Genome-Wide Analysis of Oligopeptide Transporters Gene Family in Medicago truncatula

Oligopeptide transporters (OPTs) are a group of membrane localized proteins that have a broad range of substrate transport capabilities and contribute to numerous biological processes. However, limited information has been reported on OPTs in higher plants. In this study, a comprehensive analysis of the OPT gene family in Medicago truncatula was performed. A total of 26 OPT genes (MtOPT01-MtOPT26) have been identified in the Medicago truncatula genome. Phylogenetic analyses indicated that MtOPTs consisted of two distinct subgroups, 12 MtOPTs belonged to the peptide transport subgroup (PT-OPT) based on their predicted amino acid sequences containing the two highly conserved motifs (NPG and KIPPR) and 14 MtOPTs belonged to yellow stripe subgroup (YS-OPT). The MtOPTs distributed on each of 8 chromosomes in Medicago truncatula. Sequence analysis verified that MtOPTs significant similar to those in other plants. The copy number of MtOPTs was low and the multiply of MtOPTs was simple relatively. Gene structure analysis showed that most of the MtOPTs have various numbers of introns. The multiple of MtOPTs might play different biological roles which were supported by the fact that MtOPTs have a distinct tissue-specific expression pattern. The data obtained in this study will help to better understand the complexity of the MtOPTs gene family and provide new evidence for the function and evolution of the OPT gene family in higher plants.


INTRODUCTION
The material transport of the organism is tightly related to biosynthesis, cell communication, nutrition metabolism, pathogen defense and a variety of physiological or biochemical activities [1] .Transport protein has the function of specifically carrying the molecules which is essential for growth and development and regulating the distribution of substance [2][3][4] .Peptide transport involves in the translocation of peptides (2-6 residues in length) across the cellular membrane with an energy-dependent manner.The OPT family have been found in bacteria, archaea and plants.OPT is a kind of peptide transport protein with transport various substrates ability belong to one of the three peptide transporter protein families in plant.Multiple OPTs have been identified in variety plant genome including Arabidopsis, rice, maize, poplar, grape, thlaspi caerulescens and brachypodium distachyon, from which OPTs in Arabidopsis and rice were well characterized [5][6][7] .Previous studies showed that the evolution relationship is very close among these OPTs in plant.According to the difference of protein motifs, OPT family divided into two divergent subgroups, the yellow stripe (YS-OPT) and the peptide transport (PT-OPT).PT-OPT shared the highly conserved motifs of NPG (Asn-Pro-Gly; encompassing a stretch of 18 amino acids) and KIPPR (Lys-Ile-Pro-Pro-Arg; 11-14 amino acids) while YS-OPT contains no [8] .
In addition to peptide transport, OPTs have abilities to transport glutathione derivatives and metal complexes, and may be involved in stress resistance.Previous studies suggest distinct cellular roles for PT-OPTs, including nitrogen mobilization, seed germination, pollen, ovule and embryo development, glutathione transport, seed formation and metal transport [1,9,10] .Seven members of the AtOPT family transport tetra-and pentapeptides, presumably with proton cotransport [8] .AtOPT3 appears to play a key role in early embryo development following fertilization.BjGT1 (an OPT member from Indian mustard), OsGT1 (an OPT homologs in rice) and AtOPT6 transport glutathione derivatives and are induced by primisulfuron.These OPTs enable to uptake a non-protein-derived tripeptide glutathione (GSH and GSSG) and glutathione derivatives (GS conjugates) [11][12][13] .Heterologous expression in yeast indicates that AtOPT3 transport copper, manganese and iron [12] .In root tissue, AtOPT2 express response to iron and zinc deficiency and AtOPT3 response to iron, copper and manganese deficiency [9,10] .AtOPT6 and AtOPT7 transport cadmium or cadmium-glutathione conjugates [13] .Arabidopsis PT-OPTs express in dividing areas of the plant.Furthermore, the expression of AtOPT6 is sensitive to the herbicide primisulfuron to abscisic acid (ABA).AtOPT6 transport glutathione derivatives and is induced by primisulfuron.The expression of AtOPT2 is induced by salt stress [11,14] .The PT Clade of OPT was characterized in Rice.OsOPT1, OsOPT3, OsOPT4, OsOPT5, and OsOPT7 transport ferrous and/or ferric iron chelated to nicotianamine (NA), a nonpeptidyl metal-chelate.The expression of the OsOPTs was verified in roots, stems, leaves, hulls, pedicels, and embryos with different patterns [1] .Altogether, data showed that the substrate specificity and the physiological functions of OPTs may be diverse.
The YS subgroup includes the YS1 in Zea mays and yellow stripe-like (YSL) genes, described in Arabidopsis, rice [15][16][17] .Graminaceous plants acquire iron by secreting mugineic acid family phytosiderophores (PS) into the rhizosphere and taking up complexes of iron and PS.YS subfamily has capacity of transport metal chelated by NA or mugineic lysergic acid [16][17][18][19] .The first identified YSL transporter, ZmYS1 transport Fe 3+ -PS and Fe 2+ -NA complexes [17,20] .The members similar to ZmYS1 have been found in Arabidopsis thaliana and Oryza sativa genomes.Functional analysis showed that some YSL members also involved in the transport and homeostasis of other transition metals such as Zn, Ni, Mn and Cu [20,21] .
YSL members were different in their expression patterns, localization, and transport substrate specificity [22][23][24] .OsYSL2 transports Fe (II)-NA complex and Mn-NA complex [18] ; OsYSL15 transport the Fe (III)-PS complex [23] .OsYSL18 transports Fe (III)-deoxymugineic acid (DMA) [25] .Different from other YSL members, OsYSL6 is required for tolerance to excess Mn in rice [26] .The expression of HvYSL5 is strongly induced in the root by Fe deficiency and show diurnal rhythm [27] .AtYSL also transport Fe (III)-PS in yeast, although Arabidopsis does not synthesize PS.AtYSL1 and 3 are responsible for the mobilization of micronutrients such as Mn, Zn, Cu, and Fe from leaves and for loading of Fe-NA into the seeds [17,28] .A study showed that AtYSL1 and 3 are able to transport Fe (II)-NA [29] .AtYSL2 transport Cu-NA and Fe (II)-NA complexes in yeast [15][16][17] .TcYSL3 of hyperaccumulator, Thlaspi caerulescens, is involved in the entry of Ni-NA into the symplastic transport in the roots for delivery to the xylem and unloading of the Ni-NA complexes from the xylem in the leaves [19] .All these findings indicated that YSLs had diverse functions in transporting of various metals.Both YS-OPT and PT-OPT involved in different aspects of metal transport which narrowed these two subfamilies [9] .
Nitrogen, iron and phosphorus are the three nutrients most commonly limiting plant growth.OPTs appear to play important roles in acquisition of two of these key nutrients from the environment and subsequent transport around the plant [9] .However, compared with prokaryotes and animals, peptide transport received little attention in higher plants [30] .Medicago is an important forage crop which has high protein content and strong resistance.Although OPTs play important roles in the nitrogen utilization and metal transfer, its function in Medicago still did not clarify.The whole genome sequencing of Medicago truncatula, a model plant of leguminous spices in genomics research with a small genome, has completed [31] .Several gene families such as MAPKKK, WRKY were analyzed at genome-wide in Medicago truncatula [32,33] .To provide basis for the research on the function of OPT in legume and conduce better understanding the transport of nutrition and metal in plant, genome-wide bioinformatics analysis were performed on the OPT family of Medicago truncatula in this study, The data will shed new light on the possible roles of the MtOPT family.

The identification and classification of excavated OPT genes
MtOPT genes domain information(PF02669)was downloaded by Pfam database [35] and protein sequence of Medicago truncatula (Operating parameters: -E0.01) was searched by HMMER (V3.0) software [36] .Sequence alignment between the excavated genes and OPT genes of Arabidopsis were conduct.MtOPT genes were classified according to the reported classification information of Arabidopsis OPT family.The isoelectric point, molecular weight and other sequences basic nature were analyzed by ExPAVY online ProtParam tool [37] .According to genome and cDNA sequence of Medicago truncatula, structural mapping and analysis of MtOPT gene family were conducted using GSDS online tool [38] .

The chromosomal localization of MtOPT genes
Pairwise comparison analysis was according to Medicago truncatula genome sequence and CDS sequences obtained by BLAST online tools [41] .Gene duplication between each two MtOPT genes were defined when the consistency more than 85%.Combined with location information of MtOPT genes in Medicago truncatula genome, the CIRCOS software [42] was used to draw chromosomal localization analysis diagram.

Gene expression analysis of MtOPT genes
TopHat [43] and Cufflink [44] were used to analyze the gene expression of MtOPT morphology in dividing areas (roots, nodules, leaf, bud, carpel and flowers), according to the obtained transcriptome sequencing data of Medicago truncatula, SRA database of NCBI (http://www.ncbi.nlm.nih.gov,accession numbers: SRR350517-S350521, SRR350538 and SRR349692).Ggplot2 of R software (Version 3.1.0)were used for MtOPT gene expression cluster analysis.

Identification of 26 MtOPT genes in Medicago truncatula genome
A total of 26 putative OPT gene homologues were identified in the Medicago truncatula genome by Pfam database and HMMER (V3.0) (Table 1).The predicated MtOPT proteins were confirmed by the OPT domains.All of the 26 putative MtOPT homologous belonged to the OPT gene family determined by OPT domain (SPYxEVRxxVxxxDDP) [45] .The amino acid number of most MtOPTs are from 552 (MtOPT12) to 773(MtOPT07) except that which of MtOPT25, 23 and 26 are 295, 235, and 123, respectively.All of the MtOPT proteins are hydrophobic with molecular weights range from 13 (MtOPT26) to 86 (MtOPT07) KDa.The isoelectric point (IP) of these amino acid ranged from 4.67 (MtOPT26) to 9.33 (MtOPT24).In accordance with the peptide transport (PT) clade contains the NPG and KIPPR motifs, 12 MtOPTs were clustered into PT subfamily and 14 MtOPTs were clustered into YS subfamily.The structure of NPG and KIPPR motifs in PT subfamily genes was shown in Figure 1 and 2.

Phylogenetic relationship of MtOPT genes
To further analysis the phylogenetic relationship, MtOPT gene family was compared with the OPT gene families of Arabidopsis thaliana and Oryza sativa.The comparison result supported the taxonomic status and the classification of the MtOPT gene family (Figure 3).The sequence of MtOPT gene family was highly conservative and consistent with the classification of AtOPT and OsOPT gene families.Phylogenetic analysis showed genetic independence between PT and YS subfamilies, while the evolutionary relationship existed cross at MtOPT24.The sequence conservation in YS subfamily genes was higher than that in PT subfamily.The MtOPT genes could be further classified into 5 sections (I, II, III, IV and V).As Figure 4

The gene structure of MtOPTs
To obtain more information on the genomic organization of MtOPT genes, the intron boundary for the 26 MtOPTs were determined.The structure of MtOPTs was shown in Figure 4. Most of MtOPTs have introns except for MtOPT26, the shortest MtOPT gene.Introns in others MtOPT genes were from 1 to 8, and normally from 4 to 6.The most of 11 introns were found in MtOPT24.The number and length of introns were various in the whole MtOPT gene family, despite identical and similar in the genes belonging to the same subgroup.

Chromosome Location and Gene Duplication of MtOPT genes
The 26 MtOPT genes are distributed on 8 chromosomes of Medicago truncatula (Figure 5).From 1 to 5 OPT genes are distributed on every chromosome of Medicago truncatula without packing effect.Each of 5 MtOPT genes localized on chromosome 3 and 5; four MtOPT genes localized on chromosome 7 and three localized on chromosome 4; each of only 1 MtOPT gene localized on chromosome 1, 2 and 8. Multiple copy of MtOPT genes was found by gene sequences Pairwise alignments.In the PT subfamily, gene duplication was found in MtOPt04, 05 and 06 (chromosome 5) and MtOPT07, 08 and 10 (chromosome 7); and the duplication relation was found in the above 6 genes with MtOPT11 (chromosome 2) and MtOPT 12 (chromosome 4).Duplication was also found between MtOPt01 (chromosome 3) and MtOPT 09 (chromosome 8).In the YS-MtOPT subfamily, MtOPt14 and 16 (chromosome 3) was found to be have duplication relation with MtOPT13 and 25 (chromosome 5); MtOPt15 on chromosome 6 also showed duplication relation with MtOPt14 and 16.However the replication of OPT genes is relatively simple in Medicago truncatula compared with other plants.

Expression of MtOPT genes
Organ-specific of the 26 MtOPTs were obtained from SRA database extraction transcriptome data (Figure 6).Data showed that expression profile of the MtOPTs was different in various areas of the plant.Most of genes in PT-OPT subfamily expressed in all of the six areas (roots, nodules, leaf, bud, seedpod and flowers) and mostly expressed in nodule.YS-OPT subfamily genes mainly expressed in seedpod.Taken together, expression levels of YS-OPT subfamily genes was relative higher than those of PT-OPT subfamily genes.

DISCUSSION
To absorb and maintain a balance of potentially toxic metal ions, plants utilize poorly understood mechanisms involving a large number of membrane transporters and metal binding proteins with overlapping substrate specificities and complex regulation [9] .Higher plants contain a far greater number of OPT genes for these transporters than do other eukaryotes.This may be indicative of the relative importance of (oligo) peptides and their transport to plant growth and metabolism [11] .
OPT genes belong to a small gene family in plant.Total of 26 OPT gene family members were identified in Medicago truncatula genome in this study.The MtOPT gene family is significantly larger than the OPT family in Arabidopsis and rice, 9 and 16 OPT gene family members, respectively.The amino acids Length encoded by MtOPT is generally between 500 and 800.Intron number is irregularity for MtOPT29 contains 11 introns and MtOPT26 contains no.Generally, the length in YS subfamily genes is less than that of the PT subfamily.This distinction may be caused by carrying different substrates，due to the transit of oligopeptide require more complex regulatory and transporter mechanisms.In evolution, PT subfamily and YS subfamily is independent, the gene sequences between two subfamilies have great difference, thought exist similarity in partial genes.
The OPT genes have been identified in rice and Arabidopsis genome suggest that the OPT family of transporters is larger and more diverse than previously recognized.The number of MtOPTs is larger than that of rice and Arabidopsis, nevertheless the diverse is not so obviously.The evolution of MtOPTs is consistent with OsOPTs and AtOPTs which have been further divided into two clusters [18] .
OTP family transporter proteins differ in the transmission of physiological functions such as long distance distribution of nitrogen mobilization and heavy metal sequestration.These processes play important roles in development and growth of plant [5] .All of the Arabidopsis OPT family member can transport leu tetra-and pentapeptides with the exception of OPT2 and OPT3 [14,30] .Contradictory evidence exists either in support of AtOPT6 being able to transport glutathione or against such glutathione transporting activity [10][11][12] .It is well known that chelate of iron and other metals such as Zn, Cu, Ni, Mn, etc. can be transported from rhizosphere surrounding to the root and transported in the intracellular simultaneously [45] .AtOPT2 and AtOPT3 are highly induced in response to metal (iron, copper and manganese) deficiencies.Yeast complementation experiments have shown that AtOPT3 can transport Cu, Mn, and possibly Fe [12] .AtOPT6 can mediate absorb glutathione derivatives and metal complexes; suggesting that it may also involve redox balance in the body.In addition, OPT transporter protein plays an important role in the maintenance of cellular homeostasis and adaptation to environmental stress [3,4] .It was also shown that AtOPT3 is required for embryo development [10] .YS and PT subfamily were different in function, the report showing that rice PT-OPTs are capable of transporting NA-bound iron has narrowed capabilities gap between the PT and YS subfamilies [1][2][3] .AtOPT3 is likely involved in long distance movement of metal-chelate complexes in the plant while divergent in sequence from members of the YSL subfamily [10] .Due to all of titanium, PS and niacinamide are amino acid derivatives, these two types of transport protein subfamily belong to the same OPT big family.
According to the relative expression level of MtOPTs, All the MtOPTs studied express highly in root, flower, blade, seedpod, or nodules except MtOPT14 and 24.Most of the OPT genes express in vegetative tissues of and reproductive organs indicated that OPT gene family played an important role in entire life activities of Medicago truncatula.Chelate of metals can be transported from rhizosphere into root and transported in the intracellular simultaneously [45] .The presence of the MtOPT transcripts in root and blade suggests individual MtOPTs may have specific roles in the acquisition and redistribution of organic nitrogen and/or metal during plant growth.The expression in the reproductive organs, flower and seedpod, suggests the translocation function of individual MtOPTs in the plant development.Additionally, the transcript level of the gene family reveals that MtOPTs have complex functions.The expression of YS subfamily genes is significantly higher than that of PT subfamily genes in different parts.YS subfamily main in carpel tissues, followed by the roots and nodules indicating YS subfamily genes not only play an important role in the vegetative growth but also in reproductive organ development process; While PT subfamily expressed mainly in nodules which is specific in Leguminous plants may be related to nitrogen absorption in Medicago truncatula.In short, data obtained from the analysis will help to better understand the complexity of OPT family in Medicago truncatula and evolution of OPT gene family in higher plants.
OPT plays an important role in normal nutrition metabolism and metal stress response in plant.
Several of the plant OPT transporter gene families are fairly well described; however, the MtOPT family are not among them.This is the first report of MtOPT family.Moreover, the gene function and expression patterns need to be further understood.

CONCLUSION
In this study, we identified 26 OPT genes belonging to PT and YS distinct subfamilies from Medicago truncatula.The MtOPT gene family is larger than the OPT family in Arabidopsis and rice.The sequence of MtOPT gene family was highly conservative and consistent with the AtOPT and OsOPT families though the multiply of MtOPTs was relatively simple.The MtOPTs were localized on all of the chromosomes in Medicago truncatula and have various introns in number and length.Most of MtOPTs genes showed distinct tissue-specific express in all of the six areas (roots, nodules, leaf, bud, seedpod and flowers) in Medicago truncatula.The transcript level of the MtOPT gene family supported that MtOPTs involved in the transport and homeostasis of important elements which are essential for growth and development of plant.The results should provide further understand on the systematic of OPT family and important theoretical basis in function of OPT genes in higher plants.

Fig. 4
Fig. 4 The gene structure of OPT family genes in Medicago truncatula