full issue - Association of Biotechnology and Pharmacy
full issue - Association of Biotechnology and Pharmacy
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Current Trends in <strong>Biotechnology</strong> <strong>and</strong> <strong>Pharmacy</strong>Vol. 5 (1) 1064-1072 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1067methanol were auto-injected at a volume <strong>of</strong>1.0 ìl <strong>and</strong> a split ratio <strong>of</strong> 1:20 (10). Thetemperature <strong>of</strong> the injection port, MS interface,electron impact source <strong>and</strong> mass filter were280 o C, 280 o C, 230 o C, <strong>and</strong> 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 <strong>of</strong> 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 <strong>of</strong> 6 o C/min <strong>and</strong>held at this temperature for 10 min in order toelute possible interfering high boiling pointimpurities from the column.Results <strong>and</strong> DiscussionFormation <strong>of</strong> endogenous ethanol is acommon feature <strong>of</strong> plant metabolism <strong>and</strong> it isalso produced in hypoxic conditions such asflood, cold <strong>and</strong> ozone stress (11). Ethanol isconverted to acetaldehyde by alcoholdehydrogenase (ADH) <strong>and</strong> finally oxidizedinto acetate by acetaldehyde dehydrogenase(ALDH). The accumulation <strong>of</strong> NADHproduced during ethanol metabolism canimbalance redox state <strong>of</strong> cells <strong>and</strong> causeoxidative stress (12) whereas acetaldehydeinteracts with different bioactive molecules <strong>and</strong>impairs the normal function <strong>of</strong> cells (13).Overproduction <strong>of</strong> acetaldehyde in plant t<strong>issue</strong>causes necrosis (14). Thus, lethalconcentrations <strong>of</strong> ethanol may be consideredan elicitor <strong>of</strong> endogenous reactive oxygenspecies (ROS) in plant t<strong>issue</strong>s. We treatedNicotiana leaf discs with differentconcentrations <strong>of</strong> ethanol ranging from 50 mMto 250 mM to identify the threshold <strong>of</strong>resistance to ethanol in wild type plants. Weconcluded that the minimum lethalconcentration <strong>of</strong> ethanol was 175 mM. Thus,after activation tagging <strong>of</strong> leaf-discs throughAgrobacterium, the explants were immediatelysubjected to ethanol selection pressure at theconcentration <strong>of</strong> 200 mM. Few explantsproduced shoots in regeneration media orformed roots in the presence <strong>of</strong> ethanol in thehormone-free media. Finally, three mutantswere able to grow in continuous ethanolselective media <strong>and</strong> produced shoots <strong>and</strong> roots.Extracts from these survivors were analyzedfor antioxidant activity.Ascorbate (AsA) <strong>and</strong> glutathione (GSH)are key components <strong>of</strong> the non-enzymaticdefense system <strong>of</strong> plants (15-17). The cellularrecycling <strong>of</strong> ascorbate/glutathione moleculesto quench the ROS is interconnected (18,19).The ascorbate deficient mutant <strong>of</strong> Arabidopsisthaliana vtc1 shows 14-fold increasedaccumulation <strong>of</strong> 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 <strong>and</strong> ascorbatecontents compared to control plants (Fig.1).The mutant ET-R1 <strong>and</strong> ET-R3 have total GSHalmost equal to control whereas ET-R2 <strong>and</strong>ET-R4 have two- <strong>and</strong> 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 <strong>of</strong> ascorbate <strong>and</strong> glutathione(21). It is possible that the expression <strong>of</strong>connecting regulator <strong>of</strong> AsA/GSH cycle isenhanced in ET-R2 <strong>and</strong> ET-R4 mutants dueto activation tagging <strong>and</strong> the overGunjan, SK et al