BACKGROUND
flower development in plants involves tightly regulated processes starting from floral initiation to flower formation.
the underlying processes have been extensively investigated, as flower development is an important agronomic trait that determines crop yield.
various transcription factors are essential in regulating these developmental processes, including the family of mads-box transcription factors.
the mads-box transcription factors, especially the plant-specific classical  mikc-type mads-box genes, are known to play key regulatory roles in different stages of flower development.
their roles in coordinating floral developmental processes have been revealed by functional studies largely carried out in the model plant, arabidopsis thaliana.
the mikcc-type genes are characterised by a conserved structural organisation of the mads-box, intervening-, keratin-like- and c-domains.
the highly conserved mads-domain and the weakly conserved i-domain are required for dna binding, while the strongly conserved k-domain and the variable c-domain regulate protein interactions  <cit> .
genome-wide analyses of the mikcc-type genes have been carried out in arabidopsis  <cit> , rice  <cit>  and poplar  <cit> .
while arabidopsis and rice genomes have similar numbers of mikcc-type genes , poplar has  <dig> of these genes, suggesting a higher birth rate compared to arabidopsis or rice.
these mikcc-type genes can be divided into  <dig> distinct gene clades with each clade named after the first member identified  <cit> .
all but two  are found in arabidopsis  <cit> , and the flc clade may be absent from the rice genome  <cit> .
it remains unclear whether all clades are present in the poplar genome, as no tm <dig> genes were used in the phylogenetic analysis  <cit> .
the squa subfamily clade includes four arabidopsis members, apetala <dig> , cauliflower , fruitfull  and agamous-like  <dig>   <cit> .
the functions of ap <dig>  cal and ful have been characterised, indicating that they play partially redundant roles in determining floral meristem identity  <cit> .
the sepallata  family belongs to the agl <dig> clade, and there are four members documented in arabidopsis  <cit> .
all four members  play redundant functions in determining floral organ identity and floral meristem determinacy.
ap <dig> has been shown to bind directly to the sep <dig> promoter, hence increasing the expression of sep <dig> rapidly  <cit> .
the agl <dig> subfamily has a relatively small representation  in arabidopsis.
while no knockout phenotype has been described for either of these genes in arabidopsis, studies in rice, maize and petunia hybrida have largely demonstrated the roles of the agl <dig> subfamily in regulating floral organ identity and floral meristem determinacy, indicating redundant roles with closely related genes including sep <cit> .
a phylogenetic study showed that subfamilies of squa, sep and agl <dig> are always rooted together in one superclade, which may be correlated with their overlapping roles in regulating flower development.
a total of  <dig> mads-box genes were predicted in the recent genome sequence of soybean  <cit> .
earlier we reported the diversification of some gene expression and micrornas in legume sam  <cit> .
however, much remains to be learned about these genes, especially given their potential impact on crop production.
soybean is the largest legume crop in the world and accounts for greater than 50% of the global oilseed production.
in this study, we identified all the soybean mikcc-type mads-box genes using the current glyma <dig>  gene set and identified potential phylogenetic relationships to their arabidopsis, rice and poplar counterparts.
we examined the expression patterns across different soybean tissues for the entire family.
intriguingly, the results revealed a novel agl <dig> sister clade of mikcc-type genes in soybean, and we focused our subsequent analysis on members of this novel sub-clade.
RESULTS
molecular evolutionary analysis of soybean mikcc-type mads-box transcription factors
when we searched the soybean predicted gene set available at phytozome  <cit>  for sequences containing both a mads-box and k-domain, we identified a total of  <dig> sequences.
subsequent inspection revealed three of the sequences were incomplete.
we attempted to obtain a full-length sequence for these genes using gene prediction software on the genome sequence surrounding these partial sequences but did not yield any results.
therefore, we omitted these sequences from further analysis.
to investigate their phylogenetic relationships with mikcc-type genes from arabidopsis, rice and poplar, reported mikcc group protein sequences  <cit>  were retrieved from their respective databases.
a total of  <dig> conceptually translated protein sequences were used in the phylogenetic analysis.
fifteen existing clades were identified from the generated phylogenetic tree: agl <dig>  agl <dig>  squa, agl <dig>  flc, tm <dig>  agl <dig>  ag, osmads <dig>  tm <dig>  stmads <dig>  agl <dig>  ggm <dig>  def, and glo .
the relationships among the different clades are similar to other reports  <cit> .
with the exception of osmads <dig> and tm <dig>  soybean genes are found in all clades, and the number of soybean sequences within each clade varies from one  to nine , with most genes occurring in duplicate.
consistent with a previous report  <cit> , the genes in agl <dig>  squa and agl <dig> form a superclade together .
within the agl <dig> clade, a strongly supported internal branch seems to be separate from the existing agl <dig> members, suggesting it is a novel sister clade not represented in arabidopsis or rice.
this novel sub-clade consists of three soybean genes, glyma16g <dig> and a homeolog pair, glyma10g <dig>  and glyma20g <dig> .
the top blastx match of these genes is a mads-box transcription factor from garden pea, which is annotated as psmads <dig>  <cit>  .
therefore, we named it the psmads <dig> sub-clade.
we next examined whether similar orthologous members of psmads <dig> exist in species other than soybean, garden pea and poplar.
a blastp search at the ncbi public database identified potential psmads <dig> sequences from other species .
using the top matching  <dig> orthologs including sequences from the sister clade, agl <dig>  for phylogenetic analysis, three distinct groups were identified in the tree rooted with gymnosperm agl <dig> as the outgroup .
one clade groups all of the monocot sequences, whereas the other two clades contain agl <dig> and psmads <dig> sequences .
as we were only using sequences available in the public database, it is likely that similar psmads <dig> sequences exist in species other than those examined.
future studies with increased taxon sampling will help to clarify the node.
members of the psmads <dig> sub-clade are represented in not only legume species but also poplar and grape .
as the genome sequences for arabidopsis and rice are available and well annotated, we are confident that members of this psmads <dig> sub-clade are absent from these two species.
furthermore, the observation that no orthologous psmads <dig> genes is found among the top matching sequences from other monocots including wheat and maize  implies that such genes may be absent from monocot.
the psmads <dig> clade may have evolved after monocot-dicot divergence and following the emergence of the arabidopsis species.
these genes may have been overlooked previously due to their absence in arabidopsis and rice.
in fact, none of these legume genes were analysed in a recent study on eudicot agl <dig>  <cit> .
in silico analysis of soybean mikcc gene expression patterns
we previously performed high-throughput rna-sequencing on micro-dissected shoot apical meristems  undergoing the early floral initiation process  <cit> .
the samples were derived from soybean plants  subjected to different lengths of short-day  treatments , and the induction of the floral meristem identity genes including gmap <dig> occurred on 4sd.
because genes that are involved in later processes of flowering such as floral organ development or only expressed in other tissues such as roots may not be captured by our dataset, we also used the transcriptome data reported by libault and co-workers  <cit>  to include a diverse range of tissues including flower, seed pod, root, nodule and root tip in our in silico analysis .
all identified soybean mikcc-genes are expressed in at least one of the three reproductive tissues represented , except for members of the agl <dig> clade, which are only expressed in the root .
arabidopsis agl <dig> is preferentially expressed in the root, and recent loss-of-function analyses have revealed its roles in not only regulating root meristem cell proliferation but also flowering transition  <cit> .
based on the soybean agl12-like expression profile, it is tempting to speculate that their functions in floral regulation may have been lost.
a similar expression pattern was observed for most mikcc-genes clustered within a clade.
all duplicated genes are transcribed and have comparable expression profiles, especially in the reproductive sam , suggesting their functional significance.
as for the superclade consisting of agl <dig>  squa, and agl <dig>  there are some notable differences in the gene expression profiles among the three clades .
for example, all agl2-like genes except one  are absent from the sam during the early floral transition process but are expressed later in the floral developmental process in the flower and pod.
this pattern is expected as these genes are known to be activated following ap <dig> induction in arabidopsis  <cit> .
the phylogenetic tree indicates that glyma01g <dig> is the counterpart for arabidopsis sep <dig>  in addition to being found in flower and pod like the rest of the agl2-like genes, it is also expressed in the sams during the floral initiation process and very highly in nodules.
this pattern implies a likely diverged function of gmsep <dig> with additional roles in the early floral initiation process as well as in nodule formation.
glyma01g <dig>  one of the four soybean counterparts of arabidopsis ap <dig>  also likely plays a role in nodule formation.
although the expression of glyma01g <dig> is drastically induced on 4sd in the sam , the level of expression is 6-fold less than that in the nodule .
intriguingly, its homeolog glyma02g <dig> is not expressed in the nodule but rather has the highest expression in the reproductive sam , suggesting a functional divergence between this homeolog pair.
although members of soybean agl <dig> genes are expressed in the reproductive sams, changes in their transcript levels are not comparable with those of psmads3-like and squa-like genes during the early floral transition process, suggesting that the latter two clades are likely to play more prominent roles in the developmental transition process.
because there is no information available for psmads3-like genes, we focused our study on members of this novel sister clade.
expression of gmmads <dig> during the floral initiation process
we carried out rt-pcr analysis to verify the expression of this novel family in the soybean sam during the early floral initiation process .
rna was extracted from dissected sams of plants undergoing  <dig>   <dig>   <dig>   <dig> and  <dig> sd treatments.
rt-pcr was carried out with primers specific to each of the soybean members in this psmads <dig> branch  as well as to gmap <dig>  as the control for the floral induction process.
consistent with our previous study  <cit> , the induction of gmap <dig> occurs after the  <dig> short-day treatment.
all three soybean genes in the psmads <dig> clade displayed a similar expression pattern to that of gmap <dig>  consistent with the in silico analysis.
to examine the spatial expression pattern of these genes during the floral initiation process, in situ hybridisation analysis was performed.
on 4sd, gmap <dig> expression was detected in the incipient floral primordia of the inflorescence meristem .
on 6sd, newly established floral meristems became more prominent and gmap <dig> expression was detected throughout these meristems .
gmmads3a  exhibited a rather similar expression pattern to gmap <dig> on 4sd , but its expression subsequently spread to the entire inflorescence meristem as well as to the newly established floral meristems on 6sd .
as the gmmads <dig> homeolog pair is almost identical in nucleotide sequence including the utr regions , no gene-specific probe could be made, and thus the signals observed may correspond to the expression of both genes.
nevertheless, the expression of gmmads <dig> throughout the entire inflorescence and floral meristem suggests that it can serve as both an inflorescence and floral meristem identity gene.
because the expression of gmmads <dig> initially overlaps with that of gmap <dig>  it likely performs similar functions as gmap <dig>  its subsequent widespread expression in the inflorescence meristem may ensure all vegetative activities at the sam are replaced with the initiation of the floral meristem at the meristem flanks.
the expression of glyma16g <dig> is distinct from that of gmap <dig> and gmmads <dig>  a weak signal associated with its expression was detected in the centre of the inflorescence meristem ; on 6sd, its expression was also observed in the centre of the newly emerged floral meristem .
the expression of glyma16g <dig> in the centre of the meristem indicates its potential regulatory roles in orchestrating events in the inflorescence meristem.
the spatial expression pattern of the soybean psmads3-like genes supports that these genes are novel, as their expression differs markedly from the spatial expression of closely related family members such as gmap <dig>  or arabidopsis agl <dig> <cit> .
CONCLUSIONS
in contrast to arabidopsis where the initiation timing of floral whorls does not overlap, the legume soybean has a flower development system with overlapping whorls  <cit> .
furthermore, unlike arabidopsis that usually cannot undergo flowering reversion  <cit> , the soybean inflorescent meristem can revert to leaf production when the environmental growth conditions are switched from sd to ld  <cit> .
because the mikcc-type mads-box genes play key regulatory roles in different stages of flower development, it is conceivable that members of the psmads <dig> sub-clade identified in this study could contribute to developmental plasticity in cooperation with key floral regulators such as gmap <dig> or gmflc.
future studies aimed at defining the interacting partners of these genes will aid in our understanding of the floral transition process.

 flower development in plants involves tightly regulated processes starting from floral initiation to flower formation. the underlying processes have been extensively investigated, as flower development is an important agronomic trait that determines crop yield. various transcription factors are essential in regulating these developmental processes, including the family of mads-box transcription factors. the mads-box transcription factors, especially the plant-specific classical mikc-type mads-box genes, are known to play key regulatory roles in different stages of flower development. their roles in coordinating floral developmental processes have been revealed by functional studies largely carried out in the model plant, arabidopsis thaliana. the mikcc-type genes are characterised by a conserved structural organisation of the mads-box, intervening-, keratin-like- and c-domains. the highly conserved mads-domain and the weakly conserved i-domain are required for dna binding, while the strongly conserved k-domain and the variable c-domain regulate protein interactions. genome-wide analyses of the mikcc-type genes have been carried out in arabidopsis, rice and poplar. while arabidopsis and rice genomes have similar numbers of mikcc-type genes, poplar has of these genes, suggesting a higher birth rate compared to arabidopsis or rice. these mikcc-type genes can be divided into distinct gene clades with each clade named after the first member identified. all but two are found in arabidopsis, and the flc clade may be absent from the rice genome. it remains unclear whether all clades are present in the poplar genome, as no tm genes were used in the phylogenetic analysis. the squa subfamily clade includes four arabidopsis members, apetala, cauliflower, fruitfull and agamous-like. the functions of ap cal and ful have been characterised, indicating that they play partially redundant roles in determining floral meristem identity. the sepallata family belongs to the agl clade, and there are four members documented in arabidopsis. all four members play redundant functions in determining floral organ identity and floral meristem determinacy. ap has been shown to bind directly to the sep promoter, hence increasing the expression of sep rapidly. the agl subfamily has a relatively small representation in arabidopsis. while no knockout phenotype has been described for either of these genes in arabidopsis, studies in rice, maize and petunia hybrida have largely demonstrated the roles of the agl subfamily in regulating floral organ identity and floral meristem determinacy, indicating redundant roles with closely related genes including sep. a phylogenetic study showed that subfamilies of squa, sep and agl are always rooted together in one superclade, which may be correlated with their overlapping roles in regulating flower development. a total of mads-box genes were predicted in the recent genome sequence of soybean. earlier we reported the diversification of some gene expression and micrornas in legume sam. however, much remains to be learned about these genes, especially given their potential impact on crop production. soybean is the largest legume crop in the world and accounts for greater than 50% of the global oilseed production. in this study, we identified all the soybean mikcc-type mads-box genes using the current glyma gene set and identified potential phylogenetic relationships to their arabidopsis, rice and poplar counterparts. we examined the expression patterns across different soybean tissues for the entire family. intriguingly, the results revealed a novel agl sister clade of mikcc-type genes in soybean, and we focused our subsequent analysis on members of this novel sub-clade.  molecular evolutionary analysis of soybean mikcc-type mads-box transcription factors when we searched the soybean predicted gene set available at phytozome for sequences containing both a mads-box and k-domain, we identified a total of sequences. subsequent inspection revealed three of the sequences were incomplete. we attempted to obtain a full-length sequence for these genes using gene prediction software on the genome sequence surrounding these partial sequences but did not yield any results. therefore, we omitted these sequences from further analysis. to investigate their phylogenetic relationships with mikcc-type genes from arabidopsis, rice and poplar, reported mikcc group protein sequences were retrieved from their respective databases. a total of conceptually translated protein sequences were used in the phylogenetic analysis. fifteen existing clades were identified from the generated phylogenetic tree: agl agl squa, agl flc, tm agl ag, osmads tm stmads agl ggm def, and glo. the relationships among the different clades are similar to other reports. with the exception of osmads and tm soybean genes are found in all clades, and the number of soybean sequences within each clade varies from one to nine, with most genes occurring in duplicate. consistent with a previous report, the genes in agl squa and agl form a superclade together. within the agl clade, a strongly supported internal branch seems to be separate from the existing agl members, suggesting it is a novel sister clade not represented in arabidopsis or rice. this novel sub-clade consists of three soybean genes, glyma16g and a homeolog pair, glyma10g and glyma20g. the top blastx match of these genes is a mads-box transcription factor from garden pea, which is annotated as psmads. therefore, we named it the psmads sub-clade. we next examined whether similar orthologous members of psmads exist in species other than soybean, garden pea and poplar. a blastp search at the ncbi public database identified potential psmads sequences from other species. using the top matching orthologs including sequences from the sister clade, agl for phylogenetic analysis, three distinct groups were identified in the tree rooted with gymnosperm agl as the outgroup. one clade groups all of the monocot sequences, whereas the other two clades contain agl and psmads sequences. as we were only using sequences available in the public database, it is likely that similar psmads sequences exist in species other than those examined. future studies with increased taxon sampling will help to clarify the node. members of the psmads sub-clade are represented in not only legume species but also poplar and grape. as the genome sequences for arabidopsis and rice are available and well annotated, we are confident that members of this psmads sub-clade are absent from these two species. furthermore, the observation that no orthologous psmads genes is found among the top matching sequences from other monocots including wheat and maize implies that such genes may be absent from monocot. the psmads clade may have evolved after monocot-dicot divergence and following the emergence of the arabidopsis species. these genes may have been overlooked previously due to their absence in arabidopsis and rice. in fact, none of these legume genes were analysed in a recent study on eudicot agl. in silico analysis of soybean mikcc gene expression patterns we previously performed high-throughput rna-sequencing on micro-dissected shoot apical meristems undergoing the early floral initiation process. the samples were derived from soybean plants subjected to different lengths of short-day treatments, and the induction of the floral meristem identity genes including gmap occurred on 4sd. because genes that are involved in later processes of flowering such as floral organ development or only expressed in other tissues such as roots may not be captured by our dataset, we also used the transcriptome data reported by libault and co-workers to include a diverse range of tissues including flower, seed pod, root, nodule and root tip in our in silico analysis. all identified soybean mikcc-genes are expressed in at least one of the three reproductive tissues represented, except for members of the agl clade, which are only expressed in the root. arabidopsis agl is preferentially expressed in the root, and recent loss-of-function analyses have revealed its roles in not only regulating root meristem cell proliferation but also flowering transition. based on the soybean agl12-like expression profile, it is tempting to speculate that their functions in floral regulation may have been lost. a similar expression pattern was observed for most mikcc-genes clustered within a clade. all duplicated genes are transcribed and have comparable expression profiles, especially in the reproductive sam, suggesting their functional significance. as for the superclade consisting of agl squa, and agl there are some notable differences in the gene expression profiles among the three clades. for example, all agl2-like genes except one are absent from the sam during the early floral transition process but are expressed later in the floral developmental process in the flower and pod. this pattern is expected as these genes are known to be activated following ap induction in arabidopsis. the phylogenetic tree indicates that glyma01g is the counterpart for arabidopsis sep in addition to being found in flower and pod like the rest of the agl2-like genes, it is also expressed in the sams during the floral initiation process and very highly in nodules. this pattern implies a likely diverged function of gmsep with additional roles in the early floral initiation process as well as in nodule formation. glyma01g one of the four soybean counterparts of arabidopsis ap also likely plays a role in nodule formation. although the expression of glyma01g is drastically induced on 4sd in the sam, the level of expression is 6-fold less than that in the nodule. intriguingly, its homeolog glyma02g is not expressed in the nodule but rather has the highest expression in the reproductive sam, suggesting a functional divergence between this homeolog pair. although members of soybean agl genes are expressed in the reproductive sams, changes in their transcript levels are not comparable with those of psmads3-like and squa-like genes during the early floral transition process, suggesting that the latter two clades are likely to play more prominent roles in the developmental transition process. because there is no information available for psmads3-like genes, we focused our study on members of this novel sister clade. expression of gmmads during the floral initiation process we carried out rt-pcr analysis to verify the expression of this novel family in the soybean sam during the early floral initiation process. rna was extracted from dissected sams of plants undergoing and sd treatments. rt-pcr was carried out with primers specific to each of the soybean members in this psmads branch as well as to gmap as the control for the floral induction process. consistent with our previous study, the induction of gmap occurs after the short-day treatment. all three soybean genes in the psmads clade displayed a similar expression pattern to that of gmap consistent with the in silico analysis. to examine the spatial expression pattern of these genes during the floral initiation process, in situ hybridisation analysis was performed. on 4sd, gmap expression was detected in the incipient floral primordia of the inflorescence meristem. on 6sd, newly established floral meristems became more prominent and gmap expression was detected throughout these meristems. gmmads3a exhibited a rather similar expression pattern to gmap on 4sd, but its expression subsequently spread to the entire inflorescence meristem as well as to the newly established floral meristems on 6sd. as the gmmads homeolog pair is almost identical in nucleotide sequence including the utr regions, no gene-specific probe could be made, and thus the signals observed may correspond to the expression of both genes. nevertheless, the expression of gmmads throughout the entire inflorescence and floral meristem suggests that it can serve as both an inflorescence and floral meristem identity gene. because the expression of gmmads initially overlaps with that of gmap it likely performs similar functions as gmap its subsequent widespread expression in the inflorescence meristem may ensure all vegetative activities at the sam are replaced with the initiation of the floral meristem at the meristem flanks. the expression of glyma16g is distinct from that of gmap and gmmads a weak signal associated with its expression was detected in the centre of the inflorescence meristem; on 6sd, its expression was also observed in the centre of the newly emerged floral meristem. the expression of glyma16g in the centre of the meristem indicates its potential regulatory roles in orchestrating events in the inflorescence meristem. the spatial expression pattern of the soybean psmads3-like genes supports that these genes are novel, as their expression differs markedly from the spatial expression of closely related family members such as gmap or arabidopsis agl.  in contrast to arabidopsis where the initiation timing of floral whorls does not overlap, the legume soybean has a flower development system with overlapping whorls. furthermore, unlike arabidopsis that usually cannot undergo flowering reversion, the soybean inflorescent meristem can revert to leaf production when the environmental growth conditions are switched from sd to ld. because the mikcc-type mads-box genes play key regulatory roles in different stages of flower development, it is conceivable that members of the psmads sub-clade identified in this study could contribute to developmental plasticity in cooperation with key floral regulators such as gmap or gmflc. future studies aimed at defining the interacting partners of these genes will aid in our understanding of the floral transition process. 
our study implicates three mikcc-type transcription factors as novel members of the agamous like subfamily of the mikcc-type mads-box genes, and we named this sister clade psmads while similar genes were identified in other legume species, poplar and grape, no such gene is represented in arabidopsis thaliana or rice. legume flower development system differs from that in the model plant, arabidopsis. mikcc-type mads-box genes have been shown to play key regulatory roles in different stages of flower development. there is an overlap in the initiation of different floral whorls in soybean, and inflorescent meristems can revert to leaf production depending on the environmental conditions. the results from this study will contribute to a better understanding of legume-specific floral developmental processes. rt-pcr analysis on these three soybean psmads genes during early floral initiation processes revealed their temporal expression similar to that of apetala a gene known to function as a floral meristem identity gene. despite their importance in regulating developmental processes that impact crop yield, they remain largely unexplored in the major legume oilseed crop, soybean. we identified members of the psmads sub-clade in legumes that show differential spatial expression during floral initiation, indicating their potential novel roles in the floral initiation process. the classical mikc-type mads-box transcription factors comprise one gene family that plays diverse roles in the flowering process ranging from floral initiation to the development of floral organs.
 the classical mikc-type mads-box transcription factors comprise one gene family that plays diverse roles in the flowering process ranging from floral initiation to the development of floral organs. despite their importance in regulating developmental processes that impact crop yield, they remain largely unexplored in the major legume oilseed crop, soybean.  we identified mikcc-type transcription factors from soybean and determined the in silico gene expression profiles of the soybean mikcc-type genes across different tissues. our study implicates three mikcc-type transcription factors as novel members of the agamous like subfamily of the mikcc-type mads-box genes, and we named this sister clade psmads while similar genes were identified in other legume species, poplar and grape, no such gene is represented in arabidopsis thaliana or rice. rt-pcr analysis on these three soybean psmads genes during early floral initiation processes revealed their temporal expression similar to that of apetala a gene known to function as a floral meristem identity gene. however, rna in situ hybridisation showed that their spatial expression patterns are markedly different from those of apetala  legume flower development system differs from that in the model plant, arabidopsis. there is an overlap in the initiation of different floral whorls in soybean, and inflorescent meristems can revert to leaf production depending on the environmental conditions. mikcc-type mads-box genes have been shown to play key regulatory roles in different stages of flower development. we identified members of the psmads sub-clade in legumes that show differential spatial expression during floral initiation, indicating their potential novel roles in the floral initiation process. the results from this study will contribute to a better understanding of legume-specific floral developmental processes. developmentfloral meristemmads-box transcription factorsoybean 