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Journal of Microscopy and Ultrastructure 1 (2013) 57–62

Contents lists available at ScienceDirect

Journal of Microscopy and Ultrastructure

jo ur nal homep age: www.els evier .com/ locate / jmau

riginal article

icroscopic analysis of lead accumulation in tobaccoNicotiana tabacum var. Turkish) roots and leaves

ami Alkhatiba,b,∗, Emad Bsoulc, Douglas A. Blomd, Kajal Ghoshroyf,ebecca Creamera, Soumitra Ghoshroyd,e

Electron Microscopy Lab, New Mexico State University, Las Cruces, NM 88003, United StatesDepartment of Biotechnology and Genetic Engineering, Jordan University of Science and Technology, Irbid, JordanDepartment of Biology and Biotechnology, The Hashemite University, Zarqa, JordanElectron Microscopy Center, University of South Carolina, Columbia, SC 29208, United StatesDepartment of Biological Sciences, University of South Carolina, Columbia, SC 29208, United StatesDivision of Science, Mathematics and Engineering, University of South Carolina at Sumter, Sumter, SC 29150, United States

a r t i c l e i n f o

rticle history:eceived 22 April 2013eceived in revised form 10 June 2013ccepted 27 June 2013

eywords:ead nitrateoagland’s solution

nvagination

a b s t r a c t

A hydroponic experiment was carried out to localize lead accumulation in tobacco roots andleaves. Plants were grown for seven days in Hoagland’s solution supplemented with differ-ent concentrations of lead nitrate [Pb(NO3)2]: 0.0 (control), 10 �M, 100 �M, and 500 �M.Growth was inhibited in plants treated with 100 �M, and 500 �M. Light and electron micro-scopic studies showed that lead accumulated mainly in cell walls and vascular tissues ofroots of plants exposed to 100 �M, and 500 �M lead nitrate. In contrast, roots exposed to10 �M lead nitrate did not show any detectable accumulation of lead in the roots. The TEMimages confirmed the presence of lead outside the epidermis of the roots in the form ofelectron dense clusters of fine needles. No lead was detected in the leaves. However, plants

erritinhloroplast

exposed to 100 �M lead nitrate were characterized by the presence of ferritin clusters in thechloroplasts. This suggests a protective mechanism by the plant to prevent oxidative dam-age caused by Pb. Plants exposed to 500 �M lead nitrate showed mesophyll cells containingaltered chloroplasts with disrupted thylakoid systems.

© 2013 The Saudi Society of Microscopes. Production and hosting by Elsevier Ltd.All rights reserved.

. Introduction

According to the Environmental Protection AgencyEPA), lead (Pb) is the most common heavy metal

∗ Corresponding author at: Department of Biotechnology and Geneticngineering, Jordan University of Science and Technology, Irbid, Jordan.el.: +962 776319493; fax: +962 027095123.

E-mail address: ramiqfk@gmail.com (R. Alkhatib).eer review under responsibility of Saudi Society of Microscopes.

213-879X © 2013 The Saudi Society of Microscopes. Production andosting by Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.jmau.2013.06.005

contaminant in the environment [17]. Anthropogenicactivities such as mining, smelting, burning of fossil fuel,effluents from storage battery, and the manufacturing ofpesticides and fertilizers are some of the primary sources ofPb contamination of the environment [2,11,21,26]. Severalstudies showed that increased concentrations of Pb causedetrimental effects on plant growth [6]. Visible symptomsof toxicity are characterized by smaller leaves and a stuntedgrowth for both shoots and roots [25]. Leaves show somechlorosis and necrosis and roots turn black or dark brown[15,24,28]. In addition, high concentrations of Pb cause aconsiderable decrease in dry weights of shoots and roots[5,24,25]. After Pb is absorbed by the plant’s root system,

it accumulates in different parts of the plant. In general,Pb becomes highly concentrated in the root system ascompared to other parts of the plant [24]. Thus, the con-centration of Pb in the aerial parts of the plant decreases

scopy a

58 R. Alkhatib et al. / Journal of Micro

as the distance from the root increases [24]. This is dueto higher accumulation of Pb in cell walls of the root thanother parts of the plant [1,24] and the ability of Pb to bindthe carboxyl groups of the carbohydrates galacturnic acidand glucuronic acid in the root cell wall [8].

Numerous studies have shown that Pb is likely to inhibitand interfere with various physiological processes. Plantsexposed to Pb ions show decreased photosynthetic andtranspiration rates [18,24,25]. These responses are thoughtto be related to the distortion of the chloroplast ultra-structure, reduced chlorophyll content of the leaves, CO2deficiency as a result of stomatal closure, and the inhibitedactivities of the Calvin cycle enzymes [1,18,24]. Cerato-phyllum demesum exposed to Pb(NO3)2 showed obviouschanges in chloroplast fine structure [10]. Other plantsshowed a reduction or absence of starch grains whenexposed to heavy metals, including Pb [9,14,24]. In general,small quantity of Pb reaches chloroplasts and mitochondria[24].

We hypothesize that Pb will be sequestered andrestricted in the root system, mainly in the cell wall. Thiswork aimed to study the localization of accumulated Pb andcharacterize the method of accumulation in tobacco rootsand leaves. We also examined the ultrastructural changesin root and leaf cells treated with various concentrations ofPb.

2. Materials and methods

2.1. Plant material

Tobacco (Nicotiana tabacum var. Turkish) seeds weregerminated and grown in Promix for 3–4 weeks at 25 ◦C.Uniformly sized seedlings were removed from soil andtheir roots were carefully washed with distilled water toremove Promix particles. Plants of similar heights andequal number of leaves were placed in Hoagland’s solution(bio-WORLD, Dublin, OH, USA) and grown hydroponicallyin a growth chamber at a photosynthetic photon fluxdensity (PPFD) of 250–300 �mol m−2 s−1 under controlledtemperature (23–30 ◦C), humidity (60 ± 5%), and photope-riod (14 h of light). The nutrient solution was changed every3 days. Pb was added in the form of Pb(NO3)2. Plants wereplaced in Hoagland’s for 3 days before they were exposed to10, 100, and 500 �M lead nitrate mixed in Hoagland’s solu-tion. Both 100 �M and 500 �M lead nitrate were toxic toplants; meanwhile 10 �M lead nitrate was non-toxic [25].Toxicity symptoms caused by excess Pb include stunting,chlorosis and blackening of root system. It also inhibits pho-tosynthesis, upset water balance, and affects membranestructure [25]. Control plants were not exposed to any Pb.The plants were grown for 1 week in Pb solution and rootand leaf samples were collected.

2.2. Light and transmission electron microscopy

Root and leaf samples (0.5–1.0 cm) from Pb-treated

tobacco plants were fixed in 2.5% glutaraldehyde bufferedat pH 7.4 in 0.1 M cacodylate buffer for overnight at 4 ◦C.When fixed, the samples were rinsed with 0.1 M cacodylatefor five times, 2 min each. The samples were postfixed in

nd Ultrastructure 1 (2013) 57–62

cacodylate-buffered (pH 7.4) 1% osmium tetroxide, dehy-drated in a graded series of ethanol (50%, 70%, 80%, 95%,and 100%) for 10–15 min each, and embedded in freshlyprepared Spurr’s resin [3].

For light microscopy, thick sections of resin embeddedroot tissues (0.5 �m) were cut with a histo knife (Diatome,Hatfield, PA, USA), stained with 1% toluidine blue (EpoxyTissue Stain, EMS, PA, USA) and examined using a Zeiss lightmicroscope (Zeiss, Oberkochen, Germany) equipped witha digital camera (Micromax, Princeton, NJ, USA). For trans-mission electron microscopy, thin sections of root and leafsamples (70 nm) were cut with a diamond knife, stainedwith uranyl acetate [16] followed by lead citrate [7], andexamined using Hitachi H7650 and H8000. Energy disper-sive spectroscopy (EDS) analysis was performed on thinsections of root and leaf tissues using a JEOL 2100F 200 kVfield-emission gun TEM equipped with an Oxford Instru-ments 30 mm solid-state X-ray detector. Spectra werecollected with the Oxford INCA system, at several locationsin the thin sections of resin blocks.

3. Results

The plants exposed to 10 �M lead nitrate did not showany visible accumulation of Pb in the roots and the over-all appearance of the roots was very similar to that ofthe control plants. The roots exposed to 100 and 500 �Mlead nitrate showed severe reduction in overall size anddry weight when compared to that of the control (datanot shown). The light microscopic images of Pb-treatedplant root sections showed thickening of cell walls in thevascular tissue, enlargement of the intercellular space inthe cortex and severe distortion in the shape of the cellsas compared to the control root sections and 10 �M Pb-treated root sections (Fig. 1a and b). The roots of 100 and500 �M Pb-treated plants also showed the presence of Pbas dark precipitates in the vascular tissue and to someextent in the cortical and epidermal cells and cell walls(Fig. 1c and d). The TEM images confirmed the presenceof Pb outside the epidermis and also in the form of elec-tron dense clusters of fine needles primarily in the cellwalls (Fig. 2c and d). In addition, the cells in the vascu-lar tissue showed the presence of Pb as electron densedeposit in the cell walls (Fig. 3b). The EDS study (Fig. 4)confirmed the clusters as pure Pb (Pb crystals), and noPb was detected in any other parts of the cells (data notshown). The root epidermal cells in 100 and 500 �M Pb-treated plants also showed severe inward invagination ofthe cell walls (Fig. 3a). Leaves of plants exposed to 10 �Mlead nitrate did not show any visible accumulation of Pband the overall appearance of the chloroplast was very sim-ilar to that of the control plants (Fig. 5a and b). In contrast,localized black spots were observed inside the chloro-plast of plants exposed to 100 �M lead nitrate (Fig. 5c).None of these spots were observed in the chloroplasts ofthe plants treated with 500 �M lead nitrate, however, theultrastructural organization of chloroplasts was altered and

large grains of starch were observed compared to chloro-plasts from control plants (Fig. 6d). The EDS study (Fig. 6)confirmed the black spot clusters in the chloroplasts ofthe 100 �M treated plants were iron. Same black spots

R. Alkhatib et al. / Journal of Microscopy and Ultrastructure 1 (2013) 57–62 59

F n of thec ) did noa 2 mm.

w(oempsd

F(

ig. 1. Light microscopic images showing major disruption in organizatioompared to that in the control plants (a). The 10 �M Pb-treated plants (blong the cell walls in image c show presence of lead (arrow). Scale bars =

ere observed in plants treated with 50 �M lead nitratedata not shown). Pb was not detected in the chloroplastsf 500 �M Pb-treated plants, and no iron was detectedither (Fig. 6). Furthermore, this study demonstrated accu-

ulation of Pb in the form of electron dense crystalline

recipitates in various parts of the root tissue. The expo-ure to high concentrations of Pb also resulted in severeistortion of epidermal cell walls, and overall disruption in

ig. 2. TEM images of cortical cells in Pb-treated plants showing Pb in cell wall (Cb) as compared to control plant (a).

vascular bundles in 100 �M (c) and 500 �M (d) Pb-treated plants whent show any apparent disruption. The dark spots in image d and dark lines

the organization of the vascular tissue was clearly evidentin the roots.

4. Discussion

Stress caused by the presence of high Pb levels in themedium contributed to the disruption of the plant growthand development mainly in the roots. Regardless the

W) (c and d) (arrow). The 10 �M Pb-treated plant did not show any lead

60 R. Alkhatib et al. / Journal of Microscopy and Ultrastructure 1 (2013) 57–62

gination

Fig. 3. TEM images showing Pb in outside epidermal cells and inward invaN. tabacum roots.

concentrations of lead nitrate in the Hoagland’s medium,roots with symptoms were the main accumulation site forPb. It appears that the inhibition of root growth under highlevels of lead is caused by the inhibition of cell division inroot tips [29]. A reduction in root growth, mitotic irregu-larities and chromosome stickiness were observed, whenthe effect of different concentrations of lead nitrate wasstudied on root growth, cell division, chromosome mor-phology and the nucleolus of root tips of onion (Allium cepa)[22]. In this study, Pb taken up by the plant roots primar-

ily localized in the vascular tissue and cell walls, however,proximity of epidermal cells to lead nitrate induced severedamage in their cell walls. The movement of Pb in the root isprimarily via the apoplast, however, higher concentrations

Fig. 4. EDS data obtained from the analytical TEM show a strong Pb peak (arrowsd). The 10 �M Pb-treated plants did not show any Pb peak (b) as well as control (

of cell wall (a), and in vascular tissue cell (VTC) (b) of 500 �M Pb-treated

of Pb may disrupt the casparian strips of the endoder-mis allowing Pb ions to move into the vascular tissue ofthe plant. This suggests that endodermis act as a partialbarrier since some of the Pb ions move through the vascu-lar tissues [27]. Also, the strong binding of Pb to carboxylgroups of carbohydrates in cell walls of roots slows downits movement via apoplast [8]. Within the cell, the majorityof the Pb is sequestered in the vacuole [24]. In Stigeoclo-nium the formation of such vacuoles is important for thesequestration of excess metal ions [4]. Inward invagina-

tion of the cell walls of 100 �M and 500 �M treated plantssuggests a mechanism of Pb detoxification, to protect thecell contents from its effects, by forming pinocytotic vac-uoles.

) in the lead crystals in the 100 �M and 500 �M Pb-treated plants (c anda).

R. Alkhatib et al. / Journal of Microscopy and Ultrastructure 1 (2013) 57–62 61

F �M (b),a reated p

ooti

ssibsbair

Fw

ig. 5. TEM images showing chloroplasts of N. tabacum for control (a), 10nd grana (G). Ferritin clusters (arrow) were detected in the 100 �M Pb-t

In general, heavy metals decrease the concentrationf chlorophyll in leaves either by inhibiting its synthesisr by inducing its degradation [13]. In Pb-treated plants,he enhancement of chlorophyll degradation occurs due toncreased chlorophyllase activity [20].

In plants treated with 100 �M lead nitrate, the ultra-tructural organization of the chloroplast (Fig. 5c) wasimilar to those of control plants; however, accumulation ofron was observed in these chloroplasts in the form of smalllack spots (Fig. 5c). This suggests the formation of somepecific defense protein such as ferritin, which is known to

e involved in iron solubility with a high capacity of stor-ge of iron atoms. Ferritin accumulation in the chloroplasts often related to cell protection against damage due to freeadicals enhanced by oxidative stress [12]. Previous studies

ig. 6. EDS data obtained from the analytical TEM show a strong iron (Fe) peak (aas detected in the 500 �M Pb-treated plants chloroplast (b).

100 �M (c), and 500 �M (d) Pb-treated plants. Cell wall (CW), starch (S),lants chloroplast. Scale bar = 500 nm.

have indicated that transgenic plants accumulating alfalfaferritin exhibit tolerance to iron excess and necrotic dam-age caused by viral and fungal infections [19]. Wuytswinkelet al. [23] reported that tobacco plants overexpressing soy-bean ferritin exhibit paraquat-mediated oxidative stresstolerance. One of the phytotoxic effects of Pb in plantsappears to be the induction of oxidative stress due toenhanced production of reactive oxygen species (ROS) [24].The production of these ROS in larger amounts can pose asevere threat to the plant. Chloroplasts of plants treatedwith 500 �M lead nitrate exhibited a reduction in grana

stacks and usually were deformed and shrunken. This maybe related to the disturbances in transport of electronsin the photochemical phase of photosynthesis [13]. Also,chloroplasts were characterized by the presence of large

rrow) in the lead crystals in the 100 �M Pb-treated plants (a) and no Pb

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62 R. Alkhatib et al. / Journal of Micro

starch grains. This suggests both an increase of photosyn-thetic activity and the inhibition of carbohydrate transportfrom leaves to non-photosynthetic organs [14] may haveoccurred.

5. Conclusion

This study has shown that if Pb is available to a tobaccoplant, the root system will take up Pb and restricts itsmovement to the upper parts of the plant. Plants treatedwith 10 �M lead nitrate did not show any abnormalitiesor ferritin clusters formation and plants were similar tocontrol plants. This suggests that 10 �M lead nitrate isnot toxic or lethal to plants. Despite the fact that Pb wasnot detected in the leaves 500 �M lead treated plants, Pbadversely affected tobacco leaves. The ultrastructure of thechloroplasts was altered with disrupted thylakoid systems(reduction of their number, swelling, and condensation).Iron can be scavenged from abundant iron-containing pro-teins (such as plant ferredoxins), mainly in a situation ofiron deficiency; ferritin serves only as a last resort sourceof iron. The absence of ferritin in the chloroplasts of 500 �MPb-treated plants suggests a phytotoxic effect of Pb at thisconcentration; preventing plants from producing ferritin.

Conflict of interest

The authors declare that they have no conflict of inter-est. We confirm that this manuscript does not infringe anyother person’s copyright or property rights, all authors havecontributed substantially to the manuscript, and all authorshave agreed to publication of the work.

Acknowledgment

The authors gratefully acknowledge the work of NourAbdo for her assistance in experiment preparation.

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