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)1067methanol were auto-injected at a volume of1.0 ìl and a split ratio of 1:20 (10). Thetemperature of the injection port, MS interface,electron impact source and mass filter were280 o C, 280 o C, 230 o C, and 150 o C,respectively. The GC oven was programmedto hold at 120 o C for 2 min, then heated to160 o C at a rate of 10 o C/min. After holding theoven temperature at 160 o C for 1 min, the ovenwas heated to 275 o C at a rate of 6 o C/min andheld at this temperature for 10 min in order toelute possible interfering high boiling pointimpurities from the column.Results and DiscussionFormation of endogenous ethanol is acommon feature of plant metabolism and it isalso produced in hypoxic conditions such asflood, cold and ozone stress (11). Ethanol isconverted to acetaldehyde by alcoholdehydrogenase (ADH) and finally oxidizedinto acetate by acetaldehyde dehydrogenase(ALDH). The accumulation of NADHproduced during ethanol metabolism canimbalance redox state of cells and causeoxidative stress (12) whereas acetaldehydeinteracts with different bioactive molecules andimpairs the normal function of cells (13).Overproduction of acetaldehyde in plant tissuecauses necrosis (14). Thus, lethalconcentrations of ethanol may be consideredan elicitor of endogenous reactive oxygenspecies (ROS) in plant tissues. We treatedNicotiana leaf discs with differentconcentrations of ethanol ranging from 50 mMto 250 mM to identify the threshold ofresistance to ethanol in wild type plants. Weconcluded that the minimum lethalconcentration of ethanol was 175 mM. Thus,after activation tagging of leaf-discs throughAgrobacterium, the explants were immediatelysubjected to ethanol selection pressure at theconcentration of 200 mM. Few explantsproduced shoots in regeneration media orformed roots in the presence of ethanol in thehormone-free media. Finally, three mutantswere able to grow in continuous ethanolselective media and produced shoots and roots.Extracts from these survivors were analyzedfor antioxidant activity.Ascorbate (AsA) and glutathione (GSH)are key components of the non-enzymaticdefense system of plants (15-17). The cellularrecycling of ascorbate/glutathione moleculesto quench the ROS is interconnected (18,19).The ascorbate deficient mutant of Arabidopsisthaliana vtc1 shows 14-fold increasedaccumulation of ascorbate in leaves whensupplemented with 10 mM ascorbate, withoutsignificant change in redox state (20).Previously, we have seen that AsA redox statewas not favorably poised for scavenginginduced ROS generated by ozone fumigationon Arabidopsis whereas total GSH contentincreased three- fold (9). The activation taggedmutants that are resistant to ethanol (200 mM)showed high glutathione and ascorbatecontents compared to control plants (Fig.1).The mutant ET-R1 and ET-R3 have total GSHalmost equal to control whereas ET-R2 andET-R4 have two- and six-fold higher GSHconcentration, respectively. The total AsAcontents in these mutants are also variableranging from 2-6 folds higher than controlexcept in mutant ET-R1 (Fig.1a).In Arabidopsis mutants, tocopheroldeficientlines have been reported withincreased pools of ascorbate and glutathione(21). It is possible that the expression ofconnecting regulator of AsA/GSH cycle isenhanced in ET-R2 and ET-R4 mutants dueto activation tagging and the overGunjan, SK et al