BACKGROUND
multidrug and toxic compound extrusion  family is the most recent categorized multidrug efflux transporter family, which is a secondary transporter family that couples the translocation of substrates with an electrochemical gradient of cations  across the membrane  <cit> .
the x-ray structure of the mate transporter, norm from vibrio cholerae, reveals a unique topology of its predicted  <dig> transmembrane  helices, which is distinct from any other known multidrug resistance transporter  <cit> .
mate transporters are widely distributed in bacteria, fungi, mammals and plants  <cit> .
hvorup et al.
<cit>  found  <dig> sequenced proteins in the mate family, which could be divided into  <dig> subfamilies.
omote et al.
<cit>  identified  <dig> mate transporters from archaea, eubacteria and eukarya and classified them into three large subfamilies comprising  <dig> smaller subgroups.
there are  <dig>  over  <dig> and  <dig> putative mate transporters in arabidopsis thaliana  <cit> , medicago truncatula  <cit>  and oryza sativa  <cit> , respectively.
the bacterial mate transporters can export organic cations for multidrug resistance  <cit> .
in yeast , the erc <dig> mate-type transporter confers resistance to ethionine  <cit> .
the mammalian mate transporters can excrete the metabolic waste and xenobiotic organic cations in the kidney and liver  <cit> .
the plant mate family transports a broad range of substrates such as organic acids, plant hormones and secondary metabolites .
recently, the functions of many mate transporters have been illustrated in plants  <cit> , including transport of secondary metabolites such as alkaloids  <cit> , flavonoids  <cit> , and anthocyanidin , detoxification of toxic compounds or heavy metals  <cit> , regulation of disease resistance , efflux of plant hormones such as abscisic acid   <cit> , iron translocation  and aluminum  detoxification , which indicates mate transporters play important roles in a wide range of biological processes in plants.
al toxicity is considered as the main factor limiting crop yield on acidic soils  <cit> .
under al stress, root exudation of organic acids, such as malate, citrate, and oxalate, is an important mechanism in plant resistance to al toxicity  <cit> .
the genes controlling organic anion efflux from roots have been isolated from several crop species  <cit> .
mate transporters have been shown to mediate the citrate efflux to confer plant tolerance to al toxicity  <cit> .
the mate transporters involved in detoxification of al were first identified from sorghum  and barley  by map-based cloning, respectively  <cit> .
later study found that the function of hvaact <dig> protein is to release citrate to facilitate the translocation of iron from roots to shoots, and the 1-kb insertion in the upstream of the hvaact <dig> coding region in the al-tolerant barley variety enhances and alters its expression to root tips, which is important to detoxifying al and adaptation to acidic soils in barley  <cit> .
overexpression of hvaact <dig> increases citrate efflux and al tolerance in wheat and barley  <cit> .
bomate from cabbage  requires al3+ to activate citrate efflux, leading to enhanced al tolerance in a. thaliana  <cit> .
several other mate transporters, such as ecmate <dig> , osfrdl <dig> , and zmmate <dig> , are found localized to plasma membranes in the root tips and related to plant tolerance to al toxicity .
compared with other plant species, little work on mate transporters has been done in soybean  merr.
), which is an important oil crop worldwide.
to date, only one mate transporter, gmfrd3b , was reported to play a role in iron efficiency in soybean  <cit> .
with the public available whole genome sequence  <cit>  and rna-seq atlas  <cit>  of g. max, it is possible to identify the genome-wide mate genes in soybean and investigate their expression patterns and possible functions.
plant mate transporters have been shown to be involved in diverse functions including al tolerance.
by comparing the sequences of soybean mate family with the known mate transporters from other plant species, the possible roles of soybean mate transporters could be proposed and help us to further test their function.
in this study, we performed a genome-wide search of all putative mate transporters in soybean.
their chromosomal distribution, gene duplication, phylogenetic relationship, structures of genes and proteins, and expression patterns were analyzed.
soybean mate genes related to al detoxification/iron translocation were further investigated by promoter analysis and differential gene expression analysis between the root tips of al-tolerant and al-sensitive soybean cultivars in response to al.
this study would provide useful information on the research of mate transporters in soybean.
RESULTS
genome-wide identification of soybean mate transporters
a total of  <dig> genes encoding mate transporters  were identified from the soybean whole genome , which were denominated as gmmate <dig> - gmmate <dig> according to the soybean nomenclature based on their physical locations  <cit> .
the genomic sequences, coding sequences, and protein sequences of these  <dig> soybean mate members  were downloaded from phytozome   <cit> .
the details of all  <dig> soybean mate proteins, including the length, molecular weight, number of tm, isoelectric point , and predicted subcellular location, are listed in additional file 3: table s <dig>  the soybean mate proteins consist of  <dig> to  <dig> amino acids, containing  <dig> to  <dig> tms, whereas in arabidopsis, the lengths of mate proteins range from  <dig> to  <dig> amino acids and most with  <dig> tms  <cit> , indicating there are more variations within the soybean mate family.
the predicted molecular weights of soybean mate proteins range from  <dig>  to  <dig>  kda, and the predicted pi values are between  <dig>  and  <dig> .
their predicted subcellular locations include plasma membrane, chloroplast, cytoplasm, vacuole, endoplasmic reticulum, and extracellular, with  <dig>  %  located in the plasma membrane,  <dig>  %  located in chloroplast,  <dig>  % ,  <dig>  % ,  <dig>  %  and  <dig>  % located in cytoplasm, vacuole, endoplasmic reticulum, and extracellular, respectively.
chromosomal locations and duplication patterns of soybean mate genes
based on the physical positions , these  <dig> gmmate genes are unevenly distributed on  <dig> soybean chromosomes .
the number of gmmate genes on chromosomes  <dig> to  <dig> ranges from  <dig> to  <dig>  chromosome nine contains the highest number of gmmate genes , whereas chromosomes  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig> contain fewest gmmate genes .
majority of these gmmate genes are located on the chromosome arms , which are associated with high rates of recombination  <cit> .fig.
<dig> chromosomal locations and duplications of soybean mate genes.
the chromosome number is indicated above each bar and the scale on the left is in megabases .
the chromosome size is indicated by its relative length using the information from phytozome and soybase.
tandemly duplicated genes are shown in purple boxes.
each pair of segmental duplication is indicated by a green line
compared with the number of mate genes in a. thaliana  <cit> , m. truncatula  <cit> , and o. sativa  <cit> , which contains  <dig>  over  <dig>  and  <dig> mate genes, respectively, mate family in soybean is remarkably large with  <dig> members, which might result from two whole-genome duplication events in soybean  <cit> .
we further investigated the duplication patterns of soybean mate family.
the duplication analyses showed  <dig> out of  <dig> gmmate genes  were present in duplications , which indicates duplications contributed largely to the amplification of mate family in the soybean genome, supporting the previous observation that duplications play important roles in the evolution of large gene families  <cit> .
we observed  <dig>  gmmate genes with tandem duplications , and  <dig>  gmmate genes with segmental duplications .
duplication events of gmmate genes were found on all  <dig> soybean chromosomes.
phylogenetic analyses of the soybean mate family
there are  <dig> plant mate transporters that have been reported previously , including mate transporters with known functions and few sequences that were reported by zhao et al.
<cit> .
using the full-length protein sequences of the  <dig> mate transporters, including the  <dig> previously reported plant mate proteins and  <dig> soybean mate proteins, we constructed a maximum likelihood  tree .
these mate proteins could be classified into four primary clades  comprising ten smaller subgroups , including subfamily c <dig> , subfamily c <dig> , subfamily c <dig>  and subfamily c <dig> .
the functions of soybean mate proteins could be inferred from the known mate transporters according to their phylogenetic relationships.fig.
<dig> the phylogenetic tree of soybean mate family.
the phylogenetic tree was constructed by mega  <dig>  using the maximum likelihood  method.
bootstrap values in percentage  are indicated on the nodes.
different subfamilies are highlighted using different colors , and subgroups are marked with black arcs outside of the cycle tree
subfamily c <dig> contains three subgroups, c1- <dig>  c1- <dig> and c1- <dig>  c1- <dig> consists of  <dig> sequences, including  <dig> mate proteins from soybean and eight previously reported mate transporters, such as atfft   <cit> , slmate   <cit> , vvam <dig> and vvam <dig>   <cit> .
atfft is a flavonoid transporter that affects flavonoid levels in arabidopsis  <cit> .
slmate encodes a putative anthocyanin permease, which is co-regulated with ant <dig>  transcription factor, indicating it may function as an anthocyanin vacuolar transporter in tomato leaves  <cit> .
vvam <dig> and vvam <dig> were found to be involved in the transport of acylated anthocyanins into vacuoles in grapevine  <cit> .
there are  <dig> soybean mate proteins in the c1- <dig> subgroup, with no previously known mate proteins.
there are  <dig> proteins in the c1- <dig> subgroup, including five soybean mate proteins and six previously reported mate transporters such as attt <dig>   <cit> , mtmate <dig>   <cit> , ntmate <dig> and ntmate <dig>   <cit> , and vvmate <dig>   <cit> .
arabidopsis tt <dig>  the first mate transporter found to transport flavonoids  <cit> , was originally isolated during screening of mutants with altered seed coloration.
mtmate <dig> from m. truncatula was a functional ortholog of attt <dig> and localized in the tonoplast  <cit> .
ntmate <dig> and ntmate <dig> were suggested to transport alkaloids from the cytosol into the vacuole in tobacco  <cit> .
the grapevine mate transporter vvmate <dig> was involved in the accumulation of proanthocyanidins  <cit> .
the functions of the known mate transporters in this clade suggest the mate subfamily c <dig> might be involved in  transport and accumulation of flavonoids or alkaloids in plants.
there are  <dig> soybean mate proteins in subfamily c <dig>  which are divided into two subgroups.
subgroup c2- <dig> has  <dig> soybean mate proteins and a known mate protein atalf <dig> , in which mutation led to defects in lateral root formation and increased sensitivity of roots to various compounds, therefore it is thought to confer plant resistance to toxins  <cit> .
subgroup c2- <dig> contains  <dig> soybean mate members, as well as atdtx <dig>  and ntjat <dig>   <cit> .
atdtx <dig> was found to mediate the efflux of plant-derived antibiotics and other toxic compounds, and was also able to detoxify the heavy metal, cd2+  <cit> .
tobacco ntjat <dig> showed nicotine efflux activity in yeast and was suggested to function as a secondary transporter for nicotine translocation  <cit> .
therefore, subfamily c <dig> might be related to the efflux of various compounds.
subfamily c <dig> could be further classified into two subgroups.
none of the known mate proteins appears in subgroup c3- <dig> .
subgroup c3- <dig> contains  <dig> soybean mate proteins and three known mate proteins, atads <dig>   <cit> , atdtx <dig>   <cit> , and atzf <dig>   <cit> .
previous research indicated that atads1/abs <dig> is a negative regulator of plant disease resistance  <cit> .
atdtx <dig> functions as an aba efflux transporter, which regulates aba sensitivity, stomatal conductance and drought tolerance in arabidopsis  <cit> .
overexpression of atzf14/bcd1/abs <dig> increased leaf initiation rate and it is also involved in iron homeostasis  <cit> .
recently, atzf14/bcd1/abs <dig> and atads1/abs <dig> were also found to regulate hypocotyl cell elongation  <cit> .
the functions of mate subfamily c <dig> seem diversified and need further investigation.
there are three subgroups in subfamily c <dig>  subgroup c4- <dig> only contains one soybean mate protein.
subgroup c4- <dig> has six soybean mate proteins and ateds <dig>  which is essential for salicylic acid  dependent disease resistance  <cit> .
subgroup c4- <dig> contains  <dig> members, including eight soybean mate proteins , and  <dig> previously reported mate proteins from other plant species that are all related to al detoxification and/or iron translocation , indicating these eight soybean mate proteins in subgroup c4- <dig> might be involved in al detoxification/iron translocation in soybean.
gene structures and protein motifs of soybean mate family
in order to better understand the characteristics of the soybean mate genes, their structures were analyzed by comparing the genomic dna sequences with their corresponding coding sequences .
their intron-exon structures were plotted along with the order of subfamily in phylogenetic tree .
the gmmate gene structures including length and number of exons and introns are more similar within the same subfamily .
most genes in the largest subfamily c <dig> have 5– <dig> exons, except that gmmate <dig>  gmmate <dig> and gmmate <dig> have only 1– <dig> exons.
most genes in subfamily c <dig> contain 6– <dig> exons, except for gmmate <dig>  gmmate <dig> and gmmate <dig>  which only have 1– <dig> exons.
the gmmate genes in subfamily c <dig> contain only 1– <dig> exons.
the gmmate genes in subfamily c <dig> have 9– <dig> exons.fig.
<dig> the gene structures of soybean mate family.
the structures of  <dig> gmmate genes were plotted using green boxes representing exons , black lines representing introns and blue boxes indicating upstream/downstream sequences.
the scale on the bottom is in the unit of kilobase .
the genes are listed according to the order of subfamily c <dig> to c <dig> from the phylogenetic tree, and different subfamilies are highlighted in different colors : c <dig> in green, c <dig> in pink, c <dig> in blue and c <dig> in gray
next, motifs in soybean mate protein sequences were identified using meme .
the types and sequences of the motifs are similar among the first three subfamilies, c <dig>  c <dig> and c <dig>  but significant different with the fourth subfamily c <dig> .
the mate proteins in subfamily c <dig> generally have fewer motifs than the first three subfamilies.fig.
<dig> protein motifs of soybean mate family.
the motifs of soybean mate proteins are shown as colored boxes.
the scale on the bottom may be used to estimate the length of motif .
the gmmate proteins are listed according to the order of subfamily c <dig> to c <dig> from the phylogenetic tree, and different subfamilies are highlighted in different colors : c <dig> in green, c <dig> in pink, c <dig> in blue and c <dig> in gray
expression patterns of gmmate genes in different soybean tissues
the relative transcript abundance of the  <dig> gmmate genes in different soybean tissues was searched from phytozome v <dig>   <cit> .
there are four genes having the relative transcript abundance as  <dig> across all tested tissues and therefore were excluded for further analysis .
a heat map with clustering of the rest  <dig> gmmate genes  was constructed using mev software  <cit> .
most gmmate genes showed specific tissue expression patterns.
some genes  showed higher expression levels in root/root hair/nodule while lower levels in above-ground tissues .
some gmmate genes such as gmmate <dig>  gmmate <dig> and gmmate <dig> were mainly expressed in pod and developing seed, suggesting their putative roles during seed development.
some gmmate genes  showed higher expression levels in flower and pod.
the expression levels of some genes  were higher in leaf but lower  in other tissues.
there are also some genes  expressed in all tested tissues .
the relative transcript abundance of the  <dig> gmmate genes was also searched from the soybean rna-seq atlas  <cit>  in soybase  <cit> .
there are  <dig> gmmate genes have no data and  <dig> gmmate genes have the relative transcript abundance as  <dig> across all tested tissues .
the tissue expression patterns of most genes with non-zero relative transcript abundance from soybase were consistent with the phytozome.fig.
<dig> heat map of the expression profiles of gmmate genes in nine soybean tissues.
the heat map with hierarchical clustering of  <dig> gmmate genes was constructed using mev  <dig>  software by average linkage with euclidean distance.
color key represents the relative transcript abundance of the gmmate genes in nine soybean tissues.
the fpkm  values were log <dig> transformed and mean centred by genes using the mev  <dig>  software.
sam: shoot apical meristem
to cross-validate the expression patterns of gmmate genes with the rna-seq data, we performed quantitative real-time pcr  for eight representative genes randomly selected from the four subfamilies .
the relative expression of three genes  from subfamily c <dig> is higher in flower, leaf, pod, and shoot apical meristem  than root tip, which is consistent with fig.
<dig>  gmmate <dig> from subfamily c <dig> expresses in all tested tissues by qrt-pcr, which in general agrees with the rna-seq data.
gmmate <dig> and gmmate <dig> from subfamily c <dig> show high relative expression level in flower in both qrt-pcr and rna-seq data.
gmmate <dig> and gmmate <dig> from subfamily c <dig> express higher in leaf or pod by qrt-pcr but show higher expression in root hairs and nodules in rna-seq data, which might be due to the difference in the sensitivities of two methods, rna samples from two different cultivars , or different growing environments.fig.
<dig> relative expression levels of the representative gmmate genes in seven soybean tissues.
eight gmmate genes representing four subfamilies were randomly selected to validate their relative expression in different tissues by qrt-pcr.
the relative expression level in stem was set as one and the soybean gmef-1α gene was used as the internal control.
the error bars indicate the standard deviation from three replicates
characterization of putative cis-regulatory elements in the promoter regions of subgroup c4- <dig> gmmate genes
based on the phylogenetic tree, eight gmmate genes were classified into subgroup c4- <dig> , together with the previously reported genes that are all related to al detoxification and/or iron translocation.
the cis-acting regulatory elements in the promoter regions play important roles in plant response to stresses.
using the plantcare database, we identified  <dig> putative stress or hormone-responsive cis-acting elements in the 1500 bp upstream  of these eight gmmate genes , including abre , are , cgtca-motif, hse , ltr , mbs , tca-element, tc rich-repeats, tgacg-motif, wun  and w1-box.
gmmate <dig> contains only one cis-acting element , while the other seven gmmate genes have more than one predicted cis-acting elements.
two genes, gmmate <dig> and gmmate <dig>  contain ten and  <dig> cis-elements, respectively .
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 plant response to drought and high salinity in arabidopsis  <cit> , which is present in two c4- <dig> gmmate genes.
are element was found both necessary and sufficient for induction of gene expression by low oxygen stress  <cit> , which is present in four c4- <dig> gmmate genes.
hse has been found to be consistently conserved in the regulatory regions of many heat induced genes  <cit> , which is present in five c4- <dig> gmmate genes.
mbs cis-element was reported to bind to myb transcriptional factors involved in stress signaling  <cit> , and five c4- <dig> gmmate genes contain mbs.
ltr element is important for the induction of cold regulated genes  <cit> , which is present in two c4- <dig> gmmate genes.
tca-element mediates sa-signaling pathway and is sufficient for the response to stress  <cit> , which is found in two c4- <dig> gmmate genes.
tc-rich repeats element is involved in defense and stress-responsiveness  <cit> , which is present in three c4- <dig> gmmate genes.fig.
<dig> stress-responsive cis-elements of the eight gmmate genes in subgroup c4- <dig>  the phylogenetic tree  on the left was constructed using mega  <dig>  with the full sequences of the eight soybean mate proteins in subgroup c4- <dig>  the cis-elements in the 1500 bp upstream regions of the eight corresponding gmmate genes were predicted using the plantcare database, and shown in colored boxes with their names and positions  inside
another cis-acting element, ggnvs, has been identified as the dna-binding sequence of art <dig> , which regulates the expression of  <dig> genes  to confer al tolerance in rice  <cit> .
this element, ggnvs, was found in all eight c4- <dig> gmmate genes, with different numbers and positions .
gmmate <dig>  gmmate <dig>  gmmate <dig> and gmmate <dig> contain more ten ggnvs elements, while only five ggnvs element was found in gmmate <dig> table  <dig> number and position of ggnvs element in the promoter region of eight c4- <dig> soybean mate genesa
gmmate13
gmmate47
gmmate58
gmmate74
gmmate75
gmmate79
gmmate84
gmmate87
aggnvs is the abbreviation for ggnvs, a cis-acting element  of art <dig>
bthe position is relative to the start codon of each gene
expression of the subgroup c4- <dig> gmmate genes in response to al toxicity
ten out of  <dig>  mate transporters from other plant species in subgroup c4- <dig>  have been shown related to al detoxification, therefore the expression patterns of all eight subgroup c4- <dig> gmmate genes in response to al toxicity were analyzed by qrt-pcr.
intraspecific variation in al tolerance is striking in many crop species  <cit> .
previous studies showed that al-tolerance related mate gene expression in plant root tips is up-regulated by al and is significantly higher in al-tolerant genotypes  <cit> .
in this study, two soybean cultivars, kf  and gf , were treated with  <dig> and 25 μm alcl <dig> for 6 h, 12 h and 24 h, respectively.
the relative expression levels of the eight c4- <dig> gmmate genes in the root tips of soybean seedlings after al stress treatment are shown in fig.
<dig>  among these eight genes, only one gene, gmmate <dig>  showed a significantly higher relative gene expression in the al-tolerant cultivar kf  than in al-sensitive cultivar gf  at  <dig>   <dig>  and 24 h after al stress treatment.
gmmate <dig> showed a significant higher relative expression in kf  at 6 h but significant higher in gf  at 24 h after al stress treatment.
gmmate <dig>  gmmate <dig>  and gmmate <dig> showed a significantly higher level of relative expression in gf  than kf  after al stress treatment.
gmmate <dig>  gmmate <dig> and gmmate <dig> did not show significant difference in their relative expression between gf  and kf  during al stress treatment.
therefore, gmmate <dig>  which showed higher elevated transcript levels after al treatment in al-tolerant  than al-sensitive  cultivar, would be a candidate gene for soybean tolerance to al toxicity.
the expression patterns of gmmate <dig> could also be visualized by semi-quantitative rt-pcr .
under normal growth conditions without al treatment, their expression levels were very low and no difference was observed between kf  and gf .
however, under al treatment, their expression levels increased significantly, which was consistent with the qrt-pcr results .
a previous microarray study reported gma.
<dig> .a1_at, a putative mate gene, was up-regulated  by al treatment in an al-tolerant soybean variety jiyu  <dig>  <cit> , which is the same gene as gmmate <dig> we identified in this study.
in our study, the relative expression of gmmate <dig> under al stress was further compared between al-tolerant and al-sensitive varieties.
gmmate <dig> was highly up-regulated by  <dig>   <dig>  and 335-fold in kf  while  <dig>   <dig>  and 33-fold in gf  after  <dig>   <dig>  and 24 h al treatment, respectively, suggesting its role in soybean tolerance to al stress.fig.
<dig> relative expression of the eight soybean c4- <dig> mate genes in response to al toxicity.
the relative expression of the eight gmmate genes in the soybean root tips  in response to al stress  was quantified by qrt-pcr, using soybean gmef-1α gene as the internal control and 0 μm alcl <dig>  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 al stress for each time point  at p <  <dig>  and p <  <dig> , respectively
CONCLUSIONS
a comprehensive genome-wide analysis of mate family is performed in an important legume species and oil crop, soybean.
a total of  <dig> mate transporters were identified in soybean and could be classified into four subfamilies, c <dig>  c <dig>  c <dig> and c <dig>  the soybean mate family displays great variation in gene structure, protein motif, and tissue expression pattern, which indicates their diverse functions.
the expansion of soybean mate family was largely due to segmental duplications.
seven stress-responsive cis-acting elements and the cis-acting element of art <dig>  were identified in the upstream regions of eight gmmate genes in subgroup c4- <dig>  which contains previously reported mate genes related to al detoxification and/or iron translocation.
one gene from c4- <dig> subgroup, gmmate <dig>  showed differential relative transcript abundance between the root tips of al-tolerant and al-sensitive soybean cultivars in response to al treatment, indicating its potential role in soybean tolerance to al toxicity.
this study provides a foundation to further investigate the functions of soybean mate genes including the candidate gene for al tolerance in soybean.

 multidrug and toxic compound extrusion family is the most recent categorized multidrug efflux transporter family, which is a secondary transporter family that couples the translocation of substrates with an electrochemical gradient of cations across the membrane. the x-ray structure of the mate transporter, norm from vibrio cholerae, reveals a unique topology of its predicted transmembrane helices, which is distinct from any other known multidrug resistance transporter. mate transporters are widely distributed in bacteria, fungi, mammals and plants. hvorup et al. found sequenced proteins in the mate family, which could be divided into subfamilies. omote et al. identified mate transporters from archaea, eubacteria and eukarya and classified them into three large subfamilies comprising smaller subgroups. there are over and putative mate transporters in arabidopsis thaliana, medicago truncatula and oryza sativa, respectively. the bacterial mate transporters can export organic cations for multidrug resistance. in yeast, the erc mate-type transporter confers resistance to ethionine. the mammalian mate transporters can excrete the metabolic waste and xenobiotic organic cations in the kidney and liver. the plant mate family transports a broad range of substrates such as organic acids, plant hormones and secondary metabolites. recently, the functions of many mate transporters have been illustrated in plants, including transport of secondary metabolites such as alkaloids, flavonoids, and anthocyanidin, detoxification of toxic compounds or heavy metals, regulation of disease resistance, efflux of plant hormones such as abscisic acid, iron translocation and aluminum detoxification, which indicates mate transporters play important roles in a wide range of biological processes in plants. al toxicity is considered as the main factor limiting crop yield on acidic soils. under al stress, root exudation of organic acids, such as malate, citrate, and oxalate, is an important mechanism in plant resistance to al toxicity. the genes controlling organic anion efflux from roots have been isolated from several crop species. mate transporters have been shown to mediate the citrate efflux to confer plant tolerance to al toxicity. the mate transporters involved in detoxification of al were first identified from sorghum and barley by map-based cloning, respectively. later study found that the function of hvaact protein is to release citrate to facilitate the translocation of iron from roots to shoots, and the 1-kb insertion in the upstream of the hvaact coding region in the al-tolerant barley variety enhances and alters its expression to root tips, which is important to detoxifying al and adaptation to acidic soils in barley. overexpression of hvaact increases citrate efflux and al tolerance in wheat and barley. bomate from cabbage requires al3+ to activate citrate efflux, leading to enhanced al tolerance in a. thaliana. several other mate transporters, such as ecmate, osfrdl, and zmmate, are found localized to plasma membranes in the root tips and related to plant tolerance to al toxicity. compared with other plant species, little work on mate transporters has been done in soybean merr.), which is an important oil crop worldwide. to date, only one mate transporter, gmfrd3b, was reported to play a role in iron efficiency in soybean. with the public available whole genome sequence and rna-seq atlas of g. max, it is possible to identify the genome-wide mate genes in soybean and investigate their expression patterns and possible functions. plant mate transporters have been shown to be involved in diverse functions including al tolerance. by comparing the sequences of soybean mate family with the known mate transporters from other plant species, the possible roles of soybean mate transporters could be proposed and help us to further test their function. in this study, we performed a genome-wide search of all putative mate transporters in soybean. their chromosomal distribution, gene duplication, phylogenetic relationship, structures of genes and proteins, and expression patterns were analyzed. soybean mate genes related to al detoxification/iron translocation were further investigated by promoter analysis and differential gene expression analysis between the root tips of al-tolerant and al-sensitive soybean cultivars in response to al. this study would provide useful information on the research of mate transporters in soybean.  genome-wide identification of soybean mate transporters a total of genes encoding mate transporters were identified from the soybean whole genome, which were denominated as gmmate - gmmate according to the soybean nomenclature based on their physical locations. the genomic sequences, coding sequences, and protein sequences of these soybean mate members were downloaded from phytozome. the details of all soybean mate proteins, including the length, molecular weight, number of tm, isoelectric point, and predicted subcellular location, are listed in additional file 3: table s the soybean mate proteins consist of to amino acids, containing to tms, whereas in arabidopsis, the lengths of mate proteins range from to amino acids and most with tms, indicating there are more variations within the soybean mate family. the predicted molecular weights of soybean mate proteins range from to  kda, and the predicted pi values are between and. their predicted subcellular locations include plasma membrane, chloroplast, cytoplasm, vacuole, endoplasmic reticulum, and extracellular, with  % located in the plasma membrane,  % located in chloroplast,  %,  %,  % and  % located in cytoplasm, vacuole, endoplasmic reticulum, and extracellular, respectively. chromosomal locations and duplication patterns of soybean mate genes based on the physical positions, these gmmate genes are unevenly distributed on soybean chromosomes. the number of gmmate genes on chromosomes to ranges from to chromosome nine contains the highest number of gmmate genes, whereas chromosomes and contain fewest gmmate genes. majority of these gmmate genes are located on the chromosome arms, which are associated with high rates of recombination.fig. chromosomal locations and duplications of soybean mate genes. the chromosome number is indicated above each bar and the scale on the left is in megabases. the chromosome size is indicated by its relative length using the information from phytozome and soybase. tandemly duplicated genes are shown in purple boxes. each pair of segmental duplication is indicated by a green line compared with the number of mate genes in a. thaliana, m. truncatula, and o. sativa, which contains over and mate genes, respectively, mate family in soybean is remarkably large with members, which might result from two whole-genome duplication events in soybean. we further investigated the duplication patterns of soybean mate family. the duplication analyses showed out of gmmate genes were present in duplications, which indicates duplications contributed largely to the amplification of mate family in the soybean genome, supporting the previous observation that duplications play important roles in the evolution of large gene families. we observed gmmate genes with tandem duplications, and gmmate genes with segmental duplications. duplication events of gmmate genes were found on all soybean chromosomes. phylogenetic analyses of the soybean mate family there are plant mate transporters that have been reported previously, including mate transporters with known functions and few sequences that were reported by zhao et al.. using the full-length protein sequences of the mate transporters, including the previously reported plant mate proteins and soybean mate proteins, we constructed a maximum likelihood tree. these mate proteins could be classified into four primary clades comprising ten smaller subgroups, including subfamily c, subfamily c, subfamily c and subfamily c. the functions of soybean mate proteins could be inferred from the known mate transporters according to their phylogenetic relationships.fig. the phylogenetic tree of soybean mate family. the phylogenetic tree was constructed by mega using the maximum likelihood method. bootstrap values in percentage are indicated on the nodes. different subfamilies are highlighted using different colors, and subgroups are marked with black arcs outside of the cycle tree subfamily c contains three subgroups, c1- c1- and c1- c1- consists of sequences, including mate proteins from soybean and eight previously reported mate transporters, such as atfft, slmate, vvam and vvam. atfft is a flavonoid transporter that affects flavonoid levels in arabidopsis. slmate encodes a putative anthocyanin permease, which is co-regulated with ant transcription factor, indicating it may function as an anthocyanin vacuolar transporter in tomato leaves. vvam and vvam were found to be involved in the transport of acylated anthocyanins into vacuoles in grapevine. there are soybean mate proteins in the c1- subgroup, with no previously known mate proteins. there are proteins in the c1- subgroup, including five soybean mate proteins and six previously reported mate transporters such as attt, mtmate, ntmate and ntmate, and vvmate. arabidopsis tt the first mate transporter found to transport flavonoids, was originally isolated during screening of mutants with altered seed coloration. mtmate from m. truncatula was a functional ortholog of attt and localized in the tonoplast. ntmate and ntmate were suggested to transport alkaloids from the cytosol into the vacuole in tobacco. the grapevine mate transporter vvmate was involved in the accumulation of proanthocyanidins. the functions of the known mate transporters in this clade suggest the mate subfamily c might be involved in transport and accumulation of flavonoids or alkaloids in plants. there are soybean mate proteins in subfamily c which are divided into two subgroups. subgroup c2- has soybean mate proteins and a known mate protein atalf, in which mutation led to defects in lateral root formation and increased sensitivity of roots to various compounds, therefore it is thought to confer plant resistance to toxins. subgroup c2- contains soybean mate members, as well as atdtx and ntjat. atdtx was found to mediate the efflux of plant-derived antibiotics and other toxic compounds, and was also able to detoxify the heavy metal, cd2+. tobacco ntjat showed nicotine efflux activity in yeast and was suggested to function as a secondary transporter for nicotine translocation. therefore, subfamily c might be related to the efflux of various compounds. subfamily c could be further classified into two subgroups. none of the known mate proteins appears in subgroup c3-. subgroup c3- contains soybean mate proteins and three known mate proteins, atads, atdtx, and atzf. previous research indicated that atads1/abs is a negative regulator of plant disease resistance. atdtx functions as an aba efflux transporter, which regulates aba sensitivity, stomatal conductance and drought tolerance in arabidopsis. overexpression of atzf14/bcd1/abs increased leaf initiation rate and it is also involved in iron homeostasis. recently, atzf14/bcd1/abs and atads1/abs were also found to regulate hypocotyl cell elongation. the functions of mate subfamily c seem diversified and need further investigation. there are three subgroups in subfamily c subgroup c4- only contains one soybean mate protein. subgroup c4- has six soybean mate proteins and ateds which is essential for salicylic acid dependent disease resistance. subgroup c4- contains members, including eight soybean mate proteins, and previously reported mate proteins from other plant species that are all related to al detoxification and/or iron translocation, indicating these eight soybean mate proteins in subgroup c4- might be involved in al detoxification/iron translocation in soybean. gene structures and protein motifs of soybean mate family in order to better understand the characteristics of the soybean mate genes, their structures were analyzed by comparing the genomic dna sequences with their corresponding coding sequences. their intron-exon structures were plotted along with the order of subfamily in phylogenetic tree. the gmmate gene structures including length and number of exons and introns are more similar within the same subfamily. most genes in the largest subfamily c have 5– exons, except that gmmate gmmate and gmmate have only 1– exons. most genes in subfamily c contain 6– exons, except for gmmate gmmate and gmmate which only have 1– exons. the gmmate genes in subfamily c contain only 1– exons. the gmmate genes in subfamily c have 9– exons.fig. the gene structures of soybean mate family. the structures of gmmate genes were plotted using green boxes representing exons, black lines representing introns and blue boxes indicating upstream/downstream sequences. the scale on the bottom is in the unit of kilobase. the genes are listed according to the order of subfamily c to c from the phylogenetic tree, and different subfamilies are highlighted in different colors: c in green, c in pink, c in blue and c in gray next, motifs in soybean mate protein sequences were identified using meme. the types and sequences of the motifs are similar among the first three subfamilies, c c and c but significant different with the fourth subfamily c. the mate proteins in subfamily c generally have fewer motifs than the first three subfamilies.fig. protein motifs of soybean mate family. the motifs of soybean mate proteins are shown as colored boxes. the scale on the bottom may be used to estimate the length of motif. the gmmate proteins are listed according to the order of subfamily c to c from the phylogenetic tree, and different subfamilies are highlighted in different colors: c in green, c in pink, c in blue and c in gray expression patterns of gmmate genes in different soybean tissues the relative transcript abundance of the gmmate genes in different soybean tissues was searched from phytozome v. there are four genes having the relative transcript abundance as across all tested tissues and therefore were excluded for further analysis. a heat map with clustering of the rest gmmate genes was constructed using mev software. most gmmate genes showed specific tissue expression patterns. some genes showed higher expression levels in root/root hair/nodule while lower levels in above-ground tissues. some gmmate genes such as gmmate gmmate and gmmate were mainly expressed in pod and developing seed, suggesting their putative roles during seed development. some gmmate genes showed higher expression levels in flower and pod. the expression levels of some genes were higher in leaf but lower in other tissues. there are also some genes expressed in all tested tissues. the relative transcript abundance of the gmmate genes was also searched from the soybean rna-seq atlas in soybase. there are gmmate genes have no data and gmmate genes have the relative transcript abundance as across all tested tissues. the tissue expression patterns of most genes with non-zero relative transcript abundance from soybase were consistent with the phytozome.fig. heat map of the expression profiles of gmmate genes in nine soybean tissues. the heat map with hierarchical clustering of gmmate genes was constructed using mev software by average linkage with euclidean distance. color key represents the relative transcript abundance of the gmmate genes in nine soybean tissues. the fpkm values were log transformed and mean centred by genes using the mev software. sam: shoot apical meristem to cross-validate the expression patterns of gmmate genes with the rna-seq data, we performed quantitative real-time pcr for eight representative genes randomly selected from the four subfamilies. the relative expression of three genes from subfamily c is higher in flower, leaf, pod, and shoot apical meristem than root tip, which is consistent with fig.  gmmate from subfamily c expresses in all tested tissues by qrt-pcr, which in general agrees with the rna-seq data. gmmate and gmmate from subfamily c show high relative expression level in flower in both qrt-pcr and rna-seq data. gmmate and gmmate from subfamily c express higher in leaf or pod by qrt-pcr but show higher expression in root hairs and nodules in rna-seq data, which might be due to the difference in the sensitivities of two methods, rna samples from two different cultivars, or different growing environments.fig. relative expression levels of the representative gmmate genes in seven soybean tissues. eight gmmate genes representing four subfamilies were randomly selected to validate their relative expression in different tissues by qrt-pcr. the relative expression level in stem was set as one and the soybean gmef-1α gene was used as the internal control. the error bars indicate the standard deviation from three replicates characterization of putative cis-regulatory elements in the promoter regions of subgroup c4- gmmate genes based on the phylogenetic tree, eight gmmate genes were classified into subgroup c4-, together with the previously reported genes that are all related to al detoxification and/or iron translocation. the cis-acting regulatory elements in the promoter regions play important roles in plant response to stresses. using the plantcare database, we identified putative stress or hormone-responsive cis-acting elements in the 1500 bp upstream of these eight gmmate genes, including abre, are, cgtca-motif, hse, ltr, mbs, tca-element, tc rich-repeats, tgacg-motif, wun and w1-box. gmmate contains only one cis-acting element, while the other seven gmmate genes have more than one predicted cis-acting elements. two genes, gmmate and gmmate contain ten and cis-elements, respectively. 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 plant response to drought and high salinity in arabidopsis, which is present in two c4- gmmate genes. are element was found both necessary and sufficient for induction of gene expression by low oxygen stress, which is present in four c4- gmmate genes. hse has been found to be consistently conserved in the regulatory regions of many heat induced genes, which is present in five c4- gmmate genes. mbs cis-element was reported to bind to myb transcriptional factors involved in stress signaling, and five c4- gmmate genes contain mbs. ltr element is important for the induction of cold regulated genes, which is present in two c4- gmmate genes. tca-element mediates sa-signaling pathway and is sufficient for the response to stress, which is found in two c4- gmmate genes. tc-rich repeats element is involved in defense and stress-responsiveness, which is present in three c4- gmmate genes.fig. stress-responsive cis-elements of the eight gmmate genes in subgroup c4- the phylogenetic tree on the left was constructed using mega with the full sequences of the eight soybean mate proteins in subgroup c4- the cis-elements in the 1500 bp upstream regions of the eight corresponding gmmate genes were predicted using the plantcare database, and shown in colored boxes with their names and positions inside another cis-acting element, ggnvs, has been identified as the dna-binding sequence of art, which regulates the expression of genes to confer al tolerance in rice. this element, ggnvs, was found in all eight c4- gmmate genes, with different numbers and positions. gmmate gmmate gmmate and gmmate contain more ten ggnvs elements, while only five ggnvs element was found in gmmate table number and position of ggnvs element in the promoter region of eight c4- soybean mate genesa gmmate13 gmmate47 gmmate58 gmmate74 gmmate75 gmmate79 gmmate84 gmmate87 aggnvs is the abbreviation for ggnvs, a cis-acting element of art bthe position is relative to the start codon of each gene expression of the subgroup c4- gmmate genes in response to al toxicity ten out of mate transporters from other plant species in subgroup c4- have been shown related to al detoxification, therefore the expression patterns of all eight subgroup c4- gmmate genes in response to al toxicity were analyzed by qrt-pcr. intraspecific variation in al tolerance is striking in many crop species. previous studies showed that al-tolerance related mate gene expression in plant root tips is up-regulated by al and is significantly higher in al-tolerant genotypes. in this study, two soybean cultivars, kf and gf, were treated with and 25 μm alcl for 6 h, 12 h and 24 h, respectively. the relative expression levels of the eight c4- gmmate genes in the root tips of soybean seedlings after al stress treatment are shown in fig.  among these eight genes, only one gene, gmmate showed a significantly higher relative gene expression in the al-tolerant cultivar kf than in al-sensitive cultivar gf at and 24 h after al stress treatment. gmmate showed a significant higher relative expression in kf at 6 h but significant higher in gf at 24 h after al stress treatment. gmmate gmmate and gmmate showed a significantly higher level of relative expression in gf than kf after al stress treatment. gmmate gmmate and gmmate did not show significant difference in their relative expression between gf and kf during al stress treatment. therefore, gmmate which showed higher elevated transcript levels after al treatment in al-tolerant than al-sensitive cultivar, would be a candidate gene for soybean tolerance to al toxicity. the expression patterns of gmmate could also be visualized by semi-quantitative rt-pcr. under normal growth conditions without al treatment, their expression levels were very low and no difference was observed between kf and gf. however, under al treatment, their expression levels increased significantly, which was consistent with the qrt-pcr results. a previous microarray study reported gma..a1_at, a putative mate gene, was up-regulated by al treatment in an al-tolerant soybean variety jiyu, which is the same gene as gmmate we identified in this study. in our study, the relative expression of gmmate under al stress was further compared between al-tolerant and al-sensitive varieties. gmmate was highly up-regulated by and 335-fold in kf while and 33-fold in gf after and 24 h al treatment, respectively, suggesting its role in soybean tolerance to al stress.fig. relative expression of the eight soybean c4- mate genes in response to al toxicity. the relative expression of the eight gmmate genes in the soybean root tips in response to al stress was quantified by qrt-pcr, using soybean gmef-1α gene as the internal control and 0 μm alcl 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 al stress for each time point at p <  and p < , respectively  a comprehensive genome-wide analysis of mate family is performed in an important legume species and oil crop, soybean. a total of mate transporters were identified in soybean and could be classified into four subfamilies, c c c and c the soybean mate family displays great variation in gene structure, protein motif, and tissue expression pattern, which indicates their diverse functions. the expansion of soybean mate family was largely due to segmental duplications. seven stress-responsive cis-acting elements and the cis-acting element of art were identified in the upstream regions of eight gmmate genes in subgroup c4- which contains previously reported mate genes related to al detoxification and/or iron translocation. one gene from c4- subgroup, gmmate showed differential relative transcript abundance between the root tips of al-tolerant and al-sensitive soybean cultivars in response to al treatment, indicating its potential role in soybean tolerance to al toxicity. this study provides a foundation to further investigate the functions of soybean mate genes including the candidate gene for al tolerance in soybean. 
multidrug and toxic compound extrusion family is an important group of the multidrug efflux transporters that extrude organic compounds, transporting a broad range of substrates such as organic acids, plant hormones and secondary metabolites. a total of genes encoding mate transporters were identified from the whole genome sequence of soybean, which were denominated as gmmate - gmmate these gmmate genes were unevenly localized on soybean chromosomes to with both tandem and segmental duplication events detected, and most genes showed tissue-specific expression patterns. the cis-elements and expression patterns of eight soybean mate genes related to al detoxification/iron translocation were analyzed, and gmmate was identified as a candidate gene for al tolerance in soybean. differential gene expression analysis of these eight gmmate genes in response to al stress helps us identify gmmate as the candidate gene for al tolerance in soybean, whose relative transcript abundance increased at and 24 h after al treatment, with more fold changes in al-tolerant than al-sensitive cultivar, which is consistent with previously reported al-tolerance related mate genes. eight soybean mate transporters clustered together with the previously reported mate proteins related to aluminum detoxification and iron translocation were further analyzed. however, genome-wide analysis of mate family in plant species is limited and no such studies have been reported in soybean. seven stress-responsive cis-elements such as abre, are, hse, ltr, mbs, as well as a cis-element of art, ggnvs, were identified in the upstream region of these eight gmmate genes. a total of mate transporters were identified in soybean and their potential functions were proposed by phylogenetic analysis with known plant mate transporters. soybean mate family could be classified into four subfamilies comprising ten smaller subgroups, with diverse potential functions such as transport and accumulation of flavonoids or alkaloids, extrusion of plant-derived or xenobiotic compounds, regulation of disease resistance, and response to abiotic stresses. this study provides a first insight on soybean mate family and their potential roles in soybean response to abiotic stresses especially al toxicity.
 multidrug and toxic compound extrusion family is an important group of the multidrug efflux transporters that extrude organic compounds, transporting a broad range of substrates such as organic acids, plant hormones and secondary metabolites. however, genome-wide analysis of mate family in plant species is limited and no such studies have been reported in soybean.  a total of genes encoding mate transporters were identified from the whole genome sequence of soybean, which were denominated as gmmate - gmmate these gmmate genes were unevenly localized on soybean chromosomes to with both tandem and segmental duplication events detected, and most genes showed tissue-specific expression patterns. soybean mate family could be classified into four subfamilies comprising ten smaller subgroups, with diverse potential functions such as transport and accumulation of flavonoids or alkaloids, extrusion of plant-derived or xenobiotic compounds, regulation of disease resistance, and response to abiotic stresses. eight soybean mate transporters clustered together with the previously reported mate proteins related to aluminum detoxification and iron translocation were further analyzed. seven stress-responsive cis-elements such as abre, are, hse, ltr, mbs, as well as a cis-element of art, ggnvs, were identified in the upstream region of these eight gmmate genes. differential gene expression analysis of these eight gmmate genes in response to al stress helps us identify gmmate as the candidate gene for al tolerance in soybean, whose relative transcript abundance increased at and 24 h after al treatment, with more fold changes in al-tolerant than al-sensitive cultivar, which is consistent with previously reported al-tolerance related mate genes.  a total of mate transporters were identified in soybean and their potential functions were proposed by phylogenetic analysis with known plant mate transporters. the cis-elements and expression patterns of eight soybean mate genes related to al detoxification/iron translocation were analyzed, and gmmate was identified as a candidate gene for al tolerance in soybean. this study provides a first insight on soybean mate family and their potential roles in soybean response to abiotic stresses especially al toxicity. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords abiotic stressaluminum toxicitycis-elementduplicationexpression analysismatephylogenetic analysissoybeanthe national natural science foundation of china 31371645li yan the national high-tech r & d program of china2013aa102602li yan the fundamental research funds for the central universitiesthe program for changjiang scholars and innovative research team in universitypcsirt13073li yan the program for new century excellent talents in universityncet-12-0891li yan the program for high-level innovative and entrepreneurial talents in jiangsu provincethe jiangsu higher education papd programissue-copyright-statement© the author 2016 