Effect of aluminum on δ-aminolevulinic acid dehydratase from mouse blood

Environmental and Experimental Botany 57 (2006) 106–115

Effect of aluminum on �-aminolevulinic acid dehydratase (ALA-D)and the development of cucumber (Cucumis sativus)

Luciane Belmonte Pereira a, Luciane Almeri Tabaldi a, Jamile Fabbrin Goncalves a,Gladis Oliveira Jucoski b, Mareni Maria Pauletto a, Simone Nardin Weis a,

Fernando Teixeira Nicoloso b, Denise Borher a,Joao Batista Teixeira Rocha a, Maria Rosa Chitolina Schetinger a,∗

a Departamento de Quımica, Centro de Ciencias Naturais e Exatas, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazilb Departamento de Biologia, Centro de Ciencias Naturais e Exatas, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil

Received 6 January 2005; accepted 4 May 2005

Abstract

Aluminum is one of the most abundant elements on the planet. The effects of its toxicity to plants include inhibition of the growth oftatm1aogw©

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he root system and inhibition of enzymes of plant metabolism causing a delay in development. The objective of the present study was tonalyze the effects of aluminum on the activity of the enzyme �-aminolevulinic acid dehydratase (ALA-D), responsible for the formation ofhe monopyrrole porphobilinogen that is part of the chlorophyll molecule, as well as the cytochromes, in cucumbers. Plant growth was also

onitored and the roots were submitted to histological analysis. The concentrations of Al2(SO4)3 used in the growth medium ranged fromto 2000 �mol/L. Cucumber (Cucumis sativus) was chosen because it is a good bioindicator of metal pollution. Results demonstrated that

luminum inhibits this enzyme and also greatly impairs plant growth. Histological analysis of the roots indicated a delay in the developmentf the vase elements, alterations in cell shape and cellular lesions. ALA-D inhibition may be due to the fact that aluminum present in therowth medium can compete with Mg2+ or reduce the expression of ALA-D. Probably, Al3+ forms complexes with nucleotides, with the cellall and with other biomolecules, reducing the growth and development of the plant.2005 Elsevier B.V. All rights reserved.

eywords: �-Aminolevulinic acid dehydratase; Aluminum toxicity; Cucumber; Histological analysis

. Introduction

Although numerous metals are essential for the normalunctioning of all organisms, no biochemical role has beenssigned to aluminum thus far. However, this trivalent ele-ent, which is the most widespread metal in the crust of the

arth, has attracted significant attention due to its toxic influ-nce on most living systems (Yokel and McNamara, 2001;xley et al., 2002; Schetinger et al., 2002).

Micromolar concentrations of Al3+ can inhibit root growthithin minutes or hours in many agriculturally importantlant species. The subsequent effects on nutrient and water

∗ Corresponding author. Fax: +55 55 220 8031.E-mail address: [email protected] (M.R.C. Schetinger).

acquisition result in poor growth and productivity. Al3+ caninteract with multiple sites in the apoplasm and symplasm ofroot cells. Al3+ is located specifically at the root apex. Thebinding of Al3+ to these sites is probably an important factorin its toxicity (Jaffe et al., 1995; Kochian, 1995; Delhaizeet al., 2001). In fact, many plant species developed toleranceto aluminum to growth in acid mineral soils, Al-sensitiveplants absorb more aluminum than do Al-tolerant plants andthus the exclusion mechanism of aluminum is the major ideafor aluminum tolerance (Kochian, 1995; Matsumoto, 2000;Tolra et al., 2004; Yang et al., 2004).

The enzyme �-aminolevulinic acid dehydratase (ALA-D)is sensitive to metals due to its sulfhydrylic nature (Bevanet al., 1980; Rocha et al., 1995) and catalyzes the asymmet-ric condensation of two molecules of �-aminolevulinic acid

098-8472/$ – see front matter © 2005 Elsevier B.V. All rights reserved.oi:10.1016/j.envexpbot.2005.05.004

L.B. Pereira et al. / Environmental and Experimental Botany 57 (2006) 106–115 107

(ALA) to porphobilinogen (Gibson et al., 1955). The synthe-sis of porphobilinogen promotes the formation of porphyrins,hemes and chlorophylls, which are essential for adequateaerobic metabolism and for photosynthesis (Warren et al.,1998; Jaffe et al., 2000). In line with this, it has been reportedthat ALA-D activity in plants increases during chloroplastdevelopment, which is a period of rapid chlorophyll accumu-lation (Shibata and Ochiai, 1976) and a role for ALA-D inregulation of chlorophyll have been proposed by Naito et al.(1980). Furthermore, altered ALA-D activity concomitantwith reduced chlorophyll contents has been reported in manyterrestrial plants exposed to various metals (Stobart et al.,1985).

The effect of aluminum on animal ALA-D have beenextensively investigated and the results from literature arecontradictory. The majority of studies indicate that Al3+ invitro inhibit ALA-D (Abdulla et al., 1979; Schroeder andCaspers, 1996; Schetinger et al., 1999; Vieira et al., 2000;Rocha et al., 2004), whereas it activates or had no effect onthe enzyme after in vivo exposure (Chmielnicka et al., 1994,1996; Schetinger et al., 1999; Vieira et al., 2000; Farina etal., 2002).

However, studies about the effect of aluminum on plantALA-D activity are not available in the literature. The studyof Al3+ on plant ALA-D is of particular importance due tothe fact that plant enzyme has in its primary structure, a siteriwiaoiBitwal

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which is a period of rapid chlorophyll accumulation (Naitoet al., 1980; Papenbrock et al., 2000). Since the cotyledonspresented high chlorophyll contents, they were used initiallyin the in vitro experiments.

2.2. Sample preparation and biochemical assay

Cucumber cotyledons were harvested on the fifth dayof germination and homogenized in 10 mmol/L Tris–HClbuffer, pH 9.0, at the proportion of 1:1 (w/v). The homogenatewas centrifuged at 12,000 × g at 4 ◦C for 10 min to yield asupernatant that was used for the enzyme assay (S1). Forthe assay, an aluminum sulfate stock solution was preparedin deionized water and stored in plastic containers. A dilutesolution was prepared 24 h before use in the assay.

The supernatant (9 mL) was pre-treated with 5 mmol/LEDTA, 0.1% Triton X-100, 0.5 mmol/L DTT (S1 pre-treatedwith EDTA) or with 0.1% Triton X-100 and DTT (S1 con-trol). A 4.5 mL aliquot of these mixtures was applied to aSephadex G-40-50 column (30 cm × 6 cm in diameter) andeluted with 10 mmol/L Tris–HCl, pH 7.4. Fractions of 4.5 mLwere collected starting at the time when the bulk of the proteinreached the end of the column. The first fraction was desig-nated as fraction 1 (F1). The remaining 4.5 mL was reservedwithout being applied to the chromatography column. Thus,four different preparations were used in the present investi-gdc

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ich in negative charged aspartate residues, which is involvedn enzyme catalysis and is a hypothetical site for interactionith the trivalent cation of aluminum. Furthermore, ALA-D

nactivation may lead to accumulation of �-aminolevuliniccid (substrate) that can cause an overproduction of reactivexygen species (Bechara et al., 1993; Bechara, 1996), which,n turn, could explain some of the toxic effects of the metal.iochemical studies indicate that Al ions have a strong affin-

ty to biomembranes (Jones and Kochian, 1997) and causehe rigidification of the membranes (Deleers et al., 1986),hich seems to facilitate the radical chain reactions medi-

ted by iron (Fe) ions and to enhance the peroxidation ofipids (Yamamoto et al., 2003).

To contribute to a better understanding of the toxicology ofhis metal, the present study was carried out to investigate theffect of aluminum on the development of Cucumis sativus,n environmental bioindicator of ecosystems polluted withetals and its effect on ALA-D activity.

. Material and methods

.1. Plant

Cucumber seeds (C. sativus L.) obtained from Feltrin Ltd.Santa Maria, RS) were maintained in an air conditionedoom (22–25 ◦C) under controlled lighting conditions (16 hight/8 h dark) for 10 days for in vivo experiment and 5 daysor in vitro experiment. It has been reported that ALA-Dctivity in plants increases during chloroplast development,

ation: (1) S1 pre-treated with EDTA; (2) control S1; (3) F1erived from S1 pre-treated with EDTA; (4) F1 derived fromontrol S1.

ALA-D activity was assayed as described by Barbosa et al.1998) by measuring the rate of porphobilinogen formation.he incubation medium for the assays contained 100 mmol/Lris–HCl buffer, pH 9.0 and 1.3 mmol/L MgCl2, unless oth-rwise indicated. In all enzyme assays, the final concen-ration of ALA was 3.6 mmol/L. Incubation was started bydding 200 �L of the tissue preparation to a final volumef 800 �L.

The product of the reaction was determined with thehrlich reagent at 555 nm using a molar absorption coeffi-ient of 6.1 × 104 M−1 cm−1 (Sassa, 1982) for the Ehrlich-orphobilinogen salt. Protein was determined by the methodf Bradford using bovine serum albumin as a standardBradford, 1976).

.3. Morin reaction

To obtain the free and complexed aluminum concentrationn the incubation medium at pH 9.0, the Morin reaction waserformed (Ahmed and Hossan, 1995). The reaction mediumontained 100 mmol/L Tris–HCl buffer, pH 9.0, 3.6 mmol/LLA, 1–2000 �mol/L of Al2(SO4)3 with S1 or F1, with anal volume of 1000 �L. Reagent of Morin (2000 �L) wasdded together with 200 �L of H2SO4. After this, water wasdded to the medium to reach a final volume of 10 mL. Allhe readings were made at 421 nm with spectrometer Lambda6 Perkin-Elmer UV/vis.

108 L.B. Pereira et al. / Environmental and Experimental Botany 57 (2006) 106–115

2.4. In vivo treatment

Cucumber seeds were kept in a closed plastic containerwith medium containing Al2(SO4)3 diluted in a 0.5% agarsolution, pH 4.0. The agar solution was heated and cookedand the aluminum solution was then added. No nutritioussolution was added to the agar. The seedlings make use of seednutritious in initial stage of the development and it was veri-fied in an initial experiment that until the 10th day, the plantsdid not suffer severe nutrient deficiency (data not shown).Aluminum concentrations used for treatment were 1, 10, 100,500, 1000 and 2000 �mol/L in a volume of 15 mL. For eachrecipient containing 15 mL of the solution were added sixseeds, approximately 2.5 mL solution for each seed/seedling.The total number was 30 seeds for each aluminum concen-tration. Cucumber cotyledons were harvested on the 10thday of germination and homogenized in Tris–HCl, pH 7.4,10 mmol/L, at 1:1 proportion. The homogenate was cen-trifuged at 12,000 × g at 4 ◦C for 10 min and the supernatantfraction was used for enzyme assay as previously described.

2.5. Growth analysis

Cucumber growth was determined by measuring thelength of the root system (Tennant, 1975) and of the aerial part(stem and leaves). The roots were put on squared paper andtRirNpraw

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Table 1Concentration of free aluminum in fraction1 (F1) by the reaction of Morin

Added Al(�mol/L)

Absorbance Free Al detected(�mol/L)

Complexed Al detected(�mol/L)

1 nd nd nd10 nd nd nd

100 nd nd nd500 0.0129 2 498

1000 0.0413 73 9272000 0.0929 202 1798

The medium contained 100 mmol/L Tris–HCl buffer, pH 9.0, 3.6 mmol/LALA, 1–2000 �mol/L Al2(SO4)3 and F1. Morin reagent was added. Nofree aluminum was detected in the incubation medium after adding 1, 10 or100 �mol/L Al2(SO4)3 ; nd, not detected.

S1 fraction, no free aluminum was detected by the Morinreaction, even when 2000 �mol/L Al2(SO4)3 were addedexogenously to the reaction mixture (data not shown). In con-trast, the addition of Al2(SO4)3 to S1 filtered in SephadexG-50 (F1 fraction) revealed the presence of free Al3+ in theincubation medium. The actual free aluminum concentrationsin F1, in the tubes where 0, 500, 1000 and 2000 �mol/LAl2(SO4) were added, equaled to 0, 2, 73 and 202 �mol/LAl3+, respectively (Table 1). Consequently, Al2(SO4)3 didnot inhibit �-ALA-D from cucumber cotyledons when thealuminum salt was mixed directly with S1 (Fig. 1). In con-trast to the S1 fraction, enzyme activity after gel filtration(F1) was inhibited by Al2(SO4)3 (Fig. 2).

Plant �-ALA-D contains Mg2+ ions in its octamer struc-ture and the binding of Mg2+ is relatively stable but can beremoved from the enzyme with chelators, such as EDTA(Jaffe et al., 1995). Consequently, we realized that pre-treatment of the enzyme with EDTA would remove Mg2+

from the enzyme structure and leave these sites free for inter-action with Al3+. In line with this, EDTA inhibited enzymeactivity (5 mmol/L or higher concentrations), probably bythe chelation of Mg2+ from the enzyme (Fig. 3). This findingagrees with the previous finding for Pseudomonas aerugi-

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he number of intersections between the lines was counted.oot length (R) was measured by counting the number of root

ntersections with the lines of paper (N) in a randomly locatedegular area (A) with oriented lines of total length (H) and theewman (1966) formula was used (R = πNA/2H). The aerialart was measured with a ruler and fresh and dry weight wereecorded. To obtain the fresh mass, excess water was removednd the plants were weighed. To obtain dry mass, the plantsere left at 65 ◦C for 5 days.

.6. Histological analysis

For histology, 3 mm sections were obtained from root tips.en sections per group (n = 3) were prepared. The first 20ections of each root were discarded and the subsequent onesere standardized for histological analysis. Of each part of

he root with 3 mm, were made cuts of 5 �m and stained witholuidine blue.

.7. Statistical analysis

Data were analyzed by one-way analysis of variance fol-owed by the Duncan test when the F-test was significantP < 0.05) to determine the differences between the controlroup and the other groups.

. Results and discussion

The presence of free aluminum Al3+ (the toxic form) athe pH of the assay (pH 9.0) is very limited. In the crude

ig. 1. Effect of aluminum on ALA-D activity from cucumber cotyledons.luminum (1–2000 �mol/L) was incubated with S1 which was pre-treatedith 5 mmol/L EDTA. Values are expressed as mean ± S.D. of three inde-endent experiments performed in triplicate. Activity is expressed as % ofontrol (2.9 nmol porphobilinogen/h/mg of protein).

L.B. Pereira et al. / Environmental and Experimental Botany 57 (2006) 106–115 109

Fig. 2. Effect of aluminum on ALA-D activity from cucumber cotyle-dons after gel filtration (F1). Aluminum (1–2000 �mol/L) was incubatedwith F1 which was pre-treated with 5 mmol/L EDTA. Values are expressedas mean ± S.D. of three independent experiments performed in triplicate.Activity is expressed as % of control (F1, 2.0 nmol porphobilinogen/h/mgof protein). *Significantly different from control (P < 0.05).

nosa ALA-D (Frankenberg et al., 1999), where the incubationof P. aeruginosa ALA-D with EDTA resulted in the dramaticreduction of enzyme activity with 5 mM EDTA.

Pre-treatment of S1 from cucumber cotyledons with Tri-ton X-100 (0.1%) and 0.5 mmol/L DTT for 20 min, followedby incubation with either 1.3 mmol/L MgCl2 or 2 mmol/LAl2(SO4)3 (which contains approximately 202 �mol/L offree aluminum) or a combination of both ions during theALA-D assay did not modify enzyme activity (Fig. 4A).However, enzyme activity after gel filtration was reduced byabout 40% (2.0 nmol PBG/h/mg of protein) when comparedto the enzyme activity not applied to the column (2.8 nmolPBG/h/mg of protein). Addition of MgCl2 caused a four-foldincrease in ALA-D activity compared to the control enzymeactivity after column filtration (Fig. 4B). The chromato-graphic column allowed for the removal of low-molecularweight structures that are bound to the Mg2+ site, masking

enzyme activity. Exposure of the gel-chromatographedenzyme to Al3+ caused practically no change in enzymeactivity compared to the control. Simultaneous exposure ofthe gel-filtered enzyme to Mg2+ and Al3+ resulted in enzymeactivity that was about 30% lower than that observed forthe enzyme incubated in the presence of Mg2+, but higherwhen compared to the control and Al3+ groups. A secondfraction that was eluted after fraction 1 showed essentiallythe same behavior towards Al3+ and Mg2+ (data notshown).

Pre-treatment of S1 from cucumber cotyledons with0.1%Triton X-100, 0.5 mmol/L DTT and 5 mmol/L EDTA(a cation chelator) for 20 min followed by incubation in thepresence of Al3+ (2 mmol/L) or a combination of both ionspractically did not affect enzyme activity; however, Mg2+

(1.3 mmol/L) caused a small statistically significant activa-tion of the enzyme (Fig. 5A). Enzyme activity after gelfiltration was reduced by about 50% (compared to the controlactivity values of F1—1.9 nmol PBG/h/mg of protein—withthe control not applied to the column, 3.0 nmol PBG/h/mg ofprotein). Addition of Mg2+ to F1 increased enzyme activityabout five-fold when compared to control enzyme activity(Fig. 5A and B), as has been seen previously by Frankenberget al. (1999).

In contrast to the results observed when S1 was pre-incubated in the absence of EDTA, �-ALA-D was signifi-csac(

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F (100–11 as meani differen

ig. 3. Effect of EDTA on ALA-D activity from cucumber cotyledons. EDTA00% of ALA-D activity is 3.0 nmol/(h mg) of protein. Values are reporteds reported as nmol porphobilinogen (PBG)/h/mg of protein. *Significantly

antly inhibited after incubation in the presence of Al3+ andimultaneous exposure of the gel-filtered enzyme to Mg2+

nd Al3+ reduced the inhibitory effect of Al3+ and, in fact,aused a stimulation of enzyme activity above control valuesFig. 5B).

Mg2+ is not essential for plant ALA-D activity but causes aignificant increase in the Vmax of the enzyme (Jaffe, 1995).he binding data with Mg(II) indicate that plants ALA-Dan bind up to 3 Mg(II)/subunit. The kinetic data supportrequired Mg(II), an allosteric Mg(II) and an inhibitory

0000 �mol/L) was incubated with the S1 fraction. The value that represents± S.D. of three independent experiments performed in triplicate. Activity

t from control (P < 0.05).

110 L.B. Pereira et al. / Environmental and Experimental Botany 57 (2006) 106–115

Fig. 4. Effect of aluminum and magnesium on ALA-D activity from cucumber cotyledons. Aluminum (2000 �mol/L) was incubated with the S1 (A) and F1(B) fractions. Values are reported as mean ± S.D. of three independent experiments performed in triplicate. Activity is expressed as % of control (no column,2.8 nmol porphobilinogen/h/mg of protein; F1, 2.0 nmol porphobilinogen/(h mg) of protein). *Significantly different from control (P < 0.05).

Mg(II), but data are insufficient to address the individual sto-ichiometries of these three types of Mg(II) (Kervinen et al.,2000).

In the present report, we showed that in a crude preparationof plant ALA-D, Mg2+ did not stimulate the enzyme activity.However, after gel filtration, which removes low-molecularweight compounds, a significant stimulation of the enzyme

was observed. The stimulatory effect of Mg2+ was also seenafter the pre-incubation of S1 with EDTA followed by gelfiltration (F1). Taken together, these results seem to indicatethat a ligand (possibly a cation) interacts with a stimulatoryMg2+ site in vivo. The chromatographic column is efficientin removing the ligand, as, even without EDTA, an activationof enzyme activity by Mg2+ is observed.

F ber coi mean ±e f proteif

ig. 5. Effect of aluminum and magnesium on ALA-D activity from cucumncubated with the S1 (A) and F1 (B) fractions. Values are reported as thexpressed as % of control (no column, 3.0 nmol porphobilinogen/(h mg) orom control (P < 0.05).

tyledons pre-treated with 5 mmol/L EDTA. Aluminum (2000 �mol/L) wasS.D. of three independent experiments performed in triplicate. Activity is

n; F1, 1.9 nmol porphobilinogen/h/mg of protein). *Significantly different

L.B. Pereira et al. / Environmental and Experimental Botany 57 (2006) 106–115 111

Fig. 6. Effect of deferoxamine on ALA-D activity from cucumber cotyle-dons. DFO (100–10000 �mol/L) was pre-incubated with the S1 and F1fractions. Values are reported as the mean ± S.D. of three independent exper-iments performed in triplicate. Activity is expressed as % of control (nocolumn, 3.0 nmol porphobilinogen/(h mg) of protein; F1, 2.0 nmol porpho-bilinogen/h/mg of protein). *Significantly different from control (P < 0.05).

The column alone was not sufficient for aluminum toinhibit the enzyme. Only after the use of EDTA, togetherwith the column, was aluminum able to inhibit the enzyme,probably by removing Mg2+. Deferoxamine (DFO, a trivalentcation chelator, 0.1–10 mmol/L) increased ALA-D activityfrom the S1 and F1 fractions (Fig. 6). The activation washigher after gel filtration, which probably removed low-molecular weight compounds. In addition, the activation ofenzyme activity increased with higher DFO concentration.Deferoxamine has the same action as the column and EDTAtogether, as it increases the enzyme activity of S1 and F1.This suggests that DFO removed some endogenous ligandsthat could mask the Mg2+ site or that were bound to Mg2+.

Based on the results presented in Figs. 4 and 5, we proposethe following scheme (Scheme 1).

In this scheme, loss of Mg2+ after gel filtration caused areduction in enzyme activity that was compensated in part bya loss of the negative modulator Ct (possibly a cation). Addi-tion of exogenous Mg2+ to the enzyme that had lost part of theCt considerably stimulated the enzyme. The results concern-ing DFO support its proposal. In fact, DFO can chelate Al3+

but has very low affinity for Mg2+. Thus, DFO can removethe Ct (possibly Al3+ or other trivalent cation) without inter-fering with Mg2+. The additional increase in ALA-D activitycaused by DFO after gel filtration may indicate that DFOis more effective in removing Ct from the enzyme than gelfi

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Fig. 7. Effect of aluminum on Cucumis sativus wet (A) and dry weight(B). Aluminum (1–2000 �mol/L) was added to the growth medium, whichwas maintained at pH 4.0. Values are expressed as mean ± S.D. of threeindependent experiments performed in triplicate. The values obtained forthe linear regression were r2 = 0.96 to A and r2 = 0.94 to B. Dry and wetweight are expressed in g; *P < 0.05.

(Fig. 8B) and on aerial parts (stem and leaves; Fig. 8A).Data from Fig. 8B reveal hormesis-type curve of growth,these results are in accordance with data from literature,where it was found stimulation of growth by exposure tolow concentrations of Al (below toxicity threshold) (Barceloand Poschenrieder, 2002). Hormesis during the first min-utes or hours after aluminum exposure seems often relatedto alleviation of proton toxicity (Lazof and Holland, 1999).Aluminum-induced growth stimulation in the H+-sensitivevarieties may be brought about by Al3+ which, as a triva-lent cation, would reduce the cell surface negativity and, inconsequence, the H+ activity at the cell membrane surface(Kinraide, 1994). The hormetic effect and the Al-inducedalleviation of H+ toxicity is being an important starting pointfor the investigations into the mechanisms of Al- and proton-induced inhibition of root elongation in relation to Al speciesand their toxic effects on the plasma membrane (Barcelo andPoschenrieder, 2002, 2004). Probably, the growth of root cellsis affected by aluminum causing a decrease of the cell wallsynthesis because aluminum inhibits the secretory function ofthe Golgi apparatus (Furlani and Clark, 1981). Furthermore,

S al by ge

ltration through Sephadex G-50.In vivo experiments demonstrate that the exposure to

l3+ caused a concentration-dependent decrease in wet andry weight (Fig. 7A and B, respectively). Similarly, Al3+

aused a concentration-dependent reduction in root system

cheme 1. ALA-D modulation by an endogenous Ct (cation) and its removnzyme cation (Mg2+) binding site.

el filtration; ↓ or ↑ indicate a decrease or an increase in the occupation of

112 L.B. Pereira et al. / Environmental and Experimental Botany 57 (2006) 106–115

Fig. 8. Effect of aluminum on the growth of the aerial part (A) and of theroot system of the plant (B). Aluminum (1–2000 �mol/L) was added to thegrowth medium, which was maintained at pH 4.0. The values obtained for thelinear regression were r2 = 0.98 to A and r2 = 0.99 to B. Values are expressedas mean ± S.D.; n = 28; *P < 0.05.

aluminum can reduce the amount of almost all the organicnutrients of plants (Calbo and Cambraia, 1980) and inter-feres with the absorption, transport and use of several cationssuch as calcium and magnesium (Foy, 1974; Grimme, 1983).Aluminum can also lower the absorption of other minerals,including copper, zinc, manganese and iron (Cambraia et al.,1983).

Al toxicity in plants is related with inhibition of rootelongation. It was demonstrated that the addition of Mg ame-liorate Al rhizotoxicity in soybean at �M levels (Silva et al.,2001a,b,c; Ryan et al., 1997). Probably, the presence of Mgmay stimulate events that would lead to more efficient Aldetoxification, such as the Al-chelating substances (Silvaet al., 2001). These authors suggest also that Al and Mg cancompete by a sensitive binding site. We believe that in rela-tion to ALA-D activity, these cations can compete by thesame binding site, interfering with enzymatic catalysis, asobserved in this work.

The effect of Al3+ on ALA-D activity varied dependingon the tissue and on the metal concentration used. For rootenzyme, an inhibitory effect of about 50% was observed at100 �mol/L (Fig. 9B). For leaves enzyme, the effect wasbiphasic and an increase on the enzyme activity was observedat 1 �mol/L, whereas at 1000–2000 �mol/L, a significantinhibition (about 20%) was detected (Fig. 9A). This biphasiceffect could be a hormetic response (Calabrese and Baldwin,

Fig. 9. Effect of aluminum on ALA-D activity from cucumber leaves (A) androots (B). Control value for leaves and roots were 11 and 3.3 nmol/(h mg),respectively. Activity is expressed as nmol porphobilinogen (PBG)/h/mgprotein. Values are expressed as mean ± S.D. of three independent experi-ments performed in triplicate; *P < 0.05.

2001), as that observed after an in vivo treatment with alu-minum in adult mice (Vieira et al., 2000).

The plant under study, C. sativus, showed a significantdecrease in the amount of organic matter indicating a decreasein photosynthesis, which can be a consequence of reductionin chlorophyll content (Fageria et al., 1988). According toBarker (1979), aluminum directly affects the photosynthesisrate and indirectly affects the synthesis of enzymes, pigmentsand essential cofactors for the process.

The histological analysis of cucumber root tips demon-strated alterations at aluminum concentrations of 500 �mol/L(Fig. 10B), 1000 �mol/L (Fig. 10C) and 2000 �mol/L(Fig. 10D) when compared to control (Fig. 10A). Alterationssuch as loss of root hairs, irregular cell shape, accumulationof grains of starch, delay in the development of the vase ele-ments and lesions in the cytoplasm with cell emptying werethe main aspects observed in cucumber root sections. Theseresults are similar to those previously published in the lit-erature for other species of plants indicating that the moredrastic and typical effect of aluminum is the phytotoxicity tothe roots (Darko et al., 2004).

Based on the present results, we conclude that a com-pound (possibly a cation) negatively modulates the cucumberenzyme in vitro. The binding of this compound to the samesite as Mg2+ and the gel filtration of the crude enzyme prepa-rations facilitate the removal of this negative modulator andtip

he addition of Mg2+ activates the enzyme. Moreover, Al3+

nhibits �-ALA-D activity only after filtration with EDTAre-treatment. In vivo was also observed that aluminum

L.B. Pereira et al. / Environmental and Experimental Botany 57 (2006) 106–115 113

Fig. 10. (A) Cross section (×10) of cucumber root tips control (without aluminum). From left to right, the arrows indicate the root hairs and the vase elements.(B) (×10) 500 �mol/L; from left to right, the arrows indicate cellular emptying, the irregular shape of vase elements, and the different number of layers of theparenchyma. (C) (×10) 1000 �mol/L; from left to right, the arrows indicate a possible formation of grains of starch, the irregular shape of vase elements, andthe cellular emptying. (D) (×10) 2000 �mol/L; the arrow indicate cellular lesion. Ten sections per group were prepared, n = 3. The material was stained withtoluidine blue.

strongly interferes with C. sativus growth, the effect on plantgrowth is a very complex process and the Al-induced inhi-bition of growth can have multiple causes. The effect can bein part attributed to ALA-D inhibition; however, the exactextent of contribution of ALA-D inhibition to plant growthinhibition is not known.

Acknowledgements

The authors wish to thank to Conselho Nacional de Desen-volvimento Cientıfico e Tecnologico (CNPq), Coordenacao e

Aperfeicoamento de Pessoal de Nıvel Superior (CAPES) andFundacao de Amparo a Pesquisa do Estado do Rio Grandedo Sul (FAPERGS).

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Ahmed, M.J., Hossan, J., 1995. Spectrophotometric determination of alu-minum by Morin. Talanta 42, 1135–1142.

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