Role of Proline in Tobacco Cultured Cells Under. Arsenate Stress

Transcription

1 Role of Proline in Tocco Cultured Cells Under Arsente Stress 2017, 9 Mst. NurENzmun Nhr Grdute School of Environmentl nd Life Science (Doctor s Course) OKAYAMA UNIVERSITY, JAPAN 1

2 Role of Proline in Tocco Cultured Cells Under Arsente Stress A thesis Presented to Grdute School of Environmentl nd Life Science Okym University In prtil fulfillment of the requirement for the degree of Doctor of Philosophy Sumitted y Mst. NurENzmun Nhr Deprtment of Biofunctionl Chemistry Grdute School of Environmentl nd Life Science Okym University, Jpn 2017, 9 2

3 CONTENTS List of figures 8 Arevitions used 10 Chpter 1 Generl Introduction Toxicity of hevy metls Sources of rsenic Arsenic Trnsport nd Hyperccumultion in Plnts Arsenic toxicity Cell deth DNA frgmenttion Rective oxygen species (ROS) Oxidtive dmge nd ntioxidnt defense mechnism Glutthione Arsente reductse Superoxide dismutse Comptile solutes Proline Arginine Alnine Betine Lipid peroxidtion Arsenic tolernce mechnisms Purpose of the study 20 Chpter 2 Regultion of rsenteinduced chnges y exogenous proline in tocco BY2 cells 21 3

4 2.1 Astrct Introduction Mterils nd Methods Culture of tocco BY2 cells Mesurement of BY2 cell growth Estimtion of cell deth of BY2 cells Counting of BY2 totl cell numer Sttisticl nlysis Results Effects of exogenous proline on BY2 cell growth in the 25 sence of rsente Effects of exogenous rsente on BY2 cell growth Effects of exogenous proline on the inhiition of BY2 cell 28 growth y rsente Effects of exogenous proline on rsenteinduced cell deth Effects of exogenous proline on the reduction of BY2 cell 33 numer y rsente 2.5 Discussion 34 Chpter 3 Mitigtion of rsente stress y exogenous proline in cultured tocco cells under rsente stress Astrct Introduction Mterils nd Methods Culture of tocco BY2 cells Estimtion of proline content 40 4

5 3.3.3 Isoltion of genomic DNA nd gel electrophoresis Detection of ROS Mesurement of GSH content Extrctions nd mesurements of rsente reductse Extrctions nd mesurements of superoxide dismutse Mesurement of protein Sttisticl nlysis Results Proline content in response to ppliction of exogenous 44 proline DNA frgmenttion Intrcellulr ROS level Effects of exogenous proline on glutthione content t µm AsO 4 stressed BY2 cells Effects of exogenous proline on the ctivity of rsente 49 reductse in AsO 4 stressed BY2 cells Superoxide dismutse ctivity Discussion 51 Chpter 4 Comprison etween the effects of proline nd the effects of other osmolytes in BY2 cells under rsente stress Astrct Introduction Mterils nd Methods Culture of tocco BY2 cells Mesurement of BY2 cell growth 58 5

6 4.3.3 Estimtion of endogenous proline Mesurement of glutthione content Extrctions nd mesurements of rsente reductse Extrctions nd mesurements of superoxide dismutse Determintion of lipid peroxidtion Mesurement of protein Sttisticl nlysis Results Proline content in response to ppliction of exogenous 60 proline Effects of exogenous proline on glutthione content t µm AsO 4 stressed BY2 cells Effects of exogenous proline on glutthione content t µm AsO 4 stressed BY2 cells Effects of exogenous proline on the ctivity of rsente 64 reductse in AsO 4 stressed BY2 cells Superoxide dismutse ctivity Effects of exogenous rginine on BY2 cell growth in the 65 sence of rsente Effects of exogenous rginine on the inhiition of BY2 cell 67 growth y rsente Effects of exogenous lnine on BY2 cell growth in the 68 sence of rsente Effects of exogenous lnine on AsO 4 induced BY2 cell 70 growth 6

7 Effects of exogenous glutmte on BY2 cell growth Effects of exogenous glutmte on the inhiition of BY2 72 cell growth y rsente Effects of exogenous etine on BY2 cell growth in the 74 sence of rsente Effects of exogenous etine on the inhiition of BY2 cell 75 growth y rsente Effects of exogenous proline on lipid peroxidtion of 76 tocco BY2 cells in sence nd presence of rsente 4.5 Discussion 78 Summry 84 Conclusions 88 Acknowledgements 89 References 90 7

8 LIST OF FIGURES Figure 2.1 Effects of exogenous proline on BY2 cell growth 26 Figure 2.2 Effects of exogenous rsente (As) on BY2 cell growth 27 Figure 2.3 Figure 2.4 Figure 2.5 Figure 2.6 Effects of exogenous pro (Pro) on 60 µm rsente (As)induced growth inhiition of BY2 cells Effects of exogenous proline (Pro) on 40 µm rsente (As)stressed BY2 cells Effects of exogenous proline (Pro) on rsente (As)induced BY2 cell deth Reduction of BY2 cell numer y rsente (As) in the presence or sence of proline (Pro) Figure 3.1 Proline content in response to exogenous proline ppliction 44 Figure 3.2 Figure 3.3 Figure 3.4 Figure 3.5 Figure 3.6 DNA frgmenttion in rsente (As) stress BY2 cells in response to exogenous proline (Pro) ppliction Intrcellulr ROS levels in tocco BY2 suspension cells under rsente (As) stress in the presence or sence of proline (Pro) Glutthione content t 60 µm AsO 4 stressed BY2 cells in presence of lower concentrtion of exogenous proline (Pro) Arsente reductse (AR) ctivity of AsO 4 undpted tocco BY2 suspension cells induced y proline under AsO 4 stress Superoxide dismutse (SOD) ctivity of rsenteundpted tocco BY 2 suspension cells induced y different concentrtion of proline (Pro) under AsO 4 stress Figure 4.1 Proline (Pro) content in response to exogenous proline ppliction 60 8

9 Figure 4.2 Figure 4.3 Figure 4.4 Figure 4.5 Glutthione content t 60 µm rsenic (As)stressed BY2 cells in presence of higher concentrtion of exogenous proline (Pro) Glutthione content t 40 µm rsente (As)stressed BY2 cells in presence of different concentrtion of exogenous proline (Pro) Arsente reductse (AR) ctivity of rsente (As) undpted tocco BY 2 suspension cells induced y proline (Pro) under AsO 4 stress Superoxide dismutse (SOD) ctivity of rsente (As)undpted tocco BY2 suspension cells induced y higher concentrtion of proline (Pro) under AsO 4 stress Figure 4.6 Effects of exogenous rginine (Arg) on BY2 cell growth 66 Figure 4.7 Effects of exogenous rginine (Arg) on 60 µm rsente (As)stressed BY 2 cells 68 Figure4.8 Effects of exogenous lnine (Al) on BY2 cell growth 69 Figure 4.9 Effects of exogenous lnine (Al) on 60 µm AsO 4 stressed BY2 cells 70 Figure 4.10 Effects of exogenous glutmte (Glu) on BY2 cells growth 72 Figure 4.11 Effects of exogenous glutmte (Glu) on 60 µm rsente (As)stressed BY 2 cells 73 Figure 4.12 Effects of exogenous etine on BY2 cell growth 74 Figure 4.13 Effects of exogenous etine on 60 µm AsO 4 stressed BY2 cells 75 Figure 4.14 Effects of exogenous proline (Pro) on lipid peroxidtion t 60 µm AsO 4 77 Figure 4.15 stressed BY2 cells A hypotheticl flowdigrm showing the inhiition of proline ctolic process y rsenite 81 9

10 ABBREVIATIONS USED LS, Linsmier nd Skoog ROS, rective oxygen species GSH, reduced glutthione GSSG, oxidized glutthione AR, rsente reductse SOD, superoxide dismutse P5C, pyrroline5croxylte GSA, glutmte5semildehyde P5CDH, pyrroline5croxylte dehydrogense PCs, phytocheltins ATP, Adenosine triphosphte O2, superoxide nion FW, Fresh weight DW, Dry weight 10

11 CHAPTER 1 Generl Introduction 1.1 Toxicity of hevy metls Hevy metls re metllic elements which re toxic nd hve high density ( 5.0 g/cm 3 ), specific grvity or tomic weight. Arsenic, chromium, cdmium, mercury, nd led hve the gretest potentil to cuse hrm ecuse of their extensive use, nd widespred distriution in the environment (Bird nd Cnn, 2012). The toxicity of hevy metls oserved in plnts when they re present in excessive mounts due to rnge of interctions t the cellulr level. Enzymes re one of the min trgets of hevy metl in plnts, nd prolonged exposure to hevy metls results in remrkle decreses in enzyme ctivity (Tyler et l., 1989). Hevy metls cn pollute ir, wter, nd soil qulity, nd cuse toxicity to plnts nd nimls (Stnkovic nd Stnkocic, 2013). To mintin the level of essentil metls within physiologicl rnges, plnts hve evolved vriety of defense mechnisms tht control the uptke, ccumultion nd detoxifiction of metls. 1.2 Sources of rsenic The nturl source of rsenic is the erth crust. Arsenic cn e introduced into the environment from vrious sources. The sources of rsenic include oth nturl nd nthropogenic (e.g. use of insecticides, hericides, nd phosphte fertilizers) (Cozzolino et l., 2010; Verruggen et l., 2009) processes. The involvement of rsenic with griculturl prctices egn fter the green revolution when the use of pesticides nd fertilizers incresed rpidly. Arsenic lso finds its wy into the food chin (Mehrg, 2004). One of the most widespred prolems of nturlly occurring rsenic is due to leching into drinking wter quifers, which hve een reported in mny countries including Indi nd Bngldesh 11

12 (Kim et l., 2009). In Bngldesh, rseno pyrite hs een identified s the prime source of rsenic pollution (Fzl et l., 2001). 1.3 Arsenic Trnsport nd Hyperccumultion in Plnts The processes of hevy metl trnsport hve een recognized s centrl mechnism of metl detoxifiction nd tolernce. Trnsporters ply criticl role in rsenic metolism in plnts. Arsente is the dominnt species in eroic soils nd redily enters plnt roots vi phosphte trnsporters. A numer of the quporin nodulin26like intrinsic proteins re le to trnsport rsenite, the predominnt form of rsenic in reducing environments. Arsenic is primrily tken up y plnts vi root nd leves. In most plnts, rsenic is ccumulted through roots ecuse its low moility restricts its roottoroot trnsloction, except in rsenic hyperccumultors (R et l., 2005). A hyperccumultors plnt is defining s the plnt tht could tolerte nd ccumulte 1 mg metl g 1 dry mss (Brooks et l., 1977). Three min forms of rsenic in soil re ville to plnts, nmely rsente (AsO 4 ), rsenite (AsO 3 ) nd methylted rsenic. To understnd how plnt tke up nd metolize rsenic is importnt for the mitigtion. Plnt roots selectively tke up rsenic species vi distinct pthwys nd trnsporters. Hyperccumultors such s Pteris vittt L, Agrostis tenerrim Trin hve n extrordinry ility to sor hevy metls from the soil under vrying concentrtion of hevy metls (M et l., 2001). 1.4 Arsenic toxicity Arsenic is highly toxic metlloid. It is nturlly tken up y plnts from the wter nd soil. Arsenic toxicity to living cells cuses t very low concentrtion nd its toxicity symptoms in plnts rnge from growth inhiition to ultimte cell deth (Brrchin et l., 1995). Indeed, rsenic toxicity in humns hs recently ecome evident on very lrge scle in Bngldesh (Dhr et l., 1997). The cceptle level of rsenic sfe drinking wter is 0.01 mg/l (WHO, 12

13 2003). Plnts cn respond to rsenic toxicity y vriety of mechnisms such s hyperccumultion, ntioxidnt defense system, nd phytocheltion (Grg nd Singl, 2011). Arsenic intercts with sulfhydryl (SH) groups of enzymes nd proteins, leding to inhiition of severl importnt cellulr functions (Mehrg nd Hrtley Whitker, 2002). Plnt growth cn severely hve inhiited through exposure of rsenic y slowing or rresting expnsion nd iomss ccumultion. The toxicity of rsenic cuse degrdtion of plnt reproductive cpcity through losses in fertility, yield, nd fruit production. Arsenic interferes with the criticl metolic processes of plnts tht cn led to the deth of plnt (Grg nd Singl, 2011). 1.5 Cell deth Cell deth is essentil for growth nd development of eukryotes, y mintining tissue nd orgn homeostsis in concert with prolifertion, growth, nd differentition (Breusegem nd Dt, 2006). Tretment of tocco cells in suspension cultures with rsenic cused cell deth. There re two min ctegories of cell deth such s poptosis nd necrosis. The difference etween these two forms ws sed on the presence or sence of specific iochemicl nd moleculr hllmrks, such s DNA lddering, cytochrome c, cspse involvement, ATP depletion, cytoplsmic swelling, nd loss of memrne integrity (Pennell nd Lm, 1997). 1.6 DNA frgmenttion DNA frgmenttion is n importnt hllmrk of cell deth. Arsenic cused DNA dmge y ttcking on its ses nd sugr moieties, consequently DNA frgmenttion. It hs lredy een documented tht rsenic cn replce phosphorous from phosphte group of DNA nd inhiits DNA repir system which could led to genomic templte instility (Erturk et l., 2015). NClinduced poptosislike cell deth long with genomic DNA degrdtion ws lso oserved in tocco protoplsts (Lin et l., 2006). 13

14 1.7 Rective oxygen species Rective oxygen species (ROS) re produced s norml product of plnt cellulr metolism. Despite their destructive ctivity, ROS re welldescried second messengers in vriety of cellulr processes including tolernce to environmentl stresses (Desikn et l., 2001; Yn et l., 2007). The most common ROS include singlet oxygen ( 1 O 2), superoxide rdicl (O 2 ), hydrogen peroxide (H 2O 2) nd hydroxyl rdicl ( OH). ROS re lwys formed y the inevitle lekge of electrons onto O 2 from the electron trnsport ctivities of chloroplsts, mitochondri, nd plsm memrnes or s yproduct of vrious metolic pthwys loclized in different cellulr comprtments. Production nd removl of ROS must e strictly controlled in order to void oxidtive stress. When the level of ROS exceeds the defense mechnisms, cell is sid to e in stte of oxidtive stress ˮ. However, the equilirium etween production nd scvenging of ROS is pertured under numer of stressful conditions such s slinity, drought, toxicity due to metls, pthogens, nd so forth. Enhnced level of ROS cn cuse dmge to iomolecules such s lipids, proteins nd DNA. 1.8 Oxidtive dmge nd ntioxidnt defense mechnism In the cell, rsente cn e redily converted to rsenite tht is more toxic. Arsente nd rsenite disrupt plnt metolism through distinct mechnisms. Arsenite is dithiol rective compound tht inds to nd potentilly inctivtes enzymes contining closely spced cysteine residues or dithiol cofctors. Arsenic exposure generlly induces the production of ROS tht cn led to the production of ntioxidnt metolites nd numerous enzymes involved in ntioxidnt defense mechnisms. Oxidtive cron metolism, mino cid nd protein reltionships, nd nitrogen nd sulfur ssimiltion pthwys re lso impcted y rsenic exposure (Finnegn nd Chen, 2012). Metl toxicity cn cuse redox imlnce nd induce the increse of ROS concentrtion, ctivting the ntioxidnt defense mechnisms of plnts (Shrm nd Dietz, 2009). 14

15 1.9 Glutthione Glutthione (γglutmylcysteinylglycine) represents the mjor pool of reduced sulfur (Kurnert & Foyer 1993). Under norml conditions, glutthione is predominntly present in its reduced form (GSH), with only smll proportion present in its fully oxidized stte (GSSG). Glutthione ply roles in iosynthetic pthwys, detoxifiction, ntioxidnt iochemistry nd redox homeostsis. Glutthione is the precursor of phytocheltins (PCs) compounds tht re synthesized in response to hevy metls. Arsente nd rsenite re lso known to hve high ffinity for thiols such s glutthione (Jocelyn 1972). Glutthione is key ROS scvenger nd mjor cellulr redox uffer; it is crucil prt of stress signling pthwys nd hs importnt roles in the regultion of the cell cycle. Glutthione is the min nonprotein thiols of the cell nd is nonenzymtic ntioxidnt tht prticiptes of free rdicl scvenging nd modultion of the cellulr redox sttus nd thioldisulfide sttus of proteins (Cnuen et l., 2001; Foyer nd Noctor, 2011) Arsente reductse Arsente reductse (glutredoxin) (EC ) ctlyzes the following chemicl rection Arsente reductse (AR) elongs to the fmily of oxidoreductses. This group of rsente reductses elongs to the lowmoleculr weight proteintyrosine phosphtse fmily, s does group of glutthione/glutredoxin type rsente reductses. Arsente cn e reduced to rsenite nonenzymticlly, ut the process is too slow to e physiologiclly significnt (Bhttchrjee et l., 1999). The thiollinked reductses re required to confer resistnce to rsente in oth prokryotes (Ji et l., 1994) nd eukryotes (Borowicz et l., 15

16 1997). The reduction of rsente to rsenite is ctlyzed y AR which is lso considered s mechnism involved in detoxifiction ecuse rsenite cn ind with PCs. Arsente reduction cused y AR nd GSH serving s the electron donor (Ellis et l., 2006) Superoxide dismutse (SOD) It is group of metlloisozymes tht neutrlized the highly rective superoxide rdicl into O 2 nd H 2O 2 so it cn ply very importnt role in the protection of cells upon stress (Fridovich 1995). O2 + O2 + 2H + H2O2 + O2 Superoxide dismutse ctlyzes the dismuttion of the O 2 into O 2 or H 2O 2. Superoxide dismutse constitutes the first line of defense ginst ROS within cell (Alscher, 2002). Superoxide dismutse is n ntioxidnt enzyme ssocited with metl cofctors. Bsed on the metl cofctor used y the enzyme, SODs re clssified into three groups: iron SOD (Fe SOD), mngnese SOD (Mn SOD), nd copperzinc SOD (CuZn SOD). During rsenic stress, the up regultion of Cu/Zn SOD hs een reported in rice seedlings (Shri et l. 2009). The proteomic nlysis of mize root revels tht Cu/Zn SOD is one of the highly responsive enzymes to rsenic which involved in cellulr homeostsis during redox disturnce (Requejo nd Ten, 2005). Mylon et l. (1998) demonstrted tht SOD ctivity incresed in response to low rsenic concentrtion ut high concentrtion of rsenic inhiits the ccumultion of SOD mrna nd leds to decline its ctivity Comptile solutes Comptile solutes or osmoprotectnts re smll molecules tht ct s n osmolyte nd help orgnisms to survive t extreme osmotic stress. Comptile solutes re low moleculr weights, highly solule orgnic compounds tht re usully nontoxic t high cellulr 16

17 concentrtions. These solutes provide protection to plnts from stress y contriuting to cellulr osmotic djustment, ROS detoxifiction, protection of memrne integrity nd enzymes/protein stiliztion (Yncey,1994; Ashrf nd Foold, 2007). In plnts, the ccumultion of comptile solute is incresed. Exmples of comptile solutes include mino cids, glycine etine etc. These molecules ccumulte in cells nd lnce the osmotic difference etween the cells surroundings nd the cytosol. Comptile solutes hve lso een shown to ply protective role y mintining enzyme ctivity Proline Proline is one of the most ccumulted comptile solutes found in plnts s well s in other orgnisms. Most plnt species cn ccumulte proline in response to environmentl stresses including hevy metl stress. Proline is predominntly synthesized from glutmte. Bsed on its known properties proline my e involved in plnt hevy metl stress y different mechnisms, i.e. osmo nd redoxregultion, metl cheltion, nd scvenging of free rdicls. However, in some cses, exogenous proline showed its toxicity to plnts nd cused progrmmed cell deth (Hellmnn et l., 2000). Osmoregultion ppers to e common element of plnt rections to vrious iotic stress. Stress conditions upregulte the key enzyme 1pyrroline5croxylte synthetse (P5CS), for proline iosynthesis (Hre et l., 1999) tht ctlyzes the first two steps of proline iosynthesis from glutmte in plnts (Deluney nd Verm, 1993). Proline plys multiple roles in plnt stress tolernce. Spontneous P5C synthetse cycliztion P5C reductse Glutmte Glutmte Pyrroline5croxylte Proline Semildehyde (GSA) (P5C) Free proline ccumultes in plnts exposed to Cd (Shrm nd Dietz, 2006). Exogenous 17

18 ppliction of proline mitigtes slt stress (Okum et l., 2000, 2004; Hoque et l., 2007) nd cdmium stress (Islm et l., 2009) in BY2 cells Arginine Lrginine is n importnt nd unique mino cid in plnts. It is one of the most functionlly diverse mino cids in living cells nd lso serves s precursor for the synthesis of protein, nitric oxide, cretine, polymines, gmtine, nd ure (Sidney nd Morris, 2007). The rginine metolism plys importnt role in perception nd dpttion of plnt to environmentl disturnces Alnine The ppliction of mino cids cn result in etter plnt development since these molecules cn ct s signls of severl eneficil physiologicl processes of plnts. Alnine, unlike proline, follows the seprte ctolic pthwy. Alnine inhiits rginine degrdtion process nd formtion of ure from rginine Betine Glycine etine (Betine), comptile solute, is smll orgnic metolite tht re very solule in wter nd nontoxic t high concentrtions. Betine is involved in the protection of mcro components of plnt cells, such s protein complexes nd memrnes, under stress conditions. Among the comptile solutes, etine is prticulrly effective protectnt ginst iotic stress (Skmoto nd Murt, 2000;2001). Exogenous ppliction of etine cn improve the tolernce of numerous plnt species to vrious types of iotic stresses. In ddition to its functions s n osmoprotectnt, etine contriutes to stiliztion nd protection of memrnes, proteins nd enzymes ginst stresses (Ppgeorgiou nd Murt, 1995; Ashrf nd Foold, 2007). 18

19 1.13 Lipid peroxidtion Lipid peroxidtion is one of the first consequences of oxidtive dmge. Mlondildehyde (MDA), lipid peroxidtion product, hs widely een used to ssess the levels of free rdicls in living cells. The concentrtion of MDA ws used s n indictor of lipid peroxidtion. The restriction on nutrient uptke cn e ssocited to incresed lipid peroxidtion, which reduces its functionlity Arsenic tolernce mechnisms Though rsenic is not redox metl, there is significnt evidence tht exposure of plnts to inorgnic rsenic results in the genertion of ROS. Plnts hve evolved mechnisms to protect cells from the effects of ROS y using enzymtic ntioxidnts such s SOD, ctlse (CAT), nd scorte peroxidse nd nonenzymtic ntioxidnts such s scorte, glutthione, nd αtocopherol (Sirm et l., 2005; Gunes et l., 2009). Glutthione hs dul role in response to metl stress, nd it hs een suggested tht PCs production, resulting in oxidized glutthione depletion, could itself cuse oxidtive stress (Hrtley Whitker et l., 2001; Verruggen et l., 2009). One of the mjor types of rsenic tolernce/resistnce mechnisms tht hve een demonstrted in plnts is complextion of metls y PCs. Arsente cn e redily reduced to rsenite vi rsente reductse in glutthionedependent rection (Mukhopdhyy et l., 2000) nd rsenite cn susequently complex with thiols, prticulrly PCs. One potentil strtegy for plnts to protect from the toxicity of rsenic to methylte inorgnic rsenic to orgnic rsenic species (Lomx et l., 2012; Zho et l., 2013). 19

20 1.15 Purpose of the study To investigte the role of exogenous proline in plnts, the present reserch ws conducted with the following ojectives: (i) To investigte the effects of proline on rsenicinduced cell growth inhiition in tocco BY2 cultured cells. (ii) To clrify the protective mechnisms of proline in the components of the ntioxidnt defense systems ginst rsenicinduced oxidtive dmge. (iii) To compre the role of proline with other osmolytes such s rginine, lnine, glutmte, nd etine on BY2 cell growth under rsente stress. 20

21 CHAPTER 2 Regultion of rsenteinduced chnges y exogenous proline in tocco BY2 cells 2.1 ABSTRACT Arsenic, nonessentil toxic element, cuses toxicity to plnts. Plnts tke up rsenic minly s rsente. Proline is ccumulted s comptile solute in plnts under vrious stress conditions. Exogenous proline scvenges free rdicles, improves plnt metolism nd stimultes plnt growth under stress conditions. However, in some cses, exogenous proline showed its toxicity to plnts nd cused progrmmed cell deth. It is reported tht proline meliortes hevymetl toxicity in plnts. However, the role of proline in rsentestressed BY2 cells remins uncler. In this study, I investigted the effects of exogenous proline on tocco BY2 cells cultured under AsO 4 stress nd found tht proline depending on its concentrtions plys dynmic roles in BY2 cells such s mitigtion of rsente stress in response to lower concentrtions of proline (e.g., 0.05 mm), wheres sensitiztion of BY 2 cells to rsente in response to higher proline tretment (e.g., 10 mm). In this study, the effects of exogenous proline, exogenous rsente, nd the cotretment of rsente nd proline on BY2 cells growth, cell deth, nd cell numer were presented. Here, AsO 4 significntly inhiited the growth of BY2 cells t 60 µm ut not t either 40 µm or 50 µm. Proline t 0.05 mm to 10 mm did not ffect the cell growth ut delyed it t 20 mm. Therefore, for mitigting the rsenic stress, I exmined the effects of wide rnge (0.05, 0.1, 0.5, 1, nd 10 mm) of exogenous proline on the inhiition of cell growth y 60 µm rsente nd found tht proline t 0.05 mm nd 0.1 mm llevited the rsenteinduced cell growth inhiition, ut surprisingly ccelerted the growth inhiition t 1 mm nd 10 mm. Proline t 0.05 mm nd 0.1 mm significntly decresed the numer of Evns Blue stined cells ut 10 21

22 mm proline oosted the numer of stined cells. Proline t 0.05 mm nd 0.1 mm incresed the totl numer of cells, wheres 10 mm proline decresed the totl numer of cells. These results indicte tht the effects of 0.05 mm nd 0.1 mm proline on rsentestressed BY2 cells reversed with the increse of proline concentrtion to 10 mm, nd lso suggests tht the lower concentrtion of proline mitigtes rsente stress, wheres the higher concentrtion of proline sensitizes BY2 cells to rsente. To insight into the issue tht 10 mm proline enhnces the sensitivity of BY2 cells to rsente, I further investigted the effects of 10 mm proline on BY2 cells treted with 40 µm rsente nd found tht 40 µm rsente did not inhiit cell growth in the sence of proline ut inhiits it in the presence of proline, suggesting tht ppliction of 10 mm proline enhnces the dverse effects of rsente. Together, these results suggest tht proline plys two distinct roles in BY2 cells in the presence of rsente. 2.2 INTRODUCTION Arsenic, toxic metlloid, is widely distriuted in the environment nd cuses physiologicl nd structurl disorders in plnts (Shrm, 2012). Arsenic ccelertes cell deth nd inhiits plnt growth (Stoev nd Binev, 2003; Stoev et l., 2005). Now dy, the reduction of crop yield y rsenic stress hs een recognized s thret to the sustinle food production (Brmmer nd Rvenscroft, 2009; Pnullh et l., 2009). Arsenic occurs predominntly s inorgnic forms such s rsente (AsO 4 ) nd rsenite (AsO 3 ). Plnts tke up rsenic minly s rsente (Tripthi et l., 2007). The orgnic comptile solute, proline, hs een reported to ccumulte in plnts sujected to vrious iotic stresses such s slt stress (Skmoto nd Murt, 2000). Exogenous proline functions s free rdicl scvenger nd n enzyme protectnt (Tsugne et l., 1999; Hong et l., 2000). Okum et l. (2004) reported tht proline exhiits n ntioxidnt ctivity which ws proved y the 1,1diphenyl2picrylhydrzyl ssy. Proline 22

23 improves plnt metolism nd stimultes plnt growth under stress conditions (Ali et l., 1991; Fedin et l., 1993). Erlier studies suggest tht exogenous ppliction of proline confers protection ginst metls (Islm et l., 2009) nd tht proline ply diverse roles in plnts nd confer protection ginst vriety of iotic stresses (Hre et l., 1998). However, in some cses, exogenous proline showed its toxicity to plnts (Hellmnn et l., 2000) nd cused progrmmed cell deth y producing 1pyrroline5croxylte (P5C), n intermedite in iosynthesis nd degrdtion of proline, is ssumed to ply role in cell deth in plnts (Deuschle et l., 2001). Proline hs een known to ccumulte under metl stress in plnts (Shrm nd Dietz, 2006; Xu et l., 2009). In this study, I investigted the effects of exogenous proline on tocco BY2 cultured cells under rsentestress conditions. I found tht rsente inhiited the BY 2 cell growth. Exogenous proline t lower concentrtion mitigtes rsenic induced growth inhiition y decresing Evns Blue stined cells nd incresing the totl numer of cells, wheres, tht rsente ccelerted the inhiition of cell growth in the presence of higher concentrtion of proline. I lso found tht rsente oosted the numer of ded cells nd decresed the totl numer of cells in the presence of higher proline. These results indicted tht exogenous proline t higher concentrtion did not mitigte rsente stress in BY2 cells ut tht proline enhnces the sensitivity of BY2 cells to rsente. 2.3 MATERIALS AND METHODS Culture of tocco BY2 cells Suspensioncultured cells of tocco (Nicotin tcum L., cv. BY2) were used for the rsenicundpted cell lines (Murt et l., 1994, ). The modified LS medium (Linsmier nd Skoog, 1965) ws used s stndrd medium in which KH 2PO 4 nd thiminehcl were incresed to 370 nd 1 mgl 1, respectively, supplemented with 3% sucrose nd 1 µm 2,4 23

24 dichlorophenoxycetic cid (Ngt et l., 1981). The LS medium supplemented with 40 µm, 50 µm, nd 60 µm AsO 4 were regrded s the stndrd rsenic medium of 40 µm, 50 µm, nd 60 µm AsO 4, respectively. The 0.05 mm, 0.1 mm, 0.5 mm, 1 mm nd 10 mm proline medi were the 60 µm AsO 4 medi contining 0.05 mm, 0.1 mm, 0.5 mm, 1 mm nd 10 mm proline. The 0.5 mm, 1 mm nd 10 mm proline medi were the 40 µm AsO 4 medi contining 0.5 mm, 1 mm nd 10 mm proline. The BY2 cells were cultured nd mintined s descried previously (Murt et l., 1994, ). The cells were sucultured weekly nd were incuted on rotry shker t 100 rpm t 25 C in the drk Mesurement of BY2 cell growth The growth of BY2 cells ws mesured s descried previously (Murt et l., 1994, ). To mesure the BY2 cell growth, the cells were incuted in the culture medi for different dys such s 2, 4, 6, 8, nd 10 dys. After incution, the cells were collected y removing the queous solution in vcuum using nylon sieve (pore size 45 µm), nd the fresh weight (FW) of the cells ws tken. Then the cells were dried in n oven t 70 C nd the dry weight (DW) ws mesured. Both the FW nd DW of the cells were tken t different dys fter inocultion (DAI) Estimtion of cell deth of BY2 cells Ded cells were quntified y the method descried previously (Yno et l., 1998; Tktsuk et l., 2004). Cells were stined with Evns Blue solution (0.05%) for 20 min nd susequently wshed with distilled wter to remove the excess dye. Dye tht hd ound to ded cells ws soluilized in 1 ml of 50% methnol tht contined 1% sodium dodecyl sulfte followed y the incution for 50 min t 50 C. Then the sornce ws mesured t 600 nm y spectrophotometer. For clcultion, the cells prepred in the sme wy were sujected to two cycles of freezing t 20 C nd thwing t room temperture. The cells 24

25 killed in such wy were used to define 100% cell deth. The vlue otined from 4dyold cultured cells ws defined s 0% cell deth. The deth cells were clculted y compring the sornce of the smples with the sornce of 100% cell deth Counting of BY2 totl cell numer The numer of totl cells ws counted using Hemcytometer (BurkerTurk, mm 2, mm 2 ) under the microscope Sttisticl nlysis Unless stted otherwise, the significnce of differences etween the men vlues of ll prmeters ws ssessed y Tukey s test. Differences t the level of p 0.05 were considered s significnt. 2.4 RESULTS Effects of exogenous proline on BY2 cell growth in the sence of rsente To investigte the roles of exogenous proline for the mitigtion of rsente stress in BY2 cells, we exmined whether exogenous proline shows ny effects on BY2 cells. We mesured the FW nd DW of cells t 0, 2, 4, 6, 8, nd 10 DAI in response to exogenous proline in the sence of rsente. We found tht 0.05 mm, 0.1 mm, 0.5 mm, 1 mm nd 10 mm proline did not chnge BY2 cell growth compred with control ut delyed it t 20 mm s well s the cell growth curve ws drmticlly incresed t 4 to 6 DAI nd then stedily incresed up to 10 DAI (Fig.2.1A nd B). 25

26 Dry weight (g/flsk) Fresh weight (g/flsk) A Control 0.05 mm Pro 0.1 mm Pro 0.5 mm Pro 1 mm Pro 10 mm Pro 20 mm Pro Dys fter innocultion B Control 0.05 mm Pro 0.1 mm Pro 0.5 mm Pro 1 mm Pro 10 mm Pro 20 mm Pro Dys fter innocultion Figure 2.1 Effects of exogenous proline (Pro) on BY2 cell growth. A, Shows the cell growth sed on fresh weight nd B, shows the cell growth sed on dry weight in response to 0.05 mm, 0.1 mm, 0.5 mm, 1 mm, 10 mm nd 20 mm Pro t 0, 2, 4, 6, 8, nd 10 DAI. Averges of cell growth from three independent experiments (n = 3) re shown. Error rs represent SE. There were no significnt differences (p<0.05) etween control (untreted) cells nd treted cells in fresh weight or dry weight t ech time point fter inocultion. 26

27 Dry weight (g/flsk) Fresh weight (g/ Flsk) Effects of exogenous rsente on BY2 cell growth We tested the effects of rsente on BY2 cell growth. We mesured the FW nd DW of BY 2 cells t 0, 4, 6, nd 8 DAI in response to 40 µm, 50 µm, nd 60 µm AsO 4. Compred with control, rsente did not ffect the growth of BY2 cells t 40 µm nd 50 µm ut significntly inhiited it t 60 µm t ll DAI (Fig.2.2A nd B). A Control 40 µm As 50 µm As 60 µm As Dys fter inocultion B Control 40 µm As 50 µm As 60 µm As Dys fter inocultion Figure 2.2 Effects of exogenous rsente (As) on BY2 cell growth. A, Shows the cell growth sed on fresh weight nd B, shows the cell growth sed on dry weight in response to 40 µm, 50 µm nd 60 µm rsente t 0, 4, 6 nd 8 dys fter inocultion. Averges of cell 27

28 Fresh weight (g/flsk) growth from three independent experiments (n = 3) re shown Effects of exogenous proline on the inhiition of BY2 cell growth y rsente To investigte whether exogenous proline recovered the inhiition of cell growth y rsente, we exmined the effects of exogenous proline on the growth of BY2 cells cultured t 40 µm AsO 4 nd 60 µm AsO 4. The FW nd DW of cells t 0, 4, 6, nd 8 DAI were mesured. At 60 µm AsO 4 stress condition, 0.05 mm proline significntly recovered the AsO 4 induced inhiition of BY2 cell growth, wheres 0.1 mm nd 0.5 mm proline did not ffect the cell growth ut 1 mm nd 10 mm proline significntly inhiited it (Fig. 2.3A nd B). Moreover, AsO 4 t 60 µm induced more inhiition of cell growth in the presence of 1 mm nd 10 mm exogenous proline thn in the sence of exogenous proline. In the presence of 40 µm AsO 4, neither 0.5 mm proline nor 1 mm proline ffected the cell growth ut 10 mm proline inhiited it (Fig. 2.4A nd B). These results suggest tht exogenous proline t lower concentrtion mitigte the rsenteinduced growth inhiition of BY2 cells ut did not t 1 mm nd 10 mm nd further enhnces the sensitivity of BY2 cells to rsente. A Control As mm Pro As mmpro As + 10 mm Pro 60 µm As As mm Pro As + 1 mm Pro 4 2 c c d e c c c d e cc cd e c Dys fter inocultion 28

29 Dry weight (g/flsk) B Control As mm Pro As mmpro As + 10 mm Pro c c c cd d 60 µm As As mm Pro As + 1 mm Pro Dys fter inocultion c c cd d e c c cdd e Figure 2.3 Effects of exogenous pro (Pro) on 60 µm rsente (As)induced growth inhiition of BY2 cells. Increment of cell growth in the presence of 0.05 mm proline nd enhncement of rsenteinduced cell growth reduction (Fresh weight sis, A; dry weight sis, B) in the presence of oth the 1 mm nd 10 mm Pro ut not in the presence of 0.1 mm nd 0.5 mm Pro t 4, 6 nd 8 dys fter inocultion. Averges of cell growth from three independent experiments (n = 3) re shown. The error rs represent SE. For the sme inocultion dy, vlues indicted y the sme letter do not differ significntly t 5% level of significnce s determined y Tukey s test. 29

30 Dry weight (g/flsk) Fresh weight (g/flsk) A Control 40 µm As As mm Pro As + 1 mm Pro As +10 mm Pro Dys fter inocultion B 0.5 Control As mm Pro As +10 mm Pro 40 µm As As + 1 mm Pro Dys fter inocultion Fig. 2.4 Effects of exogenous proline (Pro) on 40 µm rsentestressed BY2 cells. Reduction of BY2 cell growth y rsente (FW sis, A; DW sis, B) in the presence of 10 mm Pro ut not in presence of 0.5 mm or 1 mm Pro t 4, 6 nd 8 DAI. Averges of cell growth from three independent experiments re shown. Error rs represent SE. For the sme inocultion dy, vlues indicted y the sme letter do not differ significntly t 5% level of significnce s determined y Tukey s test. 30

31 Cell deth (%) Effects of exogenous proline on rsenteinduced cell deth In this study, we exmined the cell deth of BY2 y rsente in the presence nd sence of proline. Arsente t 60 µm incresed the numer of Evns lue stined cells y 25% compred with control. In the presence of AsO 4, 0.05 mm nd 0.1 mm exogenous proline significntly recovered the AsO 4 induced cell deth. On the other hnd, rsente oosted the numer of stined cells y 45% in the presence of 10 mm proline ut not in the presence of either 0.5 mm or 1 mm proline (Fig. 2.5A). Arsente t 40 µm did not show ny effect on the cell deth wheres tht rsente in the presence of 10 mm proline significntly incresed the Evns lue positive cells ut not in the presence of 0.5 mm nd 1 mm proline (Fig. 2.5B). These results indicte tht ppliction of proline recovered the rsenteinduced cell deth t lower concentrtion ut increse the numer of ded cell y rsente t higher concentrtion. A C c c 60 µm Control As mm Pro d As mm Pro As + 1 mm Pro 0 As (µm) Pro (mm) As mm Pro As + 10 mm Pro 31

32 Cell deth (%) B D Control 40 µm As As mm Pro As + 1 mm Pro As + 10 mm Pro Figure 2.5 Effects of exogenous proline (Pro) on rsente (As)induced BY2 cell deth. A, Induction of cell deth y the cotretment of 60 µm rsente. Arsente t 60 µm induced significnt cell deth which ws recovered y 0.05 mm nd 0.1 mm Pro, nd enhnced in the presence of 10 mm Pro. B, Induction of cell deth y the cotretment of 40 µm rsente nd 10 mm Pro ut not y the individul tretment of 40 μm rsente. Averges of cell deth from three independent experiments (n = 3) re shown. The error rs represent SE. Vlues indicted y the sme letter do not differ significntly t 5% level of significnce s determined y Tukey s test. C nd D, Evns Blue stining of rsentetreted BY2 cells in the presence or sence of Pro. At 60 µm rsentestress condition, BY2 cells showed more Evns Blue positive cells compred with control. Pro t 0.05 mm nd 0.1 mm decresed the Evns Blue stined cells t AsO 4 stress ut the stined cell ws enhnced in the presence of 10 mm Pro (Fig. 2.5C). At 40 µm rsentestress condition, BY2 cells showed Evns Bluestined cells in the presence of Pro ut not in the sence (Fig. 2.5D). 32

33 Cell numer Effects of exogenous proline on the reduction of BY2 cell numer y rsente In our study, we monitored the effects of exogenous rsente t 40 µm nd 60 µm on the totl numer of BY2 cells with or without the ppliction of proline. We found tht rsente t 60 µm decresed the totl numer of cells y 3.5fold compred with control. At 60 µm rsente, proline t 0.05 mm nd 0.1 mm significntly incresed the totl numer of cell nd tht rsente in the presence of 1 mm nd 10 mm proline decresed the numer of cells y 4.5 nd 7.5fold, respectively (Fig. 2. 6A). We lso found tht rsente t 40 µm did not show ny effect on the cell numer compred with control wheres tht rsente in the presence of 10 mm proline significntly decresed the cell numer ut not in the presence of 0.5 mm nd 1 mm proline (Fig. 2.6B). These results indicte tht ppliction of proline t lower concentrtion recovered the reduction of cell numer y rsente ut higher proline level enhnces the rsenteinduced decrese of cells numer. A c c c d 0 33

34 Cell numer B Figure 2.6 Reduction of BY2 cell numer y rsente (As) in the presence or sence of proline (Pro). A, the reduction of cell numer y the cotretment of 60 µm rsente nd increment of cell numer t 0.05 mm, nd the reduction in cell numer t 10 mm Pro. B, Decrese of cell numer y the cotretment of 40 µm rsente nd 10 mm Pro ut not y the rsente lone. Averges of cell numer from three independent experiments (n = 3) re shown. 2.5 DISCUSSION Arsenic is one of the most hzrdous elements in the environment nd ecomes glol griculturl prolem. Accumultion of rsenic in plnts cuses destruction of cellulr memrnes interferes with plnt metolic processes nd reduces plnt productivity (Shrm, 2012; Singh et l., 2006). It ws reported tht proline meliortes hevymetl toxicity in plnts. However, whether proline mitigtes AsO 4 stress in BY2 cells re to e investigted. In this study, we present the AsO 4 induced growth inhiition of BY2 cells. Here, the AsO 4 induced growth inhiition ws recovered y lower concentrtion of exogenous 34

35 proline nd the incresing rte of growth inhiition y tht rsente in the presence of higher proline. We lso demonstrte tht the AsO 4 induced increment of cell deth nd the reduction of totl cell numer ws recovered y lower proline ut did not in the presence of higher proline. It is wellknown tht rsente inhiits the growth of plnts (Shri et l., 2009; Ali et l., 2014). In this study, we found tht AsO 4 significntly inhiited the growth of BY2 cells t 60 µm ut not t either 40 µm or 50 µm (Fig. 2.2A nd B). Therefore, for mitigting the rsenic stress, we exmined the effects of exogenous proline on the inhiition of cell growth y 60 µm rsente. Our results indicte tht ppliction of proline t lower concentrtion (0.05 mm) meliorted the rsenteinduced growth reduction. On the other hnd, proline t higher concentrtion (10 mm proline) did not recover rsenteinduced growth reduction ut surprisingly tht rsente enhnces the cell growth reduction in the presence of 1mM nd 10 mm proline (Fig. 2.3A nd B). To insight into this issue, we further investigted the effects of 0.5 mm to 10 mm proline on BY2 cells treted with 40 µm rsente. Arsente t 40 µm did not inhiit cell growth in the sence of proline ut inhiits it in the presence of proline (Fig. 2.4A nd B), suggesting tht ppliction of 10 mm proline enhnces the dverse effects of rsente. In contrst to our proline sensitivity enhncement results, Singh et l. (2015) reported tht exogenous proline ppliction meliorted toxic effects of rsente in Solnum melongen seedlings. This difference my come from the mjor difference in endogenous proline contents etween BY2 cells (pproximtely 3 mm) nd eggplnt seedlings (round 1 μg/gfw; lmost equivlent to out 10 μm). Choudhury et l. (2010) nd Siddiqui et l. (2015) reported tht rsente stress incresed the proline contents. However, it is uncler which inhiition of plnt growth is due to ccumultion of rsente or proline or due to dditive or synergistic effect. In plnts, hevy metls cn cuse tissue dmge, growth inhiition, nd even deth. In this study, we found tht rsente induced the numer of Evns Bluestined (ded) cells 35

36 (Fig. 2.5A) nd decresed the totl numer of BY2 cells (Fig. 2.6A), which is consistent with the previous results (Xue nd Yi, 1014). Moreover, we found tht exogenous proline t lower concentrtion recovered the rsenteinduced cell deth nd incresed the totl numer of cells t rsente stressed condition. On the contrry, proline t higher concentrtion oosted the Evns Blue stined cells s well s the reduction of cell numer y rsente is potentited in response to exogenous proline. These results suggest tht rsente decresed the numer of deth cell nd increses the totl numer of cell in the presence of lower proline level ut incresed the cell deth nd enhnces the reduction of totl cell numer in the presence of higher proline. There is no cler consensus out the role of proline in plnts s well s the mechnism y which proline mitigtes hevy metl stresses in plnts. Previous reserch reported tht proline mitigtes stresses in plnts. For exmple, ppliction of proline meliortes slt stress (Hoque et l., 2007) nd cdmium stress in BY2 cells (Islm et l., 2009). However, the negtive role of exogenous proline ws lso reported in some cses. For instnces, exogenous proline shows toxicity to plnts (Hellmnn et l., 2000; Deuschle et l., 2001) nd proline t 2 mm inhiits the growth of sltgrss (Distichlis spict) (Rodriguez nd Heyser, 1988). It ws lso reported tht the ccumultion of proline in plnts under stress condition is not ssocited with the mitigtion of stress ut it is just symptom (Liu nd Zhu, 1997) nd did not show ny protective vlue (Mofth nd Michel, 1987). In the present study, our dt showed tht proline meliortes rsenic stress t lower concentrtion nd higher proline did not mitigte rsenic stress in BY2 cells ut rsente induced more stressing effects in the presence of higher proline. The previous reports with our findings suggest tht the mitigtory role of proline might depend on some conditions such s type of stress, nd plnt species. Together, we conclude tht exogenous proline t lower concentrtion mitigtes rsente stress ut higher proline enhnces the sensitivity of BY2 cells to rsente. 36

37 CHAPTER 3 Mitigtion of rsente stress y exogenous proline in cultured tocco cells under rsente stress 3.1 ABSTRACT Arsenic, one of the most toxic hevy metls, cuses hzrd to plnt nd humn helth. Arsenic exposure generlly induces the production of ROS cusing progressive oxidtive dmge nd ultimtely cell deth tht cn led to the production of ntioxidnts. Proline llevites the dmging effect of oxidtive stress in plnts y the upregultion of the ntioxidnt defense system. In this study, we investigted the protective effects of exogenously pplied proline on cell growth, ROS production, glutthione content, nd ctivities of different ntioxidnt enzymes in cultured tocco BY2 cells exposed to rsente (AsO 4 ) stress. This study descries the moleculr mechnisms of lower proline in BY2 cells under rsente stress. Previous evidences suggest tht rsenic exposure induces the genertion of ROS. Our results indicte tht rsente stress incresed the ROS levels compred with control nd in the presence of rsente, 0.05 mm nd 0.1 mm proline decresed the intrcellulr ROS level compred with rsente stress. The mechnisms of how proline mitigtes stress in plnts re not fully understood ut pper to involve its chemicl properties nd effects on redox systems such s the glutthione pool. Glutthione is the mjor source of nonprotein thiols in plnt cells nd functions s key component of the ntioxidnt network. The incresed level of glutthione pool is generlly regrded s protective response ginst oxidtive stress. The reduced glutthione (GSH) content is ssocited with the protection to oxidtive stress in plnts. In the presence of AsO 4, 0.05 mm proline did not show ny effect on totl glutthione (Totl GSH) content compred with rsente stress. Furthermore, 0.05 mm proline decresed the GSH nd incresed the 37

38 oxidized glutthione (GSSG) contents compred with rsente stress. These results suggest tht during mitigtion process proline mintins glutthione homeostsis y decresing GSH nd incresing GSSG. Arsente is redily reduced to rsenite through rsente reductse using GSH s reductnt. In this study, t AsO 4 stress condition, compred with control, 0.05 mm proline did not show ny effects on rsente reductse ctivity. Moreover, compred with rsente stress, 0.05 mm proline significntly incresed the rsente reductse ctivity. These results suggest tht proline mitigtes rsente stress y incresing the rsente reductse ctivity which ccelertes the conversion of rsente to rsenite, leding detoxifiction nd sequestrtion of rsenite. In this study, we lso found tht the SOD ctivity is incresed t 60 µm rsenic stress compred with control. In the presence of rsente, proline t 0.05 mm did not show ny effect on the SOD ctivity compred with rsente stress. The ove dt indicte tht 0.05 mm proline in most of the prmeters positively regulted, nd recovered the stressing effects of rsente. Therefore, the mechnism of lower prolineinduced stress mitigtion is consistent with the previous reports, which is importnt to understnd the mitigtory roles of proline in BY2 cells. 3.2 INTRODUCTION Arsenic is n environmentl toxin tht is found nturlly in the environment. The min two forms of inorgnic rsenic, rsente nd rsenite, re esily tken up y plnt cell. In eroic environments, rsenic occurs mostly in its oxidized form, rsente, nd hs een reported to e tken up y plnts vi the phosphte trnsport system (Asher nd Rey, 1979; Lee, 1982; UllrichEerius et l., 1989; Mehrg nd Mcnir, 1991, 1992; Mehrg nd Hrtley Whitker, 2002; Wng et l., 2002). Once tken up y the plnt, rsente cn e redily converted to rsenite, the more toxic of the two forms. Arsenic, when not detoxified, my trigger sequence of rections leding to growth inhiition, nd stimultion of secondry metolism, nd cuses memrne degrdtion nd cell deth (Mehrg nd Hrtley 38

39 Whitker, 2002). Arsenic is lso known to induce oxidtive stress directly y generting ROS during conversion of its vlence forms y inctivting ntioxidnt molecules through inding with their sulfhydryl (SH) groups (Shrm, 2012). Arsenic interferes with vrious metolic processes nd therey dversely ffects the plnt metolism nd leds to reduced plnt productivity. In response to different stresses, plnts ccumulte lrge quntities of different types of comptile solutes (Serrj nd Sinclir, 2002). Proline is wellknown comptile solute tht ccumulte under metl stress hs een correlted with stress tolernce in plnts. Proline ply significnt functions under metl stress nd it hs een reported tht proline ccumultion my e prt of stress signl influencing dptive responses. Both glutthione (Ogw, 2005) nd proline (Lehmnn et l., 2010) perform multiple functions in plnts nd originted from common precursor Lglutmte (Mot et l., 2003). Glutthione is key component of the ntioxidnt network tht functions s mjor intrcellulr ntioxidnt inside the cell (Coett, 2000). It is reported tht glutthione cn function s n intrcellulr signling gent nd responsive to chnges in the extrcellulr environment (Fernndez et l., 1997). Previous reports suggest tht rsenite form complexes with thiol compounds in plnts, due to its high ffinity with sulfhydryl groups (R et l., 2005). The incresed level of glutthione synthesis is considered mens of metl inding cpcity s well s wy to incresed cellulr defense ginst oxidtive stress. The reduction of rsente to rsenite is key step in the rsenic detoxifiction pthwys. Arsente is redily reduced to rsenite through rsente reductse using reduced glutthione (GSH) s reductnt (Dun et l., 2005; Mukhopdhyy et l., 2000). Though the reduction mechnism is not well understood in plnts, the reduction of rsente to rsenite occurs in numer of plnt species (Pickering et l., 2000; Mehrg nd HrtleyWhitker, 2002; Slt et l., 2002; Qugheeur nd Rengel, 2003). 39

40 Under environmentl stresses, plnts often produce ROS such s superoxide, hydrogen peroxide nd hydroxyl rdicls, cusing dmge to DNA, proteins nd lipids. It is evident tht rsenic exposure leds to the genertion of ROS through the conversion of rsente to rsenite, process tht rpidly occurs in plnts (Flor, 1999; Mscher et l., 2002). To minimize the hrmful effects of ROS, plnts hve evolved n effective scvenging system composed of ntioxidnt molecules nd ntioxidnt enzymes (Mehrg, 1994). However, in some instnces this defensive mechnism ecomes indequte to withstnd ginst stressed conditions (Chndrkr et l., 2016). Report suggest tht during stress, proline decresed the denturtion of enzyme, hence cn e pplied ginst rsenic stress which redily inds with thiol groups nd inctivtes enzymes (Chndrkr et l., 2016). Therefore, reserch on the protective role of proline is mtter of scientific interest for its ppliction ginst rsenicstress. 3.3 MATERIALS AND METHODS Culture of tocco BY2 cells Suspensioncultured cells of tocco (Nicotin tcum L., cv. BY2) were used for the rsenicundpted cell lines (Murt et l., 1994, ). The modified LS medium (Linsmier nd Skoog, 1965) ws used s stndrd medium. The BY2 cells were cultured nd mintined s descried previously (Murt et l., 1994, ). The culture nd mintennce of cells were descried in Chpter Estimtion of proline content Proline ws mesured following the method of Btes et l. (1973). Aliquots of BY2 cells were wshed with 0.2 M soritol. Cells were ground nd homogenized using liquid N 2 in 10 ml of 3% queous sulfoslicylic cid. The homogente ws centrifuged t 12,000 g for 15 min t 40

41 4 0 C. Two ml of the superntnt were rected with 2 ml of cid ninhydrin (1.25 g ninhydrin dissolved in 30 ml of glcil cetic cid nd 20 ml 6M phosphoric cid) nd 2 ml of glcil cetic cid for 1 h t C, nd the rection ws then terminted in n ice th. The colored rection mixture ws extrcted with 4 ml of toluene nd the sornce ws recorded t 520 nm. The proline content ws otined from the stndrd curve Isoltion of genomic DNA nd gel electrophoresis Approximtely 200 mg of fresh BY2 cells ws ground nd homogenized with mortr nd pestle using liquid nitrogen in 500 µl of extrction uffer contining 3% cetyltrimethylmmonium romide, 1.4 M NCl, 20 mm EDTA, 100 mm TrisHCl, ph 8.0, nd 0.2 % (v/v) βmercptoethnol. The mixture ws incuted t 60 0 C for 45 min. After centrifugtion t 11,000 g for 10 min t room temperture, the superntnt ws trnsferred to new microtue nd then mixed well with n equl volume of chloroform. After centrifugtion t 11,000 g for 10 min t room temperture, the queous upper phse ws collected. The DNA ws precipitted with n equl volume of isopropnol, wshed in 70% (v/) ethnol, dried, nd suspended in sterile distilled wter. The DNA ws then electrophoresed on 1% (w/v) grose gel followed y visuliztion with ethidium romide Detection of ROS ROS in rsente stressed BY2 cells ws mesured using 2, 7 dichlorodihydroflurescein dicette (H 2DCFDA) s descried previously (Bisws nd Mno, 2015). BY2 cells were collected nd wshed with distilled wter y centrifugtion. Cells were then incuted in 20 µm H 2DCF dicette in PBS t 37 0 C for 30 min nd wshed two times with PBS. The fluorescence ws monitored using fluorescence microscope. 41

42 3.3.5 Mesurement of glutthione content GSH contents were determined ccording to Bker et l. (1990) method. An liquot of BY2 cells ws ground using liquid nitrogen (N 2) nd homogenized with extrction uffer (50 mm KH 2PO 4, 5 mm EDTA, ph 8.0). The homogente ws centrifuged t 9500g for 10 min followed y deproteiniztion with sulphoslicylic cid (4%) nd the superntnt ws collected. In ddition, for the mesurement of GSSG content, 2vinylpyridine (97%) nd triethnolmine (20%) were dded to the superntnt. Then 50 µl of the superntnt ws tken in the wells nd 100 µl of the rection mixture (NADPH, DTNB, KH 2PO 4 nd glutthione reductse) ws dded. The sornce ws recorded t 412 nm using microplte reder (Nippon BioRd Lortories (680), Tokyo, Jpn). The GSH content ws clculted y sutrcting the GSSG from the totl GSH Extrctions nd mesurements of rsente reductse For preprtion of smple solution, pproximtely 0.5 g of fresh BY2 cells ws ground with mortr nd pestle tht were chilled with liquid nitrogen nd homogenized in 3 ml uffer solution contining 50 mm MOPS ((3(Nmorpholino) propne sulfonic cid) nd 50 mm MES((2(Nmorpholino) ethne sulfonic cid), which ws djusted to ph of 6.5 with 1 M NOH. The homogente ws centrifuged t 10,000 ˣ g for 30 min t 4 0 C. The superntnt ws filtered through Qulittive Advntec 2 filter pper. The resulting filtrte ws collected nd pssed through Sephdex PD10 deslting columns (Dun et l., 2005). The finl filtrte ws the extrct contining rsente reductse. All the ove steps were crried out on ice. Arsente reductse ctivity ws ssyed using the methods descried y Shi et l. (1999) nd Dun et l. (2005). The ssy uffer ws 50 mm MOPS nd 50 mm MES, ph 6.5, with totl volume 1.5 ml contining 1.0 mm NADPH, 1 unit yest glutthione reductse, 1 mm GSH, 10 mm sodium rsente. The extrct (100 µl) ws preincuted t 30 0 C for 5 min in uffer contining yest glutthione reductse nd GSH. Smll volumes of NADPH 42

43 nd sodium rsente were then dded nd mixed thoroughly to strt the rection. All steps were crried out t 30 0 C. Arsente reductse ctivity ws monitored y the decrese in NADPH sornce t 340 nm nd the NADPH oxidtion ws clculted using molr extinction coefficient of 6200 for NADPH t 430 nm Extrctions nd mesurements of superoxide dismutse Approximtely 200 mg of fresh BY2 cells ws hrvested nd ground with mortr nd pestle tht were chilled with liquid nitrogen nd dded 500 µl of phosphte uffer solution (ph 8.0). SOD ctivity ws mesured y using SOD Test Kit (Wko). For ech SOD ctivity mesurement, 100 µl of the smple solution ws tken in microtue for smple (S) nd smple. lnk (S.BL). Hundred µl of distilled wter ws tken for lnk (BL) nd lnk. lnk (BL.BL). After tht 1.0 ml of coloring regent ws dded to ech tue nd 1.0 ml of enzyme solution ws dded for smple (S) nd lnk (BL). One ml of lnk regent ws dded for smple. lnk (S.BL) nd lnk. lnk (BL.BL). After incution t 37 0 C for 20 min, 2.0 ml rection stopper ws dded to ech tue. The sornce ws mesured t 560 nm. The ctivity ws clculted (Inhiition rte %) using the following eqution: SOD ctivity (Inhiition %)= {(E BLE BL. BL) (E SE S. BL)/ (E BLE BL. BL)} χ Mesurement of protein Protein determintions were crried out using the method of Brdford (1976) with BSA s stndrd Sttisticl nlysis Unless stted otherwise, the significnce of differences etween the men vlues of ll prmeters ws ssessed y Tukey s test. Differences t the level of p 0.05 were considered s significnt. 43

44 Proline (mg/g FW) 3.4 RESULTS Proline content in response to ppliction of exogenous proline To investigte whether exogenously pplied proline induces the ccumultion of proline content under AsO 4 stress, we mesured the intrcellulr proline contents in BY2 cells oth t rsente stress nd nonstress condition (Fig. 3.1). The proline content ws significntly incresed t 60 µm AsO 4 compred with control, showing tht AsO 4 stress induced the ccumultion of proline. Exogenously pplied 0.05 mm proline showed remrkle increse in the ccumultion of proline compred with control. In the presence of rsente, 0.05 mm proline led to significnt increse in proline content which is similr with 60 µm AsO 4 stress. Proline content ws significntly incresed t 0.1 mm oth t AsO 4 stress nd nonstress condition. These results suggest tht oth under Asstressed nd nonstressed conditions, proline content ws incresed in concentrtiondependent mnner in response to the ppliction of exogenous proline d c 5 0 Figure 3.1 Proline content in response to exogenous proline ppliction. The Proline content ws incresed oth under 60 µm AsO 4 stressed nd nonstressed conditions in concentrtiondependent mnner in response to the ppliction of exogenous proline. Averges of proline contents from three independent experiments (n = 3) re shown. 44

45 DNA migrtion DNA frgmenttion To investigte whether DNA frgmenttion occurred in BY2 cells under rsente stress, DNA ws extrcted from BY2 cells nd sujected to electrophoresis on grose gel prior to stining with ethidium romide. No DNA frgmenttion ws detected in the rsente stressed cells irrespective of the presence or sence of proline (Fig. 3.2). As (µm) Pro (mm) M Figure 3.2 DNA frgmenttion in rsente (As) stress BY2 cells in response to exogenous proline (Pro) ppliction. DNA ws extrcted nd sujected to electrophoresis on 1 % grose gel. Photogrphs shown re representtive of four independent experiments. M, 1 k DNA mrker. 45

46 ROS Level (% of control) Intrcellulr ROS level We investigted whether proline could inhiit rsenteinduced ROS ccumultion in BY2 cells (Fig. 3.3). Arsente stress resulted in significnt increse in ccumultion of intrcellulr ROS in BY2 cells compred with control. In the presence of rsente, proline t 0.05 mm nd 0.1 mm significntly inhiited the rsente induced ROS ccumultion in BY 2 cells compred with rsente stress (Fig. 3.3). A i ii iii iv B c c 0 Figure 3.3 Intrcellulr ROS levels in tocco BY2 suspension cells under rsente (As) stress in the presence or sence of proline (Pro). Fourdycultured cells were incuted in As stress medi with different concentrtion of Pro nd DCF fluorescence ws recorded using fluorescence microscope. A, Typicl photogrphs re shown: untreted cells s control (i), 60 µm As (ii), As mm Pro (iii), nd As mm Pro (iv). Br = 10 µm. B, 46