Interdependency of Reactive Oxygen Species generating and scavenging system in salt sensitive and salt tolerant cultivars of rice

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1 Kur et l. BMC Plnt Biology (2016) 16:131 DOI /s RESEARCH ARTICLE Interdependeny of Retive Oxygen Speies generting nd svenging system in slt sensitive nd slt tolernt ultivrs of rie Nvdeep Kur 1, Mnish Dhwn 1, Ish Shrm 1,2 nd Prtp Kumr Pti 1* Open Aess Astrt Bkground: Slinity stress is mjor onstrin in the glol rie prodution nd hene serious efforts re eing undertken towrds deiphering its remedil strtegies. The omprtive nlysis of differentil response of slt sensitive nd slt tolernt lines is judiious pproh to otin essentil lues towrds understnding the quisition of slinity tolerne in rie plnts. However, dpttion to slt stress is firly omplex proess nd opertes through different mehnisms. Among vrious mehnisms involved, the retive oxygen speies medited slinity tolerne is elieved to e ritil s it evokes sde of responses relted to stress tolerne. In this kground, the present pper for the first time evlutes the ROS generting nd the svenging system in tndem in oth slt sensitive nd slt tolernt ultivrs of rie for getting etter insight into slinity stress dpttion. Results: Comprtive nlysis of ROS indites the higher level of hydrogen peroxide (H 2 O 2 ) nd lower level of superoxide ions (O 2- ) in the slt tolernt s ompred to slt sensitive ultivrs. Speifi tivity of ROS generting enzyme, NADPH oxidse ws lso found to e more in the tolernt ultivrs. Further, tivities of vrious enzymes involved in enzymti nd non enzymti ntioxidnt defene system were mostly higher in tolernt ultivrs. The trnsript level nlysis of ntioxidnt enzymes were in lignment with the enzymti tivity. Other stress mrkers like proline were oserved to e higher in tolernt vrieties wheres, the level of mlondildehyde (MDA) equivlents nd hlorophyll ontent were estimted to e more in sensitive. Conlusion: The present study showed signifint differenes in the level of ROS prodution nd ntioxidnt enzymes tivities mong sensitive nd tolernt ultivrs, suggesting their possile role in providing nturl slt tolerne to seleted ultivrs of rie. Our study demonstrtes tht the ellulr mhinery for ROS prodution nd svenging system works in n interdependent mnner to offer etter slt stress dpttion in rie. The present work further highlights tht the elevted level of H 2 O 2 whih is onsidered s key determinnt for onferring slt stress tolerne to rie might hve originted through n lterntive route of phototlyti tivity of hlorophyll. Keywords: Rie, Slinity, Retive oxygen speies (ROS), NADPH oxidse, Hydrogen peroxide (H 2 O 2 ), Antioxidnt enzymes * Correspondene: pkpti@yhoo.om 1 Deprtment of Biotehnology, Guru Nnk Dev University, Amritsr , Punj, Indi Full list of uthor informtion is ville t the end of the rtile 2016 The Author(s). Open Aess This rtile is distriuted under the terms of the Cretive Commons Attriution 4.0 Interntionl Liense ( whih permits unrestrited use, distriution, nd reprodution in ny medium, provided you give pproprite redit to the originl uthor(s) nd the soure, provide link to the Cretive Commons liense, nd indite if hnges were mde. The Cretive Commons Puli Domin Dedition wiver ( pplies to the dt mde ville in this rtile, unless otherwise stted.

2 Kur et l. BMC Plnt Biology (2016) 16:131 Pge 2 of 13 Bkground Rie is n importnt erel rop tht hs the potentil to provide food seurity to the inresing world popultion. Out of the totl glol griulturl lnd, 150 million hetres is estimted to e under rie ultivtion tht leds to n nnul prodution verging to 500 million metri tons [1]. But lrge mount of the rie iomss is not hrvested under field onditions due to sensitivity of the rop to vrious ioti stresses like drought, slinity, low temperture, het shok [2, 3]. Among these stresses, slinity is mjor onstrin to rie prodution worldwide whih dversely ffet its growth nd development t the moleulr, iohemil nd physiologil level [4, 5]. It ffets more thn 20 % of totl ultivted lnd worldwide tht results in US$12 illion loss in glol griulturl prodution nd this loss is further inresing eh yer [5, 6]. As in the present senrio of glol limte hnge, the level of lnd sliniztion is expeted to inrese nd hene there is n immedite world-wide onern for development of etter slt tolernt ultivrs for future food seurity. For hieving this ojetive, thorough omprtive nlysis of slt sensitive nd tolernt ultivrs oupled with inresed understnding of the underlying mehnism involved in slt stress dpttion is muh wrrnted. Retive oxygen speies (ROS) genertion, signlling nd detoxifition re vitl omponents of slt stress dpttion mehnisms nd priority re of reserh worldwide for the omplishment of etter growth nd yield of rop under slt ffeted res [7]. ROS suh s superoxide rdil (O 2 - ), hydroxyl rdil (OH - ) nd hydrogen peroxide (H 2 O 2 ) re produed in low onentrtion s result of norml ellulr metolism in plnts nd plys importnt role in growth, development nd in dpttion to stress [8]. But, when plnt is sujeted to stress, the perturtion of ROS homeostsis tkes ple whih triggers multiple signlling responses involved in ritil funtions of plnts [9]. Hene, fine tuned lne etween ROS prodution nd svenging is ruil for the plnt survivl under unfvourle onditions. NADPH oxidses re one of the mjor enzyme systems involved in prodution of ROS in the poplsti spe under stress [10]. They tlyze the trnsfer of eletons from NADPH to moleulr oxygen (O 2 ) for the genertion of free rdil O 2- [11]. NADPH oxidses hold distint position mong the vrious ROS generting enzyme systems in plnts euse of their role in different signlling pthwys involved in plnt growth, development nd stress tolerne [12]. Superoxide ions produed s result of enzymti tivity of NADPH oxidse is onverted into H 2 O 2 vi superoxide dismutse (SOD) nd it diffuses to djent ellulr omponents where it ts s signlling moleule tht tivtes vrious stress responsive pthwys. This indites tht the oordintion of ROS generting nd svenging system might ply ritil role in slt stress dpttion mehnism. However, the ext role of NADPH oxidse in onferring slt stress dpttion is not yet properly understood. Erlier studies onduted on slt tolernt ultivr Pokli nd sensitive ultivr IR64 under the effet of slt tretment hs indited definitive link etween svenging system nd slinity tolerne [13]. However, to est of our knowledge no work hs een initited to ompre the ROS generting nd svenging system together in slt sensitive nd tolernt ultivrs. Therefore, in the present study, omprtive study of ROS dynmis ws onduted in slt tolernt (Lun Snkhi nd Lun Suvrn) nd slt sensitive (IR64 nd Pus Bsmti-1) ultivrs of rie. The informtion gined on rel time sitution on this ritil stress lleviting ROS pthwy will filitte in developing ultivrs with etter slt stress dpttion. Further, this work dds to the existing knowledge of our understnding on the omplex physiologil, iohemil nd geneti mehnisms involved in slt stress dpttion. Results Histohemil nlysis of ROS The level of different types of ROS mong slt sensitive nd tolernt ultivrs ws monitored histohemilly using three different dyes (Fig. 1). Nitrozolium lue (NBT) ssy showed higher umultion of drk lue oloured pigment tht estimtes the higher level of O 2- in the rie ultivrs IR64 nd Pus Bsmti-1. On the ontrry to this, 3, 3-diminoenzidine (DAB) showed higher level of H 2 O 2 tht ws ssessed through the intensity of drk rown oloured spots in tolernt ultivrs (Lun Snkhi nd Lun Suvrn). Similrly, stining using 2', 7 -dihlorodihydrofluoresein diette (H 2 DCFDA) whih inds to ROS (predominntly H 2 O 2 ) yielded higher intensity of green fluoresene in tolernt ultivrs s ompred to the sensitive ultivrs. NADPH oxidse tivity ssy The quntittive nlysis of ROS generting NADPH oxidse enzyme tivity showed signifint differene mong four ultivrs of rie (Fig. 2). The enzymti tivity (nmoles min -1 mg -1 ) ws found to e signifintly high in Lun Snkhi (0.424 ± 0.009) followed y Lun Suvrn ± thn the sensitive ultivr IR64 (0.182 ± 0.011) nd Pus Bsmti-1 (0.177 ± 0.028).

3 Kur et l. BMC Plnt Biology (2016) 16:131 Pge 3 of 13 Fig. 1 O2- nd H2O2 umultion in leves of different ultivrs of rie. Leves were exised t the se of stems nd were supplied with different stins. () NBT stining () DAB stining () Confol imges of H2DCFDA stining (d) 3D-view of onfol H2DCFDA stining imges (IR: IR64, PB: Pus Bsmti-1, SA: Lun Snkhi, SU: Lun Suvrn) H2O2 ontent estimtion Antioxidnt enzyme tivity The estimtion of queous H2O2 ontent in four vrieties showed onsiderle differene s expeted on the sis of histohemil ssys (Fig. 2). The ontent of queous H2O2 (units nmoles g-1 FW) ws found to e signifintly more in tolernt ultivrs Lun Snkhi ( ± 0.187) nd Lun Suvrn ( ± 0.585) s ompred to sensitive vrieties IR64 ( ± 0.76) nd Pus Bsmti-1 ( ± 0.340) whih were t pr with eh other. Signifint differenes in the speifi tivity of vrious ntioxidnt enzymes were oserved in different ultivrs (Tle 1). The overll speifi tivity of ll the ntioxidnt enzymes, exept tlse (CAT) ws found to e higher in tolernt ultivrs. SOD tht onverts superoxide ions into hydrogen peroxide showed signifintly higher speifi tivity (units mg protein-1) in Lun Snkhi ( ± ) nd Lun Suvrn ( ± ) with respet to IR64 ( ± )

4 Kur et l. BMC Plnt Biology (2016) 16:131 Pge 4 of 13 NADPH oxidse tivity (nmoles min -1 mg -1 ) IR PB SA SU IR PB SA SU Fig. 2 Comprtive nlysis of () NADPH oxidse tivity nd () H 2 O 2 ontent of 14 dys old seedlings of different rie ultivrs. (IR: IR64, PB: Pus Bsmti-1, SA: Lun Snkhi, SU: Lun Suvrn). Brs represent men ± SE (n = 3). Different letters (,, ) within ultivrs re signifintly different (Tukey LSD, p 0.05) H 2 O 2 ontent (nmoles g FW -1 ) nd Pus Bsmti-1 ( ± ). The speifi tivity of sorte peroxidse (APX) ws ugmented to signifintly higher in Lun Snkhi (26.21 ± 0.301) nd Lun Suvrn (26.13 ± 0.230) s ompred to IR64 (19.85 ± 0.563) nd Pus Bsmti-1 (18.55 ± 0.518) ut on the ontrry the speifi tivity of CAT ws more in sensitive ultivrs IR64 (0.486 ± 0.012) nd Pus Bsmti-1 (0.581 ± 0.003) in omprison to Lun Snkhi (0.277 ± 0.190) nd Lun Suvrn (0.397 ± 0.013) (Tle 1). The enzymes involved in the well known glutthionesorte pthwy lso showed onsiderle differene in their speifi tivity mong different ultivrs. The speifi tivity of glutthione redutse (GR) ws found to e higher in Lun Snkhi (5.98 ± 0.276) nd Lun Suvrn (5.91 ± 0.072) thn IR64 (3.58 ± 0.045) nd Pus Bsmti-1 (4.12 ± 0.112) (Tle 1). Similrly, guiol peroxidse enzyme (GPX) lso showed similr type of trend with higher tivity in Lun Snkhi (68.41 ± 3.45) nd Lun Suvrn (75.51 ± 1.68) followed y IR64 (51.16 ± 1.65) nd Pus Bsmti-1 (51.80 ± 0.40). Dehydrosorte redutse (DHAR) nd monodehydrosorte redutse (MDHAR) tivities were lso found to e high in tolernt ultivrs (Lun Snkhi nd Lun Suvrn) in omprison to the sensitive ones (IR64 nd Pus Bsmti-1) (Tle 1). Expression nlysis of ntioxidnt enzymes The semi-rt PCR nlysis of vrious ntioxidnt genes lso showed higher level of key ntioxidnt enzymes in tolernt ultivrs exept CAT nd Mn-SOD t the trnsript level (Fig. 3). Among the different isoforms of SOD, the trnsript level of Fe-SOD nd Cu/Zn-SOD showed muh higher umultion in Lun Snkhi nd Lun Suvrn. These results were further onfirmed t the protein level with in gel SOD ssy (Fig. 4). On the ontrry, Mn-SOD showed lesser expression in tolernt ultivrs when ompred ginst the sensitive ultivrs. The trnsript level of CAT ws lso found to e more in the sensitive ultivrs (IR64 nd Pus Bsmti-1). However, APX nd GR enzymes were found in the sme type of trend t the trnsript level s followed in their respetive enzymti tivities. Their gene expression ws lso higher in Lun Snkhi nd Lun Suvrn s ompred to the sensitive ones (IR64 nd Pus Bsmti-1). Asorte ontent A notle differene in the redued to oxidised sorte rtio ws found in 4 ultivrs of rie (Tle 1). Tolernt ultivrs Lun Suvrn nd Lun Snkhi showed signifintly higher rtio of (0.905 ± 0.033) nd (0.884 ± 0.017), respetively in ontrst to the sensitive ultivrs IR64 (0.739 ± 0.027) nd Pus Bsmti-1 (0.698 ± 0.011). Totl protein, free proline nd MDA ontent nlysis In this study, we estimted the totl protein ontent in the four ultivrs. The protein ontent (mg FW -1 ) ws found to e signifintly high in tolernt ultivrs Lun Snkhi (14.73 ± 0.275) nd Lun Suvrn (15.22 ± 0.403) with respet to the sensitive ultivrs IR64 (12.75 ± 0.275) nd Pus Bsmti-1(12.85 ± 0.352) (Tle 2). The signifintly enhned ontent of proline (μmoles g FW -1 ) ws found in Lun Snkhi (6.90 ± ) nd Lun Suvrn (6.78 ± ) ginst the sensitive ultivr IR64 (4.29 ± ) nd Pus Bsmti-1 (4.58 ± ) (Tle 2). Further, the level of lipid peroxidtion (μmoles g FW -1 ) ws determined through the estimtion of MDA equivlents

5 Tle 1 Comprtive nlysis of different ntioxidnt enzymes nd sorte ontent in 14 dys old seedlings of different rie ultivrs Cultivr SOD (units mg protein -1 ) APX (μmol min -1 mg protein -1 ) CAT (μmol min -1 mg protein -1 ) GPX (μmol min -1 mg protein -1 ) GR (μmol min -1 mg protein -1 ) DHAR (μmol min -1 mg protein -1 ) MDHAR (μmol min-1 mg protein -1 ) Redued sorte/oxidised sorte rtio IR ± d ± ± ± ± ± ± ± PB ± ± ± ± ± ± ± ± Lun Snkhi ± ± ± ± ± ± ± ± Lun Suvrn ± ± ± ± ± ± ± ± (SOD Superoxide Dismutse, APX Asorte Peroxidse, CAT Ctlse, GPX Guiol Peroxidse, GR Glutthione Redutse, DHAR Dehydrosorte Redutse, MDHAR Monodehydrosorte Redutse). Vlues represent men ± SE (n =3) Different letters (,,, d ) within ultivrs re signifintly different (Tukey LSD, p 0.05) Kur et l. BMC Plnt Biology (2016) 16:131 Pge 5 of 13

6 Kur et l. BMC Plnt Biology (2016) 16:131 Pge 6 of 13 Reltive Expression Reltive Expression Mn-SOD Cu/Zn-SOD EF1 Reltive Expression EF1 Reltive Expression Fe-SOD APX EF1 EF1 Reltive Expression Reltive Expression CAT EF1 GR EF1 Fig. 3 An ethidium romide stined grose gel hrouring produts from reverse trnsriptse-pcr of different key ntioxidnt genes of 14 dys old seedlings of different rie ultivrs. Gel shows the PCR produts fter 35 yles of PCR nd the results were first normlized to the housekeeping gene EF1α. Brs represent men ± SE (n = 3). Different letters (,,, d) within ultivrs re signifintly different (Tukey LSD, p 0.05). (IR: IR64, PB: Pus Bsmti-1, SA: Lun Snkhi, SU: Lun Suvrn) IR PB SA SU Mn-SOD Fe-SOD Cu/Zn-SOD Fig. 4 In-gel nlysis of the pttern of SOD isoenzymes in two slt sensitive nd two slt tolernt ultivrs of rie (IR: IR64, PB: Pus Bsmti-1, SA: Lun Snkhi, SU: Lun Suvrn) Tle 2 Comprtive nlysis of totl protein, proline nd MDA of 14 dys old seedlings of different rie ultivrs Cultivr Protein ontent (mg FW -1 ) Proline ontent (μmoles g FW -1 ) MDA ontent (μmoles g FW -1 ) IR ± ± ± PB ± ± ± Lun Snkhi ± ± ± Lun Suvrn ± ± ± Vlues represent men ± SE (n =3) Different letters (,, ) within ultivrs re signifintly different (Tukey LSD, p 0.05)

7 Kur et l. BMC Plnt Biology (2016) 16:131 Pge 7 of 13 ontent in plnts. The level of MDA ws found to e more in sensitive ultivrs IR64 ( ± 0.058) nd Pus Bsmti-1 ( ± 0.047) thn the tolernt ultivrs Lun Snkhi ( ± 0.206) nd Lun Suvrn ( ± 0.222) (Tle 2). Chlorophyll ontent nlysis A signifint differene in the ontent of hlorophyll, hlorophyll nd totl hlorophyll ws oserved in sensitive nd tolernt ultivrs of rie (Fig. 5). The mximum ontent of hlorophyll (μg/ml) ws found in IR64 ( ± 0.076) followed y Pus Bsmti- 1( ± 0.995) tht ws signifintly higher from the Lun Snkhi ( ± 0.128) nd Lun Suvrn ( ± 0.195). However, the highest ontent of hlorophyll ( ± 0.868) nd totl hlorophyll ( ± 0.772) ws found in Pus Bsmti-1 wheres the minimum ontent of these pigments ws found in Lun Suvrn. Disussion In the present study, it ws oserved tht the tolernt ultivrs (Lun Snkhi nd Lun Suvrn) mintin higher level of H 2 O 2 nd lower level of O 2- s ompred to the sensitive ultivrs (IR64 nd Pus Bsmti-1). H 2 O 2 ts s seondry messenger tht tivtes the vrious stress dptive signlling pthwys t oth geneti nd physiologil levels [14]. It is known to tivte vrious genes involved in the limtion nd tolerne to slt stress [15]. Further, enhned H 2 O 2 prodution in hlophytes is essentil for their slt stress prepredness [16]. Our study indites tht slt tolernt ultivrs Lun Snkhi nd Lun Suvrn re ple of onstitutive tivtion of plnt defene pthwys due to the higher sl level of H 2 O 2 tht in turn keeps the tolernt ultivrs redy for dpttion to slt stress onditions. Hene, H 2 O 2 signtures my e operting in slt stress signlling pthwys in rie tht keeps the defene pthwy networks tivted in tolernt ultivrs [17]. Furthermore, the elevted sl level of H 2 O 2 found in the tolernt ultivrs ould e ttriuted to the higher tivity of NADPH oxidse nd SOD enzyme in Lun Snkhi nd Lun Suvrn tht ws further onfirmed through the estimtion of speifi enzymti tivity of SOD. These findings suggest tht the higher genertion of O 2- in NADPH dependent mnner nd its rpid onversion into signlling moleule H 2 O 2 ould e onsidered s ritil lue in understnding the model depiting etter dpttion of tolernt ultivrs towrds slinity. One the signlling sde hs een initited, plnts re defended to the toxi effets of H 2 O 2 with the help of APX, CAT nd other peroxidses in wy similr to the C 2+ efflux system tht opertes in well-known ytosoli lium signtures [6]. Similrly, in the present work, the speifi tivity of peroxidses APX nd GPX ws found to e high in tolernt vrieties whih ould e svenging the exess of H 2 O 2 to prevent the plnt from its ill effets, wheres the speifi tivity of CAT ws found to e low. The less expression of CAT in tolernt ultivrs ould e due to the ommon sustrte of APX nd CAT nd euse of the higher ffinity of APX for hydrogen peroxide thn CAT [18]. Moreover, APX regultes the role of H 2 O 2 s signlling moleule unlike CAT whih is more involved in detoxifition [15]. Till now, the mjority of reserh on the reltion etween ROS nd slinity tolerne hs een foussed on the enzymti ntioxidnts lthough non-enzymti ntioxidnts lso ply ruil role in svenging exess of ROS nd onferring tolerne [6]. Asorte is known non enzymti ntioxidnt tht plys entrl role in Chlorophyll ontent (µg/ml) d d d IR PB SA SU 0 Chl Chl Totl Chl Fig. 5 Comprtive nlysis of hlorophyll, hlorophyll nd totl hlorophyll in 14 dys old seedlings of different rie ultivrs. Brs represent men ± SE (n = 3). Different letters (,,, d) within ultivrs re signifintly different (Tukey LSD, p 0.05). (Chl: Chlorophyll, IR: IR64, PB: Pus Bsmti-1, SA: Lun Snkhi, SU: Lun Suvrn)

8 Kur et l. BMC Plnt Biology (2016) 16:131 Pge 8 of 13 the sorte glutthione yle [19]. APX uses sorte s redutnt for the svenging of H 2 O 2 nd onverts it into dehydrosorte. But for the mintenne of ntioxidtive pity of sorte, its rpid regenertion into redued form is neessry. APX funtions in omintion with GR, DHAR nd MDHAR in glutthione-sorte pthwy for the regenertion of sorte. In our studies, the speifi tivities of GR, DHAR nd MDHAR enzymes were lso found to e high in tolernt ultivrs. Further the rtio of redued to oxidised sorte ws lso oserved to e high in Lun Snkhi nd Lun Suvrn. This ould e implied to the etter opertion of non-enzymti glutthione-sorte pthwy in tolernt ultivrs whih my e one of the mny resons for their superior slt tolerne ility [19]. Furthermore, the higher tivity of vrious ntioxidnt enzymes (SOD, CAT, GPX, APX, nd GR) in oordinted mnner in slt tolernt ultivrs suggests tht they re the mjor determinnts in the model for depiting slt tolerne. To further understnd the dynmi role of ntioxidnt enzymes in onferring slt tolerne to hlophyti vrieties of rie, their trnsript level expression study ws onduted. Among the different isoforms of SOD, the expression of Fe-SOD nd Cu/Zn-SOD ws found to e high in tolernt ultivrs wheres, the expression of Mn-SOD ws more in sensitive ultivrs. In n erlier study from our lortory, we oserved higher expression of Fe-SOD nd Cu/Zn-SOD in response to slt tretment in Pus Bsmti-1 [20]. Therefore, the higher sl expression level of Fe-SOD nd Cu/Zn-SOD in tolernt ultivrs ould e one of the mny resons for their etter dpttion in slt stress. Furthermore, to get etter insight into the mehnism onferring elevted dpttion to slinity stress in Lun Snkhi nd Lun Suvrn, some of the iohemil stress mrkers were studied. In the present study, enhned level of totl protein in Lun Snkhi nd Lun Suvrn ws oserved whih ould e ttriuted to the synthesis of new or elevted level of proteins linked to stress tolerne in these ultivrs. This result is further strengthened y the report tht enhned synthesis level of proteins is one of the mjor determinnts of stress dpttion in hlophytes [21]. Proline is known osmoprotetnt tht lso plys multiple ntioxidnt roles. Hyperumultion of free proline in plnts is known in onferring slinity tolerne [22, 23]. The higher umultion of free proline in Lun Snkhi nd Lun Suvrn my e plying n importnt role in quenhing of exess of ROS nd stiliztion of ROS-svenging enzymes long with its well known role in osmotium mintinene [24]. Further, with regrd to redued MDA ontent in tolernt ultivrs, our results re in greement to the previous studies where it hs een shown tht higher ntioxidnt enzyme tivities re responsile for low MDA ontent whih in turn inhiits the memrne dmge y ROS nd hene onfers tolerne [25, 26]. It ws interesting to note tht the ontent of hlorophyll, hlorophyll nd totl hlorophyll ws less in the tolernt ultivrs in omprison to sensitive ultivrs. Under slt stress onditions, plnts re often oserved to hve redued hlorophyll ontent [20, 27]. However, nlysis of hlorophyll ontent in slt tolernt nd sensitive ultivrs will provide the physiologil explntion to dpttion of plnt to slinity stress nd hene it is sujet of worth investigtion. In our investigtion the less hlorophyll ontents in tolernt ultivrs my e either due to the ROS medited degrdtion of holorophyll or it ould e phototlyti tivity of holorophyll itself for the prodution of H 2 O 2 [28, 29]. In our se, we presume tht in tolernt ultivrs the slt tolerne ould e ttriuted to the enhned prodution of H 2 O 2, whih my e due to the phototlyti tivity of hlorophyll [30]. Moreover, the role of light in hlorophyll degrdtion hs lso een suggested [31]. Conlusion The tolernt ultivrs of rie mintin high threshold level of H 2 O 2 due to more enzymti tivity of NADPH oxidse nd enough SOD in stok tht my onstitutively tivtes the defene pthwys resulting in higher intrinsi tivity of vrious ntioxidnt enzymes. This provides ertin dptive dvntge to hlophytes over glyophytes euse of whih slt stress does not led to oxidtive stress in tolernt ultivrs. Further, the first time omprtive nlysis of ROS generting system NADPH oxidse in hlophytes nd glyophytes provide ritil lue on understnding the model depiting slt tolerne in rie. We propose tht these two systems work together in synhroniztion with eh other for hieving slt tolerne. It will e interesting to further explore the rosstlk tht exists etween ROS generting nd svenging system for hieving slinity tolerne nd whether this rosstlk opertes in feedforwrd or feedk mnner. Furthermore, the oservtion of degrdtion of hlorophyll for produing higher level of H 2 O 2 nd in turn offering tolerne points out towrds n unhrted mehnism in slt stress dpttion iology. Methods Plnt mteril Four rie ultivrs differing in slt stress tolerne level were seleted for this work. Seeds of two slt sensitive ultivrs viz. IR64 nd Pus Bsmti-1 were proured from IARI (Indin Agriulturl Reserh Institute, New Delhi) nd the seeds of two slt tolernt ultivrs Lun Snkhi nd Lun Suvrn were olleted from CRRI (Centrl Rie Reserh Institute Cuttk, Odish, Indi).

9 Kur et l. BMC Plnt Biology (2016) 16:131 Pge 9 of 13 Lun Snkhi (CR Dhn 405) nd Lun Suvrn (CR Dhn 403) re slt tolernt vrieties of Indin origin tht n tolerte slinity stress in the rnge of 6-8 ds m -1 [31]. Surfe steriliztion nd inoultion Surfe steriliztion of the seeds ws rried out ording to Shrm s method [26]. Helthy nd mture seeds were seleted nd were sterilized using sodium hypohlorite nd meruri hloride. Sterilized seeds were then inoulted in utolved snd moistened with utolved doule distilled wter in plsti oxes nd were kept t 25 ± 2 C temperture with 14 h photoperiod under light intensity of 2000 lux for 14 dys. Histohemil ROS detetion The histohemil level of different types of ROS ws deteted using three different dyes. For the detetion of O 2-, the seond lef of 14 d seedlings ws ut into smll piees nd these piees were vuum infiltered in 6 mm NBT prepred in 10 mm of sodium itrte uffer (ph = 6) for 10 min. They were inuted for 2 h t room temperture nd the hlorophyll ontent of these piees ws then removed y oiling them in solute ethnol till the omplete removl of hlorophyll. After the removl of hlorophyll, piees were oserved under the stereo-mirosope [32]. For the detetion of H 2 O 2, two different stins were used, DAB nd H 2 DCFDA. For the DAB ssy, smll piees of seond lef of 14 d seedlings were vuum infiltered in DAB (1 mg/ml in wter) for 10 min nd were then inuted for 2 h under the drk onditions. Chlorophyll ontent ws removed nd they were oserved under the stereo-mirosope [32]. For H 2 DCFDA ssy, piees of seond lef were inuted in 10 μm H 2 DCFDA nd were vuum infiltered for 5 min. The leves were then wshed with doule distilled wter nd were oserved under the onfol mirosope using lser em of exittion 488 nm [33, 34]. NADPH oxidse ssy Isoltion of plsm memrne proteins Plsm memrne proteins were extrted from the 0.5 g of fresh tissue [13]. The plntlets were homogenized in protein extrtion uffer ontining 0.25 M surose, 50 mm HEPES (ph 7.2), 3 mm ethylenediminetetreti id (EDTA), 1 mm dithiothreitol (DTT), 3.6 mm L- ysteine, 0.1 mm MgCl 2, 0.6 % PVP nd PIC (Protese Inhiitory Coktil). The homogente ws filtered through two lyers of muslin loth nd ws entrifuged t 10,000 g for 45 min t 4 C. Superntnt ws ultrentrifuged t 1, 80, 000 g for 60 min t 4 0 C. The pellet ws resuspended in 10 mm hilled Tris-HCl. The protein ontent ws determined ording to method desried y Brdford [35] using Bovine Serum Alumin (BSA) s stndrd. Spetrophotometri ssy of NADPH oxidse tivity The O 2- generting tivity of NADPH oxidse ws mesured y following the Kundl s method [13]. The ssy mixture ws prepred with 50 mm Tris-HCl uffer (ph 7.5), 1 mm XTT (sodium, 3-[1-[phenylmino-ronyl]-3, 4-tetrzolium]-is(4-methoxy-6-nitro)enzenesulfoni id hydrte), 1 mm NADPH, nd 20 μg of memrne protein. The rte of redution of XTT y O 2 - ws determined spetrophotometrilly t 492 nm using n extintion oeffiient of 2.16 x 10 4 M -1 m -1. Estimtion of queous H 2 O 2 ontent The ontent of queous H 2 O 2 ws estimted using the modified ferrous oxidtion-xylenol ornge (FOX) ssy [13]. 0.5 g of fresh seedlings of 14 d plnts ws homogenised in tivted hrol (0.1 g) prepred in 5 ml of 5 % trihloroeti id. Homogente ws filtered through Whtmnn filter No.1 nd ws entrifuged t 8000 rpm for 10 min. The superntnt ws used for the estimtion of queous H 2 O 2. FOX regent ws prepred using 1 ml of regent (25 mm mmonium ferrous sulfte prepred in 2.5 M sulfuri id), 50 μl of regent (0.25 M xylenol ornge prepred in HPLC-grde methnol), nd 90 ml of regent (9.69 mg of utylted hydroxytoluene prepred in 90 ml of HPLC- grde methnol). The totl volume ws rised to 100 ml with distilled wter. In test tue, 0.2 ml of plnt extrt ws tken nd 1 ml FOX regent ws dded to it. The retion mix ws mixed properly nd ws inuted t room temperture for 15 min. All solutions were prepred fresh nd used within 2 h. The ontent of queous H 2 O 2 ws estimted y reording the sorne t 560 nm. Conentrtion ws lulted using stndrd urve prepred y tking vrying onentrtions of H 2 O 2. Extrtion of protein For the estimtion of ntioxidnt enzyme tivities, 1 g of fresh 14 d seedlings were homogenised in 3 ml of hilled uffer ontining 50 mm phosphte uffer (ph = 7.8), 2 mm EDTA, 1 mm DTT, 1 mm PMSF (Phenylmethylsulfonyl Fluoride), 0.5 %(v/v) Triton X- 100 nd 10 % (w/v) PVP-40 (Polyvinylpyrrolidone). The homogente ws entrifuged for 20 min t 12,000 rpm nd the superntnt ws olleted. This superntnt ws then used for vrious enzymti ssys [26]. Protein onentrtion ws determined using Brdford ssy with BSA s stndrd [35]. Estimtion of ntioxidnt enzyme tivities The enzymti tivities of different ntioxidnt enzymes were determined spetrophotometrilly. The EC (Enzyme Commission) numer mentioned ginst eh enzyme is

10 Kur et l. BMC Plnt Biology (2016) 16:131 Pge 10 of 13 unique numer in the numeril lssifition sheme for enzymes nd represent the hemil retions they tlyze. ) SOD ssy (EC ) The enzymti tivity of SOD ws determined using the method given y Beuhmp nd Fridovih [36]. 1 ml of retion mixture ws prepred in 50 mm potssium phosphte uffer using 2 μm rioflvin, 75 μm Nitrotetrzolium lue (NBT), 100 μm EDTA, 13 mm DL-methionine nd 50 μl of enzyme extrt nd the sorne ws tken t 560 nm. The retion mix ws illuminted for 20 min t 25 C for initition. 1 unit of enzyme tivity is expressed s the mount of enzymes required for 50 % inhiition of NBT redution t 25 C. ) CAT ssy (EC ) CAT speifi tivity ws estimted t 25 C using the method given y Aei [37]. Derese in sorne of H 2 O 2 ws mesured in 1 ml of retion mixture hving 10 mm H 2 O 2 nd 20 μl of enzyme extrt in 50 mm of potssium phosphte uffer (ph = 7). Speifi enzyme tivity ws expressed s l mole of H 2 O 2 deomposed mg protein -1 min -1. ) APX ssy (EC ) The speifi tivity of APX ws ssyed s the rte of oxidtion of sorte in the presene of H 2 O 2 [26]. The retion mixture of 1 ml omprised of 0.5 mm sorte, 0.1 mm H 2 O 2 nd 0.1 mm EDTA, potssium phosphte uffer(ph = 7) nd 10 μl of enzyme extrt. Derese in sorption ws oserved spetrophotometerilly t 290 nm t 25 C. One unit of enzyme tivity ws expressed s the mount of enzyme required to oxidise 1 μm of sorte/minute/g tissue. d) GR ssy (EC ) The speifi tivity of GR is determined y nlysing the derese in sorne t 340 nm [26]. 1 ml retion mixture used for the ssy hd 50 mm potssium phosphte uffer (ph = 7.8), 1 mm EDTA, 1 mm oxidized glutthione (GSSG) nd 25 μl enzyme extrt. The retion ws strted y dding 0.1 mm NADPH t lst. Enzyme tivity ws expressed s μmol of NADPH oxidised min -1 mg protein -1. e) GPX ssy (EC ) The tivity of GPX is oserved y mesuring the inrese in sorne t 436 nm [26]. The retion mixture ws prepred in 50 mm potssium phosphte uffer (ph = 7) hving 9 mm guiol, 10 mm H 2 O 2 nd 33 μl of enzyme extrt. The enzymti tivity of GPX is expressed s the mount of enzyme required to produe 1 μmol guiol dehydrogention produt min -1 mg protein -1. f) DHAR ssy (EC ) The speifi tivity of DHAR ws estimted y mesuring the inrese in sorne t 25 C due to the formtion of sorte from dehydrosorte t 265 nm [26]. The retion mixture of 1 ml onsisted of 50 mm of potssium phosphte uffer (ph = 7), 0.1 mm EDTA, 0.5 mm dehydrosorte, 2.5 mm GSH nd 25 μl of enzyme extrt. One unit of enzyme tivity ws expressed s mount of enzyme required to produe 1 μmol of sorte min -1 mg protein -1. g) MDHAR ssy (EC ) The speifi tivity of MDHAR ws determined t 25 C y mesuring the inrese in sorne t 340 nm [26]. The 1 ml retion mixture used for this estimtion onsisted of 50 mm Tris-HCl uffer (ph = 7.6), 0.15 units of sorte oxidse enzyme, 2.5 mm sori id nd 0.2 mm NADPH/NADH. One unit of enzyme tivity ws given s the mount of enzyme required to oxidse 1 μmol of NADPH min -1 mg protein -1. Expression nlysis of ntioxidnt enzymes For the gene expression studies of vrious ntioxidnt genes, semi quntittive primers were designed using the IDT softwre ( (Tle 3). Totl RNA from the 14 d plnts ws extrted using the trizol method s per the mnufturer s instrutions. A totl of 3 μg of RNA ws used for the DNA preprtion with ommeril DNA synthesis kit. Gene expression ws studied in 50 μl of polymerse hin retion (PCR) using 50 ng of DNA templte. The PCR prmeters used were: predenturtion t 94 C for 4 min, followed y 35 yles of 94 C for 1 min, 55 C for 1 min, 72 C for 1 min, with finl extension step of 72 C for 7 min. Rie elongtion ftor ef1α ws used s internl ontrol. All the PCR s were performed t lest with three independent smples nd intensity of the produts ws onfirmed with 1 % grose gel with ethidium romide. The reltive level of trnsript in eh PCR retion ws determined using the integerted density vlue (IDV), with Alph 2000TM Imge Anlyzer softwre. In-gel SOD ssy For in-gel SOD ssy, 100 mg of fresh seedling tissue of eh ultivr ws homogenized with 50 mm Tris HCl

11 Kur et l. BMC Plnt Biology (2016) 16:131 Pge 11 of 13 Tle 3 List of primers for RT-PCR S. No. Nme of the gene Dtse Aession numer Primer sequene 1 EF1α GenBnk D F: 5 0 -GTACAAGATCGGTGGTATT-3 0 R: 5 0 -GGGTACTCAGAGAAGGTCT Cu/Zn-SOD GenBnk L F: 5 0 -CCTCAAGCCTGGTCTCCAT-3 0 R:5 0- CAGCCTTGAAGTCCGATGAT Fe-SOD GenBnk AY F: 5 0 -CTTGATGCCCTGGAACCTTA-3 0 R: 5 0 -GCCAGACCCCAAAAGTGATA Mn-SOD GenBnk L F: 5 0 -GCCATTGATGAGGATTTTGG-3 0 R: 5 0 -CAAGCAGTCGCATTTTCGTA CAT GenBnk D F: 5 0 -GTTCGGTTCTCCACAGTCGT-3 0 R: 5 0 -CCCTCCATGTGCCTGTAGTT APX GenBnk D F: 5 0 -CCAAGGGTTCTGACCACCTA-3 0 R: 5 0- CAGTTCGGAGAGCTTGAGGT GR GenBnk AB F: 5 0- AACAGCCGATGGCATAAAAG-3 0 R:5 0 -CAACCACCAGTTTCATGACG-3 0 uffer (ph = 7.5). The homogente ws entrifuged t rpm for 15 min t 4 C [38]. The protein ontent ws quntified in the superntnt using Brdford ssy with BSA s stndrd. Ntive-PAGE nlysis ws rried out with 50 μg protein of eh ultivr t 80 V stking nd 100 V resolving. The gel ws then stined ording to Ruinsk s method using 2.45 mm NBT prepred in 50 mm potssium phosphte uffer for 20 min in drk [39]. The gel ws gin wshed with utolved wter followed y immersion in potssium phosphte (50 mm, ph 7.8) ontining TEMED (28 mm) nd rioflvin (3 μm) for 15 min. The gel ws then kept on dry white illumintion try till the gel eme uniformly lue exept t positions ontining SOD nd mximum ontrst etween the hromti zones nd generl lue olour ws hieved. Asorte ontent The ontent of sori id ws estimted using the Dutilleul s method with minor modifitions [40]. 1 g of fresh lef tissues of four ultivrs ws homogenized with 3 % metphosphori id (3 %) ontining EDTA (1 mm). The homogente ws entrifuged t 8000 g for 15 min. 0.1 ml of superntnt ws tken nd ws mixed with 1 ml itrte phosphte uffer (0.1 M, ph 2.1). The sorne ws tken t 265 nm nd 1 unit of APX ws dded. The solution ws inuted for 5 min t room temperture nd sorne ws gin noted t 265 nm. The differene in two redings of sorne ws lulted tht indited the redued sorte ontent utilized speifilly y APX. For the estimtion of totl sorte ontent, 0.1 ml of DTT (0.1 M) ws dded in nother set nd sorne ws red t 265 nm fter 5 min. 1 unit of APX ws dded nd sorne ws tken fter 5 min. For the determintion of oxidized sorte ontent, redued sorte ontent ws sutrted from the totl sorte ontent. Clirtion urve ws prepred using grded onentrtion of L-sori id in 3 % HPO 3. The rtio of redued sorte nd oxidized sorte ws lso determined. Proline ontent Proline ontent ws determined using the method given y Btes [41]. 0.5 g of totl seedlings were rushed in liquid nitrogen nd homogenised in 3 % queous sulfosliyli id. The smples were then entrifuged t rpm for 15 min nd 2 ml of the superntnt ws tken in test tue. Equl mount of id ninhydin nd glil eti id ws dded to the superntnt nd the retion mix ws kept in oiling wter for 1 h. The retion ws stopped y keeping the tues on ie. Added 4 ml of toluene to the retion mixture nd toluene lyer seprted from the queous phse ws olleted. The proline ontent ws determined y mesuring the sorne of the toluene t 520 nm. The mount of proline ws estimted using the proline stndrd urve nd ws expressed s μmoles gfw -1. Lipid peroxidtion The level of lipid peroxidtion ws determined using the TBARS ssy [42]. 1 g of tissue smple ws homogenised in 3 ml of 0.1 % of hilled trihloroeti id (TCA) nd 3 ml of solution ontining 0.5 % TBA (Thiorituri id) in 20 % TCA ws dded to it. The retion mixture ws inuted t 95 C for 30 min nd the retion ws stopped y keeping the tue on ie. Centrifuged the mixture t 10,000 rpm for 15 min. The superntnt

12 Kur et l. BMC Plnt Biology (2016) 16:131 Pge 12 of 13 ws olleted nd its sorne ws mesured t 532 nm, with reding t 600 nm sutrted from it to ount for nonspeifi turidity. 155 mm -1 m -1 extintion oeffiient ws used for the quntifition of MDA- TBA omplex nd ws expressed s μmoles gfw -1. Chlorophyll ontent Chlorophyll ontent ws estimted using the method given y Arnon with some modifitions [43]. 100 mg of fresh leves of eh ultivr were tken nd were homogenised in liquid nitrogen. To the homogenised tissue smple, 1.5 ml of 80 % etone ws dded nd the retion ws inuted in drk for 1 h. Smples were entrifuged t 15,000 rpm for 3 min. The superntnt ws olleted nd the sorne ws mesured spetrophotometrilly t 645 nd 663 nm ginst 80 % etone s lnk. The hlorophyll ontent ws determined s follows: Totl hlorophyll ðμg=mlþ ¼ 20:2 ða 645 Þ þ 8:02ðA 663 Þ Chlorophyll ðμg=mlþ ¼ 12:7 ða 663 Þ 2:69ðA 645 Þ Chlorophyll ðμg=mlþ ¼ 22:9 ða 645 Þ 4:68ðA 663 Þ Sttistil nlysis Dt from different experiments ws nlysed sttistilly with one-wy nlysis of vrine (ANOVA). The vlue of different prmeters is expressed s men ± SE of three independent replites. The Tukey LSD test ws pplied for multiple omprisons using Sigm stt version 3.5, nd signifine of differene etween the ultivrs were set s p Arevitions APX, sorte peroxidse; CAT, tlse; DAB, 3, 3-diminoenzidine; DHAR, dehydrosorte redutse; GPX, guiol peroxidse; GR, glutthione redutse; H 2 DCFDA, 2',7'-dihlorodihydrofluoresein diette; H 2 O 2,hydrogen peroxide; MDA, mlondildehyde; MDHAR, monodehydrosorte redutse; NADPH, niotinmide denine dinuleotide phosphte hydrogen; NBT, nitrozolium lue; O 2-, superoxide ions; PCR, polymerse hin retion; PIC, protese inhiitory oktil; PMSF, phenylmethylsulfonyl fluoride; ROS, retive oxygen speies; semi-rt, semi quntittive reverse trnsriptse; SOD, superoxide dismutse; XTT, sodium3-[1-[phenylmino-ronyl]-3,4-tetrzolium]-is(4-methoxy-6-nitro)enzenesulfoni id hydrte Aknowledgements Nvdeep Kur is thnkful to the Deprtment of Biotehnology (Government. of Indi) for Senior Reserh Fellowship. We thnk the Deprtment of Genetis IARI, New Delhi, Indi for providing the seeds of IR64 nd Pus Bsmti-1 nd Diretor, CRRI, Odish, Indi for providing the seeds of Lun Snkhi nd Lun Suvrn. Authors knowledge the Deprtment of Biotehnology, Government of Indi for finnil support (Projet No. BT/PR13965/BRB/10/883/2010). Avilility of dt nd mterils All the dt supporting the findings is ontined within the mnusript. Authors ontriutions NK, PKP nd IS oneived nd designed the experiments. NK nd MD performed the experiments. PKP nd NK nlysed nd interpreted the dt. NK rried out sttistil nlysis. PKP, NK nd IS wrote the mnusript. All uthors red nd pproved the finl mnusript. Competing interests The uthors delre tht they hve no ompeting interests. Consent for pulition Not pplile. Ethis pprovl nd onsent to prtiipte Not pplile. Author detils 1 Deprtment of Biotehnology, Guru Nnk Dev University, Amritsr , Punj, Indi. 2 Deprtment of Orl iology, August University, August, GA, USA. 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