Influence of Si Supplementation on Growth and Some Physiological and Biochemical Parameters in Salt- Stressed Tobacco (Nicotiana rustica L.

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1 Journl of Sienes, Islmi Repuli of Irn 25(3): (2014) University of Tehrn, ISSN Influene of Si Supplementtion on Growth nd Some Physiologil nd Biohemil Prmeters in Slt- Stressed Too (Niotin rusti L.) Plnts R. Hjiolnd 1,2,*, L. Cherghvreh 2 1 Center of Exellene for Biodiversity, Fulty of Nturl Sienes, University of Triz, Triz, Islmi Repuli of Irn 2 Plnt Siene Deprtment, Fulty of Nturl Sienes, University of Triz, Triz, Islmi Repuli of Irn Reeived: 24 My 2014 / Revised: 2 August 2014 / Aepted: 4 Septemer 2014 Astrt Too is slt-sensitive glyophyte rop speies. In this work effet of silione (Si) supplementtion (1 mm s N2SiO3) ws studied in Niotin rusti L. v. Bsms grown hydroponilly in growth hmer under ontrol, low (25 mm) nd high (75 mm) NCl onentrtion for two weeks. Dry mtter prodution of leves ws depressed y slinity level s low s 25 mm nd higher slt onentrtion deresed plnts dry weight y 52-82%. Si supplementtion llevited slt stress effet s ould e judged y higher dry weight of shoot nd roots in +Si plnts ompred with Si ounterprts. Lef hlorophyll nd rotenoids onentrtions nd net ssimiltion rte were higher in Si-treted plnts not only in slt-ffeted ut lso in ontrol plnts. Si tretment resulted in higher onentrtion of solule rohydrtes ut not proline. Lef trnspirtion rte, unexpetedly, ws not diminished y Si nd wter use effiieny ws rther lowered y Si in slt-treted plnts. Si pplition used slight redution of N onentrtion while inresed tht of K nd C signifintly nd resulted in higher K:N rtio in the leves, stem nd roots. Our results suggested tht Si pplition improved tolerne to slt stress in too due to n enhnement of photosynthesis, umultion of orgni osmolytes s well s improvement of K:N seletivity ut not limiting wter loss. In ddition, greter dry mtter prodution of Si-supplemented plnts in the sene of slt ws ssoited with elevted photosynthesis rte, higher K nd C uptke nd proline ontent. Keywords: Too, Slinity, Silione, K:N rtio, Orgni osmolytes. Introdution Slinity is mong the most stressful soil ftors tht limits plnts growth nd produtivity. Crop plnts re minly sensitive to slt nd under rid nd semirid limti onditions, soil slinity is the mjor onstrint for growth nd yield of rop speies [10]. * Corresponding uthor: Tel: ; Fx: ; Emil: ehsn@trizu..ir 205

2 Vol. 25 No. 3 Summer 2014 R. Hjiolnd, nd L. Cherghvreh J. Si. I. R. Irn Plnt performne is dversely influened y slinity vi different mehnisms. There re two omponents of slt stress, inlude osmoti stress nd toxi effet of ions, tht influene differentilly plnt growth nd metolism depending on slt onentrtion [35]. Osmoti stress is linked to lower slt onentrtions while ion toxiity effet impirs plnts growth under higher slinity levels [16]. The threshold onentrtion of slt for these two ontrstive effets is highly dependent on the extent of uptke nd umultion of slt nd suseptiility of plnt speies. Glyophytes re slt-sensitive speies tht re ffeted even y low onentrtions of slt nd higher levels of slinity my distur severely metolism nd growth nd use ultimtely plnts deth [36]. Silione (Si) is the seond most undnt element in the erth s rust nd soils, ut it hs not yet een tegorized s n essentil nutrient for higher plnts [4, 37]. Regrding plnts ility for Si uptke nd umultion, they re lssified into Si-umultor nd non-umultor speies [4, 37]. In the sene of Si, growth nd reprodution of umultor speies re severely depressed while non-umultor speies do not pprently require Si for optimum growth [4]. Nevertheless, Si in the growth medium of nonumultor speies meliortes detrimentl effets of vrious environmentl stresses [4, 15, 22]. Effets of different ioti stresses re llevited in the presene of Si, inlude drought [13, 42], UVrdition effets [42], hilling nd freezing [27] nd toxiity of hevy metls [19]. Effet of Si on the llevition of slt stress effets hs een reported for Siumultor speies suh s rie [31, 46] nd uumer [48] s well s in some non-umultor speies suh s tomto [39]. Vrious mehnisms re involved in the meliortive effet of Si on plnt response to ioti stresses inlude tivtion of ntioxidtive defense [21] nd improvement of plnts ility for wter nd nutrients uptke from soil [22, 39, 45]. Under slt stress, Si redues N uptke nd inreses K:N rtio thus, llevites ion toxiity effet in severl plnt speies suh s rie [31, 46] rley [23] nd tomto [39]. Too is the most importnt non-food rop speies in the world nd is highly suseptile to slt. Different ultivrs of too (Niotin spp.) elonging minly to N. toum or N. rusti speies re produed in the North nd North-Western Irn with n estimted re of h of ultivted lnd nd nnul prodution of round tons [10]. The most widely ultivted speies in the North-Western Irn is N. rusti. Lnd sliniztion is mjor limiting ftor for rop prodution in Irn [11] minly s the onsequene of low preipittion nd lking mngement strtegies of sline soils. In North-Western Irn griulturl lnds in proximity to the Slt Lke Urmi [9] re eing fed with the rise of wter tles using slt umultion in the surfe soils. Consequently, improvement of slt tolerne in glyophyte rop speies suh s too ould e regrded s n importnt strtegy for enhnement of produtivity nd yield of these speies ultivted on slinized griulturl soils. Reserh works on the effet of Si in too hs een limited to the studies on its effet on preventing infetions y pthogens [47]. Informtion is lking on the mitigtion of environmentl stresses inlude slt stress y Si in too. In umultor speies, Si deposition in the lef epidermis uses redution of trnspirtion nd improves pility of leves for wter retention [31]. In ddition, due to deposition in the root endodermis Si redues N ypss flow [14, 46]. In nonumultor speies suh s too, however, Si onentrtion is lower thn 5% [47] nd thus, it my llevite the effets of slt using other mehnisms. This work ws imed t studying the effet of Si supplementtion on growth, photosynthesis nd ion reltions in too plnts under slinity stress. In order to eluidte the effet of Si s influened y the level of slt onentrtion, two ontrstive slt levels, seleted ording to our preliminry experiment, were pplied in this work. Mterils nd Methods Plnts ulture nd tretment Seeds of too (Niotinrusti L. v. Bsms) plnts provided y the Agriulturl Reserh Center, Urmi, Irn, were surfe-sterilized using sodiumhypohlorite t 5%nd were germinted in the drk on perlite. Seven-dy-old young seedlings were preultured in Hoglnd nutrient solution [18] for three weeks. Four-weeks-old plnts were trnsferred to hydroponi medium ontining Hoglnd nutrient solution nd preultured for further one week. Therefter, Si tretments inluding without ( Si) or with (+Si) 1 mm Si (s N 2 SiO 3 ) were pplied. One week fter strting Si pplition, slinity tretments s three levels of NCl t 0 (ontrol), low (25 mm) nd high (75 mm) onentrtions were strted. Too plnts exposed to 100 mm NCl nd higher slinity died one week fter tretment, therefore, 75 mm NCl ws set s the mximum slinity level for the ultivr used in this work. In order to determine the possile effet of N s ompnying ion in the Si slt pplied to plnts, n experiment ws onduted in prllel with the min 206

3 Influene of Si Supplementtion on Growth nd Some Physiologil nd experiment with ontrol (without ddition of slt or Si) nd 2 mm NCl ontining n equivlent N with 1 mm N 2 SiO 3. Dry weight (mg plnt -1 ) of plnts under ontrol (2.35±0.16) nd 2 mm slt (2.66±0.64) ws not different signifintly (Tukey test, P<0.001). Plnts were grown under ontrolled environmentl onditions with temperture regime of 25 /18 C dy/night, 14/10 h light/drk period, reltive humidity of 70/80% nd t photon flux density of out 400 μmol m -2 s -1. Plnts hrvest Two weeks fter strting slinity tretment (3 weeks fter Si tretment, 8 weeks fter sowing) plnts were hrvested. Leves, stem nd roots were seprted, wshed with distilled wter nd lotted dry on filter pper. Plnts dry weight ws determined fter drying in 60 C for 48 h. Susmples were tken for iohemil nd ion nlyses efore nd fter drying, respetively. Before hrvest, hlorophyll fluoresene nd gs exhnge prmeters were determined in tthed leves. Determintion of hlorophyll fluoresene nd gs exhnge prmeters Chlorophyll (Chl) fluoresene prmeters were reorded using portle fluorometer (OSF1, ADC Biosientifi Ltd., UK). Mesurements were rried out on the seond youngest, fully expnded nd tthed leves of 4 plnts per tretment. An verge of 4 reords from different prts of eh individul lef ws onsidered for eh replites. Leves were limted to drk for 30 min using lef lips efore tking the mesurements for drk-dpted leves. Mximum quntum yield of PSII (Fv/Fm) ws lulted using initil (F 0 ), mximum (F m ) nd vrile (F v =F m -F 0 ) fluoresene prmeters. Clultions for light-dpted leves were undertken using initil (Ft), stedy-stte (F s ), mximum (F m '), vrile (F'v=F'm-Ft) nd F 0 '[F 0 '=F 0 /(F v /F m )+(F 0 /F m ')] fluoresene for exittion pture effiieny of open PSII (F'v/F'm),photohemil quenhing (qp) [(F m ' F s )/(F m ' F 0 ')] nd nonphotohemil quenhing (qn) [1 (F m ' F 0 ')/(F m F 0 )] [32]. CO 2 ssimiltion nd trnspirtion rtes were mesured in prllel for hlorophyll fluoresene mesurements in the sme lef with lirted portle gs exhnge system (LCA-4, ADC Biosientifi Ltd., UK) etween 10:00 A.M. nd 13:00 P.M. t hrvest. The mesurements were onduted with photosynthetilly tive rdition (PAR) intensity t the lef surfe of 400 μmol m -2 s -1. The net photosynthesis rte y unit of lef re (A, μmol CO 2 m - 2 s -1 ), trnspirtion rte (E, mmol H 2 O m -2 s -1 ) nd the stomtl ondutne to wter vpor (g s, mol m -2 s -1 ) were lulted using the vlues of CO 2 nd humidity vrition inside the hmer, oth mesured y the infrred gs nlyzer of the photosynthesis system. Determintions of lef pigments Lef onentrtion of Chl, nd rotenoids (Cr) were determined ording to Lihtenthler nd Wellurn [26]. Leves were homogenized in 80% old etone in the drk t 4 C. After 24 h, the sorption of smples ws determined t 663 (Chl ), 646 (Chl ) nd 470 (Cr) nm using spetrophotometer (Speord 200, Anlyti Jen, Jen, Germny). Determintion of nthoynins ws performed using ph differentil method t ph 1 nd ph 4.5 in the methnol/hcl (98:2, v/v) extrt nd ws expressed s mg of ynidine-3- gluoside g -1 FW [12]. Totl flvonoids ontent ws determined in the methnol extrt of leves. An liquot of 1 ml of the solution ontining 1 mg extrts in methnol ws dded to test tues ontining 0.1 ml of 10% Al(NO 3 ) 3, 0.1 ml of 1 M potssium ette nd 3.8 ml of methnol. After 40 min t room temperture, the sorne ws reorded using spetrophotometer t 415 nm. Queretin ws used s stndrd [40]. Determintions of orgni solutes For determintion of nonstruturl rohydrtes, smples were homogenized in 100 mm phosphte uffer (ph = 7.5) t 4 o C. After entrifugtion t g for 15 min, n liquot of the superntnt ws mixed with nthrone-sulfuri id regent nd inuted for 10 min t 100 C. After ooling, the sorne ws determined t 625 nm. Stndrd urve ws reted using gluose (Merk) [29]. Proline ws extrted nd determined ording to Btes et l. [2]. Lef tissues were homogenized with 3% sulfosliyli id nd the homogente ws entrifuged t 3000 g for 20 min. The superntnt ws treted with eti id nd id ninhydrin, oiled for 1 h, nd then sorne t 520 nm ws determined. Proline (Sigm) ws used for prodution of stndrd urve. Determintion of elements onentrtion For determintion of N, K, nd C ontent, ovendried smples were weighed nd shed in muffle furne t 550 ºC for 8 h, resolved in HCl, nd mde up to volume y distilled wter. Conentrtions of N, K, nd C were determined y flme photometry (PFP7, Jenwy, UK). Experimentl design nd sttistil nlyses This experiment ws undertken in rndomized 207

4 Vol. 25 No. 3 Summer 2014 R. Hjiolnd, nd L. Cherghvreh J. Si. I. R. Irn lok design with four replitions s four independent pots nd two ftors inluding slinity (S) nd Si pplition (Si). Two-wy ANOVA ws performed using Sigm Stt Differenes etween the mens were deteted using the Tukey test (P<0.05). Results Plnts growth ws inhiited y oth pplied levels of slt. Redution of dry mss y low (25 mm) slt onentrtion ws signifint only for the leves, while growth impirment y higher slt onentrtion (75 mm) ws signifint for ll three plnt frtions (Fig. 1). Si supplementtion improved plnts dry weight not only in slt-ffeted plnts, ut lso in ontrol ones. Dry weight of leves (g plnt - 1 ) A Si +Si d d Control 25 mm 75 mm Dry weight of stem (g plnt -1 ) B Si +Si Control 25 mm 75 mm Dry weight of roots (g plnt - 1 ) C Slinity levels Figure 1. Dry weight of leves (A), stem (B) nd roots (C) in too (Niotin rusti L.) plnts grown for two weeks under different levels of NCl in the sene ( Si) or presene (+Si) of 1 mm N2SiO3 in hydroponi medium. Brs indited y the sme letter re not signifintly different (P<0.05). Control 25 mm 75 mm Si d +Si 208

5 Influene of Si Supplementtion on Growth nd Some Physiologil nd Tle 1. Lef onentrtion of hlorophyll (Chl),, rotenoids (Cr), nthoynins nd flvonoids (mg g -1 FW) in too (Niotin rusti L.) plnts grown for two weeks under different levels of NCl in the sene ( Si) or presene (+Si) of 1 mm N2SiO3 in hydroponi medium. Dt of eh olumn followed y the sme letter re not signifintly different (P<0.05). Tretments Chl Chl Cr Anthoynins Flvonoids Si Control 0.49±0.03 e 0.16± ±0.00 d 7.11± ± mm 0.72±0.04 d 0.18± ±0.00 d 9.21± ± mm 0.55±0.04 e 0.13± ±0.00 d 11.6± ±0.05 +Si Control 1.41± ± ± ± ± mm 1.61± ± ± ± ± mm 2.09± ± ± ± ±0.05 Tle 2. Chlorophyll fluoresene prmeters inluding Fv/Fm (photohemil effiieny of PSII), F v/f m (exittion pture effiieny of open PSII), qp (photohemil quenhing) nd qn (non-photohemil quenhing) in the leves of too (Niotin rusti L.) plnts grown for two weeks under different levels of NCl in the sene ( Si) or presene (+Si) of 1 mm N2SiO3 in hydroponi medium. Dt of eh olumn followed y the sme letter re not signifintly different (P<0.05). Tretments Fv/Fm F v/f m qp qn Si Control 0.81± ± ± ± mm 0.84± ± ± ± mm 0.86± ± ± ±0.04 +Si Control 0.85± ± ± ± mm 0.84± ± ± ± mm 0.85± ± ± ±0.01 Ameliortive effet of Si, however, ws not signifint for roots under low slt onentrtion nd for stem under oth pplied slinity levels (Fig. 1). Lef onentrtion of Chl inresed y low ut not high slt onentrtion in the sene of Si, while inresed y oth slinity levels in Si-treted plnts. Si supplementtion resulted in higher Chl, nd Cr in ontrol s well s in slt-ffeted plnts. Conentrtion of nthoynins inresed y slinity tretments, while Si did not ffet it signifintly. Lef onentrtion of flvonoids ws not influened either y slt or Si ddition (Tle 1). Chl fluoresene prmeters were not influened y slt or Si tretments signifintly (Tle 2). In ontrst, lef gs exhnge prmeters were ffeted y oth pplied tretments (Fig. 2). Net ssimiltion rte (A) remined stle under slt tretments, while trnspirtion rte (E) nd stomtl ondutne (g s ) oth were redued y higher slt onentrtion signifintly (Fig. 2). Supplementtion of plnts with Si used onsistent inrese of ll three gs exhnge prmeters, slightly or signifintly. In the sene of slt net ssimiltion rte ws signifintly higher in Si-treted plnts, while it ws less ffeted y Si in slt-treted ones. In plnts grown under higher slt onentrtion, Si ddition inresed signifintly trnspirtion rte nd stomtl opening of leves (Fig. 2). N onentrtion of ll three plnt frtions inresed ontinuously y inresing slt onentrtion in the medium irrespetive to the Si tretment. Applied Si redued slightly N onentrtion, the signifint effet, however, ws oserved only for stem N onentrtion under high slt onentrtion (Tle 3). In ontrst to N, K onentrtion ws ontinuously deresed y oth pplied slinity levels in ll three plnt frtions without Si ddition. In Si-supplemented plnts, K onentrtion ws higher s ompred with their Si ounterprts irrespetive to the slinity tretment. This effet ws oserved in the leves nd stem more pronounedly thn in the roots (Tle 3). On the other hnd, in Si-supplemented plnts lef nd stem K onentrtion ws inresed with inresing slt onentrtion (Tle 3). C onentrtion deresed in the leves upon slinity tretments in oth Si nd +Si plnts. Si-treted plnts, however, hd signifintly higher C onentrtion ompred with their Si ounterprts irrespetive to slinity tretment. In the stem, slt tretment did not ffet C onentrtion in the Si plnts. Similr with the leves, Si pplition inresed stem C onentrtion. Similr with the leves, C onentrtion delined y slinity tretment in the roots, however, in ontrst to the leves nd stem, Si-treted plnts hd lower C onentrtion in the roots ompred with their Si ounterprts (Tle 3). The rtio of K:N ws gretly deresed y oth slt tretments in ll three plnt frtions, however, omprison of mens using t-test reveled tht this rtio t eh level of slt ws higher in +Si plnts s ompred with Si ounterprts (Tle 3). Conentrtion of solule sugrs in the leves, ut not in the roots, ws stedily inresed y inresing slt 209

6 Vol. 25 No. 3 Summer 2014 R. Hjiolnd, nd L. Cherghvreh J. Si. I. R. Irn Net ssimiltion rte (μmol m -2 s -1 ) A Si +Si Control 25 mm 75 mm Trnspirtion rte (mmol m -2 s -1 ) B Si +Si Control 25 mm 75 mm Stomtl ondutne (mol m -2 s -1 ) C Slinity levels Figure 2. Gs exhnge prmeters inluding net ssimiltion rte (A), trnspirtion rte (B) nd stomtl ondutne (C) in the leves of too (Niotin rusti L.) plnts grown for two weeks under different levels of NCl in the sene ( Si) or presene (+Si) of 1 mm N2SiO3 in hydroponi medium. Brs indited y the sme letter re not signifintly different (P<0.05). Si Control 25 mm 75 mm +Si onentrtion in the medium (Fig. 3). Si tretment inresed further lef solule sugr onentrtions, so tht the highest level ws found in the leves of plnts treted with 75 mm slt nd supplemented with Si. In the roots, in ontrst, redution of solule sugrs ws oserved in plnts under higher slt onentrtion nd Si did not ffet it (Fig. 3). Conentrtion of free proline inresed y slt eing signifint t high slinity level in the leves nd roots. Si tretment, however, rther deresed free proline onentrtion in the leves nd roots of slt-ffeted plnts. In the leves of ontrol plnts, in ontrst, Si 210

7 Influene of Si Supplementtion on Growth nd Some Physiologil nd Tle 3. Conentrtion of N, K nd C (mg g -1 DW) in the leves, stem nd roots of too (Niotin rusti L.) plnts grown for two weeks under different levels of NCl in the sene ( Si) or presene (+Si) of 1 mm N2SiO3 in hydroponi medium. Dt of N, K nd C onentrtions within eh olumn followed y the sme letter re not signifintly different (P<0.05). Dt of K:N rtio were ompred etween Si nd +Si plnts using t-test (P<0.05). Tretments Leves Stem Roots N onentrtion (mg g -1 DW) Si Control 2±0.4 9±0.9 9± mm 23±5 24±5 16±2 75 mm 46±13 54±9 32±6 +Si Control 1.4±0.5 10±3.7 d 7± mm 17±2 21±5 16±7 75 mm 40±12 34±8 30±2 K onentrtion (mg g -1 DW) Si Control 43±2 d 57±2 51±1 25 mm 34±4 e 54±3 d 42±1 d 75 mm 28±2 f 49±2 d 41±2 d +Si Control 52±3 66±2 57±3 25 mm 61±1 80±6 52±2 75 mm 72±2 78±1 46±3 C onentrtion (mg g -1 DW) Si Control 69±3 17±2 16±2 25 mm 31±2 18±1 18±3 75 mm 32±6 17±6 10±1 +Si Control 102±9 29±1 11±1 25 mm 63±12 40±6 13±3 75 mm 62±3 29±4 11±2 K:N Si Control 19± ± ± mm 1.5± ± ± mm 0.6± ± ±0.2 +Si Control 45±14 7.1± ± mm 3.2± ± ± mm 1.9± ± ±0.2 tretment inresed free proline onentrtion (Fig. 3B). Results of two-wy ANOVA reveled tht the mjority of prmeters determined in this work were influened y oth slt nd Si tretments with the exeption of Chl fluoresene prmeters. The lk of intertion etween these two ftors on plnt growth prmeters emphsized tht effet of Si ws not limited to the stress onditions (Tle 4). Disussion Plnts growth under different slt onentrtions nd Si supplementtion Too is slt-sensitive glyophyte speies. Slt tolerne of rop plnts is typilly expressed s redution of yield ssoited with given level of soil slinity i.e. threshold slinity, s ompred with yield under non-sline onditions [41]. Aording to this method, rop speies re lssified into slt-sensitive, modertely-sensitive, modertely-tolernt nd tolernt speies [41]. The yield of sensitive rop speies strts to deline t soil eletri ondutivity (EC) of 3 dsm -1 (30 mm NCl) ompred with3-6 dsm -1 for modertelysensitive, 6-8 dsm -1 for modertely-tolernt nd >8 dsm -1 in tolernt speies [41]. In this work, redution of growth ws oserved for leves t slinity level of 25 mm. Under suh low slinity ondition, dry mtter of leves ws depressed signifintly up to 30% while stem nd roots did not respond to this slinity level. It indited tht leves were the most suseptile plnt prt in too nd even low slt onentrtions my depress the yield of leves nd thus, prodution of this non-food forge rop. Higher sensitivity of leves ws likely resulted from redution of lef expnsion under lower wter potentils. Positive turgor is neessry for expnsion growth of ells, thus, plnts respond immeditely to osmoti stress s redution of the rte of lef expnsion [35]. In greement with this, lef re nd lef expnsion rte ws the most sensitive prmeter to slt in too. From the two omponents of slt stress, i.e. hyperosmoti effet nd disturne of ioni equilirium, the former is the min reson for growth redution under lower slt onentrtions [16]. High slt onentrtion, in ontrst, impired growth 211

8 Vol. 25 No. 3 Summer 2014 R. Hjiolnd, nd L. Cherghvreh J. Si. I. R. Irn Solule sugrs (mg g -1 FW) A d Free proline (nmol g -1 FW) B d Control 75 mm d 25 mm Figure 3. Conentrtion of totl solule sugrs (A) nd free proline (B) in the leves (ove of the horizontl xis) nd roots (elow of the horizontl xis) in too (Niotin rusti L.) plnts grown for two weeks under different levels of NCl in the sene ( Si, open r) or presene (+Si, drk r) of 1 mm N2SiO3 in hydroponi medium. Brs within eh plnt orgn indited y the sme letter re not signifintly different (P<0.05). of ll three plnt prts nd deresed dry weight of leves, stem nd roots up to 58%, 82% nd 62%,respetively, inditing gin higher suseptiility of this rop speies to slt. Depression of plnt growth under higher slt onentrtions is relted to toxi effets of N ions nd ioni disequilirium ut the threshold tissue N onentrtion is dependent on plnt speies [16]. In this work N onentrtion of out mg g -1 DW ws pprently toxi for too s ould e judged y signifint growth redution of plnts. Slt onentrtions higher thn 75 mm used deth of plnts in this work. Silion supplementtion improved plnts growth signifintly regrding iomss of ll three frtions not only under slinity ut lso under ontrol onditions. Too is not lssified s Si umultor speies [4] nd its positive response to Si supplementtion under optimum growth onditions hs not een reported so fr. Under sline onditions, in ontrst, enefiil effet of Si on plnts growth oserved in this work ws in greement with the results otined for other nonumultor speies suh s tomto [39]. The possile involving mehnisms for Si effet under non-stress onditions s well s Si-medited llevition of slt stress in too will e disussed lter in this setion. Lef pigment onentrtions, photohemistry nd gs exhnge s ffeted y slt nd Si supplementtion Lef Chl onentrtion ws higher under low slt onentrtion tht my e ttriutle to the onentrtion effet due to the higher redution of lef re ompred with less inhiition of Chl synthesis. Under higher slt onentrtion, however, redution of Chl onentrtion surpssed pprently tht of lef growth nd thus, its redution ws ovious. Two possile mehnisms re involved in the effet of slt 212

9 Influene of Si Supplementtion on Growth nd Some Physiologil nd Tle 4. Results of two-wy ANOVA test (men of squres) for the effet of different tretments inluding slinity (S) nd silion (Si) nd their intertions (S Si) on vrious physiologil prmeters in too plnts. *** P<0.001, ** P<0.01, * P<0.05, ns: non-signifint, ording to Tukey test. Prmeters S Si S Si Prmeters S Si S Si Lef DW *** 1.21 *** ns gs * * ns Stem DW *** * ns Lef N 3448 *** 113 * ns Root DW *** *** ns Stem N 2405 *** 303 ** 248 ** Chl *** 7.09 *** *** Root N 1079 *** 1.84 ns 8.98 ns Chl * *** *** Lef K * 4314 *** 555 *** Crotenoids * *** * Stem K * 2312 *** ** Anthoynins *** 9.70 ns 1.68 ns Root K *** *** ns Flvonoids 0.00 ns ns ns Lef C 3724 *** 6789 *** ns Fv/Fm 0.00 ns 0.00 ns 0.00 ns Stem C 90.1 ** 1377 *** * F'v/F'm 0.00 ns 0.00 ns 0.00 ns Root C *** * * qp 0.00 ns 0.00 ns 0.00 ns Lef sugrs *** *** ns qn 0.00 ns 0.00 ns 0.00 ns Root sugrs *** ** ns A 2.65 ** *** * Lef proline * 3074 *** 2644 *** E *** ** ns Root proline 444 *** 1752 *** 275 *** on redution of lef Chl onentrtion inlude enhnement of degrdtion s the onsequene of oxidtive dmge [33] nd redution of nitrogen uptke [30] s the most importnt lef minerl omponent for Chl synthesis [17] in slt-ffeted plnts. Lef nthoynins onentrtion, in ontrst, ws higher under oth slinity levels implied likely higher synthesis in slt-ffeted plnts. Anthoynins re importnt plyers in the ntioxidtive defense of leves under stress onditions [5]. Under slt stress, elevted levels of lef nthoynins hve een reported in plnts speies suh rie [7] nd tomto [3]. Si-supplemented plnts hd higher Chl, nd Cr. Higher pigments onentrtion of Si-supplied plnts hs een lso reported y other uthors [45]. This my e the result of lower destrution of pigments s the onsequene of higher protetion of leves ginst retive oxygen speies generted under slt stress. It hs een oserved tht tivity of ntioxidtive enzymes nd onentrtion of relted metolites inresed in Sitreted plnts nd memrne dmge is mitigted onsiderly [21, 27]. Enhnement of lef Cr upon Si tretment my e of gret importne to slt tolerne of plnts. Under stresses suh s drought nd slinity the imlne etween photosyntheti light pture nd NADPH utiliztion in ron fixtion my led to exess exittion energy nd dmge to photosyntheti pprtus [16]. Het dissiption of exess light energy medited y Cr is n effetive mehnism for quenhing exess photons nd proteting leves from dmging effets of exess exittion energy [34].Unexpetedly, lef Chl fluoresene prmeters were not influened y slt or Si. It my suggest tht hnges of these prmeters were under detetion limit in this work nd/or these prmeters were not the ritil omponents of lef photosynthesis in too plnts under slinity. In ontrst, lef gs exhnge prmeters were pprently the mjor ftors in plnts response to oth slt nd Si pplition. Slt t higher onentrtion depressed stomtl opening tht used in turn lower trnspirtion rte. Slinity ffets stomtl opening immeditely, firstly euse of perturtion in the wter reltions nd fterwrd due to the synthesis of ABA [35]. In onsequene, slinity my restrit net ssimiltion rte primrily euse of redution of CO 2 supply due to prtil stomtl losure. In too, however, redution of stomtl ondutne ws not pprently effetive in depression of CO 2 fixtion rte, likely euse of suffiient internl CO 2 onentrtion. The rtio of C i /C n (rtio of internl CO 2 to referene CO 2 ) ws hnged slightly from 0.92±0.03 in ontrol to 0.87±0.01 in plnts treted with 75 mm slt (dt not shown). Nevertheless, regrding gret depression of lef re in slt-treted too plnts, signifint deline of net CO 2 fixtion is expeted on whole-plnt sis. Lef stomtl ondutne ws higher in Si-treted 213

10 Vol. 25 No. 3 Summer 2014 R. Hjiolnd, nd L. Cherghvreh J. Si. I. R. Irn too plnts similr with the reports on nonumultor speies suh s tomto [39]. This effet my e the result of n improvement in plnts wter sttus (see elow) nd/or enhnement of the tivity of proton pumping nd K + urrents in the gurd ells. Higher plsm memrne H + ATPse (25) nd tonoplst V-ATPse nd V-PPise tivities [24] hs een reported in rley plnts under Si tretment. Following elevtion of stomtl ondutne, trnspirtion rte inresed in Si-treted plnts eing signifint under 75 mm slt. Contrstive response to dded Si ws reported in Si-umultor speies likely euse of Si deposition in the lef epidermis tht redues utiulr trnspirtion nd wter loss [31]. Lef Si onentrtion in too hs een reported to e in the rnge of μg g -1 DW [47], muh less thn the folir onentrtions found in umultor speies (20-40 mg g -1 DW) with epiderml Si depositions [4]. Similr with too, higher wter loss hs een reported upon Si tretment in tomto plnts [39]. However, in tomto simultneously higher photosynthesis rte resulted in higher wter use effiieny (the rtio of photosynthesis:trnspirtion) in Si-supplemented plnts [39]. Here wter use effiieny inresed y slt (75 mm) from 4.86±0.99 to 7.57±0.49 in Si plnts while redued from 6.19±0.21 to 5.63±0.51 in +Si ones (dt not shown). Our dt indited tht effet of Si on the llevition of slt stress in too under high slt onentrtion ws not mehnistilly relted either to redution of wter loss or to elevtion of wter use effiieny. It my imply lso tht Si-treted plnts hd higher ility for wter uptke likely euse of greter root surfe re. A signifint inrese of too root iomss y Si pplition (Fig. 1C) my onfirm this suggestion. Moreover, n improvement in plnts wter uptke my enle them to hve more open stomt nd thus, to reh higher photosynthesis rte. In greement with this, under ontrol nd low slinity levels, Si effet on the stomtl ondutne ws well orrelted (r=0.85, P<0.05) with tht of CO 2 fixtion rte, suggesting the fundmentl role of stomt-relted mehnisms in the enhnement of photosynthesis in Si-supplemented plnts. It ould e speulted tht, higher slt tolerne in Si-supplemented too plnts ws losely linked to Si effet on stomtl opening, thus, mintining CO 2 fixtion under slt nd providing plnts with rohydrtes for osmoti djustments nd dry mtter prodution. Under high slt onentrtion, however, n out 66% inrese in stomtl opening ws not ssoited with orrespondingly higher net CO 2 fixtion inditing likely involvement of limiting ftors, i.e. metoli impirment. Biohemil limittion of photosynthesis is likely in turn, resulted from N toxiity nd ioni imlne [6, 38] s the ommon response of sltsensitive glyophytes to higher slt onentrtions [16]. Expression of numerous genes nd tivity of severl enzymes relted with photosynthesis re depressed under these onditions [6]. Ion ytotoxiity is used y replement of K + y N + in iohemil retions nd onformtionl hnges nd loss of funtion of proteins [6]. Although meliortive effet of Si on too lef photosyntheti tivity ws not oserved under high slt onentrtion nd plnts hd higher wter loss under these onditions, dry weight of shoot orgns nd roots inresed y Si slightly or signifintly. It implies funtion of dditionl mehnisms for growth meliortion y Si under high slinity level (see elow). Orgni osmolytes nd ion reltions s ffeted y slt nd Si supplementtion In the leves, higher solule rohydrtes nd free proline onentrtions were oserved in slt-ffeted plnts. In ontrst to the leves, root solule rohydrtes onentrtion ws not higher in slttreted plnts while proline ws eqully inresed in the leves nd roots y slinity. It indited different importne of these orgni osmolytes for the osmoti djustment of the leves nd roots. In plnts sujeted to slt, in order to ommodte the ioni lne in the vuoles, ytoplsm umultes low-moleulr-weight ompounds, i.e. omptile solutes euse they do not interfere with norml iohemil retions [36]. Aumultion of orgni osmosolutes protets strutures nd osmoti lne nd supports ontinued wter influx [16, 35]. It hs een suggested tht the mjority of ronnd nitrogen ssimilted under slt onditions re lloted to the osmoti purposes [16]. Si ddition resulted in further inrese of solule rohydrte in the leves. This effet in the roots ws signifint only under high slt onentrtion. Higher net ssimiltion rte oserved in Si-treted plnts my provide exess redued ron soures for synthesis of solule sugrs. Solule rohydrtes suh s gluose, frutose, surose, frutns umulte under slt stress nd ply leding role in osmo-protetion, osmoti djustment, ron storge, nd free rdil svenging [16]. Sugrs ontriute up to 50% of the totl osmoti potentil in glyophytes sujeted to sline onditions [1]. In ontrst to rohydrtes, proline onentrtion rther lowered y Si pplition in the leves nd roots of slt-ffeted plnts. Lower proline ontent in Sitreted plnts implied likely tht they were less ffeted y osmoti stress nd thus, needed lower proline 214

11 Influene of Si Supplementtion on Growth nd Some Physiologil nd onentrtions ompred with Si ounterprts. It hs een proposed tht umultion of proline in plnts grown under slt stress ws due to slt injury nd not s n indition of slt tolerne [28]. In two sorghum genotypes ontrsting in slt tolerne proline umultion ws retion to slt stress nd not plnt response ssoited with tolerne [8]. Higher onentrtion of proline ws umulted in sensitive rie ultivrs thn in tolernt genotypes under slt stress [28]. In view of these reports, the role of proline in slt tolerne nd its use s seletion riterion in rop speies hs een questioned [1].In too, regrding onstitutively low proline onentrtion, i.e. in the nnomolr rnge, its role s n orgni osmotium does not seem to e ritil omponent of plnt response to dded Si. Similr with our results, redution of proline onentrtion in slt-stressed plnts fter tretment with Si hs een reported for whet [45] nd grpevine [43]. In ontrst, funtion of proline s free rdils svenger ws onsidered its min physiologil role under slt stress in some speies [30, 44]. Here, ontrol plnts without slt hd higher proline onentrtion in the leves tht my ontriute to the enhnement of plnt defense pity ginst free rdils. Benefiil elements re effetive in the improvement of plnts growth even under non-stress onditions euse of tivtion of ntioxidtive defense tht ould mitigte effet of ltent stress ftors [4, 15]. Conentrtion of N ws diminished only slightly y Si tretment. In ontrst, K onentrtion ws signifintly higher in Si-supplemented plnts s ompred with Si ounterprts. In Si-treted plnts, onentrtion of K in the stem nd prtiulrly in the leves ws rther inresed with inresing slt onentrtion (Tle 3). It is oviously onentrtion effet s the onsequene of lower iomss in slttreted plnts in the sene of redution of K ontent. Si supplementtion resulted in signifint inrese of K:N rtio, even though it remined muh less thn ontrol plnts. Beuse of toxi effets of N on ell metolism, the mintenne of K nd N homeostsis is importnt for the tivities of mny ytosoli enzymes nd for mintining memrne integrity [20]. The K:N rtio is n importnt riterion for slt tolerne of plnts nd mintenne of photosynthesis nd other metoli proesses is minly dependent on the higher K:N rtio in the slt-ffeted plnts [36]. Higher K uptke upon Si tretment my e the result of n improved memrne integrity nd seletivity proess in Si-supplemented plnts. Higher defense pity ginst retive oxygen speies in Si-treted plnts tht hve een reported in some plnt speies [21] is lso highly prole in our experimentl plnts. In ontrst to our results, differenes were not oserved in folir ontent of K nd C or K:N rtio in slinized tomto plnts treted with Si [39]. In whet s Si-umultor speies, however, redution of N in the leves nd roots nd inrese of K ws oserved y Si [45]. The sme effet of Si ws found on C onentrtion in the leves nd stem ut not in the roots. Higher C onentrtion in the shoot orgns of Si-treted plnts my e in turn nother mehnism for improved memrne integrity nd higher K:N seletivity [16]. Elevted trnspirtion rte my e responsile for higher shoot C onentrtion in Si-treted plnts regrding the well-known dependeny of C trnsport on trnspirtion rte [17]. This ould e onfirmed y orrespondingly lower root C onentrtion in Sitreted plnts ompred with their Si ounterprts. The ltter indited lso tht only root-shoot C trnsport ut not root uptke ws ffeted y Si. Conlusion Si-medited llevition of slt stress effets in too plnts ws relted to the higher photosynthesis rte, n improved osmoti djustment euse of higher onentrtion of orgni osmolytes nd higher K, C nd K:N onentrtion rtio in Si-supplemented plnts. Similr mehnisms were involved in the effet of Si on plnts growth under non-stress onditions. Elevted levels of CO 2 fixtion rte tht my support plnts dry mtter prodution, higher uptke of K nd C nd likely other nutrients nd higher proline synthesis s n ntioxidtive ompound, were mehnisms for growth enhnement in Si-treted plnts in the sene of slt. Referenes 1. Ashrf M., nd Hrris P.J.C. Potentil iohemil inditors of slinity tolerne in plnts. Plnt Si., 166: 3 16 (2004). 2. Btes L.S., Wldren R.P., nd Tere I.D. Rpid determintion of free proline for wter-stress studies. Plnt Soil, 39: (1973). 3. Borghesi E., Gonzlez-Miret M.L., Esudero-Gilete M.L., Mlorgio F., Heredi F.J., nd Meléndez-Mrtínez A.J. Effets of slinity stress on rotenoids, nthoynins, nd olor of diverse tomto genotypes. J. Agri. Food Chem., Doi: /jf (2014). 4. Brodley M., Brown P., Ckmk I., M J.F., Rengel Z., nd Zho F. Benefiil elements. In: Mrshner P. (Ed.), Mrshner's Minerl Nutrition of Higher Plnts, 3rd Ed., Elsevier, Oxford, U.K. pp (2012). 5. Chlker-Sott L. Environmentl signifine of nthoynins in plnt stress responses. Photohem. Photoiol., 70: 1 9 (1999). 6. Chves M.M., Flexs J., nd Pinheiro C. Photosynthesis under drought nd slt stress: regultion mehnisms from 215

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