full issue - Association of Biotechnology and Pharmacy

Current Trends in Biotechnology and PharmacyVol. 5 (1) 1064-1072 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1068Fig.1. Comparison of antioxidants and ROS levels in activationtagged mutants resistant to ethanol (200 mM) and incontrol (wild type) of Nicotiana tabaccum var. SR1. About200 mg of fresh leaf tissue was used to analyse total AsA bymeasuring the change in spectrophotometric absorbancebefore and after adding ascorbate oxidase enzyme. Similarly,a spectrophotometric assay was used for measuring totalGSH. a) Glutathione (GSH), b) Ascorbate (AsA), c) Reactiveoxygen species (ROS) was measured using general ROSsubstrate 2’,7’-dichlorodihydrofluorescein diacetate(H 2DCFDA). Fluorescence was measured using a VictorFluorescence plate reader (Perkin Elmer). Total protein wasquantified using Bradford dye (Bio-Rad). RFU is relativefluorescence units. Error bars represent the SD from threebiological replicates.1a1b1caccumulation of glutathione also increased theascorbate pool due to connected cycles. TheGSH content of ET-R3 is almost equal to wildtypeplants, but total AsA is six-fold higher(Fig. 1b). This may be due to enhancedexpression of a regulatory component of AsApathway in response to stress. It is welldocumented that plants produce large amountsof ROS in response to various biotic andabiotic stresses (22,23) and it is believed thatthe amount of different antioxidants stronglyincreases to scavenge ROS under stressfulconditions (24,25). Thus, we compared theROS level in these mutants with wild-typecontrol plants. The general ROS substrate2’,7’-dichlorodihydrofluorescein diacetate(H 2DCFDA) was used with the extracts of plantsamples (9). Our results show the ROS levelsin mutants are either equal or less than controlplant samples (Fig. 1c). Interestingly, themutant ET-R1 showed ROS levels 3- fold lessthan wild type plant but almost similarantioxidants profile.The advent of plant molecular biologyhas brought renewed interest in theunderstanding of the physiological andecological roles of secondary metabolites.Plants coordinately regulate primary andsecondary metabolism. Coordinate regulationin some plants is regulated by the stresshormone methyljasmonate (26). InCatharanthus roseus, this mechanism ismodulated by a single stress-responsivetranscription factor, ORCA 3. Likewise,alkaloid synthesis in Nicotiana is regulated bya host of developmental and environmentallyresponsive factors (26,27). A number ofstudies have demonstrated jasmonatemediatedsignaling leads to increased nicotinebiosynthesis following wounding or herbivoreattack but at present, little is known regardingthe gene regulatory mechanisms involved inSurvival selection of N. tabaccum activation tagged mutants