BACKGROUND
duplications are recurrent in the evolutionary history of plant genomes.
whole genome duplications  are described for dicotyledons and monocotyledons  <cit> .
it is estimated that the incidence of polyploidy in angiosperms is 30-80%, and ploidy changes may represent about 24% of speciation events  <cit> .
duplication generates two copies of each gene, and the fate of duplicated genes was first described by ohno: one copy should maintain the ancient function and another copy should lose function  or gain a new function   <cit> .
this model was improved, giving rise to the duplication-degeneration-complementation  model, where the duplicated copies can have complementary functions that resemble the ancestral gene's function   <cit> .
the ddc model's predictions are believed to be more accurate than the previous model, since loss-of-function changes in regulatory regions are more likely to occur than gain-of-function mutations  <cit> .
other improvements of the basic model for duplicated gene retention, involving buffering of crucial functions via conversion and crossing-over, were recently proposed  <cit> .
due to repetitive genome duplications, plants are likely to harbor relatively larger gene families, as compared to animal genomes  <cit> .
it is well established that one whole-genome duplication occurred in the cereal lineage, estimated  <dig> million years ago , preceding the radiation of the major cereal clades by  <dig> million years or more  <cit> .
recently, comparing the genomic sequences of rice  and sorghum bicolor, it was demonstrated that an early duplication occurred in the monocot lineage  <cit> .
the duplication blocks cover at least 20% of the cereals transcriptome  <cit> .
it was also shown that expression divergence between duplicate genes is significantly correlated with their sequence divergence  <cit> .
after duplication, gene pairs rapidly diverge, and only a small fraction of ancient gene pairs do not show expression divergence  <cit> .
however, for some genomic segments, concerted evolution homogenizes homologous sequences through unequal crossing-over and gene conversion, changing the estimated duplication age and gene divergence  <cit> .
rice was first described as having  <dig> duplicated segments which cover  <dig> % of its genomic sequence, and several individual gene duplications  <cit> .
more recent estimates account for  <dig> duplications in the rice genome, including  <dig> minor blocks that overlap with  <dig> major blocks  <cit> .
a duplication block between chromosomes  <dig> and  <dig> has been extensively characterized in rice and other cereals, although the age of its birth is still controversial  <cit> .
rice is a model for cereal genomic and genetics studies, due to the availability of the genome sequences from two varieties, extensive gene annotation, and mutant resources  <cit> .
rice is also a major staple food, feeding nearly half of the world's population.
however, it is a poor source of minerals such as iron  and zinc , the two mineral elements most commonly lacking in human diets  <cit> .
metal homeostasis in plants has been extensively studied in recent years, with a special focus on the transition metals zn and fe  <cit> .
thus, rice emerges both as a model species for physiological and molecular studies and as a candidate for biotechnological improvement aiming at zn and fe biofortification  <cit> .
both zn and fe are essential to mineral nutrition of plants.
zn has a key role in gene expression, cell development and replication, while fe is necessary for photosynthesis, electron transport and other redox reactions  <cit> .
although essential, both can be toxic when in excess  <cit> .
several transporters involved in uptake and translocation inside the plant were described for fe and zn  <cit> .
the zinc-induced facilitator  <dig> gene , described by haydon and cobbett, belongs to a new family of transporters, with three members in arabidopsis thaliana: atzif <dig> , atzifl <dig>  and atzifl <dig>   <cit> .
the atzif <dig> transporter is clearly involved in zn homeostasis, as the loss-of-function atzif <dig> mutant has altered zn distribution and its transcription is up-regulated by zn-excess  <cit> .
importantly, atzif <dig> proteins are expressed in the tonoplast, and probably are involved in transport of zn, zn and a ligand or a ligand alone, to the vacuole  <cit> .
besides atzif <dig>  only one similar protein had been previously characterized: the maize  zm-mfs <dig>  which is induced by infection by the pathogens cochliobolus heterostrophus and c. carbonum and to ultraviolet light  <cit> .
this gene is highly expressed in the les <dig> disease lesion mimic background and in plant tissues engineered to express flavonoids or the avirulence gene avrrxv  <cit> .
both atzif <dig> and zm-mfs <dig> are part of the major facilitator superfamily , which comprises the largest superfamily of secondary transport carriers found in living organisms and is subdivided in at least  <dig> families  <cit> .
more recently, atzif <dig> and atzifl <dig> were described as quantitative trait loci  candidates for zn concentrations in arabidopsis seeds  <cit> .
in barley , microarray analyses revealed that a zif1-like gene is expressed in the aleurone layer of seeds and its transcription increases in the embryo upon foliar zn application  <cit> .
therefore, it is possible that zifl genes are involved in zn translocation to the seeds.
in this work, we describe the zif-like  family of transporters.
we identified  <dig> family members from plants and reconstructed their phylogenetic relationships.
we also analyzed in detail the organization of zifl genes in the rice  genome: the motif composition, genomic organization, and promoter sequences.
we analyzed the expression of oszifl genes in different plant organs and developmental stages, as well as in response to different stresses.
this is the first attempt to describe the zifl gene family in plants, and the first expression analysis of these genes in rice.
RESULTS
zifl genes in plants
we first used the atzif <dig>  atzifl <dig> and atzifl <dig> sequences to query genomic databases to determine the distribution of this gene family among plant species.
two dicots, vitis vinifera and populus trichocarpa, one bryophyte, physcomitrella patens, one lycophyte, selaginella moellendorffii, and four monocots, sorghum bicolor, brachypodium distachyon, oryza sativa and zea mays had their genomes screened for zifl genes.
all sequences found through this search plus the three arabidopsis sequences were used to generate a hidden markov model  profile to iteratively search the same genomes .
the final dataset consists of  <dig> genes coding for proteins already annotated  and two unannotated proteins from zea mays .
all organisms queried contain zifl sequences, with predicted protein sequences ranging from  <dig> to  <dig> amino acids and an average of  <dig>  amino acids per protein.
all gene sequences begin with an initiation codon and end with a stop codon, except for the protein ppzifl <dig>  which lacks a small n-terminal portion  and was included in the analyses.
the overall structure contains  <dig> to  <dig> predicted transmembrane  domains , found in 63% of the proteins in our dataset.
fourteen putative proteins are predicted to have  <dig> tm domains, and  <dig> proteins have seven to nine tm domains .
dicot species have a small number of zifl gene copies, with v. vinifera and p. trichocarpa showing five and four paralogs of zifl in their genomes, similar to the three members of the arabidopsis zifl gene family  <cit> .
conversely, monocot species show a higher number of zifl genes, with s. bicolor having the highest number of members , followed by rice , b. distachyon  and z. mays .
p. patens and s. moellendorffii harbor two and seven zifl genes, respectively.
clearly, monocot species have a higher number of zifl family paralogs than dicots.
the seven zifl genes found in s. moellendorffii seem to be closely related and not originated from the same expansion which originated the monocot zifl genes.
zifl proteins are a distinct family of mfs transporters
the zifl proteins are all part of the major facilitator superfamily  clan of transporter proteins , composed by  <dig> families.
they show similarity to the mfs_ <dig> family , which is the largest family within the mfs clan.
we used the mfs_ <dig> hmm profile to isolate the mfs_ <dig> proteins from all dicot and monocot genomes analyzed in this work.
phylogenetic trees reconstructing the evolutionary history of mfs_ <dig> and zifl proteins for each species were generated using the neighbor-joining method .
we observed that in all cases the zifl proteins clustered in a separate group from all other mfs_ <dig> members.
this result could be an indication that zifl is a distinct family of mfs transporters.
simmons et al suggested that sequences similar to zm-mfs <dig>  could be a distinct group of mfs proteins found in plants  <cit> .
this was based on comparison of signature sequences of nine plant sequences to bacterial and fungal mfs sequences.
to confirm this hypothesis, we searched for signatures in the zifl hmm profile and aligned them to the mfs_ <dig> hmm profile.
we found the canonical mfs signature, located in the cytoplasmic loop between tm <dig> and tm <dig>  as well as the antiporter signature in tm <dig> .
when aligning these signatures to the mfs_ <dig> hmm profile, we noticed that the zifl mfs signature g-x-d--x-g-r- has a conserved tryptophan  before the first glycine position, which is not observed in mfs_ <dig> .
the antiporter signature, s-x-g-x-g-p-x-g-g, is also slightly different, having preference for serine in the first position, instead of glycine, as observed by simmons et al   <cit> .
the presence of these conserved positions indicates that zifl transporters share structural and functional similarities with mfs antiporters, although they show specific features that are not common to other mfs proteins.
the zifl sequences also show signatures that are not shared with mfs_ <dig> proteins.
the conserved positions in the loop between tm <dig> and tm <dig>  -x-g-p--x-r, previously reported by simmons et al, were confirmed in our dataset with a few changes   <cit> .
importantly, we found two new conserved signatures that are specific for the zifl proteins.
one of them is a cysteine -containing motif c--g-c in the cytoplasmic n-terminal loop of zifl proteins, and the second one is a histidine -containing motif -e---h-x- in the cytoplasmic loop between tm <dig> and tm <dig>  before the beginning of a variable region .
from our dataset of  <dig> zifl proteins,  <dig> have the cys motif, with only three proteins showing a serine residue in the second position instead of a proline .
for the histidine motif,  <dig> zifl proteins have the conserved residues .
from these,  <dig> have the most conserved residues p-e-t-l-h-x-h, while the other  <dig> zifl members contain the same motif with no more than one residue substitution .
considering that the mfs_ <dig> family has  <dig>  proteins with very low overall similarity between them, and that zifl proteins share both high similarity and unique signatures, we suggest that zifl proteins comprise a distinct family of transporters.
zifl gene expansion is lineage specific
to address the hypothesis of a lineage specific expansion of zifl genes in monocot species, we generated an alignment using the amino acid sequences of the  <dig> zifl genes found and reconstructed the phylogenetic relationships of these protein sequences using two methods: neighbor-joining and bayesian analysis .
although some nodes are not in agreement comparing the two methods, our bootstrap values and posterior probabilities support all the major nodes of the tree, indicating that the reported group relationships are reliable .
proteins from bryophyte and lycophyte species grouped together, with paralogs from each species in a separate cluster.
the zifl proteins from dicots also formed a distinct group .
however, there was no clear separation into sub-groups of orthologous sequences within the dicots group .
species-specific gene duplications are observed in arabidopsis , v. vinifera  and p. trichocarpa  .
the zifl paralogs from monocot species were grouped in three distinct groups, named monocot i, monocot ii and monocot iii.
all three zifl protein groups from the monocots contain paralogs from the four species included in our analysis.
the monocot i group contains  <dig> zifl proteins, while the monocot ii and monocot iii groups contain  <dig> proteins each .
both the number of sequences found in monocot species and the tree topology strongly suggest that the zifl gene family experienced an expansion in the monocot lineage, and that the last common ancestor of the monocots already had zifl paralogs of the three groups .
thus, the split of the four monocot species used in this work probably occurred after the expansion of the zifl family observed in monocots, and this expansion is not shared with other plant lineages.
zifl paralogs are unequally distributed in the rice genome
the identification of the zifl gene chromosome locations revealed that they are not evenly distributed in the rice genome, but rather arranged in clusters .
the same trend is observed in s. bicolor and b. distachyon, but not in z. mays .
rice zifl genes were named zifl <dig> to  <dig> based on their genomic locations.
two zifl genes, oszifl <dig> and oszifl <dig> are located in chromosome  <dig>  and oszifl <dig> is located in chromosome  <dig>  oszifl <dig>  oszifl <dig>  oszifl <dig>  oszifl <dig> and oszifl <dig> are found in chromosome  <dig>  while oszifl <dig>  oszifl <dig>  oszifl <dig>  oszifl <dig> and oszifl <dig> are located in chromosome  <dig>  interestingly, the zifl genes arranged in tandem in chromosomes  <dig> and  <dig> are closely related, with oszifl <dig> being very similar to oszifl <dig> , oszifl <dig> to oszifl <dig> , oszifl <dig> to oszifl <dig> , oszifl <dig> to oszifl <dig>  and oszifl <dig> to oszifl <dig>  .
we used the gata tool to align the  <dig> kb regions that include oszifl genes in chromosomes  <dig> and  <dig> .
the regions of chromosomes  <dig> and  <dig> where these genes are located have already been described as a recent segmental duplication in the rice genome, what would explain the high number of matches between these regions   <cit> .
however, the same duplication was recently found in s. bicolor, indicating that this segmental duplication is ancient to the split of these species  <cit> .
we observed that s. bicolor chromosomes  <dig> and  <dig> , which are homologous to rice chromosomes  <dig> and  <dig> , harbor three and two zifl genes, respectively   <cit> .
an incomplete sequence related to zifl is also found in chromosome  <dig> .
it is possible to observe that an inversion has occurred when comparing the orientation of zifl regions in sb <dig> and sb <dig> .
the alignment between rice and s. bicolor homologous chromosomes os <dig> with sb <dig> and os <dig> with sb <dig> demonstrate that the s. bicolor zifl region in sb <dig> is inverted, since the alignment of os <dig> with sb <dig> is in direct orientation  while the alignment of os <dig> with sb <dig> is in reverse .
therefore, although in homologous regions, the zifl gene cluster in sb <dig> is differentially oriented in relation to rice.
a rice zifl gene pairs from chromosomes  <dig> and 12
oszifl genes organization is highly conserved
we aligned the genomic and coding sequence  of each zifl gene from rice and determined the exon-intron organization .
the exon sizes of each gene pair, oszifl4-oszifl <dig>  oszifl5-oszifl <dig>  oszifl6-oszifl <dig>  oszifl7-oszifl <dig> and oszifl8-oszifl <dig> are nearly identical, with very few variations in sequences.
we observed that oszifl <dig> and oszifl <dig> are probably originated from duplication, since they share a similar exon-intron organization.
however, their amino acid sequences are only 57% identical .
this duplication probably occurred in the common ancestor of monocots, as orthologs from s. bicolor, b. distachyon and z. mays were found for both oszifl <dig> and oszifl <dig> .
oszifl <dig> is suggested to be originated from a partial duplication of the oszifl8-oszifl <dig> pair last common ancestor , and shares more identities to oszifl <dig> sequence  than to oszifl <dig> .
thus, it is clear that duplications were of major importance in the zifl family expansion in rice, especially the segmental duplication observed in chromosomes  <dig> and  <dig>
protein motif composition reveals a variable region in the zifl family
we aligned the  <dig> rice zifl proteins and observed that they share large similarity .
to search for functional sites shared by oszifl putative proteins, we used meme  to identify conserved motifs in their amino acid sequences  <cit> .
we found eleven motifs shared by almost all  <dig> oszifl proteins, with few exceptions .
seven motifs matched the general mfs_ <dig> motif in interproscan  , while four showed no hits  .
the zifl signatures cys motif and his motif are located within the motif  <dig> and motif  <dig>  respectively .
amfs
banalysis performed with whole set of zif proteins
oszifl <dig>  oszifl <dig>  oszifl <dig>  oszifl <dig>  oszifl <dig> and oszifl <dig> have all eleven motifs, while the duplicated pair oszifl8-oszifl <dig> and their duplicated copy in chromosome  <dig>  lack several motifs .
some of these motifs are located in regions predicted to be transmembrane .
further characterization is needed to determine if the duplicated rice zifl genes are becoming pseudogenes or acquiring new functions.
the oszifl <dig> duplicated copy oszifl <dig> lacks the n-terminal motif  <dig> and the c-terminal motif 10; oszifl <dig> lacks motif  <dig> and its duplicated copy oszifl <dig> lacks motif  <dig> and motif 10; the duplicated pair oszifl <dig> and oszifl <dig> only differ by the c-terminal motifs  <dig> and  <dig>  which are absent in oszifl <dig> .
these differences suggest a divergence process between duplicated pairs.
moreover, it is clear that the central motifs are more conserved than those located at the n- and c-terminal regions of oszifl proteins .
we also observed a variable region between motifs  <dig> and  <dig> which did not show significant pattern conservation in oszifl proteins .
this region is located between transmembrane regions  <dig> and  <dig>  and is a cytoplasmic loop according to conpred ii predictions .
the variable region is preceded by the conserved his motif p-e-t-l-h-x-h .
variable regions are found in transporters and could be involved in transport or sensing functions  <cit> .
the whole set of  <dig> zifl proteins used in this work was submitted to meme analysis to find any conserved motifs specifically in the variable region.
three motifs were found in this region and named motifs a, b and c .
none matched any known motif in the interpro database .
we indicated proteins that contain each motif in our phylogenetic tree  and showed their positions in rice zifl protein sequences .
rice zifl proteins contain motifs a and b in their variable region, but not motifs c.
motif a is present in proteins from the monocot i, monocot ii, dicot and bryophyte-lycophyte groups .
this motif shows low amino acid conservation .
the negatively charged glutamic acid  residue in the seventh position of the motif is the most conserved residue.
conserved negatively charged residues are also found in the fourth position .
between these positions, two non-polar residues, alanine  and leucine  are also conserved .
other positions containing a positively-charged residue of lysine , a negatively charged glutamic acid , and residues of leucine  and glycine , although less conserved, are present .
charged positions could be involved in transporter specificity, as already described for cation diffusion facilitator  proteins  <cit> .
motif b is shared only by a sub-group of six proteins from monocot ii .
the fifth and seventh positions of this motif contain one positively charged residue and one hydrophobic residue, lysine  and leucine  .
polar residues of serine , glutamine  and tyrosine , non-polar tryptophan  and proline  are also observed .
the motif c is common to  <dig> proteins from the monocot i group , and is similar to motif a, showing the two glutamic acids  separated by one instead of two non-polar residues .
however, since only a small number of proteins share motifs b and c, we should be cautious on making assumptions about the functionality of conserved amino acids found in these motifs, as their conservation could be an effect of phylogenetic relatedness and not of evolutionary constraints.
importantly, it is possible to observe the high divergence of the variable region even when comparing these three motifs.
the variability is much higher in this region than in the whole sequence of zifl proteins, as meme analysis revealed several motifs shared by all the  <dig> zifl proteins .
therefore, these motifs in the cytoplasmic loop could be involved in specific functions of different zifl proteins.
expression of oszifl genes in rice vegetative and reproductive organs
we analyzed the expression levels of oszifl transcripts in several rice organs by qpcr, including roots, culms and shoots ; flag-leaves and whole panicles , both during r <dig> , r <dig>  and r <dig>  stages .
throughout our qpcr experiments, oszifl <dig>  oszifl <dig>  oszifl <dig>  oszifl <dig> and oszifl <dig> transcripts were not detected or were detected below a confidence threshold for analysis.
the expression levels of oszifl genes varied considerably, with some genes reaching higher expression levels  and others showing very low expression .
oszifl <dig> and oszifl <dig>  although not resultant of a duplication event, share a similar pattern of expression: both are more expressed in leaves and also accumulate in the later stages of flag-leaf development, reaching the highest levels in r <dig> .
when analyzing gene pairs, we observed that oszifl <dig> is almost specifically expressed in roots, showing only little expression in panicles during the r <dig> stage , while its duplicated copy oszifl <dig> is not expressed in vegetative tissues nor in flag-leaves, but is detected at low levels in panicles during r <dig> and at higher levels during r <dig> .
transcripts from the oszifl5-oszifl <dig> pair show similar patterns of expression, especially when considering the reproductive organs flag-leaves and panicles .
oszifl <dig> and oszifl <dig> are both induced from r <dig> to r <dig> in flag-leaves, maintaining high levels at r <dig>  in panicles, they are also induced from r <dig> to r <dig>  although oszifl <dig> transcript levels are further induced from r <dig> to r <dig> .
in vegetative tissues, oszifl <dig> levels are higher in roots, while oszifl <dig> is more expressed in shoots .
the genes from the oszifl7-oszifl <dig> pair also show similar expression patterns in the organs analyzed.
oszifl <dig> is more expressed in culms and leaves, accumulates from r <dig> to r <dig> in flag-leaves and decreases its expression from r <dig> to r <dig> during panicle development .
the oszifl <dig> transcript accumulates in leaves and also increases from r <dig> to r <dig> in flag-leaves and decreases from r <dig> to r <dig> in panicles .
taken together, our gene expression data demonstrates that, even after duplication and divergence, most oszifl genes still share similar expression patterns in rice organs within gene pairs.
the fe-deficiency element ide <dig> is enriched in promoters of oszifl genes
to investigate the presence of conserved cis-elements in promoter regions of oszifl genes, we used the poco tool  <cit> .
this approach consisted in comparing the - <dig>  to + <dig> regions of oszifl genes to several random samples of promoters from the entire arabidopsis genome with the same size .
if a cis-element is more often found in the promoters of oszifl genes than in a random set of promoters, this cis-element is enriched in these sequences.
the poco analysis revealed that the sequence catgc is enriched in our promoter set when compared to arabidopsis promoters.
this sequence is the core binding site of idef <dig> , a transcription factor of the abi3/vp <dig> family involved in fe-deficiency response in rice  <cit> .
as arabidopsis is not closely related to rice and thus the motif frequency in promoters could vary between these species, we confirmed the enrichment by counting the average number of catgc boxes in nearly  <dig>  promoters of rice downloaded from osiris   <cit> .
while the average number of the catgc sequences in rice promoters was  <dig> , in promoters of the thirteen oszifl genes it was  <dig>  boxes per promoter.
some promoters are highly enriched for catgc boxes, such as oszifl <dig> , oszifl <dig> , oszifl <dig>  and oszifl <dig>  .
genes that were not detected in our qpcr experiments such as oszifl <dig> and oszifl <dig> also have promoters enriched in catgc boxes  .
oszifl <dig>  oszifl <dig> and oszifl <dig> promoters show  <dig> boxes each .
since the catgc box is the core motif of ide <dig>  we searched for ide1-like sequences in promoters of oszifl genes following the method described by kobayashi et al.
<cit> .
we found eleven ide1-like motifs distributed in seven gene promoters, oszifl <dig>  oszifl <dig>  oszifl <dig>  oszifl <dig>  oszifl <dig>  oszifl <dig> and oszifl <dig> .
oszifl <dig> shows three sequences, two of them overlapping with catgc boxes, while oszifl <dig> and oszifl <dig> show two ide1-like motifs .
considering that the motif is  <dig> bp long, it is surprising to find such a high number of ide1-like motifs in our promoter set.
the enrichment for catgc and ide1-like sequences in promoters of oszifl genes suggests that they are possibly regulated by fe-deficiency.
zn-excess and fe-deficiency regulate oszifl expression mainly in rice roots
it has been demonstrated that atzif <dig> is up-regulated by zn-excess in roots and leaves of arabidopsis plants, as well as by fe-deficiency  <cit> .
as promoters of oszifl genes are enriched for fe-deficiency cis-elements, we submitted rice plants to zn-excess  for three days and to fe-deficiency  for seven days.
oszifl mrna expression level was evaluated by qpcr in roots and leaves from both experiments.
several oszifl genes were up-regulated in roots of zn-excess treated plants: oszifl <dig>  oszifl <dig>  oszifl <dig>  oszifl <dig>  oszifl <dig> and oszifl <dig> .
expression of oszifl <dig>  oszifl <dig>  oszifl <dig> and of the duplicated pairs oszifl6-oszifl <dig> and oszifl8-oszifl <dig> was not detected.
expression of oszifl <dig>  which is nearly root-specific , is induced  <dig> -fold by zn-excess .
both oszifl <dig> and oszifl <dig>  a duplicated pair, are also up-regulated by 2- and 3-fold, respectively .
oszifl <dig> and oszifl <dig> show different patterns of induction, with oszifl <dig> induced by almost 14-fold in comparison to control levels .
oszifl <dig>  although induced by zn-excess in roots, is up-regulated only by 3-fold .
to confirm that our treatment was effective, we used osnas <dig> and osiro <dig> , two genes up-regulated by zn-excess in rice roots  <cit> .
therefore, the oszifl genes which are expressed in roots are up-regulated under zn-excess.
a very different expression pattern of oszifl genes was observed in leaves under zn-excess: expression of oszifl <dig>  oszifl <dig>  oszifl <dig> and oszifl <dig> was not affected .
only oszifl <dig> and oszifl <dig> mrna levels were altered under zn-excess:  <dig> -fold and  <dig> -fold higher than in the control treatment, respectively .
the oszifl <dig> and oszifl <dig> genes are a duplicated pair and are also up-regulated by zn-excess in roots, suggesting a strong co-regulation under these conditions in both organs.
however, most oszifl genes seem to be differentially regulated in leaves compared to roots when plants are under excessive zn concentrations.
oszifl expression was also regulated in roots of plants under fe-deficiency.
expression of oszifl <dig> and oszifl <dig> was not significantly increased by the treatment .
oszifl <dig>  oszifl <dig>  oszifl <dig> and oszifl <dig>  however, were up-regulated by  <dig>  to 2-fold .
this effect occurred in parallel with increased expression of osirt <dig> , a gene already described as responsive to fe-deficiency in rice roots  <cit> .
this demonstrates that the plants were indeed under fe-deficient conditions.
moreover, all four genes regulated by fe-deficiency in roots were also induced by zn-excess , confirming a trend for common responses to both stresses in this organ, as previously reported  <cit> .
a completely different response to fe-deficiency was observed in leaves.
none of the oszifl genes showed up-regulation under this condition , although expression of the osiro <dig> gene, was up-regulated by  <dig> -fold .
it is known that osiro <dig> is induced by fe-deficiency in leaves  <cit> .
this is, however, similar to oszifl gene expression in leaves of zn-excess-treated plants : although six oszifl genes were expressed, only oszifl <dig> and oszifl <dig> were up-regulated, while all other family members did not change their expression levels.
considering the results obtained with zn-excess and fe-deficiency, it is possible to suggest that transcriptional regulation of most oszifl genes is more important in roots than in leaves, regardless of the level of expression in control conditions.
oszifl duplicated pairs are co-expressed in specific plant organs and in response to stresses
to analyze the expression pattern of oszifl genes based on microarray meta-analysis, we used genevestigator  <cit> .
affymetrix unique probes used for expression analyses of oszifl <dig>  oszifl <dig>  oszifl <dig>  oszifl <dig>  oszifl <dig>  oszifl <dig>  oszifl <dig> and oszifl <dig> are listed in additional file  <dig>  the available data on expression of oszifl genes in different organs of rice plants is shown in figure  <dig>  clearly, the expression pattern within each one of the duplicated gene pairs oszifl5-oszifl <dig> and oszifl7-oszifl <dig> cluster together, indicating their overlapping expression.
according to microarray data, oszifl <dig> and oszifl <dig> are highly expressed in seed tissues, while oszifl <dig> and oszifl <dig> are expressed in reproductive organs and shoot tissues .
similarly, our qpcr experiments showed higher expression of both oszifl <dig> and oszifl <dig> in flag leaves and panicles and lower in roots .
the pair oszifl <dig> and oszifl <dig>  which had no detected expression in our qpcr experiments, was evaluated using specific probes.
while oszifl <dig> showed no expression, low expression of oszifl <dig> was observed in shoot tissues.
although qpcr will never generate the large amount of data that is achieved by cdna microarrays, pcr has the advantage of unparalleled sensitivity, and therefore slight discrepancies are expected  <cit> .
oszifl duplicated pairs also show co-expression under stress conditions .
oszifl <dig> and oszifl <dig> are highly up-regulated by arsenate in roots of an arsenate-tolerant  and an arsenate-sensitive  cultivars   <cit> .
this suggests that these transporters could be responsive to general stress, as they are also up-regulated by zn-excess and fe-deficiency .
oszifl <dig> is also responsive to arsenate .
oszifl <dig> and oszifl <dig> are also up-regulated under drought and salt stresses .
oszifl <dig> and oszifl <dig> are mostly co-expressed, although no marked increase or decrease in expression was observed for both genes .
the microarray results indicate a strong co-expression of the recently duplicated gene pairs oszifl7-oszifl <dig> and oszifl5-oszifl <dig>  in accordance with our qpcr data.
discussion
zifl expansion through segmental duplication
phenotypic variation is not necessarily the result of entirely new genes.
instead, redundancy generated through gene duplication can be the source of evolutionary novelty.
plants are highly susceptible to duplication events, as most  have experienced whole-genome duplication events in their evolutionary past, as well as tandem and segmental duplications  <cit> .
after duplication, gene copies can follow  neofunctionalization, where one copy maintains the ancestral function and the other can explore new evolutionary terrain;  pseudogenization, where one copy accumulates mutations and lose function while the other maintains the ancestral function;  subfunctionalization, in which deleterious mutations make one copy to be partially functional, but complementary to the other   <cit> .
as deleterious mutations are expected to be more common than beneficial ones, subfunctionalization is considered to be a more common fate for duplicated copies than neofunctionalization, and examples are already known  <cit> .
these mutations are also more common in regulatory regions  than in functional motifs, where selective pressure is stronger; therefore, changes in expression patterns and/or changes in the responses to stimuli are probably more frequent  <cit> .
in this work, we described the zifl protein family in plants, which is part of the mfs superfamily.
we suggested that zifl proteins experienced an expansion in the monocot lineage, as we found three to four gene copies in dicots and eight up to thirteen in monocots, with all monocot paralogs grouping together .
we further characterized the genomic organization of zifl genes in rice, and found that ten out of thirteen copies are located in a duplicated region of chromosomes  <dig> and  <dig> .
this region was first described as a recent segmental duplication, estimated from five to seven mya  <cit> .
this estimation was based on the high degree of similarity between terminal segments of both chromosomes .
however, recent data showed that the duplication of this genomic segment is ancestral to the split of s. bicolor, b. distachyon and rice  <cit> .
wang et al.
proposed that three rounds of unequal crossing-over events have produced the high similarity observed  <cit> .
thus, variation in sequence similarity within these regions reflects rather the antiquity of the unequal crossing-over events, than the date of segmental duplication as suggested earlier  <cit> .
gene conversion is also occurring at high frequencies within this region, further contributing for the maintenance of high similarity  <cit> .
using paralog pairs within the  <dig> mb of chromosomes  <dig> and  <dig> from all species in the oryza genus, a recent work demonstrated that concerted evolution is recurrent in this region for oryza species  <cit> .
gene conversion was specifically found between oszifl <dig> and oszifl <dig> in indica rice, suggesting that concerted evolution has participated in the evolution of zifl genes  <cit> .
we also demonstrated that the region where s. bicolor zifl genes are located in chromosome  <dig> is inverted in relation to its homologous region in rice chromosome  <dig> .
this inverted region was recently described for both s. bicolor and b. distachyon, encompassing  <dig>  mb  <cit> .
s. bicolor zifl gene pairs are not as similar as rice paralogs, indicating that s. bicolor zifl genes probably did not undergo the same degree of concerted evolution as rice paralogs .
in agreement with that, wang et al.
used paralog quartets from rice and s. bicolor  to search for gene conversions.
they found that oszifl <dig> and oszifl <dig> went through whole gene conversion after the split between rice and s. bicolor, while s. bicolor homologs did not show conversion   <cit> .
inversions are known to reduce the probability of recombination and to facilitate the maintenance of differences between interbreeding populations  <cit> .
these results suggest that the inversion observed in s. bicolor reduced the probability of concerted evolution in the sbzifl genes when compared to rice paralogs.
sequence and expression analyses suggest new functional sites in oszifl proteins and insights about duplicated gene pairs
our analysis on motif composition of oszifl proteins also revealed interesting features of this family in rice.
together with the exon-intron organization , motif composition of duplicated genes oszifl <dig> and oszifl <dig> and their partial duplicated copy oszifl <dig> suggests that these genes are diverging in a higher rate when compared to other oszifl paralogs.
they all show no zifl signature cys motif c-p-g-c .
oszifl <dig> and oszifl <dig> also lack the mfs signature and oszifl <dig> lacks both mfs and antiporter signatures .
oszifl <dig> expression was detected in leaves, but at relatively low levels .
oszifl <dig> and oszifl <dig> transcripts were not detected in any of our qpcr experiments, and cdnas corresponding to them are not present at the kome database .
however, microarray metadata showed low expression of oszifl <dig> in shoots, although no expression of oszifl <dig> was detected in all plant organs evaluated .
further experiments should clarify if these genes are gaining new functions or accumulating mutations to become pseudogenes.
a variable region, which corresponds to a cytoplasmic loop, occupies a central position in the oszifl proteins .
there is very low amino acid conservation within this loop.
for this reason, we were able to find conserved motifs within the variable region only when using the whole zifl protein dataset in our analyses .
variable regions are often found in transporters  <cit> .
in the zip family , a variable region is considered to be the metal-binding site, as these loops are rich in histidine residues  <cit> .
our motif analysis in the zifl variable region detected some residues in conserved positions.
in the cdf family, substrate specificity was proposed to be determined by few amino acids, normally histidine  or aspartic acid , which are, respectively, positively and negatively charged  <cit> .
in oszifl proteins, lysine  and glutamic acid , also positive and negative residues, seem to be conserved in the variable loop , although aspartic acid  and leucine  are also frequent .
this region and its conserved residues emerge as candidates for mutagenesis studies to clarify their importance in substrate transport, although no substrate was proven to be transported by zifl proteins  <cit> .
moreover, we described conserved motifs specific to zifl proteins , which also contain candidate residues for site-directed mutagenesis studies.
we characterized the expression of oszifl genes in rice vegetative and reproductive organs  and compared the expression patterns of three duplicated gene pairs, oszifl4-oszifl <dig>  oszifl5-oszifl <dig> and oszifl7-oszifl <dig> .
oszifl <dig> and oszifl <dig> are both expressed in panicles at r <dig> stage, but only oszifl <dig> is expressed in roots .
this partial overlap suggests that their ancestral gene was at least expressed in panicles at r <dig> and in roots, as deleterious mutations could be subfunctionalizing oszifl <dig>  while oszifl <dig> maintains both panicle and root expression.
however, neofunctionalization of oszifl <dig> cannot be discarded.
in agreement with that, throude et al.
showed that, from  <dig> duplicated gene pairs, the vast majority have been neofunctionalized or subfunctionalized, as 88%, 89% and 96% of duplicates, respectively expressed in grain, leaf and root, show distinct expression patterns  <cit> .
a recent work in rice showed that the average number of conserved motifs between duplicated gene pairs declines with increased expression diversity, partially supporting the subfunctionalization model  <cit> .
this is in accordance with the observed divergent expression of oszifl <dig> and oszifl <dig> and their motif composition, because oszifl <dig> has lost one n-terminal and two c-terminal motifs .
expression patterns within the gene pairs oszifl5-oszifl <dig> and oszifl7-oszifl <dig> are similar , and both oszifl <dig> and oszifl <dig> have the same motif composition .
oszifl <dig> has two c-terminal motifs which are lacking in oszifl <dig> , but expression of oszifl <dig> and oszifl <dig> is quite similar, as both are up-regulated in roots and leaves under zn-excess  and in roots under fe-deficiency .
microarray data also shows that the oszifl duplicated pairs oszifl5-oszifl <dig> and oszifl7-oszifl <dig> are co-expressed in the same plant organs and under the same treatments .
yim et al.
showed that duplicated gene pairs with high local similarity  segments show higher expression correlations than gene pairs without these segments  <cit> .
this probably results in an increased likelihood of gene conversion in promoters of gene pairs harboring hls  <cit> .
as gene conversion is known to homogenize sequences in multigene families, this probably explains the similar expression patterns of oszifl pairs, although it is established that duplicated gene pairs tend to rapidly diverge in their expression patterns  <cit> .
expression of oszifl genes is involved in the partially overlapping pathways of zn-excess and fe-deficiency responses
ten out of thirteen oszifl genes are found in two tandem groups of five genes in rice chromosomes  <dig> and  <dig>  probably as a result of repeatedly tandem duplication events.
this size of tandemly arrayed genes was estimated to be very rare, as only 7% of gene arrays in the rice genome have more than three genes  <cit> .
tandem duplication events have a tendency to be retained when involving genes for which fluctuation in copy number is unlikely to affect downstream genes, such as those at the end of or in flexible steps of pathways  <cit> .
in arabidopsis and rice, tandemly arrayed genes are enriched for membrane proteins and genes with function on abiotic and biotic stresses  <cit> .
moreover, tandemly arrayed genes often share regulatory cis-elements and tend to be expressed in a coordinated manner, as well as family members with hls generated through gene conversion  <cit> .
these observations are in accordance with the up-regulation of oszifl members under zn-excess or fe-deficiency, some of which show strong up-regulation upon stress imposition, mostly in roots .
it also agrees with the enrichment observed for catgc and ide1-like elements in oszifl promoter sequences .
enrichment for the catgc-box is related to fe-deficiency responses in rice  <cit> .
the rice specific gene osmir is strongly up-regulated by fe-deficiency and shows  <dig> catgc-boxes in its promoter sequence  <cit> .
in another work, catgc was shown to be enriched in promoters of genes regulated by osidef <dig>  an upstream transcription factor involved in the early response to fe-deficiency  <cit> .
thus, oszifl genes which are responsive to fe-deficiency are potentially under the same control network, although more data is necessary to confirm this hypothesis.
moreover, a similar up-regulation pattern is also observed in the arabidopsis atzif <dig> gene, which is also responsive to both zn-excess and fe-deficiency  <cit> .
this suggests that oszifl genes which are responsive to both stresses could have conserved regulatory sequences in comparison to atzif <dig>
partial overlap between zn-excess and fe-deficiency response has been reported  <cit> .
zn-excess treated plants show much higher concentrations of fe in roots, but slightly decreased fe in shoots and inhibited expression of osfer <dig>  <cit> .
this indicates that zn-excess causes fe-deficiency due to mislocalization of the available fe  <cit> .
on the other hand, fe-deficiency can cause zn-excess, as fe regulated transporters such as osirt <dig> are suggested to transport zn and arabidopsis plants under fe-deficiency accumulate excessive zn  <cit> .
it was also demonstrated that  <dig> % of the zn-excess up-regulated genes in roots are also up-regulated by fe-deficiency, further indicating an overlap between these stresses  <cit> .
excessive zn was also shown to induce more genes in rice roots than in shoots, as  <dig> genes were induced in roots, while only  <dig> in shoots of arabidopsis plants under zn-excess  <cit> .
osiro <dig>  a bhlh  transcription factor induced by fe-deficiency, is the regulator of fe-deficiency responsive genes in roots, such as the genes osnas <dig> , osnas <dig>  osnaat <dig> , osdmas <dig>  and the dma-fe3+ transporter osysl <dig>  <cit> .
expression of osiro <dig> was shown to be up-regulated by and to control the induction of these genes under zn-excess  <cit> .
however, osirt <dig>  a classical fe-deficiency-regulated gene, is not regulated by osiro <dig>  <cit> , and is not up-regulated under zn-excess  <cit> .
these results indicate that osiro <dig> is in the crosslink between zn-excess and fe-deficiency responses.
the osiro <dig> binding site cacgtgg is not found in oszifl promoters, but our qpcr data shows that oszifl <dig>  oszifl <dig>  oszifl <dig> and oszifl <dig> are up-regulated in roots by both stresses .
considering our results, it is possible to suggest that oszifl genes are part of the overlapping pathway that links fe-deficiency and zn-excess, although regulators different from osiro <dig> may control their expression.
one of these regulators could be idef <dig>  <cit> .
CONCLUSIONS
as the first description of the zifl family in plants, this work is the basis for functional studies, especially in rice.
we have shed light onto the unusual genomic distribution of oszifl genes, and made suggestions about the evolutionary forces that shaped the high degree of similarity between them.
we also characterized in detail the motif composition of rice oszifl genes and the expression patterns in different rice organs and under stress conditions.
more functional data, such as loss-of-function mutants, sub cellular localization and ligand specificity, are necessary to uncover the specific roles of each protein and to know to what extent they are functionally redundant, as well as to clarify the roles of oszifl genes in the homeostasis of zn and fe in rice.

 duplications are recurrent in the evolutionary history of plant genomes. whole genome duplications are described for dicotyledons and monocotyledons. it is estimated that the incidence of polyploidy in angiosperms is 30-80%, and ploidy changes may represent about 24% of speciation events. duplication generates two copies of each gene, and the fate of duplicated genes was first described by ohno: one copy should maintain the ancient function and another copy should lose function or gain a new function. this model was improved, giving rise to the duplication-degeneration-complementation model, where the duplicated copies can have complementary functions that resemble the ancestral gene's function. the ddc model's predictions are believed to be more accurate than the previous model, since loss-of-function changes in regulatory regions are more likely to occur than gain-of-function mutations. other improvements of the basic model for duplicated gene retention, involving buffering of crucial functions via conversion and crossing-over, were recently proposed. due to repetitive genome duplications, plants are likely to harbor relatively larger gene families, as compared to animal genomes. it is well established that one whole-genome duplication occurred in the cereal lineage, estimated million years ago, preceding the radiation of the major cereal clades by million years or more. recently, comparing the genomic sequences of rice and sorghum bicolor, it was demonstrated that an early duplication occurred in the monocot lineage. the duplication blocks cover at least 20% of the cereals transcriptome. it was also shown that expression divergence between duplicate genes is significantly correlated with their sequence divergence. after duplication, gene pairs rapidly diverge, and only a small fraction of ancient gene pairs do not show expression divergence. however, for some genomic segments, concerted evolution homogenizes homologous sequences through unequal crossing-over and gene conversion, changing the estimated duplication age and gene divergence. rice was first described as having duplicated segments which cover % of its genomic sequence, and several individual gene duplications. more recent estimates account for duplications in the rice genome, including minor blocks that overlap with major blocks. a duplication block between chromosomes and has been extensively characterized in rice and other cereals, although the age of its birth is still controversial. rice is a model for cereal genomic and genetics studies, due to the availability of the genome sequences from two varieties, extensive gene annotation, and mutant resources. rice is also a major staple food, feeding nearly half of the world's population. however, it is a poor source of minerals such as iron and zinc, the two mineral elements most commonly lacking in human diets. metal homeostasis in plants has been extensively studied in recent years, with a special focus on the transition metals zn and fe. thus, rice emerges both as a model species for physiological and molecular studies and as a candidate for biotechnological improvement aiming at zn and fe biofortification. both zn and fe are essential to mineral nutrition of plants. zn has a key role in gene expression, cell development and replication, while fe is necessary for photosynthesis, electron transport and other redox reactions. although essential, both can be toxic when in excess. several transporters involved in uptake and translocation inside the plant were described for fe and zn. the zinc-induced facilitator gene, described by haydon and cobbett, belongs to a new family of transporters, with three members in arabidopsis thaliana: atzif, atzifl and atzifl. the atzif transporter is clearly involved in zn homeostasis, as the loss-of-function atzif mutant has altered zn distribution and its transcription is up-regulated by zn-excess. importantly, atzif proteins are expressed in the tonoplast, and probably are involved in transport of zn, zn and a ligand or a ligand alone, to the vacuole. besides atzif only one similar protein had been previously characterized: the maize zm-mfs which is induced by infection by the pathogens cochliobolus heterostrophus and c. carbonum and to ultraviolet light. this gene is highly expressed in the les disease lesion mimic background and in plant tissues engineered to express flavonoids or the avirulence gene avrrxv. both atzif and zm-mfs are part of the major facilitator superfamily, which comprises the largest superfamily of secondary transport carriers found in living organisms and is subdivided in at least families. more recently, atzif and atzifl were described as quantitative trait loci candidates for zn concentrations in arabidopsis seeds. in barley, microarray analyses revealed that a zif1-like gene is expressed in the aleurone layer of seeds and its transcription increases in the embryo upon foliar zn application. therefore, it is possible that zifl genes are involved in zn translocation to the seeds. in this work, we describe the zif-like family of transporters. we identified family members from plants and reconstructed their phylogenetic relationships. we also analyzed in detail the organization of zifl genes in the rice genome: the motif composition, genomic organization, and promoter sequences. we analyzed the expression of oszifl genes in different plant organs and developmental stages, as well as in response to different stresses. this is the first attempt to describe the zifl gene family in plants, and the first expression analysis of these genes in rice.  zifl genes in plants we first used the atzif atzifl and atzifl sequences to query genomic databases to determine the distribution of this gene family among plant species. two dicots, vitis vinifera and populus trichocarpa, one bryophyte, physcomitrella patens, one lycophyte, selaginella moellendorffii, and four monocots, sorghum bicolor, brachypodium distachyon, oryza sativa and zea mays had their genomes screened for zifl genes. all sequences found through this search plus the three arabidopsis sequences were used to generate a hidden markov model profile to iteratively search the same genomes. the final dataset consists of genes coding for proteins already annotated and two unannotated proteins from zea mays. all organisms queried contain zifl sequences, with predicted protein sequences ranging from to amino acids and an average of amino acids per protein. all gene sequences begin with an initiation codon and end with a stop codon, except for the protein ppzifl which lacks a small n-terminal portion and was included in the analyses. the overall structure contains to predicted transmembrane domains, found in 63% of the proteins in our dataset. fourteen putative proteins are predicted to have tm domains, and proteins have seven to nine tm domains. dicot species have a small number of zifl gene copies, with v. vinifera and p. trichocarpa showing five and four paralogs of zifl in their genomes, similar to the three members of the arabidopsis zifl gene family. conversely, monocot species show a higher number of zifl genes, with s. bicolor having the highest number of members, followed by rice, b. distachyon and z. mays. p. patens and s. moellendorffii harbor two and seven zifl genes, respectively. clearly, monocot species have a higher number of zifl family paralogs than dicots. the seven zifl genes found in s. moellendorffii seem to be closely related and not originated from the same expansion which originated the monocot zifl genes. zifl proteins are a distinct family of mfs transporters the zifl proteins are all part of the major facilitator superfamily clan of transporter proteins, composed by families. they show similarity to the mfs_ family, which is the largest family within the mfs clan. we used the mfs_ hmm profile to isolate the mfs_ proteins from all dicot and monocot genomes analyzed in this work. phylogenetic trees reconstructing the evolutionary history of mfs_ and zifl proteins for each species were generated using the neighbor-joining method. we observed that in all cases the zifl proteins clustered in a separate group from all other mfs_ members. this result could be an indication that zifl is a distinct family of mfs transporters. simmons et al suggested that sequences similar to zm-mfs could be a distinct group of mfs proteins found in plants. this was based on comparison of signature sequences of nine plant sequences to bacterial and fungal mfs sequences. to confirm this hypothesis, we searched for signatures in the zifl hmm profile and aligned them to the mfs_ hmm profile. we found the canonical mfs signature, located in the cytoplasmic loop between tm and tm as well as the antiporter signature in tm. when aligning these signatures to the mfs_ hmm profile, we noticed that the zifl mfs signature g-x-d--x-g-r- has a conserved tryptophan before the first glycine position, which is not observed in mfs_. the antiporter signature, s-x-g-x-g-p-x-g-g, is also slightly different, having preference for serine in the first position, instead of glycine, as observed by simmons et al. the presence of these conserved positions indicates that zifl transporters share structural and functional similarities with mfs antiporters, although they show specific features that are not common to other mfs proteins. the zifl sequences also show signatures that are not shared with mfs_ proteins. the conserved positions in the loop between tm and tm -x-g-p--x-r, previously reported by simmons et al, were confirmed in our dataset with a few changes. importantly, we found two new conserved signatures that are specific for the zifl proteins. one of them is a cysteine -containing motif c--g-c in the cytoplasmic n-terminal loop of zifl proteins, and the second one is a histidine -containing motif -e---h-x- in the cytoplasmic loop between tm and tm before the beginning of a variable region. from our dataset of zifl proteins, have the cys motif, with only three proteins showing a serine residue in the second position instead of a proline. for the histidine motif, zifl proteins have the conserved residues. from these, have the most conserved residues p-e-t-l-h-x-h, while the other zifl members contain the same motif with no more than one residue substitution. considering that the mfs_ family has proteins with very low overall similarity between them, and that zifl proteins share both high similarity and unique signatures, we suggest that zifl proteins comprise a distinct family of transporters. zifl gene expansion is lineage specific to address the hypothesis of a lineage specific expansion of zifl genes in monocot species, we generated an alignment using the amino acid sequences of the zifl genes found and reconstructed the phylogenetic relationships of these protein sequences using two methods: neighbor-joining and bayesian analysis. although some nodes are not in agreement comparing the two methods, our bootstrap values and posterior probabilities support all the major nodes of the tree, indicating that the reported group relationships are reliable. proteins from bryophyte and lycophyte species grouped together, with paralogs from each species in a separate cluster. the zifl proteins from dicots also formed a distinct group. however, there was no clear separation into sub-groups of orthologous sequences within the dicots group. species-specific gene duplications are observed in arabidopsis, v. vinifera and p. trichocarpa. the zifl paralogs from monocot species were grouped in three distinct groups, named monocot i, monocot ii and monocot iii. all three zifl protein groups from the monocots contain paralogs from the four species included in our analysis. the monocot i group contains zifl proteins, while the monocot ii and monocot iii groups contain proteins each. both the number of sequences found in monocot species and the tree topology strongly suggest that the zifl gene family experienced an expansion in the monocot lineage, and that the last common ancestor of the monocots already had zifl paralogs of the three groups. thus, the split of the four monocot species used in this work probably occurred after the expansion of the zifl family observed in monocots, and this expansion is not shared with other plant lineages. zifl paralogs are unequally distributed in the rice genome the identification of the zifl gene chromosome locations revealed that they are not evenly distributed in the rice genome, but rather arranged in clusters. the same trend is observed in s. bicolor and b. distachyon, but not in z. mays. rice zifl genes were named zifl to based on their genomic locations. two zifl genes, oszifl and oszifl are located in chromosome and oszifl is located in chromosome oszifl oszifl oszifl oszifl and oszifl are found in chromosome while oszifl oszifl oszifl oszifl and oszifl are located in chromosome interestingly, the zifl genes arranged in tandem in chromosomes and are closely related, with oszifl being very similar to oszifl, oszifl to oszifl, oszifl to oszifl, oszifl to oszifl and oszifl to oszifl. we used the gata tool to align the kb regions that include oszifl genes in chromosomes and. the regions of chromosomes and where these genes are located have already been described as a recent segmental duplication in the rice genome, what would explain the high number of matches between these regions. however, the same duplication was recently found in s. bicolor, indicating that this segmental duplication is ancient to the split of these species. we observed that s. bicolor chromosomes and, which are homologous to rice chromosomes and, harbor three and two zifl genes, respectively. an incomplete sequence related to zifl is also found in chromosome. it is possible to observe that an inversion has occurred when comparing the orientation of zifl regions in sb and sb. the alignment between rice and s. bicolor homologous chromosomes os with sb and os with sb demonstrate that the s. bicolor zifl region in sb is inverted, since the alignment of os with sb is in direct orientation while the alignment of os with sb is in reverse. therefore, although in homologous regions, the zifl gene cluster in sb is differentially oriented in relation to rice. a rice zifl gene pairs from chromosomes and 12 oszifl genes organization is highly conserved we aligned the genomic and coding sequence of each zifl gene from rice and determined the exon-intron organization. the exon sizes of each gene pair, oszifl4-oszifl oszifl5-oszifl oszifl6-oszifl oszifl7-oszifl and oszifl8-oszifl are nearly identical, with very few variations in sequences. we observed that oszifl and oszifl are probably originated from duplication, since they share a similar exon-intron organization. however, their amino acid sequences are only 57% identical. this duplication probably occurred in the common ancestor of monocots, as orthologs from s. bicolor, b. distachyon and z. mays were found for both oszifl and oszifl. oszifl is suggested to be originated from a partial duplication of the oszifl8-oszifl pair last common ancestor, and shares more identities to oszifl sequence than to oszifl. thus, it is clear that duplications were of major importance in the zifl family expansion in rice, especially the segmental duplication observed in chromosomes and protein motif composition reveals a variable region in the zifl family we aligned the rice zifl proteins and observed that they share large similarity. to search for functional sites shared by oszifl putative proteins, we used meme to identify conserved motifs in their amino acid sequences. we found eleven motifs shared by almost all oszifl proteins, with few exceptions. seven motifs matched the general mfs_ motif in interproscan, while four showed no hits. the zifl signatures cys motif and his motif are located within the motif and motif respectively. amfs banalysis performed with whole set of zif proteins oszifl oszifl oszifl oszifl oszifl and oszifl have all eleven motifs, while the duplicated pair oszifl8-oszifl and their duplicated copy in chromosome lack several motifs. some of these motifs are located in regions predicted to be transmembrane. further characterization is needed to determine if the duplicated rice zifl genes are becoming pseudogenes or acquiring new functions. the oszifl duplicated copy oszifl lacks the n-terminal motif and the c-terminal motif 10; oszifl lacks motif and its duplicated copy oszifl lacks motif and motif 10; the duplicated pair oszifl and oszifl only differ by the c-terminal motifs and which are absent in oszifl. these differences suggest a divergence process between duplicated pairs. moreover, it is clear that the central motifs are more conserved than those located at the n- and c-terminal regions of oszifl proteins. we also observed a variable region between motifs and which did not show significant pattern conservation in oszifl proteins. this region is located between transmembrane regions and and is a cytoplasmic loop according to conpred ii predictions. the variable region is preceded by the conserved his motif p-e-t-l-h-x-h. variable regions are found in transporters and could be involved in transport or sensing functions. the whole set of zifl proteins used in this work was submitted to meme analysis to find any conserved motifs specifically in the variable region. three motifs were found in this region and named motifs a, b and c. none matched any known motif in the interpro database. we indicated proteins that contain each motif in our phylogenetic tree and showed their positions in rice zifl protein sequences. rice zifl proteins contain motifs a and b in their variable region, but not motifs c. motif a is present in proteins from the monocot i, monocot ii, dicot and bryophyte-lycophyte groups. this motif shows low amino acid conservation. the negatively charged glutamic acid residue in the seventh position of the motif is the most conserved residue. conserved negatively charged residues are also found in the fourth position. between these positions, two non-polar residues, alanine and leucine are also conserved. other positions containing a positively-charged residue of lysine, a negatively charged glutamic acid, and residues of leucine and glycine, although less conserved, are present. charged positions could be involved in transporter specificity, as already described for cation diffusion facilitator proteins. motif b is shared only by a sub-group of six proteins from monocot ii. the fifth and seventh positions of this motif contain one positively charged residue and one hydrophobic residue, lysine and leucine. polar residues of serine, glutamine and tyrosine, non-polar tryptophan and proline are also observed. the motif c is common to proteins from the monocot i group, and is similar to motif a, showing the two glutamic acids separated by one instead of two non-polar residues. however, since only a small number of proteins share motifs b and c, we should be cautious on making assumptions about the functionality of conserved amino acids found in these motifs, as their conservation could be an effect of phylogenetic relatedness and not of evolutionary constraints. importantly, it is possible to observe the high divergence of the variable region even when comparing these three motifs. the variability is much higher in this region than in the whole sequence of zifl proteins, as meme analysis revealed several motifs shared by all the zifl proteins. therefore, these motifs in the cytoplasmic loop could be involved in specific functions of different zifl proteins. expression of oszifl genes in rice vegetative and reproductive organs we analyzed the expression levels of oszifl transcripts in several rice organs by qpcr, including roots, culms and shoots; flag-leaves and whole panicles, both during r, r and r stages. throughout our qpcr experiments, oszifl oszifl oszifl oszifl and oszifl transcripts were not detected or were detected below a confidence threshold for analysis. the expression levels of oszifl genes varied considerably, with some genes reaching higher expression levels and others showing very low expression. oszifl and oszifl although not resultant of a duplication event, share a similar pattern of expression: both are more expressed in leaves and also accumulate in the later stages of flag-leaf development, reaching the highest levels in r. when analyzing gene pairs, we observed that oszifl is almost specifically expressed in roots, showing only little expression in panicles during the r stage, while its duplicated copy oszifl is not expressed in vegetative tissues nor in flag-leaves, but is detected at low levels in panicles during r and at higher levels during r. transcripts from the oszifl5-oszifl pair show similar patterns of expression, especially when considering the reproductive organs flag-leaves and panicles. oszifl and oszifl are both induced from r to r in flag-leaves, maintaining high levels at r in panicles, they are also induced from r to r although oszifl transcript levels are further induced from r to r. in vegetative tissues, oszifl levels are higher in roots, while oszifl is more expressed in shoots. the genes from the oszifl7-oszifl pair also show similar expression patterns in the organs analyzed. oszifl is more expressed in culms and leaves, accumulates from r to r in flag-leaves and decreases its expression from r to r during panicle development. the oszifl transcript accumulates in leaves and also increases from r to r in flag-leaves and decreases from r to r in panicles. taken together, our gene expression data demonstrates that, even after duplication and divergence, most oszifl genes still share similar expression patterns in rice organs within gene pairs. the fe-deficiency element ide is enriched in promoters of oszifl genes to investigate the presence of conserved cis-elements in promoter regions of oszifl genes, we used the poco tool. this approach consisted in comparing the - to + regions of oszifl genes to several random samples of promoters from the entire arabidopsis genome with the same size. if a cis-element is more often found in the promoters of oszifl genes than in a random set of promoters, this cis-element is enriched in these sequences. the poco analysis revealed that the sequence catgc is enriched in our promoter set when compared to arabidopsis promoters. this sequence is the core binding site of idef, a transcription factor of the abi3/vp family involved in fe-deficiency response in rice. as arabidopsis is not closely related to rice and thus the motif frequency in promoters could vary between these species, we confirmed the enrichment by counting the average number of catgc boxes in nearly promoters of rice downloaded from osiris. while the average number of the catgc sequences in rice promoters was, in promoters of the thirteen oszifl genes it was boxes per promoter. some promoters are highly enriched for catgc boxes, such as oszifl, oszifl, oszifl and oszifl. genes that were not detected in our qpcr experiments such as oszifl and oszifl also have promoters enriched in catgc boxes. oszifl oszifl and oszifl promoters show boxes each. since the catgc box is the core motif of ide we searched for ide1-like sequences in promoters of oszifl genes following the method described by kobayashi et al.. we found eleven ide1-like motifs distributed in seven gene promoters, oszifl oszifl oszifl oszifl oszifl oszifl and oszifl. oszifl shows three sequences, two of them overlapping with catgc boxes, while oszifl and oszifl show two ide1-like motifs. considering that the motif is bp long, it is surprising to find such a high number of ide1-like motifs in our promoter set. the enrichment for catgc and ide1-like sequences in promoters of oszifl genes suggests that they are possibly regulated by fe-deficiency. zn-excess and fe-deficiency regulate oszifl expression mainly in rice roots it has been demonstrated that atzif is up-regulated by zn-excess in roots and leaves of arabidopsis plants, as well as by fe-deficiency. as promoters of oszifl genes are enriched for fe-deficiency cis-elements, we submitted rice plants to zn-excess for three days and to fe-deficiency for seven days. oszifl mrna expression level was evaluated by qpcr in roots and leaves from both experiments. several oszifl genes were up-regulated in roots of zn-excess treated plants: oszifl oszifl oszifl oszifl oszifl and oszifl. expression of oszifl oszifl oszifl and of the duplicated pairs oszifl6-oszifl and oszifl8-oszifl was not detected. expression of oszifl which is nearly root-specific, is induced -fold by zn-excess. both oszifl and oszifl a duplicated pair, are also up-regulated by 2- and 3-fold, respectively. oszifl and oszifl show different patterns of induction, with oszifl induced by almost 14-fold in comparison to control levels. oszifl although induced by zn-excess in roots, is up-regulated only by 3-fold. to confirm that our treatment was effective, we used osnas and osiro, two genes up-regulated by zn-excess in rice roots. therefore, the oszifl genes which are expressed in roots are up-regulated under zn-excess. a very different expression pattern of oszifl genes was observed in leaves under zn-excess: expression of oszifl oszifl oszifl and oszifl was not affected. only oszifl and oszifl mrna levels were altered under zn-excess: -fold and -fold higher than in the control treatment, respectively. the oszifl and oszifl genes are a duplicated pair and are also up-regulated by zn-excess in roots, suggesting a strong co-regulation under these conditions in both organs. however, most oszifl genes seem to be differentially regulated in leaves compared to roots when plants are under excessive zn concentrations. oszifl expression was also regulated in roots of plants under fe-deficiency. expression of oszifl and oszifl was not significantly increased by the treatment. oszifl oszifl oszifl and oszifl however, were up-regulated by to 2-fold. this effect occurred in parallel with increased expression of osirt, a gene already described as responsive to fe-deficiency in rice roots. this demonstrates that the plants were indeed under fe-deficient conditions. moreover, all four genes regulated by fe-deficiency in roots were also induced by zn-excess, confirming a trend for common responses to both stresses in this organ, as previously reported. a completely different response to fe-deficiency was observed in leaves. none of the oszifl genes showed up-regulation under this condition, although expression of the osiro gene, was up-regulated by -fold. it is known that osiro is induced by fe-deficiency in leaves. this is, however, similar to oszifl gene expression in leaves of zn-excess-treated plants: although six oszifl genes were expressed, only oszifl and oszifl were up-regulated, while all other family members did not change their expression levels. considering the results obtained with zn-excess and fe-deficiency, it is possible to suggest that transcriptional regulation of most oszifl genes is more important in roots than in leaves, regardless of the level of expression in control conditions. oszifl duplicated pairs are co-expressed in specific plant organs and in response to stresses to analyze the expression pattern of oszifl genes based on microarray meta-analysis, we used genevestigator. affymetrix unique probes used for expression analyses of oszifl oszifl oszifl oszifl oszifl oszifl oszifl and oszifl are listed in additional file the available data on expression of oszifl genes in different organs of rice plants is shown in figure clearly, the expression pattern within each one of the duplicated gene pairs oszifl5-oszifl and oszifl7-oszifl cluster together, indicating their overlapping expression. according to microarray data, oszifl and oszifl are highly expressed in seed tissues, while oszifl and oszifl are expressed in reproductive organs and shoot tissues. similarly, our qpcr experiments showed higher expression of both oszifl and oszifl in flag leaves and panicles and lower in roots. the pair oszifl and oszifl which had no detected expression in our qpcr experiments, was evaluated using specific probes. while oszifl showed no expression, low expression of oszifl was observed in shoot tissues. although qpcr will never generate the large amount of data that is achieved by cdna microarrays, pcr has the advantage of unparalleled sensitivity, and therefore slight discrepancies are expected. oszifl duplicated pairs also show co-expression under stress conditions. oszifl and oszifl are highly up-regulated by arsenate in roots of an arsenate-tolerant and an arsenate-sensitive cultivars. this suggests that these transporters could be responsive to general stress, as they are also up-regulated by zn-excess and fe-deficiency. oszifl is also responsive to arsenate. oszifl and oszifl are also up-regulated under drought and salt stresses. oszifl and oszifl are mostly co-expressed, although no marked increase or decrease in expression was observed for both genes. the microarray results indicate a strong co-expression of the recently duplicated gene pairs oszifl7-oszifl and oszifl5-oszifl in accordance with our qpcr data. discussion zifl expansion through segmental duplication phenotypic variation is not necessarily the result of entirely new genes. instead, redundancy generated through gene duplication can be the source of evolutionary novelty. plants are highly susceptible to duplication events, as most have experienced whole-genome duplication events in their evolutionary past, as well as tandem and segmental duplications. after duplication, gene copies can follow neofunctionalization, where one copy maintains the ancestral function and the other can explore new evolutionary terrain; pseudogenization, where one copy accumulates mutations and lose function while the other maintains the ancestral function; subfunctionalization, in which deleterious mutations make one copy to be partially functional, but complementary to the other. as deleterious mutations are expected to be more common than beneficial ones, subfunctionalization is considered to be a more common fate for duplicated copies than neofunctionalization, and examples are already known. these mutations are also more common in regulatory regions than in functional motifs, where selective pressure is stronger; therefore, changes in expression patterns and/or changes in the responses to stimuli are probably more frequent. in this work, we described the zifl protein family in plants, which is part of the mfs superfamily. we suggested that zifl proteins experienced an expansion in the monocot lineage, as we found three to four gene copies in dicots and eight up to thirteen in monocots, with all monocot paralogs grouping together. we further characterized the genomic organization of zifl genes in rice, and found that ten out of thirteen copies are located in a duplicated region of chromosomes and. this region was first described as a recent segmental duplication, estimated from five to seven mya. this estimation was based on the high degree of similarity between terminal segments of both chromosomes. however, recent data showed that the duplication of this genomic segment is ancestral to the split of s. bicolor, b. distachyon and rice. wang et al. proposed that three rounds of unequal crossing-over events have produced the high similarity observed. thus, variation in sequence similarity within these regions reflects rather the antiquity of the unequal crossing-over events, than the date of segmental duplication as suggested earlier. gene conversion is also occurring at high frequencies within this region, further contributing for the maintenance of high similarity. using paralog pairs within the mb of chromosomes and from all species in the oryza genus, a recent work demonstrated that concerted evolution is recurrent in this region for oryza species. gene conversion was specifically found between oszifl and oszifl in indica rice, suggesting that concerted evolution has participated in the evolution of zifl genes. we also demonstrated that the region where s. bicolor zifl genes are located in chromosome is inverted in relation to its homologous region in rice chromosome. this inverted region was recently described for both s. bicolor and b. distachyon, encompassing mb. s. bicolor zifl gene pairs are not as similar as rice paralogs, indicating that s. bicolor zifl genes probably did not undergo the same degree of concerted evolution as rice paralogs. in agreement with that, wang et al. used paralog quartets from rice and s. bicolor to search for gene conversions. they found that oszifl and oszifl went through whole gene conversion after the split between rice and s. bicolor, while s. bicolor homologs did not show conversion. inversions are known to reduce the probability of recombination and to facilitate the maintenance of differences between interbreeding populations. these results suggest that the inversion observed in s. bicolor reduced the probability of concerted evolution in the sbzifl genes when compared to rice paralogs. sequence and expression analyses suggest new functional sites in oszifl proteins and insights about duplicated gene pairs our analysis on motif composition of oszifl proteins also revealed interesting features of this family in rice. together with the exon-intron organization, motif composition of duplicated genes oszifl and oszifl and their partial duplicated copy oszifl suggests that these genes are diverging in a higher rate when compared to other oszifl paralogs. they all show no zifl signature cys motif c-p-g-c. oszifl and oszifl also lack the mfs signature and oszifl lacks both mfs and antiporter signatures. oszifl expression was detected in leaves, but at relatively low levels. oszifl and oszifl transcripts were not detected in any of our qpcr experiments, and cdnas corresponding to them are not present at the kome database. however, microarray metadata showed low expression of oszifl in shoots, although no expression of oszifl was detected in all plant organs evaluated. further experiments should clarify if these genes are gaining new functions or accumulating mutations to become pseudogenes. a variable region, which corresponds to a cytoplasmic loop, occupies a central position in the oszifl proteins. there is very low amino acid conservation within this loop. for this reason, we were able to find conserved motifs within the variable region only when using the whole zifl protein dataset in our analyses. variable regions are often found in transporters. in the zip family, a variable region is considered to be the metal-binding site, as these loops are rich in histidine residues. our motif analysis in the zifl variable region detected some residues in conserved positions. in the cdf family, substrate specificity was proposed to be determined by few amino acids, normally histidine or aspartic acid, which are, respectively, positively and negatively charged. in oszifl proteins, lysine and glutamic acid, also positive and negative residues, seem to be conserved in the variable loop, although aspartic acid and leucine are also frequent. this region and its conserved residues emerge as candidates for mutagenesis studies to clarify their importance in substrate transport, although no substrate was proven to be transported by zifl proteins. moreover, we described conserved motifs specific to zifl proteins, which also contain candidate residues for site-directed mutagenesis studies. we characterized the expression of oszifl genes in rice vegetative and reproductive organs and compared the expression patterns of three duplicated gene pairs, oszifl4-oszifl oszifl5-oszifl and oszifl7-oszifl. oszifl and oszifl are both expressed in panicles at r stage, but only oszifl is expressed in roots. this partial overlap suggests that their ancestral gene was at least expressed in panicles at r and in roots, as deleterious mutations could be subfunctionalizing oszifl while oszifl maintains both panicle and root expression. however, neofunctionalization of oszifl cannot be discarded. in agreement with that, throude et al. showed that, from duplicated gene pairs, the vast majority have been neofunctionalized or subfunctionalized, as 88%, 89% and 96% of duplicates, respectively expressed in grain, leaf and root, show distinct expression patterns. a recent work in rice showed that the average number of conserved motifs between duplicated gene pairs declines with increased expression diversity, partially supporting the subfunctionalization model. this is in accordance with the observed divergent expression of oszifl and oszifl and their motif composition, because oszifl has lost one n-terminal and two c-terminal motifs. expression patterns within the gene pairs oszifl5-oszifl and oszifl7-oszifl are similar, and both oszifl and oszifl have the same motif composition. oszifl has two c-terminal motifs which are lacking in oszifl, but expression of oszifl and oszifl is quite similar, as both are up-regulated in roots and leaves under zn-excess and in roots under fe-deficiency. microarray data also shows that the oszifl duplicated pairs oszifl5-oszifl and oszifl7-oszifl are co-expressed in the same plant organs and under the same treatments. yim et al. showed that duplicated gene pairs with high local similarity segments show higher expression correlations than gene pairs without these segments. this probably results in an increased likelihood of gene conversion in promoters of gene pairs harboring hls. as gene conversion is known to homogenize sequences in multigene families, this probably explains the similar expression patterns of oszifl pairs, although it is established that duplicated gene pairs tend to rapidly diverge in their expression patterns. expression of oszifl genes is involved in the partially overlapping pathways of zn-excess and fe-deficiency responses ten out of thirteen oszifl genes are found in two tandem groups of five genes in rice chromosomes and probably as a result of repeatedly tandem duplication events. this size of tandemly arrayed genes was estimated to be very rare, as only 7% of gene arrays in the rice genome have more than three genes. tandem duplication events have a tendency to be retained when involving genes for which fluctuation in copy number is unlikely to affect downstream genes, such as those at the end of or in flexible steps of pathways. in arabidopsis and rice, tandemly arrayed genes are enriched for membrane proteins and genes with function on abiotic and biotic stresses. moreover, tandemly arrayed genes often share regulatory cis-elements and tend to be expressed in a coordinated manner, as well as family members with hls generated through gene conversion. these observations are in accordance with the up-regulation of oszifl members under zn-excess or fe-deficiency, some of which show strong up-regulation upon stress imposition, mostly in roots. it also agrees with the enrichment observed for catgc and ide1-like elements in oszifl promoter sequences. enrichment for the catgc-box is related to fe-deficiency responses in rice. the rice specific gene osmir is strongly up-regulated by fe-deficiency and shows catgc-boxes in its promoter sequence. in another work, catgc was shown to be enriched in promoters of genes regulated by osidef an upstream transcription factor involved in the early response to fe-deficiency. thus, oszifl genes which are responsive to fe-deficiency are potentially under the same control network, although more data is necessary to confirm this hypothesis. moreover, a similar up-regulation pattern is also observed in the arabidopsis atzif gene, which is also responsive to both zn-excess and fe-deficiency. this suggests that oszifl genes which are responsive to both stresses could have conserved regulatory sequences in comparison to atzif partial overlap between zn-excess and fe-deficiency response has been reported. zn-excess treated plants show much higher concentrations of fe in roots, but slightly decreased fe in shoots and inhibited expression of osfer. this indicates that zn-excess causes fe-deficiency due to mislocalization of the available fe. on the other hand, fe-deficiency can cause zn-excess, as fe regulated transporters such as osirt are suggested to transport zn and arabidopsis plants under fe-deficiency accumulate excessive zn. it was also demonstrated that % of the zn-excess up-regulated genes in roots are also up-regulated by fe-deficiency, further indicating an overlap between these stresses. excessive zn was also shown to induce more genes in rice roots than in shoots, as genes were induced in roots, while only in shoots of arabidopsis plants under zn-excess. osiro a bhlh transcription factor induced by fe-deficiency, is the regulator of fe-deficiency responsive genes in roots, such as the genes osnas, osnas osnaat, osdmas and the dma-fe3+ transporter osysl. expression of osiro was shown to be up-regulated by and to control the induction of these genes under zn-excess. however, osirt a classical fe-deficiency-regulated gene, is not regulated by osiro, and is not up-regulated under zn-excess. these results indicate that osiro is in the crosslink between zn-excess and fe-deficiency responses. the osiro binding site cacgtgg is not found in oszifl promoters, but our qpcr data shows that oszifl oszifl oszifl and oszifl are up-regulated in roots by both stresses. considering our results, it is possible to suggest that oszifl genes are part of the overlapping pathway that links fe-deficiency and zn-excess, although regulators different from osiro may control their expression. one of these regulators could be idef.  as the first description of the zifl family in plants, this work is the basis for functional studies, especially in rice. we have shed light onto the unusual genomic distribution of oszifl genes, and made suggestions about the evolutionary forces that shaped the high degree of similarity between them. we also characterized in detail the motif composition of rice oszifl genes and the expression patterns in different rice organs and under stress conditions. more functional data, such as loss-of-function mutants, sub cellular localization and ligand specificity, are necessary to uncover the specific roles of each protein and to know to what extent they are functionally redundant, as well as to clarify the roles of oszifl genes in the homeostasis of zn and fe in rice. 
sequences of sixty-eight zifl genes, from nine plant species, were comparatively analyzed. monocots harbor a larger number of zifl genes in their genomes than dicots, probably a result of a lineage-specific expansion. the genomic organization of the rice zifl genes seems to be highly influenced by segmental and tandem duplications and concerted evolution, as rice genome contains five highly similar zifl gene pairs. we suggest that zifl genes have different evolutionary histories in monocot and dicot lineages. here, we describe the zinc-induced facilitator-like gene family in plants, and characterize the genomic structure and expression of rice paralogs, which are highly affected by segmental duplication. most rice zifl promoters are enriched for the core sequence of the fe-deficiency-related box ide gene expression analyses of different plant organs, growth stages and treatments, both from our qpcr data and from microarray databases, revealed that the duplicated zifl gene pairs are mostly co-expressed. moreover, this is the first description of the zifl gene family in plants and the basis for functional studies on this family, which may play important roles in zn and fe homeostasis in plants. duplications are very common in the evolution of plant genomes, explaining the high number of members in plant gene families. in rice, concerted evolution affected zifl duplicated genes, possibly maintaining similar expression patterns between pairs. new genes born after duplication can undergo pseudogenization, neofunctionalization or subfunctionalization.
 duplications are very common in the evolution of plant genomes, explaining the high number of members in plant gene families. new genes born after duplication can undergo pseudogenization, neofunctionalization or subfunctionalization. rice is a model for functional genomics research, an important crop for human nutrition and a target for biofortification. increased zinc and iron content in the rice grain could be achieved by manipulation of metal transporters. here, we describe the zinc-induced facilitator-like gene family in plants, and characterize the genomic structure and expression of rice paralogs, which are highly affected by segmental duplication.  sequences of sixty-eight zifl genes, from nine plant species, were comparatively analyzed. although related to msf_ proteins, zifl protein sequences consistently grouped separately. specific zifl sequence signatures were identified. monocots harbor a larger number of zifl genes in their genomes than dicots, probably a result of a lineage-specific expansion. the rice zifl paralogs were named oszifl to oszifl and characterized. the genomic organization of the rice zifl genes seems to be highly influenced by segmental and tandem duplications and concerted evolution, as rice genome contains five highly similar zifl gene pairs. most rice zifl promoters are enriched for the core sequence of the fe-deficiency-related box ide gene expression analyses of different plant organs, growth stages and treatments, both from our qpcr data and from microarray databases, revealed that the duplicated zifl gene pairs are mostly co-expressed. transcripts of oszifl oszifl oszifl and oszifl accumulate in response to zn-excess and fe-deficiency in roots, two stresses with partially overlapping responses.  we suggest that zifl genes have different evolutionary histories in monocot and dicot lineages. in rice, concerted evolution affected zifl duplicated genes, possibly maintaining similar expression patterns between pairs. the enrichment for ide boxes in rice zifl gene promoters suggests a role in zn-excess and fe-deficiency up-regulation of zifl transcripts. moreover, this is the first description of the zifl gene family in plants and the basis for functional studies on this family, which may play important roles in zn and fe homeostasis in plants. 