BACKGROUND
the mads-box family members, identified initially as floral homeotic genes, are one of the most extensively studied transcription factor genes in plants  <cit> .
the word mads finds its origin from the first letters of its founding members, mini chromosome maintenance  <dig>  of yeast   <cit> , agamous  of arabidopsis   <cit> , deficiens  of snapdragon   <cit>  and serum response factor  of humans   <cit> .
mads-box transcription factors are characterized by the presence of an approximately  <dig> amino acids dna binding domain, known as the mads-box domain, located in the n-terminal region of the protein.
the plant-specific mikc-type mads-box proteins include three additional domains followed by the mads domain, viz.
a less-conserved intervening region of ~ <dig> amino acids, a moderately conserved keratin-like domain of ~ <dig> amino acids mainly involved in heterodimerization, and a highly variable c-terminal region of variable length implicated in transcriptional activation and higher-order complex formation  <cit> .
the mads-box family has been divided into two main groups.
the type i consists of arg80/srf-like genes of animals and fungi, also designated as m-type genes in plants, and type ii contains mef2-like genes of animals and yeast as well as mikc-type genes of plants.
it is proposed that an ancestral duplication before the divergence of plants and animals gave rise to these groups  <cit> .
the mikc-type genes are also characterized by the presence of k domain that could have evolved after the divergence of these lineages.
the type ii genes have been categorized into mikcc- and mikc*-type based on structural features  <cit> .
the mikcc genes have been further classified into  <dig> clades based on phylogeny  <cit> .
type i genes have also been categorized into m- and n-type based on the protein motifs identified using the meme search tool  <cit>  and also as mα, mβ, mγ and mδ, based on the phylogenetic relationships between mads-box regions  <cit> .
the mδ group, however, corresponds to the mikc* class described in this report and elsewhere  <cit> .
the most striking feature of the mads-box gene family is the diverse functions taken up by its members in different aspects of plant growth and development.
these include flowering time control, meristem identity, floral organ identity, formation of dehiscence zone, fruit ripening, embryo development as well as development of vegetative organs such as root and leaf  <cit> .
genome-wide identification and phylogenetic analyses of mads-box genes have revealed  <dig> and  <dig>  rice pseudomolecule release  <dig> database) genes in arabidopsis and rice, respectively  <cit> .
though a large amount of expression data based on sage, microarrays and other high-throughput transcriptome analysis techniques is available in public databases, the studies involving expression of the entire mads-box family have so far been restricted to northern blot analysis or reverse transcriptase pcr at limited stages of development  <cit> .
recently, the comparison of expression profiles resulting from a  <dig> k rice cdna microarray-based transcriptome analysis of early panicle development in rice was used to implicate three mads-box genes, osmads <dig>   <dig> and  <dig>  in panicle branching  <cit> .
the use of high-throughput genome-wide transcriptome analysis provides an insight into changes in the entire transcriptome across a variety of biological conditions.
in combination with the whole genome sequence data and comparative expression analysis with genes of known functions, the transcriptomic data can become an initiation point for systematic investigations into structure-function relationships.
with an overall objective to understand regulation of reproductive organ development in indica rice, we have initiated a program on microarray-based expression profiling of transcription factors and signal transduction components.
here, we report a comprehensive account of identification and phylogenetic analysis of  <dig> members of mads-box gene family in rice and their expression profiling during  <dig> stages of panicle and seed development along with three abiotic stress conditions and  <dig> stages of vegetative development.
this analysis is based on tigr rice pseudomolecule release  <dig> and kome  rice full-length cdna database.
we have identified  <dig> new members belonging to this gene family besides confirming  <dig> previously identified genes.
out of  <dig> previously identified genes by nam and coworkers  <cit> , six were not found in version  <dig> of tigr.
our analysis also suggests the existence of mβ-type genes in rice, which was earlier thought to be absent in monocots  <cit> .
the results of expression profiling have been discussed in light of phylogenetic relatedness of the genes and their known functions in rice as well as other systems.
RESULTS
identification, organization and structure of mads-box genes
hmm analysis and name search resulted in the identification of  <dig> mads-box genes in rice genome.
since, gene names from osmads <dig> to osmads <dig> representing  <dig> genes already existed in the literature , newly identified genes were named from osmads <dig> to osmads <dig> .
the individual genes were localized on chromosomes based on the 5' and 3' coordinates for respective gene models in tigr database .
the maximum number of genes  were found to be localized on chromosome  <dig>  whereas, chromosomes  <dig> and  <dig> had only one mads-box gene each.
out of five types of mads-box genes, the mγ genes were confined to chromosome  <dig>   <dig> and  <dig>  while mβ genes were present only on chromosome  <dig>  no chromosomal bias was observed in the distribution of mikcc, mikc* and mα genes .
analysis of the tigr rice segmental duplication database revealed  <dig> mads-box genes within the duplicated segments of rice chromosomes.
only  <dig> genes, however, were found to have their counterparts on duplicated segments .
most of the duplicated genes belonged to the mikcc group.
several mads-box genes, especially m-type, were also found juxtaposed on chromosomes  <dig>   <dig>   <dig>   <dig>   <dig> and  <dig>  twenty such genes showed significant sequence identity.
*molecular wt and pi of osmads <dig> has been calculated using gene runner
** tigr model represents only k box protein.
similar to that reported in arabidopsis, distribution of introns in rice mads-box family genes was also found bimodal with mikcc and mikc* genes containing multiple introns and the mα, mβ and mγ genes usually having no or occasionally up to  <dig> introns .
the length of mads-box proteins varied from  <dig> to  <dig> amino acids, with few exceptionally longer or smaller proteins .
for details on other parameters of nucleic acid and protein sequences, refer to table  <dig>
evolutionary relationships between rice and arabidopsis mads-box family genes
to examine the evolutionary relationships of mads-box genes in rice  and arabidopsis, a tree was constructed using only the conserved mads-box domain.
five groups, as described by parenicova and coworkers  <cit>  were identified containing representative genes of both rice and arabidopsis.
all the arabidopsis proteins were found to lie in groups similar to those identified previously  <cit> , except agl <dig> and agl <dig>  which instead of forming a basal branch of the mβ, grouped with mγ proteins in our analysis as shown in supplementary figure s <dig> .
osmads <dig> grouped separately with agl <dig> of arabidopsis, which does not cluster with any of the mads groups described above .
a separate phylogenetic tree was also generated from complete protein sequences of all the mads-box genes in rice and arabidopsis .
of the  <dig> rice mads-box genes,  <dig> grouped with mikcc, six with mikc*, nine with mβ,  <dig> with mα and  <dig> grouped with mγ-type arabidopsis genes.
in case of m-type genes, barring osmads <dig>   <dig> and  <dig>  all other genes exhibited similar groupings as in the mads-domain-specific phylogenetic analysis .
mikcc proteins were further divided into  <dig> clades.
representatives of both rice and arabidopsis could be identified in all the clades except osmads <dig> and the flc clade, which are exclusive to rice and arabidopsis, respectively .
phylogenetic trees with bootstrap values, for complete protein sequences as well as coding sequences of rice and arabidopsis mads-box genes have been shown as supplementary figures s <dig> and s <dig>  respectively .
distribution of conserved motifs
the meme motif search tool was employed to identify the conserved motifs present in mads-box proteins .
motifs  <dig>   <dig> or  <dig> specifying the mads domain were found in all the members of the mads-box family.
all proteins belonging to mikcc and mikc* groups had motif 1-type mads domain.
most mα proteins also had the motif 1-type mads domain except osmads <dig> and  <dig> which contained motif  <dig>  motif  <dig> was found to be the most common type of mads domain in mβ-type proteins.
distinctively, in case of mγ proteins a larger mads domain of  <dig> amino acids was detected, followed by a coiled coil region and a region of unknown complexity as indicated by simple modular architecture research tool  version  <dig> .
together these components formed a larger conserved region of  <dig> amino acids denoted as motif  <dig>  the conserved motifs  <dig>   <dig> and  <dig>  specifying k domain, were found mainly in the mikcc group members.
of the  <dig> mikcc proteins,  <dig> had all three conserved regions, whereas, motif  <dig> was absent in case of osmads <dig> and 57; motif  <dig> in osmads <dig> and 55; and the complete k domain was missing in osmads <dig>   <dig> and  <dig>  the partial k domain was also detected in some of the non-mikcc proteins including osmads <dig> and  <dig> of mγ, osmads <dig> and  <dig> of mikc*, osmads <dig> and osmads <dig> of mα group.
certain unknown motifs, numbered  <dig>   <dig>   <dig>   <dig>   <dig> and  <dig>  besides the mads  and the k  domains in mikcc proteins, were also revealed by meme motif search.
motif  <dig> corresponded to the intervening region between the mads and the k domains, whereas, the rest were found to be distributed in the c-terminal regions.
the mads domain in five m-type proteins, osmads <dig>   <dig>   <dig>   <dig> and  <dig>  was found to be preceded by an n-terminal domain .
in mβ proteins, the mads domain was represented by motif  <dig> except in osmads <dig> and  <dig>  in addition, other motifs, viz.
<dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> were also detected, of which motifs  <dig>   <dig>   <dig>   <dig> and  <dig> were exclusive to the mβ class of proteins.
the sequences and lengths of all the motifs are given in supplementary table s <dig> .
expression profiling of mads-box genes during vegetative and reproductive development and stress
for expression analysis, the rice panicle and seed development stages were divided into  <dig> and  <dig> broad categories, respectively, based on landmark developmental events as described by itoh and coworkers  <cit> ; information available at oryzabase  <cit>  and our preliminary histochemical analysis .
seedlings subjected to three stress conditions, viz.
desiccation, cold and salt stress, were also included in this analysis.
transcriptome profiling of these stages along with mature leaf, root and 7-day-old seedlings was carried out by using genechip® rice genome arrays.
the raw data from  <dig> chips representing three biological replicates each from  <dig> samples was normalized by gene chip robust multiarray analysis  algorithm  <cit> .
since five tandemly duplicated mγ genes osmads <dig>   <dig>   <dig>   <dig> and  <dig> showed very high sequence identity , the unique probe sets for these genes were not available on the genechip®.
therefore, a cumulative expression profile for these genes is presented .
expression profiles for osmads <dig> and  <dig>  which were also not represented on the chip, were studied using qpcr .
the primers used in this study are listed in supplementary table s <dig> .
the initial analysis revealed all but one  gene to be expressing in at least one of the experimental stages analyzed .
*molecular wt and pi of osmads <dig> has been calculated using gene runner
** tigr model represents only k box protein.
based on expression profiles during panicle, seed and vegetative development, mads-box genes were classified into eight groups.
figure  <dig> shows mean expression profiles for each of the groups.
group i consisted of  <dig> genes  most of which showed high transcript accumulation in all the stages analyzed.
however, eight genes  of group ii showed high expression preferentially in panicle and seed with more than  <dig> to  <dig> folds expression in majority of the reproductive tissues in comparison to mature leaf.
osmads <dig> and  <dig> were the most highly expressed genes in panicle and seed stages with more than 1000-fold transcript accumulation in s <dig> stage of seed development.
group iii comprised of the genes osmads <dig>   <dig>   <dig> and  <dig>  which showed high expression during early stages of panicle development.
the expression declined gradually as the panicles matured.
in contrast, the expression of group iv genes  increased with the development of the panicles and continued to increase during stages of seed development; the highest expression being  <dig> and  <dig> folds for osmads <dig> and  <dig>  respectively.
five genes, osmads <dig>   <dig>   <dig>   <dig> and  <dig> , expressed preferentially during seed development.
the expression of osmads <dig> and  <dig> was more than  <dig> folds in s1-s <dig> stages, whereas, the expression of osmads <dig> increased up to  <dig> folds in s <dig> to s <dig> stages.
osmads <dig> and  <dig> transcripts showed s3-s <dig> stage-specific accumulation .
group vi comprised of five genes, osmads <dig>   <dig>   <dig>   <dig> and  <dig> that expressed predominantly in vegetative tissues.
the maximum expression of osmads <dig> was observed in mature leaves, whereas, osmads <dig> showed higher expression in roots as well.
three genes  that constituted group vii showed low levels of expression in most stages of panicle and seed development.
the peak expression for these genes was observed either in p <dig> , s1/s <dig>  or s <dig>  stages.
the remaining genes  showed very low levels of expression and were clustered together as group viii .
for normalized expression values and calculated fold change values with respect to mature leaf refer to supplementary tables s <dig> and s <dig> .
eight genes showing discrete expression patterns were selected for validation by qpcr analysis.
figure  <dig> shows a comparison of the qpcr and microarray analysis.
the expression patterns obtained for all eight genes using qpcr were similar to that derived from the microarrays.
moreover, most of the characterized genes showed similar expression patterns to that already described in the literature, which strengthens the reliability of the data obtained by using microarrays  <cit> expression levels of four mads-box genes  were up regulated by more than two folds in response to cold and dehydration stress treatments .
three genes  showed more than 2-fold down regulation in response to dehydration and salt stress.
the fold change values with respect to seedlings are given in supplementary table s <dig>
expression profiles of putative orthologs of arabidopsis mikcc-type genes in rice
comparative expression profiles of phylogenetically related mikcc class genes in rice and arabidopsis is shown in figure  <dig>  these genes have been considered orthologs in arabidopsis and rice  <cit> .
in glo-like clade, the expression of pi and osmads <dig> was found to be restricted to reproductive tissues, whereas, osmads <dig> showed significant expression in seedlings as well.
the expression of another b-class gene, ap <dig>  was also found to be specific to reproductive tissues in arabidopsis; however its rice ortholog, osmads <dig>  also expressed in vegetative tissues besides showing peak expression in reproductive tissues.
the expression of ag was found to be restricted to the stages of floral development and initial stages of seed development in arabidopsis.
in rice, its putative orthologs osmads <dig> and osmads <dig> were found to have comparable expression profiles to that of ag, with osmads <dig> showing relatively low level transcript accumulation.
in ag clade, the expression profiles of an arabidopsis d-class gene, agl <dig> and its rice counterpart osmads <dig> showing 53% identity at amino acid level were also comparable.
suppressor of constans <dig>  and its putative ortholog osmads <dig> in ricewere found to exhibit low level ubiquitous expression in the stages analyzed.
the members of agl2-like clade, osmads7/ <dig> and osmads8/ <dig> are duplicated genes with high level of sequence homology to agl2/sep <dig> and agl14/sep <dig>  respectively.
these genes, along with osmads <dig>   <dig> and  <dig> were found to exhibit similar expression patterns as those of sep genes.
in squa-like clade, ap <dig> gene of arabidopsis and its putative rice orthologs osmads <dig> ,  <dig> and osmads <dig> show high sequence similarity.
in reproductive tissues, the expression profiles of osmads <dig>   <dig> and  <dig> were found to be very similar to that of ap <dig>  but unlike ap <dig>  the rice genes also expressed in mature leaves.
in agl6-like clade, duplicated genes, osmads <dig> and osmads <dig>  exhibit similar expression patterns as that of agl <dig>  both the genes show more than 50% identity with agl <dig> at amino acid level suggesting that these could be putative orthologs of agl <dig> in rice.
expression profiles of duplicated genes
analysis of the tigr rice segmental duplication database revealed  <dig> mads-box genes that were localized on the duplicated segments of the rice genome.
a comparison of expression profiles of the duplicated gene pairs, as obtained from microarray data, revealed that except for  <dig> gene pairs, viz.
osmads7: <dig>  osmads3: <dig> and osmads2:4; expression patterns of other duplicated genes had diverged significantly .
chromosome  <dig> was found to have four tandemly duplicated genes within a  <dig> kb region.
two groups of tandemly duplicated genes with three genes each were localized on chromosome  <dig>  incidentally, this region overlapped with an intra-chromosomal duplicated segment suggesting that these six genes probably evolved from a single ancestral gene by a combination of segmental and tandem duplication events.
all four tandemly duplicated mikcc-type genes showed varied expression patterns.
osmads <dig> and  <dig> as well as osmads <dig> and  <dig>  although fulfilling our selection criteria, were not considered as being tandemly duplicated because of the high level of sequence divergence, which was evident from their placement in different clades of mikcc-type genes.
discussion
involvement of mads-box genes in panicle and seed development in rice
for over a decade, investigations leading to the understanding of genetic and molecular basis of floral development in model eudicots, arabidopsis and antirrhinum, have revealed involvement of a number of mads-box genes in specifying floral organ identity  <cit> .
attempts have been made to predict the function of mads-box genes in diverse species based on sequence similarities  <cit> .
however, identification of additional paralogs with very similar sequences and existence of duplicated genes with different expression patterns made it difficult to predict the function based on sequence data alone.
similarity in temporal and spatial expression patterns in combination with the sequence comparisons, however, was found to be a better criterion for establishing orthologous relationships.
in this paper, we have presented a comprehensive expression profiling for all the mads-box genes in rice along with an account of their phylogenetic relationships with the arabidopsis genes.
of the  <dig> genes analyzed in this study, more than  <dig> were found to exhibit either specific or preferential transcript accumulation during stages of panicle and seed development.
some of these genes have already been characterized as orthologs of arabidopsis abcde class genes, viz.osmads <dig> and  <dig> are apetala <dig> orthologs; osmads <dig> and  <dig> are pistillata orthologs; osmads <dig> is an apetala <dig> ortholog; osmads <dig> and  <dig> are agamous orthologs; d class gene, osmads <dig> is putative ortholog of agl11; osmads <dig> and  <dig> are orthologous to sepallata <dig> and  <dig>  respectively  <cit> .
most of the putative orthologous genes in rice and arabidopsis exhibit similar expression patterns .
it was, however, observed that the rice counterparts had a general tendency to express in vegetative organs as well, whereas, the expression of arabidopsis genes was restricted to reproductive tissues.
from the expression data and per cent identity, it seems that duplicated genes, osmads <dig> and osmads <dig>  are orthologous to agl <dig>  further experimentation would be required to verify if these genes have similar functions as well.
in addition to some of the well characterized genes described above, there are several others, e.g.
osmads <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and  <dig>  that show specific up-regulation in panicles but have not yet been functionally validated in rice.
this list also includes mikc*- and m-type genes along with mikcc genes.
most of the functionally validated mads-box family genes belong to the mikcc class, while functions of most of the m-type genes are not yet known in any system.
therefore, this study provides a solid base to select genes for functional validation.
in  <dig>  parenicova and coworkers showed that  <dig> of the  <dig> arabidopsis mads-box genes expressed in siliques  <cit> .
later, by using high-density transcription factor filter arrays, almost all the mads-box genes were found to express during silique development  <cit> .
these results suggested that besides being involved in the development of flowers, the mads-box gene family could be involved in the process of seed development as well.
in rice, we have also found that transcripts for almost all the mads-box genes are expressed in at least one of the seed development stages analyzed.
interestingly, the highest expression values for osmads <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> were observed for seed stages, suggesting that these genes could be involved in development of seeds.
four of these genes belong to type ii , whereas, the remainder are type i .
since, two of the type i  genes, agl <dig> and pheres, have previously been implicated in seed development, it might be interesting to investigate the role of other type-i genes showing up-regulation during development of seeds  <cit> .
rice mikcc genes
the mikcc genes have been sub-grouped into  <dig> clades in arabidopsis.
representatives of all but the flc clade were also found in rice.
six genes belonging to the flc clade have been implicated in control of flowering by vernalization and autonomous pathways in arabidopsis.
since rice does not require vernalization for flowering, this clade has been suggested to be lost in rice  <cit> .
recently, zhao and coworkers  have reported a new monocot-specific clade, osmads32-like clade, consisting of osmads <dig> of rice and taagl <dig> and  <dig> of wheat  <cit> .
the expression of taagl <dig> and  <dig> was detected in most vegetative stages along with inflorescence and seeds.
in contrast, the osmads <dig> transcripts were found to be restricted to early stages of panicle and late seed development, suggesting that the osmads32-like clade might have evolved to cater for diverse monocot-specific functions.
a comparison of phylogenetic relationships and expression profiles between rice and arabidopsis mads-box genes suggests that although most of the basic abcde functions have been retained in rice, acquisition of new functions and subfunctionalization of existing gene functions is also apparent.
mβ-like genes are represented in rice
in earlier studies, no gene of rice could be assigned to the mβ group of m-type mads-box genes, hence it was suggested that probably mβ genes have not been retained in the rice genome  <cit> .
in this study, we have identified nine new genes that grouped with arabidopsis mβ type genes.
although bootstrap values are low, separation of this clade from the rest of m-type genes of rice and the presence of conserved motifs in arabidopsis mβ protein and the newly identified group is suggestive of the existence of mβ group in rice as well.
duplication seems to have played major role in diversification of mads-box family of genes
arabidopsis has been reported to have  <dig> mads-box genes  <cit> .
however, in rice that has a genome size almost three times as that of arabidopsis  <cit> , the number of mads-box genes was found to be only  <dig>  the reason for this could be the variable status of whole genome duplications in arabidopsis and rice  <cit> .
surprisingly, however, the number of mikcc-type genes in both arabidopsis and rice was found to be similar at  <dig> and  <dig>  respectively.
therefore, the difference in the total number is mainly due to the variation in the number of m-type genes, which are  <dig> in rice and  <dig> in arabidopsis.
it seems that duplication events have contributed significantly towards evolution of m-type genes.
our analysis revealed  <dig> m-type genes, which could have originated because of tandem duplications .
phylogenetic analysis suggests that rice and arabidopsis mγ genes probably had a common ancestor and the expansion occurred independently after divergence of monocots and dicots.
mads-box genes seem to have evolved mainly through gene duplication events followed by neofunctionalization, subfunctionalization or in some cases pseudogenization of the duplicated gene  <cit> .
however, redundancy being one of the fates of duplication is also common in mads-box family.
we found  <dig> mads-box genes lying on segmental duplicated regions of rice chromosomes while only  <dig> were found to have been retained, suggesting that considerable changes may have taken place following segmental duplication leading to loss of some of the genes.
except one, all paralogous gene pairs belong to mikcc-type of mads-box family.
expression data show that most of these duplicated genes have divergent expression patterns that may be because they have undergone neofunctionalization or subfunctionalization, though sufficient experimentation is required to prove this hypothesis.
interestingly, three genes, viz.
osmads <dig>   <dig> and  <dig>  lying on duplicated segments of chromosomes  <dig>   <dig> and  <dig>  respectively, show collinearity in gene order.
on the other hand, osmads <dig> lying on chromosome  <dig> shows synteny with only one of these genes, i.e.
osmads <dig>  they may all have resulted due to duplication of a segment on chromosome  <dig>  but thereafter, evolution of these genes may have been quite independent resulting in loss of micro-collinearity between the duplicated regions.
stress responsive mads-box genes in rice
mads-box genes have been shown to be affected by low temperature stress in tomato  <cit>  and by application of hormones like cytokinins, gibberellins  <cit> , ethylene  <cit>  and auxins  <cit>  in other plants.
seven mads-box genes exhibited differential expression in response to cold, salt and/or desiccation stress in rice.
so far, none of these genes has been implicated in stress response.
amongst stress-induced genes, osmads <dig> is a member of ap1/squa group that has been shown to express widely during development with its transcripts accumulating at high levels specifically in meristematic tissues  <cit> .
it has been shown to interact with osmads <dig>  8/ <dig>  7/ <dig>  and  <dig>  <cit>  and in our analysis its expression pattern was found to overlap with those of osmads <dig>   <dig> and  <dig> in reproductive tissues and with osmads <dig> during vegetative development, suggesting that it might be interacting with different partners during reproductive development and stress.
recently, tardif and coworkers showed that a large number of genes involved in flower development are associated with abiotic stress responses in wheat  <cit> .
our preliminary analysis involving transcript profiling during reproductive development and abiotic stress conditions has also revealed approximately  <dig> genes that are up regulated during panicle/seed development and three stress conditions, viz.
cold, salt, and dehydration .
it would be, therefore, interesting to undertake specific investigations, which could establish the interactions of biochemical pathways that are activated during reproductive development and stress response.
CONCLUSIONS
contribution of mads-box gene family in flower organ specification is well documented in eudicots; however, functions of many gene members of this class have not been elucidated in rice.
a comparison of phylogenetic relationships and expression profiles between rice and arabidopsis mads-box genes suggests that although most of the basic abcde functions have been retained in rice, acquisition of new functions and subfunctionalization of existing gene functions is also not uncommon.
furthermore, the role of mads-box transcription factors in seed development and during stress response also needs to be explored.
the new information generated is expected to help in selection of appropriate candidate genes for further functional characterization.

 the mads-box family members, identified initially as floral homeotic genes, are one of the most extensively studied transcription factor genes in plants. the word mads finds its origin from the first letters of its founding members, mini chromosome maintenance of yeast, agamous of arabidopsis, deficiens of snapdragon and serum response factor of humans. mads-box transcription factors are characterized by the presence of an approximately amino acids dna binding domain, known as the mads-box domain, located in the n-terminal region of the protein. the plant-specific mikc-type mads-box proteins include three additional domains followed by the mads domain, viz. a less-conserved intervening region of ~ amino acids, a moderately conserved keratin-like domain of ~ amino acids mainly involved in heterodimerization, and a highly variable c-terminal region of variable length implicated in transcriptional activation and higher-order complex formation. the mads-box family has been divided into two main groups. the type i consists of arg80/srf-like genes of animals and fungi, also designated as m-type genes in plants, and type ii contains mef2-like genes of animals and yeast as well as mikc-type genes of plants. it is proposed that an ancestral duplication before the divergence of plants and animals gave rise to these groups. the mikc-type genes are also characterized by the presence of k domain that could have evolved after the divergence of these lineages. the type ii genes have been categorized into mikcc- and mikc*-type based on structural features. the mikcc genes have been further classified into clades based on phylogeny. type i genes have also been categorized into m- and n-type based on the protein motifs identified using the meme search tool and also as mα, mβ, mγ and mδ, based on the phylogenetic relationships between mads-box regions. the mδ group, however, corresponds to the mikc* class described in this report and elsewhere. the most striking feature of the mads-box gene family is the diverse functions taken up by its members in different aspects of plant growth and development. these include flowering time control, meristem identity, floral organ identity, formation of dehiscence zone, fruit ripening, embryo development as well as development of vegetative organs such as root and leaf. genome-wide identification and phylogenetic analyses of mads-box genes have revealed and rice pseudomolecule release database) genes in arabidopsis and rice, respectively. though a large amount of expression data based on sage, microarrays and other high-throughput transcriptome analysis techniques is available in public databases, the studies involving expression of the entire mads-box family have so far been restricted to northern blot analysis or reverse transcriptase pcr at limited stages of development. recently, the comparison of expression profiles resulting from a k rice cdna microarray-based transcriptome analysis of early panicle development in rice was used to implicate three mads-box genes, osmads and in panicle branching. the use of high-throughput genome-wide transcriptome analysis provides an insight into changes in the entire transcriptome across a variety of biological conditions. in combination with the whole genome sequence data and comparative expression analysis with genes of known functions, the transcriptomic data can become an initiation point for systematic investigations into structure-function relationships. with an overall objective to understand regulation of reproductive organ development in indica rice, we have initiated a program on microarray-based expression profiling of transcription factors and signal transduction components. here, we report a comprehensive account of identification and phylogenetic analysis of members of mads-box gene family in rice and their expression profiling during stages of panicle and seed development along with three abiotic stress conditions and stages of vegetative development. this analysis is based on tigr rice pseudomolecule release and kome rice full-length cdna database. we have identified new members belonging to this gene family besides confirming previously identified genes. out of previously identified genes by nam and coworkers, six were not found in version of tigr. our analysis also suggests the existence of mβ-type genes in rice, which was earlier thought to be absent in monocots. the results of expression profiling have been discussed in light of phylogenetic relatedness of the genes and their known functions in rice as well as other systems.  identification, organization and structure of mads-box genes hmm analysis and name search resulted in the identification of mads-box genes in rice genome. since, gene names from osmads to osmads representing genes already existed in the literature, newly identified genes were named from osmads to osmads. the individual genes were localized on chromosomes based on the 5' and 3' coordinates for respective gene models in tigr database. the maximum number of genes were found to be localized on chromosome whereas, chromosomes and had only one mads-box gene each. out of five types of mads-box genes, the mγ genes were confined to chromosome and while mβ genes were present only on chromosome no chromosomal bias was observed in the distribution of mikcc, mikc* and mα genes. analysis of the tigr rice segmental duplication database revealed mads-box genes within the duplicated segments of rice chromosomes. only genes, however, were found to have their counterparts on duplicated segments. most of the duplicated genes belonged to the mikcc group. several mads-box genes, especially m-type, were also found juxtaposed on chromosomes and twenty such genes showed significant sequence identity. *molecular wt and pi of osmads has been calculated using gene runner ** tigr model represents only k box protein. similar to that reported in arabidopsis, distribution of introns in rice mads-box family genes was also found bimodal with mikcc and mikc* genes containing multiple introns and the mα, mβ and mγ genes usually having no or occasionally up to introns. the length of mads-box proteins varied from to amino acids, with few exceptionally longer or smaller proteins. for details on other parameters of nucleic acid and protein sequences, refer to table evolutionary relationships between rice and arabidopsis mads-box family genes to examine the evolutionary relationships of mads-box genes in rice and arabidopsis, a tree was constructed using only the conserved mads-box domain. five groups, as described by parenicova and coworkers were identified containing representative genes of both rice and arabidopsis. all the arabidopsis proteins were found to lie in groups similar to those identified previously, except agl and agl which instead of forming a basal branch of the mβ, grouped with mγ proteins in our analysis as shown in supplementary figure s. osmads grouped separately with agl of arabidopsis, which does not cluster with any of the mads groups described above. a separate phylogenetic tree was also generated from complete protein sequences of all the mads-box genes in rice and arabidopsis. of the rice mads-box genes, grouped with mikcc, six with mikc*, nine with mβ, with mα and grouped with mγ-type arabidopsis genes. in case of m-type genes, barring osmads and all other genes exhibited similar groupings as in the mads-domain-specific phylogenetic analysis. mikcc proteins were further divided into clades. representatives of both rice and arabidopsis could be identified in all the clades except osmads and the flc clade, which are exclusive to rice and arabidopsis, respectively. phylogenetic trees with bootstrap values, for complete protein sequences as well as coding sequences of rice and arabidopsis mads-box genes have been shown as supplementary figures s and s respectively. distribution of conserved motifs the meme motif search tool was employed to identify the conserved motifs present in mads-box proteins. motifs or specifying the mads domain were found in all the members of the mads-box family. all proteins belonging to mikcc and mikc* groups had motif 1-type mads domain. most mα proteins also had the motif 1-type mads domain except osmads and which contained motif motif was found to be the most common type of mads domain in mβ-type proteins. distinctively, in case of mγ proteins a larger mads domain of amino acids was detected, followed by a coiled coil region and a region of unknown complexity as indicated by simple modular architecture research tool version. together these components formed a larger conserved region of amino acids denoted as motif the conserved motifs and specifying k domain, were found mainly in the mikcc group members. of the mikcc proteins, had all three conserved regions, whereas, motif was absent in case of osmads and 57; motif in osmads and 55; and the complete k domain was missing in osmads and the partial k domain was also detected in some of the non-mikcc proteins including osmads and of mγ, osmads and of mikc*, osmads and osmads of mα group. certain unknown motifs, numbered and besides the mads and the k domains in mikcc proteins, were also revealed by meme motif search. motif corresponded to the intervening region between the mads and the k domains, whereas, the rest were found to be distributed in the c-terminal regions. the mads domain in five m-type proteins, osmads and was found to be preceded by an n-terminal domain. in mβ proteins, the mads domain was represented by motif except in osmads and in addition, other motifs, viz. and were also detected, of which motifs and were exclusive to the mβ class of proteins. the sequences and lengths of all the motifs are given in supplementary table s. expression profiling of mads-box genes during vegetative and reproductive development and stress for expression analysis, the rice panicle and seed development stages were divided into and broad categories, respectively, based on landmark developmental events as described by itoh and coworkers; information available at oryzabase and our preliminary histochemical analysis. seedlings subjected to three stress conditions, viz. desiccation, cold and salt stress, were also included in this analysis. transcriptome profiling of these stages along with mature leaf, root and 7-day-old seedlings was carried out by using genechip® rice genome arrays. the raw data from chips representing three biological replicates each from samples was normalized by gene chip robust multiarray analysis algorithm. since five tandemly duplicated mγ genes osmads and showed very high sequence identity, the unique probe sets for these genes were not available on the genechip®. therefore, a cumulative expression profile for these genes is presented. expression profiles for osmads and which were also not represented on the chip, were studied using qpcr. the primers used in this study are listed in supplementary table s. the initial analysis revealed all but one gene to be expressing in at least one of the experimental stages analyzed. *molecular wt and pi of osmads has been calculated using gene runner ** tigr model represents only k box protein. based on expression profiles during panicle, seed and vegetative development, mads-box genes were classified into eight groups. figure shows mean expression profiles for each of the groups. group i consisted of genes most of which showed high transcript accumulation in all the stages analyzed. however, eight genes of group ii showed high expression preferentially in panicle and seed with more than to folds expression in majority of the reproductive tissues in comparison to mature leaf. osmads and were the most highly expressed genes in panicle and seed stages with more than 1000-fold transcript accumulation in s stage of seed development. group iii comprised of the genes osmads and which showed high expression during early stages of panicle development. the expression declined gradually as the panicles matured. in contrast, the expression of group iv genes increased with the development of the panicles and continued to increase during stages of seed development; the highest expression being and folds for osmads and respectively. five genes, osmads and, expressed preferentially during seed development. the expression of osmads and was more than folds in s1-s stages, whereas, the expression of osmads increased up to folds in s to s stages. osmads and transcripts showed s3-s stage-specific accumulation. group vi comprised of five genes, osmads and that expressed predominantly in vegetative tissues. the maximum expression of osmads was observed in mature leaves, whereas, osmads showed higher expression in roots as well. three genes that constituted group vii showed low levels of expression in most stages of panicle and seed development. the peak expression for these genes was observed either in p, s1/s or s stages. the remaining genes showed very low levels of expression and were clustered together as group viii. for normalized expression values and calculated fold change values with respect to mature leaf refer to supplementary tables s and s. eight genes showing discrete expression patterns were selected for validation by qpcr analysis. figure shows a comparison of the qpcr and microarray analysis. the expression patterns obtained for all eight genes using qpcr were similar to that derived from the microarrays. moreover, most of the characterized genes showed similar expression patterns to that already described in the literature, which strengthens the reliability of the data obtained by using microarrays expression levels of four mads-box genes were up regulated by more than two folds in response to cold and dehydration stress treatments. three genes showed more than 2-fold down regulation in response to dehydration and salt stress. the fold change values with respect to seedlings are given in supplementary table s expression profiles of putative orthologs of arabidopsis mikcc-type genes in rice comparative expression profiles of phylogenetically related mikcc class genes in rice and arabidopsis is shown in figure these genes have been considered orthologs in arabidopsis and rice. in glo-like clade, the expression of pi and osmads was found to be restricted to reproductive tissues, whereas, osmads showed significant expression in seedlings as well. the expression of another b-class gene, ap was also found to be specific to reproductive tissues in arabidopsis; however its rice ortholog, osmads also expressed in vegetative tissues besides showing peak expression in reproductive tissues. the expression of ag was found to be restricted to the stages of floral development and initial stages of seed development in arabidopsis. in rice, its putative orthologs osmads and osmads were found to have comparable expression profiles to that of ag, with osmads showing relatively low level transcript accumulation. in ag clade, the expression profiles of an arabidopsis d-class gene, agl and its rice counterpart osmads showing 53% identity at amino acid level were also comparable. suppressor of constans and its putative ortholog osmads in ricewere found to exhibit low level ubiquitous expression in the stages analyzed. the members of agl2-like clade, osmads7/ and osmads8/ are duplicated genes with high level of sequence homology to agl2/sep and agl14/sep respectively. these genes, along with osmads and were found to exhibit similar expression patterns as those of sep genes. in squa-like clade, ap gene of arabidopsis and its putative rice orthologs osmads, and osmads show high sequence similarity. in reproductive tissues, the expression profiles of osmads and were found to be very similar to that of ap but unlike ap the rice genes also expressed in mature leaves. in agl6-like clade, duplicated genes, osmads and osmads exhibit similar expression patterns as that of agl both the genes show more than 50% identity with agl at amino acid level suggesting that these could be putative orthologs of agl in rice. expression profiles of duplicated genes analysis of the tigr rice segmental duplication database revealed mads-box genes that were localized on the duplicated segments of the rice genome. a comparison of expression profiles of the duplicated gene pairs, as obtained from microarray data, revealed that except for gene pairs, viz. osmads7: osmads3: and osmads2:4; expression patterns of other duplicated genes had diverged significantly. chromosome was found to have four tandemly duplicated genes within a kb region. two groups of tandemly duplicated genes with three genes each were localized on chromosome incidentally, this region overlapped with an intra-chromosomal duplicated segment suggesting that these six genes probably evolved from a single ancestral gene by a combination of segmental and tandem duplication events. all four tandemly duplicated mikcc-type genes showed varied expression patterns. osmads and as well as osmads and although fulfilling our selection criteria, were not considered as being tandemly duplicated because of the high level of sequence divergence, which was evident from their placement in different clades of mikcc-type genes. discussion involvement of mads-box genes in panicle and seed development in rice for over a decade, investigations leading to the understanding of genetic and molecular basis of floral development in model eudicots, arabidopsis and antirrhinum, have revealed involvement of a number of mads-box genes in specifying floral organ identity. attempts have been made to predict the function of mads-box genes in diverse species based on sequence similarities. however, identification of additional paralogs with very similar sequences and existence of duplicated genes with different expression patterns made it difficult to predict the function based on sequence data alone. similarity in temporal and spatial expression patterns in combination with the sequence comparisons, however, was found to be a better criterion for establishing orthologous relationships. in this paper, we have presented a comprehensive expression profiling for all the mads-box genes in rice along with an account of their phylogenetic relationships with the arabidopsis genes. of the genes analyzed in this study, more than were found to exhibit either specific or preferential transcript accumulation during stages of panicle and seed development. some of these genes have already been characterized as orthologs of arabidopsis abcde class genes, viz.osmads and are apetala orthologs; osmads and are pistillata orthologs; osmads is an apetala ortholog; osmads and are agamous orthologs; d class gene, osmads is putative ortholog of agl11; osmads and are orthologous to sepallata and respectively. most of the putative orthologous genes in rice and arabidopsis exhibit similar expression patterns. it was, however, observed that the rice counterparts had a general tendency to express in vegetative organs as well, whereas, the expression of arabidopsis genes was restricted to reproductive tissues. from the expression data and per cent identity, it seems that duplicated genes, osmads and osmads are orthologous to agl further experimentation would be required to verify if these genes have similar functions as well. in addition to some of the well characterized genes described above, there are several others, e.g. osmads and that show specific up-regulation in panicles but have not yet been functionally validated in rice. this list also includes mikc*- and m-type genes along with mikcc genes. most of the functionally validated mads-box family genes belong to the mikcc class, while functions of most of the m-type genes are not yet known in any system. therefore, this study provides a solid base to select genes for functional validation. in parenicova and coworkers showed that of the arabidopsis mads-box genes expressed in siliques. later, by using high-density transcription factor filter arrays, almost all the mads-box genes were found to express during silique development. these results suggested that besides being involved in the development of flowers, the mads-box gene family could be involved in the process of seed development as well. in rice, we have also found that transcripts for almost all the mads-box genes are expressed in at least one of the seed development stages analyzed. interestingly, the highest expression values for osmads and were observed for seed stages, suggesting that these genes could be involved in development of seeds. four of these genes belong to type ii, whereas, the remainder are type i. since, two of the type i genes, agl and pheres, have previously been implicated in seed development, it might be interesting to investigate the role of other type-i genes showing up-regulation during development of seeds. rice mikcc genes the mikcc genes have been sub-grouped into clades in arabidopsis. representatives of all but the flc clade were also found in rice. six genes belonging to the flc clade have been implicated in control of flowering by vernalization and autonomous pathways in arabidopsis. since rice does not require vernalization for flowering, this clade has been suggested to be lost in rice. recently, zhao and coworkers have reported a new monocot-specific clade, osmads32-like clade, consisting of osmads of rice and taagl and of wheat. the expression of taagl and was detected in most vegetative stages along with inflorescence and seeds. in contrast, the osmads transcripts were found to be restricted to early stages of panicle and late seed development, suggesting that the osmads32-like clade might have evolved to cater for diverse monocot-specific functions. a comparison of phylogenetic relationships and expression profiles between rice and arabidopsis mads-box genes suggests that although most of the basic abcde functions have been retained in rice, acquisition of new functions and subfunctionalization of existing gene functions is also apparent. mβ-like genes are represented in rice in earlier studies, no gene of rice could be assigned to the mβ group of m-type mads-box genes, hence it was suggested that probably mβ genes have not been retained in the rice genome. in this study, we have identified nine new genes that grouped with arabidopsis mβ type genes. although bootstrap values are low, separation of this clade from the rest of m-type genes of rice and the presence of conserved motifs in arabidopsis mβ protein and the newly identified group is suggestive of the existence of mβ group in rice as well. duplication seems to have played major role in diversification of mads-box family of genes arabidopsis has been reported to have mads-box genes. however, in rice that has a genome size almost three times as that of arabidopsis, the number of mads-box genes was found to be only the reason for this could be the variable status of whole genome duplications in arabidopsis and rice. surprisingly, however, the number of mikcc-type genes in both arabidopsis and rice was found to be similar at and respectively. therefore, the difference in the total number is mainly due to the variation in the number of m-type genes, which are in rice and in arabidopsis. it seems that duplication events have contributed significantly towards evolution of m-type genes. our analysis revealed m-type genes, which could have originated because of tandem duplications. phylogenetic analysis suggests that rice and arabidopsis mγ genes probably had a common ancestor and the expansion occurred independently after divergence of monocots and dicots. mads-box genes seem to have evolved mainly through gene duplication events followed by neofunctionalization, subfunctionalization or in some cases pseudogenization of the duplicated gene. however, redundancy being one of the fates of duplication is also common in mads-box family. we found mads-box genes lying on segmental duplicated regions of rice chromosomes while only were found to have been retained, suggesting that considerable changes may have taken place following segmental duplication leading to loss of some of the genes. except one, all paralogous gene pairs belong to mikcc-type of mads-box family. expression data show that most of these duplicated genes have divergent expression patterns that may be because they have undergone neofunctionalization or subfunctionalization, though sufficient experimentation is required to prove this hypothesis. interestingly, three genes, viz. osmads and lying on duplicated segments of chromosomes and respectively, show collinearity in gene order. on the other hand, osmads lying on chromosome shows synteny with only one of these genes, i.e. osmads they may all have resulted due to duplication of a segment on chromosome but thereafter, evolution of these genes may have been quite independent resulting in loss of micro-collinearity between the duplicated regions. stress responsive mads-box genes in rice mads-box genes have been shown to be affected by low temperature stress in tomato and by application of hormones like cytokinins, gibberellins, ethylene and auxins in other plants. seven mads-box genes exhibited differential expression in response to cold, salt and/or desiccation stress in rice. so far, none of these genes has been implicated in stress response. amongst stress-induced genes, osmads is a member of ap1/squa group that has been shown to express widely during development with its transcripts accumulating at high levels specifically in meristematic tissues. it has been shown to interact with osmads 8/ 7/ and and in our analysis its expression pattern was found to overlap with those of osmads and in reproductive tissues and with osmads during vegetative development, suggesting that it might be interacting with different partners during reproductive development and stress. recently, tardif and coworkers showed that a large number of genes involved in flower development are associated with abiotic stress responses in wheat. our preliminary analysis involving transcript profiling during reproductive development and abiotic stress conditions has also revealed approximately genes that are up regulated during panicle/seed development and three stress conditions, viz. cold, salt, and dehydration. it would be, therefore, interesting to undertake specific investigations, which could establish the interactions of biochemical pathways that are activated during reproductive development and stress response.  contribution of mads-box gene family in flower organ specification is well documented in eudicots; however, functions of many gene members of this class have not been elucidated in rice. a comparison of phylogenetic relationships and expression profiles between rice and arabidopsis mads-box genes suggests that although most of the basic abcde functions have been retained in rice, acquisition of new functions and subfunctionalization of existing gene functions is also not uncommon. furthermore, the role of mads-box transcription factors in seed development and during stress response also needs to be explored. the new information generated is expected to help in selection of appropriate candidate genes for further functional characterization. 
the expression profiles of all the mads-box genes have been analyzed under temporal stages of panicle and seed development, three abiotic stress conditions, along with three stages of vegetative development. mads-box transcription factors, besides being involved in floral organ specification, have also been implicated in several aspects of plant growth and development. furthermore, owing to the role of mads-box family in flower development, an analysis involving structure, expression and functional aspects of mads-box genes in rice and arabidopsis was required to understand the role of this gene family in reproductive development. transcripts for genes accumulate preferentially in the reproductive phase, of which, genes are specifically expressed in seeds, and six genes show expression specific to panicle development. though there have been some studies in rice as well, an analysis of the complete mads-box family along with a comprehensive expression profiling was still awaited after the completion of rice genome sequencing. chromosomal localization of these genes reveals that mads-box genes, mostly mikcc-type, are located within the duplicated segments of the rice genome, whereas most of the m-type genes, in all, seem to have resulted from tandem duplications. in recent years, there have been reports on genomic localization, protein motif structure, phylogenetic relationships, gene structure and expression of the entire mads-box family in the model plant system, arabidopsis. a comprehensive annotation and transcriptome profiling undertaken in this investigation adds to our understanding of the involvement of mads-box family genes during reproductive development and stress in rice and also provides the basis for selection of candidate genes for functional validation studies. an attempt has been made to gain insight into plausible functions of rice mads-box genes by collating the expression data of functionally validated genes in rice and arabidopsis. nine members belonging to the mβ group, which was considered absent in monocots, have also been identified. differential expression of seven genes under stress conditions is also evident. using a thorough annotation exercise, mads-box genes have been identified in rice and categorized into mikcc, mikc*, mα, mβ and mγ groups based on phylogeny.
 mads-box transcription factors, besides being involved in floral organ specification, have also been implicated in several aspects of plant growth and development. in recent years, there have been reports on genomic localization, protein motif structure, phylogenetic relationships, gene structure and expression of the entire mads-box family in the model plant system, arabidopsis. though there have been some studies in rice as well, an analysis of the complete mads-box family along with a comprehensive expression profiling was still awaited after the completion of rice genome sequencing. furthermore, owing to the role of mads-box family in flower development, an analysis involving structure, expression and functional aspects of mads-box genes in rice and arabidopsis was required to understand the role of this gene family in reproductive development.  a genome-wide molecular characterization and microarray-based expression profiling of the genes encoding mads-box transcription factor family in rice is presented. using a thorough annotation exercise, mads-box genes have been identified in rice and categorized into mikcc, mikc*, mα, mβ and mγ groups based on phylogeny. chromosomal localization of these genes reveals that mads-box genes, mostly mikcc-type, are located within the duplicated segments of the rice genome, whereas most of the m-type genes, in all, seem to have resulted from tandem duplications. nine members belonging to the mβ group, which was considered absent in monocots, have also been identified. the expression profiles of all the mads-box genes have been analyzed under temporal stages of panicle and seed development, three abiotic stress conditions, along with three stages of vegetative development. transcripts for genes accumulate preferentially in the reproductive phase, of which, genes are specifically expressed in seeds, and six genes show expression specific to panicle development. differential expression of seven genes under stress conditions is also evident. an attempt has been made to gain insight into plausible functions of rice mads-box genes by collating the expression data of functionally validated genes in rice and arabidopsis.  only a limited number of mads genes have been functionally validated in rice. a comprehensive annotation and transcriptome profiling undertaken in this investigation adds to our understanding of the involvement of mads-box family genes during reproductive development and stress in rice and also provides the basis for selection of candidate genes for functional validation studies. 