KOCHIAN, L. V.

LIU, J.

MAGALHAES, J. V. de

SHAFF, J.

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Aluminum-Activated Citrate and Malate Transporters from theMATE and ALMT Families Function Independently to ConferArabidopsis Aluminum ToleranceJiping Liu1 , Jurandir V Magalhaes2 , Jon shaff and Leon V Kocbian1•1. u. s. Plant Soil and Nutrition Laboratory, USDA-ARS, Cornell University, Ithaca, NY 14853, USA.2. Embrapa Maize and Sorghum, Rod. MG 424, Km 65, 35701 - 970, Sete Lagoas - MG, Brazil,* Corresponding author: Leon. Kochian@ ars. usda, govABSTRACTAluminum (AI) activated root malate and citrate exudation play an important role in AI tolerance in many plant species.AtAlMTl, an Al-activated malate transporter, is a major contributor to Arabidopsis AI tolerance. Here, we demonstrate thatAtMATE encodes an Arabidopsis Al-activated citrate transporter. AtMATE is expressed primarily in roots and is induced by AI.A loss-of-function AtMATE mutant line lacks Al-activated root citrate exudation. An AtAlMTl AtMATE double mutant lacks bothAl-activated root malate and citrate exudation and exhibits greater AI sensitivity than the single AtALMTl mutant. Therefore,AtMATE makes a significant although smaller contribution to Arabidopsis AI tolerance.AtMATE shares the highest sequence identity withSbMATEIn Arabidopsis, the MATE family contains at least 56members, which can be further classified into severalclusters based on sequence similarity (Li et ai , 2002;Rogers and Guerinot, 2002). AtMATE is the ArabidopsisMATE family member with the highest sequence similarity toSbMATE, the recently cloned sorghum Al tolerance gene( Magalhaes et ai , 2007) (Figure 1). The AtFRD3 cladecontains 4 members, including AtFRD3 (At3g08040),AtMATE (Atlg51340), At2g38330 and At4g38380.~;;;~~~~~~~~AtMATE(Atlg5J340)r SbMATEI 1~lW~~63G08040).76.2 , , , , , , , ,At4G383807060504030 20100Amino Acid Substitu1ion.s(x 1(0)Figure 1 AtMATE shares the highest sequence similaritywith SbMATE in ArabidopsisAtMATE expression in roots is induced by AItreatmentBy conducting semi-quantitative reverse-transcriptase( RT ) -PCR analysis, we demonstrated that within theAtFRD3 clade, AtMATE is the only member whose geneexpression is induced by AI and is localized primarily to theroot, which is the site where AI tolerance must occur(Figure 2).AtMATE is responsible for AI-activated root citrateexudationIn Arabidopsis, Al induces a high leveI of malate andlower levei of root citrate exudation (WT, Figure 3a, b).The AtAlMTl knock-out line (AtAlMTI-KO) and theAtMATE-KO line lack Al-activated root malate and citrateexudation, respectively. The AtALMTl AtMATE doubleknock-out (double-KO) line lacks both Al-activated rootmalate and root citrate exudation.WT AtMATE-KO AtALMTI-KOR S·" R S" R S'~ ,-----,,------,r----1 r---t ~~M~ld~~~M~M~ld~~~M~M~ld~~~M~M~~~~~M~M~ld~~~M~M~ld~~~Mt AtMATEJ J AtALMT1I i AtFrd3At2g38330At4g38380I I AtACTFigure 2 Spatial and temporal gene expression pattems forAtMATE and other members of the AtFRm clade in theArabidopsis MATE family, as well as the major AI tolerancegene, A1AI.MTI. The 6-day-old Arabidopsis seedlings, wildtype (WT), AtMATE knock-out (AtMATE-KO) and A1AI.MTIknock-out (A1AI.MTl-KO) lines, were treated with AI3+for Oday (Od, the control) , 1 day (ld) , 3 days (3d) , and 6 days( 6d). Total RNA was extracted from roots (R) and shoots(S).The AtMATE AtALMTI double mutant is highlyhypersensitive to AI stressAlthough AtAlMTI is clearly the major determinant ofAI tolerance in Arabidopsis as knocking out this gene caused69an approximately 60% reduction in AI tolerance with respectto the wild type at 1. 5 11MAf + activity (Hoekenga et alo ,2006) , AtMATE also contribute a moderate (approx. 30%)component to the overall Arabidopsis Al tolerance. Thedouble AtMATE AtALMTl T-DNA knock-out (Double-KO)line displayed more AI sensitivity compared 10 the AtALMTlsingIe mutant (Figure 4) .(a)2.5,-------------,fD-Ail~WT AL4.L~nJ-KOAL\·l~TE-KODouble-KO(b) 2.5.-------------,~2.0§~~~ 1.5~c:1.0~~H 0.5.s0.0Figure 3 Root malate (a) and citrate (b) exudation under+/- Al conditions for the wild-type (WT) control, the AtMATEknock-out (AtMATE-KO) line, the AtAlMTl knock-out(AtAIMTI-KO) line, and the AtMATE, AtAlMTl douhleknock-out (Double-KO) lineDISCUSSIONIt appears that AtAlMTl-mediated AI-activated malateexudation and AtMATE-mediated Al-actívated citrateexudation evolved and function independently in conferringthe fuIl range of Arabidopsis Al tolerance.70(a)WT AIAlMIi-KO AIMATE-KODouble-KO(O}f,1MAI"(b) IOOr-------~~~~908070~ 60Z~ 50~ 40.,..- 302010O+--L----'~wr ArAIMfI-KO A1MAl'E-KO Double-KOFigure 4 The AtMATE, AtAIMTl douhle mutanl IShypersensitive 10 AI stress. (a) Aluminum (AI) tolerance(root growth in nutrition solutions containing IO I 11M, 11. O I11M, or 11. 51 11MAI' + ) for the wild-type (WT) control, theAtAIMTI-KO, the AtMATE-KO and the double-KO. (b)Aluminum tolerance as measured by percentage of relative netroot growth (% RNRG = rool growth in AI / control rootgrowth x 100) in wild type and the three knock-out linesREFEHENCEHoekenga OA, Maron LG, Piãeros MA, et al. 2006. ProcNat Acad Sei USA, 103: 9738 - 9743.li L, He Z, Pandey GK, et alo 2002. J Biol Chem, 277:5360 -5368.Magalhaes JM, Liu J, Guimares CT et al. 2007. NatureCenet , 39: 1156-116l.Rogers EE, Guerinot ML. 2002. Plant CeU, 14: 1787- 1791.

Aluminum-activated citrate and malate transporters from the MATE and ALMT families function independently to confer arabidopsis aluminum tolerance.

http://www.alice.cnptia.embrapa.br/alice/bitstream/doc/491930/1/Aluminumactivated.pdf

Aluminum-activated citrate and malate transporters from the MATE and ALMT families function independently to confer arabidopsis aluminum tolerance.

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