen identified using this approach (3).Activation tagging is especially usefulin manipulating plants with complex,relatively intractable metabolic pathways,such the biosynthetic pathways found in manyspecies of medicinal plants. For mostlaboratories, conventional biochemicalanalyses of the hundreds of thousands ofmutants required for effective saturation of themajority of any given plant genome usingATM is prohibitive. In some cases, physicaltraits, such as flower color have been used toidentify prospective mutants of interest.Overproduction of the plant pigmentanthocyanin in arabidopsis flowers was usedto identify mutants resistant to the lethal effectsof ultra-violet radiation (4). Unfortunately,most valuable plant metabolites are“secondary” and confer no obvious survivaladvantage within a population, thus survivalselection strategies are difficult to apply tolarge mutant plant populations, and most ofthe genes involved in plant secondarymetabolism remain undiscovered.Ethanol metabolism is not known to be adirect precursor to known secondarymetabolite production in Nicotiana species. Inthis attempt to alter secondary metabolitesusing survival selections, we exposed a largepopulation of activation tagged leaf disccultures to high concentrations of ethanol. Inboth plants and mammals, ethanol ismetabolized by alcohol dehydrogenase.Alcohol dehydrogenase activity in plants isincreased by anaerobic conditions (5) and thismetabolic adaptation is important in variousstages of the life cycle such as flowerformation, or adaptation to stressful conditionssuch as cold tolerance during early seedlingdevelopment. The byproduct of ethanol,acetaldehyde, is toxic to plants and animal cellsvia oxidative damage. We hypothesize thatATM (activation tagged mutagenesis)Nicotiana mutants resistant to ethanol willcontain elevated levels of antioxidantscompared to wild-type plants and ultimatelyfound that mutant survivors of ethanolselection contain higher antioxidants activitycompared to non-mutant control plants.Activation tagging mutagenesis has beenused in Catharanthus roseus to isolate a masterregulatory gene involved in terpenoid indolealkaloid synthesis (TIAs), ORCA3. Overexpressionof ORCA3 results in increasedtranscription of several genes directly involvedin the Catharanthus TIA pathway (6). Thisstudy used resistance to the toxic substrate 4-MT (4-methyltryptophan) of the TIAbiosynthetic enzyme tryptophandecarboxylase to identify mutants withenhanced TIA synthesis and presumablypossessing the metabolic capacity to detoxify4-MT. Because Nicotiana possesses arelatively large genome, activation taggingmutagenesis might uncover quiescent genesthat may impact upon tryptophan metabolismand reveal novel synthetic routes not apparentin the native plant. These altered syntheticroutes may be revealed in activation taggedmutants surviving exposure to toxictryptophan analogs such as 4MT. In this studywe hypothesize that survivors of 4-MTtoxicity may possess increased nicotine levelscompared to non-mutant controls. We foundthat mutant survivors of these selectionscontain 3-5 fold greater nicotineconcentrations compared to control plants.Gunjan, SK et al
Current Trends in Biotechnology and PharmacyVol. 5 (1) 1064-1072 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1066Materials and MethodsPlant material and activation tagging byAgrobacterium infection: Nicotiana tabacumvar. SR1 was used for all studies detailed here.Leaf discs from 3 week old magenta boxgrownplants were transformed withAgrobacterium tumefaciens 3850 harbouringthe activation tagging T-DNA constructpPCVICEn4HPT. Agrobacterium wascultured in Luria-Bertani (LB) mediumcontaining 100 mg l -1 Ampicillin. Bacterialculture was initiated by inoculating singlecolony of A. tumefaciens into 25 ml flaskcontaining 5 ml of liquid media and kept at200 rpm on a rotary shaker at 28ºC for oneday. Subsequently, 2 ml of overnight grownbacterial culture was transferred to 500 ml flaskcontaining 200 ml of liquid media. When theoptical density (OD) of the culture reachedaround 0.6 at 660 nm, 100 µM acetosyringonewas added. Bacterial culture was spun downin a centrifuge at 2500 g, when culture reachedaround 1.0 OD. The pellet was gentlyresuspended in 2 ml MS medium and used asbacterial stock for infection. A finalconcentration of 100 µM acetosyringone wasadded to the bacterial culture and leaf explantswere infected for approximately 20 min. Afterinfection, explants were blot-dried onto sterilefilter paper and transferred onto platescontaining solid MS media. The infectedexplants were co-cultivated for 2 days in theculture room set at 25 ºC and then transferredonto shoot induction media. Ethanol (200mM)or 4-MT was added directly to culture media.Regenerated shoots were excised andtransferred to rooting medium (MS mediumwithout hormones) containing either ethanolor 4-MT along with hygromycin andcefotaxime. The resistant mutants weretransferred to compost substrate and grown ina greenhouse.Antioxidant assays: 200 mg of fresh leaftissue was ground to a fine powder underliquid nitrogen. The powder was homogenizedin 2 mL of 2% (w/v) metaphosphoric acid and2 mM ethylenediaminetetraacetic acid(EDTA), and transferred to eppendorf tubes.The supernatant was collected aftercentrifugation at 14,000xg for 10 min at 4 o C.The supernatants were then split into two tubesof 900 uL each and neutralized with 600 uLof 10% sodium citrate (7). Total AsA wasanalysed with 500 uL of neutralized extractby measuring the change inspectrophotometric absorbance before andafter adding ascorbate oxidase enzyme (8).Similarly, a spectrophotometric assay wasused for measuring total GSH (7).ROS measurement: About 300 mg of leaftissue was ground in liquid nitrogen andhomogenized with 1 mL of TRIS(hydroxymethylaminomethane) buffer (pH7.2). The supernatant was collected aftercentrifugation at 14,000xg for 10 min at 4 o C.800 uL of this extract was mixed with 1 mM2’,7’-dichlorodihydrofluorescein diacetate(H 2DCFDA) and incubated in darkness for 10min at room temp (7,9). Fluorescence wasmeasured using a Victor Fluorescence platereader (Perkin Elmer). Total protein wasquantified using Bradford dye (Bio-Rad).Gas chromatography-mass spectrometry (GC-MS) analysis: Tobacco alkaloid content wasdetermined using an Agilent 6890 GC/Agilent5973 mass-selective detector (MSD) with anAgilent 7683 Autosampler. The GC was fittedwith a 30m x 0.2 mm X 0.25 µm HP-5MSglass capillary column and a helium flow rateof 1.0 ml/ min. Extracts from survivorsselected from mutant populations at aconcentration of 100 mg dried material/ mlSurvival selection of N. tabaccum activation tagged mutants
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