BACKGROUND
nutrient transport is essential to life and occurs in both prokaryotes and eukaryotes.
it is well established that peptide transporters play an important role in the nutrition of bacteria, yeasts and animals  <cit> .
however, the role of transporters of small peptides  in plants is less well defined  <cit> .
peptide transporter  family , also called the proton-dependent oligopeptide transport  family  <cit>  is a family consists of di-/tripeptides transporters  <cit> .
besides, some plant ptrs acted as nitrate transporters are termed as the members of nitrate transporter  <dig> family .
ptr proteins in plants show significant sequence homology and contain several transmembrane  regions, with a large hydrophilic loop among tms.
generally, the identified ptr members in plants fall into three types based on the nature of their substrates: di-/tripeptides transporter, nitrate transporter and other substrates transporter  <cit> .
atptr <dig>  was the first identified di-/tripeptides transporter in arabidopsis  <cit>  and had high mrna expression levels in 3-d-germinating seed, root and young leaf  <cit> .
the antisense plants of atptr <dig> exhibited delayed flowering time and arrested seed development  <cit> .
atptr <dig>  transported di-/tripeptides with low selectivity as well as substrates lacking a peptide bond.
atptr <dig> was expressed in the vascular tissue throughout the plant, indicative of a role in long-distance transport  <cit> .
atptr <dig>  was a salt stress and mechanical wounding inducible gene, ja and sa were both involved in regulation of it  <cit> .
atptr <dig>  which mediated high-affinity transport of dipeptides was most likely supplying peptides to maturating pollen, developing ovules and seeds.
overexpression of it resulted in enhanced shoot growth and increased n content  <cit> .
the barley peptide transporter gene, hvptr <dig>  had seed-specific expression.
transport activity of hvptr <dig> was regulated by phosphorylation in response to rising levels of amino acids in the germinating grain  <cit> .
functional di-/tripeptides transporters also have been reported in nepenthes , faba bean  and hakea actites .
although ptr is the abbreviation of peptide transporter, many cloned ptr members in plants are nitrate transporters, especially in arabidopsis.
atnrt <dig>  , a dual-affinity nitrate transporter, was regulated by phosphorylation and involved in nascent organ development  <cit> .
atnrt <dig>   encoded a low-affinity nitrate transporter.
rnai plants of it exhibited reduced nitrate-induced membrane depolarization and nitrate uptake activities  <cit> .
the characterization of atnrt <dig>   revealed the special role of petiole in nitrate homeostasis  <cit> .
atnrt <dig>   and atnrt <dig>   were both plasma membrane proteins.
atnrt <dig>  was expressed in root pericycle cells close to the xylem and participated in root xylem loading of nitrate  <cit>  while atnrt <dig>  was only expressed in reproductive tissue and involved in delivering nitrate from maternal tissue to the developing embryo  <cit> .
csnitr <dig>  a nitrite transporter in cucumis sativus, had two isoforms: csnitr1-s and csnitr1-l. in contrast to that csnitr1-s enhanced the efflux of nitrite from the cell, csnitr1-l might load cytosolic nitrite into chloroplast stroma  <cit> .
amino acids and other substrates can also be transported by some ptrs.
bnnrt <dig> , isolated from brassica napus could transport both nitrate and histidine  <cit> .
agdcat <dig> localized at the symbiotic interface was a dicarboxylates transporter in alnus glutinosa and the mrna of agdcat <dig> was only detected in the nodules  <cit> .
in rice, only two ptr genes have been functionally verified.
osnrt <dig> , which displayed low-affinity nitrate transport activity was constitutively expressed in the most external layer of the root, epidermis and root hair  <cit> .
sp <dig>  which determined the panicle size had high expression level in the phloem of the branches of young panicles.
phylogenetic analysis implied that sp <dig> might be a nitrate transporter, however, neither nitrate nor other compounds transport activity could be obtained from it  <cit> .
as the main staple food for a large segment of the world population, rice also serves as a model plant for monocotyledon species  <cit> .
nutrient transport is critical for plant growth and development, in which ptr family members may play important roles.
this work focused on a comprehensive identification, characterization, phylogenetic and evolutionary analysis of all the ptr family members in rice  as well as their whole-life expression profiling.
moreover, it has been demonstrated that the expression of some osptr genes is regulated phytohormone  and light/dark treatments.
RESULTS
collection and identification of ptr genes in rice
domain search  of msu rice genome annotation project  database released  <dig> sequences, out of which  <dig> were unique.
keywords "ptr", "peptide transporter" and "proton-dependent oligopeptide transport" searching identified  <dig>   <dig> and  <dig> genes respectively.
blastp and tblastn searches against rice genome in three databases: msu rgap, ncbi and kome released  <dig>   <dig> and  <dig> sequences respectively.
by removal of the same sequences and different transcripts of the same gene, we initially identified  <dig> putative ptr genes in rice.
protein sequences of these genes were subjected to pfam and smart searches for the presence of ptr domains.
as a result, seven genes whose protein contained only partial ptr domain and two genes annotated as retrotransposons were excluded from further analysis.
taken together, a total of  <dig> genes were predicted to encode ptr proteins in rice.
eighty-three genes had corresponding locus ids in msu rgap database and one gene  was only found in ncbi.
for convenience, all the "loc_" prefix of msu rgap locus ids were omitted representing osptr genes or proteins for further analysis.
meanwhile, similar methods were used and  <dig> ptr family members in arabidopsis  were identified.
the detailed information about the full-length cdna, transcript, chromosomal position, bac accession, gene structure for each osptr gene and characteristics of corresponding proteins can be found in additional file  <dig>  protein structures predicted by smart are shown in additional file  <dig>  the putative transmembrane  regions in each osptr protein are listed in additional file  <dig>
chromosomal localization and gene duplication
to further investigate the relationship between the genetic divergence within the ptr family and gene duplication in rice, the chromosomal location of each ptr gene was determined from the genomic sequence of rice.
the  <dig> osptr genes were dispersed on all the rice chromosomes except chromosome  <dig>  their chromosomal distribution pattern revealed that certain chromosomes and chromosomal regions had a relatively high density of osptr genes.
for instance, chromosome  <dig> had the highest density with  <dig> members, followed by chromosome  <dig> had  <dig> members.
ten genes were located on chromosome  <dig>  eight genes on chromosome  <dig>   <dig> and  <dig>  five genes on chromosome  <dig> and four genes on chromosome  <dig> and  <dig>  and only two genes on chromosome  <dig> and  <dig>  for most chromosomes, osptr genes were present only on half arm of the chromosome or in clusters, i.e., no osptr gene appeared on the short arm of chromosome  <dig>   <dig> and  <dig> and most of the genes on chromosome  <dig> and  <dig> were clustered together .
analysis of the msu rgap segmental duplication database revealed that  <dig> osptr genes  could be assigned to rice segmental duplication blocks .
os02g <dig> participated in two segmental duplications with os04g <dig> and os10g <dig> respectively.
the overall identities of the protein sequences of these genes ranged from  <dig> % to  <dig> %.
a total of  <dig> genes  were considered as tandem duplications according to the criterion adopted in our analysis, including the largest cluster of nine genes on chromosome  <dig>  two four-gene clusters on chromosome  <dig> and  <dig>  five three-gene clusters on chromosome  <dig>   <dig>   <dig> and  <dig>  six two-gene clusters on chromosome  <dig>   <dig>   <dig>   <dig> and  <dig> .
the protein homology of these genes varied from  <dig> % to  <dig> %.
interestingly, all the tandemly duplicated genes in the same cluster had the same direction of transcription except os06g <dig> and os06g <dig>  this might suggest the conserved behavior of tandem duplications in this family.
of the  <dig> osptr genes,  <dig> %  involved in the duplication events.
moreover, most of the duplicated genes had relative close phylogenetic relationship .
therefore, segmental and large-scale tandem duplication events appeared to have exclusively contributed to the expansion of the osptr gene family.
similar manner of member duplication had also been described in some large gene families in rice, for instance, the ankyrin repeat gene family  <cit>  and the bhlh family  <cit> .
phylogenetic analysis and multiple sequence alignment
to examine in detail the phylogenetic relationship and functional divergence of osptr members, the aligned  <dig> osptr protein sequences were used to construct the joint unrooted phylogenetic tree.
our result suggested that the osptr proteins could be classified into five major subfamilies designated from i to v, with high bootstrap value support .
this exercise resulted in five distinct subfamilies similar to that in arabidopsis reported by waterworth and bray  <cit> .
within the five subfamilies formed, subfamily i, ii and iii were found to be more closely related to each other in comparison to subfamily iv and v. subfamily i had the largest  <dig> ptr members and fell into three groups.
subfamily ii, containing  <dig> ptr members was further divided into two groups.
subfamily iii also including two groups contained  <dig> members.
subfamily iv had  <dig> members and subfamily v consisted of six members which had relative phylogenetic distance from other members.
we identified  <dig> pairs of osptr genes that were close paralogs on the terminal node of phylogenetic tree.
tandem duplication contributed to  <dig> pairs of them and segmental duplication contributed to three pairs.
these results furthermore illustrated that segmental and tandem duplication events were the dominant pattern in the expansion of osptr family members.
to well understand the evolutionary history of osptr family, the amino acid sequences of ptr domains in each osptr protein were used for further phylogenetic analysis and similar tree was obtained.
this result indicated that the main characteristic of osptr family was determined by the ptr domain.
in order to investigate the relationship between the function and phylogenetic subfamily formation of ptr members as well as identify some orthologous genes, a combined phylogenetic tree with osptr and atptr proteins was also established .
as a result, similar subfamilies were formed compared to the tree of osptr.
each subfamily contained both arabidopsis and rice ptr members.
however, most of the members were clustered in species-specific distinct clades, and only four pairs of orthologs  could be figured out.
this result indicated that the main characteristics of ptr family in rice and arabidopsis were formed before the split of monocotyledonous and dicotyledonous plants and then evolved separately in a species-specific manner.
moreover, the difference in the total number of osptr and atptr was mainly due to the variation in the number of subfamily i genes, which were  <dig> in rice and eight in arabidopsis.
of the  <dig> subfamily i ptrs in rice,  <dig> were tandemly or segmentally duplicated genes.
the alignments and comparison of the osptr full-length protein sequences and the ptr domain sequences illustrated that most of the amino acids in tm regions in or out of the ptr domain in members of the same phylogenetic subfamily were very conservative.
on the contrary, the amino acids outside the tm regions were in great variation.
in addition, some loop areas between the tm regions in the ptr domain of different members varied significantly both in length and amino acid composition.
the alignment of the subfamily iii members is shown in figure  <dig> as an example.
due to the conservatism of the ptr domain and function variety of the ptr members, the diversity of the amino acids in the loop and beyond the ptr domain areas might be the major force of functional discrepancy of each ptr member.
from the comparison of the protein sequences and sequences alignment, we found three special motifs that were highly conserved in most of the osptr members.
motif  <dig>  was found nearly before the n terminal of the ptr domain.
motif  <dig> whose sequence was lylxxxgxggxkxxxxxfgadqfd was located at the end of the first tm region of the ptr domain or stretched into the following hydrophilic loop before the second tm region.
following the motif  <dig>  before/in or at the end of the second tm region of the ptr domain, motif  <dig>  was identified.
the hmm logos of these motifs in osptr are shown in figure 4a and their locations can be found in figure  <dig>  in most atptr members, similar conserved motifs could also be figured out .
however, motif  <dig> and  <dig> in atptr were located between the second and third tm regions of the ptr domain.
sequence screening of motif  <dig> in interproscan and uniprot databases retrieved only ptr family members.
therefore, we supposed that this motif was a newly identified signature sequences associated to the plant ptr family.
expression profiling of osptr genes in whole-life cycle of rice
probes for  <dig> of  <dig> osptr genes could be found in our expression database.
to analyze the expression profiles of the osptr genes, the signal values of the  <dig> osptr genes represented on the array were extracted.
average signal values for the  <dig> osptr genes from two or three biological replicates of each sample are given in additional file  <dig>  a hierarchical cluster displayed of logarithm of average signal values for the  <dig> osptr genes in selected tissues in minghui  <dig>  is presented in figure 5a.
three genes  had no corresponding probe set.
therefore, real-time pcr was used to study the expression pattern of these genes .
based on hierarchical clustering, expression pattern of osptr genes could be divided into five major groups.
group  <dig> consisted of  <dig> genes which showed high expression levels in all the tissues expect six genes that showed relatively low expression in certain tissues .
conversely, group  <dig> contained  <dig> genes that showed low expression level in the whole life cycle.
group  <dig> was comprised of  <dig> genes that showed relatively high expression level in both vegetative tissues and hull.
all these genes had low expression level in early panicle and endosperm stages.
group  <dig> comprised of  <dig> genes with medium expression level in all the tissues except that os04g <dig> had high expression in root, os01g <dig> in e <dig>  os11g <dig> in e1-e <dig> and os01g <dig> in e1-e <dig>  the rest  <dig> genes belonged to group  <dig> although these genes had different expression patterns.
for example, os04g <dig> had high expression signal in root and stamen, os04g <dig> showed high expression in fl <dig>  os11g <dig> had high expression in panicle and stem.
interestingly, os01g <dig>  os01g <dig>  os11g <dig>  os10g <dig> and os06g <dig> all showed relatively high expression in endosperms.
the expression patterns of most osptr genes in zhenshan  <dig>  were similar to that in mh <dig> and the corresponding hierarchical cluster was shown in additional file  <dig>
for further study regarding the expression pattern and looking inside the putative functions of osptr family members, we employed the analysis of preferential and tissue-specific expression genes in mh <dig> and zs <dig>  the identified preferential expression genes were mainly expressed in stamen and root.
os04g <dig> was preferentially expressed in stamen of one day before flowering and os04g <dig> was in root of seedling with two tillers in mh <dig>  in zs <dig>  os01g <dig> and os12g <dig> were identified as stamen preferential expression genes, while os02g <dig> and os04g <dig> were preferentially expressed in root .
the check of our whole genome expression profile of rice for different tissues obtained several tissue-specific expression genes including os04g <dig> in root of mh <dig> and in flag leaf of 14-day after flowering in mh <dig> as well as zs <dig>  os02g <dig> in root of mh <dig> and zs <dig> and os01g <dig> in endosperm of 14-day after pollination in zs <dig> .
the hierarchical cluster of the atptr genes in  <dig> development stages released the expression profile of  <dig> genes except at1g <dig>  at3g <dig> and at3g <dig> .
obviously, the expression patterns of atptr genes fell into the similar groups as osptr genes.
some tissue-specific expression atptr genes could also be identified, for example, at1g <dig>  at4g <dig> and at3g <dig> in seed and siliques, at3g <dig> and at3g <dig> in root.
most of the atptr genes had relatively low expression level in mature pollen except at1g <dig>  at5g <dig> and at5g <dig>  these genes were most likely supplying nutrition to maturating pollen.
some of the orthologous osptr and atptr genes showed the similar expression pattern such as os06g <dig> and at1g <dig>  os05g <dig> and at2g <dig>  they both had high expression level in all the tissues.
however, no more apparent relationship between the expression pattern and sequence similarities of osptr and atptr members could be pointed out.
expression regulation of osptr genes under phytohormone and light/dark treatments
to determine the osptr genes in response to phytohormone treatments, microarray analysis was performed.
we identified six and nine osptr genes that were differentially expressed under one or more of the phytohormone treatments compared with the control in mh <dig> and zs <dig>  respectively .
among these genes, only one gene  was in common for two genotypes and it was down-regulated under three phytohormone treatments.
the expression profile of the remaining genes in two genotypes was different.
for instance, in mh <dig>  os01g <dig> and os10g <dig> were down-regulated under naa and ga <dig> treatments while os04g <dig> and os06g <dig> were down-regulated specifically to kt and ga <dig> treatment, respectively.
os01g <dig> was up-regulated specifically to naa treatment.
five genes were up-regulated and four genes were down-regulated in zs <dig>  the up-regulated genes were os01g <dig>  os01g <dig>  os01g <dig>  os01g <dig> and os04g <dig> while the down-regulated genes were os01g <dig>  os06g <dig>  os11g <dig> and os12g <dig>  os01g <dig> and os12g <dig> were both down-regulated under three phytohormone treatments.
os01g <dig> and os04g <dig> were both up-regulated under naa and kt treatments.
os01g <dig> was up-regulated under naa and ga <dig> treatments.
os01g <dig> and os11g <dig> showed differential expression specifically to kt treatment while os01g <dig> and os06g <dig> showed differential expression to ga <dig> treatment.
the fold changes with respect to control of differential expression genes under phytohormone treatments are given in additional file  <dig>
to investigate possible light regulation of osptr genes, the expression profile of osptr genes in seedlings  treated with light or dark for  <dig> hours after emergence was also analyzed.
twelve and seven genes were differentially expressed under light compared to dark in plumule and radicle, respectively .
eight genes  showed differential expression in plumule and four genes  in radicle in mh <dig>  interestingly, all the differentially expressed genes were up-regulated in plumule under light compared to dark while two of four genes in radicle were down-regulated.
os01g <dig> was up-regulated nearly 10-fold under light compared to dark in plumule.
in zs <dig>  five genes  were differentially expressed in plumule and three genes  in radicle.
interestingly, two genes  were differentially expressed in both plumule and radicle in zs <dig>  the fold changes with respect to control of light/dark regulation genes are listed in additional file  <dig>
ka/ks analysis and express pattern divergence of paralogous osptr genes involved in duplication
to explore the mechanism of gene divergence after duplication, the coding sequences of the paralogous osptr genes involved in duplications were used to calculate the synonymous  and non-synonymous  substitution rate after duplication.
the ka/ks ratio provides information about the degree of positive selection across all nucleotides in a gene since divergence.
generally, ka/ks =  <dig> means neutral selection, ka/ks <  <dig> indicates purifying selection and ka/ks >  <dig> shows the accelerated evolution with positive selection  <cit> .
the ka/ks analysis demonstrated that ka/ks ratios for all the paralogous osptr genes were always less than one, most of them even close to zero, suggesting that purifying selection might be largely responsible for function maintenance of this family .
paralog
gence
ka: non-synonymous substitution rate.
ks: synonymous substitution rate.
nf: neofunctionalization.
sf: subfunctionalization.
nof: nonfunctionalization.
d: just after duplication.
comparison of expression pattern shifts of the duplicated paralogous genes can reflect the divergence hypotheses that a duplicate gene pair might be involved in: nonfunctionalization, subfunctionalization and neofunctionalization  <cit> .
the expression profile of the  <dig> pairs of paralogous osptr genes with corresponding probes was extracted for analysis.
obviously, the  <dig> pairs of paralogous osptr genes had divergent expression patterns.
it seemed that all the three hypotheses influence the fate of the duplicated genes.
two pairs of genes  showed almost the same expression pattern in both copies, indicating they were just formed due to duplication .
the fate of four pairs in figure 9b could be described as nonfunctionalization, where one copy of the paralog lost expression in all tissues.
subfunctionalization explained for five pairs of paralogous genes, in which the expression patterns of both copies were complementary to the pattern before duplication .
the divergence hypotheses of the rest six pairs of paralogous genes were assigned as neofunctionalization, where the expression of one copy had an obvious increase in one or more tissues .
discussion
di-/tripeptides and nitrate transport is important for various aspects of plant growth and development, in which peptide transporter family members may play important roles.
considering the potential functional diversity of ptr members associated with nutrient transport and only a few ptr family members have been described in higher plants, it was timely and quite relevant for us to characterize the rice ptr family.
combination of the whole genome sequence data, sequences alignment and high-throughput expression analysis undertaken in this investigation provides an insight into all the  <dig> members of ptr family in rice.
duplication played an important role in the expansion of the osptr family
it is well known that gene duplication is important for gene family evolution and diversity, which can occur via three major mechanisms: segmental duplication, tandem duplication and retroposition  <cit> .
due to repetitive plant genome duplications, certain plant genomes are likely to harbor relatively large members of a gene family, as compared to animal genomes  <cit> .
the ptr families in rice and arabidopsis are very much in line with the statement and showed increased family members in order to adapt for specific functions.
the number of  <dig> osptr and  <dig> atptr members is far greater as compared to the low number of six in human, four in c. elegans, three in drosophila and two in yeast  <cit> .
it may indicate the importance of these transporters in plant growth and metabolism.
nearly 70% of the  <dig> osptr genes are duplicated genes,  <dig> genes  are found to be involved in the segmental duplication while  <dig>  participated in tandem duplication.
our analysis also indicated that in subfamily i, duplication events were largely responsible for the number difference of the member between osptr and atptr.
it demonstrates that the osptr family may undergo extensive and rapid genomic changes which can maintain relative large number of member and lead to expression divergences between duplicate genes, further contributing to the establishment of novel gene functions  <cit> .
phylogenetic relationship and functional diversity among the osptr members
the phylogenetic analysis reveals that the osptr family can be further divided into five subfamilies.
the combined phylogenetic tree of osptr and atptr showed similar subfamily formation whereas most ptr members were clustered in species-specific clades and only four pairs of orthologs could be identified.
it suggests that the members of the ptr family have diverged from each other primarily due to speciation and then undergone significant changes since the monocot/dicot split.
however, highly conserved motifs have been identified in most members of atptr and osptr.
we further adopted the sequence alignment including the ptr members in plant, animal, human and prokaryotic organisms.
it was surprising to find that the three motifs could be found in most eukaryotic ptr members but changed a lot or lost in the prokaryotic ptr members, especially motif  <dig> and motif  <dig>  moreover, some members such as os01g <dig>  os04g <dig>  os12g <dig> and at2g <dig> which had far phylogenetic relationship from other ptr members, had great amino acid variation in motif  <dig> and motif  <dig>  we inferred that motif  <dig> and motif  <dig> might be important characteristic logos of the eukaryotic ptr members and played considerable roles in the function formation of the ptr family.
further research may employ the point mutations introduced into these motifs, which should cause a loss of function of the corresponding protein.
this experimentation could indicate the essentiality of these motifs for the formation of the transporter pore and the interaction with the substrates.
the sequence similarity among the osptr members seemed to show little relationship with their putative transporter type classification.
in addition, we found that gene structures, intron phases of osptr genes and distribution of tm regions in the proteins had no relationship with their phylogenetic subfamily and transporter type classifications.
as most osptr proteins had ptr as their only recognizable domain , we adopted the meme method  <cit> , in order to identify some new motifs or domains.
however, the conserved motifs investigated by meme were all tm regions .
we infer that the substrate selection of osptr proteins may be decided by their three dimensional structures.
the determination of the di-/tripeptide, nitrate and other type transporters in this family by mutagenesis or crystal structure studies should solve the puzzle that di-/tripeptides, nitrate and other substrates of ptr proteins are very different in structure but they share the same transporter family - ptr  <cit> .
expression pattern divergence of the osptr genes
microarray provides a rich resource for investigating the evolution of gene expression.
the temporal and spatial expression patterns of osptr genes are useful information for establishing their putative functions.
our microarray analysis indicated that the expression patterns of the  <dig> osptr genes could be classified into five groups.
we also identified some preferential or tissue-specific expression osptr genes.
of the  <dig> genes analyzed in this study, only seven were found to exhibit either preferential or tissue-specific expression during different development stages of mh <dig> and zs <dig>  interestingly, these genes were most preferentially or specifically expressed in root and stamen.
os02g <dig>  os04g <dig> and os04g <dig> were preferentially expressed in root.
moreover, these three genes were in the same phylogenetic clade that belonged to the group three of subfamily i. in arabidopsis, the expression of atnrt <dig>  was found concentrated in the tips of primary and lateral roots  <cit> .
atnrt <dig>  was primarily expressed in root hairs and the epidermis in both young and mature regions of roots  <cit> .
osnrt <dig> was constitutively expressed in the most external layer of the root, epidermis and root hair  <cit> .
our data showed that osnrt <dig> had high expression levels in all the tissues especially in root.
os03g <dig> and os03g <dig> were the paralogs of osnrt <dig>  moreover, these three genes formed a tandem duplication cluster.
os03g <dig> and os03g <dig> showed similar expression patterns with osnrt <dig> and os03g <dig> had the highest expression level in root.
it can be inferred that these root-preferentially or -specifically expressed osptr genes might be nitrate transporters and play important roles in root formation and development.
atnrt <dig>  was expressed in the leaf petiole and participated in regulating leaf nitrate homeostasis  <cit> .
the ortholog of atnrt <dig>  in rice was os01g <dig> which continuously expressed in vegetative tissues with high level in leaves.
expression of os04g <dig> was specific in flag leaf of 14-day after flowering both in mh <dig> and zs <dig>  therefore, we infer that os01g <dig> and os04g <dig> might be participated in the development of leaf as well as the nutrient transporting in and/or out of leaf.
in addition, we also identified three genes  that were preferentially expressed in stamen and one gene  in endosperm.
one interesting phenomenon in our dendrogram was that three-quarter of the osptr genes had significant low expression level in panicles of different development stages.
sp <dig> whose transcription accumulated in panicle was one of the exceptions.
sp <dig> was highly expressed in the phloem of the branches of young panicles, which was consistent with the predicted function of sp <dig> and the phenotype of sp <dig> mutant  <cit> .
it can be demonstrated that some ptr members might also play roles in nutrient transport in reproductive organ development.
potential involvement of osptr members in phytohormone and light/dark regulations
little attention has been paid on the functions of ptr members in phytohormone and light/dark regulations.
until now, atnrt <dig>  was the only reported ptr gene that was involved in these regulations.
atnrt <dig>  was regulated by auxin in both shoots and roots and involved in light-induced stomatal opening  <cit> .
however, our microarray data showed that the expression patterns of more than one-third osptr genes changed significantly under phytohormone and light/dark treatments.
fourteen osptr genes are regulated by at least one phytohormone and  <dig> osptr genes are involved in the regulation of light/dark treatment in mh <dig> and zs <dig>  from these results, it can be speculated that these differentially expressed osptr genes may play an important role in the transport of regulatory molecules  and other metabolites induced by regulation of phytohormone and light/dark treatments.
for example, iaa is an important plant growth phytohormone and 95% of the iaa pool is sequestered in an inactivated conjugated form, e.g.
iaa can be conjugated to single amino acids, peptides or proteins via amide bonds.
these conjugates may be involved in regulating phytohormone activity in response to plant growth and development, as well as facilitating hormone transport  <cit> .
it seems that the proteins of those osptr genes regulated by naa might have iaa conjugates transport activity.
fate of the paralogous osptr genes after duplication
from the ka/ks analysis of the paralogous osptr genes, it is obvious that purifying selection plays an important role in function maintenance of this family.
however, comparison of the expression data implied that after duplication, the paralogous pairs may have been evolved in different hypotheses to gain functional divergence.
expression data shows that some of these duplicated genes have undergone subfunctionalization and neofunctionalization, which contribute to the possibility of maintenance and divergence of a paralogous set of genes.
some paralogs have adopted nonfunctionalization which contributes to the likelihood of loss of one member in a pair of paralogous genes.
however, sufficient experimentation is required to prove these hypotheses.
CONCLUSIONS
in conclusion, the results of this study display the genomic framework, characterization, conserved motifs, duplication manner and evolutionary analysis of the  <dig> osptr members, along with their expression profiles during whole-life stages as well as under phytohormone and light/dark treatments.
these data will provide the basis for understanding the evolutionary history of ptr members and their roles in rice growth and development.
the findings in our work would be useful in selecting candidate genes for functional validation studies of ptr members in rice.
however, future research by adopting rnai/overexpression strategies or insertion mutagenesis is required to elucidate the precise role of individual osptr gene.

 nutrient transport is essential to life and occurs in both prokaryotes and eukaryotes. it is well established that peptide transporters play an important role in the nutrition of bacteria, yeasts and animals. however, the role of transporters of small peptides in plants is less well defined. peptide transporter family, also called the proton-dependent oligopeptide transport family is a family consists of di-/tripeptides transporters. besides, some plant ptrs acted as nitrate transporters are termed as the members of nitrate transporter family. ptr proteins in plants show significant sequence homology and contain several transmembrane regions, with a large hydrophilic loop among tms. generally, the identified ptr members in plants fall into three types based on the nature of their substrates: di-/tripeptides transporter, nitrate transporter and other substrates transporter. atptr was the first identified di-/tripeptides transporter in arabidopsis and had high mrna expression levels in 3-d-germinating seed, root and young leaf. the antisense plants of atptr exhibited delayed flowering time and arrested seed development. atptr transported di-/tripeptides with low selectivity as well as substrates lacking a peptide bond. atptr was expressed in the vascular tissue throughout the plant, indicative of a role in long-distance transport. atptr was a salt stress and mechanical wounding inducible gene, ja and sa were both involved in regulation of it. atptr which mediated high-affinity transport of dipeptides was most likely supplying peptides to maturating pollen, developing ovules and seeds. overexpression of it resulted in enhanced shoot growth and increased n content. the barley peptide transporter gene, hvptr had seed-specific expression. transport activity of hvptr was regulated by phosphorylation in response to rising levels of amino acids in the germinating grain. functional di-/tripeptides transporters also have been reported in nepenthes, faba bean and hakea actites. although ptr is the abbreviation of peptide transporter, many cloned ptr members in plants are nitrate transporters, especially in arabidopsis. atnrt, a dual-affinity nitrate transporter, was regulated by phosphorylation and involved in nascent organ development. atnrt encoded a low-affinity nitrate transporter. rnai plants of it exhibited reduced nitrate-induced membrane depolarization and nitrate uptake activities. the characterization of atnrt revealed the special role of petiole in nitrate homeostasis. atnrt and atnrt were both plasma membrane proteins. atnrt was expressed in root pericycle cells close to the xylem and participated in root xylem loading of nitrate while atnrt was only expressed in reproductive tissue and involved in delivering nitrate from maternal tissue to the developing embryo. csnitr a nitrite transporter in cucumis sativus, had two isoforms: csnitr1-s and csnitr1-l. in contrast to that csnitr1-s enhanced the efflux of nitrite from the cell, csnitr1-l might load cytosolic nitrite into chloroplast stroma. amino acids and other substrates can also be transported by some ptrs. bnnrt, isolated from brassica napus could transport both nitrate and histidine. agdcat localized at the symbiotic interface was a dicarboxylates transporter in alnus glutinosa and the mrna of agdcat was only detected in the nodules. in rice, only two ptr genes have been functionally verified. osnrt, which displayed low-affinity nitrate transport activity was constitutively expressed in the most external layer of the root, epidermis and root hair. sp which determined the panicle size had high expression level in the phloem of the branches of young panicles. phylogenetic analysis implied that sp might be a nitrate transporter, however, neither nitrate nor other compounds transport activity could be obtained from it. as the main staple food for a large segment of the world population, rice also serves as a model plant for monocotyledon species. nutrient transport is critical for plant growth and development, in which ptr family members may play important roles. this work focused on a comprehensive identification, characterization, phylogenetic and evolutionary analysis of all the ptr family members in rice as well as their whole-life expression profiling. moreover, it has been demonstrated that the expression of some osptr genes is regulated phytohormone and light/dark treatments.  collection and identification of ptr genes in rice domain search of msu rice genome annotation project database released sequences, out of which were unique. keywords "ptr", "peptide transporter" and "proton-dependent oligopeptide transport" searching identified and genes respectively. blastp and tblastn searches against rice genome in three databases: msu rgap, ncbi and kome released and sequences respectively. by removal of the same sequences and different transcripts of the same gene, we initially identified putative ptr genes in rice. protein sequences of these genes were subjected to pfam and smart searches for the presence of ptr domains. as a result, seven genes whose protein contained only partial ptr domain and two genes annotated as retrotransposons were excluded from further analysis. taken together, a total of genes were predicted to encode ptr proteins in rice. eighty-three genes had corresponding locus ids in msu rgap database and one gene was only found in ncbi. for convenience, all the "loc_" prefix of msu rgap locus ids were omitted representing osptr genes or proteins for further analysis. meanwhile, similar methods were used and ptr family members in arabidopsis were identified. the detailed information about the full-length cdna, transcript, chromosomal position, bac accession, gene structure for each osptr gene and characteristics of corresponding proteins can be found in additional file protein structures predicted by smart are shown in additional file the putative transmembrane regions in each osptr protein are listed in additional file chromosomal localization and gene duplication to further investigate the relationship between the genetic divergence within the ptr family and gene duplication in rice, the chromosomal location of each ptr gene was determined from the genomic sequence of rice. the osptr genes were dispersed on all the rice chromosomes except chromosome their chromosomal distribution pattern revealed that certain chromosomes and chromosomal regions had a relatively high density of osptr genes. for instance, chromosome had the highest density with members, followed by chromosome had members. ten genes were located on chromosome eight genes on chromosome and five genes on chromosome and four genes on chromosome and and only two genes on chromosome and for most chromosomes, osptr genes were present only on half arm of the chromosome or in clusters, i.e., no osptr gene appeared on the short arm of chromosome and and most of the genes on chromosome and were clustered together. analysis of the msu rgap segmental duplication database revealed that osptr genes could be assigned to rice segmental duplication blocks. os02g participated in two segmental duplications with os04g and os10g respectively. the overall identities of the protein sequences of these genes ranged from % to %. a total of genes were considered as tandem duplications according to the criterion adopted in our analysis, including the largest cluster of nine genes on chromosome two four-gene clusters on chromosome and five three-gene clusters on chromosome and six two-gene clusters on chromosome and. the protein homology of these genes varied from % to %. interestingly, all the tandemly duplicated genes in the same cluster had the same direction of transcription except os06g and os06g this might suggest the conserved behavior of tandem duplications in this family. of the osptr genes, % involved in the duplication events. moreover, most of the duplicated genes had relative close phylogenetic relationship. therefore, segmental and large-scale tandem duplication events appeared to have exclusively contributed to the expansion of the osptr gene family. similar manner of member duplication had also been described in some large gene families in rice, for instance, the ankyrin repeat gene family and the bhlh family. phylogenetic analysis and multiple sequence alignment to examine in detail the phylogenetic relationship and functional divergence of osptr members, the aligned osptr protein sequences were used to construct the joint unrooted phylogenetic tree. our result suggested that the osptr proteins could be classified into five major subfamilies designated from i to v, with high bootstrap value support. this exercise resulted in five distinct subfamilies similar to that in arabidopsis reported by waterworth and bray. within the five subfamilies formed, subfamily i, ii and iii were found to be more closely related to each other in comparison to subfamily iv and v. subfamily i had the largest ptr members and fell into three groups. subfamily ii, containing ptr members was further divided into two groups. subfamily iii also including two groups contained members. subfamily iv had members and subfamily v consisted of six members which had relative phylogenetic distance from other members. we identified pairs of osptr genes that were close paralogs on the terminal node of phylogenetic tree. tandem duplication contributed to pairs of them and segmental duplication contributed to three pairs. these results furthermore illustrated that segmental and tandem duplication events were the dominant pattern in the expansion of osptr family members. to well understand the evolutionary history of osptr family, the amino acid sequences of ptr domains in each osptr protein were used for further phylogenetic analysis and similar tree was obtained. this result indicated that the main characteristic of osptr family was determined by the ptr domain. in order to investigate the relationship between the function and phylogenetic subfamily formation of ptr members as well as identify some orthologous genes, a combined phylogenetic tree with osptr and atptr proteins was also established. as a result, similar subfamilies were formed compared to the tree of osptr. each subfamily contained both arabidopsis and rice ptr members. however, most of the members were clustered in species-specific distinct clades, and only four pairs of orthologs could be figured out. this result indicated that the main characteristics of ptr family in rice and arabidopsis were formed before the split of monocotyledonous and dicotyledonous plants and then evolved separately in a species-specific manner. moreover, the difference in the total number of osptr and atptr was mainly due to the variation in the number of subfamily i genes, which were in rice and eight in arabidopsis. of the subfamily i ptrs in rice, were tandemly or segmentally duplicated genes. the alignments and comparison of the osptr full-length protein sequences and the ptr domain sequences illustrated that most of the amino acids in tm regions in or out of the ptr domain in members of the same phylogenetic subfamily were very conservative. on the contrary, the amino acids outside the tm regions were in great variation. in addition, some loop areas between the tm regions in the ptr domain of different members varied significantly both in length and amino acid composition. the alignment of the subfamily iii members is shown in figure as an example. due to the conservatism of the ptr domain and function variety of the ptr members, the diversity of the amino acids in the loop and beyond the ptr domain areas might be the major force of functional discrepancy of each ptr member. from the comparison of the protein sequences and sequences alignment, we found three special motifs that were highly conserved in most of the osptr members. motif was found nearly before the n terminal of the ptr domain. motif whose sequence was lylxxxgxggxkxxxxxfgadqfd was located at the end of the first tm region of the ptr domain or stretched into the following hydrophilic loop before the second tm region. following the motif before/in or at the end of the second tm region of the ptr domain, motif was identified. the hmm logos of these motifs in osptr are shown in figure 4a and their locations can be found in figure in most atptr members, similar conserved motifs could also be figured out. however, motif and in atptr were located between the second and third tm regions of the ptr domain. sequence screening of motif in interproscan and uniprot databases retrieved only ptr family members. therefore, we supposed that this motif was a newly identified signature sequences associated to the plant ptr family. expression profiling of osptr genes in whole-life cycle of rice probes for of osptr genes could be found in our expression database. to analyze the expression profiles of the osptr genes, the signal values of the osptr genes represented on the array were extracted. average signal values for the osptr genes from two or three biological replicates of each sample are given in additional file a hierarchical cluster displayed of logarithm of average signal values for the osptr genes in selected tissues in minghui is presented in figure 5a. three genes had no corresponding probe set. therefore, real-time pcr was used to study the expression pattern of these genes. based on hierarchical clustering, expression pattern of osptr genes could be divided into five major groups. group consisted of genes which showed high expression levels in all the tissues expect six genes that showed relatively low expression in certain tissues. conversely, group contained genes that showed low expression level in the whole life cycle. group was comprised of genes that showed relatively high expression level in both vegetative tissues and hull. all these genes had low expression level in early panicle and endosperm stages. group comprised of genes with medium expression level in all the tissues except that os04g had high expression in root, os01g in e os11g in e1-e and os01g in e1-e the rest genes belonged to group although these genes had different expression patterns. for example, os04g had high expression signal in root and stamen, os04g showed high expression in fl os11g had high expression in panicle and stem. interestingly, os01g os01g os11g os10g and os06g all showed relatively high expression in endosperms. the expression patterns of most osptr genes in zhenshan were similar to that in mh and the corresponding hierarchical cluster was shown in additional file for further study regarding the expression pattern and looking inside the putative functions of osptr family members, we employed the analysis of preferential and tissue-specific expression genes in mh and zs the identified preferential expression genes were mainly expressed in stamen and root. os04g was preferentially expressed in stamen of one day before flowering and os04g was in root of seedling with two tillers in mh in zs os01g and os12g were identified as stamen preferential expression genes, while os02g and os04g were preferentially expressed in root. the check of our whole genome expression profile of rice for different tissues obtained several tissue-specific expression genes including os04g in root of mh and in flag leaf of 14-day after flowering in mh as well as zs os02g in root of mh and zs and os01g in endosperm of 14-day after pollination in zs. the hierarchical cluster of the atptr genes in development stages released the expression profile of genes except at1g at3g and at3g. obviously, the expression patterns of atptr genes fell into the similar groups as osptr genes. some tissue-specific expression atptr genes could also be identified, for example, at1g at4g and at3g in seed and siliques, at3g and at3g in root. most of the atptr genes had relatively low expression level in mature pollen except at1g at5g and at5g these genes were most likely supplying nutrition to maturating pollen. some of the orthologous osptr and atptr genes showed the similar expression pattern such as os06g and at1g os05g and at2g they both had high expression level in all the tissues. however, no more apparent relationship between the expression pattern and sequence similarities of osptr and atptr members could be pointed out. expression regulation of osptr genes under phytohormone and light/dark treatments to determine the osptr genes in response to phytohormone treatments, microarray analysis was performed. we identified six and nine osptr genes that were differentially expressed under one or more of the phytohormone treatments compared with the control in mh and zs respectively. among these genes, only one gene was in common for two genotypes and it was down-regulated under three phytohormone treatments. the expression profile of the remaining genes in two genotypes was different. for instance, in mh os01g and os10g were down-regulated under naa and ga treatments while os04g and os06g were down-regulated specifically to kt and ga treatment, respectively. os01g was up-regulated specifically to naa treatment. five genes were up-regulated and four genes were down-regulated in zs the up-regulated genes were os01g os01g os01g os01g and os04g while the down-regulated genes were os01g os06g os11g and os12g os01g and os12g were both down-regulated under three phytohormone treatments. os01g and os04g were both up-regulated under naa and kt treatments. os01g was up-regulated under naa and ga treatments. os01g and os11g showed differential expression specifically to kt treatment while os01g and os06g showed differential expression to ga treatment. the fold changes with respect to control of differential expression genes under phytohormone treatments are given in additional file to investigate possible light regulation of osptr genes, the expression profile of osptr genes in seedlings treated with light or dark for hours after emergence was also analyzed. twelve and seven genes were differentially expressed under light compared to dark in plumule and radicle, respectively. eight genes showed differential expression in plumule and four genes in radicle in mh interestingly, all the differentially expressed genes were up-regulated in plumule under light compared to dark while two of four genes in radicle were down-regulated. os01g was up-regulated nearly 10-fold under light compared to dark in plumule. in zs five genes were differentially expressed in plumule and three genes in radicle. interestingly, two genes were differentially expressed in both plumule and radicle in zs the fold changes with respect to control of light/dark regulation genes are listed in additional file ka/ks analysis and express pattern divergence of paralogous osptr genes involved in duplication to explore the mechanism of gene divergence after duplication, the coding sequences of the paralogous osptr genes involved in duplications were used to calculate the synonymous and non-synonymous substitution rate after duplication. the ka/ks ratio provides information about the degree of positive selection across all nucleotides in a gene since divergence. generally, ka/ks = means neutral selection, ka/ks < indicates purifying selection and ka/ks > shows the accelerated evolution with positive selection. the ka/ks analysis demonstrated that ka/ks ratios for all the paralogous osptr genes were always less than one, most of them even close to zero, suggesting that purifying selection might be largely responsible for function maintenance of this family. paralog gence ka: non-synonymous substitution rate. ks: synonymous substitution rate. nf: neofunctionalization. sf: subfunctionalization. nof: nonfunctionalization. d: just after duplication. comparison of expression pattern shifts of the duplicated paralogous genes can reflect the divergence hypotheses that a duplicate gene pair might be involved in: nonfunctionalization, subfunctionalization and neofunctionalization. the expression profile of the pairs of paralogous osptr genes with corresponding probes was extracted for analysis. obviously, the pairs of paralogous osptr genes had divergent expression patterns. it seemed that all the three hypotheses influence the fate of the duplicated genes. two pairs of genes showed almost the same expression pattern in both copies, indicating they were just formed due to duplication. the fate of four pairs in figure 9b could be described as nonfunctionalization, where one copy of the paralog lost expression in all tissues. subfunctionalization explained for five pairs of paralogous genes, in which the expression patterns of both copies were complementary to the pattern before duplication. the divergence hypotheses of the rest six pairs of paralogous genes were assigned as neofunctionalization, where the expression of one copy had an obvious increase in one or more tissues. discussion di-/tripeptides and nitrate transport is important for various aspects of plant growth and development, in which peptide transporter family members may play important roles. considering the potential functional diversity of ptr members associated with nutrient transport and only a few ptr family members have been described in higher plants, it was timely and quite relevant for us to characterize the rice ptr family. combination of the whole genome sequence data, sequences alignment and high-throughput expression analysis undertaken in this investigation provides an insight into all the members of ptr family in rice. duplication played an important role in the expansion of the osptr family it is well known that gene duplication is important for gene family evolution and diversity, which can occur via three major mechanisms: segmental duplication, tandem duplication and retroposition. due to repetitive plant genome duplications, certain plant genomes are likely to harbor relatively large members of a gene family, as compared to animal genomes. the ptr families in rice and arabidopsis are very much in line with the statement and showed increased family members in order to adapt for specific functions. the number of osptr and atptr members is far greater as compared to the low number of six in human, four in c. elegans, three in drosophila and two in yeast. it may indicate the importance of these transporters in plant growth and metabolism. nearly 70% of the osptr genes are duplicated genes, genes are found to be involved in the segmental duplication while participated in tandem duplication. our analysis also indicated that in subfamily i, duplication events were largely responsible for the number difference of the member between osptr and atptr. it demonstrates that the osptr family may undergo extensive and rapid genomic changes which can maintain relative large number of member and lead to expression divergences between duplicate genes, further contributing to the establishment of novel gene functions. phylogenetic relationship and functional diversity among the osptr members the phylogenetic analysis reveals that the osptr family can be further divided into five subfamilies. the combined phylogenetic tree of osptr and atptr showed similar subfamily formation whereas most ptr members were clustered in species-specific clades and only four pairs of orthologs could be identified. it suggests that the members of the ptr family have diverged from each other primarily due to speciation and then undergone significant changes since the monocot/dicot split. however, highly conserved motifs have been identified in most members of atptr and osptr. we further adopted the sequence alignment including the ptr members in plant, animal, human and prokaryotic organisms. it was surprising to find that the three motifs could be found in most eukaryotic ptr members but changed a lot or lost in the prokaryotic ptr members, especially motif and motif moreover, some members such as os01g os04g os12g and at2g which had far phylogenetic relationship from other ptr members, had great amino acid variation in motif and motif we inferred that motif and motif might be important characteristic logos of the eukaryotic ptr members and played considerable roles in the function formation of the ptr family. further research may employ the point mutations introduced into these motifs, which should cause a loss of function of the corresponding protein. this experimentation could indicate the essentiality of these motifs for the formation of the transporter pore and the interaction with the substrates. the sequence similarity among the osptr members seemed to show little relationship with their putative transporter type classification. in addition, we found that gene structures, intron phases of osptr genes and distribution of tm regions in the proteins had no relationship with their phylogenetic subfamily and transporter type classifications. as most osptr proteins had ptr as their only recognizable domain, we adopted the meme method, in order to identify some new motifs or domains. however, the conserved motifs investigated by meme were all tm regions. we infer that the substrate selection of osptr proteins may be decided by their three dimensional structures. the determination of the di-/tripeptide, nitrate and other type transporters in this family by mutagenesis or crystal structure studies should solve the puzzle that di-/tripeptides, nitrate and other substrates of ptr proteins are very different in structure but they share the same transporter family - ptr. expression pattern divergence of the osptr genes microarray provides a rich resource for investigating the evolution of gene expression. the temporal and spatial expression patterns of osptr genes are useful information for establishing their putative functions. our microarray analysis indicated that the expression patterns of the osptr genes could be classified into five groups. we also identified some preferential or tissue-specific expression osptr genes. of the genes analyzed in this study, only seven were found to exhibit either preferential or tissue-specific expression during different development stages of mh and zs interestingly, these genes were most preferentially or specifically expressed in root and stamen. os02g os04g and os04g were preferentially expressed in root. moreover, these three genes were in the same phylogenetic clade that belonged to the group three of subfamily i. in arabidopsis, the expression of atnrt was found concentrated in the tips of primary and lateral roots. atnrt was primarily expressed in root hairs and the epidermis in both young and mature regions of roots. osnrt was constitutively expressed in the most external layer of the root, epidermis and root hair. our data showed that osnrt had high expression levels in all the tissues especially in root. os03g and os03g were the paralogs of osnrt moreover, these three genes formed a tandem duplication cluster. os03g and os03g showed similar expression patterns with osnrt and os03g had the highest expression level in root. it can be inferred that these root-preferentially or -specifically expressed osptr genes might be nitrate transporters and play important roles in root formation and development. atnrt was expressed in the leaf petiole and participated in regulating leaf nitrate homeostasis. the ortholog of atnrt in rice was os01g which continuously expressed in vegetative tissues with high level in leaves. expression of os04g was specific in flag leaf of 14-day after flowering both in mh and zs therefore, we infer that os01g and os04g might be participated in the development of leaf as well as the nutrient transporting in and/or out of leaf. in addition, we also identified three genes that were preferentially expressed in stamen and one gene in endosperm. one interesting phenomenon in our dendrogram was that three-quarter of the osptr genes had significant low expression level in panicles of different development stages. sp whose transcription accumulated in panicle was one of the exceptions. sp was highly expressed in the phloem of the branches of young panicles, which was consistent with the predicted function of sp and the phenotype of sp mutant. it can be demonstrated that some ptr members might also play roles in nutrient transport in reproductive organ development. potential involvement of osptr members in phytohormone and light/dark regulations little attention has been paid on the functions of ptr members in phytohormone and light/dark regulations. until now, atnrt was the only reported ptr gene that was involved in these regulations. atnrt was regulated by auxin in both shoots and roots and involved in light-induced stomatal opening. however, our microarray data showed that the expression patterns of more than one-third osptr genes changed significantly under phytohormone and light/dark treatments. fourteen osptr genes are regulated by at least one phytohormone and osptr genes are involved in the regulation of light/dark treatment in mh and zs from these results, it can be speculated that these differentially expressed osptr genes may play an important role in the transport of regulatory molecules and other metabolites induced by regulation of phytohormone and light/dark treatments. for example, iaa is an important plant growth phytohormone and 95% of the iaa pool is sequestered in an inactivated conjugated form, e.g. iaa can be conjugated to single amino acids, peptides or proteins via amide bonds. these conjugates may be involved in regulating phytohormone activity in response to plant growth and development, as well as facilitating hormone transport. it seems that the proteins of those osptr genes regulated by naa might have iaa conjugates transport activity. fate of the paralogous osptr genes after duplication from the ka/ks analysis of the paralogous osptr genes, it is obvious that purifying selection plays an important role in function maintenance of this family. however, comparison of the expression data implied that after duplication, the paralogous pairs may have been evolved in different hypotheses to gain functional divergence. expression data shows that some of these duplicated genes have undergone subfunctionalization and neofunctionalization, which contribute to the possibility of maintenance and divergence of a paralogous set of genes. some paralogs have adopted nonfunctionalization which contributes to the likelihood of loss of one member in a pair of paralogous genes. however, sufficient experimentation is required to prove these hypotheses.  in conclusion, the results of this study display the genomic framework, characterization, conserved motifs, duplication manner and evolutionary analysis of the osptr members, along with their expression profiles during whole-life stages as well as under phytohormone and light/dark treatments. these data will provide the basis for understanding the evolutionary history of ptr members and their roles in rice growth and development. the findings in our work would be useful in selecting candidate genes for functional validation studies of ptr members in rice. however, future research by adopting rnai/overexpression strategies or insertion mutagenesis is required to elucidate the precise role of individual osptr gene. 
ka/ks analysis of the paralogous osptr genes indicates that purifying selection plays an important role in function maintenance of this family. under phytohormone and light/dark treatments, and osptr genes are differentially expressed respectively. meanwhile, expression profile of osptr genes has been analyzed by using affymetrix rice microarray and real-time pcr in two elite hybrid rice parents, minghui and zhenshan seven genes are found to exhibit either preferential or tissue-specific expression during different development stages of rice. these investigations add to our understanding of the importance of osptr family members and provide useful reference for selecting candidate genes for functional validation studies of this family in rice. chromosomal distribution and sequence analysis indicate that nearly 70% of osptr members are involved in the tandem and segmental duplication events. highly conserved motifs were identified in most of the osptr members. peptide transporter family whose member can transport di-/tripeptides and nitrate is important for plant growth and development. in this study, we report a comprehensive identification, characterization, phylogenetic and evolutionary analysis of ptr family members in rice as well as their whole-life expression patterns. although the rice genome has been sequenced for a few years, a genomic survey, characterization and expression profile analysis of the ptr family in this species has not been reported.
 peptide transporter family whose member can transport di-/tripeptides and nitrate is important for plant growth and development. although the rice genome has been sequenced for a few years, a genomic survey, characterization and expression profile analysis of the ptr family in this species has not been reported.  in this study, we report a comprehensive identification, characterization, phylogenetic and evolutionary analysis of ptr family members in rice as well as their whole-life expression patterns. chromosomal distribution and sequence analysis indicate that nearly 70% of osptr members are involved in the tandem and segmental duplication events. it suggests that genome duplication might be a major mechanism for expansion of this family. highly conserved motifs were identified in most of the osptr members. meanwhile, expression profile of osptr genes has been analyzed by using affymetrix rice microarray and real-time pcr in two elite hybrid rice parents, minghui and zhenshan seven genes are found to exhibit either preferential or tissue-specific expression during different development stages of rice. under phytohormone and light/dark treatments, and osptr genes are differentially expressed respectively. ka/ks analysis of the paralogous osptr genes indicates that purifying selection plays an important role in function maintenance of this family.  these investigations add to our understanding of the importance of osptr family members and provide useful reference for selecting candidate genes for functional validation studies of this family in rice. 