翻译原文

DOI:10.1093/jxb/erh030Advanced Access publication28November,2003RESEARCH PAPERIdenti®cation of aluminium-regulated genes by cDNA-AFLP in rice(Oryza sativa L.):aluminium-regulated genes for the metabolism of cell wall componentsChuanzao Mao,Keke Yi*,Ling Yang*,Bingsong Zheng*,Yunrong Wu,Feiyan Liu and Ping Wu²The State Key Laboratory of Plant Physiology and Biochemistry,College of Life Sciences,Zhejiang University, Hangzhou310029,PR ChinaReceived20July2003;Accepted17October2003AbstractAluminium(Al)toxicity is the major factor limiting crop productivity in acid soils.To investigate the molecular mechanisms of Al toxicity and Al tolerance of rice,cDNA-ampli®ed fragment length polymorph-ism(cDNA-AFLP)was used for identifying Al-regu-lated genes in roots of an Al-tolerant tropical upland rice,Azucena,and an Al-sensitive lowland rice, IR1552.Nineteen function-known genes were found among34transcript-derived fragments(TDFs)regu-lated by Al stress.The results indicate that Al stress could induce the biosynthesis of lignin and other cell wall components in roots.Temporal expression pat-terns of14genes were identi®ed between the two varieties.In silico mapping was performed for all the 33unique genes.Two genes for a function-unknown protein and for a ubiquitin-like protein,respectively, were mapped on the interval with the common QTL (quantitative trait loci)for Al tolerance in rice on chromosome1.Key words:Aluminium stress,cDNA-AFLP,cell wall components,Oryza sativa L.IntroductionAluminium(Al)is the most abundant metal in the earth's crust and is the major factor limiting crop productivity in acid soils,which comprises up to40%of the world's arable lands(Kochian,1995).The abundance of Al products in the environment and the potential of Al as a plant growth inhibitor make it necessary to understand the mechanisms of Al phytotoxicity and Al tolerance of plant (Kochian,1995).The major symptom of Al toxicity is a rapid inhibition of root growth.Al accumulates rapidly in the apoplast,in the plasma membrane,and eventually enters the cytosol (Lazof et al.,1994).Many different mechanisms of Al toxicity have been hypothesized,including Al interactions with the root cell wall and Al interactions with symplasmic constituents such as the cytoskeleton etc.(Kochian,1995). Alhough the mechanisms of Al toxicity have been studied by many researchers,the speci®c mechanism by which Al inhibits root elongation is still elusive(Matsumoto,2000). It is still not clear whether Al affects the root symplasti-cally or apoplastically,but the increased evidence supports the view that the apoplast plays the major role in Al perception(Horst,1995).Al tolerance has been speculated to be the result of either exclusion of Al from the root apex and/or the tolerance for symplasmic Al.Detoxi®cation of Al in the rhizosphere by releasing organic acids to chelate Al has been reported in wheat and maize,while the detoxi®cation of Al internally was also found by forming complexes with organic acids in plants(Ma et al.,2001).Rice was the most Al tolerance species among small-grain cereal crops(Foy, 1988).However,information on Al tolerance mechanisms in rice is limited.Ma et al.(2002)reported that no organic acid was induced by Al exposure,except citrate in small amounts in rice,and there was no signi®cant effect on Al detoxi®cation in both Al-tolerant and Al-sensitive var-ieties.It means that rice may have a different Al tolerance mechanism other than the release of organic acids.Up to now,a number of Al-regulated genes has been reported from the roots of wheat(Snowden and Gardner,*These authors contributed equally to this study.²To whom correspondence should be addressed.Fax:+8657186971323.E-mail:docpwu@ Journal of Experimental Botany,Vol.55,No.394,ãSociety for Experimental Biology2004;all rightsreserved by guest on August 25, 2011 Downloaded from1993;Richards et al .,1994;Hamel et al .,1998;Sasaki et al .,2002),Arabidopsis (Richards et al .,1998),rye (Milla et al .,2002),and sugarcane (Watt,2003).However,most of these genes were also found to be responsive to other toxic metals,low Ca 2+levels,physical wounding (Snowden et al .,1995),oxidative stress (Watt,2003),and pathogens (Hamel et al .,1998),and were expressed equally well in both Al-tolerant and Al-sensitive genotypes (Hamel et al .,1998).Up to now,there has been no Al-regulated gene reported in rice.cDNA-ampli®ed fragment length polymorphism (cDNA-AFLP)(Bachem et al .,1996)is an extremely ef®cient method for the isolation of differentially ex-pressed genes,which gave reproducible results that were con®rmed using RNA gel blot analysis (Bachem et al .,1996,1998).It is a genome-wide expression analysis tool which does not require prior sequence information and therefore constitutes a useful tool for gene discovery (Ditt et al .,2001).In this study,cDNA-AFLP was used to identify differentially expressed genes from rice subjected to Al stress treatment.Thirty-four Al-regulated transcript derived fragments (TDFs)were isolated.The information from the genes may be helpful for a better understanding of the mechanisms of Al toxicity and the Al tolerance of rice and other plant species.Materials and methodsPlant material and culture experimentAccording to previous work (Wu et al .,2000),an Al-tolerant upland tropical japonica (Oryza sativa L.)rice,Azucena,and an Al-sensitive indica rice,IR1552,were used in this study.Uniform seeds were rinsed with distilled water,and incubated with distilled water in the dark at 30°C for 2d.Germinated seeds were grown in distilled water for another 2d at 27T 2°C.Seedlings were then transferred to plastic trays that were covered by a PVC sheet with a nylon mesh.Half-strength nutrient solution was used (Yoshida et al .,1976).The pH of the solution was adjusted daily to 4.0with 1mol l ±1NaOH or 1mol l ±1HCl.Four-day-old seedlings were used for the Al-stress treatment.Seedlings were exposed to a 0.5mmol l ±1CaCl 2solution (pH 4.0)for 2h and then exposed to a 0.5mmol l ±1CaCl 2solution (pH 4.0)containing 0or 183m mol l ±1AlCl 3,with a free active Al 3+concentration of 100m mol l ±1as determined by the Geochem-PC program (Parker et al .,1995).Roots and shoots of seedlings sampled at 0,0.5,2,12,24,and 48h were cut,quickly frozen in liquid nitrogen,and stored at ±70°C for RNA extraction.The experiment was conducted in a greenhouse under a diurnal photoperiod of 12h light (158m mol m ±2s ±1).The daily maximum and minimum temperatures were 28°C and 22°C,respectively.The relative humidity ranged from 65%to 85%.Root length measurementsThe longest root of each seedling was measured after 60h of growth in the control or Al stress solutions.The relative root elongation (RRE )was calculated asRRE =(T Al ±T initial )/(C control ±C initial )where T and C refer to the measured root lengths under Al-stress and control conditions,respectively.cDNA-AFLP analysisTotal RNA was extracted from the roots,sampled at the ®ve indicated time points,using Trizol reagent (Gibco,Germany)and poly (A)+RNA was puri®ed with the Oligotex mRNA Mini Kit (Qiagen,Germany).Treated RNA and control RNA of Azucena and IR1552were prepared by pooling equal amounts of RNA at the four time points (0.5,2,12,and 24h).Double-stranded cDNA was synthesized using the SMART ÔcDNA Library Construction Kit (Clontech,USA)according to the manufacturer's instructions,puri®ed by QIAquick PCR Puri®cation Kit (Qiagen,Germany)and digested by the Taq I/Ase I enzyme combination.AFLP reactions were performed according to the methods of Bachem et al .(1996).DNA fragments were visualized by silver staining according to the Silver Sequence ÔDNA Sequencing System Technical Manual (Promega,USA).Isolation and sequencing of TDFsThe Al-regulated TDFs were recovered by PCR under the same conditions used for the pre-ampli®cation.Puri®ed PCR products were ligated to the pUCm-T vector.The clones were sequenced using MegaBACE Ô1000(Amersham Pharmacia,USA).Northern blot analysis and densitometryTotal RNA (20m g)was separated by electrophoresis on a 1.2%formaldehyde agarose gel followed by blotting onto nylon mem-brane (Hybond-N +,Amersham Pharmacia,USA).Hybridization was performed as described previously (Sambrook and Russell,2001).The hybridization signals were scanned by Typhoon 8600scanner (Molecular Dynamics,USA)and quanti®ed using the ImageQuant 5.0software (Molecular Dynamics,USA).The signals obtained from the genes were weighted against those obtained from the 18s rRNA to correct for minor differences in RNA loading and normalized to that at 0h of Azucena,which was set at 1for each gene.Gene function analysisDatabase searches were performed using the BLAST Network Service (NCBI,National Center for Biotechnology Information)(/BLAST).The sequence of each TDF was searched against all sequences in the non-redundant databases using the BLASTN,BLASTX and TBLASTX algorithm,and in the EST database using the BLASTN program in turn.Sequences that Returned with no signi®cant homology were compared again against genomic sequence databases using the BLASTN program or Genomic BLAST pages.The retrieved genomic sequences were further annotated at the web site (http://ricegaas.dna.affrc.go.jp/)or analysed using the Genscan program (http://bioweb.pasteur.fr/seqanal/interfaces/genscan.html).The function of function-known genes (BLASTX and TBLASTX,E values less than 1e ±5)(Ditt et al .,2001)was classi®ed according to the putative function.In silico mappingThe sequence of markers near the common QTL for Al tolerance in rice were obtained from the website (/).Rice bacterial arti®cial chromosome (BAC)clones were found,based on the sequence information or accession number of 33TDFs and the markers,and were anchored to the Rice Genetic Map in silico (/tdb/e2k1/osa1/sequencing.shtml).ResultsTolerance performance of the two varietiesAn Al-tolerant upland japonica rice,Azucena,and an Al-sensitive lowland indica rice,IR1552,were used for the Al138Mao et al .by guest on August 25, 2011 Downloaded fromstress treatments using 100m mol l ±1active Al 3+in this work.Root elongation was signi®cantly inhibited by Al stress in the ®rst 12h in both varieties with RRE (relative root elongation)of 76%in Azucena and 34%in IR1552.The RRE was 89%in Azucena and 29%in IR1552after the 48h stress treatment.Identi®cation of Al-regulated genesTo analyse genes responsive to Al stress,cDNA-AFLP analysis was performed on roots of Azucena and IR1552subjected to Al stress by a non-radioactive procedure.The differentially expressed fragments were investigated by selective ampli®cation using 35primer combinations.More than 2100bands were generated and all the bands longer than 100bp in length were compared in all four treatments:Azucena +/±Al and IR1552+/±Al.Fragments up-regulated or down-regulated by Al in Azucena or IR1552,or in both were identi®ed as Al-regulated TDFs.Thirty-four signi®cantly Al-regulated TDFs ranging in length from 100to 600bp were cloned and sequenced,including three up-regulated TDFs in Azucena,one in IR1552,29in both varieties,and one down-regulated TDF in Azucena (Table 1).The clones corresponding to different TDFs were renamed as Os AR (Oryza sativa Al-regulated).OsAR7and OsAR8showed homology with different parts of p -coumarate 3-hydroxylase,suggesting that these two TDFs may be the same gene.Nineteen of the33unique genes showed signi®cant homology with function-known genes by BLAST searches (Table 1).Ten TDFs with no signi®cant homology with function-known protein and four TDFs with no match in the database were classi®ed into a function-unknown gene and an unknown gene,respectively.Of the 19function-known genes,seven genes were possibly involved in the metabolism of cell wall components,including lignin synthesis (OsAR4,OsAR5,OsAR6,and OsAR7),hemicellulose (OsAR9),glycoprotein (OsAR11),and other components (OsAR10).One gene is involved in oxidative stress (OsAR13).Nine genes are related to cellular metabolism (OsAR1,OsAR2and OsAR3),retroelement (OsAR19),transcription (OsAR20),protein metabolism (OsAR14,OsAR15and OsAR16),and the cell cycle (OsAR17).All the 17genes were up-regulated by Al in both Azucena and IR1552(Table 1).One TDF (OsAR18),up-regulated only in Azucena,showed homology to KN1-like protein.Another TDF (OsAR12),up-regulated in IR1552,is for the biosynthesis of taxol (Table 1).Among the ten function-unknown genes and the four unknown genes,two genes (OsAR24and OsAR34)and one gene (OsAR25)were up-regulated and down-regulated in Azucena,respectively,and 11other genes were up-regulated in both Azucena and IR1552(Table 1).Table 1.Function analysis of Al-regulated TDFs and their expression patterns obtained by cDNA-AFLPTDFSize Expression a Homology Function category(bp)A I Os AR1255++Dihydrolipoamide S -acetyltransferase Cellular metabolism Os AR2283++2-oxoglutarate dehydrogenase Os AR3334++Aspartate aminotransferaseOs AR4333++4-coumarate:CoA ligase isoform 2Lignin synthesisOs AR5128++Phenylalanine ammonia-lyaseOs AR6133++Putative cinnamyl-alcohol dehydrogenase Os AR7272++p -coumarate 3-hydroxylase Os AR8232++p -coumarate 3-hydroxylase Os AR9256++Xylose isomerase Cell wall-related Os AR10241++Beta-1,3-glucanaseOs AR11194++UDP-N -acetylglucosamine pyrophosphorylase Os AR12322+O -deacetylbaccatin III-10-O -acetyl transferase Secondary metabolism Os AR13265++Quinone oxidoreductase Oxidative stress Os AR14286++Proteinase inhibitor Protein metabolism Os AR15227++Elongation factor EF-2Os AR16112++SUMO-1Os AR17572++MCT-1protein-like OthersOs AR18253+Rice KN1-like proteinsOs AR19268++Putative retroelement pol polyprotein Os AR20239++Histone H4Os AR21±23++Unknown protein Os AR24+Unknown protein Os AR25±Unknown protein Os AR26±30++Unknown proteinOs AR31±33++No match b Os AR34+No match ba A:Azucena;I:IR1552;(+)means up-regulated;(±)means down-regulated bNo signi®cant sequence homology found in genome,EST and protein database.Identi®cation of Al-regulated genes in rice 139by guest on August 25, 2011 Downloaded fromTemporal expression of some Al-regulated genesTo validate the Al-regulated genes and to analyse the temporal expression patterns,17genes,including seven genes for cell-wall-related functions (OsAR4to OsAR11),one for taxol synthesis (OsAR12),one for cellular metab-olism (OsAR1),two for protein metabolism (OsAR15,OsAR16),two for quinone oxidoreductase (OsAR13)and KN1-like protein (OsAR18),and four for unknown proteins (OsAR24,OsAR25,OsAR28,and OsAR29)with different expression patterns,were used for northern blotting analysis.The results were comparable to the expression patterns revealed by cDNA-AFLP except for OsAR25,which was inhibited by Al in both Azucena and IR1552,but no inhibition was revealed in IR1552by cDNA-AFLP (Table 1;Figs 1,2).The transcripts of three genes (OsAR1,OsAR12and OsAR24)could not be detected in the northern blotting.For genes involved in cell wall metabolism (Fig.1),three genes (OsAR9to OsAR11)for the synthesis of hemicellulose,glycoprotein and other cell wall components,showed the same expression patterns in both Al-tolerant and Al-sensitive varieties with the highest up-regulation at 12h (OsAR9)and 48h (OsAR10and OsAR11).Four genes (OsAR4to OsAR7)for lignin synthesis showed different expression patterns in Al-tolerant and Al-sensitive varieties.OsAR6and OsAR7were up-regulated gradually up to 48h in Azucena,but were up-regulated in IR1552within 2h then decreased.OsAR4and OsAR5showed a biphasic regulation in Azucena,transiently up-regulated within 2h and decreased at 12h and 24h and then up-regulated again;in IR1552,they were transiently up-regulated within 0.5h and then decreased and up-regulated again for OsAR5.For genes involved in other metabolism pathways (Fig.2),four (OsAR13,OsAR16,OsAR28,and OsAR29)showed similar expression patterns in Azucena and IR1552,with OsAR13and OsAR16up-regulated gradually after Al treatment,OsAR25down-regulated,and OsAR29showed a biphasic regulation which was up-regulated within 2h and after 24h,but decreased at 12h.Three genes showed different temporal expression patterns in Azucena and IR1552.The gene for KN1-like protein (OsAR18)was up-regulated in Azucena,but not in IR1552.A function-unkown gene (OsAR28)was more strongly induced within 2h in IR1552than in Azucena and was more strongly induced at 48h in Azucena than in IR1552.By contrast,the gene of the elongation factor EF-2(OsAR15)was induced within 2h in Azucena,but induced up to 48h in IR1552.DiscussioncDNA-AFLP is an ef®cient method for the isolation of differentially expressed genes and this was con®rmed bynorthern blotting analysis of 14genes (42%of the 33Al-regulated genes).Three of the 33Al-regulated genes found are reported to be Al-regulated in wheat (Snowden and Gardner,1993;Snowden et al .,1995;Cruz-Ortega et al .,1997),including phenylalanine ammonia-lyase,proteinase inhibitor and b -1,3-glucanase.The result indicates that different crops may have similar Al-responsive mechan-isms.Al stress-induced genes for cell wall componentsNineteen function-known genes isolated here are involved in different metabolic pathways,which indicates thatAlFig. 1.Temporal expression analysis of Al-regulated cell-wall-related-genes.(A,C)Different expression pro®les during Al treatment of rice varieties Azucena and IR1552over a 48h period.The ®rst lanes (0h)of each variety correspond to unstressed plants.The 18s rRNA shows the RNA integrity and uniform loading.(B,D)Quantitation of mRNA levels.The hybridization signals obtained from the genes were weighted against those obtained from the 18s rRNA to correct for minor differences in RNA loading,normalized to that at 0h of Azucena (which was set at 1for each gene)and plotted against time to compare changes in gene expression.140Mao et al .by guest on August 25, 2011 Downloaded fromtoxicity can affect different physiological and biochemical pathways.Seven of the 19genes may function in modifying cell wall components (Table 1).Morphological and histological studies have revealed that Al stress could increase the amounts of certain cell wall components,such as polysaccharides and lignin (Eleftheriou et al .,1993;Sasaki et al .,1996).Four genes (OsAR4,OsAR5,OsAR6,and OsAR7)encoding the enzymes catalysing different steps of lignin biosynthesis according to the lignin roadmap (Humphreys and Chapple,2002)were up-regulated by Al stress in this work.Northern blotting analysis indicated that transcripts of these genes were accumulated to a much higher extent in roots than in shoots (data not shown).The Al-induced up-regulation of these genes was within 2h in the Al-sensitive variety IR1552,and this was earlier than in the Al-tolerant variety Azucena.The transcripts of phenylala-nine ammonia-lyase (OsAR5),a key enzyme that catalyses the ®rst step of the phenylpropanoid pathway leading to lignin synthesis,was accumulated more in IR1552than inAzucena (Fig.1).Lignin is the principal structural component of plant cell walls.Various stress factors,including ion de®ciency,invasion by fungal pathogens and wounding,can induce the deposition of lignin in cell walls.Research by Sasaki et al .(1996)indicated that the extent of growth inhibition is closely correlated with the extent of lignin deposition in both Al-tolerant and Al-sensitive varieties,and Al-sensitive varieties would accumulate more lignin in the roots than Al-tolerant varieties.This was in accordance with the expression of the genes.Three genes for xylose isomerase (OsAR9),b -1,3-glucanase (OsAR10)and UDP-N -acetylglucosamine (UDP-GlcNAc)pyrophosphorylase (OsAR11),were up-regulated by Al stress in both Al-tolerant and Al-sensitive varieties in this study.Xylose isomerase (OsAR9)catalyses the conversion of D -xylulose to D -xylose (http://www.brenda.uni-koeln.de/),which is a major constituent of hemicellulose.It coincided with the physiological phenomenon that Al can induce hemicellulose deposition in cell walls (Tabuchi and Matsumoto,2001).b -1,3-glucanase plays an important role in plant defence against pathongen attack.It is strongly induced when plants respond to wounding or infection by fungal,bacterial or viral pathogens (Leubner-Metzger and Meins,1999).It was also induced by Al stress in wheat (Cruz-Ortega et al .,1997).It has been hypothesized that b -1,3-glucanase contributes to the hydrolysis of cell wall components during seed germination (Leubner-Metzger and Meins,1999)and suggested that it participates in the modi®cation of cell wall components under Al stress.The UDP-N -acetylglucosamine (UDP-GlcNAc)pyrophosphorylase re-versibly catalyses the synthesis of UDP-GlcNAc from UDP-GlcNAc-1-P and UTP (http://www.brenda.uni-koeln.de/).UDP-GlcNAc acts as a speci®c glycosyl donor for the biosynthesis of N-and O-linked glycopro-teins (Piro et al .,1994),which is one of the plant cell wall components.The transcripts accumulated of the two genes,OsAR10and OsAR11,increased with time of Al treatment,and the transcripts accumulation in the Al-sensitive variety was higher than that in Al-tolerant variety after 48h (Fig.1).It suggested that the up-regulation of the two genes should associate with the extent of Al toxicity.Tabuchi and Matsumoto (2001)suggested that Al modi®es the metabolism of cell wall components and thus makes the cell wall thick and rigid,which is supported by these results.Lignin,hemicellulose,glycoprotein,and other secondary metabolites deposition in the cell wall may thicken the cell wall and prevent Al from entering into the plasma membrane.However,it may also result in the arrest of root growth and cellular elongation (Le Van et al .,1994)and impose oxidative stress on the plant,which was implied by the up-regulation of the gene for quinone oxidoreductase (OsAR13),an enzyme believed to protect an organism against oxidativestress.Fig.2.Temporal expression analysis of some other Al-regulated genes.Experimental conditions and indications are as Fig.1.Identi®cation of Al-regulated genes in rice 141by guest on August 25, 2011 Downloaded fromTemporal expression patterns of the genes result from Al toxicityAmong the 33Al-regulated genes detected in this study,28genes were regulated by Al in both Al-tolerant and Al-sensitive varieties,suggesting that these genes result from Al-toxicity.Temporal expression patterns of 14genes responsive to Al,including seven genes for cell wall metabolism,were revealed by northern blotting analysis (Figs 1,2).Most of the blotted genes (OsAR9to OsAR11,OsAR13,OsAR16,OsAR25,OsAR29)were regulated similarly in both Al-tolerant and Al-sensitive varieties and were induced after 12h of Al stress,except for the gene for xylose isomerase (OsAR9)which was induced before 12h,suggesting that they could be involved in the secondary mechanisms of Al toxicity.Different temporal expression patterns of the Al-induced genes in the Al-tolerant and Al-sensitive varieties were also found.This could be because of the different tolerance levels of the two rice varieties.On the other hand,the two rice varieties with different Al sensitivity were subjected to the same level of Al and this factor may also be contributing to the differences observed in expression patterns.Candidate genes for Al toleranceThirty-three unique genes regulated by Al were isolated in this study,but no gene involved in organic acid synthesis and release was found.This result is consistent with other reports that no organic acid was released other than a small amount of citrate in rice under Al stress (Ma et al .,2002).It is reported that Al-regulated genes may have protective roles (Ezaki et al .,2000,2001),which implies that Al-regulated genes can be tolerance genes.Up to now,severalstudies have reported QTL analysis for Al tolerance in rice.It will be very helpful for selecting candidate Al tolerance genes in rice from the Al-regulated genes.All the Al-regulated genes were used for in silico mapping and 26were mapped (data not shown).Two genes for a function-unknown protein (OsAR28)and for SUMO-1(small ubiquitin-like modi®er-1,OsAR16)were in silico mapped on the common QTL interval for Al tolerance in rice on chromosome 1(Fig.3).OsAR16(112bp)is for a ubiquitin-like protein SUMO-1(a member of the SUMO conjugation system).The SUMO conjugation system appears to be a complex and functionally hetero-geneous pathway for protein modi®cation in plants.It may have important functions in stress protection and/or repair (Kurepa et al .,2003).However,it was expressed equally well in both Al-tolerant and Al-sensitive varieties (Fig.2),and might be the result of Al toxicity.OsAR28(181bp)showed different expression patterns between Azucena and IR1552,its transcript accumulation increased grad-ually up to 48h in Azucena,whereas in IR1552,it was up-regulated early (0.5h)and then decreased gradually almost to the level of CK (0h)(Fig.1).It suggests that OsAR28could be a candidate gene for Al tolerance from its different temporal expression patterns and its location on the QTL interval.To investigate the gene further for possible tolerance to Al toxicity in rice,transgenic work is being carried out.AcknowledgementThis research was funded by The National Science Foundation of China (No.30070070).Fig.3.In silico mapping of two Al-regulated genes.(A)Integrated map of the common QTL interval for Al tolerance in rice on chromosome 1by alignment of RFLP markers with an RGP physical map (http://rgp.dna.affrc.go.jp/).Sequences of the alignment markers were obtained from NCBI (/).Two Al-regulated TDFs were shaded.The vertical black bars denotes the comparable QTL positions identi®ed by Wu et al .(2000)(B),Nguyen et al .(2002)(C),Nguyen et al .(2001)(D),and Ma et al .(2002)(E).142Mao et al .by guest on August 25, 2011 Downloaded fromReferencesBahem CW,Oomen RJF,Visser RG.1998.Transcript imaging with cDNA-AFLP:a step-by-step protocol.Plant Molecular Biology Reporter16,157±173.Bachem CW,van der Hoeven RS,de Bruijn SM,Vreugdenhil D,Zabeau M,Visser RG.1996.Visualization of differential gene expression using a novel method of RNA®ngerprinting based on AFLP:analysis of gene expression during potato tuber development.The Plant Journal9,745±753.Cruz-Ortega R,Cushman JC,Ownby JD.1997.cDNA clones encoding1,3-beta-glucanase and a®mbrin-like cytoskeletal protein are induced by Al toxicity in wheat roots.Plant Physiology114,1453±1460.Ditt RF,Nester EW,Comai L.2001.Plant gene expression response to Agrobacterium tumefaciens.Proceedings of the National Academy of Sciences,USA98,10954±10959. Eleftheriou E,Moustakas M,Fragiskos N.1993.Aluminate-induced changes in morphology and ultrastructure of Thinopyrum roots.Journal of Experimental Botany44,427±436.Ezaki B,Gardner RC,Ezaki Y,Matsumoto H.2000.Expression of aluminium-induced genes in transgenic Arabidopsis plants can ameliorate aluminium stress and/or oxidative stress.Plant Physiology122,657±665.Ezaki B,Katsuhara M,Kawamura M,Matsumoto H.2001. Different mechanisms of four aluminium(Al)-resistant transgenes for Al toxicity in Arabidopsis.Plant Physiology127, 918±927.Foy CD.1988.Plant adaptation to acid,aluminium-toxic soils. Communications in Soil Science and Plant Analysis19,959±987. Hamel F,Breton C,Houde M.1998.Isolation and characterization of wheat aluminium-regulated genes:possible involvement of aluminium as a pathogenesis response elicitor. Planta205,531±538.Horst WJ.1995.The role of the apoplast in aluminium toxicity and resistance of higher plants:a review.The Journal of Plant Nutrition and Soil Science158,419±428.Humphreys JM,Chapple C.2002.Rewriting the lignin roadmap. Current Opinion in Plant Biology5,224±229.Kochian LV.1995.Cellular mechanisms of aluminium toxicity and resistance in plants.Annual Reveiw of Plant Physiology and Plant Molecular Biology46,237±260.Kurepa J,Walker JM,Smalle J,Gosink MM,Davis SJ, Durham TL,Sung DY,Vierstra RD.2003.The small ubiquitin-like modi®er(SUMO)protein modi®cation system in Arabidopsis.Accumulation of SUMO-1and-2conjugates is increased by stress.Journal of Biological Chemistry278,6862±6872.Lazof DB,Goldsmith JG,Rufty TW,Linton RW.1994.Rapid uptake of aluminium into cells of intact soybean root tips(a microanalytical study using secondary ion mass spectrometry). Plant Physiology106,1107±1114.Le Van H,Kuraishi S,Sakurai N.1994.Aluminium-induced rapid root inhibition and changes in cell-wall components of squash seedlings.Plant Physiology106,971±976.Leubner-Metzger G,Meins FJ.1999.Functions and regulation of plant b-1,3-glucanases(PR-2).In:Datta SK,Muthukrishnan S, eds.Pathogenesis-related proteins in plants.Boca Raton,FL: CRC Press,49±76.Ma JF,Ryan PR,Delhaize E.2001.Aluminium tolerance in plants and the complexing role of organic acids.Trends in Plant Science 6,273±278.Ma JF,Shen R,Zhao Z,Wissuwa M,Takeuchi Y,Ebitani T,Yano M.2002.Response of rice to Al stress and identi®cation of quantitative trait loci for Al tolerance.Plant and Cell Physiology43,652±659.Matsumoto H.2000.Cell biology of aluminium toxicity and tolerance in higher plants.International Review of Cytology200,1±46.Milla MAR,Butler E,Huete AR,Wilson CF,Anderson O, Gustafson JP.2002.Expressed sequence tag-based gene expression analysis under aluminium stress in rye.Plant Physiology130,1706±1716.Nguyen VT,Burow MD,Nguyen HT,Le BT,Le TD,PatersonAH.2001.Molecular mapping of genes conferring aluminium tolerance in rice(Oryza sativa L.).Theoretical and Applied Genetics102,1002±1010.Nguyen VT,Nguyen BD,Sarkarung S,Martinez C,PatersonAH,Nguyen HT.2002.Mapping of genes controlling aluminium tolerance in rice:comparison of different genetic backgrounds. Molecular Genetics and Genomics267,772±780.Parker D,Norvell W,Chaney R.1995.Geochem-PC:a chemical speciation program for IBM-compatibles.In:Loeppert RH, Schwab AP,Goldberg S,eds.Chemical equilibrium and reaction models.Madison,WI:Soil Science Society of America,253±269.Piro G,Buffo M,Dalessandro G.1994.Membrane-bound glucosamine acetyltransferase in coleoptile segments of Avena sativa.Physiologia Plantarum90,181±186.Richards KD,Schott EJ,Sharma YK,Davis KR,Gardner RC. 1998.Aluminium induces oxidative stress genes in Arabidopsis thaliana.Plant Physiology116,409±418.Richards KD,Snowden KC,Gardner RC.1994.Wali6andwali7,genes induced by aluminium in wheat(Triticum aestivumL.)roots.Plant Physiology105,1455±1456.Sambrook J,Russell DW.2001.Molecular cloning:a laboratory manual.New York:Cold Spring Harbor Laboratory Press.Sasaki M,Yamamoto Y,Matsumoto H.1996.Ligin deposition induced by aluminium in wheat(Triticum aestivum)roots. Physiologia Plantarum96,193±198.Sasaki T,Ezaki B,Matsumoto H.2002.A gene encoding multidrug resistance(MDR)-like protein is induced by aluminiumand inhibitors of calcium¯ux in wheat.Plant and Cell Physiology43,177±185.Snowden KC,Gardner RC.1993.Five genes induced by aluminium in wheat(Triticum aestivum L.)roots.Plant Physiology103,855±861.Snowden KC,Richards KD,Gardner RC.1995.Aluminium-induced genes(induction by toxic metals,low calcium,and wounding and pattern of expression in root tips).Plant Physiology107,341±348.Tabuchi A,Matsumoto H.2001.Changes in cell-wall propertiesof wheat(Triticum aestivum)roots during aluminium-induced growth inhibition.Physiologia Plantarum112,353±358.Watt DA.2003.Aluminium-responsive genes in sugarcane:identi®cation and analysis of expression under oxidative stress. Journal of Experimental Botany54,1163±1174.Wu P,Liao CY,Hu B,Yi KK,Jin WZ,Ni JJ,He C.2000.QTLsand epistasis for aluminium tolerance in rice(Oryza sativa L)at different seedling stages.Theoretical and Applied Genetics100,1295±1303.Yoshida S,Forno D,Cock J,Gomez boratory manualfor physiological studies of rice.Manila,Philippines: International Rice Research Institute.Identi®cation of Al-regulated genes in rice143by guest on August 25, 2011Downloaded from。