HOEKENGA, O. A.

KOCHIAN, L.

LIU, J.

MAGALHAES, J. V. de

PINEROS, M. A.

Repository Open Access to Scientific Information from Embrapa

Recent Advances on the Molecular Basis of Crop Aluminum ResistanceLeon Kochianl• , Jurandír V Magalhaes' , Jiping Linl , Owen A Hoekenga' and Miguel A Pífíeros'1. Robert W Holley Center for Agriculture and Health, USDA-ARS, Comell University, lthaca, NY 14853, USA.2. Embrapa, Maize and Sorghum, Rod. MG 424, Km 65,35701-970, Sete Lagoas-MG, Brazil.* Corresponding author , lvkI @ comell, eduThe considerable genetic variability for aluminum (AI)resistance within many crop plant species has been utilizedby plant breeders for a number of years to enhance AIresistance. But beyond this, genetic variability in AIresistance has been an excellent experimental resource that isbeing mined by researchers to elucidate the molecular basisfor this trait. Because of the agronomic importance of AItoxicity, research on the identification of crop AI resistancegenes has attracted significant interest from a number oflaboratories around the world. There has been considerablerecent research progress on identifying the genes underlyinga primary mechanism of AI resistance , the release of AIchelating molecules such as low molecular weight organicacids from the root system, which consequently immobilizeA13• at the root surface, preventing it from entering rootcells. To date, the AI resistance genes that have beenidentified are from two different families of membranetransporters mediating organic acid efflux. For both types oftransporters, Al-inducible regulation of transporter geneexpression plays an important role in differential AIresistance. lt is likely that differences in protein structureand function also play a role in differential resistance,although to date there is no data supporting this. Theidentification of genes conferring AI resistance now providesthe necessary molecular tools to more effectively address aworldwide agronomic problem that is only exceeded bydrought stress with regards to abiotic limitations to cropproduction (von Uexküll and Mutert, 1995).The first AI resistance gene was identified in wheat,and was originally named ALMT1, for aluminum-activatedmembrane transporter (Sasaki et al., 2004). ln thisseminal publication, it was shown that AIMTl ISpreferentially expressed in wheat root tips and is expressed atconstitutively higher levels in AI resistant wheat genotypes.Functional studies in Xenopus oocytes suggested thatTaALMTl encodes a novel transporter that is activated in thepresence of extracellular Al3 + and is permeable to malate(Sasaki et aI., 2004; Piãeros et al., 2oo8a) .Subsequently, studies in Arabidopsis, rape and maizeidentified TaALMTl homologues (AtALMT1, BnALMT1 andZmALMTl , respectively). The homologs in Arabidopsis andrape were also shown to encode Al-activated organic acidanion transporters when expre~sed in Xenopus oocytes(Hoekenga et alo , 2006; Ligaba et alo , 2006) , while themaize homolog was shown to not be involved in maize AIresistance and did not transport organic acids (Pifieros etalo , 2oo8b). lnstead it appears to mediate mineral anionefflux and is involved in mineral nutrition.Identification of a Second AI Resistance GeneWe have been using sorghum as a model system for AIresistance research as it is the fifth most important cerealcrop grown worldwide ( http://faostat.fao.org),is amongthe most tolerant of the major cereals to a number of abioticstresses, and its genoine has been fully sequenced. Of th(major grain crops, it is the closest relative of maize and lik~'maize, sorghum AI resistance is associated with Al-activatedroot citrate exudation. Magalhães et alo (2007) used highresolution mapping to clone the gene responsible for themajor sorghum AI resistance locus, AltSB• This mappingapproach identified two flanking markers that delineated a 25kb region containing three putative open-reading frames( ORFs). Two of the ORFs (sucrose phosphate synthase andan unknown protein) have putative functions inconsistentwith known physiological mechanisms of AI resistance, whilethe third ORF is a member of the MATE or multi-drug andtoxic compound extrusion family of mernbrane transportersinvolved in the efflux of small organic solutes, which wouldbe consistent with a role in root citrate release. Subsequentwork confirmed that this transporter is in fact the sorghum AIresistance gene based on the following evidence , 1) Thetransporter gene is highly expressed in the root apices (thesite of AI resistance) of a tolerant NIL derived from a crossbetween the tolerant and sensitive parents, but it is notexpressed in the root apices of an AI sensitive NIL; 2)Expression of this gene is induced by AI only in the tolerantNIL; 3) AltSB expression increases significantly over a 3 - 6day period of AI exposure, with a parallel increase of AI-activated root citrate exudation and AI resistance over thesame time period; 4) The other 2 ORFs are expressed solelyin the shoot, and thus are not expressed in the site where AIresistance mechanisms must operate (the root apex }: 5)Transformation in AI sensitive Arabidopsis and wheatgenerated AI resistant transgenic plants that showedincreased citrate release.Due to the large difference in AltSB expression observedbetween AI tolerant and AI sensitive NILs and the lack ofpolymorphisms in the coding sequence of the gene, it washypothesized that the primary difference between the tolerantand sensitive lines was due to changes in regulatory regionsof AltsB' Thus the entire 25 kb region within the flankingmarkers was scanned in these lines for polymorphisms.Several differences between these two alleles were identified,including a miniature inverted repeat transposable elementinsertion (MITB-type transposable element) in the promoterregion of the gene at - 2. O kb from the transcriptioninitiation site , 6 single nucleotide polymorphisms (SNPs)and one insertion/ deletion (indel) in the second intron ofAltsB, and 2 SNPS/l indel and a 19 bp indel located afterthe stop codon, in the 3 regions of the gene. It should benoted that the set of polymorphisms we described above wasdetected between two sorghum lines only, and that manymore polymorphisms important for function might be found ina diverse and relatively large sorghum pane!. In fact, wehave recently identified another resistance source that has themost effective AltsB allele we have identified to date,exhibiting the largest Al-activated root citrate release andgreatest AI resistance we have observed (Magalhães,unpublished results). This line harbors a non-conservativeamino-acid substitution in the coding region of the gene,suggesting this amino acid may play a critical role in citratetransporto Altogether, these findings support the notion thatidentification of superior AltsB haplotypes can only begathered through a comprehensive analysis on a relativelylarge and diverse sorghum panel.Our sequence scanning was subsequently expanded intoa broader investigation of AltsB diversity using a panel of 47sorghum accessions of diverse origins. These studies indicatethat the MITE containing region in the promoter is highlystructured, with the MITE and flanking sequences repeatedbetween 1 and 5 times. There is a significant and positivecorrelation between the number of repeats in this region,AltsB expression and AI resistance , with most of the AItolerant lines harboring larger number of repeats. However,a number of interesting outliers have also been found usingNILs generated with different sources of AltsB' Significantallelic variation was detected with these NILs within a singleMITE insertion class, suggesting that the degree ofphenotypic expression may depend on interactions amongpolymorphisms in the 25 kb region.As with the discovery of ALMT1 in wheat, the discoveryof a second AI resistance gene from a different family ofmembrane transporters provides a new avenue of inquiry intocandidate AI resistance genes in other species, It should benoted that the first member of the MATE family to be shownto be involved in plant AI resistance was HvAACTl, which2was shown to be the Al-activated root citrate transporter inbarley (Furukawa et alo'., }007). Additionally, we havefound a MATE family homolog that has the same function inArabidopsis AI resistance (Liu et alo , 2008), and wecurrently are investigating a MATE homolog in maize thatappears to be involved in AI resistance (Maron et alo ,2008 ). It is interesting that there is convergent evolutionregarding a mechanism of AI resistance based on organic acidrelease in different plant species that involves genes from twocompletely unrelated families of transporters. ,This isdepicted in Figure 1, where Al-activated malate and citraterelease are shown to be mediated by members of the ALMT'and MATE transporter families, respectively.MATE ALMTAp·Act\vation.,....Organic Acid-AlComplexesII,Pyruvatet Oxal~cetatePfGLYCOLYSISFigure 1 Model illustrating a major AI resistance mechanismbasedon AI-activated release of organic acids into the rhizosphere, wherethey chelate AI3• ions and prevent their entry into the root tipo Themodel suggests that members of the ALMT family of membranetransporters mediate malate release in certain plant species , whileMATEtransporters are involved in Al-activated citrate exudationREFERENCESFurukawa et al. 2007. Plant Cell Physiol, 48: 1081 -1091.Hoekenga et alo 2006. Proc National Acad Sci USA, 103:9738 -9743.Ligaba et alo 2006. Plant Physiol, 142: 1294 -1303.Liu et al. 2008. Plant 1. (in press)Magalhães, et alo 2007. Nature Genet, 39: 1156 - 1161.Maron, et aL 2008. Proceedings-7thPSILPH Symposium.(in press )Piãeros , et alo 2oo8a. Plant Physiol. (in press)Piüeros , et aI. 2oo8b. Plant J, 53: 352 - 367.Sasaki, et alo 2004. Plant J, 37: 645 -653.von Uexküll , Mutert. 1995. Plant Soil , 171: 1 -15.

Recent advances on the molecular basis of crop aluminum resistance.

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