Plant Responses to Arsenic: the Role of Nitric Oxide

Plant Responses to Arsenic: the Role of Nitric Oxide

Fernanda S. Farnese & Juraci A. de Oliveira &

Grasielle S. Gusman & Gabriela A. Leão &

Cleberson Ribeiro & Luhan I. Siman & José Cambraia

Received: 25 February 2013 /Accepted: 16 July 2013 /Published online: 6 August 2013# Springer Science+Business Media Dordrecht 2013

Abstract Arsenic (As) toxicity and the effects of nitricoxide (NO), supplied as sodium nitroprusside (SNP),were analyzed in Pistia stratiotes. The plants, whichwere grown in nutrient solution at pH 6.5, were exposedto four treatments for 24 h: control; SNP (0.1 mg L−1);As (1.5mg L−1); and As + SNP (1.5 and 0.1 mg L−1). Asaccumulated primarily in the roots, indicating the lowtranslocation factor of P. stratiotes. The As accumula-tion triggered a series of changes with increasing pro-duction of reactive oxygen intermediates and damage tocell membranes. The application of SNP was able tomitigate the harmful effects of As. This attenuation wasprobably due to the action of the SNP as an antioxidant,reducing the superoxide anion concentration, and as asignaling agent. Acting as a signal transducer, SNPincreased the activity of enzymatic antioxidants (POX,CAT, and APX) in the leaves and stimulated the entirephytochelatins biosynthetic pathway in the roots (in-creased sulfate uptake and synthesis of amino acids,

non-proteinthiols, and phytochelatins). The As alsostimulated the phytochelatins biosynthesis, but this ef-fect was limited, probably because plants exposed onlyto pollutant showed small increments in the sulfateuptake. Thus, NO also may be involved in gene regula-tion of sulfate carriers.

Keywords Antioxidants . Toxicity . Phytochelatins .

Cellular Signaling . Sulfate Uptake

1 Introduction

Arsenic (As) occurs naturally in the earth’s crust andhas been detected at toxic concentrations in water andsoil; primarily reflecting the anthropic actions. Due tothe high toxicity and increasing environmental concen-trations of As, many research studies have beenconducted to control and/or remove this pollutant fromthe environment. Among the techniques developed,the use of accumulating plants has been highlightedas a simple and viable solution (Yang et al. 2005).Several plant species have effectively demonstratedthe phytoremediation of As-contaminated environ-ments, and the aquatic macrophyte Pistia stratiotes L.(Araceae) has been identified as a good candidate inaquatic environments (Mufarrege et al. 2010).

The chemical form of As in surface water is arsenate(AsV) (Mandal and Suzuki 2002), and because of itschemical similarity to phosphate, plants easily absorb

Water Air Soil Pollut (2013) 224:1660DOI 10.1007/s11270-013-1660-8

F. S. Farnese :G. A. LeãoDepartment of Plant Biology, Federal University of Viçosa,Viçosa, Brazil

J. A. de Oliveira (*) : C. Ribeiro : L. I. Siman : J. CambraiaDepartment of General Biology,Federal University of Viçosa,Viçosa, Minas Gerais 36570-000, Brazile-mail: [email protected]

G. S. GusmanCollege of Pharmacy, Federal University of Minas Gerais,Belo Horizonte, Minas Gerais 31270-901, Brazil

AsV, causing direct and indirect cellular damage(Gusman et al. 2013). The indirect toxic effects of thisabsorption primarily result from the oxidative stresscaused by the increased production of reactive oxygenintermediates (ROIs) (Zhang and Qui 2007).

The increased production of ROIs alters the normalmetabolism of plants and might cause cell death,depending on the extent of ROI-induced damage(Zhang and Qui 2007). However, plants have developedmechanisms to mitigate these effects using enzymaticantioxidants, such as superoxide dismutase (SOD), per-oxidases (POX), catalases (CAT), ascorbate peroxidases(APX), as well as non-enzymatic antioxidants, such asglutathione and thiols (Singh et al. 2009). Another im-portant strategy is the chelation of heavy metals. One ofthe principal classes of heavy metal chelators known inplants are phytochelatins (PCs), a family of Cys-richpeptides and the most abundant class of non-proteinthiols (Zhang et al. 2012).

The activation of response mechanisms to heavymetals involves a complex network of stimuli andsignaling molecules, like hormones and the nitric oxide(NO) (Leitner et al. 2009). NO is a small molecule thatparticipates as a signal in several biochemical andphysiological processes in plants. Indeed, NO plays afundamental role in controlling physiological functionsduring plant growth and development besides mediat-ing plants responses to abiotic stresses, such as heavymetal toxicity (González et al. 2012). However, littleinformation is available concerning the role of NO inthe regulation of As-induced stress (Leterrier et al.2012). Therefore, the aim of this study was to examinethe role of NO in the attenuation of As-induced oxida-tive stress in P. stratiotes.

2 Material and Methods

Specimens of P. stratiotes collected in non-polluteddams at the Federal University of Viçosa, Viçosa,Minas Gerais State, Brazil, were used in all experi-ments. Plants of similar size (about 4.0 g fresh weight)were surface sterilized with 1 % sodium hypochloritefor 1 min and then extensively rinsed with running tapwater and demineralized water, and maintained indemineralized water for 24 h. Then, they were trans-ferred to polyethylene flasks with 10 L of Clark’snutrient solution, pH 6.5 (Clark 1975), and maintainedin a growth room with controlled temperature and

irradiance (25±2 °C; 230 μmol m−2 s−1), under a pho-toperiod of 16 h, for an adaptation period of 3 days.

After the adaptation period, plants were transferred to1.0-L polyethylene pots containing 0.5-L Clark’s nutri-ent solution, pH 6.5, with 1/2 of the full ionic strengthand exposed to four treatments: control (nutrient solutiononly), sodium nitroprusside (SNP) (0.1 mg L−1), As(1.5 mg L−1) and As + SNP (1.5 and 0.1 mg L−1),respectively. Sodium nitroprusside is a substance that iscommonly used in biochemical studies as an NO donor.The concentration of As chosen was the maximum con-centration at which the plant still showed positive growthrate (in higher concentrations the growth rate was nega-tive due the loss of root system) and the concentration ofSNP selected was one in which the index of tolerance toarsenic was approximately 50 %. The plants weremaintained under these conditions for 24 h.

2.1 Determination of Arsenic

The leaves and roots of P. stratiotes were separated,washed in deionized water, and placed in a conven-tional oven at 80 °C until a constant dry weight wasobtained. The dry plant material was crushed anddigested in a mixture of nitric acid and perchloric acid(Marin et al. 1993), and the concentrations of As wasdetermined through inductively coupled plasma emis-sion spectroscopy (Optima 3300 DV, Perkin-Elmer,Norwalk, CT). The accuracy of the method was veri-fied by analysis of certified reference materials. Thereference material, Lemna minor (an aquatic plant)(BCR-670), from the National Institute of Standardsand Technology (Gaithersburg, MD, USA), was usedfor As determination.

2.2 Concentration of Reactive Oxygen Intermediates

To determine the concentration of the superoxide anion(O2

−), 50 mg of leaf samples were incubated in anextraction medium consisting of 100-μM ethylenedia-minetetraacetic acid (EDTA) disodium salt, 20-μMNADH, and 20-mM sodium phosphate buffer, pH 7.8(Mohammadi and Karr 2001). The reaction was initi-ated by adding 100 μL of 25.2-mM epinephrine in0.1-N HCl. The samples were incubated at 28 °C understirring for 5 min. The absorbance was read at 480 nmfor 5 min. Superoxide anion production was assessedby determining the accumulated adenochrome, using

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the molar absorption coefficient of 4.0×103 M−1

(Boveris et al. 2002).The hydrogen peroxide (H2O2) concentration was

determined using 200 mg of leaf samples that werehomogenized in an extraction medium consisting of50-mM potassium phosphate buffer, pH 6.5, contain-ing 1-mM hydroxylamine and centrifuged at 10,000×gfor 15 min at 4 °C. Subsequently, 50-μL aliquots of thesupernatant were added to a reaction medium contain-ing 100-μM FeNH4SO4, 25-mM sulfuric acid,250-μM xylenol orange, and 100-mM sorbitol (Gayand Gebicki 2000). The samples were kept in the darkfor 30 min, and the absorbance was read at 560 nm.The H2O2 concentrations were estimated based on acalibration curve prepared with H2O2 standards.

2.3 Cellular Damage

The cellular damage was evaluated through an assess-ment of cell membrane integrity, quantifying electro-lyte leakage according to Lima et al. (2002). Leaf discsand root apices were obtained after each treatment,rinsed thoroughly in distilled water, and maintainedin 10 mL of distilled water in sealed flasks for 6 h atroom temperature. The electrolyte leakage was esti-mated from the electrical conductivity in the solutioncontaining the root apices and leaf discs using anelectrical conductivity meter (DM31, Digimed, SantoAmaro, Brazil). The conductivity was expressed as apercentage of the total conductivity measured afterincubating the vials at 90 °C for 2 h.

2.4 Assessment of Enzymes Activityof the Antioxidant System

To assess the activity of the antioxidants and metabo-lism enzymes, 0.3 g of the roots and leaf fresh matterwere homogenized in extraction medium comprising0.1-M potassium phosphate buffer, pH 6.8, 0.1-mMEDTA, 1-mM phenylmethanesulfonyl fluoride, and1 % polyvinylpyrrolidone (Peixoto et al. 1999). Thehomogenate was centrifuged at 12,000×g for 15 min at4 °C. The resulting supernatant was used as a crudeextract for the assessment of SOD, POX, APX, andCAT activities.

The SOD activity (SOD, EC 1.15.1.1) was mea-sured as the inhibition of p-nitro tetrazolium photore-duction according to the method of Giannopolitis andRies (1977). The enzymatic activity was expressed in

SOD units corresponding to the amount of enzymerequired to inhibit 50 % of the p-nitro blue tetrazoliumphotoreduction (Beauchamp and Fridovich 1971).

The POX activity (POX, EC 1.11.1.7) was assessedthrough the production rate of purpurogallin at 420 nmaccording to the proposed method of Nakano andAsada (1981) with a molar extinction coefficient of2.47 mmol−1 L cm−1. The enzymatic activity wasexpressed in micromoles purpurogallin min−1 g−1 freshweight (FW).

The APX activity (APX, EC 1.11.1.11) was assessedas the rate of ascorbate oxidation at 290 nm (Nakanoand Asada 1981) using a molar extinction coefficient of2.8 mmol−1 L cm−1. The enzymatic activity wasexpressed in micromoles ascorbate min−1 g−1 FW.

The CAT activity (CAT, EC 1.11.1.6) was estimatedthrough the decomposition of H2O2 during the first min-ute of the reaction at 240 nm (Havir and McHale 1987)using a molar extinction coefficient of 36 mol−1 L cm−1.The enzymatic activity was expressed in micromolesH2O2 min−1 g−1 FW.

2.5 Effect of Arsenic in Sulfate Uptake

Plants were transferred to 0.5-L polyethylene pots con-taining Clark’s nutrient solution, pH 6.5. Uptake ofsulfate by the plants was assessed by Claassen andBarber’s depletion method (Claassen and Barber1974). Twenty-four hours before evaluation of thekinetic constants, the nutrient solution was renewedat every 6 h to obtain the steady state uptake. Finally,the nutrient solution was renewed again, and half of thepots received 1.5 mg L−1of As, adding then 1.0 mL ofcarrier freeNa2

35SO4 (0.042 MBq mL−1). After theintroduction of the plants, the absorption solution wassampled periodically during a period of 12 h. Theamount of radioactivity in each aliquot, after additionof 10 mL of a liquid scintillation cocktail was deter-mined in a Beckman LS 6500 Scintillation System.The kinetic parameters Km and Vmax and the ratioVmax/Km were estimated by graphic-mathematical ap-proach suggested by Ruiz (1985).

2.6 Amino Acids Content

Amino acid content was determined by the method ofMoore and Stein (1948). 0.5 g of plant sample washomogenized in 10 mL of 80 % ethanol. The homog-enate was centrifuged 800 rpm for 10 min. One

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milliliter of the extract was taken in the test tube and1 mL of 0.1 N HCl was added to neutralize the sample.To this, 1 mL of ninhydrin reagent was added andheated for 20 min in a boiling water bath. Later, 5 mLof the diluent solution was added and heated again inwater bath for 10 min. The test tubes were cooled andread the absorbance at 570 nm in a spectrophotometer.

2.7 Glutathione, Thiols, and Phytochelatins Content

Extraction and estimation of total glutathione (GSH) wasperformed according to Griffith’s method (Griffith 1980).The leaf and root tissue was homogenized in 5 % (m/v)sulfosalicylic acid and the homogenate was centrifuged at10,000×g for 10 min. A volume of 1 ml of supernatantwas neutralized with 0.5 ml of potassium phosphatebuffer (pH 7.5). Total GSH content was measured byadding 1ml neutralized supernatant to a standard solutionmixture consisting of 0.5 ml of 0.1 M sodium phosphatebuffer (pH 7.5) containing 1-ml EDTA, 0.2 ml of 6-mM5,5-dithiobis-2-nitrobenzoic acid, 0.1 ml of 2-mMNADPH, and 0.1 ml of 1-U yeast GR Type III. Changein absorbance was measured at 412 nm and followed at25±2 °C until the absorbance reached 0.5 U.

To assess the thiols content, 0.3 g of the roots and leaffresh matter were homogenized in extraction mediumcomprising 0.1-M Tris–HCl buffer, pH 8.0, 1-mMEDTA, and 1 % ascorbic acid. The homogenate wascentrifuged at 10,000×g for 10 min at 4 °C. Theresulting supernatant was used for determination of total

thiols, protein thiols, and non-protein thiols (Sedlak andLindsay 1968). The estimated concentration of total PCswas calculated as PCs = non-protein thiols − total GSH(Hartley-Whitaker et al. 2001).

2.8 Statistical Analyses

The experiment was conducted as a completely ran-domized design with five replicates. The data wereanalyzed using ANOVA, and the means were calculat-ed using Tukey’s test at 5 % probability. The statisticalanalyses were conducted with the statistical programSAS (Cary, NC, United States).

3 Results

3.1 Plant Arsenic Concentration and TranslocationFactor

The plants exposed to As accumulated large amountsof the pollutant, and no changes were observed afterthe addition of SNP (Fig. 1). Arsenic accumulationoccurred mainly in the roots with little translocationto the leaves (Fig. 1).

3.2 Concentration of Reactive Oxygen Intermediates

In contrast to the values observed in the control, Asexposure increased the O2

− concentration in the leaves

Fig. 1 Concentration of ar-senic in the leaves (black)and roots (gray) of Pistiastratiotes. Means followedby the same uppercase letter,between treatments for thesame organ, and by the samelowercase letter, betweenorgans for the same treat-ment, were not significantlydifferent according toTukey’s test at 5 %probability


and roots of P. stratiotes, and SNP treatment had noeffect on the concentrations of this anion in the leavesor roots (Fig. 2a). The application of SNP in combina-tion with As, reduced the anion O2

− concentrationapproximately 31.8 and 10.7 % in the leaves and roots,respectively, compared with plants exposed to As only.

The H2O2 concentration was also increased in theleaves and roots of plants after exposure to As, and thisincrease was higher in the roots (Fig. 2b). The SNPtreatment alone had no effect on the H2O2 concentration.However, the application of this exogenous source of NO

(SNP) to As-treated plants induced a 28.5 and 21.7 %reduction of the H2O2 concentration in the leaves androots, respectively.

3.3 Electrolyte Leakage

Plant treatments with As exhibited increased electricalconductivity, which is indicative of increased electrolyteleakage (Fig. 3). There was no difference between thedamage generated in the roots and leaves, despite theelevated As concentration in the roots. Treatment with

Fig. 2 Effect of arsenic, alone or in combination with SNP, onthe concentration of superoxide anion (a) and hydrogen peroxide(b) in the leaves (black) and roots (gray) ofPistia stratiotes. Meansfollowed by the same uppercase letter, between treatments for the

same organ, and by the same lowercase letter, between organs forthe same treatment, were not significantly different according toTukey’s test at 5 % probability

Fig. 3 Electrolyte leakagein the leaves (black) androots (gray) of Pistiastratiotes. Means followedby the same uppercase letter,between treatments for thesame organ, and by the samelowercase letter, betweenorgans for the same treat-ment, were not significantlydifferent according toTukey’s test at 5 %probability


SNP alone had no effect on electrolyte leakage; however,when added to plants in combination with As, this treat-ment attenuated the As damage in the leaves and roots.

3.4 Antioxidant Enzyme Activity

The activities of SOD, CAT, and POX significantlyincreased in the leaves of As-treated plants (Fig. 4).However, only increased SOD activity was observed inthe roots of plants exposed to this pollutant (Fig. 4a).The application of SNP alone had no effect on enzymeactivity compared with the control. However, the As-treated plants exposed to SNPmaintained SOD activitysimilar to the control. In contrast, the activities of POX,APX, and CAT were increased after the combinedtreatment with As and SNP, showing higher enzymatic

activity than that observed in plants exposed only to As(Fig. 4b–d). The increment in the activity of theseenzymes was much more intense in leaves. In fact,the greatest increase in enzymatic activity was observed

Fig. 4 Activities of SOD (a), POX (b), APX (c), and CAT (d) inthe leaves (black) and roots (gray) of Pistia stratiotes. Meansfollowed by the same uppercase letter, between treatments for

the same organ, and by the same lowercase letter, betweenorgans for the same treatment, were not significantly differentaccording to Tukey’s test at 5 % probability

Table 1 Estimated values of Vmax (in micromoles per hour pergrain fresh weight) and Km (in mole per liter) for absorption ofsulfate in Pistia stratiotes

Treatments Vmax (μmol h−1 g−1 FW) Km (mol L−1)

Control 0.13 c 9.78 a

SNP 0.11 c 11.3 a

As 0.19 b 8.61 a

As + SNP 0.27 a 8.93 a

Means followed by the same letter were not significantly differ-ent according to Tukey’s test at 5 % probability


with CAT in the leaves of plants exposed to As alone orin combination with SNP. However, the CAT activityremained unaltered in the roots of these plants.

3.5 Effect of Arsenic in Sulfate Uptake

The kinetic constants of sulfate uptakewere changed afterAs treatment. The presence of the metalloid in combina-tion with SNP, however, intensified the increase of Vmax

for sulfate, but had no significant effect on Km (Table 1).

3.6 Effect of Arsenic in Glutathione, Amino Acids,Thiols, and Phytochelatins

The concentrations of GSH were decreased by As(Fig. 5), while the levels of amino acids (Fig. 6), totalthiols, non-protein thiols (Fig. 7a and b), and PCs (Fig. 8)increased. The application of SNP intensified all theresponses in roots, inducing changes even more signifi-cantly. In the leaves, the concentration of GSH and PCswere decreased while the concentration of protein thiolsincreased after the application of SNP (Fig. 7c).

Fig. 5 Effect of arsenic,alone or in combinationwith SNP, on the concentra-tion of total glutathione inthe leaves (black) and roots(gray) of Pistia stratiotes.Means followed by the sameuppercase letter, betweentreatments for the same or-gan, and by the same lower-case letter, between organsfor the same treatment, werenot significantly differentaccording to Tukey’s test at5 % probability

Fig. 6 Effect of arsenic,alone or in combination withSNP, on the concentration ofamino acid in the leaves(black) and roots (gray) ofPistia stratiotes. Meansfollowed by the same up-percase letter, betweentreatments for the same or-gan, and by the same lower-case letter, between organsfor the same treatment, werenot significantly differentaccording to Tukey’s test at5 % probability


Fig. 7 Concentration of total thiols (a), non-protein thiols (b), andprotein thiols (c) in the leaves (black) and roots (gray) of Pistiastratiotes. Means followed by the same uppercase letter, between

treatments for the same organ, and by the same lowercase letter,between organs for the same treatment, were not significantlydifferent according to Tukey’s test at 5 % probability

Fig. 8 Effect of arsenic,alone or in combination withSNP, on the concentration ofphytochelatins in the leaves(black) and roots (gray) ofPistia stratiotes. Meansfollowed by the same up-percase letter, betweentreatments for the same or-gan, and by the same lower-case letter, between organsfor the same treatment, werenot significantly differentaccording to Tukey’s test at5 % probability


4 Discussion

The results of the present study suggest that the macro-phyte P. stratiotes L. (Araceae), compared with otheraquatic species, has great potential for the phytore-mediation of aquatic environments polluted with As.Besides the large accumulation of As, it was observedthat P. stratiotes has high tolerance to this pollutant.

Most of the absorbed As was retained in the roots ofP. stratiotes, as demonstrated by a translocation factorvalue lower than 1, indicating great retention in theroots and lowmetalloid translocation to the shoots. Thetranslocation factor depends not only on the plantspecies but also on the absorbed toxic element. P.stratiotes plants exposed to Hg, for example, showeda translocation factor of four times greater than thatobserved with As (Mishra et al. 2009). The ability tomaintain the toxic element in the roots is considered asa key mechanism of adaptation to contaminated areas(Mishra et al. 2009) and typically reflects the presenceof molecules rich in –SH groups, which chelate As andprevent the translocation of this pollutant to the leaves(Singh and Agrawal 2007). In fact, high concentrationsof thiols were observed in roots.

Given the difficulty of removing the roots from thesoil, pollutant retention in the roots might be problematicfor the phytoremediation of land environments. Howev-er, in aquatic environments, this problem is minimizedthrough the removal of the entire plant from the environ-ment. Furthermore, the roots are more tolerant to As thanthe leaves, despite showing higher pollutant concentra-tions, suggesting that changes in lipid peroxidation in theroots are lower than those observed in the shoots.

The P. stratiotes plants exposed to As experiencedoxidative stress, as demonstrated by the increase in ROIsconcentration and membrane damage, causing electro-lyte leakage. The increase in ROIs production mightreflect the conversion of arsenate into arsenite, which ispart of the mechanism of pollutant tolerance (Mehargand Hartley-Whitaker 2002) through the direct reductionof molecular oxygen or the alteration of proteins in themitochondria, chloroplasts, and peroxisomes (Zhang andQui 2007). The toxic effect of As, however, was attenu-ated after the addition of NO (supplied as SNP), whichacted as both a direct antioxidant, eliminatingO2

−, and asa signal transducer, enhancing the response of enzymaticantioxidants and the sulfate uptake.

With certain types of abiotic stress, SNP acts as acellular signal transducer to increase SOD activity and

thereby reduces the concentration of O2− (Arasimowicz

and Floryszak-Wieczorek 2007). In P. stratiotes, how-ever, no significant changes in SOD activity were ob-served in the presence of As and SNP, suggesting thatSNP acted directly as an antioxidant (Singh et al. 2009;Xiong et al. 2010). For CAT, POX, and APX, the NOreleased from SNP acted as a signal transducer (Singhet al. 2009), inducing an increase in the enzyme activityand a reduction in the H2O2 concentration. These in-creases were much more significant in the leaves thanthat in the roots.

The activity of antioxidant enzymes is essential toeliminate the ROIs, which are an indirect and harmfuleffect of absorption of As. However, in addition torepairing the damage caused by As, plants resistant topollutants also must provide mechanisms to inactivatethe toxic compound, thus preventing further damage(Zhang et al. 2012). The main strategy for As detoxifi-cation in plant cells is based on chelation by PCs andsubsequent compartmentalization of the As–PCs com-plex (Zhang et al. 2012).

PCs are the most abundant class of non-protein thiolsand their synthesis involves the participation of aminoacids and glutathione (Zhang et al. 2012). In the presentinvestigation, the increases in the levels of amino acids,non-protein thiols and PCs, as well as depletion of GSHpools, suggests a stimulation of the entire PC biosyn-thetic pathway by the NO in the roots. These resultswere not observed in the leaves, where the major modeof action of SNP was the increment in enzyme activity.Although the As also has been able to stimulate the PCsbiosynthetic pathway in the roots and in the leaves, theobserved increase was limited and was not able toprevent the damage triggered by the pollutant. Thedefense mechanism involving PCs results in an increas-ing demand for sulfur, making necessary increases insulfate uptake (Nocito et al. 2006). The plants exposedonly to As showed small increments in sulfate uptake,which was probably the main factor that limited thesynthesis of PCs. Transcriptional regulation of the genesencoding high-affinity sulfate transporters is linked tothe availability of sulfate, the demand for reduced sulfurcompounds, and the supply of a C/N skeleton precursorneeded in the assimilatory pathway (Nocito et al. 2006).Our results suggest that NO may be involved in generegulation of carriers sulfate, since the NO was able toincrease the uptake of this ion.

Taken together, this data suggests that exogenous NOmitigates As-induced damage in P. stratiotes plants. The


beneficial effects of NO most likely reflect the directelimination of ROIs, the increased activity of enzymesin the antioxidant system, and the stimulation of the PCbiosynthetic pathway. In the latter case, NO is probablyinvolved in the up-regulation of genes involved in theabsorption of sulfate.

Acknowledgments The authors are grateful to the UniversidadeFederal de Viçosa, CNPq, and FAPEMIG.

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Plant Responses to Arsenic: the Role of Nitric Oxide