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Environmental and Experimental Botany 66 (2009) 514–521

Contents lists available at ScienceDirect

Environmental and Experimental Botany

journa l homepage: www.e lsev ier .com/ locate /envexpbot

ffect of the polycyclic aromatic hydrocarbon phenanthrene on root exudation oforghum bicolor (L.) Moench

nna Muratovaa,∗, Sergey Golubeva, Lutz Wittenmayerb, Tatyana Dmitrievaa, Anastasia Bondarenkovaa,rank Hircheb, Wolfgang Merbachb, Olga Turkovskayaa

Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, Prospekt Entuziastov 13, 410049 Saratov, RussiaInstitute of Agricultural and Nutritional Sciences, Martin-Luther-University Halle-Wittenberg, Adam-Kuckhoff-Str. 17b, D-06108 Halle/Saale, Germany

r t i c l e i n f o

rticle history:eceived 30 November 2008ccepted 3 March 2009

eywords:olycyclic aromatic hydrocarbonsoot exudatesorghum bicolor

a b s t r a c t

The objective of this work was to study the effect of two concentrations (10 and 100 mg kg−1) of phenan-threne, a ubiquitous polycyclic aromatic hydrocarbon (PAH), on root exudation of the remediating plantSorghum bicolor (L.) Moench under controlled conditions in a pot experiment. It was found that thephenanthrene concentration of 10 mg kg−1 did not cause significant effects on plant survival and growthbut had little stimulating effect on carbohydrate exudation. The contamination with phenanthrene at100 mg kg−1 inhibited accumulation of plant shoot and root biomass, decreasing the carboxylic acid,carbohydrate, and amino acid amounts released by sorghum root into the rhizosphere. However, root exu-dation per unit of root surface was not changed significantly with increasing phenanthrene concentration.There were no differences in qualitative composition of root exudates under the influence of PAH werefound. The observed alterations in the ratio between the main root-exuded components are assumed tomanifest adaptive alterations occurring in the plant as a response to pollutant stress. The activity of three

oxidoreductases (oxidase, peroxidase, and tyrosinase) released by sorghum roots was clearly progressiveto the increasing phenanthrene concentration in the substrate. Under the influence of phenanthrene, thepopulation of phenanthrene-degrading microorganisms in sorghum root zone increased, and their sharein the total number of culturable heterotrophs increased as well. The main promotional factor was thepollutant; however, the stimulating effect of the plant root exudates was also involved. The increased

obialodegr

pollutant-degrading micrbe important for the rhiz

. Introduction

On the basis of the promise offered by application of plantsnd their associated rhizospheric microorganisms to remediationf soils contaminated with organic substances, special attention inurrent biotechnology is given to studying the basics of interactionsf plants and microorganisms with technogenic environmental pol-

utants (Pilon-Smits, 2005). In this process, plant root exudates areconnecting link in the complex plant – microorganism – pollutantystem, playing an important role in the degradation of pollutantsJoner et al., 2002; Rentz et al., 2005; Chaudhry et al., 2005; Bais etl., 2006).

Up to one-fifth of total photosynthetically fixed carbon is trans-

erred to the rhizosphere via root exudation (Marschner, 1995).oot exudates are commonly divided into low- and high-molecular-eight compounds. Amino acids, carboxylic acids, sugars, phe-olic compounds, and various secondary metabolites belong

∗ Corresponding author. Tel.: +7 8452 97 04 03; fax: +7 8452 97 03 83.E-mail address: amuratova@yahoo.com (A. Muratova).

098-8472/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.envexpbot.2009.03.001

population and activity of the extracellular root enzymes are presumed toadation of PAH.

© 2009 Elsevier B.V. All rights reserved.

to the group of low-molecular-weight compounds, whereashigh-molecular-weight exudates primarily include mucilage (high-molecular-weight polysaccharides) and proteins (Vancura, 1988;Walker et al., 2003). The multicomponent composition of rootexudates determines their complex and various effects on biodegra-dation of pollutants in the rhizosphere, including: (i) stimulationof microbial number and activity (Buyer et al., 2002; Muratovaet al., 2003; Corgie et al., 2004); (ii) induction of microbialcatabolic enzymes (Fletcher and Hegde, 1995; Harvey et al., 2002);(iii) provision of cosubstrates for microbial pollutant degrada-tion (Rasolomanana and Balandreau, 1987; Donnelly et al., 1994;Fletcher and Hegde, 1995; Rentz et al., 2005); (iv) modification ofsoil conditions so as to improve pollutant bioavailability (Reilley etal., 1996; Joner et al., 2002); and (v) direct participation of the plantenzymes released by the roots in pollutant degradation (Schnooret al., 1995). Thus, there is a substantial impact of root exudates

in contaminated environments, depending on the nature of bothexudates and pollutants.

Polycyclic aromatic hydrocarbons (PAHs) are widespread, recal-citrant, and hazardous contaminants released into the environmentvia oil spills and wastes from the oil-processing and chemical indus-

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A. Muratova et al. / Environmental an

ries. The effectiveness of vegetation for the bioremediation ofAH-contaminated soils has previously been reported (Walton etl., 1994; Binet et al., 2000; Muratova et al., 2003). Joner et al.2002) and Yoshitomi and Shann (2001) have shown that phy-oremediation of PAH-contaminated soils may be enhanced bylant root exudates, which stimulate microbial PAH degradation

n rhizosphere. Studies by Rentz et al. (2004) and Kamath et al.2004) observed repression of PAH-catabolic genes by root exu-ates and demonstrated greater PAH degradation by bacteria grownn root products compared to PAH, supporting the notion thatrolific microbial growth provides improved degradation in thehizosphere. Consequently, the mechanisms responsible for PAHegradation in the rhizosphere require to be deeper understand-

ng. The only undeniable fact is that the metabolic activity ofhe rhizomicroflora is closely linked to plant root exudates andepends on their composition; hence, no investigation of pollutantegradation in the rhizosphere is possible without understandinghe peculiarities of root exudation under contamination condi-ions. Quantitative and qualitative shifts in the exudation profilef sugars, carboxylic acids and amino acids, predominantly used byicroorganisms as nutrients, may affect their physiological activ-

ty in PAH degradation. Unfortunately, no data are available abouthanges in root exudation patterns in response to PAH contamina-ion.

The objective of this work was to reveal quantitative and/or qual-tative alterations in plant root exudation in response to presencef PAHs in the environment in order to reveal possible mecha-isms of promotion of pollutant biodegradation in the rhizosphere.aking into account the marked toxic effects of PAHs on plantrowth and development (Henner et al., 1999; Baek et al., 2004;lkio et al., 2005; Song et al., 2005), we hypothesize that in aAH-contaminated environment, plants reduce the release of root-erived nutrients into the rhizosphere. This, in combination withollutant pressure, should stimulate root-associated pollutant-egrading microorganisms. In addition, the pollutant presencehould induce plant responses, including the root exudation ofxtracellular oxidoreductases into the rhizosphere (Criquet et al.,000). Both expected processes should make an important contri-ution to pollutant degradation in the rhizosphere.

The subjects of our research were the three-ring polycyclic aro-atic hydrocarbon phenanthrene, a ubiquitous PAH and Sorghum

icolor (L.) Moench, a plant species previously applied to the phy-oremediation of PAH-contaminated soil (Schwab et al., 1995;anks et al., 2003).

. Materials and methods

.1. Plant cultivation

Seeds of sorghum (Sorghum bicolor (L.) Moench) were obtainedrom the Science Research Institute of Agriculture in the South-ast (Saratov, Russia). Calibrated seeds were surface sterilized withmixture of ethanol and hydrogen peroxide (1:1) for 3 min, placed

n petri dishes filled with quartz sand, and wetted with a saturatedaSO4 solution. The seeds were germinated for 3 days at 18–20 ◦C.en seedlings were selected by root length (about 1 cm) and werelanted in each pot.

Sorghum plants were cultivated in uncontaminated (control)nd two phenanthrene containing substrates (10 and 100 mg kg−1).he two phenanthrene concentrations were applied to heat-

terilized quartz sand (particle size 1–2 mm) by spraying 0.15 and.5% (w/V) acetone solution to final phenanthrene concentrations of0 and 100 mg kg−1, respectively. The control substrate was treatedith pure acetone of equal volume. After solvent evaporation, the

ubstrates were transferred to pots containing 1.5 kg of quartz sand.

rimental Botany 66 (2009) 514–521 515

Each pot was equipped with a proliferated plastic bag of appropri-ate size. At harvest by lifting the bag out of the pot, the completeroots system with attending substrate could be transferred.

Plants were cultivated in a growth chamber with a 14/10 hday/night regimen (light intensity: 450 �mol m−2 s−1; tempera-ture: 24/20 ◦C; relative humidity: 70%) for 4 weeks. The watercontent of the quartz sand was maintained at 80% of the maxi-mum water-holding capacity by adding deionized water or Ruakuranutrient solution, according to Smith et al. (1983).

2.2. Collection of root exudates

After 4 weeks of cultivation, the plants were lifted out of thepots and were transferred into a 2-L beaker. The plastic bags wereremoved, and 1 L of deionized water was added. The plant rootswere washed gently for 2 min to release rhizosphere solution andsubstrate particles. Root exudates were collected according to Egleet al. (2003). The sorghum plants were placed in a beaker contain-ing 0.05 mmol L−1 CaCl2 solution. The beaker was wrapped withtin foil in order to keep the roots in the dark, whereas the plantshoots were exposed to light in the growth chamber under condi-tions described above. To avoid collection of cell contaminants, theCaCl2 solution was discarded after 30 min and replaced it by a freshportion of 0.05 mmol L−1 CaCl2 containing Micropur®. The plantswere exposed for 4 h under the same conditions. Micropur® is anAg+-containing substance that prevents microbial changes duringsampling. After filtration through glass wool, the root-exudate solu-tion was frozen in liquid nitrogen and was lyophilized. Dry residuesof root exudates were kept at −20 ◦C until analysis. For assessmentof the amount of exuded compounds, sorghum root exudation wasexpressed as mg or �g h−1 (total amount). For evaluation of the exu-dation intensity, the root exudation was expressed as mg or �g h−1

per root surface (dm2).

2.3. Measurements of plant biomass and root surface

After collection of root exudates, the shoots were separated fromthe roots, and were dried at 70 ◦C until constant dry weight. Theroot surface was estimated by the methylene-blue method accord-ing to Sattelmacher et al. (1983). After measurement of the rootsurface, the roots were frozen in liquid nitrogen, lyophilized, andweighed.

2.4. Fractionation of root exudates by ion-exchangechromatography

Root exudates were separated into acidic, neutral, and basiccompounds by ion-exchange chromatography (Johnson et al., 1996)with modifications in the solvent volumes. For this purpose,lyophilized samples (10 mg) were dissolved in 2 mL of 50% aque-ous ethanol. The acidic fraction from each samples was separatedfrom the neutral and basic fractions with an anion-exchange col-umn (SAX Bond Elut columns, 500 mg, LRC). The cartridge waspreconditioned with 2 mL of pure methanol and was activatedwith 4 mL of 50% aqueous ethanol. Thereafter, the dissolved samplewas loaded onto the cartridge, washed with 4 mL of 50% aqueousethanol (neutral plus basic fractions), and eluted with 4 mL of 2%HCl in methanol (acidic fraction). After anion exchange, the neutralplus basic fractions were dried in vacuo at 35 ◦C, and the residuewas resuspended in 1 mL of 50% aqueous ethanol and was acidi-

fied to pH 2. SCX Bond Elut LRC extraction cartridges (Varian) wereused to separate the neutral and basic fractions. The SCX cartridgeswere conditioned with 2 mL of methanol followed by 2 mL of 2%HCl in absolute ethanol. The entire sample was loaded, washedwith 4 mL of 50% aqueous ethanol (neutral fraction), and eluted

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16 A. Muratova et al. / Environmental an

ith 10 mL of 28% acetonitrile and 2% HCl in methanol (basic frac-ion).

The dried acidic and neutral residues were redissolved in0 mmol L−1 HClO4 and in deionized water, respectively, and wereept at −20 ◦C.

.5. Qualitative and quantitative determinations of singleompounds

Acidic compounds (carboxylic acids) were determined on anminex HPX-87H column (300 mm × 7.8 mm) with a cartridgeolumn (BioRad, Hercules, CA, USA). The mobile phase was0 mmol L−1 HClO4 (flow rate, 0.6 mL min−1). The column tem-erature was 30 ◦C. Individual acidic compounds were detectedt 215 nm with an L-4000 UV detector (Merck Hitachi) (Saarniot al., 2004). Neutral compounds (carbohydrates) were separatedn a Rezex RCM-Monosaccharide column (300 mm × 7.8 mm) withcartridge column (Phenomenex, Torrance, CA, USA). The eluentas deionized water with a flow rate of 0.6 mL min −1 at 85 ◦C. AnI 71 refractive index detector (Merck) was used for the measure-ents (Saarnio et al., 2004). The concentrations of free amino acidsere measured as isoindole derivatives by high-performance liquid

hromatography (1100 series, Agilent Technologies) according tochuster (1988), by using a Hypersil ODS column (250 mm × 4 mm;article size, 5 �m; Agilent Technologies, Waldbronn, Germany).he column temperature was 42 ◦C, and the flow rate was.8 mL min−1. Separation was done with a gradient of acetateuffer–acetonitrile as the mobile phase. Amino acid isoindoles wereuantified by fluorescence detection at an excitation wavelength of37 nm and an emission wavelength of 454 nm. Precolumn deriva-ization was conducted according to Teerlink et al. (1994).

.6. Measurement of enzymatic activities

The protein content of the freeze-dried root-exudate samplesas determined according to Bradford (1976).

Enzymatic activities in the root exudates were measuredpectrophotometrically. For this purpose, 10 mg of a lyophilizedample was dissolved in distilled water (1 mL). Laccase activ-ty was measured with 30 �mol L−1 syringaldazine (Leonowicznd Grzywnowicz, 1981), or 5 �mol L−1 catechol (Jung et al.,002), or 0.5 mmol L−1 2,6-dimethoxyphenol (Slomczynski etl., 1995), or 1 mmol L−1 2,2′-azinobis-(3-ethylbenzo-thiazoline--sulphonate) (ABTS) (Niku-Paavola et al., 1988), or 23 �mol L−1

,7-diaminofluorene (2,7-DAF) (Criquet et al., 2000), by using0 mmol L−1 sodium tartrate buffer (pH 3.5), 50 mmol L−1 sodiumcetate buffer (pH 5.0), 50 mmol L−1 Na–K phosphate buffer (pH.0), and 50 mmol L−1 Tris–HCl buffer (pH 7.5). The oxidation ratesf the substrates were monitored at � = 525, 410, 468, 436, and00 nm, respectively. The activity of peroxidase was measuredy monitoring the oxidation rate of the same substrates and athe same pH values, in the presence of 0.1 mmol L−1 H2O2. Thectivity of monophenol monooxygenase (tyrosinase) was mea-ured in 4 mmol L−1 3,4-dihydroxy-dl-phenylalanine (dl-DOPA)nd 50 mmol L−1 sodium–potassium phosphate buffer at pH 6.0Criquet et al., 2000). The oxidation rate of the substrate was mon-tored at � = 475 nm. Catechol-dioxygenase activity was measured

ith 0.67 �mol L−1 catechol and 50 �mol L−1 Tris–HCl buffer at pH.5. Catechol-1,2-dioxygenase activity was measured according to

ayaishi et al. (1957) and monitored at � = 257 nm. Catechol-2,3-ioxygenase activity was measured according to Kojima et al. (1961)nd was measured at � = 375 nm.

All the activities are expressed in enzyme units (U), defineds �mol oxidized substrate min−1 and mg−1 of root exudate dryeight or mg−1 protein.

erimental Botany 66 (2009) 514–521

2.7. Enumeration of microorganisms

The total number of culturable heterotrophic microorganismsin the bulk substrate and in the plant rhizosphere and rhizoplanewas determined by the plating dilution technique by using Nutri-ent agar medium (Difco), as described previously (Muratova et al.,2003). For isolation of PAH-degrading microorganisms, Kiyohara’splate test with phenanthrene as a substrate was used as describedpreviously (Muratova et al., 2003). The colony-forming units (CFUs)were calculated after 2–3 days of incubation.

2.8. Phenanthrene analysis

Air-dried sand samples were extracted with chloroform andwere analyzed for phenanthrene with a La Chrom® HPLC system(Merck, Germany) containing an L-7480 fluorescence detector, anL-7100 pump, an L-7350 column oven, an L-7614 vacuum degasser,and D-7000 HPLC System manager software. HPLC analysis was car-ried out with an Aqua C18 column (250 mm × 4.6 mm ID; particlesize 5 �m; 125 Å pore size; Phenomenex, Torrance, USA). The col-umn temperature was 30 ◦C, and the flow rate was 0.3 mL min−1.Elution was done with a gradient of water–acetonitrile. The meanphenanthrene recovery was 95% in recently spiked sand.

2.9. Statistics

The experiment design was completely randomized and hadthree replications. Analysis of variance (ANOVA) was performedwith SAS software version 9.1.3 (SAS Institute Inc., Cary, NC, USA),and means were compared by using LSD (P = 0.05) Tukey’s test.

3. Results

The phenanthrene concentration of 10 mg kg−1 did not causesignificant effects on plant survival and was little (14%) inhibitoryto root-biomass accumulation (Table 1). The 10-fold increase inphenanthrene concentration was hazardous and was toxic tosorghum: plant survival was decreased by almost 50%, and biomassproduction in each plant was 2.6 times (shoots) and 2.8 times (roots)less than that in the control plants, grown in uncontaminated sub-strate. The total reduction in plant growth in this treatment waseven higher. Additionally, in heavily phenanthrene-contaminatedsubstrate, the average root surface of each plant was also two timessmaller than that in the control variant. The total reduction washigher than two-thirds. These findings indicate severe inhibition indevelopment of the plant root system in the heavily phenanthrene-contaminated substrate.

Root exudation was not changed significantly at 10 mg kg−1

phenanthrene, but at 100 mg kg−1 it was strongly affected byphenanthrene-induced damage of the root system. The totalamount of exuded compounds was reduced by 78% (Table 1), thisresulting from both decreased plant number per pot and inhibitedbiomass production by each plant. The intensity of root exudationwas not changed significantly with increasing phenanthrene con-centration. There were no differences in qualitative composition ofroot exudates under the influence of the PAH.

Table 2 shows the results from analysis of exuded acidic com-pounds. In S. bicolor, malate, trans-aconitate, and citrate were thepredominant organic acids. Oxalate, lactate, fumarate, and succi-nate were also measured but were found to be present in smaller

amounts (in the order of one to two magnitudes). Alterations incarboxylate exudation were significant only at 100 mg kg−1: in theaverage, the total exuded amount of each acid was reduced by 77%,whereas exudation per unit root surface was unchanged. Thus, theorganic acid transfer into the rhizosphere depended mainly on the

A. Muratova et al. / Environmental and Experimental Botany 66 (2009) 514–521 517

Table 1Effect of phenanthrene on plant survival, plant growth, and root exudation in S. bicolor (L.) Moench.

Parameter Unit Phenanthrene, mg kg−1 LSD (P = 0.05)

0 10 100

Plant survival, % Total 93.3 100.0 56.7 9.1

Shoot dry weight, gTotal 2.91 2.86 0.67 0.55Plant−1 0.311 0.286 0.119 0.065

Root dry weight, gTotal 1.37 1.26 0.30 0.15Plant−1 0.147 0.127 0.052 0.012

Root surface, dm2 Total 9.7 10.4 2.8 2.3Plant−1 1.04 1.04 0.49 0.26

Root exudation, mg h−1 Total 12.4 12.0 2.7 2.6dm−2 1.28 1.16 1.03 0.30

Table 2Effect of phenanthrene on the exudation of carboxylic acids in S. bicolor (L.) Moench.

Compound Unit Phenanthrene, mg kg−1 LSD (P = 0.05)

0 10 100

Malate�g h−1 1532.5 1411.6 393.3 514.0�g h−1 dm−2 157.9 137.2 148.7 101.9

trans-Aconitate�g h−1 478.5 477.6 56.5 311.4�g h−1 dm−2 49.3 46.4 25.9 31.9

Citrate�g h−1 157.2 166.4 20.7 48.5�g h−1 dm−2 16.2 16.3 8.6 6.0

Oxalate�g h−1 15.8 19.5 5.6 1.9�g h−1 dm−2 1.6 1.9 2.0 0.9

Lactate�g h−1 15.7 11.6 7.5 9.0�g h−1 dm−2 1.6 1.1 2.6 1.4

F�g h−1 6.9 7.9 0.9 3.5

S

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C

F

G

M

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umarate �g h−1 dm−2 0.7

uccinate�g h−1 2.9�g h−1 dm−2 0.3

otal root biomass and was less affected by the exudation inten-ity. No substantial differences in composition of the acidic fractionetween control and phenanthrene-affected plants were observed.

The neutral exudate fraction from the control plants consistedf fructose (53%), glucose (27%), maltose (12%), and galactose8%) (Table 3). Similarly to the acidic-fraction case, application of00 mg kg−1 phenanthrene reduced sugar exudation into the rhi-osphere by 84%, primarily because of the root-biomass inhibitionnd, to a lower extent, the reduced exudation intensity. The lowhenanthrene concentration seemed to stimulate carbohydratexudation, but a significant increase was found for maltose only (by

2% and 6% for the amount and intensity of exudation, respectively).he effect of phenanthrene on each detected compound was sim-lar. The composition of the neutral exudate fraction did not differignificantly between treatments.

able 3ffect of phenanthrene on the exudation of carbohydrates in S. bicolor (L.) Moench.

ompound Unit Phenanthrene, mg kg−1 LSD (P = 0.05)

0 10 100

ructose�g h−1 217.9 406.0 32.2 222.8�g h−1 dm−2 22.5 39.2 12.7 20.8

lucose�g h−1 110.0 184.2 18.3 95.6�g h−1 dm−2 11.4 17.8 7.2 9.3

altose�g h−1 50.1 56.2 4.9 5.7�g h−1 dm−2 5.2 5.5 2.0 0.3

alactose�g h−1 33.2 47.8 8.6 21.9�g h−1 dm−2 3.4 4.6 3.3 2.2

0.8 0.4 0.3

1.7 0.4 1.90.2 0.2 0.2

Among the detected amino acids, alanine, leucine, serine,and glycine were most important quantitatively (Table 4). The10 mg kg−1 concentration did not significantly affect their amount,whereas 100 mg kg−1 concentration brought about a substantialdecrease in amino acid exudation (by 78%). The exudation of aminoacids per unit root surface was unchanged. These effects were simi-lar for each compound. Thus, the relative composition of the aminoacid fraction did not change in plants exposed to both phenanthreneconcentrations.

The protein content in the S. bicolor rhizosphere was decreasedby phenanthrene application (Table 5). In comparison to the con-trol plants, the protein content decreased by more than 70% in thetreatment with 100 mg kg−1 phenanthrene, both as a part of plantdry weight and as part of the root exudates.

Using test substrates, we revealed the enzymatic activities ofoxidase, peroxidases, and tyrosinase in the sorghum root exudates(Table 5). Oxidase activity was revealed only with ABTS as a sub-strate and was not found with syringaldazine as a laccase testsubstrate, independently of the pH value used (3.6, 5.0, 6.0, and7.5). Thus, it was not possible to claim that the enzyme revealedwas laccase. Oxidase activity trended to increase as a result ofphenanthrene treatments. Peroxidase activity was detected withABTS (pH 3.6), syringaldazine (pH 7.5), and 2,7-DAF (pH 6.0) assubstrates. Peroxidase activity was highest in the ABTS-oxidationreaction in all treatments. Related to mg of protein in root exu-

dates, the enzymatic turnover of peroxidase(s) increased 5.7 and8.3 times as a result of treatments 10 and 100 mg kg−1, respectively.This trend was observed independently of the enzyme substrateused. Tyrosinase activity also increased (by 2.3 and 3.3 times for 10and 100 mg kg−1, respectively). Neither catechol-1,2-dioxygenase

518 A. Muratova et al. / Environmental and Experimental Botany 66 (2009) 514–521

Table 4Effect of phenanthrene on the exudation of amino acids in S. bicolor (L.) Moench.

Compound Unit Phenanthrene, mg kg−1 LSD (P = 0.05)

0 10 100

Alanine�g h−1 22.4 24.4 6.5 11.0�g h−1 dm−2 2.3 2.4 2.5 1.4

Leucine�g h−1 11.8 11.7 1.5 7.4�g h−1 dm−2 1.2 1.1 0.6 0.7

Serine�g h−1 11.3 8.4 2.0 5.5�g h−1 dm−2 1.2 0.8 0.8 0.7

Glycine�g h−1 10.9 10.4 1.8 5.9�g h−1 dm−2 1.1 1.0 0.7 0.6

Tyrosine�g h−1 6.4 6.9 1.0 3.1�g h−1 dm−2 0.7 0.7 0.4 0.3

Phenylalanine�g h−1 6.3 7.0 1.2 3.5�g h−1 dm−2 0.7 0.7 0.5 0.4

Threonine�g h−1 5.9 6.8 1.1 2.3�g h−1 dm−2 0.6 0.7 0.4 0.3

Isoleucine�g h−1 5.2 6.0 0.9 3.0�g h−1 dm−2 0.5 0.6 0.3 0.3

Asparagine�g h−1 4.6 5.1 2.0 4.3�g h−1 dm−2 0.5 0.5 0.8 0.6

Glutamate�g h−1 3.2 3.3 1.0 2.9�g h−1 dm−2 0.3 0.3 0.4 0.3

Tryptophane�g h−1 1.3 1.0 0.2 1.0�g h−1 dm−2 0.1

Glutamine�g h−1 0.7�g h−1 dm−2 0.1

Table 5Effect of phenanthrene on the protein content and enzyme activities in the rootexudates of S. bicolor (L.) Moench.

Parameter Phenanthrene, mg kg−1 LSD (P = 0.05)

0 10 100

Protein contentmg per g root dry weight 0.144 0.086 0.041 0.050mg per mg root exudates 0.016 0.010 0.004 0.006

Oxidasea (ABTS, pH 3.6) 0.058 0.264 0.184 0.311

Peroxidasea

ABTS (pH 3.6) 0.085 0.485 0.706 0.318Syringaldazine (pH 7.5) 0.004 0.034 0.092 0.0802,7-Diaminofluorene (pH 6.0) 0.001 0.09 0.017 0.006

T

nm

rait

exposure (Henner et al., 1999; Holoubek et al., 2000; Baek et al.,

TD

P

011

L

yrosinasea (DL-DOPA, pH 7.5) 0.075 0.174 0.248 0.037

a U per mg protein.

or catechol-2,3-dioxygenase activities was found in either treat-ent.Microbiological analysis of bulk substrate and the plant

oot zone showed (Table 6) that the total number of cultur-ble heterotrophic and phenanthrene-degrading microorganismsncreased as the distance from the plant root decreased. At the sameime, the number of heterotrophs was changed differently under

able 6istribution of culturable heterotrophs and phenanthrene degraders in the root zone of S

henanthrene, mg kg−1 Heterotrophs (×106)

Bulk substrate Rhizosphere Rhi

2.45 27.73 1790 1.54 28.60 19500 0.67 6.26 170

SD (P = 0.05) 0.45 19.42 18

0.1 0.1 0.1

1.0 0.3 0.70.1 0.1 0.1

the influence of pollutant. At 10 and 100 mg kg−1 phenanthrenein the bulk substrate it was 37 and 73% smaller than the bacte-rial number in the uncontaminated control. In the rhizosphere,10 mg kg−1 phenanthrene did not significantly affect the numberof heterotrophic microorganisms, whereas 100 mg kg−1 reduced itby 77%. On the root surface (rhizoplane), the number of culturableheterotrophic microorganisms was largest and was not changed sig-nificantly. Under the influence of phenanthrene, the population ofPAH degraders increased 4.2- and 2.2-fold in the rhizosphere and4.4- and 3.1-fold in the rhizoplane, for 10 and 100 mg kg−1 phenan-threne contamination, respectively.

The residual concentrations of phenanthrene in the rhizosphericsubstrate were 0.04 and 4.5 mg kg−1 for 10 and 100 mg kg−1, respec-tively.

4. Discussion

The toxic effects of PAHs on plants depend both on environmen-tal factors and on pollutant chemical structure, concentration and

2004; Song et al., 2005). The phytotoxicity of PAHs was reportedto be mainly determined by their lipophilicity, water solubility andbioavailability (Thygesen and Trapp, 2002). Physical–chemical char-acteristics of phenanthrene (Mr, 178.2; water solubility, 1.29 mg L−1

. bicolor (L.) Moench (CFU per gram of dry root or substrate).

PAH-degraders (×105)

zoplane Bulk substrate Rhizosphere Rhizoplane

.42 0.35 0.46 4.42

.14 0.95 1.94 19.33

.30 0.68 1.03 13.85

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t 25 ◦C; log Kow, 4.57) determine its low uptake by the plant rootsnd some phytotoxic effects of this pollutant (Gao and Zhu, 2004;lkio et al., 2005).

Plants exhibit responses to toxic compounds only after beingxposed to the ‘threshold concentration’ (Holoubek et al., 2000),hich varies for different compounds and plant species. Baek et

l. (2004) observed no effect of low (10 mg kg−1) concentrations ofaphthalene and phenanthrene, but not pyrene, on the biomass ofhaserolus nipponessis and Zea mays grown in soil, whereas a con-entration of these PAHs of 100 mg kg−1 inhibited plant growth.ccording to Song et al. (2005), the lowest observable concentra-

ion that caused adverse effect was 10 mg kg−1 for phenanthrenehen tested with green onion and 50 mg kg−1 for pyrene when

ested with wheat. In our experiment with sorghum, the lowhenanthrene concentration (10 mg kg−1) had a slight toxic effectn plant root biomass. The higher concentration of phenanthrene100 mg kg−1) caused a distinct phytotoxic effect manifested asigns of chlorosis, decreased plant survival, shoot and root-biomassroduction.

Alterations of biochemical and physiological characteristics astress responses of plants appear earlier than alterations in plantorphology become visible (Radetski et al., 2000; Alkio et al.,

005). With this in mind, distinct changes in processes in planthysiological and biochemical processes could be expected for both

evels of phenanthrene application (10 mg kg−1 [slight morpholog-cal alterations] and with 100 mg kg−1 [pronounced symptoms ofntoxication]). Among these processes, root exudation is of fun-amental importance for phytoremediation and is accessible foronitoring and measurement in studies on the basic aspects of

hytoremediation of PAH contamination.Significant alterations in plant root exudation have been

evealed in studies of biochemical and physiological responses oflants to environmental stress (Wittenmayer and Merbach, 2005).s a whole, it is believed that unfavourable environmental con-itions cause an increase in plant root exudation (Neumann andömheld, 2000). This idea was confirmed by a study of changes

n plant root exudation in response to pollutant stress (Porteoust al., 2000). Using the lux-marked rhizobacterium Pseudomonasuorescens as a biosensor, Porteous et al. showed that herbicideparaquat) stress increased the C flow from the roots of Plantagoanceolata. Unfortunately, data concerning alterations in plant rootxudation under the influence of organic pollutants are limited.ccording to the results obtained in this study, the presence ofhenanthrene in substrate did not significantly affect the inten-ity (mg h−1 dm−2) of plant root exudation. However, the overallhytotoxic effect of the pollutant, manifested as a reduction in totaliomass accumulation, caused a decrease in the release of plant rootxudates into the rhizosphere (mg h−1). Walton et al. (1994) spec-lated that when a chemical stress is present in soil, a plant mayespond by changing exudation to the rhizosphere, which mod-fies rhizospheric microflora composition or activity. As a result,he microbial community might increase the transformation ratesf the toxicant. Our data showed that under the influence of theAH in the sorghum root zone, the population of phenanthrene-egrading microorganisms increased and their share in the totalumber of culturable heterotrophs increased as well (from 0.2%o 0.7 and 1.7% in the uncontaminated and contaminated [10 and00 mg kg−1] rhizospheres, respectively). The main promotionalactor was the pollutant, both at the low and at the high concen-ration. However, the stimulating effect of the plant root exudatesas also observed: the number of PAH degraders was maximal

n the rhizoplane, providing a consistently high level of exudatesven in the presence of the pollutant and in the little contam-nated rhizosphere, where the root-exudate content had not yeteen decreased by the pollutant. The total number of heterotrophicicroorganisms as a whole strongly depended on the amount of

rimental Botany 66 (2009) 514–521 519

root exudates, ensuring the maximal microbial population on theplant root surface (rhizoplane) and in the uncontaminated and littlecontaminated rhizospheres of sorghum. Strong inhibition of het-erotrophic microorganisms at 100 mg kg−1 phenanthrene was dueby the rhizosphere content of root exudates, which was low becauseof the pollutant-induced damage of the root system.

Phenanthrene application caused slight qualitative alterationsin sorghum root exudation. In the control treatment, the quanti-tative ratios between fractions containing carboxylic acids, sugarsand amino acids were 81.1%:15.5%:3.4%. The low and high phenan-threne concentrations shifted the ratios to 72.1%:24.6%:3.3% and85.5%:11.0%:3.5%, respectively. The 10 mg kg−1 phenanthrene con-centration was associated with an increase (significant only formaltose) in carbohydrate exudation. The increased exudation ofcarbohydrates may be explained by the hypothesis as to a regulatoryfunction of catabolic products appearing under stress (Usmanov etal., 2001). According to this hypothesis, the breakdown productsof biopolymers (oligonucleotides, oligopeptides, oligosaccharides,and oxygenic products of lipid metabolism) degradation under theinfluence of external factors should be a trouble signal, and theresponse to that should be a correction of synthetic processes. Weassume that the increased exudation of carbohydrates is a mani-festation of adaptive alterations in plant metabolism in response topollutant action.

It was reported (Keuth and Rehm, 1991; Wong et al., 2002) thatcarbohydrates at concentrations of up to 450 mg L−1 may stimulatephenanthrene degradation by bacteria. On the other hand, repres-sion of phenanthrene degradation by carbohydrates (e.g., glucose)was observed (Rentz et al., 2004) to be less than that by carboxylicacids (e.g., lactate) found in marked quantities in the sorghumroot exudates (Table 2). Thus, an increase in the carbohydrate fluxinto the rhizosphere may enhance the population of phenanthrenedegraders, which was observed in our experiment at 10 mg kg−1

pollutant. The absence of such an effect at 100 mg kg−1 may becaused by dose–response relationships between the stress symp-tom and the pollutant concentration. We assume that dependingon the level of contamination, there may be two mechanisms con-ducive to colonization of a phenanthrene-contaminated root zoneby degradative bacteria: through an increase in carbohydrate exu-dation at 10 mg kg−1 pollutant and through a decrease in the releaseof root-derived substances at 100 mg kg−1 pollutant.

It is known that plants increase root exudation of carboxylicacids in response to different environmental stresses (Jones, 1998).This increase is connected with the fact that carboxylic acids mod-ify soil conditions and, thus, the bioavailability to microbes andplants of deficient mineral nutrients, toxic metals, and even someorganic pollutants (Yang et al., 2001; Joner et al., 2002; White andKottler, 2002). Within this in mind, we speculate that carboxylicacids may be involved in plant—pollutant interactions. In contrastto what happens in soil, exudation of carboxylic acids in quartzsand may not affect the bioavailability of phenanthrene becauseof a lag of lipophilic compartments, as described by Joner et al.(2002). This may explain the absence of significant effect on theintensity of exudation of carboxylic acids observed in our study. Inaddition, the decrease in their share in the whole composition ofroot exudates (at 10 mg kg−1) suggests that carboxylic acids did notplay a key role in the sorghum—phenanthrene interactions. In spiteof the decrease in exudation of carboxylic acids (as well as sugarsand amino acids), caused by the 100 mg kg−1 phenanthrene con-centration, the dominance of carboxylic acids in the root exudatesin all treatments suggests their general role as a nutrient source

and promoter of co-oxidation of pollutants for soil microorganisms(Schwab et al., 1995). The importance of ranking of acids accordingto their exuded amounts and their specific effect on PAH degra-dation in the rhizosphere is not well understood yet (Rentz et al.,2004, 2005).

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20 A. Muratova et al. / Environmental an

In all treatments, the share of amino acids in the root exu-ates was the lowest and was not changed significantly underhe influence of phenanthrene. Among the identified amino acids,ryptophan (a precursor of auxin phytohormones), asparagines,nd alanine (which affect auxin production in the rhizosphere)Frankenberger and Arshad, 1995) were of particular interest. Theryptophan and asparagine contents in the root exudates releasedy the tested sorghum cultivar were relatively small but the ala-ine content was largest in all treatments. On the basis of thebtained data, we assume that the tested cultivar of S. bicolorL.) Moench could be competent for bacterial auxin productionnd, thus, the application of plant-growth-promoting rhizobacte-ia (PGPR) to enhance the phytoremediation of PAH-contaminatedoils with this plant seems promising.

In our study of sorghum root exudates, we did not analyzehenolics—another important class of compounds released by thelant roots, the importance of which for phytoremediation of PAH-ontamination has been noted by some researchers (Fletcher andegde, 1995; Singer et al., 2003; Narasimhan et al., 2003). How-ver, the low proportion of these compounds in the plant rootroducts suggests that easily degradable substrates, such as organiccids, amino acids, and sugars, might be more readily accessible foricrobial degradation of pollutants, masking potential inductionechanisms by phenolics (Rentz et al., 2004). The root exudation of

econdary compounds is believed to be important for the establish-ent of microbial degraders in the rhizosphere of plants growing in

AH-contaminated soil. The plant exudates may contain moleculeshat can be used by microorganisms not only as a substrate but alsos factors that induce PAH biodegradation. Therefore, further inves-igations of the role of root exudates in phytoremediation shouldlso include compounds derived from secondary metabolism.

Under the influence of phenanthrene, the exudation of pro-ein decreased as well. The decrease in the protein content wasccompanied by an increase in the activity of oxidoreductases. Wepeculate that the most of the proteins released were enzymes.

Analysis of the enzymatic activity of sorghum root exudatesevealed three oxidoreductases—oxidase, peroxidases, and tyrosi-ase. The main oxidative system in the sorghum root exudatesas that of peroxidase, which has also been shown for otherlant species (Gramss and Rudeschko, 1998; Gramss, 2000; Criquett al., 2000). According to Criquet et al. (2000), the expressionf 2,7-diaminofluorene peroxidases in the rhizosphere and rootsf lucerne was stimulated by the PAH anthracene, added to therowth medium at 500 mg kg−1. In our study, the activities of per-xidases and another oxidoreductase enzyme, tyrosinase, werelso clearly progressive to the increase in the substrate phenan-hrene concentration. Involvement of peroxidase and tyrosinasento the conversion of PAHs and PAH derivatives was previouslytudied by Günther et al. (1998) and by Kraus et al. (1999). Günthert al. (1998) showed that extracellular monophenol oxygenases,uch as mushroom tyrosinase and horseradish peroxidase, do notatalyze conversions of unsubstituted PAHs, but the enzyme ortho-ydroxylated PAH phenols. On the basis of these findings, weuppose that the increase in exudation of these enzymes mayave been caused by the appearance of hydroxylated PAH deriva-ives (e.g., of microbial origin) in the rhizosphere of sorghum. Theevealed enzymatic activity of the root exudates of sorghum mayndicate the participation of plant oxidoreductases released by theoots in the rhizospheric degradation of PAHs and/or their deriva-ives.

Thus, in this work, priority data on the effect of the three-

ing PAH phenanthrene on root exudation in S. bicolor (L.) Moenchave been obtained. We have shown that contamination withhenanthrene at 100 mg kg−1 inhibited accumulation of plantiomass, causing decreases in carboxylic acid, carbohydrates, andmino acids in the rhizosphere of sorghum. A low concentra-

erimental Botany 66 (2009) 514–521

tion of phenanthrene (10 mg kg−1) did not effect significantly onplant growth and total amount of root exudation, but stimu-lated a release of carbohydrates. These were accompanied byincreases in the activity of oxidoreductases such as peroxidase,tyrosinase, and oxidase released by the plant roots and in thenumber of phenanthrene-degrading microorganisms. The observedquantitative changes in the ratio between the main root-exudatecomponents may manifest adaptive alterations occurring in theplant as a response to pollutant stress. The increased pollutant-degrading microbial population and activity of extracellular rootenzymes are presumed to make an important contribution to therhizodegradation of PAHs. This is confirmed by the reduction inphenanthrene content in the sorghum root zone. Study of theeffect of the detected individual root-exudate components on thebiodegradation of phenanthrene in the rhizosphere of sorghum,both under the influence of plant enzymes and with the assistanceof rhizospheric microorganisms, is the purpose of further research.

The choice of quartz sand for cultivation of sorghum plantsallowed us to reveal precisely PAH-dependent plant responses. Weassume that the above-described responses of the plants to PAHcontamination under natural conditions should be the same as inquartz sand, but the effect should be less marked, first of all becauseof soil organic matter, and it should be observed at higher PAHconcentrations. This may be connected with (i) increased root exu-dation due to enhanced plant biomass accumulation in soil and/orincreased exudates efflux under the influence of soil organic matter(Haller et al., 1985; Canellas et al., 2008) and (ii) decreased in phy-totoxicity of PAHs in soil due to the binding of soil organic matterto PAHs (Chiou, 1998). In addition, alterations in the ratio betweenthe main root-exudate constituents affected by soil organic mattermay be expected in natural soil (Canellas et al., 2008).

Acknowledgements

The authors gratefully acknowledge support from theDeutscher Akademischer Austauschdienst (DAAD, Leonhard-Euler-Stipendienprogramm) for making this collaborative work possible.We thank Dr. Natalia Pozdnyakova (IBPPM RAS) for her kindassistance in the analysis of enzymatic activity of the root exudates.

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