A protective role for nitric oxide and salicylic acid for arsenite phytotoxicity in rice (Oryza sativa L.)

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1 Aepted Mnusript A protetive role for nitri oxide nd sliyli id for rsenite phytotoxiity in rie (Oryz stiv L.) Amit Pl Singh, Grim Dixit, Amit Kumr, Seem Mishr, Nvin Kumr, Smeer Dixit, Prdyumn Kumr Singh, Snjy Dwivedi, Prodh Kumr Trivedi, Vivek Pndey, Om Prksh Dhnkher, Greth J. Norton, Desis Chkrrty, Rudr Deo Tripthi PII: S (17) DOI: /j.plphy Referene: PLAPHY 4816 To pper in: Plnt Physiology nd Biohemistry Reeived Dte: 2 Deemer 2016 Revised Dte: 22 Ferury 2017 Aepted Dte: 22 Ferury 2017 Plese ite this rtile s: A.P. Singh, G. Dixit, A. Kumr, S. Mishr, N. Kumr, S. Dixit, P.K. Singh, S. Dwivedi, P.K. Trivedi, V. Pndey, O.P. Dhnkher, G.J. Norton, D. Chkrrty, R.D. Tripthi, A protetive role for nitri oxide nd sliyli id for rsenite phytotoxiity in rie (Oryz stiv L.), Plnt Physiology et Biohemistry (2017), doi: /j.plphy This is PDF file of n unedited mnusript tht hs een epted for pulition. As servie to our ustomers we re providing this erly version of the mnusript. The mnusript will undergo opyediting, typesetting, nd review of the resulting proof efore it is pulished in its finl form. Plese note tht during the prodution proess errors my e disovered whih ould ffet the ontent, nd ll legl dislimers tht pply to the journl pertin.

2 A Protetive role for Nitri oxide nd Sliyli id for Arsenite Phytotoxiity in Rie (Oryz stiv L.) Amit Pl Singh, Grim Dixit, Amit Kumr, Seem Mishr, Nvin Kumr, Smeer Dixit, Prdyumn Kumr Singh, Snjy Dwivedi, Prodh Kumr Trivedi, Vivek Pndey, Om Prksh Dhnkher, Greth J Norton, Desis Chkrrty, Rudr Deo Tripthi * CSIR-Ntionl Botnil Reserh Institute, Rn Prtp Mrg, Luknow , Uttr Prdesh, Indi Deprtment of Botny, Luknow University, Luknow , Indi. Stokridge Shool of Agriulture, University of Msshusetts, Amherst, MA , U.S.A. d Institute of Biologil nd Environmentl Sienes, University of Aerdeen, Cruikshnk Building, St. Mhr Drive, Aerdeen, AB24 3UU, UK Amit Pl Singh: mitomrde@gmil.om; Grim Dixit: griim21@gmil.om; Amit Kumr: mit_gene@yhoo.om; Seem Mishr: seem_mishr2007@yhoo.o.in; Prdyumn Kumr Singh: pkdk2009@gmil.om; Nvin Kumr: nvinms@gmil.om Smeer Dixit: smeerdxt646@gmil.om; Snjy Dwivedi: drs_dwived@yhoo.o.in; Desis Chkrrty : hkrrtyd@nri.res.in; Prodh K. Trivedi: prodht@hotmil.om Vivek Pndey: v.pndey@nri.res.in; Om Prksh Dhnkher: prksh@umss.edu Greth J Norton: g.norton@dn..uk * Corresponding uthor Dr. Rudr Deo Tripthi, FNAS Chief Sientist & Professor, Plnt Eology nd Environmentl Siene Division CSIR-Ntionl Botnil Reserh Institute, Rn Prtp Mrg, Luknow , Indi. Ph: ; Fx: E-mil: tripthi_rd@rediffmil.om; tripthird@gmil.om 1

3 Astrt Nitri oxide (NO) nd sliyli id (SA) re importnt signling moleules in plnt system. In the present study oth NO nd SA showed protetive role ginst rsenite (As III ) stress in rie plnts when supplied exogenously. The pplition of NO nd SA llevited negtive impt of As III on plnt growth. Nitri oxide supplementtion to As III treted plnts gretly deresed rseni (As) umultion in the roots s well s shoots/ roots trnslotion ftor. Arsenite exposure in plnts deresed the endogenous levels of NO nd SA. Exogenous supplementtion of SA not only enhned endogenous level of SA ut lso the level of NO through enhned nitrte redutse (NR) tivity, whether As III ws present or not. Exogenously supplied NO deresed the NR tivity nd level of endogenous NO. Arseni umultion ws positively orrelted with the expression level of OsLsi1, trnsporter responsile for As III uptke. The endogenous level of NO nd SA were positively orrelted to eh other either when As III ws present or not. This lose reltionship indites tht NO nd SA work in hrmony to modulte the signling response in As III stressed plnts. Key words: Arseni, Rie, Nitri oxide, Sliyli id, Trnsporters the 2

4 Introdution Arseni (As) poses serious thret to humn eings, it is ssoited with severl helth risks suh s skin lesions, ner, nd rdiovsulr nd renl diseses (Rtnike 2003). Contminted wter is the prinipl soure of As exposure. Rie is prtiulrly effiient in As umultion nd toxi onentrtions of As in rie grins hs een reported (M et l. 2008). As rie is trded ommodity it thus, serves s n entry route for As in the food hin for regions where there is no As ontmintion (Mondl et l. 2010). Arseni exposure to plnts uses oxidtive stress tht dmges tissue through enhned prodution of retive oxygen speies (ROS). Although the ext mehnism is not known, there re inresing evidenes to suggest tht As leds to distured ell redox stte (Dixit et l. 2015). To ope with inresed ROS in ells, plnt system is proteted y vrious rdil svengers, suh s ntioxidnt enzymes s well s non enzymti ompounds like glutthione, sorte, rotenoids nd α toopherol (Mittler 2002; Gupt & Ahmd, 2014; Kumr et l. 2015). Nitri oxide (NO) is short lived gseous signling moleule, whih hve vriety of funtions in plnts, inluding ioti stress tement (Arsimowiz & Floryszk-Wiezorek, 2007; Neill et l. 2008). Two mehnisms hve een postulted for NO-medited stress mitigtion. The first mehnism is tht NO is free rdil, therefore, it n diretly svenge ROS (Lmttin et l. 2003). Seond, it n serve s ntioxidnt induer y triggering ntioxidnt gene expression or tivting ntioxidnt enzymes (Grün et l. 2006; Groβ et l. 2013) y post trnsltionl modifition of these ntioxidnt enzymes (Grennn 2007; Tnou et l. 2009). Applition of exogenous NO donor (sodium nitroprusside, SNP) hs een shown to onfer resistne to vrious ioti stresses suh s slt (Tnou et l. 2009) nd hevy metls (Zhng et l. 2011, Singh et l., 2016). Nitri oxide hs lso een shown to 3

5 improve internl iron (Fe) vilility y forming Fe-nitrosyl omplexes (Grzino et l. 2002). Sliyli id (SA) is phenoli ompound, it serves s growth regultor nd hs ruil role in vrious physiologil proesses suh s germintion, flowering nd het prodution in thermogeni plnts (Rivs-Sn Viente nd Plseni 2011). Sliyli id medited defense signling hs een widely studied in plnts during the lst dede, lrgely ginst ioti stresses (Yng et l. 2004; Chen et l. 1993). Vrious reports indited tht exogenously pplied SA mitigted the Hg nd Cd medited toxiity in plnts (Zhou et l. 2009: Metwlly et l. 2003). Rie shoots hs extremely high level of SA (5-30 µg g -1 fresh weight) in omprison to other plnts so endogenous level of SA in shoots is lrgely insensitive to exogenous SA ut rie roots hve low level of endogenous SA tht mkes them sensitive to exogenous pplition of SA (Yng et l. 2004; Chen et l ). The reltionship etween NO nd SA signling hs een studied under ioti stress onditions. Nitri oxide tretment ws shown to indue sustntil inrese in the levels of totl SA, while NO tivity hve een lso shown to e dependent endogenous level of SA (Song nd Goodmn 2001). Sliyli id my indues NO synthesis vi lium nd sein kinse 2 pthwy (Zottini et l. 2007). Thus, there ppers omplementry reltionship etween NO nd SA. In rie roots, rsenite (As III ) is known to e trnsported through silii (Si) id trnsporter, OsLsi1. While nother trnsporter OsLsi2 medites efflux of As III nd Si towrds the xylem (M et l. 2008). Reent reports hve showed tht the mehnism of Fe uptke orreltes with the As umultion nd ffets As trnsport in plnts (Tiwri et l. 2014). There hve een lrge numer of trnsporters identified in plnts tht hve the ility to trnsport iron. Nturl Resistne Assoited Mrophge Protein (NRAMPs), primrily identified s Fe trnsporters, lso retin trnsport ility for other hevy metls. 4

6 OsNRAMP5 is polrly lolized t the distl side of oth exodermis nd endodermis ells, nd uptkes Fe s well s mngnese (Mn) nd dmium (Cd) (Ishimru et l. 2006). Reently, overexpression of OsNRAMP1 in Aridopsis thlin hs een reported to e involved in trnsport of As nd Cd (Tiwri et l. 2014). Grmineous plnts soluilize soil Fe y sereting Fe(III) heltors lled mugeni id (MAs) from their roots. The resulting Fe(III)-MA omplexes re then sored into the roots y Yellow Stripe Like (YSL) trnsporters (Bshir et l. 2010; Koyshi nd Nishizw 2012). However, rie lso possesses the ility to trnsport Fe(II) through the trnsporters OsIRT1 nd OsIRT2. One the Fe is loted in the plnt numer of trnsporters re involeved in trnsport of the Fe from the roots to the shoots. OsFRDL1, itrte effluxer, lolized on the roots periyle ells is required for effiient Fe trnslotion to shoots (Yokosho et l. 2009). OsYSL2 is responsile for long-distne trnsport of niotinminehelted Fe nd Mn into sink tissues inluding leves nd grins (Koike et l. 2004). A numer of key regultors of Fe trnsport hve een identified. For exmple, OsIRO2 positively regultes vrious genes responsile for Fe trnsport (OsNAS1, OsNAS2, OsNAAT1, OsDMAS1, TOM1 nd OsYSL15). OsIRO2 lso ffets the expression of some Fe defiienyinduile trnsription ftors, whih might e involved in the indiret regultion of OsIRO2- downstrem genes (Ogo et l. 2007). In the present study, the role of NO nd SA ws evluted in mitigting As III toxiity in hydroponilly grown rie. The dependeny of NO nd SA on eh other during As III stress nd their effets on As nd Fe trnsporters nd minerl nutrition were investigted. Oxidtive stress relted prmeters were nlyzed to explore the stress mitigtion mehnisms. MATERIAL AND METHODS 5

7 Growth onditions nd experimentl design Seeds of Oryz stiv (v. Srjoo52) olleted from Msin Reserh Center, Pvt. Ltd., Bihr (Indi) were surfe sterilized using 10% hydrogen peroxide for 30 s nd wshed with doule distilled wter. Seeds were germinted in moist pre-sterilized lotting sheets on try in seed germintor for 4 d t 25 o C t 65% reltive humidity. The 4 d old seedlings were trnsferred to perforted ups with 10 seedlings/up. The ups were pled in trys holding 3 L of fullstrength Hewitt nutrient medium prepred in Milli-Q wter. The seedlings were grown for 10 d under 210 µ mol m -2 s -1 (16/8 h dy/night) white floresent light in ulture room mintined t C. After 10 d, As III (25µM) s NAsO 2, SA (40µM) nd SNP (30µM; NO donor) were supplemented in the nutrient medium. The rie seedlings grown in 25µM of As III were revited s As III, 40µM of SA s SA nd 30µM SNP s NO. Similrly, 25µM As III with 40µM SA s As III +SA nd 25µM As III with 30µM SNP s As III +NO nd seedlings grown in Hewitt nutrient medium served s ontrol. The nutrient medi ws hnged fter every 48 h. The treted seedlings were hrvested for nlysis fter 7 d of exposure to the tretments. All the hemils were purhsed from Sigm Aldrih (USA) or s mentioned seprtely. For germintion studies, seeds were surfe sterilized s desried oved nd 50 seeds trnsferred to Petri plte. Seeds were soked with the ove mentioned tretments in Milli-Q wter for 5 d nd germintion rte ws oserved. Biohemil nlysis For hlorophyll estimtion, 100 mg fresh leves were rushed in 5 ml 80% ie old etone nd entrifuged t 10,000 g for 10 min. Chlorophyll ontent in superntnt ws estimted s desried y the method of Arnon (1949). For thiorituri id retive sustnes (TBARS) nd hydrogen peroxide estimtion, 300 mg of roots or shoots were rushed in 3 ml 6

8 of 0.2% trihloroeiti id (TCA) (w/v), entrifuged t 10,000 g for 10 min, the superntnt ws olleted for further estimtion. TBARS nd hydrogen peroxide were estimted s desried y Heth nd Pker (1968) nd Velikov et l. (2000), respetively. For nlysis of enzyme tivities, leves or roots (300 mg eh) were ground using liquid nitrogen in hilled mortr nd pestle nd extrted with 3 ml of ie-old 100 mm potssium phosphte uffer (ph 7.5) ontining 1% (w/v) poly-vinylpyrrolidone (PVP). The homogente ws entrifuged t 10,000 g for 15 min nd the superntnt ws used for enzyme ssys. The tivity of superoxide dismutse (SOD) ws mesured using the method s desried y Beuhmp nd Fridovish et l.(1971), sorte peroxidse (APX) y Nkno nd Asd (1981), guiol peroxidse (GPX) y Kto nd Shimizu (1987), CAT y Sndlios et l. (1983) nd NR y Hgemn nd Reed (1980). Enzymti tivities were lulted in per unit protein estimted y the method of Lowry et l.(1951). Element nlysis The elementl omposition of the plnts mteril ws determined following method of Mllik et l. (2013). Plnts were wshed three times in 1 mm phosphte uffer (4 o C; ph 5.6). Roots nd shoots were seprted nd oven dried t 70 0 C. Dried plnt tissues (lef nd root, 100 mg eh) were digested using HNO 3 : HClO 4 (3:1). Digested smples were filtered (Whtmn No 42) nd the volume ws mde up to 10 ml using Milli-Q wter. Arseni ws nlyzed y AAS (GBS Avnt, Austrli) supported with hydride genertor (MDS 2000) using NH 2 BO 4 nd NOH (3 M) nd HCl (3 M) nd other elements were nlyzed y AAS (GBC Avnt, Austrli). The vlues re presented in µg per g dry weight. Superoxide nd hydrogen peroxide Stining 7

9 Superoxide nd hydrogen peroxide were determined in the leves y stining using nitro lue tetrzolium (NBT) nd 3,3-diminoenzidine (DAB), respetively, s desried previously y Thordl Christensen et l. (1997) nd Orozo-Cárdens et l. (1999). Rie leves were exised t the se with rzor lde nd NBT (1 mg ml -1 ) or DAB (0.5 mg ml -1 ) solutions were supplied through the ut ends for 8 h. Leves were then deolorized in oiling ethnol (95%) for 15 min. At lest 3 leves were used for eh tretment. Nitri Oxide Detetion For NO detetion, roots of pproximtely equl thikness were inuted for 30 min t 25 o C in the drk with 10 mm DAF-FM-DA (Cliohem; exittion t 495 nm, emission t 515 nm) prepred in 10 mm Tris-HCl (ph 7.4) uffer, s desried y Sndlio et l. (2008). For negtive ontrol, roots were inuted with 1 mm PTIO, NO svenger. Then roots were wshed 3 times for 10 min eh with the sme uffer nd DAF-FM-T, fluoresene ws visulized y onfol mirosopy (Crl Zeiss LSM510 Met, Germny). Fluoresene intensity ws estimted y mesuring the verge pixel intensity. For eh tretment, triplite nlysis ws performed. Sliyli Aid Estimtion Presene of SA in the smple ws nlyzed y HPLC (Dionex Ultimte 3000, USA y using Chromeleon 6.8) using UV detetor t 210 nm following the method of Pn et l. (2010). The moile phse ws progrmmed with liner grdient of A (0.1% of formi id in methnol) nd B (0.1% of formi id in wter) progrmmed s 0-20 min; % A, min; 100% A nd then min; % of A. Flow rte ws mintined t 0.3 ml min -1, the retention time for SA ws reorded t 22.4 min. 8

10 Gene Expression Anlysis Using Quntittive RT-PCR 100 mg roots smple ws rushed in liquid N 2 nd RNA were isolted y using RNesy Plnt Mini Kit (Qigen) following the mnufturer s instrtion. Qulity nd quntity of RNA ws ssessed y spetrophotometer (Nno Drop, USA). Approximtely 5 µg of RNse free DNse-treted totl RNA isolted from roots of rie plnts exposed to vrious tretments nd ontrol ws reverse-trnsried using SuperSriptII (Ferments, USA), following the mnufturer s reommendtion. The synthesized DNA ws diluted 1:5 in RNse free wter nd sujeted to quntittive RT-PCR (qrt-pcr) nlysis. The qrt-pcr ws performed using n ABI 7500 instrument (ABI Biosystems, USA) using gene speifi primers (Supplementl Informtion Tle S1). Eh qpcr retion ontined 5 µl of SYBR Green Supermix (ABI Biosystems, USA), 1 µl of the diluted DNA retion mixture (orresponding to 5 ng of strting mount of RNA) nd 10 pm of eh primer in totl retion volume of 10 µl. qpcr retions were performed under the following onditions: 10 min t 95 C nd 40 yles of the one step therml yling of 3 s t 95 C, 30 s t 60 C in 96-well retion plte. The rie Atin1 gene ws used s n internl ontrol to estimte the reltive trnsript levels of the trget gene. Speifiity of mplions generted in qpcr retions ws verified y melting urve nlysis. Eh qpcr retion ws performed in triplite (tehnil replites) for eh iologil replite (three for eh tretment). Reltive gene expression ws lulted using - CT method (Livk nd Shmittgen 2001). Sttistil nlysis Anlysis of vrine (ANOVA), Dunn s multiple rnge test (DMRT) nd Person s orreltion nlysis were performed to determine the signifint differene etween tretments t the 95% onfidene level. 9

11 RESULTS Morphologil hnges In the sene of As III, SA or NO hs no signifint effet on seed germintion. Arsenite tretment drstilly redued the seed germintion potentil. Nitri oxide or SA tretment of the seeds deresed the negtive impt tht As III on seed germintion potentil (Supplementl Informtion Fig. S1). Nitri oxide nd SA tretment enhned the shoot, root length nd totl hlorophyll ontent in omprison to ontrol. A more prominent growth response ws oserved in SA treted plnts thn NO (Tle -1 nd Supplementl Informtion Fig. S2 nd S3). Exposure to As III deresed the shoot nd root length y 27% nd 47%, respetively, ompred to the ontrol. Arsenite tretment lso hd similr ffet on totl iomss. The hlorophyll ontent ws deresed y 44% in the As III exposed plnts. Root nd lef growth ws deresed under As III tretment (Supplementl Informtion Fig. S4 nd S5). Nitri oxide nd SA supplementtion long with As III, prtilly restored over ll plnt growth, prtiulrly shoot length. Growth of root, lef nd totl hlorophyll ontent were ompletely restored upto ontrol levels. In sene of As III, SA ws more responsive for growth enhnement, while in presene of As III, NO performed etter. Oxidtive stress nd ntioxidnt enzymes Arsenite exposure inresed hydrogen peroxide nd TBARS onentrtions in roots y. 4 nd 2.5 fold nd shoots y. 2 nd 2.5 fold ompred to the ontrols (Fig. 1A, B nd Supplementl Informtion Fig S6 A, B). Sliyli id tretment signifintly inresed the level of hydrogen peroxide in the roots ompred to the ontrols. Supplementtion of SA or NO long with As III resulted in signifintly deresed the prodution of hydogen peroxide nd TBARS in omprison to As III lone treted plnts (Fig. 1A, B nd Supplementl Informtion Fig S6 A, B). Histohemil stining lso showed higher level of superoxide 10

12 rdils in As III stressed plnts thn As III nd NO treted or As III nd SA treted plnts (Supplementl Informtion Fig. S7). In the As III treted plnts, the tivity of ntioxidnt enzymes SOD, CAT, GPX, APX nd GR were signifintly inresed in oth the roots nd shoots ompred to the ontrol plnts. Sliyli id tretment deresed the CAT tivity (40%) in the roots while in the shoots it remined unltered in omprison to the ontrol. Nitri oxide deresed CAT nd APX tivity in oth the roots nd the shoots signifintly ompred to the ontrol. Nitri oxide or SA supplementtion to As III treted plnts deresed the tivity of ll the ntioxidnt enzymes in roots nd shoots ompred to the As III tretment. However, GR tivity ws enhned in roots t As III +SA treted plnts in omprison to As III lone. (Fig. 1C-G nd Fig S6C-G). Endogenous levels of NO, SA nd NR tivity The endogenous level of NO in roots did not hnge signifintly y exogenous pplition of NO ut exogenous pplition of SA enhned the endogenous level of SA. Arsenite stress, however, used signifint redution in the onentrtion of NO whih ws deresed to hlf of the ontrol. Nitri oxide nd SA supplementtion long with As III enhned the level of endogenous NO y 35% nd 77%, respetively ompred to As III lone exposed roots, lthough the levels were still lower thn ontrol (Fig.2A nd Fig.3). The endogenous level of SA showed signifint inrese y exogenous pplition of oth NO nd SA (56 nd 250% respetively) in roots. In As III exposed roots, endogenous SA level ws deresed y 63% thn ontrol roots. Nitri oxide supplementtion long with As III did not hnge endogenous SA level while SA supplementtion enhned the endogenous SA level y 324% thn As III stressed roots (Fig. 2B). 11

13 NR plys ruil role in NO synthesis in plnt systems. In NO treted plnts, NR tivity deresed y 6 fold while SA tretment enhned the tivity y round 9 fold ompred to the ontrols. In As III stressed roots, NR tivity enhned y. 2 fold ompred to ontrols. Sliyli id supplementtion further enhned the NR tivity y. 2 fold while NO supplementtion deresed the NR tivity. 5 fold ompred to As III treted roots (Fig. 2C). Aumultion of rseni nd other elements Arsenite exposed plnts umulted signifintly higher mount of As. Most of the As (90%) ws onfined to roots, pproximtely 10% ws trnsported to shoots (Tle 2 A, B nd C). Nitri oxide supplementtion long with As III signifintly deresed As umultion in roots (35%) nd shoots (61%) ompred to plnts treted with just As. Sliyli id tretment to AsIII stressed plnts, deresed the As umultion in the shoots (27%) while it showed no signifint impt on roots As umultion thn As III lone treted plnts. Nitri oxide tretment lso redued the shoots/roots trnslotion ftor (TF; 0.06) y ~50% in omprison to As III lone treted plnts (TF; 0.1). In the As III treted plnt roots, Fe umultion ws enhned 92%, however, the level of Fe in shoots remined unltered ompred to ontrol. Nitri oxide or SA lone did not ltered Fe umultion signifintly in the roots while in the shoots, Fe umultion ws enhned y NO (15%) nd SA (18%) ompred to the ontrols. Nitri oxide supplementtion to As III treted plnts used signifint redution (21%) in the level of Fe in the roots, while inresed Fe (21%) in shoots in ompred to the plnts treted with As III only. Thus, NO supplementtion to As III treted plnts enhned the shoots/roots TF thn As III treted plnts (Tle 2C). Wheres, SA supplementtion to As III treted plnts did not 12

14 ffet the level of Fe in roots signifintly, while in the shoots it enhned the Fe umultion s well s inresed the shoots/roots TF in omprison to As III lone tretment. Nitri oxide nd SA tretment lone did not signifintly lter the level of C nd Zn in shoots nd roots. Arsenite lso showed no signifint effet on C nd Zn umultion oth in roots nd shoots, exept the C umultion ws lowered in the roots of As III treted plnts ompred to the ontrol plnts. Aumultion of Mn ws enhned signifintly in the roots y NO nd SA tretments ut there ws no impt on Mn umultion in shoots. As III lone or in omintion with NO or SA hve no signifint impt ompred to the ontrols on Mn umultion. Impt of NO nd SA on Fe nd As trnsporters In NO treted plnts, OsLsi1 expression level ws lowered y 20%, while the SA treted plnts hd enhned expression (~2 fold) of OsLsi1 ompred to the ontrol plnts. In As III exposed plnts, OsLsi1 expression ws higher thn ontrol plnts. NO supplementtion to As III treted plnts deresed the expression of OsLsi1, wheres, SA supplementtion in omintion with As III signifintly enhned the expression of OsLsi1 ompred to the plnts treted with just As III. In NO treted plnts the expression level of OsLsi2 ws enhned y more thn 2 fold while SA tretment hve no signifint impt on OsLsi2 ompred to the ontrol plnts. In As III treted plnts, OsLsi2 expression level ws lso more thn 5 fold greter thn the ontrol. Nitri oxide nd SA supplementtion to As III treted plnts deresed the OsLsi2 expression when ompred to the plnts just treted with As III, lthough more redution ws oserved in SA treted plnts (Fig. 4A, B). Both NO nd SA tretments enhned the expression of ll the Fe trnsporters studied, exept OsFRDL1 whih ws not enhned in response to NO. OsIRO2, positive 13

15 trnsription regultor, ws unltered y NO nd SA tretments. Arsenite tretment lone signifintly enhned the expression of OsIRT1 nd OsYSL2 (11 nd 10 folds, respetively) nd inhiited the expression of OsFRDL1 (77%) in omprison to the ontrol. When NO ws pplied long side As III, the expression levels of OsIRT1, OsIRO2 nd OsYSL2 were deresed, wheres, OsFRDL1 nd OsNRAMP5 remined unltered ompred to the plnts just treted with As III. When SA ws used in onjuntion to the As III treted plnts there ws redution in the expression of OsIRT1, OsYSL2 nd OsIRO2 expression levels ut OsFRDL1 ws enhned in omprison to As III only treted plnts (Fig. 4C-G). Disussion The present study nlyze the effet of SA nd NO on rie plnts exposed to toxi onentrtion of As III. Under As III stress, NO nd SA levels shrply delined. Plnt growth ws lso signifintly hmpered. Nitri oxide nd SA show protetive role ginst As III toxiity in rie plnts nd enhned the plnt growth. Exogenously supplied NO deresed the As umultion in roots nd its trnsport to shoots. Arsenite tretment inhiits seed germintion nd growth, shoots, roots length nd totl hlorophyll ontent (Dixit et l. 2015,; Kumr et l. 2014,, 2016) these were mostly restored y NO nd SA pplition (Tle 1; Supplementl Informtion Fig. S1, S2, S3, S4 nd S5). The roots re the first orgn whih ome in ontt with As III, it ws oserved tht As III inhited the growth of roots nd root hirs. However, NO nd SA supplementtion ompletely restored the root length nd root hir growth (Tle 1 nd Supplementl Informtion Fig. S2, S4). Nitri oxide tretment not only signifintly redues As umultion in roots, ut lso restrits its trnslotion from roots to shoots. This is the first report on As III nd NO intertions in plnts nd my e the ruil ftor for NO-medited protetion ginst As III stress. Both NO nd SA re reported to tivte ABC trnsporters tht re responsile for 14

16 hevy metl sequestrtion to vuole nd restriting its entry into shoots (Grun et l. 2006; Eihhorn et l. 2006). However, in our study, SA did not ffet the umultion of As in roots ut deresed its umultion in shoots (Singh et l. 2015). This might e due to the tivtion of ABC trnsporters in the roots hene sequestering more As in root vuole while restriting its entry to the shoots. Nitri oxide tretment, however, drstilly deresed As umultion in the roots s well s its trnslotion to shoots. OsLsi1expression whih is responsile for internlistion of As III lso showed positive orreltion with As umultion in the roots (R=0.98, p 0.05) while OsLsi2 expression, tht is responsile for roots to shoots trnsport, ws lso positively orrelted with As umultion in the shoots (Supplementl Informtion Tle S2). The present study showed tht As III tretment resulted in oxidtive stress in terms of enhned TBARS, hydrogen peroxide nd superoxide rdils. Enhned prodution of ROS nd peroxidtion of memrne lipids (i.e. inrese in the level of TBARS) y As exposure hs previously een reported in rie (Singh et l. 2015). It ws oserved tht ntioxidnt enzymes suh s SOD, CAT, APX GPX nd GR were enhned in oth roots nd shoots under As III stress, potentilly ould e to ountert the oxidtive stress used y As III. Nitri oxide or SA supplementtion with As III used sustntive redution in the level of TBARS nd hydrogen peroxide, whih indites the meliorting effet of NO nd SA on As III indued oxidtive stress (Singh et l. 2009). Furthermore NO nd SA supplementtion with As III, deresed the tivity of ntioxidnt enzymes ompred to As III lone exposed plnts. It gin onfirms the protetive role of NO nd SA ginst As III indued oxidtive stress. Protetive role of SA nd NO supplementtion hs een previously oserved under Cd stress in Medigo stiv nd under As stress in rie respetively (Zhou et l. 2009; Singh et l. 2009). 15

17 Arsenite stress signifintly deresed NO-dependent fluoresene ompred to ontrol roots (Fig. 3A, 6). Singh et l. (2009) lso reported signifint derese in NO dependent fluoresene in rie root under As stress. A similr derese in NO dependent fluoresene in shoot of pe plnt ws oserved under Cd stress (Rodriguez-Serrno et l. 2009). In ontrst, Besson-Brd et l. (2008) demonstrted n inrese in NO dependent floresene in oth roots nd shoots of A. thlin under Cd stress. During plnt responses to hevy metl stress, NO my inrese or derese nd t s n induer or inhiitor of stress tolerne, depending on plnt speies nd experimentl setup (Arsimowiz-Jelonek et l. 2011). In the present study, endogenous NO did not inrese y exogenous NO ut exogenous pplition of SA enhned the endogenous level of NO. Sliyli id medited enhnement of endogenous NO ws reported in A. thlin (Zottini et l. 2007). It ppers tht there is n optiml level of endogenous NO in rie roots tht ould not e further elevted. However, when the endogenous level of NO ws deresed, e.g. under As III stress, exogenous supplementtion of NO or SA used n inrese in endogenous NO onentrtions in the roots. Arsenite exposure in rie signifintly deresed the endogenous SA level. This is ontrsting ehviour thn ioti stress where endogenous SA level re enhned (Vlot et l. 2009). Enhnement of endogenous SA y pplition of exogenous SA hs een reported whih is termed s SA dependent mplifition iruit (Xio et l. 2003). Nitri oxide is well known to indue SA synthesis (Klessig et l. 2000; Hung et l. 2004). Genomi studies hve shown tht NO indues phenyllnine mmoni lyse (PAL) gene t the trnsriptome level (Hung et l. 2004; Delledonne et l. 1998) these genes ply ruil role in SA iosynthesis vi innmte pthwy. However, under As III stress there ws no inrese in SA level y exogenous supplementtion of NO, this indites tht As III distured the SA iosynthesis pthwy. 16

18 A positive orreltion etween SA nd NO ws found, with As III (R=0.89, p 0.05) or without As III (R=0.88, p 0.05), whih mens NO nd SA work synergistilly (Supplementl Informtion Tle S2). Song nd Goodmn (2001) lso onluded similr hypothesis tht NO is fully dependent on the funtion of SA while NO is required for full funtioning of SA in too under ioti stress. Exogenous NO supplementtion resulted in drsti derese in NR tivity. This might e due to the feedk inhiition of NR tivity in the presene of high level of NO. Though, under As III stress, NR tivity ws enhned, ut the level of NO ws muh lower thn ontrols. This might e due to the onsumption of surplus NO (due to enhned NR tivity) in the neutrliztion of As III indued ROS. In SA treted plnts NR tivity ws mny folds higher thn ontrol nd lso level of NO ws orrespondingly higher, inditing tht SA tretment indues NO synthesis through NR. Nitri oxide tretment with or without As III stress enhned Fe umultion in the shoots, while As III lone tretment enhned the Fe umultion only in the roots ut not in the shoots. Fe defiieny results in hlorosis (Vsonelos nd Grusk 2014). In the present study, As III stressed leves eomes hloroti, though there ws no hnge in the onentrtion of Fe in shoots in omprison to ontrol. Thus, the redution in hlorophyll ontent my e result of its inhiited synthesis or As III indued degrdtion (Rhmn et l. 2007). Another possiility is tht As III stress my hve n effet on vilility of Fe, suh s due to redution in endogenous NO level. Iron homeostsis strongly depends on the iron storge protein ferritins whih re uiquitous iron storge proteins. Nitri oxide hs een reported to influene the ferritins level (Murgi et l., 2002; Grun et l., 2006). There is in vitro evidene for the first mehnism tht NO n remove iron from horse spleen ferritin (Grzino nd Lmttin, 2005). So, NO is elieved to inrese the internl Fe vilility nd revert the hloroti symptoms. Nitri oxide hs een reported to inrese internl Fe vilility nd reverts hloroti symptoms (Grzino et l. 2002). 17

19 It hs een reported previously tht OsIRO2 serves s positive regultor of genes involved in iron uptke nd utiliztion (Ogo et l. 2007), ws found to negtive orrelted with shoots Fe umultion irrespetive of presene of As III showing its role in Fe defiieny. OsNRAMP5, tht is responsile for Mn uptke long with Fe (Ishimru et l. 2006), in presene of As III Mn umultion in shoots ws negtively orrelted (R= -0.95, p 0.05) suggesting its role in Mn trnsporttion (Supplementl Informtion Tle S2). The trnsripts of Fe uptke trnsporter OsIRT1, ws negtively orrelted with the Fe umultion in shoots in presene of As III, OsIRT1 is known to strongly indued with Fe defiieny (Koyshi nd Nishizw 2012). OsYLS2 ws negtively orrelted (R= -0.98, p 0.05) with shoots Fe level in presene of As III, suggesting its role s long distne Fe trnsporter (Koyshi nd Nishizw 2012). Conlusion NO nd SA oth showed protetive role ginst As III stress. Arsenite stresse led to oxidtive stress urst nd onsequently tivity of ntioxidnt enzymes ws inresed. Exogenous pplition of NO nd SA llevited AsIII medited oxidtive stress. Nitri oxide deresed the As umultion in roots proly through the down regultion OsLsi1 trnsporter while NO nd SA oth deresed the As umultion in shoots potentilly through the down regultion of OsLsi2. Nitri oxide pplition enhned the Fe umultion in shoots nd overme the As III medited hlorosis. Aknowledgements The uthors re thnkful to Diretor, CSIR-Ntionl Botnil Reserh Institute (CSIR- NBRI), Luknow for the filities nd for the finnil support from the network projets (CSIR-INDEPTH), New Delhi, Indi. APS is thnkful to CSIR New Delhi, Indi 18

20 respetively, for the wrd of Reserh Assoiteship. RDT is grtefully thnkful to Awrd of Emeritus Sientist (CSIR). GD is thnkful to SERB-DST, New Delhi for wrd of NPDF. AK is thnkful to UGC for wrd of DSKPDF. Awrd of Fst Trk Sientist to SM from DST is grtefully knowledged. We re lso thnkful to Mr. Dilip Chkrorty for tehnil ssistne. Appendix A. Supplementry dt Supplementry dt ssoited with this rtile n e found in the online version. 19

21 Additionl informtion Competing finnil interests The uthors delre no ompeting finnil interests. Authors Contriution RDT, PKT, VP, designed experiments nd reviewed mnusript. APS, GD performed experimentl work nd prepred figures. AK performed sttistil nlysis. SD1 nd NK operted onfol mirosope nd AAS respetively. DC, SM, SD2, PKS, GJN, nd OPD reviewed mnusript. All uthors hve red nd pproved the mnusript. SD1: Smeer Dixit SD2: Snjy Dwivedi 20

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30 Figure legends Fig. 1: Effet of different omintions of NO, SA nd As III on (A) H 2 O 2, (B) TBARS, (C) SOD, (D) CAT, (E) APX, (F) GPX nd (G) GR tivity in roots of Oryz stiv fter 7 d tretment. P vlue mrked with sme lphets re not signifintly different (DMRT, p 0.05). All the vlues re men of three replites ±SD. Fig. 2: Effet of different omintions of NO, SA nd As III on (A) endogenous NO dependent fluoresene, (B) endogenous level of SA nd (C) nitrte redutse tivity in roots of Oryz stiv fter 7 d tretment. P vlue mrked with sme lphets re not signifintly different (DMRT, p 0.05). All the vlues re the men of three replites ±SD. Fig. 3: Imging of NO prodution in Oryz stiv y CLSM. Imges re showing the NOdependent DAF-FM 2DA fluoresene (green; exittion t 495 nm, emission t 515 nm) fter 7 d tretment with different omintions of NO, SA nd As III. Negtive ontrol is treted with 10Mm PTIO, NO svenger nd prior to stining with DAF-FM2DA. Fig. 4: Quntittive rel-time PCR nlysis to study the expression gene pttern. Y-xis represents reltive fold hnge expression of mrna level in different omintions of NO, SA nd As III on (A) OsLsi1, (B) OsNRAMP5, (C) OsIRT1, (D) OsIRO2, (E) OsFRDL1 nd (F) OsYSL2 trnsporters roots of Oryz stiv fter 7 d tretment. Effet P vlue mrked with sme lphets re not signifintly different (DMRT, p 0.05). All the vlues re the men of three replites ±SD. 29

31 Tle 1. Effet on root, shoot, iomss nd totl hlorophyll ontent of Oryz stiv fter 7d tretment with different omintions of NO, SA nd As III. P vlue mrked with sme lphet is not signifintly different (DMRT, p 0.05). All the vlues re men of three replites ±SD. Tretments Root (m) Shoot (m) Biomss (g) Totl Chlorophyll (mg g -1 ) Control 7.20 fg ± ± ± ±0.05 NO 7.58 gh ± de ± e ± ±0.03 SA 8.18 h ± e ± f ± e ±0.06 As III 3.75 ± ± ± ±0.03 As III +NO 5.09 ± d ± ± ±0.07 As III +SA 4.54 ± ± ± ±0.09

32 Tle 2: Effet on vrious metl umultions (µg g -1 ) in roots (A), shoot (B) nd shoot/root Trnslotion Ftor (C) of Oryz stiv fter 7d tretment with different omintions of NO, SA nd As III. P vlues mrked with sme lphets re not signifintly different (DMRT, p 0.05). All the vlues re men of three replites ±SD. Tle: 2A Tretments Fe As C Zn Mn Control ± ± ± ±27.4 NO ± ± ± ±33.6 SA ± ± ± ±24.2 As III d ± ± ± ± ±28.2 As III nd NO ± ± ± ± ±19.6 As III nd SA d ± ± ± ± ±12.4 Tle: 2B Tretments Fe As C Zn Mn Control ± ± ± ±72.2 NO ± ± ± ±84.6 SA ± ± ± ±91.1 As III ± ± ± ± ±64.9 As III nd NO ± ± ± ± ±116.3 As III nd SA ± ± ± ± ±62.7 Tle: 2C: Tretments Fe As C Zn Mn Control NO SA As III As III nd NO As III nd SA

33 APX (Units mg -1 protein) SOD (Units mg -1 protein) H 2 O 2 (n mol g-1 fw) GR (Units mg -1 protein) e A d C E G Control NO d d e d d TBARS (m mol g -1 fw) CAT (Units mg -1 protein) 16 B d SA AsIII Tretments e AsIII+NO AsIII+SA GPX (Units mg -1 protein) D F Control NO SA d d AsIII Tretments AsIII+NO AsIII+SA Fig. 1.

34 25 A 5 B d Nitri oxide (Reltive floresene) NR Root (µm min -1 mg -1 P) C Control d e NO SA AsIII Tretments AsIII+NO d AsIII+SA Sliyli id (µg -1 fw) Control NO SA AsIII Tretments AsIII+NO AsIII+SA Fig. 2.

35 Fig. 3.

36 Fig OsNRAMP5 C e d OsLsi1 A OsIRO2 E d e Control NO SA AsIII AsIII+NO AsIII+SA OsYSL2 Tretments G d OsLsi2 B e d OsIRT1 D d e Control NO SA AsIII AsIII+NO AsIII+SA OsFRDL1 Tretments F

37 Highlights 1. Nitri oxide (NO) deresed the rseni (As) umultion in oth root nd shoot 2. Sliyli id (SA) deresed the root to shoot trnslotion of As. 3. NO nd SA mitigted the As-medited oxidtive stress. 4. NO nd SA were worked in mutully oordintion mnner irrespetive of As presene.