Ecotoxicology and Environmental Safety 113 (2015) 133–144

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Ecotoxicology and Environmental Safety

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journal homepage: www.elsevier.com/locate/ecoenv

Silicon-mediated alleviation of Cr(VI) toxicity in wheat seedlings asevidenced by chlorophyll florescence, laser induced breakdownspectroscopy and anatomical changes

Durgesh Kumar Tripathi a, Vijay Pratap Singh b, Sheo Mohan Prasad c,Devendra Kumar Chauhan d,n, Nawal Kishore Dubey a, Awadhesh Kumar Rai e,n

a Center of Advance Studies, Department of Botany, Banaras Hindu University, Varanasi 221005, Indiab Govt. RamanujPratap Singhdev Post Graduate College, Baikunthpur, Korea-497335, Chhattisgarh, Indiac Ranjan Plant Physiology and Biochemistry Laboratory, Department of Botany, University of Allahabad, Allahabad 211002, Indiad D.D. Pant Interdisciplinary Research Laboratory, Department of Botany, University of Allahabad, Allahabad 211002, Indiae Laser Spectroscopy Research Lab, Department of Physics, University of Allahabad, Allahabad, India

a r t i c l e i n f o

Article history:Received 2 July 2014Received in revised form24 September 2014Accepted 25 September 2014

Keywords:AnatomyChlorophyll fluorescenceChromium toxicityICAP-AESLIBSSilicon

x.doi.org/10.1016/j.ecoenv.2014.09.02913/& 2014 Elsevier Inc. All rights reserved.

viations: F0, minimal fluorescence; Fm, maxleaves; Fv (Fm�F0), variable fluorescence in dfluorescence/maximum fluorescence ratio inm/minimal fluorescence ratio in dark adaptel quenching; qP,, photochemical quenching; IClasma-atomic emission spectroscopy; LIBS,, lacopyesponding author.ail addresses: dktripathiau@gmail.com (D.K. Ttap.au@gmail.com (V.P. Singh),rasad@gmail.com (S.M. Prasad),anau@yahoo.com (D.K. Chauhan),ybhu@gmail.com (N. Kishore Dubey),shkrai@rediffmail.com (A.K. Rai).

a b s t r a c t

Silicon (Si)-mediated alleviation of Cr(VI) toxicity was examined in wheat seedlings using an in vivoapproach that involves chlorophyll fluorescence, laser induced breakdown spectroscopy (LIBS) andanatomical changes. Exposure to Cr(VI) significantly reduced the growth and photosynthetic activities(chlorophyll fluorescence) in wheat which was accompanied by remarkable accumulation of this elementin tissues. However, addition of Si to the growth medium alleviated the effects of Cr(VI). The LIBS spectrawere used as a fingerprint of the elemental compositions in wheat seedlings, which showed a reductionin Cr accumulation following Si addition. Nutrient element levels (Ca, Mg, K and Na) declined in wheatfollowing the addition of Cr (VI), as recorded by LIBS and inductively coupled plasma atomic emissionspectroscopy (ICAP-AES). However, addition of Si along with Cr(VI) increased the contents of nutrientelements in wheat. LIBS, ICAP-AES and AAS showed a similar distribution pattern of elements measuredin wheat. Anatomical observations of leaf and root revealed that Cr(VI) affected internal structures whileSi played a role in protection from toxic effects. The results showed the suitability of chlorophyll fluor-escence as a parameter and appropriateness of LIBS technique and anatomical procedures to elucidate Si-mediated alleviation of Cr(VI) toxicity. Furthermore, our results suggest that the measured parametersand techniques can be used non-invasively for monitoring the growth of crops under different en-vironmental conditions

& 2014 Elsevier Inc. All rights reserved.

imum fluorescence in darkark adapted leaves; Fv/Fm,dark adapted leaves; Fm/F0,d leaves; NPQ,, non-photo-AP-AES,, inductively coupledser induced breakdown

ripathi),

1. Introduction

In the present time, scientific research is advancing towardsmultidisciplinary approaches and opulently achieving prodigiousmomentum day by day. Different conventional spectroscopictechniques such as colorimetry, spectrometry, high temperaturealkaline dissolution and electro-thermal vaporization togetherwith inductively coupled plasma atomic emission spectroscopy(ICP-AES), ion mobility spectrometry (IMS) and graphite furnaceatomic absorption spectrometry (GFAAS) are being used to gatherinformation related to the elemental distribution in plants undervaried environmental conditions. However, these methods seemto be tedious. For instance, sample analysis requires sample de-struction, expensive chemicals for sample preparation beside thisit also involves a great deal of time, and hence regarded as non-ecofriendly (Novozamsky et al., 1984; Elliott and Snyder, 1991;Saito et al., 2005; Haysom et al., 2006; Masson et al., 2007).

D.K. Tripathi et al. / Ecotoxicology and Environmental Safety 113 (2015) 133–144134

Laser-induced breakdown spectroscopy (LIBS) is a versatilespectroscopic technique which has capability to detect multipleelements in plants even present in the trace amount within frac-tion of second. This technique is based on optical emission fol-lowed by pulsed laser ablation of a sample (Bulatov et al., 1998;Pasquini et al., 2007). In recent years, LIBS is being used as aprominent tool for qualitative and quantitative analysis of variouselements in different plant materials (Kaiser et al., 2009; Chauhanet al., 2011; Tripathi et al., 2011; Tripathi et al., 2012a,b). Thus, LIBSmay also be applied to acquire information of spatially distributedelements in plants which is practically not possible with any otherconventional method of elemental analysis.

Chlorophyll fluorescence is one of the most rapid and non-in-vasive tools for monitoring photosynthetic performance of plantsunder biotic and abiotic stress (Lu and Zhang, 1999; Lu et al.,2001). Thus, it is considered as one of the efficient, authoritativeand extensively used techniques for plant physiologists and eco-physiologists (Biehler and Fock, 1995;Govindjee, 1995; Maxwelland Johnson, 2000; Barbagallo et al., 2003; Zhang et al., 2010).Theplant response to changing environmental condition or any kind ofstress may be studied at physiological, morphological, cellular,biochemical, anatomical or even at molecular level (Bohnert et al.,1995; Sairam and Tyagi, 2004; De Micco and Aronne, 2012; Ha-sanuzzaman et al., 2013). Though anatomical studies have beengiven much importance to correlate functional attributes of theplants from earlier time, however, less attention has been given tointerpret the anatomical adaptation with regards to changing en-vironmental conditions. Under stress condition, plants showplasticity which reflects in terms of morphological and anatomicaladaptations to contribute stress tolerance (Deng et al., 2009; Tri-pathi et al., 2012a,b; Vaculík et al., 2012).

Over past few decades, heavy metal contamination in waterand soil has become a major problem of serious global concern.Among heavy metals, chromium (Cr) is regarded as extremelytoxic element even at lower concentrations. It gets released intothe environment via imprudent effluents discharge mostly fromthe industries related to metallurgy, production of paints andpigments, electroplating, tanning and wood preservation. Amongits various forms, Cr(VI) is known to be the most toxic to livingorganism because of its ability to pass through the membranes,penetrate into the cytoplasm and react with the intracellularmaterials (Shanker et al., 2004; Singh et al., 2013a,b). Cr(VI) is alsoknown to induce cutaneous allergy, which becomes carcinogenicon its prolonged exposures (Yokel et al., 2006). Being highly toxicCr(VI) soon gets incorporated in to the food chain thereby posingsubstantial risk to the human health throughout. Cr(VI) affectsseveral physiological processes of plants such as seed germination,growth, photosynthesis, status of mineral elements, water balance,and nitrogen metabolism (Gangwar and Singh, 2011; Singh et al.,2013a,b). Thus, safer food production in changing environmentalcondition is the subject of major concern. Therefore, methods areneeded to alleviate Cr(VI) toxicity in plants to raise the crop pro-ductivity as well as to decrease the Cr content in crops which maybe helpful in minimizing health risks. In this regards, silicon (Si)application could be one of the suitable ways to overcome heavymetal toxicity problems.

Silicon (Si) is the second most abundant element in soils, and isavailable to plant in the form of silicic acid (Epstein, 1999). Al-though Si has not yet been considered as an essential element forplants, however, many studies have demonstrated the beneficialrole of Si in enhancing the plant tolerance against various abioticand biotic stresses (Lux et al., 2002; Nwugo and Huerta, 2008;Tripathi et al., 2012a; Ali et al., 2013; Farooq et al., 2013; Mitani-Ueno et al., 2014; Sanglard et al., 2014). Therefore, Si can be re-garded as an extremely efficient element for boosting growth anddevelopment in higher plants under stress conditions.

Whilst the impacts of Cr(VI) on plants and crops are welldocumented, however, study related to Si and Cr(VI) interaction inplants still needs more concern. In recent years, Si alleviative effecthas been demonstrated by various in vitro methods, but in vivoobservation of Si-alleviation of heavy metal toxicity has not yetbeen investigated using multidisciplinary approaches. Therefore,in the present study the alleviative effect of Si on Cr(VI) toxicity inwheat seedlings has been investigated directly by correlating thedata obtained from chlorophyll florescence, LIBS and anatomicalstudies. Furthermore, the data (Cr, Si and mineral elements) ob-tained from LIBS were authenticated by the results of AAS andICAP-AES.

2. Material and methods

2.1. Plant material and growth conditions

Wheat (Triticum aestivum L.) seeds were purchased from thecertified supplier of Allahabad district, India. Before the use, uni-form sized seeds were surface sterilized with 10% (v/v) sodiumhypochlorite solution for 10 min, washed thoroughly with distilledwater and soaked for 4 h. After sterilization and soaking, healthylooking uniform sized seeds were kept in Petri plates (150 mm,Riviera TM) lined with Whatman No.1 filter papers moistenedwith half-strength Hoagland's solution (Arditti and Dunn, 1969).After this, seeds were allowed to germinate in darkness at2872 °C for 4 days. Thereafter, seedlings were grown under aphoton flux density (PFD) of 350 mmol photons m�2 s�1 and re-lative humidity of 50–60% with a day/night cycle of 12/12 h at2872 °C for 8 days in a growth chamber. After this, uniform sizedseedlings were selected and transferred in half-strength Hoa-gland's solution to acclimatize them for 7 days. After acclimati-zation, Si (Na2SiO3; 10 mM) and Cr(VI) (K2Cr2O7; 100 mM) treat-ments were given to the seedlings. The selection of 10 mM of Si wasbased on dose response curve because this dose stimulates max-imum growth in wheat seedlings. The selection of 100 mM of Cr(VI)was based on the result that showed significant reduction ingrowth of wheat seedlings. Furthermore, the selected dose of Cr(VI) is environmentally relevant and found at Cr-contaminatedsites. Silicon and Cr(VI) treatments alone as well as in combinationwere given for 7 days. Silicon and Cr(VI) concentrations wereprepared in half-strength Hoagland's solution. During 7 days of Siand Cr(VI) treatments, the respective solution was changed twiceand aerated daily to avoid root anoxia. In the present study, fol-lowing combinations of Si and Cr(VI) were made: only half-strength Hoagland's solution (control), 10 mM Si, 100 mM Cr(VI) and10 mM Siþ100 mM Cr(VI). After 7 days of Si and Cr(VI) treatments,samples from each set were harvested and different parameterswere analyzed in root and shoot.

2.2. Estimation of growth and photosynthetic pigments

For the measurement of growth, seedlings from control andtreated samples were harvested randomly, divided into root andshoot and then their fresh weight and length were determined.For root and shoot dry weight measurement, the samples werewrapped in butter paper and oven dried for 48 h at 65–75 °C thenweighed. Leaf area of treated and untreated seedlings was mea-sured by leaf area meter (Model 211, Systronics, India). For themeasurement of total chlorophyll, fresh leaves (25 mg) from con-trol and treated seedlings were extracted in 80% (v/v) acetone. Theamount of total chlorophyll was calculated by using the method ofLichtenthaler and Welburn (1983).

D.K. Tripathi et al. / Ecotoxicology and Environmental Safety 113 (2015) 133–144 135

2.3. Chlorophyll fluorescence measurements

For the assessment of photosynthetic performance, chlorophyllfluorescence measurements were taken in dark adapted leaves ofcontrol and treated seedlings using hand held leaf fluorometer(FluorPen FP 100, Photon System Instruments, Czech Republic).The following fluorescence parameters: minimum fluorescence(F0), maximum fluorescence (Fm), variable fluorescence (Fv), vari-able/maximum fluorescence ratio (Fv/Fm), variable/minimumfluorescence ratio (Fv/F0) and maximum/variable fluorescence ra-tio (Fm/Fv) were assessed. Measurements were taken in second leafof three different plants of each treatment.

2.4. In vivo analysis of the wheat plant by LIBS

The in vivo LIBS spectra of different parts of the wheat plantwere recorded using LIBS (Fig. S1D). The second harmonic(532 nm) of Nd:YAG laser (Continuum, Surelite III-10, USA) wasfocused on the surface of different parts (root and shoot) of thewheat plant using a 15 cm converging lens. Emission from theplasma was collected using a collecting lens and was finally fed tothe spectrometer through a fiber bundle. The spectrometer (OceanOptics, LIBS 2000þ , USA), equipped with charged coupled device(CCD), is a four channel spectrometer in which three channels arehaving high resolution (FWHM is 0.1 nm; from 200 nm to 500 nm)and fourth channel having low resolution (FWHM is 0.75 nm; from200 nm to 900 nm). LIBS spectrum of each part of the plant wasrecorded under optimized experimental condition i.e. at 10 mJlaser energy, repetition rate 4 Hz and pulse width 4 ns. The col-lection lens was set to get maximum emission signal from theplasma plume. Three seedlings were taken for the LIBS study andaverage of 10 spot on each part i.e. root and shoot of a wheatseedling was taken to produce the data.

2.5. Estimation of Cr and nutrients content by AAS and ICAP-AES

For the determination of mineral elements, Si and Cr, dried rootand shoot samples (50 mg) from control and treated seedlingswere digested in mixed acid (HNO3:HClO4; 85:15, v/v) untiltransparent solution was obtained. The volume of digested samplewas maintained up to 30 ml with double distilled water. Thecontent of mineral elements in digested samples was determinedby using inductively coupled argon plasma-atomic emissionspectrometry (ICAP-AES) while Cr and Si accumulation in rootsand shoots of wheat seedlings was estimated by an atomic ab-sorption spectrometer (AAS).

2.6. Anatomy of root and leaf

Hand sections of leaf blades and roots of treated and untreatedwheat seedlings were made and stained with 50% safranin andthen mounted in glycerin. Images were taken by using a digitalcamera linked to an optical microscopy using the software image

Table 1Effect of exogenous Si addition on root and shoot fresh and dry weight (mg seedling�1)fresh weight) of wheat seedlings exposed to Cr (VI) toxicity. Data are means7standard(n¼9). Values with different letters within same column show significant differences a

Treatments Fresh weight Dry weight

Shoot Root Shoot Root

Control 495711.4b 21076.7b 74.371.8b 31.571.0Si 543717.4a 27078.1a 86.973.1a 43.271.6Cr (VI) 31077.7d 12574.0d 40.371.1d 16.370.4SiþCr (VI) 355711.0c 17574.6c 53.371.6c 28.071.1

tool. Leaf blades and roots sections were analyzed for mesophyllcells, xylem and phloem by using a digital camera coupled with anoptical microscopy on 40� .

2.7. Statistical analysis

Results were statistically analyzed by analysis of variance(ANOVA). Duncan's multiple range test was applied for mean se-paration for significant differences among treatments at Po0.05significance level. The results presented are the means7standarderror of three independent experiments with three replicates ineach experiment (n¼9) to check the reproducibility of the results.

3. Results

3.1. Effect of Si on growth and chlorophyll under Cr(VI) stress

Results related to growth (fresh weight, dry weight, length ofshoots and roots, and leaf area) of treated and untreated seedlingsare shown in Table1. Treatment of the wheat seedlings with Cr(VI)resulted into significant (Po0.05) decline in growth of root andshoot as fresh weight reduced by 40% and 37%, dry weight by 48%and 46%, and length by 40% and 40%, respectively (Table 1, Fig.S1A). Furthermore, Cr(VI) decreased leaf area by 47% compared tothe control. Treatment with Si, however, caused significant(Po0.05) enhancement in growth parameters of untreated controlseedlings. Besides this, addition of Si together with Cr(VI) ex-hibited significant (Po0.05) alleviation in Cr(VI)-induced reduc-tions in growth parameters (Table 1, Fig. S1A) as seedlings showeda reduction of 17% and 28% in fresh weight, 11% and 28% in dryweight, and 27% and 32% in length of root and shoot, respectivelycompared to the respective control (Table 1).

Results showed that due to the toxicity of Cr(VI), total chlor-ophyll was decreased by 19% compared to the control. Addition ofSi increased chlorophyll content by 7% over the value of control.When the seedlings were treated with Cr(VI) together with Sishowed significant improvement in chlorophyll contents (Table 1).

3.2. Chlorophyll fluorescence

Results pertaining to chlorophyll fluorescence are depicted inFig. 1. Cr(VI) significantly (Po0.05) declined Fv, Fm, Fv/F0, Fv/Fm,Fm/F0 and qP by 47%, 34%, 55%, 13%, 48% and 54%, respectivelycompared to the control (Fig. 1B–G). However, the Si additionsignificantly (Po0.05) alleviated Cr(VI)-mediated decline in Fv, Fm,Fv/F0, Fv/Fm, Fm/F0 and qP by 13%, 7%, 23%, 6%, 18%, and 16%, re-spectively compared to the Cr(VI) treatment alone (Fig. 1). Incontrast to Fv, Fm, Fv/F0, Fv/Fm, Fm/F0 and qP, F0 and NPQ wereenhanced by Cr(VI) treatment. Cr(VI) treatment enhanced F0 andNPQ by 27% and 54%, respectively compared to the control (Fig. 1Aand H). However, the Si addition along with Cr(VI) appreciably

and length (cm seedling�1), leaf area (mm cm2) and total chlorophyll (Chl; mg g�1

error of three independent experiments with three replicates in each experimentt Po0.05 level between treatments according to the Duncan's multiple range test.

Length Leaf area Total Chl

Shoot Root

b 19.871.0b 7.070.5b 3.470.050b 1.6970.06ba 22.670.8a 9.270.3a 4.570.28a 1.8170.08ad 11.970.19d 4.270.2d 1.870.10d 1.3670.04dc 13.470.34c 5.170.3c 2.570.10c 1.4270.09c

A B

C D

E F

G H

Fig. 1. Effect of Si on F0 (A), Fm (B), Fv (C), Fv/Fm (D), Fv/F0 (E), Fm/F0 (F), photochemical quenching (G) and non-photochemical quenching (H) in wheat seedlings exposed to Cr(VI) toxicity. Data are means7standard error of three independent experiments with three replicates in each experiment (n¼9). Bars followed by different letters showsignificant differences at Po0.05 significance level between treatments according to the Duncan's multiple range test.

D.K. Tripathi et al. / Ecotoxicology and Environmental Safety 113 (2015) 133–144136

(Po0.05) reduced the Cr(VI)-mediated enhancement in F0 andNPQ compared to the Cr(VI) alone treated seedlings (Fig.1A and H).

3.3. Detection of Si and Cr accumulation by LIBS and AAS

LIBS spectra of shoot and root treated with Cr(VI) and Cr(VI)þSiwere recorded in the spectral range of 200–500 nm and are shownin the Fig. 2A and C. The spectrum clearly reveals the appearanceof various elements in wheat seedlings. LIBS spectra in the spectralrange 357–361 nm as shown in Fig. 2B and D represent the pre-sence of Cr in root and shoot of wheat seedlings grown undertreatment of Cr(VI) and Cr(VI)þSi. Fig. 2B and D interpret that theintensity of spectral line of Cr and show reduction in Cr in seedling

grown under Cr(VI) þSi combination in comparison to the seed-ling grown in the presence Cr(VI) alone. These results reveal thatthe addition of Si decreases Cr accumulation in shoots and roots.For the comparative study of Cr accumulation in roots and shoots,LIBS spectra were recorded in the spectral range from 356 to362 nm in the seedling grown under Cr(VI) treatment. The resultsshow that Cr accumulation was higher in roots than in shoots(Fig. 2E). The variation of Si accumulation in seedlings was alsostudied by measuring intensity of the spectral line of Si (288.2 nm)(Fig. 2F). Results show that accumulation of Si was higher in Sitreated seedlings followed by Cr(VI)þSi, control and Cr(VI) alone.

We have also measured the normalized intensity (signal/back-ground) of the spectral line of different elements which are shown

A B

C D

EF

Fig. 2. Single shot LIBS spectra of fresh root and shoot (A and C) showing different elements and their enlarged spectra (B and D) showing a peak of Cr (356–362 nm). Graphshowing Cr accumulation in root and shoot (E). Spectrum depicts variation of intensity/concentration of atomic line of Si (288.15 nm) in wheat seedling grown under singleand combined treatment of Si and Cr(VI) (F).

D.K. Tripathi et al. / Ecotoxicology and Environmental Safety 113 (2015) 133–144 137

in Fig. 2 A –H. Difference in spectral line intensity of a particularelement is due to its different concentration in the sample becausespectral intensity of a line ( λI

ki) at wavelength lambda corre-sponding to the transition between the upper level i and the lowerlevel k of an element is directly proportional to the perceptual

concentration (C) of the emitting atomic species i.e:

=λ

−I FCA

g e

U T( ) (1)ki

kik

E K T/k B

D.K. Tripathi et al. / Ecotoxicology and Environmental Safety 113 (2015) 133–144138

where Aki is the transition probability, gk is the degeneracy U (T) isthe partition function of that species at plasma temperature T, Ek is

A

C

E

G

Fig. 3. Pattern of Si, Cr, Mg, Ca, K and Na accumulation based on data obtained from ICAwith three replicates in each experiment (n¼9). Bars followed by different letters are sigrange test.

the upper energy level, kb is the Boltzmann constant, λ is thewavelength of the transition and F is an experimental parameter.

B

D

F

H

P-AES and LIBS. Data are means7standard error of three independent experimentsnificantly different at Po0.05 significance level according to the Duncan's multiple

D.K. Tripathi et al. / Ecotoxicology and Environmental Safety 113 (2015) 133–144 139

But before using the intensity of spectral line of an element to inferits concentration in the sample, the following conditions have tobe satisfied:- (i) the plasma should be stoichiometric, (ii) theplasma should be optically thin, and (iii) the local thermal equi-librium must holds in the laser induced plasma (Fig. S1C, D).

Temperature of the laser induced plasma is calculated usingEq. 1and it is found to be equal to 7000 K (Fig. S1C, D). In the

Fig. 4. Impact of Si addition on leaf structures in wheat seedlings exposed to Cr(VI) toxicbundle sheath, b: bulliform cells, px: protoxylem, mx: metaxylem, ph: phloem, ue: upp

present experiment, laser irradiance is 1012 Wcm�2 which is suf-ficient for the stoichiometric ablation (Singh and Thakur, 2007).

The laser induced plasma is said to be optically thin when theratio of the intensity of the two spectral lines are equal to the ratioof the product of transition probability (Aki) and statistical weight (

ity. Sh: silicified hairs, Bc: bundle cap, Le: lower epidermis, mc: mesophyll cells, bs:er epidermis, pxc: protoxylem cavity, Msc: mesophyll cells.

D.K. Tripathi et al. / Ecotoxicology and Environmental Safety 113 (2015) 133–144140

gk) and inverse ratio of their wavelength.

′′′′′′

′′′′′′

′′′′′′

′′′′′′

λλ

=A gI

I A g (2)ki

ki

k

k

In the present experiment, the optically thin plasma is verifiedby measuring the above ratio for two spectral lines of Ca (II)315.8 nm and Ca (II) 317.9 nm. For these two lines, the value ofintensity ratio I′/I″¼1.8 and the value of (A0

ki g0k λ00)/(A00ki g00k λ0)¼1.7

showing that the plasma is optically thin (Fig. S1 C and D).For the local thermal equilibrium LTE, electron density of

plasma as calculated experimentally by stark broadening, shouldbe higher than the lower limit as given by Griem (1963).

≥ × ΔN T E1.6 10 (3)e12 1/2 3

Where Ne (cm�3) is electron density, T (K) is plasma temperatureand ΔE (eV) is largest energy difference between two adjacentlevels for allowed transition. The electron density of an element inlaser-induced plasma is related with the FWHM of stark broad-ening by the following equation:

⎡⎣⎢

⎤⎦⎥λΔ ≈ w

N2

10 (4)e

1/2 16

Where Δλ1/2 (nm) is stark broadening, w (Å) is electron im-pact parameter, and Ne (cm�1) is electron density. Temperature ofplasma is calculated from the Eq. 1.

Lower limit of electron density as calculated from Eq. (2) is2.28�1016 (cm�3) whereas the electron density calculated fromEq. (3) and by measuring the FWHM of the stark broadened line, isfound to be equal to 8.35�1017 which verifies the existence of LTEin laser induced plasma. All these assumptions should be ne-cessarily satisfied for laser induced plasma. Thus, the spectral lineintensity of an element can be compared to its concentration inthe sample.

3.4. Detection of Si and Cr(VI) effects on nutrients uptake by LIBS andICAP-AES

In order to recognize whether there was a resemblance in theobservation pattern of data acquired by the ICAP-AES and LIBSinstruments, we also evaluated the data related to nutrients (Mg,Ca, K and Na) accumulation. The results indicate that data re-corded by both the instruments regarding nutrients accumulationin wheat seedlings show more or less similar pattern in roots aswell as shoots (Fig. 3A–H).

3.5. Effect of Si and Cr(VI) on leaf and root structure

Anatomical observations show that Cr(VI) adversely affectedthe internal structures of leaf compared to the control. However,addition of Si with Cr(VI) improved the damaged parts of the cellscompared to the Cr(VI) treatment alone (Fig. 4). Observation ofcontrol leaf section shows that leaf is dorsi-ventral with parallelvenation. Midrib vascular bundle is larger in size with the patchesof sclerenchymatous cells on both the sides while the smallerbundles are generally without any sclerenchyma (Fig.4A and B).Epidermal cells are well cutenised and silicified with a number ofsilicified trichomes on both the surfaces (Fig.4A and B). Further-more, each vascular bundle is surrounded by two layered bundlesheath, outer one is parenchymatous and the cells of the innerbundle sheath shows suberization towards the inner tangentialwalls. Protoxylem is represented by a protoxylem cavity and twometaxylem vessels are lignified. Mesophyll cells are well devel-oped with full of chloroplast (Fig.4A and B). However, in Cr(VI)treated seedlings the internal structures are different from controlsuch as trichomes are less in number and were less silicified as

compared to the control. Mesophyll cells are more or less dis-organized with less number of chloroplast. Sclerenchymatouspatches are less developed on both sides of median vascularbundle and protoxylem is completely dissolved and metaxylemvessels are less lignified (Fig. 4C and D). On the other hand, in Sitreated seedlings leaf section shows the contrasting characterscompared to the control and Cr(VI) treated seedlings. For instance,trichomes are well developed and silicified, mesophyll cells arewell developed with high amount of chloroplast, suberization ofbundle sheath cells is similar to the control plants, vessels ele-ments are more lignified than the Cr(VI) treated plants and similarto the control (Fig. 4E and F). Besides this, addition of Si togetherwith the Cr(VI) successfully improved the damaging impact of Cr(VI) on leaf when compared to the Cr(VI) treatment alone (Fig. 4Gand H). Furthermore, frequency of trichomes and mesophyll cellsare more developed when compared to the Cr(VI) treated plants.Moreover, similar to the control plants, lignifications and sub-erization of bundle sheath cells and lignifications of metaxylemvessels are well developed as compared to the Cr(VI) treatedplants.

Root anatomical characters also show the variable changes inthe internal structures under different treatments. The internalstructures of control root demonstrated that root hairs are longwith slightly bulbous tip, air spaces are absent in the cortex,suberization of endrodermal cells is high towards the inner tan-gential walls, roots are polyarch, more than 6 protoxylem points,3–4 metaxylem vessels are present in the center and surroundedby thick walled conjunctive tissue (Fig. 5A and B). However, underCr(VI) treatments, roots hairs are small with thick walled tipcompared to control, air spaces are present in the cortex due to thedissolution of cells, suberization of endrodermal cells is less,number of metaxylem vessels reduced and generally one centrallyplaced metaxylem vessel is surrounded by conjunctive tissue,lignification of xylem vessels walls is less in both metaxylem andprotoxylem and conjunctive tissue is less developed (Fig. 5CandD). At the same time, it has been observed that in Si treated roots,root hairs are long and pointed, air spaces are also present in thecortex but less in number, xylem and phloem well developed withprominent metaxylem and protoxylem points, generally with 4–5metaxylem vessels, vessels are thick and lignified and suberizationof endrodermal cells is high as compared to the Cr(VI) treatedroots (Fig. 5E and F). Moreover, under Cr(VI) treatment along withthe Si, it has been observed that root hairs are well developed andelongated with reduced air spaces in the cortex, suberization ofendrodermal cells is well developed, xylem is well developed withmany protoxylem points and meta xylem elements, xylem ele-ments are well lignified and conjunctive tissue is also well de-veloped (Fig. 5G and H).

4. Discussion

This study explores the damaging impact of Cr(VI) on wheatseedlings and damage modulating nature of Si using in vivo ap-proach. Results show that Cr(VI) significantly (Po0.05) declinesthe growth parameters such as leaf area, length, fresh and drymass of root and shoot of wheat seedlings which could be corre-lated with significant (Po0.05) accumulation of Cr in roots andshoots (Table 1; Figs. 2 and 3). Cr(VI) is potentially hazardousphytotoxic environmental pollutant that is preferentially absorbedby plants through roots hence; it induces several discernibletoxicity symptoms like reduced growth, chlorosis, blackening ofthe root system and modification in several plant processes(Godbold and Kettner, 1991). Besides this, it has also been reportedthat Cr(VI) induces escalated production of reactive oxygen speciesleading to the enhanced oxidative damage to lipids, protein/

Fig. 5. Impact of Si addition on root structures in wheat seedlings exposed to Cr(VI) toxicity. Ct: cunjective tissue, Pc: pericycle, Mx: metaxylem, Px: protoxylem, Cb:casperian bands, End: endodermis, Ph: phloem, Cavi: cavity.

D.K. Tripathi et al. / Ecotoxicology and Environmental Safety 113 (2015) 133–144 141

enzymes and nucleic acids which may also be correlated with theadverse effects of Cr(VI) on growth and metabolism of plants(Sharma et al., 2012). However, Si addition along with the Cr(VI),significantly restricts the entry or accumulation of Cr in shoots androots of wheat seedlings and consequently mitigated Cr(VI) toxi-city on growth parameters of wheat (Table 1; Figs. 2 and 3). It wasalso noticed that Si treatment alone appreciably increased the

growth of wheat seedling as compared to the control (Table 1). Theresults of present study could be correlated with the previousfindings in which it has been noticed that under varied stressconditions Si appeared to be beneficial element for the growth anddevelopment of wheat seedlings (Moharana et al., 2012).

In this study, in vivo and rapid detection tool i.e. chlorophyllfluorescence was used to detect stress-induced effects on light

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utilization efficiency of the photosynthetic machinery (Maxwelland Johnson, 2000; Roháček, 2002; Nwugo and Huerta, 2008).Results of the present study revealed that Cr(VI) treatment in-creased F0 and decreased Fm which resulted into a decline in Fv/Fm,Fv/F0 and Fm/F0 ratios (Fig. 1A). The F0 represents the optimal es-timation of the relative size of the antenna pigments of the PS IIcomplex (Huang et al., 2004). In addition to this, it also representsthe damaging symptom of PS II reaction center which results indecreased level of absorption to light and subsequent increase inun-used emitted light (Schnettger et al., 1994). Pfundel (1998)reported that increased level of F0 during stress conditions wasdue to a contribution of chlorophyll fluorescence emission from PSI. Thus, the results related with increased F0 level is most possibledue to the impact of Cr(VI) on PS II reaction center as well as re-duction in transfer efficiency of the energy absorbed in antennachlorophyll a to reaction center of PS II (Briantais et al., 1986). Dueto Cr(VI) toxicity, the decreased level of Fm, Fv/Fm, F0/Fm, Fm/F0,Fv/F0 (Fig. 1B–F) is in accordance with earlier studies (Atal et al.,1991, Sanglard et al., 2014). Ralph and Burchett (1998) showedthat the Fm reduction in Halophilaovalis was observed due to achange occurred in the ultrastructure of the thylakoid membrane.Further, Vassilev and Manolov (1999) and Sanglard et al. (2014)noticed that value of Fv/Fm which is used to assess the maximumphotochemical efficiency of PS II in non-stressed as well as instressed plants was found to be reduced in barley and rice plantsunder Cd and As stress which might be due to down regulation ofPS II in order to avoid an over-reduction of QA, and thus reducingthe load on the electron transport chain. Furthermore, Roháček(2002) observed that decline in Fv/Fm ratio under stress conditionsis often an indicator of photoinhibition or another kind of injury toPS II components. The Fv/F0 designates the activity of PS II while Fm/F0 indicates electron transport rate through PS II. Thus, the pre-sent study showed that metal stress influences the activities of PSII and thus decreases these ratios (Xing et al., 2010). Decreasedlevel of qP (Fig. 1G) under Cr(VI) stress also suggested the struc-tural and functional photodynamic damages of PS II. Similarly,Sanglard et al. (2014) have also noticed decline in qP under arsenicstress in rice. However, under Cr(VI) stress increased NPQ valuesshow the dissipation of surplus energy as a heat through xan-thophyll cycle in order to protect seedlings from photodynamicdamages (Fig. 1H). In contrast, addition of Si along with the Cr(VI)alleviated Cr(VI)-induced negative impacts on photosyntheticparameters such as Fm, Fv/Fm, Fv/F0, Fm/F0, qP, F0 and NPQ as sup-ported by better growth of wheat seedlings grown under Cr(VI)and Si combination than Cr(VI) treatment alone (Fig. 1A–H).

It is well known that mineral nutrients play a conspicuous rolein the growth and development of plants and further the rationaldelivery of nutrient at appropriate period of growth of plants isessential for better growth performance and yield (Tripathi et al.,2012a,b; Singh et al., 2013a,b). Mineral elements are the majorcomponents of the plant body which provide biochemical, me-chanical and physical strengths to the plants.Therefore, qualitativeand quantitative information about the mineral deposition as wellas its transport may provide a deeper insight of cellular physiologyand also facilitate more efficient information based on nutrientprofile of plants which involved in various metabolic processes.

For metal as well as nutrients (Si, Cr, Mg, Ca, K and Na) de-tection, we used in vivo approach using LIBS. Similar to the resultsof the present study (Fig. 2), Samek et al. (2006) and Kaiser et al.(2007) also recorded the accumulation of Pb and Cd in the biolo-gical structure of plants roots and leaves as well as Fe deposition inthe veins of plant leaves using LIBS. Krizkova et al. (2008) alsoobserved Agþ ions induced stress response in the sunflower byLIBS. Furthermore, Galiová et al. (2007) reported the special dis-tribution of Pb in plants leaves by LIBS. The LIBS spectra show thatCr accumulation increased in root and shoot on Cr(VI) treatment,

however, Si application with the Cr(VI) reduces the transportationof Cr in the root as well as aerial parts of the plants which might bea major strategy for Si-alleviation of Cr(VI) toxicity in wheatseedlings (Fig. 2B, D and E).

Numbers of studies have evaluated the resemblance of LIBSdata with ICAP-AES and/or AAS. To ascertain this fact, we recordeddistribution of Si, Cr, Mg, Ca, K and Na by LIBS and AAS (Si and Cr)and ICAP-AES and found their similar pattern of distribution(Fig. 3). Galiová et al. (2008) observed the competences of LIBS todetect the Ag and Cu distribution in sunflower and the resultswere verified with LA-ICP-MS. Kaiser et al. (2009) demonstratedPb mapping with the LIBS and compared results with LA-ICP-MSand found a close pattern with both the instruments. In anotherstudy, Galiová et al. (2007) reported the effect of Pb on transport,distribution and concentration of K and Mn. Similarly, in this studywe have also analyzed the Cr(VI) and Si interaction, accumulationas well as effects on Mg, Ca, K and Na distribution in wheatseedlings recorded by LIBS, AAS and ICAP-AES and pattern of datafound to have close similarity (Fig. 3). This shows the greaterability of LIBS over the AAS and ICAP-AES techniques and even so,LIBS can be an alternative technique to the existing recommendedmethods that needed a harmful acid digestion steps.

Anatomical observations of leaf and root show contrastingcharacters under different treatments of Si, Cr(VI), Cr(VI)þSi andcontrol of wheat seedlings (Figs. 4 and 5). It has been reported thatSi works as a supportive element that maintains the internalstructures injuries by the metal toxicity (Singh et al. 2011; Tripathiet al. 2012a,b; Vaculík et al., 2012). However, limited attempts havebeen made in this regard. Fleck et al. (2011) reported that Si en-hances suberization and lignification in roots of rice. Similarly,Schoelynck et al. (2010) also suggested that Si modulates the lig-nification in macrophytes which may be correlated with the cel-lulose contents. Results of the present study also support theearlier observations which show that in leaf lignification, sub-erization, mesophyll cells, xylem and phloem were differentamong control, Si, Cr(VI)þSi and Cr(VI) treated wheat seedlings(Figs. 4 and 5). In Cr(VI) treated seedlings, mesophyll cells con-taining chloroplasts show severe chlorosis, lignification highlyreduced and the structural integrity of xylem and phloemwas alsoaffected. However, Si alone treated wheat seedlings did not showmuch variation in leaf and root structures as compared to thecontrol (Figs. 4 and 5). Further, it was also noticed that underSiþCr(VI) treatment, suberization and lignification is again welldeveloped and xylem and phloem are well arranged compared tothe Cr(VI) alone treated seedlings (Figs. 4 and 5). Further, results ofthis study showed that under Cr(VI) treatments, Si addition pro-tects leaf and root internal structures and these results were cor-related with Si-mediated increase in the growth of wheat seed-lings. It has also been observed that under Si and SiþCr(VI)treatments, the root hairs frequency was increased and this maybe an adaptive strategy of roots to absorb extra elements to sustainthe essential activities of seedlings (Tripathi et al., 2012a,b).

5. Conclusion

This study is the first attempt in which the reliable and quickanalytical processing technique like LIBS and chlorophyll fluores-cence parameters and anatomy have been used to gain informa-tion regarding Cr(VI) toxicity alleviative nature of Si in wheatseedlings. Overall conclusion of this study is divided into threeparts (1) the addition of Si alleviates Cr(VI) toxicity by decreasingCr accumulation, protecting photosynthesis and enhancing someof the nutrients (Mg, Ca, K and Na), (2) comparative study of LIBSwith AAS and ICAP-AES showed the same pattern of mineral ele-ments distribution in wheat suggesting that LIBS can preferably be

D.K. Tripathi et al. / Ecotoxicology and Environmental Safety 113 (2015) 133–144 143

used rather than the AAS and ICAP-AES to get the rapid and quickinformation of spatial distribution of elements in plants withoutpollution and (3) use of chlorophyll fluorescence and anatomicalobservations may further provide a new insight in investigatingthe damage alleviative nature of Si against metal stress in cropplants.

Acknowledgements

Authors are thankful to University Grants Commission, NewDelhi for providing financial assistance to carry out this work.Dr. Durgesh Kumar Tripathi is grateful to UGC for providing fi-nancial support as Dr. D. S. Kothari Post Doctoral Fellowship. AKRare thankful to BRNS, BARC, Mumbai (No. 2009/37/30/BRNS/2063)for financial assistance. Authors are also thankful to Dr. DhanwinderSingh, Sr. Soil Scientist, Department of Soil, Punjab AgricultureUniversity, Ludhiana, for providing ICAP-AES facility to determinemineral elements. Authors also extended their thanks to Mr. RohitKumar, for his help during manuscript preparation.

Appendix A. Supplementary material

Supplementary data associated with this article can be found inthe online version at http://dx.doi.org/10.1016/j.ecoenv.2014.09.029.

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