REGULAR ARTICLE

Aluminum resistance mechanisms in oat (Avena sativa L.)

Lorien Radmer & Mesfin Tesfaye &

David A. Somers & Stephen J. Temple &

Carroll P. Vance & Deborah A. Samac

Received: 11 April 2011 /Accepted: 24 July 2011 /Published online: 12 August 2011# Springer Science+Business Media B.V. (outside the USA) 2011

AbstractBackground and aims Enhanced aluminum (Al)resistance has been observed in dicots over-expressingenzymes involved in organic acid synthesis; however,this approach for improving Al resistance has not beeninvestigated in monocots. Among the cereals, oat(Avena sativa L.) is considered to be Al resistant, butthe basis of resistance is not known.Methods A hydroponic assay and hematoxylin stain-ing for Al accumulation in roots were used to evaluateAl resistance in 15 oat cultivars. Malate and citraterelease from roots was measured over a 24 h period.A malate dehydrogenase gene, neMDH, from alfalfa(Medicago sativa L.) was used to transform oat.

Results Oat seedlings were highly resistant to Al, as aconcentration of 325 μM AlK(SO4)2 was needed tocause a 50% decrease in root growth. Most oatcultivars tested are naturally resistant to high concen-trations of Al and effectively excluded Al from roots.Al-dependent release of malate and Al-independentrelease of citrate was observed. Al resistance wasenhanced in a transgenic oat line with the highestaccumulation of neMDH protein. However, overallroot growth of this line was reduced and expression ofneMDH in transgenic oat did not enhance malatesecretion.Conclusions Release of malate from oat roots wasassociated with Al resistance, which suggests that

Plant Soil (2012) 351:121–134DOI 10.1007/s11104-011-0937-1

Responsible Editor: Hans Lambers.

L. Radmer :M. TesfayeDepartment of Plant Biology,University of Minnesota,1445 Gortner Ave.,St. Paul, MN 55108, USA

D. A. Somers : S. J. TempleDepartment of Agronomy and Plant Genetics,University of Minnesota,1991 Upper Buford Circle,St. Paul, MN 55108, USA

C. P. VanceUSDA-ARS-Plant Science Research Unit andDepartment of Agronomy and Plant Genetics,University of Minnesota,1991 Upper Buford Circle,St. Paul, MN 55108, USA

D. A. Samac (*)USDA-ARS-Plant Science Research Unit andDepartment of Plant Pathology,University of Minnesota,1991 Upper Buford Circle,St. Paul, MN 55108, USAe-mail: dasamac@umn.edu

Present Address:D. A. SomersMonsanto Company,700 Chesterfield Village Parkway,Chesterfield, MO 63017, USA

Present Address:S. J. TempleForage Genetics International,N5292 S. Gills Coulee Road,West Salem, WI 54669, USA

malate plays a role in Al resistance of oat. Over-expression of alfalfa neMDH enhanced Al resistancein some lines but was not effective alone for cropimprovement.

Keywords Aluminium . Aluminum resistance . Avenasativa . Citrate . Malate dehydrogenase .Malatesecretion . Oat . Sugarcane bacilliform badnavirus(ScBV) promoter

Introduction

Aluminum (Al) is widespread in agricultural soils andAl toxicity is a major limiting factor in crop productionthroughout the world (vonUexküll andMutert 1995). Insoils with a pH of 5.5 and below, Al becomes solubleand forms phytotoxic cations, Al(H2O)6

3+ (also knownas Al3+) and Al(OH)2

+, that are rapidly taken up byplant roots (Kinraide 1991, 1997). An estimated 30–50% of the world’s potentially arable land is acidic andcurrent agricultural practices, along with acid rain, arecausing a rise in the rate of soil acidification (Tarkalsonet al. 2006; von Uexküll and Mutert 1995). Aluminumuptake by plant roots results in stunted root growthcausing a decrease in the plant’s ability to absorb waterand nutrients from the soil (Andersson 1988). Addi-tional mineral ion toxicities, particularly Mn, alsonegatively affect plant growth in acidic soil. At lowpH, many nutrients essential to plant growth are notreadily available, particularly P, but also Ca, Mg, and tosome extent N and K (Lambers et al. 1998).Alleviating Al toxicity in plants will not necessarilysolve these problems but will moderate a majorinhibiting factor for plant growth in acidic soil.

Identifying Al resistant plants and understandingthe mechanisms underlying resistance has been amajor focus of research in many laboratories(Kochian et al. 2005). Resistance to Al has beenfound to occur when Al is excluded from the rootapex, the main target of Al toxicity. Additionally,some plants have been shown to have mechanismsthat allow them to tolerate Al in the symplasm. Apredominant mechanism of Al resistance identified incereal crops is secretion of organic acids, whichchelate Al outside the root and prevent it from beingtaken up by root cells (Kochian et al. 2005; Ryan andDelhaize 2010). In Al resistant wheat (Triticumaestivum L.) seedlings, Al treatment rapidly stimu-

lates secretion of malate and succinate (Delhaize et al.1993a). The majority of malate efflux in resistantseedlings is from the root apex, although highamounts of malate accumulate behind the root tip(Ryan et al. 1995). Genetic analysis of Al resistance inwheat found that the majority of resistance isconditioned by the Alt1 locus. A gene at this locus,TaALMT1 (Al-activated malate transporter 1), enco-des a malate transporter that is localized in the plasmamembrane and mediates Al-activated release ofmalate from resistant wheat roots (Sasaki et al.2004). Expression of this gene in barley (Hordeumvulgare L.) greatly enhances malate secretion fromroots upon exposure to Al and increases Al resistancein both hydroponic and acid soil culture (Delhaize etal. 2004). More recently, Pereira et al. (2010) trans-formed an aluminum-sensitive wheat cultivar withTaALMT1 and found that resulting T2 lines showedincreased Al3+ resistance. Al-induced secretion ofcitrate has been associated with Al resistance in rice(Oryza sativa L.), maize (Zea mays L.), sorghum(Sorghum bicolor (L.) Moench), and rye (Secalecereale L.) (Kochian et al. 2005).

Oat (Avena sativa L.) is considered to be moreresistant to Al than many other cereal crops includingwheat, maize, and barley (Nava et al. 2006). However,only a few oat cultivars have been characterizedspecifically for their response to Al stress resulting ina lack of knowledge of the extent of Al resistance inoat. In one study, oat cultivars exhibited a range oftolerance to acidic Al-containing soil, which wasassumed to reflect their relative Al resistance (Foy etal. 1987). In solution culture, six oat cultivars wereshown to vary in Al tolerance (Wheeler et al. 1992).Inheritance studies conducted with Brazilian Alresistant cultivated oat genotypes found resistanceto be conditioned by single genes (Castilhos et al.2011; Nava et al. 2006). Zheng et al. (1998) foundthat oat plants release malate and citrate afterexposure to 50 μM Al. The amount of organic acidssecreted was low compared to the amount of organicacids produced by Al resistant wheat, which led theauthors to suggest that additional resistance mecha-nisms may be present in oat. In the diploid oat, A.strigosa Schreb., four QTLs for Al tolerance wereidentified (Wight et al. 2006). Since oat is expectedto have highly effective mechanisms of resistance ortolerance, further investigation of the response of oatto Al stress is warranted.

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Overexpression of genes encoding enzymes involvedin organic acid production in transgenic plants has beenshown to enhance Al tolerance. In alfalfa (Medicagosativa L.), overexpression of a novel form of malatedehydrogenase (neMDH) resulted in an increase inmalate concentration in roots, release of greateramounts of malate from roots, and enhanced Altolerance compared to nontransgenic control plants(Tesfaye et al. 2001). neMDH is normally expressed inalfalfa root nodules and has a much higher specificityfor oxaloacetate and NADH than other MDH isoforms,which drives the reaction towards malate production(Miller et al. 1998). Likewise, overexpression of citratesynthase was reported to increase Al tolerance inArabidopsis (Koyama et al. 2000), Brassica napus L.(Anoop et al. 2003), and alfalfa (Barone et al. 2008).Whether overexpression of genes involved in organicacid synthesis will enhance Al resistance in a cerealcrop has not been tested.

The objectives of this research were to developscreening methods for identifying Al tolerance orresistance mechanisms in oat and to test if malaterelease can be enhanced by gene overexpression. Inthis research, oat (cv. Belle) embryos were trans-formed, using the neMDH gene controlled by theSugarcane bacilliform badnavirus (ScBV) promoter.In oat, the ScBV promoter conveys constitutive geneexpression in most tissues of the plant with thehighest expression in the vascular tissue (Al-Saadyet al. 2004; Tzafrir et al. 1998). Plants were assayedfor Al tolerance in a hydroponic system and byhematoxylin staining to evaluate Al exclusion fromroot tips. Using the same methods, 15 oat cultivarswere evaluated for root growth, relative Al tolerance,and Al exclusion. In contrast to previously publishedreports we found that oat seedlings secreted largeamounts of malate when grown in hydroponic culturecontaining a high Al concentration and that exudationcorrelated with Al resistance.

Materials and methods

Plant material

Oat cultivars Baker, Belle, Drumlin, Esker, Kame,Leonard, Moraine, Morton, Reeves, Richard, Riser,Sesqui, Spurs, Wabasha, and Winona were tested forAl tolerance. These were the most widely grown

cultivars in the Midwestern U.S. at the time of theexperiment. Seed was obtained fromDr. Deon Stuthman,Department of Agronomy and Plant Genetics, Universityof Minnesota, St. Paul, MN.

Hydroponic assay of Al tolerance

Seed was vernalized for 24 h at 4°C in Petri platesbetween moist pieces of filter paper. Plates were thenmoved to a 30°C incubator for 24 h. Germinatedseeds with roots of approximately equal length wereplaced individually in a 48 mm plastic cup with anylon mesh bottom with 3 mm openings. Hydroponicchambers were constructed using 15 L plasticcontainers (ClearView Storage Boxes, Sterilite, Town-send, MA) fitted with a plexiglass insert with holes toaccommodate 12 plastic cups. The cups were sus-pended so that the mesh touched the surface of theassay solution. The chambers were filled with 7.3 Lof preconditioning solution consisting of 0.4 mMCaCl2 adjusted to pH 4.4 using 1M HCl. Seedlingswere grown for 24 h in the preconditioning solutionunder constant aeration with ambient room light andthen the length of the three longest roots of eachseedling was measured using a mm ruler. Seedlingswere then moved to chambers that contained freshcontrol solution, 0.4 mM CaCl2 at pH 4.4, or anexperimental solution containing 0.4 mM CaCl2 anddifferent amounts of AlK(SO4)2 at pH 4.4. Theexperimental and control solutions were adjusted topH 4.4 using 1M HCl. After 24 h under constantaeration in control and test solutions, the length of thethree longest roots was measured. The average rootlength was calculated for each plant. The relative rootgrowth ratio (RRGR) for each Al concentration wascalculated using:

RRGR ¼ RAf � RAi

� �

RCf � RCi

� �

in which RAf was the final aluminum-grown rootlength, RAi the initial aluminum-grown root length,RCf the final control root length, and RCi the initialcontrol root length.

Sixteen seeds were germinated from each trans-genic and control line and seedlings placed in thepreconditioning treatment as described above. Half ofthe plants from each line were then moved to ahydroponic chamber containing an experimental

Plant Soil (2012) 351:121–134 123

solution: 0.4 mM CaCl2, 325 μM AlK(SO4)2, pH 4.4.The remaining seedlings were placed in fresh pre-conditioning solution. The length of the three longestroots was measured. After 24 h under ambient labconditions and constant aeration, the length of thethree longest roots was measured a second time. Thisexperiment was done three times and RRGR wascalculated using the mean values of each lineaveraged across seedlings in each biological replicate.Statistical analysis of RRGR was performed using theGeneral Linear Model procedure in SAS (SASInstitute 2003).

Twenty-five seeds of each of 15 cultivars (Baker,Belle, Drumlin, Esker, Kame, Leonard, Moraine,Morton, Reeves, Richard, Riser, Sesqui, Spurs,Wabasha, and Winona) were vernalized, germinated,and grown in preconditioning solution as describedabove. The 18 seedlings with the longest roots werechosen. Nine seedlings were placed in a controlsolution of 0.4 mM CaCl2 pH 4.4 and another nineseedlings were placed in a 325 μM AlK(SO4)2,0.4 mM CaCl2, pH 4.4 solution. Cups containingseedlings were randomized before being placed in thehydroponic chambers. The three longest roots of eachseedling were measured and seedlings were incubatedin hydroponic chambers for 24 h under ambient labconditions and constant aeration. After a further 24 hgrowth, the three longest roots of each seedling weremeasured again and the relative root growth ratio forthe cultivar was calculated as described above. At theend of the Al experiment, the roots of the seedlingswere stained with hematoxylin as described below.The experiment was done three times and data wereanalyzed as described above.

Detection of Al uptake in oat roots

To detect Al uptake in Al-treated and control plants,roots were rinsed in distilled water for 30 min toremove any nutrient growth solution, and then stainedwith a solution of 0.1% hematoxylin (Sigma-Aldrich,St. Louis, MO) and 0.01% KI for 30 min (Canaado etal. 1999). Excess hematoxylin stain was washed offwith a 30 min rinse in distilled water. Finally, rootswere examined with a dissecting microscope andphotographed.

To examine Al within roots, seedlings were grownin 0 mM Al or 325 μM Al, and then stained with100 μM morin (Sigma-Aldrich) (Larsen et al. 1996).

Approximately 1 cm of root tip was excised fromeach seedling, embedded in 3% agarose, and 50 μmcross-sections were made from approximately 0.5–1 mm from the root tip using a vibratome. Sectionswere placed on a microscope slide and examined withan Olympus IX70 inverted microscope by bright fieldand epifluorescence (440 nm excitation and 510 nmemission).

Measuring malate and citrate exudation

To determine the amount of malate exuded from roottips of seedlings of cv. Belle, 10 germinating seedswere placed in a 35 mm plastic cup with a 3 mmnylon mesh bottom in a 50 mL sterile conicalpolypropylene tube (Corning Incorporated, Corning,NY). Seedlings were grown in 50 mL preconditioningsolution (0.4 mM CaCl2, pH 4.4), with constantshaking at 100 rpm for 24 h. The preconditioningsolution was replaced with 50 mL of 0.4 mM CaCl2with 0 mM, 100 μM, 200 μM, or 300 μM AlK(SO4)2at pH 4.4. Seedlings were grown for an additional24 h with constant shaking at 100 rpm. In a separatestudy, malate and citrate exudates from seedlings offour additional cultivars showing a range of Alresistance were collected in the same way using325 μM AlK(SO4)2 at pH 4.4.

The solutions were frozen at −80°C, lyophi-lized, and material dissolved in distilled water.Aliquots of the concentrated samples were used foranalysis of malate using an L-malic acid enzymaticassay (Megazyme, Wicklow, Ireland). Cations wereremoved from samples using Empore chelatingdisks (3 M Company, Eagan, MN) before measur-ing citrate content with the Megazyme citric acidenzymatic assay. Three to four root exudatesamples were measured for each treatment andthe experiment was done twice.

Oat transformation and characterization of transgeniclines

The plasmid pScBV3-MDH11 used for transforma-tion consisted of a full-length cDNA of neMDH fromalfalfa (Miller et al. 1998) inserted behind the ScBVpromoter in pRT106’-pSCBV3-BB (Tzafrir et al.1998). Embyogenic tissue cultures derived frommature embryos of the oat cv. Belle were cobom-barded with pScBV3-MDH11 and pH24, a plasmid

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with a paromomycin selectable marker (Fromm et al.1986), as described previously (Torbert et al. 1998).Seed from paromomycin resistant regenerated T0

plants were grown in the greenhouse and seedcollected from individual self-pollinated plants.

Forty-two T2 lines were assayed as follows toidentify lines putatively homozygous for the neMDHtransgene. RNA was extracted from seedling rootsusing the RNeasy kit (Qiagen, Valencia, CA). Reversetranscription- (RT) PCR was performed using theAccess RT-PCR System (Promega Corp., Madison,WI) with the primers M11F 5′-GACCTGCATCTCTATGATATCG-3′ and MRTR 5′-CAACAACTGGAACATCCACATC-3′ corresponding to positions 384–405 and 801–822 of the neMDH cDNA sequence(Genbank accession AF020273). The PCR amplifica-tion conditions were: 1 cycle of 48°C for 45 min, 1cycle of 94°C for 2 min, followed by 40 cycles of 94°C for 30 s, 53°C for 30 s, and 68°C for 1 min, with afinal cycle of 68°C for 7 min. Products were separatedon 1% agarose gels and stained with ethidiumbromide. Lines in which all eight seedlings testedproduced mRNA of the transgene were saved forfurther testing. Where possible, sister lines from thesame T0 plant but in which the transgene was notexpressed were identified using the same technique.

To estimate transgene copy number, Southern blotanalysis was done using genomic DNA digested withXba1, which has a single site in pScBV3-MDH11.Blots were probed with a 1.65 kbp fragment ofneMDH as described previously (Tesfaye et al. 2001).

Accumulation of neMDH protein in oat roots wasdetected by immunoblotting as described previously(Tesfaye et al. 2001) using protein extracted fromplant roots grown hydroponically in a 0.4 mM CaCl2solution.

Malate and citrate concentration in root exudateswere measured as described above.

Results

Response of seedlings from the cultivar Belle to Altreatment

When cv. Belle seedlings were grown in increasing Alconcentrations the relative root ratio decreased in aconcentration-dependent manner, indicating that therewas greater root growth inhibition at the higher Al

concentrations (Fig. 1a). However, inhibition of rootgrowth was similar at 250, 300, and 350 μM AlK(SO4)2. These concentrations result in 80 μM, 90 μMand 98 μM free Al activity, respectively, as deter-mined by GEOCHEM-EZ (Shaff et al. 2010). It wasnot possible to test higher concentrations of Albecause additional Al decreased the assay solutionbelow pH 4.4. Addition of base to raise the pH wouldalter the composition of Al ions in the solution. TheAl concentration at which cv. Belle plants reproduc-ibly displayed approximately 50% root growth inhi-bition under hydroponic conditions was 325 μM AlK(SO4)2 in 0.4 mM CaCl2, pH 4.4, which results in94 μM free Al activity. This concentration was usedfor further comparative tests.

Hematoxylin is an Al-specific dye that has a purplecolor when it forms a complex with Al (Polle et al.1978). After exposure to Al for 24 h, root tips of cv.Belle plants had low to moderate amounts of stainingwith hematoxylin (Fig. 1b). An increase in stainingoccurred with increasing Al concentration. Moststaining occurred in epidermal cells, the root tip, andmaterial loosely associated with the roots withrelatively little stain associated with root corticalcells. Roots grown in the control solution did notstain. Little distortion or growth abnormalities of theroot tips were observed, even at the highest Alconcentration.

Cross sections of root tips exposed to 325 μM AlK(SO4)2 for 24 h were made and stained with morin toidentify locations in the root were Al accumulated(Fig. 2). There was a small amount of backgroundfluorescence across the root sections in roots grown inthe control solution after morin staining (Fig. 2b). Afew root border cells and other cells detached fromthe root tip fluoresced brightly, without exposure toAl. Roots exposed to Al displayed a moderate numberof brightly fluorescing cells (Fig. 2d). Fluorescencewas observed in distinct patches of cells in the outercell layer and several underlying layers of corticalcells. Fluorescence was often associated with dam-aged cells visible by light microscopy (Fig. 2c). Agreater number of root border cells fluoresced after Alexposure than in the control treatment.

Malate exudation was examined in roots of cv.Belle seedlings at three Al concentrations. Theamount of malate in root exudates increased with Alconcentration (Fig. 3). The Al treatments increasedmalate in exudates 22- to 41-fold, from an average of

Plant Soil (2012) 351:121–134 125

1.24 nmol seedling−1 in the control treatment of 0 μMAl to 27.8 nmol seedling−1 in the 100 μM AlK(SO4)2treatment and 51 nmol seedling−1 in the 300 μM AlK(SO4)2 treatment.

Response of seedlings from 15 oat cultivars to Al

The 15 oat cultivars tested in the hydroponic assaywith 325 μM AlK(SO4)2 varied in response to thetreatment. Roots from the comparatively Al sensitivecultivar ‘Sesqui’ had a mean RRGR of 0.33 whichwas significantly different (p<0.05) from the meanRRGR of the most Al resistant cultivar ‘Baker,’ witha mean RRGR of 0.70 (Fig. 4a). The majority ofcultivars had a mean RRGR between 0.4 and 0.6,similar to roots from ‘Belle’ plants. The RRGR of

plants from ‘Belle’ and ‘Sesqui’ were not significant-ly different (p<0.05).

The amount of malate released by roots of thefive cultivars tested showed significant differences(p<0.05) with the Al treatment (Fig. 4b). Plants fromthe more Al tolerant cultivars such as ‘Baker’ and‘Esker’ released the greatest amount of malate, 52 and68 nmol seedling−1, respectively. Seedlings of cv.Sesqui, the most Al sensitive, exuded the leastamount of malate, 29 nmol seedling−1, over a 24 hperiod of Al exposure. In contrast, roots releasedcitrate in both the control 0.4 mM CaCl2 solution andin the Al treatment (Fig. 4c). Although citrate contentwas greater in the Al treatment for all cultivars, thedifference between the control and Al treatment wassignificant (p<0.05) only for ‘Belle’ seedlings. There

Fig. 1 Response of cv. Belle seedlings to Al-containing hydro-ponic solution culture. a The relative root growth ratio of cv.Belle seedlings. Growth in Al treatment solutions (pH 4.4) wascompared to growth in the 0.4 mM CaCl2 pH 4.4 control solution

over 24 h. Error bars indicate standard error. b Cultivar Belleroot tips grown in a range of Al concentrations and stained withhematoxylin. Scale bar indicates 1 mm

126 Plant Soil (2012) 351:121–134

was no significant difference among cultivars forcitrate release.

Roots of cv. Sesqui seedlings exposed to Al staineddarker with hematoxylin than similarly treated cv.Baker seedlings (Fig. 5a). As was observed with cv.Belle seedlings, much of the stain was associated withmaterial loosely associated with roots. In roots of cv.Sesqui seedlings, a moderate amount of stainingoccurred in the root apex and in epidermal cellsapproximately 2 mm from the root tip. However,

darker hematoxylin staining was not consistent withgreater Al sensitivity in all oat cultivars. Roots ofplants from the more resistant cultivars, Wabasha andKame, stained darker than roots from cv. Mortonplants, all of which have a similar RRGR (Fig. 5b).Although staining was generally consistent amongseedlings within a cultivar, the extent of stainingvaried markedly from seedling to seedling in cvs.Esker and Richard after Al treatment (Fig. 5c).

Effect of neMDH overexpression on malate secretionand Al resistance

Forty-two T2 lines originating from 11 paromomycinresistant regenerated T0 plants were screened for thepresence of the neMDH transcript using reversetranscription PCR. Of these, four putatively homozy-gous lines and 10 heterozygous lines were identifiedthat were derived from four T0 plants. The remaining28 lines from six T0 plants did not have measurableneMDH transcripts. Three of the homozygous posi-tive lines (L1A, L2A, L3A) and three transformationcontrol lines lacking the neMDH transcript (L1B,L3B, L5B) were selected for further characterization.Lines L1A and L1B were derived from the same T0

plant as were L3A and L3B. The third control line(L5B) was independent of the other lines and waspaired with L2A.

Fig. 3 Malate release from cv. Belle oat seedling roots.Seedlings were grown in a 0.4 mM CaCl2 pH 4.4 solutionwith the indicated concentration of Al for 24 h. Bars indicatestandard error

Fig. 2 Morin staining ofroot cross-sections fromcontrol and Al-treated cv.Belle seedlings. Root cross-sections were made approx-imately 0.5 mm behind theroot tip, stained and viewedunder (a, c) bright-fieldconditions and (b, d) UVlight. a and b, root sectionfrom control treatment. cand d, root section from325 μM Al treatment

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Fig. 4 The response of 15oat cultivars to exposure to325 μM Al pH 4.4 for 24 hcompared to plants in acontrol 0.4 mM CaCl2pH 4.4 solution. a Relativeroot growth ratio of culti-vars comparing growth inAl treatment to growth inthe control solution over24 h. b Malate exudation ofcultivars. c Citrate exuda-tion of cultivars. Bars indi-cate standard error. Meanswith the same letter are notsignificantly different

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Southern blots of genomic DNA digested withXbaI showed that the three transgenic lines were fromindependent transformation events (data not shown).Lines L1A and L2A each had a single band ofdifferent molecular weight while L3A had multiplebands, indicating insertion of multiple copies of theneMDH transgene.

neMDH protein was detected in the three trans-genic lines (Fig. 6). Line L3A appeared to accumulatemore neMDH protein than L1A and L2A, whichaccumulated similar amounts of neMDH protein.

Seedlings from transgenic lines, transformationcontrol lines and cv. Belle were exposed to 325 μMAlK(SO4)2 over a 24 h period and root growthcompared to seedlings exposed to 0.4 mM CaCl2.Root growth in the control solution was similar in all

lines except L3A, in which root growth was signif-icantly reduced (p<0.05) (Fig. 7a). The Al treatmentreduced root growth in all lines by about 50% ormore, except for line L3A for which the RRGR was0.76 (Fig. 7b).

Hematoxylin staining of roots of transgenic linesL1A, L2A and transformation control lines after 24 hexposure to 325 μM AlK(SO4)2 was similar to that ofroots from cv. Belle seedlings (data not shown). Incontrast, roots of seedlings from line L3A stainedmuch darker, although staining appeared to beassociated primarily with material and cells looselyassociated with the root surface.

The amount of malate produced by roots oftransformation control and transgenic lines in theabsence of Al was small and varied little among lines

Fig. 5 Hematoxylin stain-ing of Al-treated andcontrol roots. Scale barindicates 1 mm. a The mostAl resistant oat cultivar,Baker, and the most Alsensitive cultivar, Sesqui. bThree cultivars of similar Alresistance show differenthematoxylin staining pat-terns when grown in Al. cIntracultivar variation inhematoxylin staining pat-terns of roots grown in anAl solution

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Fig. 6 Western blots of protein from roots of transgenic oat andcontrol lines probed with neMDH antibody. L1A, L2A, andL3A are neMDH transgenic lines. L1B, L5B, and L3B aretransformation control lines. Belle is the wild type. The

neMDH antibody sometimes detected a faint signal in proteinextracted from control lines and from roots of cv. Belleseedlings, which may be due to non-specific cross reactionwith an oat protein

(Fig. 8). After Al treatment, malate in root exudatesincreased dramatically in all lines. The amount ofmalate produced varied among lines but was notassociated with the presence of the neMDH transgene.Notably, the amount of malate in exudates of lineL3A, which had a high amount of neMDH protein,was not significantly different (p<0.05) from the

amount in exudates in other lines either with orwithout Al treatment. The amount of citrate inexudates was similar for plants of line L3A and thecontrol (data not shown).

Discussion

Among the cereal crops, oat is considered to be one ofthe most Al resistant. However, mechanisms ofresistance in oat have not been investigated and thereis little information available on Al resistance incurrently cultivated oat cultivars. Exposure of seed-ling roots to Al in a hydroponic system has been usedextensively to assess Al tolerance in many species.Using a hydroponic assay with a simple nutrientsolution, we found that cv. Belle seedlings couldtolerate high concentrations of Al. An AlK(SO4)2concentration greater than 300 μM (free Al activitygreater than 90 μM) was needed to inhibit root growthby 50%. In support of our results, two oat cultivarswere found to be among the most Al tolerant cerealsin long-term experiments using a low ionic strengthnutrient solution (Wheeler et al. 1992).

Fig. 8 Malate release from roots of transgenic and control oatlines over 24 h. Error bars represent standard error. Seedlingswere grown in a 0.4 mM CaCl2 pH 4.4 solution with or without325 μM AlK(SO4)2 pH 4.4 for 24 h. L1A, L2A, and L3A areneMDH transgenic lines. L1B and L5B are transformationcontrol lines. Belle is the wild type

Fig. 7 Root growth ofneMDH transgenic andcontrol seedlings. a Plantswere grown over 24 h in a0.4 mM CaCl2 pH 4.4 con-trol solution or 325 μM AlK(SO4)2 pH 4.4 treatmentsolution. b Relative rootgrowth ratio comparinggrowth in the treatmentsolution to growth in thecontrol solution over 24 h.L1A, L2A, and L3A areneMDH transgenic lines.L1B, L3B, and L5B aretransformation control lines.Cultivar Belle is the wildtype. Results shown arefrom eight plants in threeexperiments (24 plants) ineach growth solution. Barsindicate standard error

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In the Midwestern U. S., soil acidity is managed byliming, and most agricultural soils do not have toxiclevels of Al ions. Therefore, oat plants are notroutinely exposed to high levels of Al nor arecultivars selected for Al tolerance. Nonetheless, ofthe 15 cultivars tested that are widely grown in theMidwest, 13 cultivars had Al tolerance similar to cv.Belle. Only seedlings of cv. Baker were significantlymore Al tolerant, and seedlings of cv. Sesqui weremore sensitive than seedlings of the other oatcultivars. The prevalence of Al tolerance in unselect-ed oat cultivars suggests that tolerance is linked to adesirable agronomic trait common to these cultivarsor that the mechanism is associated with a commonbasic metabolic function(s) in these cultivars. Assess-ment of Al tolerance in diverse oat germplasm iswarranted to gain a better understanding of the originof Al tolerance in cultivated oat.

Two broad types of Al tolerance mechanisms havebeen identified in plants, exclusion and internaldetoxification. Exclusion is the most widely identifiedmechanism in cereals. Hematoxylin staining is asimple means of determining the extent of Alabsorbed by roots and has been widely used toidentify Al resistant plants (Polle et al. 1978; Canaadoet al. 1999). Sensitive plants accumulate Al in the roottip and epidermal cells (Delhaize et al. 1993b). Afterexposure to Al for 24 h, oat root cells had little purplecoloration from the hematoxylin stain, even at high Alconcentrations. This indicates that oat has a highlyeffective Al exclusion mechanism. The stainingobserved was uniform along the seedling rootsuggesting that resistance is rapidly induced uponexposure to Al or is constitutively expressed. Thestaining pattern of root cross sections stained withmorin also indicated that little Al accumulated inepidermal cells of oat roots. However, hematoxylinstaining was associated with mucilage-like materialand cells loosely associated with the root tip. Morinstaining was also more prevalent in root border cellsafter Al treatment, although some border cells fromcontrol treatments fluoresced after staining. Root bordercells have been shown to influence Al tolerance inwheat, barley, and common bean (Phaseolus vulgarisL.) (Miyasaka and Hawes 2001; Zhu et al. 2003;Tamás et al. 2005). Mucilage from wheat and maizestrongly binds Al but its role in exclusion is not clear(Archambault et al. 1996; Li et al. 2000). Our resultssuggest that oat root border cells and root mucilage

bind Al, but their role in excluding Al from oat rootsremains to be tested.

We found that hematoxylin staining alone is not anaccurate indicator of relative Al tolerance among oatcultivars. In a number of instances, the degree ofhematoxylin staining in roots after Al exposure didnot correlate with Al resistance as measured by theRRGR. For example, seedlings from cvs. Morton,Kame, and Wabasha had very similar RRGRs after Alexposure but very different hematoxylin stainingpatterns. This could be due to variation in exclusionby seedlings in these cultivars or in the amount of rootmucilage and border cells produced that accumulatedAl and stained with hematoxylin. Also, seedling rootsfrom cvs. Richard and Esker showed different stain-ing patterns within the two cultivars, althoughvariation in RRGR was low. Therefore, cultivarselection based on staining alone would not accuratelyidentify Al resistant plants or cultivars.

In other cereals Al resistance is correlated with Al-induced secretion of organic acids (Kochian et al.2005). In wheat, exclusion of Al from roots is dueprimarily to Al-induced secretion of large amounts ofmalate by root tips and formation of Al-malatechelates outside of the root, which are not taken upby root cells (Delhaize et al. 1993b). Recently, Ryanet al. (2009) reported constitutive efflux of citratefrom roots of certain Al-resistant wheat cultivars as asecond major Al resistance mechanism, and that thegene for citrate efflux co-segregated with expressionof a MATE gene. Our results showed that oat seedlingroots had a similar pattern of constitutive release ofcitrate and an Al-dependent release of malate. Theamount of malate in root exudates increased with anincrease in external Al concentration and differentialresistance in oat cultivars was associated with malaterelease. These results support studies in diploid oatthat found a major QTL for Al resistance is possiblyorthologous to Alt1 (TaALMT1), an Al-dependentmalate transporter, and a minor QTL is possiblyorthologous with the maize tolerance gene Alm2,likely a multidrug and toxin efflux (MATE) proteininvolved in citrate efflux (Ninamango-Cárdenas et al.2003; Maron et al. 2010; Wight et al. 2006). However,as discussed above, our results indicate that exclusionof Al in oat is associated with multiple mechanisms.Yamaji et al. (2009) determined that in rice, anorganism with a high aluminum-tolerance, severalgenes working at multiple cellular levels are regulated

Plant Soil (2012) 351:121–134 131

in response to aluminum. This paper highlightedinternal tolerance mechanisms and also suggestedthat aluminum tolerance is not achieved by organicacid secretion alone. Similarly, organic acid exudationwas determined to be unable to completely explainthe aluminum resistance of six maize genotypes(Piñeros et al. 2005).

Expression of the alfalfa neMDH cDNA from theScBV promoter resulted in expression of the neMDHprotein in oat roots. However, this did not increasemalate secretion of transformed seedlings comparedto transformation control seedlings or cv. Belle seed-lings. One possibility for the lack of enhanced malatesecretion in transgenic oat seedlings is the inability ofoat malate transporters to export malate from cells at anincreased rate due to increased malate production, butinstead to export malate from cells at a fixed rateregardless of internal malate concentration. In triti-cale (x Triticosecale Wittmark), internal organic acidlevels or the capacity of root cells to synthesizeorganic acids has little bearing on exudation oforganic acids from the root (Hayes and Ma 2003). Itwas suggested that the difference in Al tolerancebetween tolerant and sensitive triticale lines is dueto the up-regulation of a gene or genes thatregulate the export of organic acids from the cell.Piñeros et al. (2005) found that changes in internalconcentrations of citrate in maize roots did notcorrelate with the rate of citrate release. Over-expression of TaALMT1, the malate transporter fromwheat was shown to enhance the amount of malatesecreted from barley (Delhaize et al. 2004). Possibly,overexpression of both a transporter and neMDHcould increase Al resistance in oat and other cerealcrops.

An alternative possibility for the lack of enhancedmalate secretion in transgenic oat lines is the lack ofactivity of neMDH in oat. Although neMDH tran-scripts were detected in oat and protein was extractedfrom roots that were recognized by the neMDHantibody, proper protein folding that gives neMDHits enzymatic properties may not occur in oat.Delhaize et al. (2001) speculated that one of thepossible factors that led to decreased protein expres-sion of a citrate synthase gene from Pseudomonasaeruginosa in tobacco was incorrect folding of theprotein. The neMDH antibody detected a smallamount of oat protein in control plants. In alfalfa,the active form of neMDH is a dimeric protein (Miller

et al. 1998). It is possible that a subunit of neMDHcould associate with a subunit from oat, creating aprotein that, while not functional, was recognized bythe antibody.

The expression pattern of the promoter used toexpress neMDH may be an important factor inexplaining the discrepancy between the molecularand physiological results. Genes expressed from theScBV promoter are constitutively expressed through-out much of the oat plant but the strongest expressionis in the vascular system (Tzafrir et al. 1998; Al-Saady et al. 2004). Transformed oat plants may beproducing enhanced amounts of malate due to thetransgene but are unable to export it from the rooteffectively due to neMDH being expressed in the steleof the root.

Line L3A was found to have multiple copies of theneMDH transgene, produced a greater amount ofneMDH protein and had enhanced RRGR in Alcompared to other lines but did not demonstrateenhanced exudation of malate. This line also hadsignificantly reduced root growth in control solutionscompared to other transgenic and control lines. Wecannot determine whether slower root growth wasdue to expression of the transgene or to amutation. Shoot growth was also reduced in thisline, and time to flowering increased. Hematoxylinstaining of epidermal cells was markedly greater inthis line than other transgenic lines. It is possiblethat the malate produced accumulated in epidermalcells and chelated Al in epidermal cells, preventingit from damaging the root.

This work showed that oat cultivars grown in theMidwestern U.S. are highly Al resistant, withoutdirect selection for resistance. A 24 h exposure to Alin a hydroponic assay system is sufficient to deter-mine relative resistance among entries. Hematoxylinstaining is not advisable as a plant selection tool asseedling resistance and staining patterns are notalways correlated. The primary mechanism of Alresistance in oat appears to be exclusion of Al fromthe root. The dose-dependent Al-induced secretion ofmalate from roots suggests that malate secretion playsa role in resistance although secretion of other organicacids and the role of other exclusion mechanisms arelikely important. The most resistant oat cultivarsreleased large amounts of malate in response to Al,hence the malate transporter gene from resistant oatcultivars may be useful for enhancing Al resistance in

132 Plant Soil (2012) 351:121–134

other cereals and for identifying gene promoterregulatory elements or protein amino acid sequencesthat result in greater induced malate release.

Acknowledgements Mention of any trade names or commer-cial products in this article is solely for the purpose ofproviding specific information and does not imply recommen-dation or endorsement by the U. S. Department of Agriculture.This paper is a joint contribution from the Plant ScienceResearch Unit, USDA-ARS, and the Minnesota AgriculturalExperiment Station. We gratefully acknowledge the assistanceof Kim Torbert for production of transformed oat plants, DeonStuthman and Roger Caspars for oat seed, and Karen Hilburnfor assistance with lyophilization.

References

Al-Saady NA, Torbert KA, Smith L, Makarevitch I, BaldridgeG, Zeyen RJ, Muelbauer GJ, Olszewski NE, Somers DA(2004) Tissue specificity of the sugarcane bacilliformvirus promoter in oat, barley and wheat. Mol Breed 14:331–338

Andersson M (1988) Toxicity and tolerance of aluminum invascular plants. Water Air Soil Pollut 39:439–462

Anoop VM, Bas U, McCammon MT, McAlister-Henn L, TaylorGJ (2003) Modulation of citrate metabolism alters alumi-num tolerance in yeast and transgenic canola overexpress-ing a mitrochondrial citrate synthase. Plant Physiol132:2205–2217

Archambault DJ, Zhang G, Taylor GJ (1996) Accumulation ofAl in root mucilage of an Al-resistant and an Al-sensitivecultivar of wheat. Plant Physiol 112:1471–1478

Barone P, Rosellini D, LaFayette P, Bouton J, Veronesi F, ParrottW (2008) Bacterial citrate synthase expression and soilaluminum tolerance in transgenic alfalfa. Plant Cell Rep27:893–901

Canaado GMA, Loguercio LL, Martins PR, Parentoni SN,Paiva E, Borém A, Lopes MA (1999) Hematoxylinstaining as a phenotypic index for aluminum toleranceselection in tropical maize (Zea mays L.). Theor ApplGenet 99:747–754

Castilhos G, Farias JG, de Bernardi SA, de Oliveira PH, NicolosoFT, Schetinger MRC, Delatorre CA (2011) Aluminum-stressresponse in oat genotypes with monogenic tolerance.Environ Exp Bot. doi:10.1016/j.envexpbot.2011.05.007

Delhaize E, Craig S, Beaton CD, Bennet RJ, Jagadish VC,Randall PJ (1993a) Aluminum tolerance in wheat (Triti-cum aestivum L.). I. Uptake and distribution of aluminumin root apices. Plant Physiol 103:685–693

Delhaize E, Hebb DM, Ryan PR (2001) Expression of aPseudomonas aeruginosa citrate synthase gene in tobaccois not associated with either enhanced citrate accumulationor efflux. Plant Physiol 125:2059–2067

Delhaize E, Ryan PR, Hebb DM, Yamamoto Y, Sasaki T,Matusmoto H (2004) Engineering high-level aluminumtolerance in barley with the ALMT1gene. Proc Natl AcadSci U S A 101:15249–15254

Delhaize E, Ryan PR, Randall PJ (1993b) Aluminum tolerancein wheat (Triticum aestivum L.). II. Aluminum-stimulatedexcretion of malic acid from root apices. Plant Physiol103:695–702

Foy CD, Smith DH, Briggle LW (1987) Tolerances of oatcultivars to and acid soil high in exchangeable aluminum.J Plant Nutr 10:1163–1174

Fromm ME, Taylor LP, Walbot V (1986) Stable transformationof maize after gene transfer by electroporation. Nature319:791–793

Hayes JE, Ma JF (2003) Al-induced efflux of organic acidanions is poorly associated with internal organic acidmetabolism in triticale roots. J Exp Bot 54:1753–1759

Kinraide TB (1991) Identity of the rhizotoxic aluminiumspecies. In: Wright RJ, Baligar VC, Murrmann RP (eds)Plant-soil interactions at low pH. Dordrecht, Boston, pp717–728

Kinraide TB (1997) Reconsidering the rhizotoxicity of hydroxyl,sulphate, and fluoride complexes of aluminum. J Exp Bot48:1115–1124

Kochian LV, Piñeros MA, Hoekenga OA (2005) The physiology,genetics and molecular biology of plant aluminum resistanceand toxicity. Plant Soil 274:175–195

Koyama H, Kawamura A, Kihara T, Hara T, Takita E, Shibata A(2000) Overexpression of mitochondrial citrate synthase inArabidopsis thaliana improved growth on a phosphorus-limited soil. Plant Cell Physiol 41:1030–1037

Lambers H, Chapin FS, Pons TL (1998) Plant physiologicalecology. Springer, New York

Plant Soil (2012) 351:121–134 133

Larsen PB, Tai CH, Kochian LV, Howell SH (1996) Arabidopsismutants with increased sensitivity to aluminum. PlantPhysiol 110:743–751

Li XF, Ma JF, Hiradate S, Matsumoto H (2000) Mucilagestrongly binds aluminum but does not prevent rootsfrom aluminum injury in Zea mays. Physiol Plant 108:152–160

Maron LG, Piñeros MA, Guimarães CT, Magalhaes JV, PleimanJK, Mao C, Shaff Jm Belicuas SNJ, Kochian LV (2010)Two functionally distinct members of the MATE (multi-drugand toxic compound extruxion) family of transporterspotentially underlie two major aluminum tolerance QTLsin maize. Plant J 61:728–740

Miller SS, Driscoll BT, Gregerson RG, Gantt JS, Vance CP(1998) Alfalfa malate dehydrogenase (MDH): molecularcloning and characterization of five different forms revealsa unique nodule-enhanced MDH. Plant J 15:173–184

Miyasaka SC, Hawes MC (2001) Possible role of root bordercells in detection and avoidance of aluminum toxicity.Plant Physiol 125:1978–1987

Nava IC, Delatorre CA, de Lima Duarte IT, Pacheco MT,Federizze LC (2006) Inheritance of aluminum toleranceand its effects on grain yield and grain quality in oats(Avena sativa L.). Euphytica 148:353–358

Ninamango-Cárdenas FE, Guimarães CT, Martins PR, ParentoniSN, Carneiro NP, Lopes MA, Moro JR, Paiva E (2003)Mapping QTLs for aluminum tolerance in maize. Euphytica130:223–232

Pereira JF, Zhou G, Delhaize E, Richardson T, Zhou M, RyanPR (2010) Engineering greater aluminum resistance inwheat by over-expressing TaALMT1. Ann Bot 106:205–214

Piñeros MA, Shaff JE, Manslank HS, Alves VMC, Kochian LV(2005) Aluminum resistance in maize cannot be solelyexplained by root organic acid exudation. A comparativephysiological study. Plant Physiol 137:231–241

Polle E, Konzak CF, Kittrick JA (1978) Visual detection ofaluminum tolerance levels in wheat by hematoxylinstaining of seedling roots. Crop Sci 18:823–827

Ryan PR, Delhaize E (2010) The convergent evolution ofaluminium resistance in plants exploits a convenientcurrency. Funct Plant Biol 37:275–284

Ryan P, Delhaize E, Randall P (1995) Characterization of Al-stimulated efflux of malate from the apices of Al-tolerantwheat lines. Planta 196:103–110

Ryan PR, Raman H, Gupta S, Horst WJ, Delhazie E (2009) Asecond mechanism for aluminum resistance in wheat relieson the constitutive efflux of citrate from roots. PlantPhysiol 149:340–351

SAS Institute (2003) Version 9.1 ed. SAS Inst, CarySasaki T, Yamamoto Y, Ezaki B, Katsuhara M, Ahn SJ, Ryan PR,

Delhaize E, Matsumoto H (2004) Awheat gene encoding analuminum-activating transporter. Plant J 37:645–653

Shaff JE, Schultz BA, Craft EJ, Clark RT, Kochian LV (2010)GOECHEM-EZ: a chemical speciation program withgreater power and flexibility. Plant Soil 330:207–214

Tamás L, Budíková S, Huttová J, Mistrík I, Šimonovičová M,Široká B (2005) Aluminum-induced cell death of barley-root border cells is correlated with peroxidase- and oxalateoxidase-mediated hydrogen peroxide production. PlantCell Rep 24:189–194

Tarkalson DD, Payero JO, Hergert GW, Cassman KG (2006)Acidification of soil in a dry land winter wheat-sorghum/corn-fallow rotation in the semiarid U.S. Great Plains.Plant Soil 283:367–379

Tesfaye M, Temple SJ, Allan DL, Vance CP, Samac DA (2001)Overexpression of malate dehydrogenase in transgenicalfalfa enhances organic acid synthesis and conferstolerance to aluminum. Plant Physiol 127:1836–1844

Torbert KA, Rines HW, Kaeppler HF, Menon GR, Somers DA(1998) Genetically engineering elite oat cultivars. Crop Sci38:1685–1687

Tzafrir I, Torbert KA, Lockhart BEL, Somers DA, OlszewskiNE (1998) The sugarcane bacilliform badnavirus promoteris active in both monocots and dicots. Plant Mol Biol38:347–356

von Uexküll HR, Mutert E (1995) Global extent, develop-ment and economic impact of acid soils. Plant Soil171:1–15

Wheeler DM, Edmeades DC, Christie RA, Gardner R (1992)Effect of aluminium on growth of 34 plant species: asummary of results obtained in low ionic strength solutionculture. Plant Soil 146:61–66

Wight CP, Kibite S, Tinker NA, Molnar SJ (2006) Identificationof molecular markers for aluminium tolerance in diploidoat through comparative mapping and QTL analysis.Theor Appl Genet 112:222–231

Yamaji N, Huang CF, Nagao S, Yano M, Sato Y, Nagamura Y,Ma JF (2009) A zinc finger transcription factor ART1regulates multiple genes implicated in aluminum tolerancein rice. Plant Cell 21:3339–3349

Zheng SJ, Ma JF, Matsumoto H (1998) Continuous secretion oforganic acids is related to aluminum resistance duringrelatively long-term exposure to aluminum stress. PhysiolPlant 103:209–214

Zhu M, Ahn S, Matsumoto H (2003) Inhibition of growth anddevelopment of root border cells in wheat by Al. PhysiolPlant 117:359–367

134 Plant Soil (2012) 351:121–134