BACKGROUND
auxin signaling plays a vital role in plant growth and development processes like, in apical dominance, tropic responses, lateral root formation, vascular differentiation, embryo patterning and shoot elongation  <cit> .
at the molecular level, most of these processes are controlled by the auxin-response genes  <cit> , and auxin responsiveness is conferred to these genes by conserved promoter elements, including tga-element , auxrr-core  and auxre .
among these, the auxre promoter elements are bound and activated by a plant-specific transcription factors which are called as auxin response factors   <cit> .
an arf protein contains a dna-binding domain  in the n-terminal region, a middle region that functions as an activation domain  or repression domain   <cit> , and a carboxyl-terminal dimerization domain  that are similar to those found in the c terminus of aux/iaas, which is a protein-protein interaction domain that mediates the homo- and hetero- dimerization of arfs and also the hetero-dimerization of arf and aux/iaa proteins  <cit> .
it has been reported that, the arf proteins are encoded by a large gene family, with  <dig> and  <dig> members in arabidopsis and rice, respectively  <cit> .
expression analysis suggested that these genes are, in general, transcribed in a wide variety of tissues and organs, with an exception of arf gene cluster on arabidopsis chromosome  <dig>  which appears to be restricted to embryo genesis/seed development  <cit> .
classical genetic approaches have led to the identification of arf gene functions in plant growth and development.
for example, arf mutations caused the change in gynoecium patterning   <cit> , impaired hypocotyls response to blue light, growth and auxin sensitivity   <cit> , formation of vascular strands and embryo axis formation   <cit> , suppression of hookless phenotype and hypocotyl bending   <cit> , hypocotyl elongation, and auxin homeostasis   <cit> .
moreover, the mutants of atarf sister pairs generally exhibit a much stronger phenotype than that of single mutants, suggesting that closely related atarfs have somewhat redundant roles in arabidopsis  <cit> .
in rice, antisense phenotype of osarf <dig> gene showed stunted growth, low vigor, curled leaves and sterility, suggesting that the gene is essential for vegetative and reproductive development  <cit> .
genetic divergence between arabidopsis and rice arf gene family investigated by genome-wide analysis revealed that most of the rice osarfs are related to arabidopsis arfs and fall into sister pairs as in arabidopsis  <cit> .
the first assembly of maize genome sequence has recently been published  <cit> , however, to the best of our knowledge, the maize arf gene family  has not been characterized in detail.
in this article, we provide detailed information on the genomic structures, chromosomal locations, sequence homology and expression patterns of  <dig> maize arf genes.
in addition, the phylogenetic relationship between arf genes in arabidopsis, rice and maize were also compared, which will help future studies for elucidating the precise roles of zmarfs in maize growth and development.
RESULTS
identification and chromosomal localization of maize arf genes
extensive searches of public and proprietary transcript and genomic databases, by using all previously reported arf proteins from other species as blast queries, identified a total of  <dig> maize arf genes that have complete sequences in respective bacterial artificial chromosome  clones.
among these full-length coding sequences of  <dig> zmarf genes  were further confirmed by rt-pcr amplification, cloning and sequencing .
the full length coding sequences of the zmarf genes ranged from  <dig> bp  to  <dig> bp  with the deduced proteins of  <dig> to  <dig> amino acids .
gene
a systematic designation given to maize arfs in this work.
b length of open reading frame in base pairs.
c the number of amino acids, molecular weight , and isoelectric point  of the deduced polypeptides.
d location of the zmarf genes to the chromosome bins.
e bac name, dna accession number where the zmarf gene is present.
f nearest marker to the zmarf gene.
g representative est/cdna in genbank corresponding to zmarf gene.
h location of the zmarf genes to the physical blocks  <cit> .
i location of the zmarf genes to the block pairs  <cit> .
six zmarf sister pairs are mapped on the same duplicated chromosomal blocks: zmarf <dig> and  <dig> in 10la, zmarf <dig> and  <dig> in 4l, zmarf <dig> and  <dig> in 4l, zmarf <dig> and  <dig> in 4l, zmarf <dig> and  <dig> in 1l, zmarf <dig> and  <dig> in 1l.
the nearest genetic markers for each arf gene were determined from maize bac contigs and positioned on maize genetic map.
it was found that these  <dig> zmarfs were mapped on  <dig> out of  <dig> maize chromosomes, except for chromosome  <dig>  six zmarfs were present on chromosome 3;  <dig> on chromosomes  <dig>   <dig>   <dig>   <dig> and 10;  <dig> on chromosomes  <dig> and 8; only one on chromosome  <dig> .
in addition, six zmarf sister pairs were mapped on the same duplicated chromosomal blocks that has been described previously  <cit>  .
the predicted molecular weights of the  <dig> deduced zmarf proteins ranged from  <dig>  kda  to  <dig>  kda  .
pair-wise analysis of zmarf protein sequences indicated that the overall identity fell in a range within  <dig> %  to  <dig> %  .
moreover, multiple protein sequence alignments were performed using the clustal_x program to examine structural features of these  <dig> maize arf genes.
the results revealed that all the zmarf proteins contained a highly conserved region of about  <dig> amino acid residues in their n-terminal portion corresponding to the dna-binding domain  of arabidopsis arf family.
except zmarf  <dig> and  <dig>  the other zmarf proteins contained a carboxyl-terminal domain  related to domains iii and iv found in aux/iaa proteins .
it has been reported that the middle region of arfs function as activation or repression domains  <cit> .
transfection assays with plant protoplasts indicated that atarf <dig>   <dig>   <dig>   <dig>  and  <dig> are repressors  <cit> , among which atarf <dig> contain middle region rich in proline , serine  and threonine .
atarf <dig>   <dig>   <dig>   <dig> and  <dig>  which contain middle region rich in glutamine , are activators  <cit> .
the detailed sequence analysis of all  <dig> deduced zmarf proteins revealed that pst rich middle regions were found in zmarf <dig>   <dig>   <dig>   <dig>   <dig> and  <dig>  indicating that these genes are more likely to act as repressors.
while q rich regions were found in zmarf <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and  <dig>  implying that these genes are probable transcriptional activators .
phylogenetic analysis and genomic structure of zmarfs
bayesian phylogenetic analysis was performed and the  <dig> zmarf proteins were classified into six classes: class i , ii , iii , iv, v and vi, with each class containing  <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> members, respectively .
it was worthy to note that in the joint phylogenetic tree most of the zmarf proteins fell as related sister pairs , viz.
zmarf <dig> and  <dig>   <dig> and  <dig>   <dig> and  <dig>   <dig> and  <dig>   <dig> and  <dig>   <dig> and  <dig>   <dig> and  <dig>   <dig> and  <dig>   <dig> and  <dig>   <dig> and  <dig>  or triplets  and quadruplets in the case of zmarf <dig>   <dig>   <dig> and  <dig>
the intron/exon structures of zmarf genes were determined by alignment of cdna to genomic sequences.
this sequence analysis revealed that introns were found in coding sequences of all the arf genes and the number of exons varied from  <dig> to  <dig> .
as expected, most zmarf genes in the same sister pair or triplets showed similar distribution of intron/exon, whereas the others were more divergent in genomic structure, suggesting that these sister pair genes lies in duplicated genomic regions .
the annotated arf gene family in arabidopsis and rice enabled us to determine the phylogenetic relationship between dicot and monocot arf proteins.
a phylogenetic tree constructed using the protein sequences of  <dig> zmarfs,  <dig> osarfs and  <dig> atarfs depicted that all of these  <dig> arf proteins were also divided into six classes .
prediction of potential targets for small rna
previous studies have revealed that several arf genes are targets for small rnas.
for example, atarf <dig> and  <dig> are mir <dig> targets  <cit>  while atarf <dig>   <dig>  and  <dig> are mir <dig> targets  <cit> .
atarf <dig>   <dig> and  <dig> are targets for trans-acting-small interfering rna  <dig>   <cit> .
in this study, putative small rna target sites were searched by using the miranda software.
<dig> out of  <dig> zmarf genes were predicted to be the potential targets of small rna and the number of target genes for mir <dig>  mir <dig>  tas <dig> ta-sirna was  <dig>   <dig> and  <dig>  respectively .
to determine whether the increased mir <dig> level could cause the degradation of transcripts of the six potential targets , we generated mir <dig> overexpressing zong <dig> lines and the presence of transgene was confirmed by pcr amplifications of bar gene .
we then analyzed mrna levels of these genes in roots of 8-day-old seedling in wild type and mir <dig> overexpressing zong <dig> lines .
the results of real-time rt-pcr exhibited that mrna abundance of zmarf <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> in three independent transgenic lines decreased markedly as compared with wild type, indicating that post-transcriptional regulation by small rnas may play an important role in regulating the expression of zmarf genes .
auxin inducibility and promoter motif prediction of zmarf genes
to determine the response of zmarf genes to exogenous auxin stimuli, their expression patterns in seedling roots at  <dig>   <dig> and  <dig> hours after  <dig> μm αnaa treatment were investigated using real-time rt-pcr and fold induction relative to water-treated controls for each time point were calculated.
this analysis revealed that, with an exception of zmarf <dig>  all other genes were expressed in seedling roots, among which  <dig> genes  were up-regulated and  <dig> genes  were down-regulated by exogenous auxin treatment across all time points.
in addition  <dig> genes  were up-regulated by auxin after  <dig> h treatment but down-regulated later.
in contrast,  <dig> genes  were down-regulated over the first  <dig> h of treatment but up-regulated after  <dig> h of auxin treatment.
the other  <dig> zmarf genes also displayed diverse expression pattern in response to auxin treatment, indicating the complexity of auxin-regulated gene expression of zmarf genes .
interestingly, the relative mrna abundance of some arf genes, for example zmarf <dig>   <dig> and  <dig>  were also altered dramatically in water treatment, indicating that these genes may be responsive to water logging.
putative promoter sequences  of zmarfs were obtained from the draft maize genome sequence.
database search of plant promoters   <cit>  detected one or more auxin response elements in some of these putative promoters.
notably, three auxin response elements  were detected in the promoter region of zmarf <dig> gene, consistent with its significantly increased mrna accumulation following auxin treatment.
however, out of  <dig> zmarfs genes, auxin response elements were detected only in promoter regions of  <dig> zmarfs genes .
thus, the relationship between auxin-response elements and auxin inducibility of zmarf genes remains unclear and need to be further investigated.
expression profiling of zmarf genes in embryos during seed development and germination
recently, functional analysis has revealed that some arf genes in arabidopsis play important role in seed development and germination  <cit> .
as a result, we focused on the study of expression patterns of zmarf genes in embryos during seed development and germination .
expressions of all  <dig> zmarf genes were detected but they exhibited dynamic expression patterns, of which  <dig> genes  showed peak expression in embryos at  <dig> h after imbibition.
in addition, the expression patterns of duplicated zmarf genes also varied considerably.
for example, the expression level of zmarf <dig> gene was much higher than that of its sister gene zmarf <dig> and the time point of peak expression was also different for zmarf <dig> and zmarf <dig>  it is worthy to note that the relative mrna abundances of  <dig> zmarf genes in dry mature embryos were higher than in immature embryos which further increased during seed germination .
we observed that the mrna accumulation of zmarf <dig> gene peaked in dry mature embryo but declined after seed imbibition .
rt-pcr analysis of embryos  and two vegetative tissues  detected transcripts of zmarf genes with an exception of zmarf <dig> in 8-day-old seedling leaves and roots .
discussion
characterization of an expanded arf gene family in maize
it is believed that the long evolutionary periods experienced by a particular organism is the cause of having multiple members in the specific gene family  <cit> .
several rounds of whole genome duplication have been reported in the maize genome  <cit> .
in this study, we identified and characterized  <dig> maize arf genes through genome-wide analysis, suggesting that maize arf gene family was expanded compared to arabidopsis  and rice   <cit> .
phylogenetic analysis revealed that maize arf gene family contained ten sister pairs, two triplets and one quadruplet.
however, none of these pairs were genetically linked to each other on their corresponding chromosomal locations.
on the other hand, all closely linked zmarf loci such as zmarf <dig> and zmarf <dig> on chromosome 2; zmarf <dig>   <dig>   <dig> and  <dig> on chromosome  <dig> and zmarf <dig>   <dig> and  <dig> on chromosome  <dig> were not paired together into the same sister groups.
moreover, at least six zmarf sister pairs mapped on the same duplicated chromosomal blocks   <cit> .
thus, the expansion of maize arf gene family could be explained by the ancient tetraploid ancestry of maize, in which genome duplication occurred after divergence from the common ancestor of rice and maize, followed by subsequent diploidization en route to modern maize  <cit> .
expression divergence between duplicated zmarf genes
the presence of duplicated zmarf genes raises the question about their functional redundancy.
according to the evolutionary models, duplicated genes may undergo different selection processes: nonfunctionalization where one copy loses the function, hypofunctionalization where expression/function of one copy decreases, neofunctionalization where one copy gains a novel function, or subfunctionalization where the two copies partition or specialize into distinct functions  <cit> .
these evolutionary fates may result in divergence of expression patterns or protein structure.
evidence for divergence between the duplicate genes could be inferred from expression pattern of zmarf <dig> and  <dig> genes.
zmarf <dig> was highly expressed in seed embryos during germination but the transcript of zmarf <dig> was very low.
in addition, possible subfunctionalization shifts the expression pattern trends of gene pairs.
for example, the mrna abundance of zmarf <dig> peaked in dry mature embryo but zmarf <dig> was highly expressed in embryos at  <dig> h after seed imbibition.
regulation of zmarf gene expression
since arfs are transcription factors that regulate expression of auxin response genes, it would be interesting to determine the response of zmarf gene to auxin treatment.
it has been reported that arabidopsis arf <dig>   <dig>   <dig> and  <dig> and rice osarf <dig> and  <dig> transcripts increased slightly in response to auxin, while osarf <dig>   <dig> and  <dig> decreased marginally  <cit> .
in the present study, we found that most of the zmarf genes were responsive to exogenous auxin treatment but the auxin responsive elements were only detected in promoter region of  <dig> zmarf genes.
therefore, the underlying mechanism for auxin inducibility of zmarf genes needs to be elucidated.
there is a growing body of work showing the posttranscriptional regulation of arf transcript abundance by micro-rnas  and trans-acting-small interfering rnas   <cit> .
regulation of atarf <dig> and  <dig> by mir <dig> is important for development of anthers and ovules  <cit> , regulation of atarf <dig> by mir <dig> is important for arabidopsis growth and development  <cit>  and regulation of atarf <dig> and  <dig> by mir <dig> plays a role in root cap formation  <cit> .
the regulation of atarf <dig> and  <dig> targeted by tas <dig> ta-sirnas is required for proper leaf development  <cit>  and juvenile to adult phase changes  <cit> .
the target sites of these three small rnas were also detected in maize arf genes, and are widely conserved in dicots and monocots.
furthermore, our transgenic analysis exhibited that increased mir <dig> level could cause degradation of transcripts for six potential targets , indicating that zmarf genes showed posttranscriptional regulation.
however, we found that the expression patterns of zmarf genes which contained the same target sites varied considerably in embryos during seed development and germination, for example the expression of zmarf <dig>   <dig>   <dig>   <dig> and  <dig> with two potential tas <dig> ta-sirnas recognition sites.
thus, we speculate that some unidentified factors may also play a role in regulating expression of these genes, which could be highly specific to a selected tissue type or developmental program with the possibility that mirna and tasirna may have functions in very discreet regions.
potential functions of zmarf genes during seed development
seed development is considered as a physical link between parents and sporophytic generation in plants  <cit> .
auxin signaling is thought to play an important role in embryo development.
for example, a higher level auxin is detected in root apex and ends of cotyledon primordia from heart to mature embryo in arabidopsis  <cit> .
defects in arabidopsis embryo patterning was observed in arf <dig> mutants, which enhanced in arf5arf <dig> double mutants  <cit> .
in this study, seven zmarf genes, including zmarf <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and  <dig>  appeared to be constitutively expressed in developing embryos, whereas the transcripts of other zmarf genes exhibited dynamic expression patterns, suggesting the partitioning of functions between these genes in embryo development.
in most flowering plants, seed germination is the first and may be the foremost growth stage in the plant's life cycle.
genetic evidence supporting a role of arf genes in germination has been obtained from the analysis of regulation of atarf <dig> by mir <dig>  <cit> .
it has been reported that transgenic seeds of arabidopsis expressing a mir160-resistant form of atarf <dig>  are hypersensitive to germination inhibition by exogenous aba, whereas ectopic expression of mir <dig> results in a reduced sensitivity to aba  <cit> .
in the present study, zmarf <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> are predicted to be the targets for mir <dig>  which falls in the same subfamily with atarf <dig>   <dig> and  <dig>  expression analysis demonstrated that mrna accumulation of zmarf <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> gradually increased during seed germination, reaching its peak in embryos after  <dig> h of seed imbibition and decreased in later stage, whereas zmarf <dig> showed high transcript accumulation in dry seed embryos.
in addition, dynamic expression patterns were also observed for other zmarf genes.
collectively, we speculate that zmarf genes may be involved in diverse aspects of developmental processes during seed germination.
CONCLUSIONS
maize arf gene family is expanded as compared to arabidopsis and rice reflecting a succession of maize genomic rearrangements and expansions due to extensive duplication and diversification that frequently occurred in the course of evolution.
the expressions of maize arf genes are regulated by auxin and small rnas.
dynamic expression patterns of zmarf genes in embryo at different stages were observed, which suggest that these genes may be involved in seed development and germination.

 auxin signaling plays a vital role in plant growth and development processes like, in apical dominance, tropic responses, lateral root formation, vascular differentiation, embryo patterning and shoot elongation. at the molecular level, most of these processes are controlled by the auxin-response genes, and auxin responsiveness is conferred to these genes by conserved promoter elements, including tga-element, auxrr-core and auxre. among these, the auxre promoter elements are bound and activated by a plant-specific transcription factors which are called as auxin response factors. an arf protein contains a dna-binding domain in the n-terminal region, a middle region that functions as an activation domain or repression domain, and a carboxyl-terminal dimerization domain that are similar to those found in the c terminus of aux/iaas, which is a protein-protein interaction domain that mediates the homo- and hetero- dimerization of arfs and also the hetero-dimerization of arf and aux/iaa proteins. it has been reported that, the arf proteins are encoded by a large gene family, with and members in arabidopsis and rice, respectively. expression analysis suggested that these genes are, in general, transcribed in a wide variety of tissues and organs, with an exception of arf gene cluster on arabidopsis chromosome which appears to be restricted to embryo genesis/seed development. classical genetic approaches have led to the identification of arf gene functions in plant growth and development. for example, arf mutations caused the change in gynoecium patterning, impaired hypocotyls response to blue light, growth and auxin sensitivity, formation of vascular strands and embryo axis formation, suppression of hookless phenotype and hypocotyl bending, hypocotyl elongation, and auxin homeostasis. moreover, the mutants of atarf sister pairs generally exhibit a much stronger phenotype than that of single mutants, suggesting that closely related atarfs have somewhat redundant roles in arabidopsis. in rice, antisense phenotype of osarf gene showed stunted growth, low vigor, curled leaves and sterility, suggesting that the gene is essential for vegetative and reproductive development. genetic divergence between arabidopsis and rice arf gene family investigated by genome-wide analysis revealed that most of the rice osarfs are related to arabidopsis arfs and fall into sister pairs as in arabidopsis. the first assembly of maize genome sequence has recently been published, however, to the best of our knowledge, the maize arf gene family has not been characterized in detail. in this article, we provide detailed information on the genomic structures, chromosomal locations, sequence homology and expression patterns of maize arf genes. in addition, the phylogenetic relationship between arf genes in arabidopsis, rice and maize were also compared, which will help future studies for elucidating the precise roles of zmarfs in maize growth and development.  identification and chromosomal localization of maize arf genes extensive searches of public and proprietary transcript and genomic databases, by using all previously reported arf proteins from other species as blast queries, identified a total of maize arf genes that have complete sequences in respective bacterial artificial chromosome clones. among these full-length coding sequences of zmarf genes were further confirmed by rt-pcr amplification, cloning and sequencing. the full length coding sequences of the zmarf genes ranged from bp to bp with the deduced proteins of to amino acids. gene a systematic designation given to maize arfs in this work. b length of open reading frame in base pairs. c the number of amino acids, molecular weight, and isoelectric point of the deduced polypeptides. d location of the zmarf genes to the chromosome bins. e bac name, dna accession number where the zmarf gene is present. f nearest marker to the zmarf gene. g representative est/cdna in genbank corresponding to zmarf gene. h location of the zmarf genes to the physical blocks. i location of the zmarf genes to the block pairs. six zmarf sister pairs are mapped on the same duplicated chromosomal blocks: zmarf and in 10la, zmarf and in 4l, zmarf and in 4l, zmarf and in 4l, zmarf and in 1l, zmarf and in 1l. the nearest genetic markers for each arf gene were determined from maize bac contigs and positioned on maize genetic map. it was found that these zmarfs were mapped on out of maize chromosomes, except for chromosome six zmarfs were present on chromosome 3; on chromosomes and 10; on chromosomes and 8; only one on chromosome. in addition, six zmarf sister pairs were mapped on the same duplicated chromosomal blocks that has been described previously. the predicted molecular weights of the deduced zmarf proteins ranged from kda to kda. pair-wise analysis of zmarf protein sequences indicated that the overall identity fell in a range within % to %. moreover, multiple protein sequence alignments were performed using the clustal_x program to examine structural features of these maize arf genes. the results revealed that all the zmarf proteins contained a highly conserved region of about amino acid residues in their n-terminal portion corresponding to the dna-binding domain of arabidopsis arf family. except zmarf and the other zmarf proteins contained a carboxyl-terminal domain related to domains iii and iv found in aux/iaa proteins. it has been reported that the middle region of arfs function as activation or repression domains. transfection assays with plant protoplasts indicated that atarf and are repressors, among which atarf contain middle region rich in proline, serine and threonine. atarf and which contain middle region rich in glutamine, are activators. the detailed sequence analysis of all deduced zmarf proteins revealed that pst rich middle regions were found in zmarf and indicating that these genes are more likely to act as repressors. while q rich regions were found in zmarf and implying that these genes are probable transcriptional activators. phylogenetic analysis and genomic structure of zmarfs bayesian phylogenetic analysis was performed and the zmarf proteins were classified into six classes: class i, ii, iii, iv, v and vi, with each class containing and members, respectively. it was worthy to note that in the joint phylogenetic tree most of the zmarf proteins fell as related sister pairs, viz. zmarf and and and and and and and and and and or triplets and quadruplets in the case of zmarf and the intron/exon structures of zmarf genes were determined by alignment of cdna to genomic sequences. this sequence analysis revealed that introns were found in coding sequences of all the arf genes and the number of exons varied from to. as expected, most zmarf genes in the same sister pair or triplets showed similar distribution of intron/exon, whereas the others were more divergent in genomic structure, suggesting that these sister pair genes lies in duplicated genomic regions. the annotated arf gene family in arabidopsis and rice enabled us to determine the phylogenetic relationship between dicot and monocot arf proteins. a phylogenetic tree constructed using the protein sequences of zmarfs, osarfs and atarfs depicted that all of these arf proteins were also divided into six classes. prediction of potential targets for small rna previous studies have revealed that several arf genes are targets for small rnas. for example, atarf and are mir targets while atarf and are mir targets. atarf and are targets for trans-acting-small interfering rna. in this study, putative small rna target sites were searched by using the miranda software. out of zmarf genes were predicted to be the potential targets of small rna and the number of target genes for mir mir tas ta-sirna was and respectively. to determine whether the increased mir level could cause the degradation of transcripts of the six potential targets, we generated mir overexpressing zong lines and the presence of transgene was confirmed by pcr amplifications of bar gene. we then analyzed mrna levels of these genes in roots of 8-day-old seedling in wild type and mir overexpressing zong lines. the results of real-time rt-pcr exhibited that mrna abundance of zmarf and in three independent transgenic lines decreased markedly as compared with wild type, indicating that post-transcriptional regulation by small rnas may play an important role in regulating the expression of zmarf genes. auxin inducibility and promoter motif prediction of zmarf genes to determine the response of zmarf genes to exogenous auxin stimuli, their expression patterns in seedling roots at and hours after μm αnaa treatment were investigated using real-time rt-pcr and fold induction relative to water-treated controls for each time point were calculated. this analysis revealed that, with an exception of zmarf all other genes were expressed in seedling roots, among which genes were up-regulated and genes were down-regulated by exogenous auxin treatment across all time points. in addition genes were up-regulated by auxin after h treatment but down-regulated later. in contrast, genes were down-regulated over the first h of treatment but up-regulated after h of auxin treatment. the other zmarf genes also displayed diverse expression pattern in response to auxin treatment, indicating the complexity of auxin-regulated gene expression of zmarf genes. interestingly, the relative mrna abundance of some arf genes, for example zmarf and were also altered dramatically in water treatment, indicating that these genes may be responsive to water logging. putative promoter sequences of zmarfs were obtained from the draft maize genome sequence. database search of plant promoters detected one or more auxin response elements in some of these putative promoters. notably, three auxin response elements were detected in the promoter region of zmarf gene, consistent with its significantly increased mrna accumulation following auxin treatment. however, out of zmarfs genes, auxin response elements were detected only in promoter regions of zmarfs genes. thus, the relationship between auxin-response elements and auxin inducibility of zmarf genes remains unclear and need to be further investigated. expression profiling of zmarf genes in embryos during seed development and germination recently, functional analysis has revealed that some arf genes in arabidopsis play important role in seed development and germination. as a result, we focused on the study of expression patterns of zmarf genes in embryos during seed development and germination. expressions of all zmarf genes were detected but they exhibited dynamic expression patterns, of which genes showed peak expression in embryos at h after imbibition. in addition, the expression patterns of duplicated zmarf genes also varied considerably. for example, the expression level of zmarf gene was much higher than that of its sister gene zmarf and the time point of peak expression was also different for zmarf and zmarf it is worthy to note that the relative mrna abundances of zmarf genes in dry mature embryos were higher than in immature embryos which further increased during seed germination. we observed that the mrna accumulation of zmarf gene peaked in dry mature embryo but declined after seed imbibition. rt-pcr analysis of embryos and two vegetative tissues detected transcripts of zmarf genes with an exception of zmarf in 8-day-old seedling leaves and roots. discussion characterization of an expanded arf gene family in maize it is believed that the long evolutionary periods experienced by a particular organism is the cause of having multiple members in the specific gene family. several rounds of whole genome duplication have been reported in the maize genome. in this study, we identified and characterized maize arf genes through genome-wide analysis, suggesting that maize arf gene family was expanded compared to arabidopsis and rice. phylogenetic analysis revealed that maize arf gene family contained ten sister pairs, two triplets and one quadruplet. however, none of these pairs were genetically linked to each other on their corresponding chromosomal locations. on the other hand, all closely linked zmarf loci such as zmarf and zmarf on chromosome 2; zmarf and on chromosome and zmarf and on chromosome were not paired together into the same sister groups. moreover, at least six zmarf sister pairs mapped on the same duplicated chromosomal blocks. thus, the expansion of maize arf gene family could be explained by the ancient tetraploid ancestry of maize, in which genome duplication occurred after divergence from the common ancestor of rice and maize, followed by subsequent diploidization en route to modern maize. expression divergence between duplicated zmarf genes the presence of duplicated zmarf genes raises the question about their functional redundancy. according to the evolutionary models, duplicated genes may undergo different selection processes: nonfunctionalization where one copy loses the function, hypofunctionalization where expression/function of one copy decreases, neofunctionalization where one copy gains a novel function, or subfunctionalization where the two copies partition or specialize into distinct functions. these evolutionary fates may result in divergence of expression patterns or protein structure. evidence for divergence between the duplicate genes could be inferred from expression pattern of zmarf and genes. zmarf was highly expressed in seed embryos during germination but the transcript of zmarf was very low. in addition, possible subfunctionalization shifts the expression pattern trends of gene pairs. for example, the mrna abundance of zmarf peaked in dry mature embryo but zmarf was highly expressed in embryos at h after seed imbibition. regulation of zmarf gene expression since arfs are transcription factors that regulate expression of auxin response genes, it would be interesting to determine the response of zmarf gene to auxin treatment. it has been reported that arabidopsis arf and and rice osarf and transcripts increased slightly in response to auxin, while osarf and decreased marginally. in the present study, we found that most of the zmarf genes were responsive to exogenous auxin treatment but the auxin responsive elements were only detected in promoter region of zmarf genes. therefore, the underlying mechanism for auxin inducibility of zmarf genes needs to be elucidated. there is a growing body of work showing the posttranscriptional regulation of arf transcript abundance by micro-rnas and trans-acting-small interfering rnas. regulation of atarf and by mir is important for development of anthers and ovules, regulation of atarf by mir is important for arabidopsis growth and development and regulation of atarf and by mir plays a role in root cap formation. the regulation of atarf and targeted by tas ta-sirnas is required for proper leaf development and juvenile to adult phase changes. the target sites of these three small rnas were also detected in maize arf genes, and are widely conserved in dicots and monocots. furthermore, our transgenic analysis exhibited that increased mir level could cause degradation of transcripts for six potential targets, indicating that zmarf genes showed posttranscriptional regulation. however, we found that the expression patterns of zmarf genes which contained the same target sites varied considerably in embryos during seed development and germination, for example the expression of zmarf and with two potential tas ta-sirnas recognition sites. thus, we speculate that some unidentified factors may also play a role in regulating expression of these genes, which could be highly specific to a selected tissue type or developmental program with the possibility that mirna and tasirna may have functions in very discreet regions. potential functions of zmarf genes during seed development seed development is considered as a physical link between parents and sporophytic generation in plants. auxin signaling is thought to play an important role in embryo development. for example, a higher level auxin is detected in root apex and ends of cotyledon primordia from heart to mature embryo in arabidopsis. defects in arabidopsis embryo patterning was observed in arf mutants, which enhanced in arf5arf double mutants. in this study, seven zmarf genes, including zmarf and appeared to be constitutively expressed in developing embryos, whereas the transcripts of other zmarf genes exhibited dynamic expression patterns, suggesting the partitioning of functions between these genes in embryo development. in most flowering plants, seed germination is the first and may be the foremost growth stage in the plant's life cycle. genetic evidence supporting a role of arf genes in germination has been obtained from the analysis of regulation of atarf by mir. it has been reported that transgenic seeds of arabidopsis expressing a mir160-resistant form of atarf are hypersensitive to germination inhibition by exogenous aba, whereas ectopic expression of mir results in a reduced sensitivity to aba. in the present study, zmarf and are predicted to be the targets for mir which falls in the same subfamily with atarf and expression analysis demonstrated that mrna accumulation of zmarf and gradually increased during seed germination, reaching its peak in embryos after h of seed imbibition and decreased in later stage, whereas zmarf showed high transcript accumulation in dry seed embryos. in addition, dynamic expression patterns were also observed for other zmarf genes. collectively, we speculate that zmarf genes may be involved in diverse aspects of developmental processes during seed germination.  maize arf gene family is expanded as compared to arabidopsis and rice reflecting a succession of maize genomic rearrangements and expansions due to extensive duplication and diversification that frequently occurred in the course of evolution. the expressions of maize arf genes are regulated by auxin and small rnas. dynamic expression patterns of zmarf genes in embryo at different stages were observed, which suggest that these genes may be involved in seed development and germination. 
auxin signaling is vital for plant growth and development, and plays important role in apical dominance, tropic response, lateral root formation, vascular differentiation, embryo patterning and shoot elongation. dynamic expression patterns of zmarf genes in embryo at different stages were detected which suggest that maize arf genes may be involved in seed development and germination. the zmarf genes were predicted to be the potential targets of small rnas. transgenic analysis revealed that increased mir level could cause degradation of transcripts of six potential targets. the first draft of full maize genome assembly has recently been released, however, to our knowledge, the arf gene family from maize has not been characterized in detail. maize arf gene family is expanded as compared to arabidopsis and rice. dynamic expression patterns of zmarf genes were observed in different stages of embryo development. the arf genes are represented by a large multigene family in plants. it was shown that maize arf genes fall into related sister pairs and chromosomal mapping revealed that duplication of zmarfs was associated with the chromosomal block duplications. out of these zmarf genes, possess auxin-responsive element in their promoter region, among which appear to show small or negligible response to exogenous auxin. the expressions of maize arf genes are responsive to exogenous auxin treatment. the expression of these genes in maize is regulated by auxin and small rnas. auxin response factors are the transcription factors that regulate the expression of auxin responsive genes.
 auxin signaling is vital for plant growth and development, and plays important role in apical dominance, tropic response, lateral root formation, vascular differentiation, embryo patterning and shoot elongation. auxin response factors are the transcription factors that regulate the expression of auxin responsive genes. the arf genes are represented by a large multigene family in plants. the first draft of full maize genome assembly has recently been released, however, to our knowledge, the arf gene family from maize has not been characterized in detail.  in this study, maize genes that encode arf proteins were identified in maize genome. it was shown that maize arf genes fall into related sister pairs and chromosomal mapping revealed that duplication of zmarfs was associated with the chromosomal block duplications. as expected, duplication of some zmarfs showed a conserved intron/exon structure, whereas some others were more divergent, suggesting the possibility of functional diversification for these genes. out of these zmarf genes, possess auxin-responsive element in their promoter region, among which appear to show small or negligible response to exogenous auxin. the zmarf genes were predicted to be the potential targets of small rnas. transgenic analysis revealed that increased mir level could cause degradation of transcripts of six potential targets. the expressions of maize arf genes are responsive to exogenous auxin treatment. dynamic expression patterns of zmarf genes were observed in different stages of embryo development.  maize arf gene family is expanded as compared to arabidopsis and rice. the expression of these genes in maize is regulated by auxin and small rnas. dynamic expression patterns of zmarf genes in embryo at different stages were detected which suggest that maize arf genes may be involved in seed development and germination. 