Environmental and Experimental Botany 65 (2009) 403–409

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Environmental and Experimental Botany

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Ecophysiological responses of water hyacinth exposed to Cr3+ and Cr6+

Luisa Brito Paivaa, Jurandi Goncalves de Oliveirab, Ricardo A. Azevedoc,Douglas Rodrigues Ribeiroa, Marcelo Gomes da Silvad, Angela P. Vitóriaa,∗

a Laboratório de Ciências Ambientais, Centro de Biociência e Biotecnologia, Universidade Estadual do Norte Fluminense, Brazilb Laboratório de Melhoramento Genético Vegetal, Centro de Ciências e Tecnologias Agropecuárias, Universidade Estadual do Norte Fluminense, Brazilc Departamento de Genética, Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, Brazild Laboratório de Ciências Físicas, Centro de Ciência e Tecnologia, Universidade Estadual do Norte Fluminense, Brazil

a r t i c l e i n f o

Article history:Received 31 October 2008Accepted 29 November 2008

Keywords:ChromiumEichhornia crassipesEcophysiologyPhotosynthesisChlorophyllHeavy metals

a b s t r a c t

Due to its wide industrial use, chromium (Cr) is considered a serious environmental pollutant of aquaticbodies. In order to investigate the ecophysiological responses of water hyacinth [Eichhornia crassipes(Mart.) Solms] to Cr treatment, plants were exposed to 1 and 10 mM Cr2O3 (Cr3+) and K2Cr2O7 (Cr6+)concentrations for two or 4 days in a hydroponic system. Plants exposed to the higher concentrationof Cr6+ for 4 days did not survive, whereas a 2 days treatment with 1 mM Cr3+ apparently stimulatedgrowth. Analysis of Cr uptake indicated that most of the Cr accumulated in the roots, but some wasalso translocated and accumulated in the leaves. However, in plants exposed to Cr6+ (1 mM), a highertranslocation of Cr from roots to shoots was observed. It is possible that the conversion from Cr6+ toCr3+, which immobilizes Cr in roots, was not total due to the presence of Cr6+, causing deleterious effectson gas exchange, chlorophyll a fluorescence and photosynthetic pigment contents. Chlorophyll a wasmore sensitive to Cr than chlorophyll b. Cr3+ was shown to be less toxic than Cr6+ and, in some caseseven increased photosynthesis and chlorophyll content. This result indicated that the Fv/F0 ratio wasmore effective than the Fv/Fm ratio in monitoring the development of stress by Cr6+. There was a linear

relationship between qP and Fv/Fm. No statistical differences were observed in NPQ and chlorophyll a/bratio, but there was a tendency to decrease these values with Cr exposure. This suggests that there werealterations in thylakoid stacking, which might explain the data obtained for gas exchanges and other

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. Introduction

In most natural environments, the heavy metal content of theoil and water is low and does not cause significant phytotoxicity.owever, the increasing contamination and subsequent accumula-

ion of heavy metals in the environmental, due to human activitiesuch as mining, extensive use of fertilizers and sewage waste pro-uction may have serious consequences for normal plant growthVernay et al., 2007).

Heavy metal phytotoxicity is controlled by a number of factors,ncluding the element’s uptake site, bioavalability, competition forinding sites and ionic speciation (Ralph and Burchett, 1998; Pandand Choudhury, 2005). This phytotoxicity can lead to the produc-

ion of reactive oxygen species (ROS), which can be dismutated byntioxidant enzymes (Vitória et al., 2001; Gratão et al., 2005; Tamást al., 2008). Krupa and Basynski (1995) discussed some hypothesesoncerning the possible mechanism of heavy metal toxicity on pho-

∗ Corresponding author. Tel.: +55 22 27261475; fax: +55 22 27261472.E-mail address: apvitoria@uenf.br (A.P. Vitória).

098-8472/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.envexpbot.2008.11.012

ameters.© 2008 Elsevier B.V. All rights reserved.

tosynthesis and presented a list of key enzymes of photosyntheticcarbon reduction, which were inhibited in heavy metal-treatedplants.

Anthropogenic activities have led to Cr contamination in aquaticand terrestrial ecosystems. Cr is the seventh most abundant metal inthe earth’s crust (Panda and Choudhury, 2005). Cr occurs in severaloxidation states ranging from Cr2+ to Cr6+, with the trivalent (Cr3+)and hexavalent (Cr6+) states being the most stable and common.Cr6+ usually occurs associated with oxygen as chromate (CrO2−

4)or dichromate (Cr2O2−

7) and is considered to be more mobile andtoxic than Cr3+, which on the other hand, is less soluble in waterand is required in trace amounts as an inorganic nutrient for ani-mals (Lien et al., 2001). Cr6+ and Cr3+ are taken up by plants andmany organisms (anaerobic bacteria and plants). Plants are capableof reducing Cr6+ to Cr3+ and there is also evidence that no conver-sion occurs for Cr species in the nutrient solution before uptake

by plant roots (Shanker et al., 2005). Although Cr3+ is less toxicthan Cr6+, it too induces oxidative stress (Panda and Choudhury,2005).

Cr phytotoxicity can result in inhibition of nutrient balance,changes in antioxidant enzymes activities, degradation of pigment,

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lteration of chloroplast and membrane ultrastructure, decrease inO2 assimilation and modification of chlorophyll a fluorescencearameters (Vajpayee et al., 2000; Panda and Choudhury, 2005;rduini et al., 2006; Vernay et al., 2007). Cr stress can also affecthotosynthesis in terms of CO2 fixation, electron transport, pho-ophosphorylation and enzyme activities (Shanker et al., 2005).

aintaining heavy metals (e.g. Cr) in the root system appearso maintain the level of biomass production and reduce harmfulymptoms in photosynthesis (Soltan and Rashed, 2003). Decreasesn total chlorophyll, chlorophyll a and b, and carotenoids haveeen well documented under Cr stress in plants (Panda and Khan,003; Vernay et al., 2007). Carotenoids and chlorophyll absorbadiant energy and part of this is emitted as chlorophyll fluores-ence. The proportion of radiant energy emitted in the form ofuorescence is low under plant optimum conditions. However, inany situations, fluorescence increases under stress conditions,

nd there are also changes in the characteristics related to fluo-escence.

In terrestrial plants, the negative action of Cr on photosyn-hesis is well documented (Shanker et al., 2005; Vernay et al.,007), whilst for aquatic plants their potential in removing met-ls ions from aquatic environments has received more attentionLu et al., 2004; Mangabeira et al., 2004). A group of plantpecies (termed hyperaccumulators) have the ability to accumu-ate non-essential metals, such as Cr, and apparently do not showamage. Water hyacinth [Eichhornia crassipes (Mart.) Solms] isfloating macrophyte hyperaccumulator species native of Southmerica that, due to its fast growth and large biomass produc-

ion, is particularly useful in the phytoremediation process andonitoring of heavy metals in aquatic environments (Lu et al.,

004).Disorganization of the chloroplast ultrastructure and inhibition

f electron transport processes due to Cr and a diversion of electronsrom the electron-donating side of PSI to Cr6+ is a possible explana-ion for the Cr-induced decrease in photosynthetic rate (Shanker etl., 2005). There is little reported evidence of a correlation betweenSII activity, CO2 assimilation and heavy metal accumulation underonditions of excess Cr in aquatic plants. Moreover, most reportsn Cr in plants have concentrated on its effects on growth, uptake,oxicology and translocation. We investigated the effect of addedr6+ and Cr3+ on the responses of the photosynthetic apparatus

n water hyacinth, both in C02 assimilation, as measured by leafs exchanges, and for the function of photosynthetic apparatus,s assessed by chlorophyll a fluorescence. Additionally, photosyn-hetic pigment contents and Cr translocation from roots to shootsere simultaneously analyzed.

. Material and methods

.1. Plant material and chromium treatments

Water hyacinth [Eichhornia crassipes (Mart.) Solms] samplesere collected from the Imbé River located at 21◦01′ 08′′ S,

4◦19′52′′W in the southeast region of Brazil, in May, June anduly 2007 and transferred to 10 L capacity polyethylene pots con-aining 7 L of nutrient solutions (Hoagland and Arnon, 1950) in alasshouse. The pots were placed in a randomized position and twoifferent forms of Cr were used in the experiments: Cr2O3 (Cr3+)nd K2Cr2O7 (Cr6+) at 1 and 10 mM concentrations for 0, 2 and 4

ays. The plants were grown under greenhouse conditions with ahotoperiod of 11 h (light period) and 13 h of darkness, mean tem-eratures of 25 ◦C during the day (light) time and 18 ◦C during theight (dark) time. The relative humidity was kept at 70 ± 5% andphotosynthetic photon flux density (PPFD) of 1500 �mol m−2 s−1

as used.

imental Botany 65 (2009) 403–409

2.2. Chromium analysis

After 4 days of Cr exposure, plants samples were washed gen-tly and exhaustively with distilled–deionized water to removeadsorbed culture medium. Plants were divided into roots andleaves, frozen in liquid nitrogen and freeze-dried for 48 h (LAB-CONCO 260337 Freeze Dry System). All plant parts were digested asdescribed by Klumpp et al. (2002) and resuspended in 0.5 M HNO3.An atomic absorption spectrometer (AA-120 Varian Techtron) wasused to determine the Cr content. The values were expressed in�g g−1 dry matter.

2.3. Chlorophyll a fluorescence measurement

The measurements of the chlorophyll a fluorescence werecarried out between 9:30 a.m. and 11:00 a.m. using a pulseamplitude modulation fluorimeter (FMS2, Hansatech InstrumentsLtd., Norfolk, UK). Ten intact and healthy leaves from each treat-ment were kept in the dark for 30 min and then exposed tothe weak, modulated beam light (approximately 6 �mol m−2 s−1

at 660 nm), followed by exposure for 0.8 s of high intensity(10000 �mol m−2 s−1) actinic white light, as adapted by Genty et al.(1989). The minimal fluorescence (F0), the maximum fluorescence(Fm) and extinction coefficients: qP (photochemical quenching) andNPQ (non-photochemical quenching) were measured. The vari-able fluorescence (Fv = Fm − F0), maximum quantum yield of PSII(Fv/Fm) and variable chlorophyll fluorescence ratio (Fv/F0) were cal-culated according to Van Kooten and Snel (1990). The values arepresented as the mean of ten measurements, representing ten repli-cates.

2.4. Gas exchange measurements

Net photosynthetic rate (Pn), stomatal conductance (gs)and substomatal CO2 concentration (Ci) were determined after0, 2 and 4 days after Cr exposure. The treatments weremeasured with a wearable infrared gas analyzer (Ciras 2, PP-System, UK) with clamp-on leaf cuvette that exposed 2.4 cm2

of leaf area. Light (PPFD), temperature and humidity were1800 �mol m−2 s−1, 25 ± 2◦C and 75%, respectively. CO2 wasmaintained at a constant level of 380 �mol mol−1. Light wasimposed using the Ciras 2 LED light source (PP-System). Eachmeasurement was carried out on five newly maturated leavesper plant and was repeated on five plants for each treatment(n = 5).

2.5. Photosynthetic pigments

Three discs were taken from each treatment and used toquantify the photosynthetic pigments. The three discs weresliced and placed in plastic tubes in the dark with a lidcontaining 5 ml dimethylsulfoxide reagent (DMSO) as organicsolvent. After 5 days, the extract was analyzed in a spec-trophotometer at wavelengths of 480 nm, 649 nm and 665 nm.The photosynthetic pigments were quantified for the sam-ples from 4 days using the equations by Wellburn (1994) forcarotenoids, chlorophyll a and chlorophyll b. The total chloro-phyll, chlorophyll a/b and total chlorophyll/carotenoid ratioswere calculated. All the laboratory procedures were carriedout in a low light environment. The values were expressed in�mol cm−2.

2.6. Statistical analysis

The results of the Cr accumulation, chlorophyll a fluorescencevariables, gas exchange and photosynthetic pigments were ana-

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yzed by one-way analyses of variance (ANOVA), and the meansere compared using Tukey’s test (P < 0.05).

. Results

.1. Visual aspects

The visual aspects observed in water hyacinth exposed to the dif-erent oxidation states and concentrations of Cr are shown in Fig. 1.lants exposed to 10 mM Cr6+ for 4 days did not survive (Fig. 1J),hereas treatment for 2 days with 1 mM Cr3+ apparently stimulated

rowth (Fig. 1C and D).

ig. 1. Visual aspects of water hyacinth exposed to Cr6+ and Cr3+. Left column, 2 days of C) plants exposed to 1 mM Cr3+, (E and F) plants exposed to 10 mM Cr3+, (G and H) plants

imental Botany 65 (2009) 403–409 405

3.2. Chromium contents

Analysis of Cr accumulation in water hyacinth revealed a higherconcentration in the roots, when compared to the shoots (Table 1).Cr uptake was also shown to be higher for Cr6+ even at 1 mM, andthe translocation rate from roots to the shoots was also significantlyhigher for Cr6+ than for Cr3+ (Table 1).

3.3. Gas exchange measurements

The responses of net photosynthetic rate (Pn), stomatal con-ductance (gs) and substomatal CO2 concentration (Ci) in the Cr

r exposure and right column, 4 days of Cr exposure. (A and B) control plants, (C andexposed to 1 mM Cr6+, (I and J) plants exposed to 10 mM Cr6+.

406 L.B. Paiva et al. / Environmental and Exper

Table 1Concentration of Cr (�g g−1 dry matter) in water hyacinth exposed to 1 mM and10 mM Cr3+ for 2 days and 1 mM Cr6+ for 4 days. Upper case letters: same treatmentamong the different plant parts. Lower case letters: comparison among differenttreatments in the same plant part. Values followed by different letters differ signif-icantly (P < 0.05).

Plant parts Treatments

SR

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Control 1 mM Cr3+ 10 mM Cr3+ 1 mM Cr6+

hoots 3 Bb 3 Bb 51 Bb 1560 Baoots 142 Ab 417 Ab 1258 Ab 3475 Aa

tressed plants are shown in Fig. 2. Cr6+ were more harmfulhan Cr3+ to the photosynthetic process. After 2 days of expo-ure to Cr6+, a large decrease in Pn (20.97 �mol CO2 m−2 s−1

n the control; 3.27 �mol CO2 m−2 s−1 at the 1 mM Cr6+ and1.43 �mol CO2 m−2 s−1 at the 10 mM Cr6+) and an increase

n Ci (269 �mol mol−1 in the control; 306 �mol mol−1 at themM Cr6+ and 332 �mol mol−1 at the 10 mM Cr6+), were

bserved (Fig. 2A and B, respectively). However, plants exposedor 2 days to Cr3+(1 and 10 mM) exhibited a better Pn20.97 �mol CO2 m−2 s−1 in the control; 27.53 �mol CO2 m−2 s−1

nd 22.3 �mol CO2 m−2 s−1, respectively). On the fourth day, the

ig. 2. Effects of Cr3+ and Cr6+ concentrations on the net photosynthetic rateA), substomatal CO2 concentration (B) and stomatal conductance (C) of wateryacinth leaves. Plants were cultivated with 0 (©), 1 mM Cr3+ (�), 10 mM Cr3+ (�),mM Cr6+ (�) and 10 mM Cr6+ (�) for 2 and 4 days. Pn = net photosynthetic rate,i = substomatal CO2 concentration, gs = stomatal conductance. Each point repre-ents the means ± S.E. (n = 5).

imental Botany 65 (2009) 403–409

Pn of 1 mM Cr3+ treated plants (23.97 �mol CO2 m−2 s−1) remainedgreater than the control (20.33 �mol CO2 m−2 s−1), but at 10 mMCr3+ treatment, the Pn decreased from 20.33 �mol CO2 m−2 s−1

(control) to 18.5 �mol CO2 m−2 s−1. With regard to 4-day 1 mMCr6+ treated plants, a decrease was observed resulting in nega-tive values of Pn (from 20.33 �mol CO2 m−2 s−1 in the control to−1.23 �mol CO2 m−2 s−1), showing more respiratory than photo-synthetic activity.

Plant responses of gs (Fig. 2C) to the different Cr oxidationstates and concentrations revealed increases in all treatments atthe second day and decreases in all treatments on the fourthday of treatment, although statistically there was no differencebetween the control and Cr3+ treatments on the fourth day oftreatment. The 1 mM Cr6+ at 4 days of treatment also showed adecrease in gs (488 mM H2O m−2 s−1), when compared to the con-trol (1.134 mM H2O m−2 s−1).

3.4. Chlorophyll a fluorescence parameters analysis

In parallel to the measurement of gas exchange, the photochem-ical parameters of photosynthesis were analyzed after zero, 2 and4 days in response to Cr exposure (Table 2). The F0, Fm, Fv andFv/Fm ratios were statistically stable for the majority of the treat-ments. However, Cr6+ treatments, particularly at 10 mM, resulted ina significant decrease in the photochemical parameters.

The variable chlorophyll fluorescence ratio (Fv/F0) was more sen-sitive to Cr6+ treatments than Fv/Fm (Table 2). No alterations in theFv/Fm ratio at 1 or 10 mM Cr3+ treatments were observed when com-pared to the control (Table 2), whilst a decrease in the 1 mM Cr6+

treatment (from 0.82 – control to 0.68 after 4 days) was verified,which was more pronounced at 10 mM Cr6+ (from 0.83 to 0.38, after2 days).

A significant decrease in Fv/F0 ratio values was observed for allplants treated with both concentrations of Cr6+ studied. However,the decrease was remarkable 2 days after an application of Cr6+ con-sidering 10 mM concentration. On the other hand, Cr3+ treatmentsexhibited contrasting effects when compared to the control withincreases, that were not statistically significant, in the Fv/F0 ratio inCr3+ treatments (1 and 10 mM).

The chlorophyll fluorescence quenching analysis parametersshowed a decrease in qP. The reduction in qP was observed only for10 mM Cr6+. For non-photochemical quenching (NPQ), no statisti-cal difference was observed (Table 2), but it is possible to observea tendency towards decrease after all treatments (Cr3+ and Cr6+) atday 2 and a slight increase at day 4, but always below that of day 0.

3.5. Photosynthetic pigment contents

Table 3 presents the photosynthetic pigment contents. Sig-nificant differences were observed in the chlorophyll and totalchlorophyll contents at 1 mM Cr6+ after 4 days. Although therewas an approximately 50% lower chlorophyll a/b ratio at 1 mMCr6+ treatment (1391 �mol cm−2), when compared to the control(2604 �mol cm−2), statistically we were been unable to con-firm this differences. For all photosynthetic pigments (chlorophyllb, carotenoids and other ratios), no statistical differences wereobserved. However, morphologically it was possible to verify thatthe Cr6+ treatment plants exhibited a clear reduction in green color(Fig. 1).

4. Discussion

Water hyacinth plants responded to the Cr6+ supply in nutri-ent solution by developing visible symptoms of stress, such as leafchlorosis, petiolar chlorosis and necrosis (Fig. 1G–J). On the other

L.B. Paiva et al. / Environmental and Experimental Botany 65 (2009) 403–409 407

Table 2Chlorophyll a fluorescence of water hyacinth leaves (adapted to the dark) exposed to 1 mM and 10 mM Cr3+ and 1 mM Cr6+ for 2 and 4 days and 10 mM Cr6+ for 2 days. Valuesfollowed by different letters differ significantly (P < 0.05) for same parameter (n = 10).

Treatments Cr concentration (mM) Time (days) F0 Fm Fv Fv/Fm Fv/F0 qP NPQ

Control 0 0 274 A 1678 ABC 1479 AB 0.81 A 5.12 A 0.889 AB 0.358 A2 291 A 1639 ABC 1598 A 0.83 A 5.48 A 0.916 A 0.345 A4 290 A 1554 BC 1564 AB 0.82 A 5.19 A 0.900 A 0.336 A

Cr3+ 1 0 247 AB 1678 ABC 1312 AB 0.78 A 5.32 A 0.863 AB 0.349 A2 247 AB 1618 ABC 1371 AB 0.85 A 5.55 A 0.865 AB 0.169 A4 265 A 1843 A 1578 AB 0.86 A 5.95 A 0.924 A 0.181 A

Cr3+ 10 0 270 A 1560 BC 1276 B 0.82 A 5.53 A 0.888 AB 0.359 A2 260 A 1604 ABC 1344 AB 0.84 A 5.16 A 0.944 A 0.231 A4 263 A 1798 AB 1535 AB 0.85 A 5.82 A 0.882 AB 0.266 A

Cr6+ 1 0 268 A 1507 C 1352 AB 0.82 A 5.32 A 0.886 AB 0.349 A2 319 A 1722 ABC 1403 AB 0.81 A 4.41 A 0.924 A 0.167 A

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and, at 10 mM Cr3+, the plants developed symptoms of toxicityFig. 1F), as did plants that received Cr6+ treatments. This visi-le symptom of Cr3+ toxicity was verified by Barbosa et al. (2007)

n Genipa americana. Vernay et al. (2007), studying Lolium perene,bserved a noticeable wilting in these plants. Water hyacinth, how-ver, may have not exhibited wilting due to the fact that it is ayperaccumulator plant species.

Uptake and accumulation of Cr by various plant species are wellocumented (Shanker et al., 2005; Barbosa et al., 2007; Vernay etl., 2007). In this study, analysis of water hyacinth revealed a higheroncentration of Cr in the roots than in the shoots (Table 1). Thisreferential Cr accumulation was described by Barbosa et al. (2007)

n Genipa Americana, by L., Arduini et al. (2006) in miscanthus (Mis-anthus sinensis L.), and Mangabeira et al. (2004) in water hyacinth,s well as by a number of other authors. Soltan and Rashed (2003)nd Vesk and Allaway (1997) reported that this accumulation couldave been due to the co-precipitation of metals in the plaques of

ron (Fe) and manganese (Mn) on the roots.Many researchers have indicated that water hyacinth is a suit-

ble species for heavy metal accumulation in plants (Lu et al.,004; Mangabeira et al., 2004). Hyperaccumulator plants are con-entionally defined as species capable of concentrating more than000 ppm in aerial parts. In this study, water hyacinth exhib-ted 1560 �g g−1 Cr dry matter concentration in the leaves whenxposed for 4 days at 1 mM Cr6+. Other higher plants such asymphaea alba, Sutera fodina and Dicoma niccolifera have beenhown to be able to accumulate up to 2233, 2400 and 1500 �g g−1 Crry matter in the leaves, respectively (Vajpayee et al., 2000; Ghoshnd Singh, 2005). Although Cr remains mainly in the roots (Table 1),he rates of uptake and translocation were reduced when water

yacinth plants were exposed to Cr3+ when compared to plantsxposed to Cr6+ (Table 1).

Since both Cr6+ and Cr3+ must cross the endodermis via the sym-last, the Cr6+ in the cell is probably readily reduced to Cr3+, which

able 3igment contents (�mol cm−2), chlorophyll a/b and total chlorophyll/carotenoid ratios (mays. Chlo = chlorophyll, caro = carotenoid. Values followed by different letters differ signi

igments Treatments

Control 1 mM Cr3+

hlo a 0.049 ± 0.002 A 0.049 ± 0.0hlo b 0.019 ± 0.002 A 0.026 ± 0.0otal chlo 0.069 ± 0.003 A 0.076 ± 0.0aro 0.018 ± 0.000 A 0.019 ± 0.0hlo a/Chlo b 2.604 ± 0.181 A 1.959 ± 0.4otal chlo/Caro 3.807 ± 0.178 A 3.970 ± 0.2

D 796 C 0.68 AB 3.27 B 0.914 A 0.236 A

ABC 1356 AB 0.81 A 5.53 A 0.896 A 0.337 AE 89.0 D 0.38 B 0.86 B 0.788 B 0.056 A

is retained in the root cortex cells under low concentrations of Cr6+.This could explain, in part, the lower toxicity of Cr3+ (Fig. 1). Onehypothesis that may explain the major Cr translocation when waterhyacinth were exposed to Cr6+ is that the conversion from Cr6+ toCr3+ was not total since there were still many molecules of Cr6+

present. Thus, it appears that at least part of Cr6+, which is notreduced to Cr3+, is translocated and its effect is lethal to the plant(Fig. 1). Vajpayee et al. (2000) suggested that the reduction of Cr6+

to Cr3+ reduces the mobility of Cr from roots to shoots in Nymphaeaalba, due to the formation of Cr3+ complexes with –COOH groups,which inhibit the translocation of the metal. In roots, Cr6+ is activelytaken up by the sulfate carrier and immediately converted to Cr3+.Another possible explanation is that that Cr6+ is more soluble thanCr3+ and forms chromate and dichromate in aqueous solution. Thetoxic action of Cr6+ is due to the negatively charged hexavalent Crion complexes, which can easily cross cellular membranes by meansof sulfate ionic channels, penetrate the cytoplasm and react withthe intracellular material leading to the formation of various reac-tive intermediates (Gikas and Romanos, 2006). Indeed, Cr3+ tendsto accumulate in the cell membrane and the inhibition of ion trans-port is considered as one of the major causes of its toxicity in plants(Arduini et al., 2006).

In plants a decrease in photosynthesis, promoted by increasedCr concentration in nutrient solution, is associated with bio-chemical changes, causing inhibition of chlorophyll synthesis(Boonyapookana et al., 2002). In this study (Table 3), although sta-tistical differences were observed only for chlorophyll a and totalchlorophyll, the chlorophyll a/b ratio demonstrated 50% lower con-tents in plants exposed to Cr6+. The decrease in the chlorophylla/b ratio, brought about by Cr, indicates that Cr toxicity possibly

reduces the size of the peripheral part of the antenna complex(Shanker, 2003). Decreases in total chlorophyll have been well doc-umented under Cr stress (Panda and Choudhury, 2005). Vajpayee etal. (2000) observed decreases in chlorophyll a and total chlorophyll

ean ± S.D. n = 3) in water hyacinth exposed to 1 mM and 10 mM Cr3+ and Cr6+ for 4ficantly (P < 0.05).

10 mM Cr3+ 1 mM Cr6+

01 A 0.050 ± 0.001 A 0.027 ± 0.003 B06 A 0.026 ± 0.003 A 0.024 ± 0.011 A05 A 0.075 ± 0.003 A 0.050 ± 0.009 B01 A 0.019 ± 0.001 A 0.013 ± 0.005 A85 A 1.989 ± 0.295 A 1.391 ± 0.827 A95 A 3.853 ± 0.075 A 4.803 ± 2.911 A

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n Nymphaea alba grown in the presence of various levels of Cr6+.hese authors showed that chlorophyll a was more sensitive thanhlorophyll b to Cr6+ toxicity. The inactivation of enzymes involvedn the chlorophyll biosynthetic pathway could contribute to theeneral reduction in chlorophyll content in most plants under Crtress (Shanker et al., 2005). The decrease in total chlorophyll inater hyacinths exposed to Cr6+ suggests that decreases in CO2

xchange were due, at least in part, to decreased photosyntheticapacity. The variations in Pn, measured for both Cr species andoncentration (Fig. 2A), seem to be correlated with the gs (Fig. 2C),xcept for the plants exposed for 2 days to Cr6+. Cr3+ and Cr6+ expo-ure for 4 days resulted in a decrease in Pn and gs (Fig. 2A and). In some cases, 2 days of Cr3+ exposure resulted in an increase

n photosynthetic parameters (Fig. 2). Plants exposed for 2 dayso 1 mM Cr6+ exhibited values of Ci that contrasted to those of Pnnd gs (Fig. 2), but the value of Fv/Fm (0.81) (Table 2) suggests thataximum quantum yield of PSII was not altered. It is possible that

ome problems in the biochemistry of CO2 assimilation occurred,uch as the inhibition of certain enzymes of the Calvin–Bensonycle (Shanker et al., 2005). The substitution of Mg by heavy metalsKüpper et al., 1998) decreases the affinity of Rubisco for CO2. Inolium perene, Zn treatment significantly inhibited RuBP carboxy-ase capacity, whereas RuBP oxygenase capacity was not decreasedMonnet et al., 2001).

After 4 days, one other reason for the decrease in Pn caused byr6+ (Fig. 2A) was probably the damage suffered by the photosyn-hetic apparatus based on the decreases in the Fv/Fm and Fv/F0 ratiosTable 2). According to Jakl and Bolhar-Nordenkampf (1991), valuesf Fv/Fm of between 0.75 and 0.85 show that the photosyntheticpparatus is highly efficient. In this study, we obtained a reduc-ion (0.38) in the Fv/Fm ratio after 4 days of treatment in Cr6+.he decrease in the Fv/F0 ratio is an indicator of structural dam-ge, which occurs in the thylakoids and affects the photosyntheticransport of electrons (Vernay et al., 2007). This parameter reflected

ore precisely the changes in the photosynthesis that Fv/Fm ratio.ecreases in Fv/F0 ratios were observed in other treatments in addi-

ion to 10 mM Cr6+, as also seen in the presence of 1 mM Cr6+ afterdays (Table 2). Pereira et al. (2000) reported that Fv/F0 ratio wasbetter parameter than Fv/Fm to evaluate the responses of citruslants subjected to aluminum stress. Similarly to the observationsy Pereira et al. (2000), our results showed that the decrease in Fv/F0atio was due mainly to a decrease in Fv, and not to an increase in0. Ralph and Burchett (1998) and Monnet et al. (2001) suggestedhat Fm decrease is due to a change in the ultrastructure of thehylakoid membrane, affecting the electron transport rate. How-ver, Joshi and Mohanty (2004) suggested that a decrease in Fv/F0,rrespective of whether it arises from Fv or F0, is an indicator oftructural alterations in PSII.

Energy use by PSII is monitored by the photochemical quenchingoefficient qP. Our data showed that qP decrease only at 10 mM Cr6+

fter 2 days (Table 2), suggesting alterations in the electron trans-ort rate due the ability of reoxidation of the primary Qa electroncceptor. According to Genty et al. (1989), there is a linear relation-hip between qP and Fv/Fm, which was also verified in this studyTable 2).

Events associated with non-photochemical dissipation of excessxcitation energy are measured by non-photochemical quenchingNPQ). NPQ (Table 2) and chlorophyll a/b ratio (Table 3) did notemonstrate statistical difference between treatments, althoughhere was a tendency towards reduction in these values for all treat-

ents (Cr3+ and Cr6+) for both parameters analyzed after 4 days. The

ositive correlations between chlorophyll a/b ratio, carotenoids andPQ were observed by Brugnoli et al. (1998) for Hedera helix andea mays. These authors clearly demonstrated that both NPQ andarotenoids were linearly related to chlorophyll a/b ratio, althoughhe slope of the relationship varied between species. It is known

imental Botany 65 (2009) 403–409

that the chlorophyll a/b ratio is linearly corrected with the degreeof thylakoid stacking (Anderson and Aro, 1994).

In conclusion, despite some symptoms of stress, such as necro-sis and chlorosis, a decrease in leaf gas exchange, chlorophyll afluorescence parameters and photosynthetic pigments contents,the water hyacinth plants were more sensitive to Cr6+ than Cr3+.The uptake of Cr6+ was higher than the uptake of Cr3+ from nutri-ent solution with consequently greater translocation to shoots.It was also possible to confirm the main accumulation of Crin the roots, independently of Cr species utilized. The presenceof Cr in plants suggests that structural alterations in thylakoidscould explain our data for gas exchange, chlorophyll a fluores-cence and photosynthetic pigments contents. Cr6+ exhibited amuch higher toxicity and more molecules had been found in theaerial part where photosynthetic symptoms of stress have beenobserved.

Acknowledgments

The authors thank the Fundacão de Amparo à Pesquisa do Estadode São Paulo (FAPESP), the Fundacão Carlos Chagas Filho de Amparoà Pesquisa do Estado do Rio de Janeiro (FAPERJ), the ConselhoNacional de Desenvolvimento Científico e Tecnológico (CNPq) andthe Universidade Estadual do Norte Fluminense “Darcy Ribeiro” forfinancial support.

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