Liu et al. BMC Genomics (2016) 17:223 DOI 10.1186/s12864-016-2559-8

RESEARCH ARTICLE Open Access

Genome-wide analysis of MATEtransporters and expression patternsof a subgroup of MATE genes in responseto aluminum toxicity in soybean

Juge Liu, Yang Li, Wei Wang, Junyi Gai and Yan Li*

Abstract

Background: Multidrug and toxic compound extrusion (MATE) family is an important group of the multidrug effluxtransporters that extrude organic compounds, transporting a broad range of substrates such as organic acids, planthormones and secondary metabolites. However, genome-wide analysis of MATE family in plant species is limitedand no such studies have been reported in soybean.

Results: A total of 117 genes encoding MATE transporters were identified from the whole genome sequenceof soybean (Glycine max), which were denominated as GmMATE1 - GmMATE117. These 117 GmMATE genes wereunevenly localized on soybean chromosomes 1 to 20, with both tandem and segmental duplication events detected,and most genes showed tissue-specific expression patterns. Soybean MATE family could be classified into four subfamiliescomprising ten smaller subgroups, with diverse potential functions such as transport and accumulation of flavonoids oralkaloids, extrusion of plant-derived or xenobiotic compounds, regulation of disease resistance, and response to abioticstresses. Eight soybean MATE transporters clustered together with the previously reported MATE proteins related toaluminum (Al) detoxification and iron translocation were further analyzed. Seven stress-responsive cis-elements such asABRE, ARE, HSE, LTR, MBS, as well as a cis-element of ART1 (Al resistance transcription factor 1), GGNVS, were identifiedin the upstream region of these eight GmMATE genes. Differential gene expression analysis of these eight GmMATEgenes in response to Al stress helps us identify GmMATE75 as the candidate gene for Al tolerance in soybean, whoserelative transcript abundance increased at 6, 12 and 24 h after Al treatment, with more fold changes in Al-tolerant thanAl-sensitive cultivar, which is consistent with previously reported Al-tolerance related MATE genes.

Conclusions: A total of 117 MATE transporters were identified in soybean and their potential functions were proposedby phylogenetic analysis with known plant MATE transporters. The cis-elements and expression patterns of eightsoybean MATE genes related to Al detoxification/iron translocation were analyzed, and GmMATE75 was identifiedas a candidate gene for Al tolerance in soybean. This study provides a first insight on soybean MATE family andtheir potential roles in soybean response to abiotic stresses especially Al toxicity.

Keywords: Abiotic stress, Aluminum toxicity, cis-element, Duplication, Expression analysis, MATE, Phylogeneticanalysis, Soybean

* Correspondence: yanli1@njau.edu.cnNational Key Laboratory of Crop Genetics and Germplasm Enhancement,National Center for Soybean Improvement, Key Laboratory for Biology andGenetic Improvement of Soybean (General, Ministry of Agriculture), JiangsuCollaborative Innovation Center for Modern Crop Production, NanjingAgricultural University, Nanjing 210095, China

© 2016 Liu et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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BackgroundMultidrug and toxic compound extrusion (MATE) familyis the most recent categorized multidrug efflux transporterfamily, which is a secondary transporter family thatcouples the translocation of substrates with an electro-chemical gradient of cations (such as H+ or Na+ ions)across the membrane [1, 2]. The X-ray structure of theMATE transporter, NorM from Vibrio cholerae, reveals aunique topology of its predicted 12 transmembrane (TM)helices, which is distinct from any other known multidrugresistance transporter [3].MATE transporters are widely distributed in bacteria,

fungi, mammals and plants [4]. Hvorup et al. [5] found203 sequenced proteins in the MATE family, which couldbe divided into 15 subfamilies. Omote et al. [4] identified861 MATE transporters from Archaea, Eubacteria andEukarya and classified them into three large subfamiliescomprising 14 smaller subgroups. There are 56, over 40and 53 putative MATE transporters in Arabidopsisthaliana [6], Medicago truncatula [7] and Oryza sativa[8], respectively.The bacterial MATE transporters can export organic

cations for multidrug resistance [9, 10]. In yeast (Saccha-romyces cerevisiae), the Erc1 MATE-type transporter con-fers resistance to ethionine [11]. The mammalian MATEtransporters can excrete the metabolic waste and xeno-biotic organic cations in the kidney and liver [12, 13]. Theplant MATE family transports a broad range of substratessuch as organic acids, plant hormones and secondary me-tabolites [14–16]. Recently, the functions of many MATEtransporters have been illustrated in plants [17], includ-ing transport of secondary metabolites such as alkaloids[18], flavonoids [7, 19], and anthocyanidin [20–23], de-toxification of toxic compounds or heavy metals [6, 24],regulation of disease resistance [25–27], efflux of planthormones such as abscisic acid (ABA) [28], iron transloca-tion [29–31] and aluminum (Al) detoxification [32–35],which indicates MATE transporters play important rolesin a wide range of biological processes in plants.Al toxicity is considered as the main factor limiting crop

yield on acidic soils [36]. Under Al stress, root exudationof organic acids, such as malate, citrate, and oxalate, is animportant mechanism in plant resistance to Al toxicity[37, 38]. The genes controlling organic anion efflux fromroots have been isolated from several crop species [39,40]. MATE transporters have been shown to mediate thecitrate efflux to confer plant tolerance to Al toxicity [41].The MATE transporters involved in detoxification of Alwere first identified from sorghum (Sorghum bicolor,SbMATE) and barley (Hordeum vulgare, HvAACT1) bymap-based cloning, respectively [14, 32]. Later studyfound that the function of HvAACT1 protein is to releasecitrate to facilitate the translocation of iron from roots toshoots, and the 1-kb insertion in the upstream of the

HvAACT1 coding region in the Al-tolerant barley varietyenhances and alters its expression to root tips, which isimportant to detoxifying Al and adaptation to acidic soilsin barley [33]. Overexpression of HvAACT1 increasescitrate efflux and Al tolerance in wheat and barley [34].BoMATE from cabbage (Brassica oleracea) requires Al3+

to activate citrate efflux, leading to enhanced Al tolerancein A. thaliana [35]. Several other MATE transporters,such as EcMATE1 (Eucalyptus camaldulensis), OsFRDL4(O. sativa), and ZmMATE1 (Zea mays), are found local-ized to plasma membranes in the root tips and related toplant tolerance to Al toxicity [42–44].Compared with other plant species, little work on MATE

transporters has been done in soybean (Glycine max (L.)Merr.), which is an important oil crop worldwide. To date,only one MATE transporter, GmFRD3b (G. max ferricreductase defective 3b), was reported to play a role in ironefficiency in soybean [45]. With the public available wholegenome sequence [46] and RNA-seq Atlas [47] of G. max,it is possible to identify the genome-wide MATE genes insoybean and investigate their expression patterns and pos-sible functions. Plant MATE transporters have been shownto be involved in diverse functions including Al tolerance.By comparing the sequences of soybean MATE family withthe known MATE transporters from other plant species,the possible roles of soybean MATE transporters could beproposed and help us to further test their function. In thisstudy, we performed a genome-wide search of all putativeMATE transporters in soybean. Their chromosomaldistribution, gene duplication, phylogenetic relationship,structures of genes and proteins, and expression patternswere analyzed. Soybean MATE genes related to Al detoxifi-cation/iron translocation were further investigated bypromoter analysis and differential gene expression analysisbetween the root tips of Al-tolerant and Al-sensitivesoybean cultivars in response to Al. This study wouldprovide useful information on the research of MATE trans-porters in soybean.

Results and discussionGenome-wide identification of soybean MATEtransportersA total of 117 genes encoding MATE transporters(Additional file 1: Table S1) were identified from thesoybean whole genome (see details in Methods), whichwere denominated as GmMATE1 - GmMATE117 accord-ing to the soybean nomenclature based on their physicallocations [48]. The genomic sequences, coding sequences,and protein sequences of these 117 soybean MATE mem-bers (Additional file 2) were downloaded from Phytozome(http://phytozome.jgi.doe.gov/pz/portal.html) [49].The details of all 117 soybean MATE proteins, includ-

ing the length, molecular weight, number of TM, iso-electric point (pI), and predicted subcellular location, are

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listed in Additional file 3: Table S2. The Soybean MATEproteins consist of 80 to 593 amino acids, containing 2 to13 TMs, whereas in Arabidopsis, the lengths of MATEproteins range from 400 to 700 amino acids and most with12 TMs [6], indicating there are more variations within thesoybean MATE family. The predicted molecular weights ofsoybean MATE proteins range from 8.71 to 64.28 kDa, andthe predicted pI values are between 5.13 and 9.70. Theirpredicted subcellular locations include plasma membrane,chloroplast, cytoplasm, vacuole, endoplasmic reticulum,and extracellular, with 82.91 % (97 out of 117 MATEproteins) located in the plasma membrane, 7.69 % (9 out of117) located in chloroplast, 5.12 % (6 out of 117),2.56 % (3 out of 117), 0.85 % (1 out of 117) and 0.85 %

Fig. 1 Chromosomal locations and duplications of soybean MATE genes. Ththe left is in megabases (Mb). The chromosome size is indicated by its relaTandemly duplicated genes are shown in purple boxes. Each pair of segme

located in cytoplasm, vacuole, endoplasmic reticulum,and extracellular, respectively.

Chromosomal locations and duplication patterns ofsoybean MATE genesBased on the physical positions (Additional file 1: Table S1),these 117 GmMATE genes are unevenly distributed on 20soybean chromosomes (2n = 40, Fig. 1). The number ofGmMATE genes on chromosomes 1 to 20 ranges from 4 to12. Chromosome nine contains the highest number ofGmMATE genes (12), whereas chromosomes 4, 6, 8, 11, 12,14, and 15 contain fewest GmMATE genes (four on each).Majority of these GmMATE genes are located on the

e chromosome number is indicated above each bar and the scale ontive length using the information from Phytozome and SoyBase.ntal duplication is indicated by a green line

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chromosome arms (Fig. 1), which are associated with highrates of recombination [50].Compared with the number of MATE genes in A. thali-

ana [6], M. truncatula [7], and O. sativa [8], which con-tains 56, over 40, and 53 MATE genes, respectively,MATE family in soybean is remarkably large with 117members, which might result from two whole-genomeduplication events in soybean [46]. We further investi-gated the duplication patterns of soybean MATE family.The duplication analyses showed 96 out of 117 GmMATEgenes (82.05 %) were present in duplications (Fig. 1;Additional file 4: Table S3), which indicates duplicationscontributed largely to the amplification of MATE familyin the soybean genome, supporting the previous observa-tion that duplications play important roles in the evolutionof large gene families [51]. We observed 25 (21.37 %)GmMATE genes with tandem duplications (on chro-mosomes 3, 5, 7, 9, 10, 11, 13, 16, 17 and 20), and71 (60.68 %) GmMATE genes with segmental duplica-tions (Additional file 4: Table S3). Duplication eventsof GmMATE genes were found on all 20 soybeanchromosomes.

Phylogenetic analyses of the soybean MATE familyThere are 35 plant MATE transporters that have beenreported previously (Additional file 5: Table S4), includ-ing MATE transporters with known functions and fewsequences that were reported by Zhao et al. [52]. Usingthe full-length protein sequences of the 152 MATEtransporters, including the 35 previously reported plantMATE proteins and 117 soybean MATE proteins, weconstructed a maximum likelihood (ML) tree (Fig. 2).These MATE proteins could be classified into four pri-mary clades (subfamilies) comprising ten smaller sub-groups (Fig. 2), including subfamily C1 (subgroups C1-1,C1-2, C1-3), subfamily C2 (subgroups C2-1 and C2-2),subfamily C3 (subgroups C3-1, C3-2,) and subfamily C4(subgroups C4-1, C4-2, C4-3). The functions of soybeanMATE proteins could be inferred from the known MATEtransporters according to their phylogenetic relationships.Subfamily C1 contains three subgroups, C1-1, C1-2 and

C1-3. C1-1 consists of 19 sequences, including 11 MATEproteins from soybean and eight previously reportedMATE transporters, such as AtFFT (A. thaliana, flowerflavonoid transporter) [16], SlMATE (Solanum lycopersi-cum MATE) [23], VvAM1 and VvAM3 (Vitis viniferaanthoMATE1 and 3) [21, 22]. AtFFT is a flavonoid trans-porter that affects flavonoid levels in Arabidopsis [16].SlMATE encodes a putative anthocyanin permease, whichis co-regulated with ANT1 (anthocyanin) transcriptionfactor, indicating it may function as an anthocyaninvacuolar transporter in tomato leaves [23]. VvAM1 andVvAM3 were found to be involved in the transport of ac-ylated anthocyanins into vacuoles in grapevine [21, 22].

There are 21 soybean MATE proteins in the C1-2 sub-group, with no previously known MATE proteins. Thereare 11 proteins in the C1-3 subgroup, including five soy-bean MATE proteins and six previously reported MATEtransporters such as AtTT12 (A. thaliana transparenttesta 12) [19], MtMATE1 (M. truncatula MATE1) [7],NtMATE1 and NtMATE2 (Nicotiana tabacum, MATE1and 2) [18], and VvMATE1 (V. vinifera, MATE1) [20].Arabidopsis TT12, the first MATE transporter found totransport flavonoids [19], was originally isolated duringscreening of mutants with altered seed coloration.MtMATE1 from M. truncatula was a functional orthologof AtTT12 and localized in the tonoplast [7]. NtMATE1and NtMATE2 were suggested to transport alkaloids fromthe cytosol into the vacuole in tobacco [18]. The grapevineMATE transporter VvMATE1 was involved in the accu-mulation of proanthocyanidins [20]. The functions of theknown MATE transporters in this clade suggest theMATE subfamily C1 might be involved in (vacuolar)transport and accumulation of flavonoids or alkaloids inplants.There are 34 soybean MATE proteins in subfamily C2,

which are divided into two subgroups. Subgroup C2-1 has13 soybean MATE proteins and a known MATE proteinAtALF5 (A. thaliana aberrant lateral root formation 5), inwhich mutation led to defects in lateral root formationand increased sensitivity of roots to various compounds,therefore it is thought to confer plant resistance to toxins[24]. Subgroup C2-2 contains 21 soybean MATE mem-bers, as well as AtDTX1 (A. thaliana detoxification 1) andNtJAT1 (N. tabacum jasmonate-inducible alkaloid trans-porter 1) [6, 53]. AtDTX1 was found to mediate the effluxof plant-derived antibiotics and other toxic compounds,and was also able to detoxify the heavy metal, Cd2+ [6].Tobacco NtJAT1 showed nicotine efflux activity in yeastand was suggested to function as a secondary transporterfor nicotine translocation [53]. Therefore, subfamily C2might be related to the efflux of various compounds.Subfamily C3 could be further classified into two

subgroups. None of the known MATE proteins appears insubgroup C3-1 (six soybean MATE proteins). SubgroupC3-2 contains 29 soybean MATE proteins and threeknown MATE proteins, AtADS1 (A. thaliana activateddisease susceptibility 1, which is also known asArabidopsis abnormal shoot 3, ABS3) [25, 54], AtDTX50(A. thaliana detoxification efflux carrier 50) [28], andAtZF14 (also known as bush and chlorotic dwarf 1, BCD1or Arabidopsis abnormal shoot 4, ABS4) [15, 54, 55]. Pre-vious research indicated that AtADS1/ABS3 is a negativeregulator of plant disease resistance [25]. AtDTX50 func-tions as an ABA efflux transporter, which regulates ABAsensitivity, stomatal conductance and drought tolerance inArabidopsis [28]. Overexpression of AtZF14/BCD1/ABS4increased leaf initiation rate and it is also involved in iron

Fig. 2 The phylogenetic tree of soybean MATE family. The phylogenetic tree was constructed by MEGA 6.0 using the Maximum Likelihood (ML)method. Bootstrap values in percentage (1000 replicates) are indicated on the nodes. Different subfamilies are highlighted using different colors(C1 in green, C2 in pink, C3 in blue and C4 in gray), and subgroups are marked with black arcs outside of the cycle tree

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homeostasis [15, 55]. Recently, AtZF14/BCD1/ABS4 andAtADS1/ABS3 were also found to regulate hypocotyl cellelongation [54]. The functions of MATE subfamily C3seem diversified and need further investigation.There are three subgroups in subfamily C4. Subgroup

C4-1 only contains one soybean MATE protein. SubgroupC4-2 has six soybean MATE proteins and AtEDS5, whichis essential for salicylic acid (SA) dependent disease resist-ance [26, 27]. Subgroup C4-3 contains 22 members,

including eight soybean MATE proteins (one of which,GmMATE47, is the known GmFRD3 that has beenreported to play a role in iron efficiency [45]), and 14previously reported MATE proteins from other plantspecies that are all related to Al detoxification and/or irontranslocation (Additional file 5: Table S4), indicatingthese eight soybean MATE proteins in subgroup C4-3might be involved in Al detoxification/iron transloca-tion in soybean.

Fig. 3 The gene structures of soybean MATE family. The structuresof 117 GmMATE genes were plotted using green boxes representingexons (coding DNA sequence, CDS), black lines representing intronsand blue boxes indicating upstream/downstream sequences. Thescale on the bottom is in the unit of kilobase (kb). The genes are listedaccording to the order of subfamily C1 to C4 from the phylogenetictree, and different subfamilies are highlighted in different colors (sameas Fig. 2): C1 in green, C2 in pink, C3 in blue and C4 in gray

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Gene structures and protein motifs of soybean MATEfamilyIn order to better understand the characteristics of thesoybean MATE genes, their structures were analyzed bycomparing the genomic DNA sequences with their cor-responding coding sequences (Additional file 2). Theirintron-exon structures were plotted along with the orderof subfamily in phylogenetic tree (Fig. 3). The GmMATEgene structures including length and number of exonsand introns are more similar within the same subfamily(Fig. 3). Most genes in the largest subfamily C1 have 5–10 exons, except that GmMATE45, GmMATE59 andGmMATE83 have only 1–2 exons. Most genes in sub-family C2 contain 6–11 exons, except for GmMATE26,GmMATE64 and GmMATE99, which only have 1–3exons. The GmMATE genes in subfamily C3 containonly 1–3 exons. The GmMATE genes in subfamily C4have 9–17 exons.Next, motifs in soybean MATE protein sequences

were identified using MEME (Fig. 4). The types and se-quences of the motifs are similar among the first threesubfamilies, C1, C2 and C3, but significant different withthe fourth subfamily C4 (Fig. 4). The MATE proteins insubfamily C4 generally have fewer motifs than the firstthree subfamilies.

Expression patterns of GmMATE genes in differentsoybean tissuesThe relative transcript abundance of the 117 GmMATEgenes in different soybean tissues was searched fromPhytozome v10.3 [49]. There are four genes having therelative transcript abundance as 0 across all tested tis-sues and therefore were excluded for further analysis(Additional file 6: Table S5). A heat map with clusteringof the rest 113 GmMATE genes (Fig. 5) was con-structed using MeV software [56]. Most GmMATEgenes showed specific tissue expression patterns. Somegenes (e.g. GmMATE107 and GmMATE27) showed higherexpression levels in root/root hair/nodule while lowerlevels in above-ground tissues (Fig. 5). Some GmMATEgenes such as GmMATE44, GmMATE81 and GmMATE36were mainly expressed in pod and developing seed,suggesting their putative roles during seed development.Some GmMATE genes (e.g. GmMATE1 and GmMATE39)showed higher expression levels in flower and pod. The

Fig. 4 Protein motifs of soybean MATE family. The motifs ofsoybean MATE proteins are shown as colored boxes. The scale onthe bottom may be used to estimate the length of motif (unit:amino acid). The GmMATE proteins are listed according to the orderof subfamily C1 to C4 from the phylogenetic tree, and differentsubfamilies are highlighted in different colors (same as Fig. 2): C1 ingreen, C2 in pink, C3 in blue and C4 in gray

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expression levels of some genes (e.g. GmMATE62 andGmMATE7) were higher in leaf but lower (or zero) inother tissues. There are also some genes (e.g.GmMATE69, GmMATE72, GmMATE76, GmMATE80and GmMATE117) expressed in all tested tissues(Fig. 5). The relative transcript abundance of the 117GmMATE genes was also searched from the soybeanRNA-Seq Atlas [47] in SoyBase [57]. There are 21GmMATE genes have no data and 19 GmMATE geneshave the relative transcript abundance as 0 across alltested tissues (Additional file 7: Table S6). The tissueexpression patterns of most genes with non-zero rela-tive transcript abundance from SoyBase were consistentwith the Phytozome.To cross-validate the expression patterns of GmMATE

genes with the RNA-seq data, we performed quantitativereal-time PCR (qRT-PCR) for eight representative genesrandomly selected from the four subfamilies (Fig. 6).The relative expression of three genes (GmMATE49,GmMATE91, and GmMATE117) from subfamily C1 ishigher in flower, leaf, pod, and shoot apical meristem(SAM) than root tip, which is consistent with Fig. 5.GmMATE36 from subfamily C2 expresses in all testedtissues by qRT-PCR, which in general agrees with theRNA-seq data. GmMATE86 and GmMATE93 from sub-family C3 show high relative expression level in flowerin both qRT-PCR and RNA-seq data. GmMATE13 andGmMATE75 from subfamily C4 express higher in leaf orpod by qRT-PCR but show higher expression in roothairs and nodules in RNA-seq data, which might be dueto the difference in the sensitivities of two methods,RNA samples from two different cultivars (Williams 82for RNA-seq and KF for qRT-PCR), or different growingenvironments.

Characterization of putative cis-regulatory elements inthe promoter regions of subgroup C4-3 GmMATE genesBased on the phylogenetic tree, eight GmMATE geneswere classified into subgroup C4-3 (Fig. 2), together withthe previously reported genes that are all related to Aldetoxification and/or iron translocation. The cis-actingregulatory elements in the promoter regions play im-portant roles in plant response to stresses. Using thePlantCARE database, we identified 11 putative stress orhormone-responsive cis-acting elements in the 1500 bpupstream (Additional file 8) of these eight GmMATE

Fig. 5 (See legend on next page.)

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(See figure on previous page.)Fig. 5 Heat map of the expression profiles of GmMATE genes in nine soybean tissues. The heat map with hierarchical clustering of 113 GmMATEgenes was constructed using MeV 4.9 software by average linkage with Euclidean distance. Color key represents the relative transcript abundanceof the GmMATE genes in nine soybean tissues. The FPKM (fragments/kilobase/million) values were log10 transformed and mean centred by genesusing the MeV 4.9 software. SAM: shoot apical meristem

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genes (Fig. 7, Additional file 9: Table S7), includingABRE (ABA-responsive element), ARE (anaerobic-re-sponsive element), CGTCA-motif, HSE (heat stress-responsive element), LTR (low temperature responsiveelement), MBS (MYB binding site), TCA-element, TCrich-repeats, TGACG-motif, WUN (wound-responsiveelement) and W1-Box. GmMATE13 contains only onecis-acting element (MBS), while the other sevenGmMATE genes have more than one predicted cis-act-ing elements. Two genes, GmMATE79 and GmMATE84,contain ten and 11 cis-elements, respectively (Fig. 7).Seven cis-elements, ABRE, ARE, HSE, LTR, MBS, TCA-element and TC-rich repeats, are stress responsive.ABRE element is important in ABA signaling and plantresponse to drought and high salinity in Arabidopsis[58], which is present in two C4-3 GmMATE genes.ARE element was found both necessary and sufficientfor induction of gene expression by low oxygen stress[59], which is present in four C4-3 GmMATE genes.HSE has been found to be consistently conserved in theregulatory regions of many heat induced genes [60],which is present in five C4-3 GmMATE genes. MBS cis-element was reported to bind to MYB transcriptionalfactors involved in stress signaling [61], and five C4-3GmMATE genes contain MBS. LTR element is import-ant for the induction of cold regulated genes [62], whichis present in two C4-3 GmMATE genes. TCA-elementmediates SA-signaling pathway and is sufficient for the

Fig. 6 Relative expression levels of the representative GmMATE genes in sevewere randomly selected to validate their relative expression in different tissuethe soybean GmEF-1α gene was used as the internal control. The error bars in

response to stress [63], which is found in two C4-3GmMATE genes. TC-rich repeats element is involved indefense and stress-responsiveness [64], which is presentin three C4-3 GmMATE genes.Another cis-acting element, GGN(T/g/a/C)V(C/A/g)S(C/

G), has been identified as the DNA-binding sequence ofART1 (Al resistance transcription factor 1), which regulatesthe expression of 31 genes (including MATE) to conferAl tolerance in rice [65]. This element, GGNVS, wasfound in all eight C4-3 GmMATE genes, with differentnumbers and positions (Table 1). GmMATE47, GmMATE75, GmMATE79 and GmMATE87 contain more tenGGNVS elements, while only five GGNVS element wasfound in GmMATE58.

Expression of the subgroup C4-3 GmMATE genes inresponse to Al toxicityTen out of 14 (71 %) MATE transporters from otherplant species in subgroup C4-3 (Fig. 2, Additional file 5:Table S4) have been shown related to Al detoxification,therefore the expression patterns of all eight subgroupC4-3 GmMATE genes in response to Al toxicity wereanalyzed by qRT-PCR. Intraspecific variation in Al toler-ance is striking in many crop species [14, 66, 67]. Previ-ous studies showed that Al-tolerance related MATEgene expression in plant root tips is up-regulated by Aland is significantly higher in Al-tolerant genotypes [43,44]. In this study, two soybean cultivars, KF (Al tolerant)

n soybean tissues. Eight GmMATE genes representing four subfamiliess by qRT-PCR. The relative expression level in stem was set as one anddicate the standard deviation from three replicates

Fig. 7 Stress-responsive cis-elements of the eight GmMATE genes in subgroup C4-3. The phylogenetic tree (maximum likelihood) on the left wasconstructed using MEGA 6.0 with the full sequences of the eight soybean MATE proteins in subgroup C4-3. The cis-elements in the 1500 bp upstreamregions of the eight corresponding GmMATE genes were predicted using the PlantCare database, and shown in colored boxes with their names andpositions (relative to the start codon) inside

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and GF (Al sensitive), were treated with 0 and 25 μMAlCl3 for 6 h, 12 h and 24 h, respectively. The relativeexpression levels of the eight C4-3 GmMATE genes inthe root tips of soybean seedlings after Al stress treat-ment are shown in Fig. 8. Among these eight genes, onlyone gene, GmMATE75, showed a significantly higherrelative gene expression in the Al-tolerant cultivar KF(T) than in Al-sensitive cultivar GF (S) at 6, 12, and 24 hafter Al stress treatment. GmMATE13 showed a signifi-cant higher relative expression in KF (T) at 6 h butsignificant higher in GF (S) at 24 h after Al stress treat-ment. GmMATE58, GmMATE74, and GmMATE84showed a significantly higher level of relative expressionin GF (S) than KF (T) after Al stress treatment.GmMATE47, GmMATE79 and GmMATE87 did notshow significant difference in their relative expressionbetween GF (S) and KF (T) during Al stress treatment.Therefore, GmMATE75, which showed higher elevatedtranscript levels after Al treatment in Al-tolerant (T)than Al-sensitive (S) cultivar, would be a candidate genefor soybean tolerance to Al toxicity. The expressionpatterns of GmMATE75 could also be visualized by

Table 1 Number and position of GGNVS element in the promoter r

Genes No. of GGNVS P

GmMATE13 7 -

GmMATE47 12 -

GmMATE58 5 -

GmMATE74 7 -

GmMATE75 12 -

GmMATE79 15 -

GmMATE84 9 -

GmMATE87 11 -aGGNVS is the abbreviation for GGN(T/g/a/C)V(C/A/g)S(C/G), a cis-acting element (tabThe position is relative to the start codon of each gene

semi-quantitative RT-PCR (Additional file 10: Figure S1).Under normal growth conditions without Al treatment,their expression levels were very low and no differencewas observed between KF (T) and GF (S). However,under Al treatment, their expression levels increasedsignificantly, which was consistent with the qRT-PCRresults (Fig. 8). A previous microarray study reportedGma.8768.1.A1_at, a putative MATE gene, was up-regulated (approximately 23-fold change) by Al treatmentin an Al-tolerant soybean variety Jiyu 70 [68], which is thesame gene as GmMATE75 we identified in this study. Inour study, the relative expression of GmMATE75 under Alstress was further compared between Al-tolerant and Al-sensitive varieties. GmMATE75 was highly up-regulated by127, 274, and 335-fold in KF (T) while 10, 39, and 33-foldin GF (S) after 6, 12, and 24 h Al treatment, respectively,suggesting its role in soybean tolerance to Al stress.

ConclusionsA comprehensive genome-wide analysis of MATE familyis performed in an important legume species and oilcrop, soybean. A total of 117 MATE transporters were

egion of eight C4-3 soybean MATE genesa

osition of GGNVSb

639,-943,-944,-1149,-1150,-1398,-1451

232,-541,-548,-670,-769,-834,-1026,-1043,-1044,-1183,-1417,-1457

42,-122,-133,-1447,-1481

85,-287,-768,-1210,-1354,-1386,-1390

383,-609,-611,-612,-616,-884,-885,-1002,-1003,-1015,-1351,-1449

139,-143,-144,-260,-489,-513,-560,-590,-637,-724,-725,-990,-1139,-1283,-1446

271,-772,-893,-1150,-1177,-1287,-1443,-1444,-1445,

141,-376,-515,-516,-636,-671,-813,-1010,-1162,-1235,-1491

rget DNA-binding sequence) of ART1 (Al Resistance Transcription Factor 1)

Fig. 8 Relative expression of the eight soybean C4-3 MATE genes in response to Al toxicity. The relative expression of the eight GmMATE genesin the soybean root tips (0–2 mm) in response to Al stress (25 μM AlCl3) was quantified by qRT-PCR, using soybean GmEF-1α gene as the internalcontrol and 0 μM AlCl3 (control) as reference. The error bars indicate the standard deviation from three replicates. T: aluminum-tolerant cultivar,KF; S: aluminum-sensitive cultivar, GF. * and ** indicates significant difference in the relative expression level between T and S in response to Alstress for each time point (student’s t-test) at P < 0.05 and P < 0.01, respectively

Liu et al. BMC Genomics (2016) 17:223 Page 11 of 15

identified in soybean and could be classified into foursubfamilies, C1, C2, C3 and C4. The soybean MATEfamily displays great variation in gene structure, proteinmotif, and tissue expression pattern, which indicatestheir diverse functions. The expansion of soybean MATEfamily was largely due to segmental duplications. Sevenstress-responsive cis-acting elements and the cis-actingelement of ART1 (GGNVS) were identified in the up-stream regions of eight GmMATE genes in subgroupC4-3, which contains previously reported MATE genesrelated to Al detoxification and/or iron translocation.One gene from C4-3 subgroup, GmMATE75, showeddifferential relative transcript abundance between theroot tips of Al-tolerant and Al-sensitive soybean culti-vars in response to Al treatment, indicating its potentialrole in soybean tolerance to Al toxicity. This study pro-vides a foundation to further investigate the functions ofsoybean MATE genes including the candidate gene forAl tolerance in soybean.

MethodsIdentification of MATE transporters in soybeanA total of 57 MATE (Pfam: PF01554) protein sequencesin Arabidopsis were collected from Phytozome v10.3[49] (http://phytozome.jgi.doe.gov/pz/portal.html). Soy-bean putative MATE protein sequences were retrievedby BLASTP searches against the target (Glycine maxWm82.a2.v1) proteome at Phytozome v10.3 using 57 A.thaliana MATE protein sequences as queries (E-valuewas ≤ 1e-7). These putative MATE sequences were fil-tered by the presence of conserved MATE domain

(Pfam: PF01554) using the Pfam (http://pfam.xfam.org/)[69] and the Simple Modular Architecture Research Tool(SMART, http://smart.embl-heidelberg.de/smart/batch.pl)[70], and a total of 117 soybean MATE proteins withMATE domain were identified. Data files containing theinformation of the final 117 soybean MATE (includingtheir corresponding physical locations on soybean chromo-somes, genomic sequences, coding sequences, and proteinsequences) were downloaded from Phytozome (http://phy-tozome.jgi.doe.gov/pz/portal.html) [49] (Additional file 1:Table S1 and Additional file 2). Theoretical isoelectricpoint (pI) and molecular weight (MW) of soybean MATEproteins were computed by ExPASy Compute pI/Mw tool(http://www.expasy.ch/tools/pi_tool.html) [71–73]. Thesubcellular localizations of the MATE proteins were pre-dicted using WoLF PSORT (http://www.genscript.com/wolf-psort.html) [74] and the numbers of transmembranehelices were predicted by TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/) [75].

Phylogenetic and structural analyses of MATEtransporters in soybeanThe full protein sequences of 117 soybean MATE(Additional file 1: Table S1 and Additional file 2) and35 previously reported MATE from other plant species(Additional file 5: Table S4) were used for multiplesequence alignments by ClustalW in MEGA 6.0 [76]. Theunrooted phylogenetic tree was then constructed byMEGA 6.0 [76] using the Maximum Likelihood (ML)algorithm with 1000 bootstraps, where the amino acidsubstitution model was equal input model with uniform

Liu et al. BMC Genomics (2016) 17:223 Page 12 of 15

rates among sites, using partial deletion (95 % site cover-age as cutoff) for gaps and missing data. Gene structureanalysis was performed using the Gene Structure DisplayServer (GSDS) program with default settings [77]. Motifsin MATE proteins were statistically identified using theonline tool of Multiple EM for Motif Elicitation (MEME)[78] (http://meme-suite.org/) with default settings: MotifWidth: between 6 and 50 wide (inclusive). Site Distribu-tion: zero or one occurrence (of a contributing motif site)per sequence. The maximum number of motif was set at12 [4].

Chromosomal locations and gene duplication analysisThe chromosomal locations of GmMATE genes wereillustrated by MapChart [79]. Segmental and tandemduplication events of the soybean MATE family wereidentified using the Multiple Collinearity Scan toolkit(MCScan) [80] from the Plant Genome DuplicationDatabase [81] with default settings: BLASTP was used tosearch for potential anchors (E <1e-5, top 5 matches)between every possible homolougous pair, and thesepairs were used as the input for MCscan. Syntenicblocks were identified using the E-value ≤ 1e − 10 as asignificance cutoff. Tandem duplication was defined ashomologous genes with less than ten gene loci in-between and >50 % similarity at protein level on a singlechromosome [82].

Characterization of putative cis-elements in the promoterregions of subgroup C4-3 soybean MATE genesThe 1500 bp upstream sequences (Additional file 8) relativeto the translation start codon of the eight C4-3 subgroupGmMATE genes were downloaded from Phytozome [49].The cis-elements in the 1500 bp upstream regions werepredicted using the PlantCare database (http://bioinforma-tics.psb.ugent.be/webtools/plantcare/html/) [83].

Tissue expression patterns of MATE genes in soybeanThe RNA-seq data of soybean MATE genes (Additionalfile 6: Table S5; Additional file 7: Table S6) were down-loaded from Phytozome v10.3 [49] and RNA-seq Atlas[47] of G. max which is available on SoyBase (http://soy-base.org/soyseq/) [57]. Since the RNA-seq Atlas onSoyBase was released in 2010 where the RNA-seq readshave been mapped only to the initial soybean genomeassembly (Wm82.a1.v1.), the 21 GmMATE genes thatwere uniquely identified in the later assembly Wm82.a2.v1are not represented in this dataset, and there are 19GmMATE genes have the relative transcript abundance as0 in all tested tissues. Therefore, we used the RNA-seqdata from Phytozome to investigate the tissue expressionpatterns of soybean MATE family. The heat map withhierarchical clustering of 113 GmMATE genes fromPhytozome (excluding 4 GmMATE genes with the relative

transcript abundance as 0 across all tested tissues) wasconstructed to visualize their tissue expression patterns,using average linkage clustering with Euclidean distanceby MeV 4.9 software [56].Eight GmMATE genes from four subfamilies were ran-

domly selected to be verified by quantitative real-timePCR (qRT-PCR). Soybean tissues were collected accord-ing to the developmental stages described by MarcLibault [84]. The seeds of soybean cultivar Kefeng-1(KF) were germinated in moist sterile sand. Root tips of3-day-old seedlings were harvested. The seedlings weretransferred to the glasshouse under long-day conditions(16-h day/8-h night) at 26/24 °C temperature circula-tions. Shoot apical meristem (SAM) from V2 stageplants, first trifoliate leaves, stems, and flowers from R2stage plants, pods from R4 stage plants, and seeds fromR6 stage plants were harvested and immediately frozenin liquid nitrogen and stored at − 80 °C. The experimentwas performed in triplicates.

Al treatment and RNA isolationThe seeds of Al-tolerant soybean cultivar KF and Al-sensitive cultivar Guangfengmaliaodou (GF) (obtainedfrom the National Center for Soybean Improvement,Nanjing, China), were germinated in moist sterile sandand grown under a photoperiod of 14-h day/10-h nightand 26/24 °C (day/night) temperature circulations forthree days. Then the seedlings were transferred to 0.5 mMCaCl2 (pH = 4.3) for 24 h before Al treatment. Theseedlings were then exposed to 0.5 mM CaCl2 (pH = 4.3)solution containing either 0 μM AlCl3 (control) or 25 μMAlCl3 (treatment) for 6 h, 12 h and 24 h, respectively. Theroot tips (0–2 mm) were collected and immediately frozenin liquid nitrogen and stored at − 80 °C. The experimentwas performed in triplicates.Total RNA was extracted from all samples using TRIzol

according to the manufacturer’s protocol (Invitrogen,USA).

Real-time PCRThe first-strand cDNAs were synthesized using a Primer-Script First Strand cDNA synthesis kit (TaKaRa, Japan)following the manufacturer’s protocol, in a total of 20 μlreaction volume including 1 μg of total RNA, 4 μl 5XPrimeScript RT Master Mix, and RNAase-free ddH2O.The Semi-quantitative RT-PCR was performed in a finalvolume of 20 μl containing 2 μl of diluted cDNA, 10 μl 2XPremix Taq version 2.0 Mix (TaKaRa, Japan), and 200 nMof forward and reverse primers (Additional file 11: TableS8). The thermal cycling conditions were set as follows:different cycles of 95 °C for 30 s, 54.5–55 °C for 30 s (54.5,55 °C for GmMATE75 and GmEF-1a, respectively), and72 °C for 45 s. The number of cycles is based on the genesand designed primers, which is labeled in the results. The

Liu et al. BMC Genomics (2016) 17:223 Page 13 of 15

housekeeping gene GmEF-1α was used as the internalcontrol [85]. Electrophoresis was performed using 1 %agarose gels.

Quantitative real-time PCR (qRT-PCR)Gene-specific primers were designed using primer primer5.0 (Premier Biosoft International, USA) and synthesizedby Invitrogen (Shanghai, China). Quantitative real-timePCR was performed on a Roche 480 Realtime detectionsystem (Roche Diagnostics, Switzerland) following themanufacturer’s instructions. The qRT-PCR was performedin a final volume of 15 μl containing 2 μl cDNA, 7.5 μl 2XSYBR Premix Ex Taq (TaKaRa, Japan), and 200 nM offorward and reverse primers. The amplification programwas set as follows: initial denaturation at 95 °C for 5 min;40 cycles of denaturation at 95 °C for 10 s, annealing at58 °C for 20 s, and extension at 72 °C for 20 s. Theamplification efficiencies (E) of primer pairs for 15 genes(including the housekeeping gene GmEF-1α) wereestimated by qRT-PCR using 1X, 5X, 10X, 20X, and30X dilutions of cDNA, according to the equation:E = [10-1/slope]-1 [86]. Primers and amplification effi-ciencies of qRT-PCR reactions were shown in Additionalfile 11: Table S8. The amplicon specificity was verified bymelting curve analysis (Additional file 12: Figure S2) andagarose gel electrophoresis. Each experiment wasperformed in triplicates. The relative expression valueswere calculated by 2−ΔΔCT method according to Livak andSchmittgen [87]. The housekeeping gene GmEF-1α wasused as an internal control [85] and its invariant expres-sion under our experimental conditions were shown inAdditional file 13: Table S9 which showed relativeconstant Ct values across all samples. The relative expres-sion level of GmMATE genes in response to Al stress(treatment, 25 μM AlCl3) was in comparison to theircorresponding samples under normal conditions (control,0 μM AlCl3) at each time point.

Availability of supporting dataAll supporting datasets of this article are included asadditional files and available at doi: 10.6070/H47M05ZFthat were deposited in LabArchives [88].Phylogenetic datasets have been deposited in TreeBase

and are accessible via the URL: http://purl.org/phylo/treebase/phylows/study/TB2:S18947?x-accesscode=5794eb5d85e615eed0fe24f0500a289b&format=html.

Additional files

Additional file 1: Table S1. The nomenclature and physical locationsof 117 soybean MATE genes. (XLS 39 kb)

Additional file 2: The genomic sequences, coding sequences andprotein sequences of the 117 soybean MATE members. (DOC 852 kb)

Additional file 3: Table S2. Details of the 117 MATE proteins insoybean. (XLS 42 kb)

Additional file 4: Table S3. Duplication analysis of the 117 SoybeanMATE genes. (XLS 29 kb)

Additional file 5: Table S4. The 35 known MATE transporters in plants.(XLS 51 kb)

Additional file 6: Table S5. RNA-seq data of 117 soybean MATE genesin nine tissues as shown in fragments/kilobase/million (FPKM) fromPhytozome. (XLS 43 kb)

Additional file 7: Table S6. RNA-seq data of 117 soybean MATE genesin 14 tissues as shown in reads/kilobase/million (RPKM) normalization ofthe raw data from RNA-Seq Atlas on SoyBase. (XLS 44 kb)

Additional file 8: The 1500 bp upstream sequences of the eightsoybean C4-3 MATE genes. (DOC 43 kb)

Additional file 9: Table S7. Details of cis-acting elements in the1500 bp upstream of the eight soybean C4-3 MATE genes. (DOC 33 kb)

Additional file 10: Figure S1. Semi-quantitative RT-PCR of the candidateMATE gene for Al tolerance in soybean. The semi-quantitative RT-PCR wasperformed using the RNA from soybean root tips (0–2 mm). - representscontrol plants (0 μM AlCl3) while + represents plants treated with 25 μMAlCl3. GmEF-1α was used as the internal control. T: aluminum-tolerantcultivar, KF; S: aluminum-sensitive cultivar, GF. The number of PCR cycles isshown on the right. (PNG 569 kb)

Additional file 11: Table S8. Primers and amplification efficiencies forqRT-PCR and RT-PCR in this study. (XLS 22 kb)

Additional file 12: Figure S2. The amplicon specificities of 15 pairs ofprimers for qRT-PCR in this study. (DOC 220 kb)

Additional file 13: Table S9. The Ct values of the housekeeping geneGmEF-1α by qRT-PCR across all samples in this study. (XLS 32 kb)

Competing interestsThe authors declare that they have no competing interests.

Authors’ contributionsJL carried out the data mining, bioinformatics, gene expression analysis, anddrafted the manuscript. YL (Yang Li) performed cis-element analysis. WWparticipated in the duplication analysis. JG contributed to interpretation ofthe data. YL (Yan Li) conceived and designed the study, and revised themanuscript. All authors read and approved the final manuscript.

AcknowledgementsThis work was supported by the National Natural Science Foundation of China(31371645), the National High-tech R & D Program of China (2013AA102602),the Fundamental Research Funds for the Central Universities, the Program forChangjiang Scholars and Innovative Research Team in University (PCSIRT13073),the Program for New Century Excellent Talents in University (NCET-12-0891),the Program for High-level Innovative and Entrepreneurial Talents in JiangsuProvince, and the Jiangsu Higher Education PAPD Program.

Received: 10 January 2016 Accepted: 29 February 2016

References1. Kuroda T, Tsuchiya T. Multidrug efflux transporters in the MATE family.

Biochim Biophys Acta. 2009;1794(5):763–8.2. Shoji T. ATP-binding cassette and multidrug and toxic compound extrusion

transporters in plants: a common theme among diverse detoxificationmechanisms. Int Rev Cell Mol Biol. 2014;309:303–46.

3. He X, Szewczyk P, Karyakin A, Evin M, Hong WX, Zhang Q, et al. Structure ofa cation-bound multidrug and toxic compound extrusion transporter.Nature. 2010;467(7318):991–4.

4. Omote H, Hiasa M, Matsumoto T, Otsuka M, Moriyama Y. The MATE proteinsas fundamental transporters of metabolic and xenobiotic organic cations.Trends Pharmacol Sci. 2006;27(11):587–93.

5. Hvorup RN, Winnen B, Chang AB, Jiang Y, Zhou XF, Saier MH. Themultidrug/oligosaccharidyl-lipid/polysaccharide (MOP) exporter superfamily.Eur J Biochem. 2003;270(5):799–813.

Liu et al. BMC Genomics (2016) 17:223 Page 14 of 15

6. Li LG, He ZY, Pandey GK, Tsuchiya T, Luan S. Functional cloning andcharacterization of a plant efflux carrier for multidrug and heavy metaldetoxification. J Biol Chem. 2002;277(7):5360–8.

7. Zhao J, Dixon RA. MATE transporters facilitate vacuolar uptake ofepicatechin 3’-O-glucoside for proanthocyanidin biosynthesis in Medicagotruncatula and Arabidopsis. Plant Cell. 2009;21(8):2323–40.

8. Tiwari M, Sharma D, Singh M, Tripathi RD, Trivedi PK. Expression ofOsMATE1 and OsMATE2 alters development, stress responses and pathogensusceptibility in Arabidopsis. Sci Rep. 2014;4:3964.

9. Miyamae S, Ueda O, Yoshimura F, Hwang J, Tanaka Y, Nikaido H. A MATEfamily multidrug efflux transporter pumps out fluoroquinolones in Bacteroidesthetaiotaomicron. Antimicrob Agents Chemother. 2001;45(12):3341–6.

10. Kaatz GW, McAleese F, Seo SM. Multidrug resistance in Staphylococcus aureusdue to overexpression of a novel multidrug and toxin extrusion (MATE)transport protein. Antimicrob Agents Chemother. 2005;49(5):1857–64.

11. Shiomi N, Fukuda H, Fukuda Y, Murata K, Kimura A. Nucleotide sequenceand characterization of a gene conferring resistance to ethionine in yeastsaccharomyces cerevisiae. J Ferment Bioeng. 1991;71(4):211–5.

12. Hiasa M, Matsumoto T, Komatsu T, Moriyama Y. Wide variety oflocations for rodent MATE1, a transporter protein that mediates thefinal excretion step for toxic organic cations. Am J Physiol-cell Ph.2006;291(4):C678–86.

13. Ullrich KJ. Specificity of transporters for ‘organic anions’ and ‘organic cations’in the kidney. Biochim Biophys Acta. 1994;1197(1):45–62.

14. Magalhaes JV, Liu J, Guimaraes CT, Lana UGP, Alves VMC, Wang Y-H,et al. A gene in the multidrug and toxic compound extrusion(MATE) family confers aluminum tolerance in sorghum. Nat Genet.2007;39(9):1156–61.

15. Seo PJ, Park J, Park M-J, Kim Y-S, Kim S-G, Jung J-H, et al. A Golgi-localizedMATE transporter mediates iron homoeostasis under osmotic stress inArabidopsis. Biochem J. 2012;442:551–61.

16. Thompson EP, Wilkins C, Demidchik V, Davies JM, Glover BJ. An Arabidopsisflavonoid transporter is required for anther dehiscence and pollendevelopment. J Exp Bot. 2010;61(2):439–51.

17. Takanashi K, Shitan N, Yazaki K. The multidrug and toxic compoundextrusion (MATE) family in plants. Plant Biotechnology. 2014;31(5):417–30.

18. Shoji T, Inai K, Yazaki Y, Sato Y, Takase H, Shitan N, et al. Multidrug and toxiccompound extrusion-type transporters implicated in vacuolar sequestrationof nicotine in tobacco roots. Plant Physiol. 2009;149(2):708–18.

19. Debeaujon I, Peeters AJM, Leon-Kloosterziel KM, Koornneef M. TheTRANSPARENT TESTA12 gene of Arabidopsis encodes a multidrugsecondary transporter-like protein required for flavonoid sequestration invacuoles of the seed coat endothelium. Plant Cell. 2001;13(4):853–71.

20. Perez-Diaz R, Ryngajllo M, Perez-Diaz J, Pena-Cortes H, Casaretto JA,Gonzalez-Villanueva E, et al. VvMATE1 and VvMATE2 encode putativeproanthocyanidin transporters expressed during berry development in Vitisvinifera L. Plant Cell Rep. 2014;33(7):1147–59.

21. Gomez C, Terrier N, Torregrosa L, Vialet S, Fournier-Level A, Verries C, et al.Grapevine MATE-type proteins act as vacuolar H + −dependent acylatedanthocyanin transporters. Plant Physiol. 2009;150(1):402–15.

22. Gomez C, Conejero G, Torregrosa L, Cheynier V, Terrier N, Ageorges A. Invivo grapevine anthocyanin transport involves vesicle-mediated traffickingand the contribution of anthoMATE transporters and GST. Plant J.2011;67(6):960–70.

23. Mathews H, Clendennen SK, Caldwell CG, Liu XL, Connors K, Matheis N,et al. Activation tagging in tomato identifies a transcriptional regulator ofanthocyanin biosynthesis, modification, and transport. Plant Cell. 2003;15(8):1689–703.

24. Diener AC, Gaxiola RA, Fink GR. Arabidopsis ALF5, a multidrug effluxtransporter gene family member, confers resistance to toxins. Plant Cell.2001;13(7):1625–38.

25. Sun X, Gilroy EM, Chini A, Nurmberg PL, Hein I, Lacomme C, et al. ADS1encodes a MATE‐transporter that negatively regulates plant diseaseresistance. New Phytol. 2011;192(2):471–82.

26. Nawrath C, Heck S, Parinthawong N, Metraux JP. EDS5, an essentialcomponent of salicylic acid-dependent signaling for disease resistance inArabidopsis, is a member of the MATE transporter family. Plant Cell.2002;14(1):275–86.

27. Ishihara T, Sekine KT, Hase S, Kanayama Y, Seo S, Ohashi Y, et al.Overexpression of the Arabidopsis thaliana EDS5 gene enhances resistanceto viruses. Plant Biol. 2008;10(4):451–61.

28. Zhang H, Zhu H, Pan Y, Yu Y, Luan S, Li L. A DTX/MATE-type transporterfacilitates abscisic acid efflux and modulates ABA sensitivity and droughttolerance in Arabidopsis. Mol Plant. 2014;7(10):1522–32.

29. Durrett TP, Gassmann W, Rogers EE. The FRD3-mediated efflux of citrateinto the root vasculature is necessary for efficient iron translocation. PlantPhysiol. 2007;144(1):197–205.

30. Inoue H, Mizuno D, Takahashi M, Nakanishi H, Mori S, Nishizawa NK. A riceFRD3-like (OsFRDL1) gene is expressed in the cells involved in long-distancetransport. Soil Sci Plant Nutr. 2004;50(7):1133–40.

31. Yokosho K, Yamaji N, Ueno D, Mitani N, Ma JF. OsFRDL1 is a citratetransporter required for efficient translocation of iron in rice. Plant Physiol.2009;149(1):297–305.

32. Furukawa J, Yamaji N, Wang H, Mitani N, Murata Y, Sato K, et al. Analuminum-activated citrate transporter in barley. Plant Cell Physiol.2007;48(8):1081–91.

33. Fujii M, Yokosho K, Yamaji N, Saisho D, Yamane M, Takahashi H, et al.Acquisition of aluminium tolerance by modification of a single gene inbarley. Nat Commun. 2012;3:713.

34. Zhou G, Delhaize E, Zhou M, Ryan PR. The barley MATE gene, HvAACT1,increases citrate efflux and Al(3+) tolerance when expressed in wheat andbarley. Ann Bot. 2013;112(3):603–12.

35. Wu X, Li R, Shi J, Wang J, Sun Q, Zhang H, et al. Brassica oleracea MATEencodes a citrate transporter and enhances aluminum tolerance inArabidopsis thaliana. Plant Cell Physiol. 2014;55(8):1426–36.

36. Magalhaes JV, Maron LG, Piñeros MA, Guimarães CT, Kochian LV. Aluminumtolerance in sorghum and maize. In: Varshney RK, Tuberosa R, editors.Translational genomics for crop breeding: abiotic stress, yield and quality,vol. 2. New Jersey: Wiley Blackwell; 2013. p. 83–98.

37. Lyubenova L, Kuhn AJ, Höltkemeier A, Schröder P. Root exudation patternof Typha latifolia L. plants after copper exposure. Plant Soil. 2013;370(1–2):187–95.

38. Fernández-Aparicio M, Kisugi T, Xie X, Rubiales D, Yoneyama K. Lowstrigolactone root exudation: a novel mechanism of broomrape (orobancheand phelipanche spp.) resistance available for faba bean breeding. J AgrFood Chem. 2014;62(29):7063–71.

39. Delhaize E, Ma JF, Ryan PR. Transcriptional regulation of aluminiumtolerance genes. Trends Plant Sci. 2012;17(6):341–8.

40. Zhou G, Delhaize E, Zhou M, Ryan PR. Biotechnological solutions forenhancing the aluminium resistance of crop plants. In: Shanker AK,Venkateswarlu B, editors. Abiotic stress in plants—mechanisms andadaptations. Brisbane: InTech; 2011. p. p119–42.

41. Magalhaes JV. How a microbial drug transporter became essential forcrop cultivation on acid soils: aluminium tolerance conferred by themultidrug and toxic compound extrusion (MATE) family. Ann Bot.2010;106(1):199–203.

42. Sawaki Y, Kihara-Doi T, Kobayashi Y, Nishikubo N, Kawazu T, Kobayashi Y,et al. Characterization of Al-responsive citrate excretion and citrate-transporting MATEs in Eucalyptus camaldulensis. Planta. 2013;237(4):979–89.

43. Yokosho K, Yamaji N, Ma JF. An Al-inducible MATE gene is involved inexternal detoxification of Al in rice. Plant J. 2011;68(6):1061–9.

44. Maron LG, Piñeros MA, Guimarães CT, Magalhaes JV, Pleiman JK, Mao C,et al. Two functionally distinct members of the MATE (multi‐drug and toxiccompound extrusion) family of transporters potentially underlie two majoraluminum tolerance QTLs in maize. Plant J. 2010;61(5):728–40.

45. Rogers EE, Wu X, Stacey G, Nguyen HT. Two MATE proteins play a role iniron efficiency in soybean. J Plant Physiol. 2009;166(13):1453–9.

46. Schmutz J, Cannon SB, Schlueter J, Ma J, Mitros T, Nelson W, et al. Genomesequence of the palaeopolyploid soybean. Nature. 2010;463(7278):178–83.

47. Severin AJ, Woody JL, Bolon Y-T, Joseph B, Diers BW, Farmer AD, et al. RNA-Seq Atlas of Glycine max: a guide to the soybean transcriptome. BMC PlantBiol. 2010;10(1):160.

48. Patil G, Valliyodan B, Deshmukh R, Prince S, Nicander B, Zhao M, et al.Soybean (Glycine max) SWEET gene family: insights through comparativegenomics, transcriptome profiling and whole genome re-sequence analysis.BMC Genomics. 2015;16(1):520.

49. Goodstein DM, Shu S, Howson R, Neupane R, Hayes RD, Fazo J, et al.Phytozome: a comparative platform for green plant genomics. NucleicAcids Res. 2012;40(D1):D1178–86.

50. See DR, Brooks S, Nelson JC, Brown-Guedira G, Friebe B, Gill BS. Geneevolution at the ends of wheat chromosomes. Proc Natl Acad Sci.2006;103(11):4162–7.

Liu et al. BMC Genomics (2016) 17:223 Page 15 of 15

51. Cannon SB, Mitra A, Baumgarten A, Young ND, May G. The roles ofsegmental and tandem gene duplication in the evolution of large genefamilies in Arabidopsis thaliana. BMC Plant Biol. 2004;4(1):10.

52. Zhao J, Huhman D, Shadle G, He XZ, Sumner LW, Tang Y, et al. MATE2mediates vacuolar sequestration of flavonoid glycosides and glycosidemalonates in Medicago truncatula. Plant Cell. 2011;23(4):1536–55.

53. Morita M, Shitan N, Sawada K, Van Montagu MCE, Inze D, Rischer H, et al.Vacuolar transport of nicotine is mediated by a multidrug and toxiccompound extrusion (MATE) transporter in Nicotiana tabacum. Proc NatlAcad Sci. 2009;106(7):2447–52.

54. Wang R, Liu X, Liang S, Ge Q, Li Y, Shao J, et al. A subgroup of MATEtransporter genes regulates hypocotyl cell elongation in Arabidopsis. J ExpBot. 2015;66(20):6327–43.

55. Burko Y, Geva Y, Refael-Cohen A, Shleizer-Burko S, Shani E, Berger Y, et al.From Organelle to Organ: ZRIZI MATE-Type Transporter is an OrganelleTransporter that Enhances Organ Initiation. Plant Cell Physiol. 2011;52(3):518–27.

56. Saeed AI, Bhagabati NK, Braisted JC, Liang W, Sharov V, Howe EA, et al. TM4microarray software suite. Method Enzymol. 2006;411:134–93.

57. Grant D, Nelson RT, Cannon SB, Shoemaker RC. SoyBase, the USDA-ARSsoybean genetics and genomics database. Nucleic Acids Res. 2010;38(Database issue):D843–6.

58. Narusaka Y, Nakashima K, Shinwari ZK, Sakuma Y, Furihata T, Abe H, et al.Interaction between two cis‐acting elements, ABRE and DRE, in ABA‐dependent expression of Arabidopsis rd29A gene in response todehydration and high‐salinity stresses. Plant J. 2003;34(2):137–48.

59. Walker JC, Howard EA, Dennis ES, Peacock WJ. DNA sequences required foranaerobic expression of the maize alcohol dehydrogenase 1 gene. Proc NatlAcad Sci. 1987;84(19):6624–8.

60. Larkindale J, Vierling E. Core genome responses involved in acclimation tohigh temperature. Plant Physiol. 2008;146(2):748–61.

61. Shukla PS, Agarwal P, Gupta K, Agarwal PK. Molecular characterisation of aMYB transcription factor from a succulent halophyte involved in stresstolerance. AoB plants. 2015;7:plv054.

62. Brown A, Dunn M, Goddard N, Hughes M. Identification of a novel low-temperature-response element in the promoter of the barley (Hordeumvulgare L) gene blt101. 1. Planta. 2001;213(5):770–80.

63. Mou S, Liu Z, Guan D, Qiu A, Lai Y, He S. Functional analysis andexpressional characterization of rice ankyrin repeat-containing protein, OsPIANK1, in basal defense against Magnaporthe oryzae attack. PLoS One.2013;8:e59699.

64. Sazegari S, Niazi A, Ahmadi FS. A study on the regulatory network withpromoter analysis for Arabidopsis DREB-genes. Bioinformation. 2015;11(2):101.

65. Tsutsui T, Yamaji N, Feng MJ. Identification of a cis-acting element of ART1,a C2H2-type zinc-finger transcription factor for aluminum tolerance in rice.Plant Physiol. 2011;156(2):925–31.

66. Ma JF, Nagao S, Sato K, Ito H, Furukawa J, Takeda K. Molecular mapping of agene responsible for Al‐activated secretion of citrate in barley. J Exp Bot.2004;55(401):1335–41.

67. Famoso AN, Zhao K, Clark RT, Tung C-W, Wright MH, Bustamante C, et al.Genetic architecture of aluminum tolerance in rice (Oryza sativa)determined through genome-wide association analysis and QTL mapping.PLoS Genet. 2011;7(8):e1002221. doi:10.1371/journal.pgen.1002221.

68. You J, Zhang H, Liu N, Gao L, Kong L, Yang Z. Transcriptomic responses toaluminum stress in soybean roots. Genome. 2011;54(11):923–33.

69. Finn RD, Bateman A, Clements J, Coggill P, Eberhardt RY, Eddy SR, et al.Pfam: the protein families database. Nucleic Acids Res. 2014;42(Databaseissue):D222–30.

70. Letunic I, Doerks T, Bork P. SMART: recent updates, new developments andstatus in 2015. Nucleic Acids Res. 2015;43(D1):D257–60.

71. Bjellqvist B, Hughes GJ, Pasquali C, Paquet N, Ravier F, Sanchez JC, et al. Thefocusing positions of polypeptides in immobilized pH gradients can bepredicted from their amino acid sequences. Electrophoresis. 1993;14(10):1023–31.

72. Bjellqvist B, Basse B, Olsen E, Celis JE. Reference points for comparisons oftwo-dimensional maps of proteins from different human cell types definedin a pH scale where isoelectric points correlate with polypeptidecompositions. Electrophoresis. 1994;15(3–4):529–39.

73. Wilkins MR, Gasteiger E, Bairoch A, Sanchez JC, Williams KL, Appel RD, et al.Protein identification and analysis tools in the ExPASy Server. Methods MolBiol. 1999;112:531–52.

74. Horton P, Park KJ, Obayashi T, Fujita N, Harada H, Adams-Collier CJ, et al.WoLF PSORT: protein localization predictor. Nucleic Acids Res. 2007;35(WebServer issue):W585–7.

75. Krogh A, Larsson B, Von Heijne G, Sonnhammer E. Predictingtransmembrane protein topology with a hidden Markov model: applicationto complete genomes. J Mol Biol. 2001;305(3):567–80.

76. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecularevolutionary genetics analysis version 6.0. Mol Biol Evol. 2013;30(12):2725–9.

77. Hu B, Jin JP, Guo AY, Zhang H, Luo JC, Gao G. GSDS 2.0: an upgraded genefeature visualization server. Bioinformatics. 2015;31:1296–7.

78. Bailey TL, Bodén M, Buske FA, Frith M, Grant CG, Clementi L, et al. MEMESUITE: tools for motif discovery and searching. Nucleic Acids Res. 2009;37:W202–8.

79. Voorrips R. MapChart: software for the graphical presentation of linkagemaps and QTLs. J Hered. 2002;93(1):77–8.

80. Wang Y, Tang H, DeBarry JD, Tan X, Li J, Wang X, et al. MCScanX: a toolkitfor detection and evolutionary analysis of gene synteny and collinearity.Nucleic Acids Res. 2012;40(7):e49. doi:10.1093/nar/gkr1293.

81. Lee T-H, Tang H, Wang X, Paterson AH. PGDD: a database of gene andgenome duplication in plants. Nucleic Acids Res. 2013;41(D1):D1152–8.

82. Gupta S, Garg V, Kant C, Bhatia S. Genome-wide survey and expressionanalysis of F-box genes in chickpea. BMC Genomics. 2015;16(1):67.doi:10.1186/s12864-015-1293-y.

83. Lescot M, Dehais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y, et al.PlantCARE, a database of plant cis-acting regulatory elements and a portalto tools for in silico analysis of promoter sequences. Nucleic Acids Res.2002;30(1):325–7.

84. Libault M, Farmer A, Joshi T, Takahashi K, Langley RJ, Franklin LD, et al. Anintegrated transcriptome atlas of the crop model Glycine max, and its usein comparative analyses in plants. Plant J. 2010;63(1):86–99.

85. Liang C, Pineros MA, Tian J, Yao Z, Sun L, Liu J, et al. Low pH, aluminum,and phosphorus coordinately regulate malate exudation through GmALMT1to improve soybean adaptation to acid soils. Plant Physiol. 2013;161(3):1347–61.

86. Pfaffl MW. A new mathematical model for relative quantification in real-timeRT–PCR. Nucleic Acids Res. 2001;29(9):e45.

87. Livak KJ, Schmittgen TD. Analysis of relative gene expression data usingreal-time quantitative PCR and the 2− ΔΔCT method. Methods. 2001;25(4):402–8.

88. Liu J, Li Y. Supporting data for soybean MATE family. LabArchives. 2016.http://www.labarchives.com/. doi: 10.6070/H47M05ZF

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