Environmental and Experimental Botany 68 (2010) 139–148

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Environmental and Experimental Botany

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Effects of Glomus deserticola inoculation on Prosopis: Enhancing chromium andlead uptake and translocation as confirmed by X-ray mapping, ICP-OESand TEM techniques

Jack A. Ariasa, Jose R. Peralta-Videab, Joanne T. Ellzeyc, Minghua Rend,Marian N. Viverosc, Jorge L. Gardea-Torresdeya,b,∗

a Environmental Science and Engineering Ph.D. Program, University of Texas at El Paso, 500 West University Ave., El Paso, TX 79968, USAb Chemistry Department, University of Texas at El Paso, 500 West University Ave., El Paso, TX 79968, USAc Biology Department, University of Texas at El Paso, 500 West University Ave., El Paso, TX 79968, USAd Geology Department, University of Texas at El Paso, 500 West University Ave., El Paso, TX 79968, USA

a r t i c l e i n f o

Article history:Received 2 July 2009Received in revised form 24 August 2009Accepted 28 August 2009

Keywords:ChromiumLeadX-ray mappingMesquite

a b s t r a c t

Arbuscular mycorrhizal (AM) fungi contribute to plant growth, mediating the uptake of mineral elements.In polluted areas, AM also binds toxic heavy metals to roots. In this study, mesquite plants (Prosopis sp.),associated with Glomus deserticola, were treated for 15 days (in hydroponics) with lead at 0, 10, 50, or100 mg L−1, and chromium(III) and (VI) at 0, 20, 40, 75, or 125 mg Cr L−1. All Cr ion concentrations and thehighest Pb concentration reduced shoot size compared to the control. Toxic effects (yellowish leaves, leafdecay) were observed after seven treatment days. However, Pb and Cr(III) treated plants recovered uponconclusion of experimental period. Total amylase activity in leaves increased upon the addition of Pb andCr. The inductively coupled plasma-optical emission spectroscopy results showed that plants treatedwith Pb at 50 mg L−1 accumulated in roots, stems, and leaves: 61947, 9584, and 478 mg Pb kg−1; whereasplants treated with Cr(III) and Cr(VI) at 125 mg L−1 accumulated 28815, 6055, and 647; and 13767, 5010,

Arbuscular mycorrhizal

Transmission electron microscopy and 2530 mg Cr kg−1. The transmission electron microscopy (TEM) micrographs showed the presenceof G. deserticola within roots. X-ray mapping demonstrated higher Cr and Pb deposition in xylem and

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. Introduction

Chromium (Cr) and lead (Pb) are widely distributed in nature.owever, human activities have raised the concentration of theselements to dangerous levels in a number of sites around theorld. Chromium exists in several oxidation states, but the trivalent

Cr(III)] and hexavalent [Cr(VI)] are the most stable and abundantorms in the environment (Chandra and Kulshreshtha, 2004; Rait al., 2007). Lead is found in nature complexed with organic mat-er, adsorbed on clays and oxides, and precipitated as carbonates,ydroxides and phosphates (Epstein et al., 1999).

At low concentrations, Cr(III) is considered essential while Cr(VI)s toxic and carcinogenic for animals and humans. However, nei-her Cr(III) nor Cr(VI) has known functions in plants (James, 1996).n humans, excessive Cr may produce ulcers, allergic dermatitis,

∗ Corresponding author at: Chemistry Department, University of Texas at El Paso,00 West University Ave., El Paso, TX 79968, USA. Tel.: +1 915 747 5359;ax: +1 915 747 5748.

E-mail address: jgardea@utep.edu (J.L. Gardea-Torresdey).

098-8472/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.envexpbot.2009.08.009

hat G. deserticola improves metal tolerance/accumulation in mesquite.© 2009 Elsevier B.V. All rights reserved.

lung cancer, renal insufficiency, or liver necrosis (Srivastava et al.,2002). Chromium is used for leather tanning, paints, corrosion inhi-bition, chrome plating, steel production, and wood preservation.In the presence of manganese oxide excess, Cr(III) oxidizes intoCr(VI), which is more soluble in water and more toxic than otherCr forms (Dai et al., 2009). In animals, lead has no known func-tion in metabolic processes and is toxic even when absorbed insmall amounts (Peralta-Videa et al., 2009). In humans, lead toxic-ity affects the skin, internal organs, the nervous system, and maycause sterility and mental retardation (Peralta-Videa et al., 2009).Lead contamination is produced by mining, smelting, burning offossil fuels, and the manufacture of pesticides and fertilizers (Huand Zhang, 2005).

Chromium(III) and lead persist in the environment due to theirlow solubility and bioavailability, which changes when the pHis greater than 4.5. Chromium(VI) exists predominantly at a pH

greater than 6 (Bartlett, 1988). In general, the availability of Cr andPb depends on their oxidation state, pH, and complexation (Bartlettand James, 1983). Studies have demonstrated that both Cr species,as well as Pb reduce plant growth, alter enzymatic activity, andmineral nutrition (Shanker et al., 2005; Lopez et al., 2005, 2007).

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40 J.A. Arias et al. / Environmental and

ne of the enzymes altered by Cr and Pb toxicity is amylase (Lopezt al., 2007).

The U.S. EPA has recommended and often enforced differ-nt techniques, including physical and chemical methods, for theestoration and management of areas contaminated with heavyetals (Mulligan et al., 2001). However, these techniques are often

xpensive, labor consuming, soil disturbing and improper for largereas (Chaney et al., 1997). In the last three decades it has beenocumented that plants and associated microorganisms could beonsidered a harmless option to remove heavy metals excess fromolluted sites (Horne, 2000; Fischerova et al., 2006). However, theesponse of desert plants associated with arbuscular mycorrhizalAM) fungi under exposure to chromium and lead has not beeneported.

Mesquite is a fast proliferating shrub that grows in seeminglyarsh conditions such as heavy metals in soil and water scarcity.

t produces large biomass, is a source of stock food, and is usedor erosion control (Jeffrey and March, 1995). Sinha (1999) andischerova et al. (2006) have reported that tree species can uptakeigh amounts of metals, produce greater biomass, and are easier toarvest and manipulate compared to shrubs and herbs. The abil-

ty of Prosopis sp., Salsola kali, and Convolvulus arvensis to uptaker and Pb has been previously reported (Jeffrey and March, 1995;ldrich et al., 2003; de la Rosa et al., 2005; Montes-Holguin etl., 2006). However, to our knowledge, the accumulation of Crnd Pb by mesquite associated with arbuscular mycorrhizal (AM)ungi has not been studied. In this association, the fungus con-ributes to plant health through active nutrient absorption andesistance against pathogen attacks (Rufyikiri et al., 2002). In turn,he host plant releases metabolites critical for the fungal growthnd development (Tawaraya et al., 1996). Furthermore, studiesave demonstrated that the symbiotic interaction between plantsnd AM fungi has an effect on the tolerance and uptake of heavyetals, but little is known about it (Anderson et al., 1993). To the

nowledge of the authors, there are no reports on the effect of AMungi on enzyme activity in mesquite. In order to obtain insightbout the metabolic state of mesquite associated with endomy-orrhizal fungi, in response to Cr and Pb stress, total amyloliticctivity (TAA) was assayed in leaves at the end of the experimentaleriod.

The objectives of the present work were to determine theesponse of mesquite plants associated with the AM fungus Glo-us deserticola under Cr and Pb stress by measuring the growth

f plants, elemental absorption, metal distribution and deposi-ion, and total amylase activity (TAA). The working hypothesisas that the AM fungi would either increase the mesquite

olerance to Cr and Pb toxicity or increase the uptake ofoth toxic elements. Plants were treated with 0–125 mg L−1 ofr(III) or Cr(VI), and with 0–100 mg L−1 of Pb in hydroponics.

nstrumental techniques utilized included inductively coupledlasma/optical emission spectroscopy (ICP-OES), electron scanningicroprobe microscopy (ESM), transmission electron microscopy

TEM), scanning transmission electron microscopy (STEM), andltraviolet–visible spectroscopy (UV/VIS) to determine the elementoncentrations, metal distribution and deposition, and the amylasectivity.

. Materials and methods

.1. Seed germination, fungal inoculation, and treatment

pplication

Mesquite (Prosopis sp.) seeds were obtained from Wild SeedsTempe, Arizona). Before performing an experiment, the seeds wereterilized in commercial sodium hypochlorite solution [diluted to

imental Botany 68 (2010) 139–148

4% with sterilized deionized water (DI)] for 30 min and rinsedthree times with sterilized DI. For germination, the seeds wereplaced in sterilized paper towels dampened with Murashige andSkoog nutrient solution as described by Carrillo-Castaneda et al.(2005). Five grams of clay containing the G. deserticola spores pro-vided by Reforestation Technologies International (RTI) (Salinas,CA) were ground and diluted in 5 mL of DI. An aliquot of 1 mL of thefungal–clay solution was added to the seeds in each paper towel.When the radicle–hypocotyl axis (the axial part below cotyledons)and the G. deserticola mycelia appeared (5 days), seedlings wereplaced in 400 mL capacity Mason jars containing the MS nutrientsolution in an ENVIRCO laminar flow hood (Environmental Air Con-trol, Albuquerque, NM). In this experiment, Cr(III) [from (CrNO3)],Cr(VI) [from K2CrO4], and Pb [from Pb (NO3)2] (Sigma–Aldrich,St. Louis, MO) were used. The Cr ions were used at 0, 20, 40, 75,and 125 mg L−1 and Pb at 0, 5, 10, 50, and 100 mg L−1. These con-centrations were selected based on previous studies performedin our research group without AM fungi (Aldrich et al., 2003,2004). Three replicates (15 seedlings each) were used for eachmetal ion concentration. The glassware and DI were sterilized at120 ◦C and a pressure of 1.25 kg cm−2 for 45 min to avoid fun-gal and microbial contamination (Market Forge, Albertville, MN).

Fig. 1. Average length of mesquite roots (�), and stems (�) treated for 15 daysin hydroponics with (a) chromium(VI) at 0, 20, 40, 75, and 125 mg L−1, (b)chromium(III) at 0, 20, 40, 75, and 125 mg L−1, and (c) lead at 0, 5, 10, 50, and100 mg L−1. Uppercase letters stand for significant differences (p < 0.05) betweentreatments for the same tissue. Lowercase letters indicate significant differences(p < 0.05) between tissues of the same treatment. Error bars stand for SE.

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.2. Plant growth and elemental absorption

Fifteen days after treatment application, the seedlings wereemoved from the growth medium, washed with 0.01 M HNO3nd rinsed with DI to eliminate metal deposited on the root andyphal surfaces. Subsequently, 10 seedlings per treatment wereandomly selected and measured to determine the size of thelants. The plants and associated hyphae were separated into roots,tems, and leaves, oven dried at 70 ◦C for 2 days (Fisher Scien-ific Isotemp, Pittsburg, PA), weighed and digested using 6 mL ofrace pure HNO3 (SCP Science, New York, NY). The digestion waserformed in a CEM microwave oven (CEM Corporation Mathews,C) at 125 ◦C for 30 min following the EPA 3051 method (Kingstonnd Jassie, 1998). Afterward, 2 mL of the digested samples weredjusted to 10 mL with DI and analyzed for Pb and Cr concen-

rations using an ICP-OES (Perkin Elmer Optima 4300 DV, Perkinlmer, Shelton, CT). The operation parameters of the ICP-OES were:ebulizer flow, 1.00 L min−1; radio frequency power, 1400 W; peri-taltic pump flow rate 1.50 mL min−1; flush time, 30 s; delay time,0 s; read time, 20 s; replicates, 3; wash rate, 1.50; wash time, 30 s.

ig. 2. Concentration of chromium and lead in mesquite roots ( ), stems (�), and leavend 125 mg L−1, (c and d) chromium(III) at 0, 20, 40, 75, and 125 mg L−1, and (e and f) lep < 0.05) between treatments for the same tissue. Lowercase letters indicate significant d

imental Botany 68 (2010) 139–148 141

The instrument was calibrated from 0.005 to 0.5 mg L−1 for bothmetals and the wavelengths were 405.781 and 357.869 nm for Pband Cr, respectively. The calibration curves obtained had correla-tion coefficients of 0.9999 or greater for all ICP determinations. Theinstrument accuracy was verified every 10 samples with a samplespiked with known concentrations of Pb and Cr. The concentrationof metals in each sample was determined using the mean of tripli-cate readings and the standard error was calculated for each samplevalue.

2.3. Total amylase activity determination

The total amylase activity assay was performed as describedby Fuwa (1954). Briefly, 0.1–0.2 g of fresh leaves were ground andextracted with 2 mM imidazole buffer solution. The buffer solution

at pH 7.0 was used to prepare a 10% extract solution which wascentrifuged for 15 min at 14 000 rpm at −4 ◦C (Eppendorf 5417R,Westbury, NY). An aliquot of 400 �L of the supernatant was placedin a 2 mL Eppendorf tube containing 700 �L of starch solution at 1%and 25 ◦C. To establish the linearity of the reaction with time, the

s (�) treated for 15 days in hydroponics with (a and b) chromium(VI) at 20, 40, 75,ad at 5, 10, 50, and 100 mg L−1. Uppercase letters indicate significant differences

ifferences (p < 0.05) between tissues of the same treatment. Error bars stand for SE.

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42 J.A. Arias et al. / Environmental and

eaction was stopped by adding 150 �L from the mixture into tubesontaining 200 �L of cold trichloroacetic acid at 0, 20, 40, 60, and0 min. The samples were centrifuged and 30 �L of the supernatantere transferred to microplate wells containing 300 �L of iodine

eagent. After 1 h at 25 ◦C, the samples and the respective standardsere placed in a microplate reader and analyzed at 660 nm using

UV/VIS spectrometer (SpectraMax Plus 384, Sunnyvale, CA). TheAA was calculated using the slope of the calibration curve, samplelopes, dilution factor, and the weight of the biomass.

able 1oncentration of macronutrients in mesquite plants grown for 15 days in hydroponicverage ± SE. Lowercase letters indicate differences between treatments (p < 0.05).

Treatment (mg L−1) Concentration (mg kg−1)

Mg Ca

RootControl 3275 ± 1.7a 1798 ± 2

Cr(VI)20 1773 ± 389bc 2637 ± 440 1941 ± 47b 4061 ± 175 1086 ± 33cd 3708 ± 1125 828 ± 108d 3698 ± 1

Cr(III)20 2142 ± 179b 2236 ± 140 2061 ± 292b 2461 ± 275 1611 ± 223b 1655 ± 8125 1558 ± 54b 1618 ± 1

Pb(II)5 3576 ± 53a 2612 ± 110 3355 ± 388a 2091 ± 250 2731 ± 97ab 2160 ± 7100 1870 ± 254b 981 ± 2

StemControl 3681 ± la 7135 ± 1

Cr(VI)20 2940 ± 169b 3930 ± 440 2909 ± 10b 4546 ± 375 2508 ± 190b 5638 ± 4125 2653 ± 186b 7218 ± 7

Cr(III)20 3229 ± 211a 4772 ± 740 3259 ± 36a 4601 ± 175 2914 ± 120b 2886 ± 1125 2702 ± 45b 4276 ± 1

Pb(II)5 3709 ± 246a 5712 ± 610 3647 ± 239a 6299 ± 450 3540 ± 207a 4994 ± 4100 2823 ± 158b 3706 ± 4

LeafControl 4613 ± 1 9560 ± 1

Cr(VI)20 4761 ± 208 7440 ± 340 5369 ± 241 9174 ± 175 4923 ± 217 8192 ± 7125 4848 ± 392 8895 ± 6

Cr(III)20 4159 ± 458 8089 ± 140 3719 ± 119 7150 ± 475 4304 ± 186 9020 ± 6125 4331 ± 228 7096 ± 1

Pb(II)5 4154 ± 222 7631 ± 910 4165 ± 384 7545 ± 150 4741 ± 358 8801 ± 1100 4542 ± 347 8720 ± 7

imental Botany 68 (2010) 139–148

2.4. Statistical analysis

The jars were arranged in a completely random design withthree replicates per treatment. The data were analyzed using a one-way analysis of variance performed with the statistical package forsocial sciences version 13.0 (SPSS, Chicago, IL). A Tukey’s honestly

significant difference (HSD) test was used to separate treatmentmeans. The reference to significant differences between data isbased on a probability of p < 0.05, unless otherwise noted.

s with G. deserticola and chromium(III), chromium(VI) or lead(II). Numbers are

S P

a 10291 ± 1.3a 9034 ± 1.3a

85ab 6823 ± 950b 7430 ± 1509a

14c 7323 ± 436b 8664 ± 229a

06bc 5960 ± 242b 7017 ± 156a

37bc 5434 ± 175b 6229 ± 396a

8ab 7488 ± 175b 12668 ± 676b

33ab 7805 ± 338b 12809 ± 270b

4a 7920 ± 682b 10684 ± 98b

3a 7150 ± 352b 10047 ± 244b

36ab 7869 ± 674b 9655 ± 399ab

26ab 6866 ± 446b 8209 ± 510a

2ab 7111 ± 109b 8287 ± 198a

10a 7939 ± 252b 10026 ± 429b

.7a 6042 ± 1 10707 ± 2a

26b 8165 ± 354 10426 ± 352a

92b 8199 ± 985 10333 ± 191a

72b 8210 ± 364 9825 ± 266ab

81a 7904 ± 1152 9142 ± 481b

59b 6795 ± 419 10560 ± 736a

63b 8262 ± 882 11367 ± 345a

78C 8654 ± 992 9451 ± 311b

33b 7822 ± 284 9951 ± 389ab

92b 7007 ± 473 10553 ± 359a

72ab 7549 ± 385 10078 ± 144ab

26b 6689 ± 316 10011 ± 667ab

12c 7693 ± 699 8945 ± 120b

.7 12093 ± 1.3a 11373 ± 1.7

40 8623 ± 335b 11714 ± 38581 9004 ± 541b 12844 ± 36854 85163 ± 730b 11853 ± 75518 9440 ± 646b 11811 ± 978

337 9333 ± 729b 11477 ± 13254 8477 ± 1342b 10762 ± 34382 7621 ± 519b 10310 ± 979332 9088 ± 689b 11564 ± 682

31 8395 ± 392b 11467 ± 1467314 8772 ± 161b 11064 ± 740358 7341 ± 391b 10421 ± 991a

46 9839 ± 1415ab 11554 ± 1010

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it is possible that some Cr(III) was adsorbed to the root surface,which could explain the Cr concentration trend. The concentrationof Pb in roots (Fig. 2e) increased as the exogenous Pb increased,but the uptake was exponential instead of linear, with an equationY = 6073e0.7394x and a correlation coefficient r2 = 0.9943. The dif-

J.A. Arias et al. / Environmental and

.5. Cryosectioning of plant samples for electron microprobenalysis

The cryosectioning of mesquite root and stem was performedsing a minotome plus (Triangle Biomedical Sciences, Durham,C). Root and stem segments of 1 cm long were placed in waternd later fixed on sample holder using Tissue Tek (Sakura FinetekSA, Inc., Torrance, CA), frozen at −40 ◦C and sectioned at −20 ◦C.rozen samples were dissected (8 �m thick) and mounted ontolass microprobe slides. The slides were then stored at room tem-erature until microprobe analysis.

The electron probe micro analyzer (EPMA) has a camera SX50New York, NY) with four wavelength dispersive detectors. Theack Scattered Electron (BSE) root and stem images were acquiredt 15 kV, 20 nA, and focused beam condition. The dull time was set atms and the definition of the image at 512 × 512 pixels. The raster

ength was set based on the diameter of each cross-section of thelant (not show in the images). The elemental X-ray maps were alsoollected at 15 kV and 20 nA. The qualitative mode (identificationf the elements present in a given sample) was used to collect theeak scan spectrum. Three crystals, LIF-PET-TAP, were selected onhree spectrometers to cover the largest number of elements (fromto U) on the periodic table. The collected spectra were interpretedy the (PETcrystal) quali peak identification mode and re-plotted

n Excel.

.6. Transmission electron microscopy

Samples of 1 mm3 were fixed in 2.0% (wt/vol) paraformalde-yde and 2.5% (v/v) glutaraldehyde in 0.12 M Millong’s phosphateuffer pH 7.4 for 1 h at room temperature. Specimens were washedtimes for 15 min each with cold 0.06 M phosphate buffer and

hen post-fixed with 1% (wt/v) osmium tetroxide, 0.05 M potassiumerricyanide in 0.12 M phosphate buffer for 1 h in the refrigerator−8 ◦C). Specimens were then washed in 0.06 M phosphate bufferollowed by 3 times 15 min washes with distilled water and thenlock stained overnight in the refrigerator with 0.5% (wt/v) aqueousranyl acetate. Specimens were then dehydrated with ethanol and00% acetone and embedded in Poly Bed 812 plastic (Polysciences,arrington, PA). Thick sections (1 �m) were stained with Toluidine

lue and Fuschin to determine suitable areas to be thin sectionedith a Leica Ultracut ultramicrotome (60–90 nm). Thin sectionsere post-stained with uranyl acetate followed by Reynolds lead

itrate (Reynolds, 1963). Grids were examined and photographedn a Zeiss EM-10 transmission electron microscope (Carl Zeiss Co.,hornwood, N.Y) operating at an accelerating voltage of 60 or0 kV.

. Results and discussion

.1. Plant growth

The growth of mesquite plants inoculated with G. deserticoland treated with Cr or Pb at different concentrations is shown inig. 1(a–c). Toxic effects (yellowish leaves and leaf decay) werebserved after 7 days, mainly in plants treated with Cr(VI). Mildffects were observed on Pb and Cr(III) treated plants which recov-red by the end of the experimental period. As seen in Fig. 1(a and), plants treated with Cr(III) and Cr(VI) were significantly shorterompared to control plants. However, plants treated with Cr(VI) at

0 mg L−1 had significantly longer roots compared to the roots oflants treated with 40, 75, and 125 mg L−1; while no differencesere observed among plants exposed to Cr(III) concentrations.nly the highest Pb concentration (100 mg L−1) significantly inhib-

ted root elongation (Fig. 1c). In the case of Cr(III), the growth of

imental Botany 68 (2010) 139–148 143

plants depicted a perceptible U form that could be explained bythe presence of the hormesis phenomenon (higher reduction at lowconcentration, 40% at 40 mg L−1 and 30% at 125 mg L−1) (Calabreseand Baldwin, 2001). Reports indicate that Cr inhibits root cell divi-sion/root elongation in many plants (Athar and Ahmad, 2002). Ageneral justification for the decrease in plant growth could be theinhibition of plant cell division and a direct impact of Cr and Pbon cellular metabolic activity (Lopez et al., 2005). The responseof mesquite to Cr and Pb was similar to the results reported forTriticum aestivum and other crops plants (Athar and Ahmad, 2002;Lopez et al., 2005). More studies are needed to explore the responseto Cr(VI) at 20 mg L−1.

3.2. Absorption of Cr and Pb by mesquite plants inoculated withG. deserticola

The concentrations of Cr and Pb in the dry mass from the planttissues vs. the concentrations of heavy metals in the media areshown in Fig. 2(a–f). In all cases the amount of Cr and Pb in rootswas higher compared to stems and leaves. As shown in Fig. 2a, theaccumulation in roots of Cr(VI) was concentration dependent witha linear increase described by the equation Y = 3261x + 718.7 anda correlation coefficient r2 = 0.995. In the case of Cr(III) (Fig. 2c),the accumulation in roots also showed a linear increase describedby Y = 2697.6x + 14729 with a correlation coefficient of r2 = 0.977.However, the amount of Cr accumulated in roots from Cr(III) treat-ments was almost double compared to the accumulation fromCr(VI). Although the plants were acid washed before digestion,

Fig. 3. Total amylase activity of mesquite leaves treated for 15 days in hydroponicswith a) chromium(III) ( ) and chromium(VI) (�) at 0, 20, 40, 75, and 125 mg L−1,and (b) lead ( ) at 0, 5, 10, 50, and 100 mg L−1. Uppercase letters indicate significantdifferences (p < 0.05) between treatments. Lowercase letters indicate significant dif-ferences (p < 0.05) between Cr(III) and Cr(VI) for the same treatment. Error bars standfor SE.

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44 J.A. Arias et al. / Environmental and

erence in the trend of accumulation between Cr and Pb could behe due to several factors, but the data gathered (Table 1) suggesthat the competition for Mg uptake at root level could be the mostmportant. As shown in Table 1, in roots the reduction trend in Mgptake is similar to the accumulation trend of Pb.

The accumulation of Cr in mesquite roots, without AM, for exter-al concentrations of 75 and 125 mg Cr(VI) L−1 were 7636 ± 2463nd 10983 ± 1642, respectively (Aldrich et al., 2003, 2004). Theesults demonstrated an increment in Cr accumulation of about

ig. 4. Back scattered images and X-ray mapping of mesquite roots and stems treated foead at 100 mg L−1. Brighter spots in images indicate metal presence in mesquite tissue.

imental Botany 68 (2010) 139–148

25% in roots of plants associated with AM. In the case of Pb,plants treated with 50 mg L−1 and associated with AM accumu-lated approximately 60 000 mg Pb kg−1 in roots, while the reportedaccumulation without AM was 89 935 mg Pb kg−1. This result can-not be explained at this time. Important differences were observed

in translocation. As shown in the right side of Fig. 2(b, d and f), thetranslocation from Cr(VI) to stems and leaves increased in a concen-tration dependent manner modeled by an exponential curve withr2 = 0.989 and 0.986, respectively when AM was present. Plants

r 15 days in hydroponics with (a) control, (b) chromium(VI) at 125 mg L−1, and (c)

J.A. Arias et al. / Environmental and Experimental Botany 68 (2010) 139–148 145

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reated with Cr(VI) at 125 mg L−1 and AM had concentrations of000 and 2600 mg Cr kg−1 in stems and leaves, respectively, whilehe reported translocation in plants without AM was 2263 mg kg−1

o stems and 992 mg kg−1 to leaves (Aldrich et al., 2004). In com-arison to Cr(VI), the translocation of Cr(III) was higher in stemsut lower in leaves (Fig. 2d). In the case of Pb and AM, the high-st translocation was found in 50 ppm Pb (16% to stems and 1% toeaves), which was significantly higher compared to the translo-ation reported for plants grown without AM (4% to stems and.04% to leaves) (Aldrich et al., 2004). The lower Cr(III) translocationo leaves could be explained by the fast formation of hydroxidesn roots and stems. The low translocation of Pb compared to Crould be due to the fact that Cr has been found associated withhe early growth of xylem vessels while Pb tends to disseminatehroughout the entire xylem (Brabander et al., 1999; Cervantes etl., 2001). Results for Cr(III) were similar to the results reported forenipa americana (Barbosa et al., 2007); however, more analysesre needed to explain the accumulation trend of Pb in stems andeaves.

Fig. 4 shows a comparison between cryosectioned mesquiteoots and stems with back-scattered electron images comparedo the X-ray mapping of chromium and lead. In general, Cr con-entrations found in mesquite plants associated with G. deserticolaere more than two times higher compared to the concentrations

eported for mesquite plants grown in similar conditions withoutM (Aldrich et al., 2003). In the case of Pb, the accumulation in rootsas higher in plants treated with 50 mg Pb without AM (Aldrich et

l., 2004), but the translocation to stems and leaves was higher inlants associated with AM.

.3. Cr and Pb distribution in mesquite and associated G.eserticola

The EPMA BSE and X-ray images of mesquite roots and stemsf plants associated with G. deserticola and treated with Cr(VI) and

inued).

Pb(II) are shown in Fig. 4(a–c). The intensity of the spots showedon the X-ray scanned images corresponds to the amount of metalconcentrated in tissues. The X-ray analysis showed higher Cr and Pbdeposition in xylem and phloem structures. In addition, Fig. 4(b andc) shows spectra corresponding to the crystal PET and verifies thepresence of metals in root and stem tissues. BES and X-ray imagesshowed larger deposition of Cr and Pb in roots compared withstems. These images confirmed the higher Cr and Pb accumulationin cell walls which is probably produced by the accumulation in thefungal hyphae and mesquite cells (Aldrich et al., 2003; Fischerovaet al., 2006; Scoccianti et al., 2006). Fig. 4b and c, shows that theimage of Cr treated plants is brighter compared to Pb. As explainedabove, this could be due to fact that Cr has been found associatedwith the early growth of xylem vessels while Pb tends to dissemi-nate throughout the entire xylem (Brabander et al., 1999; Cervanteset al., 2001).

3.4. Effects of Cr and Pb on the total amylase activity of leaves(TAA)

The metabolic state of mesquite associated to AM fungi, inresponse to Cr and Pb stress, total amylolitic activity (TAA) wasassayed in leaves at the end of the experimental period. As seenin Fig. 3(a and b) both Cr(VI) and Pb significantly increased TAA;however, the increasing trend was different. In all plants treatedwith Cr(VI), TAA significantly increased compared to control plants.However, the activity decreased as the concentration of the exter-nal Cr increased, but even at the highest Cr(VI) concentrations,TAA was higher compared to the control (Fig. 3a). The reductionin TAA could be due to the increase in Cr concentration in tissues

that reduced the response capacity of the plants. In plants treatedwith Cr(III), TAA showed a moderate increase at 75 and 125 mg L−1.On the other hand, in plants treated with Pb, TAA increased withthe increasing concentrations of the external Pb, but at lower ratecompared with Cr(VI) (Fig. 3b). The differential response could be

1 Experimental Botany 68 (2010) 139–148

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Fig. 5. Transmission electron micrographs of mesquite root. (a) Cross-sections, ofGlomus (arrow) in epidermal cells of Prosopis sp. root (×3200); (b) Glomus hyphae

46 J.A. Arias et al. / Environmental and

xplained by the highest Cr translocation from Cr(VI) treatments.o deal with the toxicity produced by the high Cr concentration,he plants require more energy, which could explain the increas-ng trend in TAA found in Cr(VI) treated plants (Lopez et al., 2005,007). The lower Pb translocation could also explain the increasingate in TAA observed in Pb treated plants (Fig. 2c) (Epstein et al.,999; Cervantes et al., 2001; Lopez et al., 2007).

.5. Transmission electron microscopy

The following specimens were examined and photographedn the Zeiss EM-10 transmission electron microscope (Analyticalytology Core Facility, Biological Sciences): (1) mesquite root con-rol with G. deserticola, (2) mesquite root with Glomus and 40 ppmead, (3) mesquite root with 20 ppm chromium, (4) mesquiteoot with 40 ppm Cr(III) and(VI), (5) mesquite root with 40 ppmhromium and EDTA, (6) mesquite stem control with Glomus, (7)esquite stem with 40 ppm lead, (8) mesquite stem with 40 ppm

hromium, (9) G. deserticola growing on agar, (10) Glomus with leadnd (11) Glomus with chromium.

The endomycorrhizal fungus, G. deserticola, was observed inoth the root (Fig. 5) and stem samples of mesquite (Fig. 6). Thin sec-ions were photographed with and without the post-stain uranylcetate and lead citrate to look for heavy metal deposits withinhe cells of the mesquite roots and stems. G. deserticola was foundithin and without the epidermal cells. The fungus was also foundithin and between cortex parenchymal cells and collenchymal

ells. The membranes of parenchymal cells were found to be dis-orted in the controls and in the experimental plants. This may bessociated with growing the plants in hydroponic solution. Similaresults have been reported with Solanum nigrum grown associatedith AM in zinc-enriched medium. In this species Glomus increased

he uptake of Zn and the main deposits of this element were foundn the intercellular spaces and in the cell walls of the root tissuesMarques et al., 2007).

Some dark precipitates were observed in cells of roots andtems that were not post-stained as well as those which wereost-stained. Thicker plastic sections (100–200 nm) obtained fromhe same capsule surfaces as the thin sections (60–90 nm) werexamined in the Hitachi 5000 STEM microscope (Materials andetallurgical Engineering). More studies are needed to determine

he nature of these dark spots.

.6. Absorption of macro and microelements

The concentration of macroelements (magnesium, calcium,hosphorus, and sulfur) and microelements (manganese, zinc,opper, iron and molybdenum) did not showed a clear trendTables 1 and 2). Table 1 shows that in roots the accumulation ofulfur was reduced by all of the Cr and Pb concentrations; magne-ium was reduced by both Cr ions and the highest Pb concentration;nd no changes were observed in calcium accumulation by eitherr or Pb. However, the accumulation of phosphorus increased by allr(III) concentrations and the highest Pb concentration. In stems,o clear trend was observed of the phosphorus accumulation. Inddition, none of the treatments affected the accumulation of sul-ur; magnesium was reduced by Cr(VI) at all concentrations andalcium was reduced by the first three concentrations of Cr(VI).he highest concentrations of Cr(III) and Pb(II) also reduced theccumulation of calcium and magnesium in stems. On the otherand, none of the treatments affected the accumulation of macro

lements in leaves, except for sulfur that was reduced in all metalreated plants.

The micronutrient accumulation was differentially affected byoth Cr ions and Pb (Table 2). For instance, in roots the accumulationf Zn was significantly reduced by all metal treatments, Cu was

(arrow) within parenchymal cells of the root cortex (×3000); (c) cross-section of thexylem containing 40 ppm chromium (×3200). Scale bars: 4 �m (a); 4 �m (b); 4 �m(c).

reduced by Cr(III) at 75 and 125 mg L−1, but almost all treatments

increased the accumulation of Fe. In addition, all treatments, exceptCr(III) at 20 and 40 mg L−1, reduced the accumulation of Mo, whilethe accumulation of Mn was reduced by all treatments except byPb at 5 and 10 mg L−1.

J.A. Arias et al. / Environmental and Experimental Botany 68 (2010) 139–148 147

Fig. 6. Transmission electron micrographs of mesquite stem. (a) Cross-sectionof stem control xylem cells (×3000); (b) lead (40 ppm treatment) appears to belocalized as dark spots (arrows) on the parenchymal cell wall, as well as, in the cyto-plasm (×2000); (c) Glomus-infected mesquite stem parenchymal cells with 40 ppmchromium. The additional contrast and black precipitate within vacuoles (circled)is thought to be due to the chromium (×2000). Scale bars: 4 �m (a); 6 �m (b); 6 �m(c).

Table 2Concentration of micronutrients in mesquite plants grown for 15 days in hydro-ponics with G. deserticola and chromium(III), chromium(VI) or lead(II). Numbers areaverage ± SE. Lowercase letters indicate differences between treatments (p < 0.05).

Treatment(mg L−1)

Concentration (mg kg)

Mn Zn Cu Fe Mo

RootControl 56 ± 2a 244 ± 1a 67 ± 0.3a 568 ± 2.7a 60 ± 0.3a

Cr(VI)20 19 ± 7c 85 ± 25b 83 ± 34a 905 ± 247b 20 ± 5b

40 37 ± 0.88b 64 ± 0.7b 79 ± 10a 1718 ± 250d 26 ± 2b

75 40 ± 3b 19 ± 2d 61 ± 4a 2173 ± 58d 20 ± 2b

125 39 ± 4b 47 ± 35c 60 ± 6a 1804 ± 278d 13 ± 2C

Cr(III)20 27 ± 2b 52 ± 2c 101 ± lla 922 ± 85b 79 ± 5a

40 25 ± lb 29 ± 27d 73 ± 14ab 892 ± 47b 71 ± 3a

75 11 ± 1c 108 ± 14b 43 ± 5c 638 ± 158a 51 ± 6b

125 15 ± 2c 135 ± 17b 40 ± 5c 744 ± 117b 53 ± 3b

Pb(II)5 61 ± lla 108 ± 34b 101 ± 10ab 1008 ± 86c 112 ± 3d

10 60 ± 5a 106 ± 17b 99 ± 10b 1541 ± 29d 34 ± 3b

50 38 ± 2b 102 ± 10b 76 ± 23a 1830 ± 81d 31 ± 10b100 25 ± 2b 87 ± 5b 49 ± 17b 1033 ± 249c 54 ± 7b

StemControl 12 ± 0.7a 220 ± 1a 10 ± 0.3a 93 ± 0.3a 4 ± 0.7

Cr(VI)20 18 ± 2b 110 ± 8b 15 ± lb 109 ± 6a l ± 040 15 ± la 105 ± 11b 20 ± lc 97 ± lla 2 ± 175 14 ± 3a 72 ± 6c 22 ± 0c 105 ± 4a 4 ± 2125 23 ± 3c 57 ± 26c 22 ± 2c 128 ± 13b 4 ± 1

Cr(III)20 9 ± la 71 ± 30 34 ± 14c 151 ± 22c 6 ± 240 12 ± 2a 91 ± 19b 5 ± 2a 182 ± 29c 3 ± 275 9 ± 0.33a 73 ± 10 15 ± 0.33b 105 ± 9a 2 ± 1125 10 ± 2a 47 ± 4c 15 ± 3b 97 ± 2a 4 ± 2

Pb(II)5 20 ± 5c 112 ± 6b 16 ± 3b 84 ± 13a 8 ± 310 10 ± 0.88a 95 ± 3b 19 ± 3c 147 ± 74c 6 ± 150 12 ± 0.88a 89 ± 2b 20 ± 4c 120 ± 3b 7 ± 1100 9 ± 2a 65 ± 2c 21 ± lc 102 ± 3a 4 ± 1

LeafControl 83 ± 1 128 ± la 9 ± 0.7a 149 ± 1.7a 2 ± 0.3a

Cr(VI)20 94 ± 1 89 ± 6b 5 ± 2a 163 ± 17a 7 ± 2b

40 118 ± 11 95 ± 14b 10 ± 2a 156 ± 18a 5 ± 3a

75 104 ± 9 80 ± 9b 4 ± la 169 ± 5a 14 ± 2b

125 101 ± 18 65 ± 8c 8 ± la 142 ± 7a 11 ± 4b

Cr(III)20 72 ± 13 89 ± 20b 22 ± 13b 178 ± 29a 3 ± 2a

40 77 ± 17 74 ± 6c 12 ± 3a 192 ± 27a 6 ± la75 99 ± 14 75 ± 7c 9 ± la 173 ± 37a 6 ± la125 92 ± 7 103 ± 2a 8 ± 3a 178 ± 2a 3 ± la

Pb(II)5 100 ± 13 101 ± 24 15 ± 0.6 214 ± 44 6 ± 4

a b b a

10 84 ± 15 85 ± 9b 11 ± 1a 149 ± 25a 4 ± 3a

50 64 ± 4 87 ± 57b 3 ± 2a 146 ± 16a 4 ± 3a

100 55 ± 7 85 ± 23b 9 ± 8a 151 ± 27a 7 ± 2a

In stems, Cr(VI) at 40, 75, and 125 mg L−1, Cr(III) at 20 mg L−1

and Pb at 10, 50, and 100 mg L−1 increased the accumulation of Cu.Chromium(III) at 20 and 40 mg L−1, and Pb at 10 mg L−1 increasedFe accumulation, while Cr(VI) at 125 mg L−1 and Pb at 5 mg L−1

increased the accumulation of Mn.In leaves, Cu accumulation was increased by Cr(III) at 20 mg L−1

and Pb at 5 mg L−1, but Zn was reduced by all treatments except byPb at 5 and 10 mg L−1. In addition, Pb at 5 mg L−1 and Cr(VI) at 75and 125 mg L−1 increased the accumulation of Fe and Mo, respec-

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soil by Bacopa monnieri. Environ. Monit. Asses. 57, 253–264.

48 J.A. Arias et al. / Environmental and

ively. It has been reported elsewhere that heavy metals such as Crnd Pb reduce the uptake of nutritional elements (Cervantes et al.,001; Rai et al., 2007). In addition, researchers have also found thatM fungi increased nutrients absorption through an interchangeetween the fungi and plant cells (Anderson et al., 1993; Cervantest al., 2001; Carrillo-Castaneda et al., 2005).

This investigation has shown that Cr and Pb accumulatedhe most in xylem and phloem cells of mesquite. The resultslso suggested that G. deserticola could improve the metal tol-rance/accumulation of mesquite. Although the results seem toonfirm the hypothesis that AM fungi increased the uptake andolerance of mesquite to Cr and Pb, a mechanism for the beneficialffects of endomycorrhizal fungi within mesquite plants remainso be elucidated.

cknowledgements

The authors acknowledge the Analytical Cytology Core Facil-ty of UTEP (NIH grant 2G12RR008124). This material is basedpon work supported by the National Science Foundation and thenvironmental Protection Agency under Cooperative Agreementumber EF 0830117. Any opinions, findings, and conclusions or rec-mmendations expressed in this material are those of the author(s)nd do not necessarily reflect the views of the National Scienceoundation or the Environmental Protection Agency. This workas not been subjected to EPA review and no official endorsementhould be inferred. J.L. Gardea-Torresdey acknowledges the USDArant # 2008-38422-19138, to the LERR and STARs programs ofhe UT System and the Dudley family for the Endowed Researchrofessorship in Chemistry. J. Arias acknowledges the National Sci-nce Foundation Louis Stokes Alliance for Minority Bridge to theoctorate Project # HRD-0217691 and the Reforestation Technolo-ies International Company (Salinas, CA). We are grateful to twononymous reviewers that greatly contributed to improve of ouranuscript.

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