Al Genes Isolation

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Abstract. Using dierential screening of a root tipcDNA library prepared from an Al-tolerant wheatcultivar (Triticum aestivum L. cv. Atlas-66) exposed toAl, we have isolated and characterized several wheataluminum-regulated (War) cDNAs. Sequence compari-son revealed that genes up-regulated by Al correspondto peroxidase (war4.2), cysteine proteinase (war5.2),phenylalanine-ammonia lyase (war7.2), and oxalateoxidase (war13.2). Two wheat cultivars that dier intheir level of tolerance (cv. Atlas-66: tolerant, and cv.Fredrick: sensitive) were used to evaluate the relation-ship between the accumulation of War mRNAs and Altoxicity, as measured by root growth inhibition (RGI).The mRNA accumulation was modulated to similarlevels in both cultivars compared at equivalent RGIs.

This indicates that War mRNA accumulation is associ-ated with the toxicity of Al rather than with thecultivar's tolerance. It appears that most of the genesfound to be up-regulated by Al share homologies withgenes induced by pathogens. This suggests that Al mayact as an elicitor of a pathogenesis-related transductionpathway. The potential functions of the up-regulatedwar genes in cell wall strengthening and Al trapping arediscussed.

Key words: Aluminum toxicity Cysteine proteinase Gene regulation Oxalate oxidase Pathogenesisresponse elicitor Triticum (A1 toxicity)

Introduction

Aluminum (Al) is one of the most abundant elementspresent in the earth's crust. In acidic soils, its toxicity is

considered to be the most limiting factor for plantproductivity. Based on an estimate of the world'spotentially arable land resources, it has been estimatedthat most of the cultivable area (78.4%) is composed ofacid soils. The American continent accounts for 40.9%of the world's acidic soils (von Uexku ll and Mutert1995). The combined eect of acid precipitation andsome common agricultural practices exacerbates theoverall metal toxicity of these soils. The initial and mostdramatic symptom of Al toxicity is inhibition of rootgrowth. The abundance of Al products in the environ-ment and the potential of Al as plant growth inhibitormakes it necessary to understand the mechanisms of Alphytotoxicity and tolerance (Kochian 1995).

The toxicity of Al to plants has been attributed to

alterations in several physiological and biochemicalpathways (Rengel 1992). Aluminum accumulates rapid-ly in the apoplasm, in the plasma membrane, andeventually enters the cytosol (Lazof et al. 1994; Tayloret al. 1996). However, the plasma membrane has beenproposed to be the primary site of Al toxicity (Huanget al. 1995; Sasaki et al. 1995; Wagatsuma et al. 1995).Several reports have shown specic eects on membranepotential, Ca2+ uxes, K+ eux, ion channels, and onthe phosphoinositide signal transduction pathway(Kochian 1995; Sasaki et al. 1995; Schroeder 1995;Wagatsuma et al. 1995; Jones and Kochian 1995).However, the precise mechanism of Al toxicity is not

fully understood.Several genes have been found to be up-regulated

upon exposure to Al. These include phenylalanineammonia-lyase (wali4, Snowden and Gardner 1993),putative proteinase inhibitors (wali3, wali5, and wali6,Snowden and Gardner 1993; Richards et al. 1994), aplant metallothionein-like protein (wali1, Snowden andGardner 1993), cysteine-rich proteins of unknown func-tion (wali2 and wali7, Snowden and Gardner 1993;Richards et al. 1994), a peroxidase (al201, Ezaki et al.1996), a b-1,3-glucanase (glc1 Cruz-Ortega et al. 1997), aglutathione S-transferase (al142, Ezaki et al. 1995), andan auxin-regulated gene homolog (al111, Ezaki et al.

1995). Many of these genes were also found to be

Abbreviations: RGI root growth inhibition; War wheataluminum-regulated

Correspondence to: M. Houde; Tel: 1 (514) 987 3000 ext. 3964;

Fax: 1 (514) 987 4647; E-mail: [email protected]

Planta (1998) 205: 531538

Isolation and characterization of wheat aluminum-regulated genes:possible involvement of aluminum as a pathogenesis response elicitor

Francine Hamel, Christian Breton, Mario Houde

De partement des Sciences biologiques, Universite du Que bec a Montre al, C.P. 8888, Succ. Centre-ville, Montre al,Que bec, H3C 3P8, Canada

Received: 24 October 1997 / Accepted: 21 January 1998


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responsive to other toxic metals, low Ca2+ levels, andphysical wounding (Snowden et al. 1995).

The goal of this study was to identify new genesmodulated by Al exposure, to correlate their level ofexpression as a function of the degree of toxicity, and tounderstand their possible function. To achieve that, weisolated several Al-responsive genes from a cDNA

library prepared from root tips of the tolerant wheatcultivar Atlas-66 exposed to Al. The accumulation ofmRNA corresponding to four genes was evaluated in asensitive and a tolerant wheat cultivar at equivalent rootgrowth inhibition (RGI) levels (Parker 1995). Underthese conditions, the genetic responses were found to besimilar in both cultivars, indicating that the response ismodulated as a function of the degree of Al toxicity.These results suggest that Al-regulated genes isolated inour study do not confer particular advantages to thetolerant plants and could not be implicated in thedierence observed in the cultivars' tolerance. Inaddition, we have isolated two new genes involved inthe Al-toxicity response: oxalate oxidase and cysteineproteinase. The similarity of several Al-regulated genesto the pathogenesis-related (PR) genes suggests that Almay trigger a PR transduction pathway.

Materials and methods

Plant material and growth conditions. Two winter wheat cultivarspossessing a high (Triticum aestivum L. cv. Atlas-66) and low (T.aestivum L. cv. Fredrick) tolerance to Al were used in this study.Plants were seeded in moist vermiculite and allowed to germinatefor 5 d under a controlled environment (20 C; under continuouslight: 25 lmol mA2 sA1). For Al exposure, seedlings were care-fully washed with deionized water to remove vermiculite. Flatcontainers were lled with 5 l of solution at pH 4.15 (adjusted withHCl) containing 1 mM CaCl2, and various concentrations of Alranging from 0 to 500 lM, as described for each experiment. TheAlCl3 stock solution (0.1 M) was prepared in water maintainedunder pH 3.0 with HCl, while AlCl3 powder was slowly added.Seedlings (35 per container) were exposed (24 h) to Al via theirroots under the same temperature and light conditions as used forgermination. The root growth inhibition (RGI) was measured in allexperiments and is expressed as 100 [1 A (root growth of Al-treated seedlings divided by the root growth of control seedlings)].The pH was checked at the end of each experiment to ensure that itdid not vary by more than 0.1 pH unit.

Construction and screening of the cDNA library. PolyadenylatedRNA was isolated from root tips (5 mm) of Atlas-66 seedlings

exposed to 50 lM Al. A cDNA library was constructed in lambdaZAPII (Stratagene, La Jolla, Calif. USA) using EcoRI-NotI linkersfrom Pharmacia Biotech Inc. (Baie D'Urfe , Quebec, Canada), andtransfected into Escherichia coli strain XL-1 Blue. The cDNAlibrary was screened with 32P-labeled cDNA probes prepared frompoly(A)+ RNA isolated from the root tips of control and Al-exposed wheat plants. The screening of the cDNA library and allthe recombinant DNA techniques were performed as described bySambrook et al. (1989).

Northern blot analyses. Ribonucleic acid was isolated from roottips, roots, crown, and leaves of wheat seedlings. The RNA samples(5 lg) were separated on formaldehyde agarose gels with samplescontaining ethidium bromide to control the loads. The RNA wasthen transferred to nylon membranes MAGNA MSI (Fisher

Scientic, Nepean, Ont., Canada) and hybridized with random

32P-labeled cDNA inserts (Sambrook et al. 1989). The membraneswere washed three times for 30 min with 5 SSC (1 SSC 0.15 M NaCl; 0.015 M Na3-citrate, pH 7) at 55 C, thentwice for 30 min with 0.5 SSC at 55 C and autoradiographedwith intensifying screens at A80 C. A cDNA showing no dier-ential hybridization during library screening (control probe namedW13.3) was used as a load control to correct for minor variations inRNA loads. Densitometry using a Molecular Dynamics personaldensitometer SI and ImageQuaNT 4.2 software (Molecular Dy-namics, Sunnyvale, Calif., USA) was used to quantitate theaccumulation of each transcript under the dierent Al exposureconditions.

Analysis of DNA sequences. Sequencing (T7 DNA sequencing kit;Pharmacia Biotech Inc.) was performed on both DNA strandsusing a series of deletions. Sequence analysis and comparisons werecarried out with the Genetics Computer Group sequence analysissoftware Wisconsin package, version 9.0-Unix (Michigan State U.,Wis., USA).

Results

Eect of Al on root growth. In order to reduce thenumber of Al species in the exposure medium, 1 mMCaCl2 was used and the pH adjusted to 4.15. Underthese conditions, most Al remains in the Al3+ form(minimum of 92%), with small amounts present asAl(OH)2+ (up to 7%) over the range of Al concentra-tions used (as determined by MINEQL+; Environmen-tal Research Software, Mass., USA). Exposure todierent Al concentrations showed that the cultivarFredrick is much more sensitive than the cultivar Atlas-66 since a maximal RGI was reached at approximately50 lM Al for Fredrick compared to 500 lM for Atlas-66 (Fig. 1). At the highest concentration used (500 lM),the RGI did not exceed 75%. This indicates that rootscan continue to grow during the rst 24 h of exposure.However, there was no further root growth within thenext 24 h of exposure (data not shown), indicating thatmaximal inhibition was achieved. Under our assay

Fig. 1. Eect of Al on root growth in the winter wheat cultivarsAtlas-66 and Fredrick. Seedlings of 5-d-old Atlas-66 (s) and Fredrick(d) were exposed to varying concentrations of Al for 24 h. RGI wascalculated as 100 [1 A (root growth of Al-treated seedlings dividedby the root growth of control seedlings)]. Values are the means SE

of 30 root measurements from 3 to 10 independent experiments

532 F. Hamel et al.: Wheat aluminum-regulated genes


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conditions, half of the maximal inhibition observed(37.5% RGI) would be reached at Al concentrations of 5and 45 lM for Fredrick and Atlas-66, respectively.

Isolation and characterization of Al-responsive genes.Screening of the Atlas-66 cDNA library allowed us toisolate four wheat aluminum-regulated (War) cDNAs

that do not cross-hybridize (War4.2, War5.2, War7.2,and War13.2).The eect of four Al concentrations (0, 5, 50, and

500 lM) on the accumulation of War mRNAs in thetolerant cultivar Atlas-66 and the sensitive cultivarFredrick is shown in Fig. 2. The War4.2, War5.2,War7.2, and War13.2 mRNAs accumulated in responseto Al in both Atlas-66 (Fig. 2A) and Fredrick (Fig. 2B).In the tolerant cultivar Atlas-66, the expression of thesegenes increased at an intermediate Al concentration(50 lM) and their mRNA accumulated to a high level at500 lM of Al. These mRNAs accumulated at a lowerconcentration of Al in the sensitive cultivar Fredrickcompared to the tolerant cultivar Atlas-66. However, themaximal level of accumulation of these mRNAs appearsto be comparable between the sensitive and the tolerantcultivars (Fig. 2). If we compare the level of mRNAaccumulation between Atlas-66 exposed to 50 lM Al(Fig. 2A) and Fredrick exposed to 5 lM Al (Fig. 2B),we nd that the transcript level for the dierent genes isvery similar. At these Al concentrations, both cultivarsshow a similar RGI (see Fig. 1). These results suggest

that the transcripts of the up-regulated genes accumulatein response to Al toxicity, and is not implicated in thedierential tolerance observed between cultivars.

Quantitative analyses of gene expression. The accumula-

tion of the dierent mRNAs was characterized in detailin the sensitive and tolerant cultivars exposed to similardegrees of toxicity. We used dierent ranges of Alconcentrations in the sensitive (050 lM) and thetolerant cultivar (0500 lM) in order to achieve com-parable RGIs. Aluminum toxicity was thus expressed asRGI and divided into three groups: 0%, 3560%, and>60% RGI (Fig. 3). In most cases, there was nosignicant dierence in the accumulation of War4.2,War5.2, War7.2, and War13.2 between the tolerant andthe sensitive cultivars compared at the same RGI

Fig. 2A,B. Eect of Al on the accumulation of War mRNAs in thewinter wheat cultivars Atlas-66 and Fredrick. Northern blothybridizations of 5 lg of total RNA extracted from the root tips ofwheat seedlings Atlas-66 (A) and Fredrick (B) exposed for 24 h to0500 lM Al. Each membrane was successively hybridized with thespecied 32P-labeled cDNA, dehybridized, and rehybridized with theW13.3 control probe (shown under each War cDNA hybridization)

Fig. 3AD. Eect of comparable Al toxicity levels on War mRNAaccumulation in the winter wheat cultivars Atlas-66 and Fredrick.Comparable degrees of Al toxicity measured as RGI were obtained by

treating Atlas-66 (h) and Fredrick (j) with 0500 lM Al and 050 lM Al, respectively. The accumulation of War4.2 (A), War5.2 (B),War7.2 (C), and War13.2 (D) was quantitated by densitometry ofnorthern hybridizations, and normalized using the W13.3 controlprobe. Results are shown for the control plants (0% RGI), plantsexposed to an intermediate degree of toxicity (3560% RGI), and to ahigh degree of toxicity (>60% RGI). The relative level of mRNAaccumulation for each RGI was calculated as a percentage of themaximal level of accumulation for Atlas-66, which was set to 100% inall experiments. Relative expression values are the means + SE of 3 to12 independent measurements. The asterisk indicates a Student's t testgiving a signicant dierence between cultivars at P < 0.05;otherwise, there was no signicant dierence between cultivars

F. Hamel et al.: Wheat aluminum-regulated genes 533


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(Fig. 3). On the other hand, mRNA accumulation wassignicantly increased in the two wheat cultivars exposedto Al concentrations resulting in a moderate (3560%RGI; P < 0.005) or high (>60% RGI; P < 0.005)degree of toxicity compared to the control (0% RGI).The mRNA accumulation of up-regulated genes is thusmostly regulated by the degree of Al toxicity (asmeasured by RGI) and is independent of the cultivars'

tolerance. However, the absolute Al concentrationneeded to achieve similar RGIs, is cultivar dependent.

Tissue-specicity of up-regulated war genes. To examinethe tissue-specicity of up-regulated genes, we isolatedmRNAs from dierent parts of the plants. At dierentRGIs, the level of mRNA accumulation of War13.2increased in roots as well as in root tips (Fig. 4),suggesting that the function of this gene is closelyassociated with the eect of Al on root physiology. Therelative mRNA accumulation of the other up-regulatedgenes (war4.2, war5.2, and war7.2) was found to behigher in roots than in root tips, but was unaected by

varying RGIs in roots (data not shown). All four geneswere expressed at low levels in crown and leaf tissues andincreased slightly (but non signicantly) in leaf at highRGI. This result may suggest that a low amount of Al istranslocated between roots and other tissues during the24 h of exposure or that these genes are not responsiveto Al in other tissues.

Analysis of DNA sequences. We determined the completesequences of War4.2, War5.2, War7.2, and War13.2cDNAs. Details of mRNA size, and identity with othercDNAs are summarized in Table 1; DNA sequencecomparisons have shown that their identity with known

genes is greater than 60% in all cases.

War4.2 and War7.2 show 60.6% identity to aperoxidase and 89.8% identity to a phenylalanineammonia-lyase cDNA respectively (Table 1). These

genes have already been shown to be up-regulated byAl (Ezaki et al. 1996; Snowden and Gardner 1993) andwere not characterized in detail.

Fig. 4. Relative accumulation of War13.2 mRNA in dierent tissuesfrom wheat Atlas-66 seedlings. Total RNA was isolated from roottips, roots, crown, and leaves of 5-d-old Atlas-66 seedlings exposed for24 h to 0500 lM Al, to give RGIs of 0% (h), 3560% ( ) or >60%(j). The accumulation was quantitated by densitometry of northernhybridizations, and normalized to those obtained from the W13.3control probe. The relative level of mRNA accumulation for each

RGI was calculated as a percentage of the maximal level ofaccumulation observed in root tissue, which was set to 100% in allexperiments. Relative expression values are the means + SE of atleast three independent measurements

Table 1. Characteristics of the War cDNA clones. Sizes (in bases)of mRNAs were estimated from northern hybridizations. Identitieswere obtained from the FASTA search program of the GenBankrelease 101.0

Clone mRNA Gene Identitya

(bases) (%)

War4.2 1450 Peroxidase 60.6

War5.2 1700 Cysteine proteinase 78.5War7.2 2500 Phenylalanine ammonia-lyase 89.8War13.2 1000 Oxalate oxidase 79.0

aNucleotide identity was compared between War4.2 (AF005087),War5.2 (AF005088), War7.2 (AF005089), War13.2 (AF005084)and a peroxidase (X58396), a cysteine proteinase (D45402), aphenylalanine ammonia-lyase (X16099), and an oxalate oxidase(X93171) cDNA

Fig. 5. Comparison of cysteine proteinase protein sequences. Align-ment of the deduced amino acid sequences of the wheat (Triticumaestivum) and corn (Zea mays) cysteine proteinase proteins. Identityof 82.3% was obtained using the TFASTA search program of theGenBank release 101.0. Identities, and conservative changes areindicated by vertical lines, and colons, respectively. Dots within thesequences indicate gaps that were introduced to maximize homologybetween the two sequences. An arrow indicates the putative cleavagesite of the signal peptide. An arrowhead indicates a possible cleavagesite in post-translational processing. Also indicated are the conservedcysteine and histidine residues of the active sites (h) as well as theconserved cysteines involved in disulphide bridge formation (j). Theprotein sequences were deduced from the following cDNA sequences:

wheat (War5.2, acc. no. AF005088) and corn (Ccp1, acc. no. D45402)



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The deduced amino acid sequences of War5.2 andWar13.2 show 78.5% identity to a cysteine proteinaseand 79.0% identity to an oxalate oxidase cDNA,respectively (Table 1). The results of the search indicatethat War5.2, encoding a 41-kDa protein with a calcu-lated pI of 6.14, is homologous to Ccp1, a cysteineproteinase of corn (Zea mays) encoding a 40.3-kDa

peptide with an isoelectric point (pI) of 6.32 (Domotoet al. 1995). Figure 5 shows a comparison of thepredicted protein sequences of the wheat War5.2 withthe corn Ccp1 cDNAs. It was previously noted that theCCP1 protein contains a putative peptide signal of 23amino acids. A very similar signal was present in thewheat polypeptide. WAR5.2, as well as CCP1, containsa prepro-sequence, probably from 1M to 141G, whichwas assigned based on sequence similarity to the maturesequences of other known cysteine proteinases. Thewheat protein (Fig. 5) has conserved the relative posi-tion of the catalytic cysteine (C-166) and histidine(H-308) residues, and the cysteine residues involved indisulphide-bridge formation (C-163/C-209, C-197/C-246, C-302/C-359) compared to other mature cysteineproteinases (Linthorst et al. 1993).

In the case of War13.2, the deduced amino acidsequence contains 221 residues with a molecular weightof 24.3 kDa and a pI of 6.67. It was found that thiscDNA is highly homologous to the Bh6-903 cDNA ofbarley (Hordeum vulgare) with 79.0% identity at theDNA level and 72.4% identity at the protein level(Table 1, Fig. 6). Bh6-903 encodes an oxalate oxidase-like protein of 229 residues with a molecular weight of24.7 kDa and a pI of 6.5 (Thordal-Christensen et al.1997). Figure 6 shows a comparison of the predictedprotein sequences of the wheat War13.2 with the barley

Bh6-903 cDNAs. The proteins contain the rare HI/THPRATEI sequence that is found in oxalate oxidasesand in the spherulins 1a/b. It has been postulated that amembrane transit domain may be common to thesefunctionally disparate proteins, which are destined forthe extracellular matrix (Lane 1994).

Discussion

Wheat aluminum-regulated (war) genes show similaritieswith known PR genes. We have identied four genes(war4.2, war5.2, war7.2, and war13.2) that are up-regulated by Al exposure in two wheat cultivars dieringin their level of Al tolerance. Expression of RNA is

traditionally compared at similar Al concentrations(Snowden and Gardner 1993). Using this paradigm, weobserved that tolerant cultivars express these genes at amuch lower level than the sensitive cultivar (Fig. 2). Thisdierential sensitivity to Al toxicity could be explainedby the higher capacity of tolerant plants to exclude Al(Samuels et al. 1997). In order to compare the level ofgene expression under similar conditions, we used RGIas a measure of Al toxicity, as suggested by Parker(1995). Our results show that the dierent war genes areup-regulated proportionately to Al toxicity (Fig. 3). Thissuggests that these Al-regulated genes do not conferparticular advantages to the tolerant plants and couldnot be implicated in the dierential Al-tolerance mech-anisms of wheat cultivars. The dierent war genes up-regulated upon Al exposure are homologous to aperoxidase (war4.2), a cysteine proteinase (war5.2), aphenylalanine ammonia-lyase (war7.2), and an oxalateoxidase (war13.2) gene.

Peroxidases (such as war4.2) were previously found tobe up-regulated upon Al exposure (al201, Ezaki et al.1996). An increase in the activity of both soluble and cellwall peroxidases was observed by these authors eitherusing phosphate starvation or a combination of Alexposure and Pi starvation. Peroxidase activities occurmostly at the cell wall, where these enzymes have beensuggested to modulate cell wall rigidity and extensibility

by insolubilizing extensin monomers via a progressiveincrease in inter- and intramolecular cross-linking in-volving isodityrosine bridges (Welinger 1992), thusreducing the rate of Al diusion through the cell wall.Reinforcement of the cell wall has also been postulatedto be important to slow down pathogen invasion.Furthermore, since extensins are negatively charged,they could act as a ``y paper'' through electrostaticinteraction with Al3+ (Showalter 1993). Similarly, pec-tins which contain a large number of carboxyl groupscould also bind or chelate Al3+ ions. Analysis of cellwall components in squash root seedlings inhibited by Alexposure has shown an increase in pectin, hemicellulose,

and cellulose content after a few hours of exposure (LeVan et al. 1994). Thickening of the cell wall may lead toAl detoxication but, unfortunately, it may also result inthe arrest of root growth and cellular elongation (Le Vanet al. 1994).

We have isolated a cysteine proteinase (war5.2) genewhich, for the rst time, has been shown to be inducibleby Al. This enzyme is involved in diverse metabolicevents such as protein degradation or post-translationalprocessing of protein precursors to a mature form(Hara-Nishimura et al. 1993). It may participate in thedefense against pathogen infection since it was recentlyshown that benzothiazole, a novel class of inducer of

systemic acquired resistance, activates the induction of a

Fig. 6. Comparison of oxalate oxidase protein sequences. Alignmentof the deduced amino acid sequences of the wheat (Triticum aestivum)and barley (Hordeum vulgare) oxalate oxidase-like proteins. Identity of72.4% was obtained using the TFASTA search program of theGenBank database release 101.0. The box indicates a rare consensussequence. For other symbols, see Fig. 5. The protein sequences werededuced from the following cDNA sequences: wheat (War13.2, acc.

no. AF005084) and barley (Bh6-903, acc. no. X93171)



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cysteine proteinase gene as well as disease resistance inwheat (Go rlach et al. 1996).

Accumulation of phenylalanine ammonia-lyase(PAL; War7.2) homologs in the presence of Al waspreviously reported in wheat (Wali4, Snowden andGardner 1993). This enzyme could play a benecial rolein detoxifying Al that has entered the symplasm since

PAL was shown to catalyze the rst step of themultibranched phenylpropanoid metabolism in higherplants. The synthesis of anthocyans and avonoids,which were also referred to as catechins, is performed viaPAL. Catechins are very abundant (up to 1030% dryweight) in tea, which is known to accumulate Al inamounts reaching 30 000 ppm (Nagata et al. 1992). Thephenylpropanoid pathway and PAL are also involved inthe biosynthesis of numerous compounds (Bowles 1990).In particular, they participate in the synthesis ofprecursors such as ferulate groups (derived fromp-coumaric acid) which are used in pectin to increasethe gelling of polysaccharides and thus in strengtheningthe cell wall (Bowles 1990). Phenylalanine ammonia-lyase is a well known defense protein that has beenshown to accumulate in several dierent incompatibleplant-pathogen combinations and in response to elicitors(Ebel and Cosio 1994).

This is the rst report indicating that an mRNAhomologous to oxalate oxidase (War13.2) can accumu-late upon Al exposure. The enzyme was shown toaccumulate upon fungal infection of barley (Zhang et al.1995). Oxalate oxidase was also implicated in theresistance of transgenic rapeseed to an oxalate-secretingfungus (Lane 1994). The enzyme is involved in thedegradation of oxalate, accumulated in plant cells as thecalcium salt, to produce Ca2+, CO2, and H2O2.

The four genes identied (war4.2, war5.2, war7.2, andwar13.2) are up-regulated proportionately to the Altoxicity level (as measured by the RGI) in both cultivars,suggesting that a common signal is involved in theirregulation. An intriguing aspect of these genes is theirhomologies to genes induced in the plant defenseresponses against wounding or pathogen invasion. Inboth stresses, these genes may provide protection byincreasing cell wall thickness. To this end, oxalateoxidase would provide the H2O2 needed by the perox-idase to perform cross-linking reactions; PAL wouldprovide precursor molecules and cysteine proteinasewould be involved in the processing of new enzyme

molecules, such as peroxidase and oxalate oxidase.Atlas-66 wheat plants become much more sensitive toAl when the mucilage is removed from their root cap(Puthota et al. 1991). Furthermore several Arabidopsismutants with increased sensitivity to Al have beenisolated (Larsen et al. 1996), indicating that many genesare involved in the protection against Al toxicity. Thefour war genes isolated in our study may participate inthe strengthening of the root cell wall and in theexclusion of Al in both sensitive and tolerant plants.This inducible detoxication mechanism may involve Altrapping through an increase in the cell wall negativecharges and through the increased cross-linking. How-

ever, this hypothesis needs to be conrmed by direct

enzyme activity measurements in relationship withcytoplasmic Al-entry rates.

Relationships between Al and pathogen stresses. The plantdefense mechanisms that have been associated with theresponse to pathogens include: hypersensitive cell death,lignication, callose deposition, the synthesis of cell wall

extensins, the induction of pathogenesis-related proteinssuch as b-endoglucanases and chitinases, the accumula-tion of phytoalexins, proteins that induce cell lysis insporangia, proteinase inhibitors, and other toxic sub-stances referred to as thionins which are toxic cysteine-rich peptides produced during pathogen invasion(Hammond-Kosack and Jones 1996; Ebel and Cosio1994). Many of these responses are also associated withsymptoms of Al toxicity. The production of proteinaseinhibitors has been described by Richards et al. (1994)and several cDNAs encoding cysteine-rich peptides(similar to thionins) have been isolated (Richards et al.1994; Ezaki et al. 1995). The induction of b-1,3-glucanases by Al toxicity was recently shown in wheatroots (Cruz-Ortega et al. 1997). During pathogeninfection, the function of the inducible b-1,3-glucanasewould be to degrade fungal pathogen cell walls (Bowles1990). However, since pathogens do not appear to beinvolved in Al toxicity (unless Al increases sensitivity topathogens), the function of this enzyme may be to allowthe turnover of callose (b-1,3-glucan polymer) to pro-ceed. On the other hand, this enzyme may not be usefuland could be induced serendipitously through theelicitation of a defense-related signalling pathway.

One of the most interesting similarities between theresponse to Al exposure and the infection by pathogensis the production of active oxygen species, that partic-

ipate in the growth and development of plant cells.These molecules in concert with antioxidant metabolismhave been recently suggested as important signallingmolecules in response to pathogens or other stresses(Mehdy et al. 1996). Ezaki et al. (1995) have isolated agene induced by Al that is homologous to glutathioneS-transferase. The induction of this enzyme could limitlipid peroxidation which was previously observed insoybean root tips exposed to Al (Cakmak and Horst1991). These last authors have also shown an inductionof superoxide dismutase, indicating an increased metab-olism of oxygen free radicals. Such free radicals havebeen associated with signalling pathways involving

pathogens (Mehdy et al. 1996; Hammond-Kozack andJones 1996); they may also be involved in the response toAl toxicity since several oxidative stress genes areinduced by this metal (Richards et al. 1998). This mayexplain why many of the genes induced by Al are similarto those induced by pathogens or elicitors.

Recent studies support the possibility that variouscomponents of signalling pathways are aected by Altoxicity, since it was shown that Al could aect anionchannels (Schroeder 1995) or phosphoinositide trans-duction pathways (Jones and Kochian 1995). Aluminumwas also shown to block Ca2+ channels in the wheatroot plasma membrane (Huang et al. 1996) and, thus,

regulatory pathways involving Ca

2+

could aect the



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expression of specic genes. Furthermore, a low level ofextracellular Ca2+ was previously shown to up-regulateAl-responsive genes (Snowden et al. 1995). The induc-tion of these genes by a low level of Ca2+ is interestingsince a Ca2+ deciency was previously shown to inhibitroot growth and to enhance lipid peroxidation (Cakmakand Horst 1991). An increase in oxalate oxidase

(WAR13.2) activity would favor the release of Ca

2+

which may compensate, at least partially, for the eectof Al on calcium channels. It is plausible that accumu-lation of this mRNA caused by Al may be regulated by asignalling pathway involving Ca2+.

These dierent results indicate that Al may not onlyact through interactions with Ca2+ signalling pathwaysbut it may also trigger more than one pathway throughthe generation of several active molecules. This may thusexplain the complexity of responses observed in relationto Al toxicity.

The authors thank Dr B.F. Carver (Oklahoma State U., OK, USA)who provided the Atlas-66 seeds to initiate this project, and Dr N.

Chevrier (UQAM) for producing sucient seeds. This work wassupported by an NSERC grant (OGP0138557) to M.H. The salaryof M.H. was in part provided by the Canadian Network ofToxicology Centers via the ``Centre de Toxicologie de l'Environ-nement'' of UQAM.

References

Bowles DJ (1990) Defense-related proteins in higher plants. AnnuRev Biochem 59: 873907

Cakmak I, Horst WJ (1991) Eect of aluminium on lipidperoxidation, superoxide dismutase, catalase, and peroxidaseactivities in root tips of soybean (Glycine max). Physiol Plant83: 463468

Cruz-Ortega R, Cushman JC, Ownby JD (1997) cDNA clonesencoding 1,3-b-glucanase and a mbrin-like cytoskeletal proteinare induced by Al toxicity in wheat roots. Plant Physiol 114:14531460

Domoto C, Watanabe H, Abe M, Abe K, Arai S (1995) Isolationand characterization of two cDNA clones encoding corn seedcysteine proteinases. Biochim Biophys Acta 1263: 241244

Ebel J, Cosio EG (1994) Elicitors of plant defense responses. IntRev Cytol 148: 136

Ezaki B, Yamamoto Y, Matsumoto H (1995) Cloning andsequencing of the cDNAs induced by aluminium treatmentand Pi starvation in cultured tobacco cells. Physiol Plant 93:1118

Ezaki B, Tsugita S, Matsumoto H (1996) Expression of amoderately anionic peroxidase is induced by aluminium treat-ment in tobacco cells: possible involvement of peroxidase

isozymes in aluminum ion stress. Physiol Plant 96: 2128Go rlach J, Volrath S, Knauf-Beiter G, Hengy G, Beckhove U,

Kogel K-H, Oostendorp M, Staub T, Ward E, Kessmann H,Ryals J (1996) Benzothiadiazole, a novel class of inducers ofsystemic acquired resistance, activates gene expression anddisease resistance in wheat. Plant Cell 8: 629643

Hammond-Kosack KE, Jones JDG (1996) Resistance gene-depen-dent plant defense responses. Plant Cell 8: 17731791

Hara-Nishimura I, Takeuchi Y, Nishimura M (1993) Molecularcharacterization of a vacuolar processing enzyme related to aputative cysteine proteinase of Schistosoma mansoni. Plant Cell5: 16511659

Huang JW, Grunes DL, Kochian LV (1995) Aluminium andcalcium transport interactions in intact roots and root plasma-lemma vesicles from aluminium-sensitive and tolerant wheatcultivars. Plant Soil 171: 131135

Huang JW, Pellet DM, Papernik LA, Kochian LV (1996) Alumi-num interactions with voltage-dependent calcium transport inplasma membrane vesicles isolated from roots of aluminum-sensitive and -resistant wheat cultivars. Plant Physiol 110:561569

Jones DL, Kochian LV (1995) Aluminum inhibition of the inositol1,4,5-triphosphate signal transduction pathway in wheat roots:a role in Al toxicity? Plant Cell 7: 19131922

Kochian LV (1995) Cellular mechanisms of aluminum toxicity andresistance in plants. Annu Rev Plant Physiol Plant Mol Biol 46:237260

Lane BG (1994) Oxalate, germin, and the extracellular matrix ofhigher plants. FASEB J 8: 294301

Larsen PB, Tai CY, Kochian LV, Howell SH (1996) Arabidopsismutants with increased sensitivity to aluminium. Plant Physiol110: 743751

Lazof DB, Goldsmith JG, Rufty TW, Linton RW (1994) Rapiduptake of aluminum into cells of intact soybean root tips. PlantPhysiol 106: 11071114

Le Van H, Kuraishi S, Sakurai N (1994) Aluminum-induced rapidroot inhibition and changes in cell-wall components of squashseedlings. Plant Physiol 106: 971976

Linthorst SJM, van der Does C, Brederode FT, Bol JF (1993)Circadian expression and induction by wounding of tobacco

genes for cysteine proteinase. Plant Mol Biol 21: 685694Mehdy MC, Sharma YK, Sathasivan K, Bays NW (1996) The role

of activated oxygen species in plant disease resistance. PhysiolPlant 98: 365374

Nagata T, Hayatsu M, Kosuge N (1992) Identication of alumin-ium forms in tea leaves by 27Al NMR. Phytochemistry 31:12151218

Parker DR (1995) Root growth analysis: an underutilised approachto understanding aluminium rhizotoxicity. Plant Soil 171: 151157

Puthota V, Cruz-Ortega R, Johnson J, Ownby J (1991) Anultrastructural study of the inhibition of mucilage secretion inthe wheat root cap by aluminium. In: Wright RJ, Baligar VC,Murrmann RP (eds) Plant-soil interactions at low pH. KluwerAcademic Publishers, Dordrecht, pp. 779787

Rengel Z (1992) Role of calcium in aluminium toxicity. New Phytol121: 499513

Richards KD, Schott EJ, Sharma YK, Davis KR, Gardner RC(1998) Aluminum induces oxidative stress genes in Arabidopsisthaliana. Plant Physiol 116: 409418

Richards KD, Snowden KC, Gardner RC (1994) wali6 and wali7:genes induced by aluminum in wheat (Triticum aestivum L.)roots. Plant Physiol 105: 14551456

Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: alaboratory manual, 2nd edn. Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, NY

Samuels TD, Ku c u kakyu z K, Rinco n-Zachary M (1997) Alpartitioning patterns and root growth as related to Al sensitivityand Al tolerance in wheat. Plant Physiol 113: 527534

Sasaki M, Kasai M, Yamamoto Y, Matsumoto H (1995) Involve-ment of plasma membrane potential in the tolerance mechanism

of plant roots to aluminium toxicity. Plant Soil 171: 119124Schroeder JI (1995) Anion channels as central mechanisms for

signal transduction in guard cells and putative functions inroots for plant-soil interactions. Plant Mol Biol 28: 353361

Showalter AM (1993) Structure and function of plant cell wallproteins. Plant Cell 5: 923

Snowden KC, Gardner RC (1993) Five genes induced by aluminumin wheat (Triticum aestivum L.) roots. Plant Physiol 103: 855861

Snowden KC, Richards KD, Gardner RC (1995) Aluminum-induced genes: induction by toxic metals, low calcium, andwounding and pattern of expression in root tips. Plant Physiol107: 341348

Taylor GJ, Hunter DB, Stephens J, Bertsch PM, Elmore D, RengelZ, Reid RJ (1996) Direct measurement of Al transport acrossthe plasma membrane ofChara corallina. Proceedings of the 4th



8/8

International Symposium on Plant-Soil Interactions at LowpH. Belo Horizonte, Brazil, O15

Thordal-Christensen H, Zhang Z, Wei Y, Collinge DB (1997)Subcellular localization of H2O2 in plants: H2O2 accumulationin papillae and hypersensitive response during the barley-powdery mildew interaction. Plant J 11: 11871194

von Uexku ll HR, Mutert E (1995) Global extent, development andeconomic impact of acid soils. Plant Soil 171: 115

Wagatsuma T, Ishikawa S, Obata H, Tawaraya K, Katohda S(1995) Plasma membrane of younger and outer cells is the

primary specic site for aluminium toxicity in roots. Plant Soil171: 105112

Welinger KG (1992) Plant peroxidases: structure-function relation-ships. In: Penel C, Gaspar Th, Greppin H (eds) Plantperoxidases 19801990, topics and detailed literature onmolecular, biochemical, and physiological aspects. Universityof Geneva, Switzerland, pp. 124

Zhang Z, Collinge D, Thordal-Christensen H (1995) Germin-likeoxalate oxidase, a H2O2-producing enzyme, accumulates inbarley attackedby thepowderymildew fungus. Plant J 8: 139145


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