REGULAR ARTICLE

Differential tolerance to Mn toxicity in perennial ryegrassgenotypes: involvement of antioxidative enzymes and rootexudation of carboxylates

María de la Luz Mora & Analí Rosas &

Alejandra Ribera & Zed Rengel

Received: 21 August 2008 /Accepted: 16 December 2008 / Published online: 21 January 2009# Springer Science + Business Media B.V. 2009

Abstract Mechanisms underlying differential toleranceto Manganese (Mn) toxicity in perennial ryegrass(Lolium perenne L.) cultivars are poorly understood.We evaluated activity of antioxidative enzymes androot exudation of carboxylates in four ryegrass cultivarssubjected to increasing Mn supply under nutrient solu-tion conditions. A growth reduction caused by Mntoxicity was smaller in Jumbo and Kingston than Nuiand Aries cultivars. Shoot Mn accumulation varied inthe order Nui > Aries > Kingston > Jumbo. Ascorbateperoxidase and guaiacol peroxidase activities increasedwith Mn excess. Mn-tolerant Jumbo and Kingston hadhigh activity of these enzymes and relatively low lipidperoxidation. Kingston was most tolerant to high tissue

Mn concentrations and had the highest superoxidedismutase activity. Increased activity of antioxidativeenzymes in Mn-tolerant cultivars could protect theirtissues against oxidative stress triggered by Mn excess.Mn toxicity induced root exudation of carboxylates;oxalate and citrate may decrease Mn availability in therhizosphere, thus enhancing Mn tolerance in ryegrass.

Keywords Antioxidative enzymes . Peroxidases .

Carboxylate exudation .Manganese tolerance .

Superoxide dismutase . Ryegrass genotypes

Introduction

Large areas of the world are sown to pastures for beefcattle and dairy milk production. Perennial ryegrass(Lolium perenne L.) is one of the most importantpastures species in temperate areas of the world and isdominant forage in Southern Chile (Mora et al. 1999).In Chile, most forage plant species are cultivated involcanic ash-derived soils, like Ultisols and Andisols(Mora et al. 1999, 2002). About 50% of ChileanAndisols have high soil acidity levels, and increasedamounts of exchangeable aluminium (Al) that ishighly toxic to plants (Mora et al. 2002, 2006).Whereas the correlation between soil acidity and therelease of Al in Chilean Andisols is clear, therelationship between soil pH and the amount ofplant-available manganese (Mn) in these soils is less

Plant Soil (2009) 320:79–89DOI 10.1007/s11104-008-9872-1

Responsible Editor: Henk Schat.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11104-008-9872-1) containssupplementary material, which is available to authorized users.

M. L. Mora (*) :A. Rosas :A. RiberaCentro de Ciencias y Biotecnología de Recursos Naturales,Universidad de La Frontera,Casilla 54-D,Temuco, Chilee-mail: mariluz@ufro.cl

Z. RengelSoil Science and Plant Nutrition,School of Earth and Environment,The University of Western Australia,35 Stirling Highway,Crawley, WA 6009, Australia

understood because it also depends on redox conditions.Commonly, Mn toxicity is observed in soils withreducing conditions created by organic matter accumu-lation, compaction and/or flooding (Demirevska-Kepova et al. 2004). In addition, Mn toxicity is oneof the most important limiting factors for cropproduction in many acid soils (Carver and Ownby1995; Foy 1984; Rengel 2000).

According to our soil database (La FronteraUniversity, Soil and Plant Analysis LaboratoryServices), shoot Mn concentration can reach over1,000 mg kg-1 of dry weight in pastures grown inAndisols. Furthermore, Rosas et al. (2007) demon-strated that the critical toxicity concentration of Mn inshoots of perennial ryegrass was 421 mg kg−1. Thesedata support a suggestion that the acidity in ChileanAndisols generates an increase in soil Mn availability,resulting in Mn toxicity potential for this forage species.

In plants treated with high amounts of metals, theproduction and scavenging of reactive oxygen species(ROS) are not well regulated, causing oxidative stressthat leads to macromolecule damage, disturbance ofmetabolic pathways, and disruption of cellular ho-meostasis (Hegedus et al. 2001) and physiologicalfunctions (Ogawa and Iwabuchi 2001). Oxidativestress caused by Mn excess was recorded in severalplant species (González et al. 1998; Shi et al. 2006).

To overcome metal toxicity stress, plants have arange of potential mechanisms, like induction andactivation of the plant antioxidative defense enzymes[i.e. peroxidases (PXs), catalases (CATs) and super-oxide dismutases (SODs)] that allow detoxificationof ROS produced in response to the root uptake oftoxic metals from the soil (Schützendübel and Polle2002). Also, plant tolerance to metal toxicity issometimes associated with the lower metal uptakeand translocation to other organs (Vose and Randall1962). In roots, the exudation of organic acid anions(carboxylates) to the rhizosphere may minimizeabsorption of metals, like Al (Delhaize et al. 1993;Ma et al. 2001), nickel (Yang et al. 1997) andcadmium (Krotz et al. 1989). In the case of Mntoxicity, there are only few reports regarding therelease of organic acid anions by roots (Gonzálezand Lynch 1999, Horst et al. 1999).

Previously, we observed that the peroxidase activityand root exudation of some organic acid anionsincreased in ryegrass cv. Nui in response to Mn

excess (Rosas et al. 2007), but no work was done onthe mechanisms behind differential tolerance to Mntoxicity among ryegrass genotypes. Hence, the mainaim of this study was to determine differences in theactivity of antioxidative enzymes and root exudationof organic acid anions in genotypes of perennialryegrass differing in tolerance to Mn excess.

Materials and methods

Plant material and growth conditions

Seeds of four perennial ryegrass (Lolium perenne L.)cultivars (Aries, Jumbo, Kingston, and Nui) weregerminated on filter paper moistened with deionisedwater. After 5 d of germination, 48 seedlings weretransferred to 3-L pots containing aerated hydroponicmedium, and grown under controlled conditions(16-h light period, 20°C and 60–80% relative humidity)in a growth chamber. The hydroponic medium proposedby Taylor and Foy (1985) was chosen because it hadbeen used in studies on Al tolerance in wheat andnutrient requirements of ryegrass. Solutions werereplaced every 5 d, and the pH was adjusted daily to4.8 with diluted HCl or NaOH. The four cultivars weretested in a factorial design with five Mn treatments(control, 10, 20, 50, and 150 µM) supplied as MnCl2.Control plants were grown in a nutrient solutioncontaining 2.4 µM Mn, which was previously definedas optimal supply for ryegrass (Rosas et al. 2007).Each treatment was replicated at least four times. After15 d, shoot and root tissues were harvested forchemical and biochemical assays.

Determination of plant growth parameters and Mnconcentrations

Shoots and roots were dried in a forced-air ovenfor 2d at 65°C for dry weight determination. Inaddition, the shoot and root lengths of 20 seedlingsper pot were measured and averaged. For Mnchemical analysis, samples were dry-ashed in amuffle furnace at 500°C for 8 h and digested with2 M HCl. Mn was extracted as described bySadzawka et al. (2004), and the concentration wasdetermined by atomic absorption spectrophotometry.

80 Plant Soil (2009) 320:79–89

Determination of lipid peroxidation and antioxidativeenzymes activities

In fresh material, lipid peroxidation was measuredusing thiobarbituric acid reacting substances (TBARS)assay according to the modified method of Du andBramlage (1992). The absorbance was measured at532, 600 nm and 440 nm to correct for theinterference caused by TBARS-sugar complexes.

For antioxidative enzymes extraction, subsamples offresh shoot material were frozen in liquid nitrogen andstored at −80 °C. For obtaining the crude tissue extracts,0.1 g of frozen leaves were ground in liquid nitrogen;the resulting powder was suspended in 2.0 mL of chilledphosphate buffer (50 mM potassium phosphate,pH 7.0), centrifuged at 11,000 g for 15 min at 4°C,and the supernatant was collected. The samples werestored at −80°C until determination of antioxidativeenzyme activities. These activities were expressed onthe basis of total protein (determined spectrophotomet-rically according to Bradford 1976).

Ascorbate peroxidase (APX; EC. 1.11.1.11) activitywas assessed by measuring decomposition of ascorbateat 290 nm for 60 s. First, 40 µL of the crude extract wasdiluted in a reaction mixture that contained 1 mL ofextraction buffer, 5 µL of H2O2 (30% v/v) and 40 µLof 10 mM ascorbic acid. Enzyme activity was calcu-lated using a molar extinction coefficient of 2.8 mM−1

cm−1 (Zhao and Blumwald 1998).Guaiacol peroxidase (GPX; EC. 1.11.1.7) activity was

tested by measuring the formation of tetraguaiacol at470 nm for 60 s. Crude extract (15 µL) was added to thereaction mixture that contained 1 mL of extraction buffer,5 µL of H2O2 (30% v/v) and 5 µL of guaiacol. Theenzymatic activity was calculated using a molar extinc-tion coefficient of 26.6 mM−1 cm−1 (Pinhero et al. 1997).

Catalase (CAT; EC. 1.11.1.6) activity was tested bymeasuring decomposition of hydrogen peroxide(H2O2) at 240 nm for 120 s. An aliquot of 10 µL ofthe crude extract was added to a reaction mixture thatcontained 1 mL of extraction buffer and 3 µL of H2O2

(30% v/v). The enzymatic activity was calculated usinga molar extinction coefficient of 39.4 mM−1 cm−1

(Pinhero et al. 1997).Superoxide dismutase (SOD) (EC. 1.15.1.1) activity

was assayed by measuring the photochemical inhibi-tion of nitroblue tetrazolium (NBT) as described byGiannopolitis and Ries (1977) with minor modifica-

tions. Briefly, 30 µL of the crude extract was added toa reaction mixture that contained 50 mM potassiumphosphate buffer pH 7.0, 0.1 mM ethylenediaminete-traacetic acid (EDTA), 13 mM methionine, and322 µM NBT. The reaction was started by adding22 µM riboflavin. A complete reaction mixture with30 µL of extraction buffer was used as control.Control and sample mixtures were illuminated for 15min in glass vials. The reaction was stopped byswitching off the light, and then the tubes werecovered with aluminium foil. In parallel, blanks werekept in dark during the time course of the assay. Theabsorbance was recorded at 560 nm, and one SODunit was defined as the enzyme quantity that inhibitsthe reduction of NBT by 50%.

Sampling and analysis of carboxylates from rootexudates

Root exudates were collected 15 d after the com-mencement of treatments, as described by Neumannand Römheld (1999). Intact ryegrass plants grown inthe nutrient solution were transferred to pots contain-ing deionised water. After 2 h, root exudates ofcontrol and Mn-treated plants were collected andstored at −80°C. The collection time was selectedbecause organic acid anions secreted by plant rootsare quickly degraded by microorganisms after2 h (Jones and Darrah 1994).

To quantify the concentrations of oxalate, malate,citrate and succinate, root exudates were concentratedby lyophilisation. The lyophilized products were re-dissolved in 300 µL of deionized water and used forHPLC analysis. The chromatographic separation wasachieved on a reversed phase C-18 column (LiChrospher100, RP-18, 250×4 mm, 5-μm particle size; Merck,Darmstadt, Germany). Sample injections (20 µL) wereeluted isocratically with a mobile phase of 200 mMortho-phosphoric acid solution pH 2.1, at a flow rate of1 mL min−1 and detected at 210 nm using a UVdetector. The recovery of carboxylates was about 98%.

Data analysis

The experimental design was a randomized completeblock with four replications. The effect of Mntreatments on dry matter production, shoot and rootMn concentration, and organic anions exudation of

Plant Soil (2009) 320:79–89 81

ryegrass genotypes were assessed by two-wayANOVA using SigmaStat 3.1 software. Means werecompared using Tukey’s Test (p<0.05). In somecases, Pearson correlations were used to test therelationships between two response variables.

Results

Plant growth and Mn concentration in shootsand roots

Mn toxicity in nutrient solution reduced the dryweights (DW) of shoots and roots in all ryegrasscultivars, albeit to a different extent. Cultivars Jumboand Kingston showed a relatively small decrease in

shoot and root growth with increasing Mn supply (i.e.for Jumbo, shoot DW decreased 16% and root DW22% at 150 μM Mn). On the other hand, for Aries theDW declined 67% (shoots) and 57% (roots) and forNui 62% (shoots) and 40% (roots) (cf. Fig. 1).Furthermore, in Aries and Nui treated with 150 µMof Mn, root length decreased approximately 2-foldcompared to control plants (data not shown).

Total tissue concentrations ofMn increased graduallyfor both shoot and root in a dose-dependent manner(Table 1). Cultivar Jumbo had the lowest Mn concen-tration in both tissues, followed by Kingston and Aries.In contrast, Nui displayed a strong capacity to accumu-late Mn in roots and shoots (up to 1491 mg kg−1 DW inshoots and 1956 mg kg−1 DW in roots). Roots hadhigher concentrations of Mn than shoots in all cultivars.

Ce

Dd

Dc

Cb

Ca

AbAb

AaAaAa

Bd

Bb

Ba

Bc

Ba

Cd

Cc

CbCab

Ca

0

50

100

150

200

250

Sh

oo

t d

ry w

eig

ht

(mg

pla

nt -

1 )

Cd

Bc

BbBab

BaAb

AabAaAa

Aa

AcAc

AbAabAa

Bc

BbBb

BabBa

0

10

20

30

40

50

60

70

2.4 10 20 50 150

Mn treatments (µM )

Ro

ot

dry

wei

gh

t (m

g p

lan

t-1)

KINGSTONARIES JUMBO NUIFig. 1 Effect of Mn toxicityon dry weight of shoots androots of different ryegrasscultivars grown in nutrientsolution for 15 d. Error barsin columns indicate standarderror (SE). Different lowercase letters indicate statisti-cally significant differencesbetween Mn treatments forthe same cultivar anddifferent upper case lettersshow differences betweencultivars for the same Mntreatment (Tukey’s test,P<0.05)

82 Plant Soil (2009) 320:79–89

Lipid peroxidation and antioxidative enzyme activities

The capacity of Mn to induce oxidative stress inryegrass was evaluated bymonitoring lipid peroxidation(T-BARS accumulation) in shoots. In all cultivars, lipidperoxidation increased at increasing shoot Mn concen-trations, particularly in Mn-sensitive Aries and Nuicompared with Mn-tolerant Kingston and Jumbo (c.f.Fig. 2 and Table 1). In comparison with plants treatedwith 2.4 µM Mn, shoots of Aries and Nui grown at150 µM Mn had lipid peroxidation increased 2- and3-fold respectively, whereas for Jumbo and Kingstonthe increase was 1.4-fold.

Manganese toxicity differentially influenced theactivity of various antioxidative enzymes (Fig. 3). Ingeneral, the activity of APX and GPX increased inresponse to increasing shoot Mn concentrations, andwas greater in cultivars Kingston and Jumbo comparedwith Aries and Nui. In contrast, CAT activity dimin-ished as shoot Mn concentration increased, but washigher in Kingston than in the other three cultivars.Finally, SOD activity in Jumbo and Kingston increased(from 16 to 29 units in Jumbo and from 19 to 60 unitsin Kingston) at increasing shoot Mn concentrations. Incultivars Aries and Nui, SOD activity decreased as theshoot Mn concentration increased.

.049 4.85 5.39 5.3

87 5.10 5.4

89 5.6101 5.896 5.4146 5.6176 6.0156 6.5

150 316 6.8358 6.0338 5.9

ton 2.4 93 5.5

10

20

50434 7.5367 6.4

1508 33 8.4866 8840 8.2

.4 97 5.9

20

4

6

8

10

12

14

400 800 1200

T-B

AR

S (

nm

ol

MD

A g

-1 F

W)

r = 0.900**

r = 0.720**

0 400 800 1200 1600

Shoot manganese concentration (mg kg-1 DW)

r = 0.965**

r = 0.977**

ARIES

NUI

JUMBO

KINGSTON

a b

1600

Fig. 2 Effect of shoot Mn concentrations on lipid peroxidation(T-BARS) in tolerant (a) and sensitive (b) cultivars of ryegrassgrown in nutrient solution for 15 d. Error bars indicate standard

deviation (SD). The Pearson correlation was used to test therelationships between the response variables. Asterisks denotesignificance (*P<0.05, **P<0.01)

Table 1 Mn concentrations (mg kg−1) in shoots and roots of different ryegrass cultivars grown in nutrient solution with different Mntreatments

Mn treatments (µM) Aries Jumbo Kingston Nui

Shoot Mn concentrations (mg kg-1 DW)2.4 110 Ae 57 Bd 100 Ae 107 Ae

10 245 Ad 85 Cc 150 Bd 163 Bd

20 311 Bc 95 Cc 285 Bc 501 Ac

50 519 Bb 159 Db 398 Cb 839 Ab

150 936 Ba 337 Da 846 Ca 1491 Aa

Root Mn concentrations (mg kg-1DW)2.4 267 Ad 65 De 171 Bd 134 Ce

10 272 Bd 150 Dd 189 Cd 352 Ad

20 340 Bc 272 Cc 329 Bc 783 Ac

50 609 Bb 451 Cb 468 Cb 1081 Ab

150 1001 Ca 992 Ca 1270 Ba 1956 Aa

Different lower case letters indicate statistically significant differences between Mn treatments for the same cultivar. Different uppercase letters show differences between cultivars for the same Mn treatment. Statistical analyses for shoot and root Mn concentrationwere made separately (Tukey’s test, P<0.05)

Plant Soil (2009) 320:79–89 83

Carboxylate exudation

Root exudation rates of all carboxylates increasedproportionally to the Mn supply in nutrient solution,except for citrate and succinate exudation by cultivarAries (Fig. 4). In general, ryegrass showed 10-foldgreater exudation rates of oxalate and citrate com-pared with succinate and malate. The exudationranged from 1 to 8 µmol g−1 h−1 for oxalate and 1to 5 µmol g−1 h−1 for citrate. Kingston exhibited thehighest oxalate exudation rate, followed by Jumbo,Aries and Nui. Jumbo had the greater exudation ofcitrate. Highest succinate exudation rates were ob-served in cultivars Kingston and Nui. In the case ofNui, malate was the most prevalent organic anionexuded by roots.

Discussion

Toxic Mn amounts detected in acid Chilean Andisolsmay be an important limiting factor for perennialryegrass production. In the present study, shoot and toa lesser extent root growth of ryegrass was reduced inresponse to Mn toxicity, but differentially for variouscultivars. The more tolerant cultivars were Kingstonand Jumbo, whereas Aries and Nui were sensitive toMn excess. The genotypic variation to excess Mntolerance has been identified in several crops (Bansalet al. 1991; Fang et al. 2000). The sensitivity ofcultivar Nui detected in the present study confirmsour previous findings (Rosas et al. 2007).

Ryegrass cultivars tolerant to Mn excess (Kingstonand Jumbo) accumulated high concentrations of Mn

0.0

0.1

0.2

0.3

0 400 800 1200 1600

CA

T (

µm

ol m

in-1

mg

-1o

f p

rote

in)

0.0

0.2

0.4

0.6A

PX

(µ

mo

l min

-1 m

g-1

of

pro

tein

)

0.0

2.0

4.0

6.0

GP

X (

µm

ol m

in-1

mg

-1 o

f p

rote

in)

0

20

40

60

0 400 800 1200 1600

SO

D (

U m

g-1

of

pro

tein

)

ARIES JUMBO KINGSTON NUI

Shoot manganese concentration (mg kg-1 DW)

r = 0.554*

r = 0.674**

r = 0.985**

r = 0.917**

r = 0.906**

r = 0.896**

r = – 0.875**

r = – 0.868**r = – 0.870**

r = – 0.870**r = 0.921**

r = – 0.794**

r = 0.871**

r = 0.827**

r = 0.803**

r = – 0.829**

Fig. 3 Effect of shoot Mn concentrations on activity ofascorbate peroxidase (APX), catalase (CAT), guaiacol peroxi-dase (GPX) and superoxide dismutase (SOD) in differentryegrass cultivars grown in nutrient solution for 15 d. Error

bars indicate standard deviation (SD). Pearson correlation wasused to test the relationships between the individual enzymeactivities with shoot Mn concentrations. Asterisks denotesignificance (*P<0.05, **P<0.01)

84 Plant Soil (2009) 320:79–89

in roots and proportionally low Mn concentration inshoots, while the sensitive ones exhibited largetranslocation Mn from roots to shoots showingsimilarly high concentrations of Mn in both tissues.

This result suggests an existence of a regulatorymechanism associated with the control of Mn tissueconcentration in ryegrass. According to Vose andRandall (1962), tolerance to Mn toxicity is associated

malato Aries JumboK ingston Nui2.40 .010 .008 0.00 .010 0.0160 .010 .009 0.0220 0.0110 .008 0.01 .050 0.0120 .011 0.02 0.02

1500 .014 0.0090 .028 0.02

JumboK ingstonNui2.34 .1 .098

10 1.4112 .641 0.23 .4220 1.6753 .382 0.27 .40250 1.5344 .078 0.90 .448

1501 .003 4.99 .3 0.514

Arie Nui2. .010 0.0120 .050 0.02

15 .0

Bd

Cc

Cc

Cb

CaBa

Bb

Bc

Bd

Ad

Ab

Aa

Ac

Ad

Ae

Cb

Da

Cc

Cd

Be

0 2 4 6 8 10

2.4

10

20

50

150

Mn

tre

atm

ents

(µ

M)

Oxalate exudation (µmol g-1 h-1)

Ca

Ca

Ba

Bb

Ba

Cb

Cb

Bb

Da

Da

Cb

Cc

Cd

Ba

Ba

Ac

Ad

Ab

Ab

Aa

0 0.2 0.4 0.6 0.8

Malate exudation (µmol g-1 h-1)

Da

BCa

Da

Da

DaCa

Ca

Cab

Cb

Cc

Aa

Aab

Ab

Ac

Ac

Ba

Ba

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Bd

0.00 0.05 0.10 0.15 0.20

Succinate exudation (µmol g-1 h-1)

Cc

Bab

Bab

Bb

BaAc

Ad

Ad

Ab

AaBa

Cd

Dc

Dc

Cb

Da

Cc

Cb

Cb

Dab

0 1 2 3 4 5 6

2.4

10

20

50

150

Mn

tre

atm

ents

(µ

M)

Citrate exudation (µmol g-1 h-1)

ARIES JUMBO KINGSTON NUI

Fig. 4 Organic acid anions exuded by roots of ryegrasscultivars grown in nutrient solution with different Mn levelsfor 15 d. Error bars indicate standard error (SE). Different lowercase letters indicate statistically significant differences between

Mn treatments for the same cultivar and different upper caseletters show differences between cultivars for the same Mntreatment (Tukey’s test, P<0.05)

Plant Soil (2009) 320:79–89 85

not just with low Mn uptake, but also with relativelylittle Mn translocation from roots to shoots.

The foliar Mn concentrations in the controltreatment ranged from 57 to 110 mg kg−1 of DW invarious cultivars, which are slightly lower than thevalues reported by Rosas et al. (2007) (136 mg Mnkg−1 of DW to reach 95% of maximum growth). Incontrast, Rosas et al. (2007) reported that the criticalshoot concentration for Mn toxicity in perennialryegrass was 421 mg kg−1 of DW. In the studypresented here, Mn tolerant cultivars did not reachthis shoot concentration of Mn until 150 μM Mn innutrient solution (cv. Kingston) or not even then (cv.Jumbo), whereas Mn-sensitive genotypes exceededcritical shoot Mn concentration at 20 μM Mn (cv.Nui) or 50 μM Mn in nutrient solution (cv. Aries).These results clearly indicate that Mn-tolerant ryegrasscultivars have the capacity to keep shoot Mn concen-trations at a lower level than Mn-sensitive cultivars.

Metal toxicity may cause increased production offree radicals (FR) and reactive oxygen species (ROS),generating oxidative stress (Mittler 2002) and lipidperoxidation (Halliwell and Gutteridge 1998). In ourstudy, the Mn-tolerant cultivars Kingston and Jumboshowed less lipid peroxidation than Mn-sensitivecultivars. Hence, an important component of the Mntoxicity syndrome in ryegrass could be the severity ofthe oxidative stress. Interestingly, all cultivars showeda similar level of lipid peroxidation at around 340 mgMn kg−1 shoot DW (c.f. Fig. 3), but an increase inshoot Mn concentration in cultivars other than Jumbo(which was most Mn-tolerant cultivar whose shootMn concentration did not increase beyond that level)resulted in further increases in lipid peroxidation.

Tolerant genotypes can increase activity of anti-oxidative enzymes as a general response to excessconcentrations of trace metals in soil, resulting inprotection against homeostatic disturbance and cellulardamages (Mittler 2002). In ryegrass, we found thatcultivar Kingston had high activity of peroxidases(APX and GPX), CAT, and especially SOD (c.f.Fig. 4), which would explain the lower levels ofoxidative stress (less lipid peroxidation) detected inthis genotype caused by excess Mn (in spite of highshoot Mn concentration in that cultivar) compared withMn-sensitive cultivars Aries and Nui. Hence, wesuggest that antioxidative enzymes, mainly SOD,represent an efficient mechanism of internal toleranceto Mn toxicity in perennial ryegrass.

In agreement with the present study, Shi et al.(2005, 2006) found that the activity of APX, GPXand SOD increased in cucumber plants under Mntoxicity. In addition, it has been demonstrated thatwheat (Darkó et al. 2004) and rice (Ma et al. 2007)genotypes resistant to Al toxicity displayed signifi-cantly higher CAT, APX and SOD activity thansensitive genotypes under Al excess. In contrast,CAT activity decreased with increased metal toxicity,i.e. Mn in cucumber (Shi et al. 2005, 2006), Al in rice(Sharma and Dubey 2007) and Fe, Cu and Cd insunflower (Gallego et al. 1996). A decrease in CATactivity together with an increase in activity ofperoxidases observed in our study could be due todifferential affinity of these enzymes to H2O2 (CAThas low affinity—mM range, whereas APX has highaffinity—μM range) (Panchuk et al. 2005).

SODs are considered key enzymes in the defenseof plants against oxidative damage (Yu and Rengel1999). In this work, we measured increased SODactivity in response to Mn toxicity in ryegrass, as wasdemonstrated for cucumber (Shi et al. 2005). Similarly,Lidon and Teixeira (2000) showed that Mn-tolerantrice cultivar had high internal tolerance to Mn probablydue to the synthesis of a Mn-protein with SODfunction. Del Río et al. (1985) proposed that Mn-SOD can be induced in response to high Mnconcentrations in the leaf tissue of Pea (Pisum sativumL.). In contrast, in the present study SOD activityslightly decreased in the Mn-sensitive cultivars (Ariesand Nui) with an increase in shoot Mn concentration(Fig. 3). Further work is required to ascertain reasonsfor this observation.

Organic acid anions can contribute to metalsdetoxification both internally and externally (Delhaizeand Ryan 1995). The main carboxylates associatedwith Mn detoxification mechanisms are citrate,oxalate, malate and succinate (Horst et al. 1999). Inthe present study, exudation of oxalate and citratefrom ryegrass roots was particularly induced byexcess supply of Mn, especially in tolerant genotypes,suggesting that these ligands could contribute to Mntolerance. Hoffland et al. (2004) demonstrated thatcitrate and oxalate have a strong affinity for di- andtri-valent metals, forming complexes that decrease theactivity of free cations such as Mn in the soil solution.Moreover, citrate is the organic anion with the highestaffinity for Mn2+ (formation constants are 4.15 forcitrate, 3.95 for oxalate, and 2.24 for malate; organic

86 Plant Soil (2009) 320:79–89

anion:cation ratio 1:1 in zero ionic strength media at25°C) (Ryan et al. 2001).

As an internal detoxification mechanism in plants,a large proportion of Mn in plant tissues occurs asMn-complexes, withMn chelation by oxalate appearingto be the main detoxification strategy (González andLynch 1999; Memon and Yatazawa 1984). Here, wefound that Mn-tolerant ryegrass cultivar Kingston hada high rate of oxalate exudation in response toincreasing excess supply of Mn (Fig. 5). However,oxalate exudation failed to prevent excessive Mnuptake by Kingston; hence, this cultivar had highshoot Mn concentration in comparison with Mn-tolerant cultivar Jumbo. These findings suggest thatoxalate might be a part of the internal Mn tolerancemechanisms in Kingston via formation of Mn-oxalatecomplexes. This detoxification strategy has beenobserved in response to Al excess in buckwheat (Maet al. 2001) and in some lichen species exposed to highMn levels (Wilson and Jones 1984; Paul et al. 2003).

In this study, cultivar Jumbo had low shoot Mnconcentration and exuded citrate at the highest rates.Citrate is likely to complex Mn2+ in the rhizospheresoil solution as an exclusion strategy. The highaffinity of citrate for Mn2+ in the rizosphere and highcitrate exudation rate in cultivar Jumbo (Fig. 5) mayexplain Mn tolerance of this cultivar. Similarly, citrateexudation has been associated with increased toleranceto Al (Barceló and Poschenrieder 2002).

Cultivar Nui exhibited high rates of malate andsuccinate exudation. However, these two organic acidanions do not appear to be involved in the exclusion ofMn because this cultivar had high shoot Mn concen-trations and exhibited high sensivity to Mn excess.

Conclusions

Ryegrass cultivars showed differential tolerance toMn toxicity. Kingston and Jumbo were tolerant,whereas Aries and Nui exhibited high sensitivity.Toxic levels of Mn in the nutrient solution causedoxidative stress in ryegrass. Nevertheless, Mn-tolerantKingston had high activity of antioxidative enzymes,such as APX, GPX and especially SOD. Mn-tolerantcultivar Jumbo had a high exudation rate of citrate,which could be associated with a Mn exclusionmechanism, limiting Mn uptake by the ryegrass

plants. Mn-tolerant Kingston had higher shoot Mnconcentration than Mn-tolerant Jumbo and exhibitedincreased oxalate exudation in response to Mn toxicity.Further work is required to test whether increasedoxalate exudation is linked to increased intracellularconcentrations of this ligand that detoxify Mn bycomplexation and to clarify whether intracellularcitrate and oxalate can form stable Mn-complexes thatcontribute to overcoming Mn toxicity in ryegrass.

Acknowledgements This work was supported by the Inter-national Cooperation FONDECYT project 7060093 and by theFONDECYT project 1061262 Grants. We thank to Dr. CristiánWulff Zottele who supervised the idiomatic revision and thefigure edition of the manuscript.

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