BACKGROUND
cell proliferation is controlled by an universally conserved molecular machinery in which the key players are cyclin-dependent kinases  and cyclins .
eukaryotes have therefore evolved elaborate mechanisms for cdk regulation.
an irreversible mechanism of cdk down-regulation is destruction of cyclin subunits  <cit> .
at the g1- to s-phase and metaphase to anaphase transitions, cdks are irreversibly inactivated by ubiquitin-mediated proteolysis of cognate cyclins  <cit> .
degradation of protein substrates through the ubiquitin-proteasome pathway involves the activity of different e <dig> ligases, among them the anaphase-promoting complex .
the apc was first identified based on its role in facilitating the multiubiquitination and targeting of a- and b-type cyclins for proteasome-mediated destruction during mitosis  <cit> .
apc is a multiprotein complex conserved from plants to man and contains at least  <dig> core subunits  <cit> .
the apc is regulated in part by two associated proteins, cdc <dig> and ccs52/cdh <dig>  that can both activate the apc with proper timing and provide substrate specificity  <cit> .
the apc is activated at metaphase/anaphase transition by the cdc <dig> protein and later in telophase and g <dig> by the cdh <dig> protein.
substrates that have a destruction box , ken-box or a-box motifs are recognized and ubiquitinated by the apc  <cit> .
the apc has important functions in mitosis, meiosis, g1-s-phase and in post-mitotic differentiated cells  <cit> .
the mitosis-specific activator cdc <dig> is itself an apc substrate.
other targets of apc/c degradation are: cyclins a and b; protein kinases plk <dig>  cdc <dig>  aurora a and b; regulators of dna replication geminin, cdc6; and the anaphase inhibitor securin .
the proteolytic events triggered by the apc are required to release sister chromatides cohesion during anaphase, to control the exit from mitosis and to prevent premature entry into s-phase  <cit> .
although the role of the apc in controlling cell-cycle progression has been extensively investigated, has recent work shown the unexpected presence of apc subunits in differentiated tissues, in particular in the nervous system of vertebrates .
in plants, the expression of several apc subunits has been detected in differentiated tissues of arabidopsis  <cit> .
in addition, while both cdc27a and cdc27b genes are essential during gametogenesis, the cdc27b subunit has been implicated also in post-embryogenic differentiation at meristem levels  <cit> .
besides, it has been shown that reduced levels of apc <dig> and apc <dig> subunits in arabidopsis lead to plants with several defects in vascular development  <cit> .
these results indicate that, while the overall structure of the apc is conserved among eukaryotes, this e <dig> ligase may have assumed specialized functions in the diverse kingdoms.
gene duplication and retention in plants has been extensive and gene families are generally larger in plants than in animals  <cit> .
nevertheless, almost all studies on the apc function in plants have been carried out in the model plant arabidopsis thaliana.
comparative genomic analyses can provide valuable insights into the organization of cell cycle machinery and the evolution of these protein complexes.
in this article, we describe thorough searches for the predicted sequences of the apc subunits and the activators cdc20/ccs <dig> in plant sequence databases.
our results indicate that land plants and green algae orthologs have an ancient evolutionary origin.
we present phylogenetic analysis of tpr subunits and activators, and their orthologs from other plants.
an evolutionary analysis of arabidopsis tpr domain suggests that tandem regions could have been created from duplication of internal sequence domains.
overall, our data support the proposal that apc subunits and activators have been conserved in the course of evolution.
however, while other eukaryotes like fungi and metazoans have only one copy of each apc subunit and its regulators, gene duplication of different subunits have occurred in arabidopsis, rice and poplar, and in other plant genomes as well.
an attractive proposal is that gene duplication of apc subunits may result in the formation of apc subcomplexes that evolved to assume restricted specialized roles during plant development.
indeed, in arabidopsis, duplication of the cdc <dig> subunit led to substantial sequence divergence and specialization  <cit> .
here we show that the two copies of rice cdc <dig> and apc <dig> subunits and the activators genes are differentially expressed in plant tissues or when grown under either dark or light-mediated developmental programs, suggesting the duplicated genes could be assuming new functions in the plant.
RESULTS
identification of apc subunits and activator genes
in order to carry out the cross-species comparison of the apc and activators, we have searched for homologous sequences in the genomic research  rice genome database and doe join genome institute  poplar genome database.
the sequences of the arabidopsis apc subunits have been published  <cit> .
even after exhaustive data mining, some rice and poplar sequences were only found in the est database gene index dfci.
subsequently, the smart motif identification tool was used to identify predicted domains in all candidate proteins  <cit> .
using this strategy, we report the apc/activators in the dicotyledonous poplar and the monocotyledon rice plants .
all apc subunits and activators were found, and they contained the predicted conserved domains, providing one of many examples for the evolutionary conservation of the eukaryotic cell-cycle machinery  <cit> .
agene index project access number http://compbio.dfci.harvard.edu/tgi/
two cdc <dig> homologs have been identified in arabidopsis atcdc27a and atcdc27b  <cit> .
in poplar, two homologs of cdc <dig> were also identified: ptcdc27_ <dig> and ptcdc27_ <dig>  however, a careful inspection of the ptcdc27_ <dig> deposited sequence revealed a stop codon tag at nucleotide position 1471- <dig>  this fragment was amplified and sequenced and the presence of the stop codon was discarded.
the expression of both genes in leaves was confirmed by quantitative pcr .
we also found two homologs of cdc <dig> in the bryophyte physcomitrella patens genome  <cit> .
however, only one copy of cdc <dig> was found in the rice genome as well as other monocot plants.
on the other hand, the rice genome has two oscdc <dig> and osapc <dig> subunits  <cit> .
we examined other complete genomes and found two cdc <dig>  and apc <dig>  genes in grapevine  <cit> .
two homologs of ptapc <dig> were found in the poplar and grapevine genomes, a feature that has not been reported in any other eukaryotic species so far.
the structures of many predicted genes were considerably misannotated.
comparing est databases from plant species and using bioinformatics tools, it was possible to identify the correct gene sequences.
the genomic sequence of osapc <dig> was not found in the tigr database, but a partial cds was identified in the gene index database .
most likely, the largest subunit of the apc had come apart by misannotation.
diverse exon-intron structures needed correction in the genomics sequences.
osapc <dig> may contain an intron in the genomic orf and oscdc23_ <dig> may have a fragment of an exon involved in the formation of tpr domain erroneously annotated as an intron .
the 3'region of predicted ptapc <dig> and the 5'-3'regions of ptapc <dig> were incomplete.
however, we found fragments from 3'regions of both genes and fragments from the 5'region of ptapc <dig> in the est database .
interestingly, osapc <dig>  oscdc <dig> and osapc11_ <dig> have a divergent 5'region compared to arabidopsis and poplar genes .
the first methionine of oscdc <dig> and osapc11_ <dig> are upstream from a consensus start codon in cdc <dig> and apc <dig> genes, but only for cdc <dig> there is est support.
the opposite occurs with osapc <dig>  where the first methionine is downstream from the consensus start codon.
the apc genes atcdc <dig>  oscdc <dig>  ptcdc <dig> and osapc <dig> were identified in est databases.
arabidopsis contains three ccs <dig> genes, atccs52a <dig>  atccs52a <dig> and atccs52b; and five cdc <dig> genes  <cit> .
in addition, we found another cdc <dig> homolog in the arabidopsis genome.
this atcdc20_ <dig>  showed major differences from conserved structures of other cdc20; the cds does not have the c-box sequence element ipxr) that was first identified in the n-terminal region of cdc <dig>  although it is conserved in all known apc/c co-activators , and therefore atcdc20_ <dig> could be a pseudogene.
the rice genome has three predicted cdc <dig> genes compared to five in poplar and arabidopsis.
rice has two ccs <dig> genes compared to four and three genes in poplar and arabidopsis, respectively .
exon-intron organization of activators was analyzed and we found a number of annotation errors.
atcdc20_ <dig>  ptcdc20_ <dig>  ptccs52a <dig> and ptccs52a1_ <dig> have errors in the 5' region.
we found that only ptcdc20_ <dig> is without a c-box.
oscdc20_ <dig>  atcdc20_ <dig>  ptcdc20_ <dig>  ptcdc20_ <dig> and ptcdc20_ <dig> had mistakes in the 3' region.
this region contains the ir-tail sequence element  that occupies the c terminus of apc activators and the apc subunit apc <dig>  comparing with est databases, all sequences were deduced and the ir-tail was identified .
in ptccs52a1_ <dig> and oscdc20_ <dig> genes, introns were incorrectly included.
the opposite happened in the oscdc20_ <dig> gene, where one exon was absent.
alternative splicing variants were found in the arabidopsis, rice and poplar apc/activator genes  and could represent another layer of complexity in the organization and function of the apc.
we selected only a single variant for further analysis.
duplication of cell-cycle machinery components is rare in metazoans but it is a widespread phenomenon in plants  <cit> .it has been proposed that duplication, followed by sequence divergence of promoter and/or coding regions, leads to novel and specialized functions unique to the plant kingdom  <cit> .
still, additional file  <dig> shows that duplications of individual subunits of the apc are not present in all plants; on the contrary, they may be restricted to one or to a small group of phylogenetically related plant species and may be involved in the organization of developmental events unique to this group.
chromosomal location of genes for apc subunits and activator
the close similarity and presence of putative subunits and paralogs prompted us to investigate the genome distribution of plant apc/activators.
apc genes were found in the five arabidopsis chromosomes,  <dig> of the  <dig> rice chromosomes and  <dig> of the  <dig> poplar chromosomes  .
only one apc subunit is duplicated in the arabidopsis genome, atcdc27a and atcdc27b, and they are located on different chromosomes,  <dig> and  <dig> respectively; they share 47% identity and 64% protein similarity.
a duplication event is predicted to have occurred in this case according to the tool paralogons in arabidopsis  <cit> .
arabidopsis cdc <dig> and ccs <dig> genes are located on chromosomes  <dig> and  <dig>  atccs52a <dig> and atccs52a <dig> appear to be the result of a recent duplication and atcdc20_ <dig> and atcdc20_ <dig> are present in tandem on chromosome  <dig>  <cit> .
the  <dig> rice apc and activator genes are distributed among all chromosomes, except chromosomes  <dig> and  <dig>  based on the sequence consensus, rice genome, segmental duplication information from the tigr database was used to identify paralog genes.
each pair of paralogs located in the corresponding segmental duplication regions share high sequence similarity.
two subunits - cdc <dig> and apc <dig> - are duplicated.
oscdc23_ <dig> and oscdc23_ <dig> are located on chromosomes  <dig> and  <dig>  respectively and share 92% identity.
osapc11_ <dig> and osapc11_ <dig> are located on chromosome  <dig> and  <dig>  respectively and share 97% identify.
no evidences for recent duplication events in rice activators were found.
in-silico chromosome mapping revealed that poplar apc/activators are scattered throughout the genome.
paralogs are also located on different chromosomes or scaffolds.
it is possible, however, that some of the apparently closely related genes are in fact alleles from unassembled haplotypes, which are potential artifacts from shotgun assembly of this highly heterozygous genome.
however, the apparent co-orthologs are divergent at the nucleotide level, as well as in the flanking gene order, and they are identified in the syntenic blocks which argue against the classification of the scaffold as a haplotype.
duplications of two apc subunits were identified; cdc <dig> and apc <dig>  ptcdc27_ <dig> is located on linkage group viii and ptcdc27_ <dig> on scaffold_ <dig>  both are very similar to each other - 88% identity and 85% similarity in their amino-acid sequences, respectively.
ptapc13_ <dig> is 95% identical to ptapc13_ <dig> and they are located on linkage groups iv and xi, respectively.
poplar ccs <dig> genes are closely related, suggesting a recent genomic duplication event.
linkage groups iii and i and viii and x share large megabase-blocks in complete colinearity.
ptcdc20_ <dig> and ptcdc20_ <dig> are located in chromosomal regions that might represent paralog segments - linkage groups xiii and xix.
other cdc <dig> genes are located on linkage group xvi and scaffold_ <dig>  the scattered distribution of apc/activators is in good agreement with previous studies that described large-scale duplication events in poplar  <cit> .
phylogenetic analysis of apc tprs subunits and activators proteins
an important question is why the apc is composed of many different subunits, while most e <dig> ligases are composed of one to three subunits.
most of the apc subunits are conserved in all eukaryotes and remain tightly associated throughout the cell cycle  <cit> .
it seems probable that the complexity of multiprotein domains is related to the intricate topology of apc  <cit> .
one important domain in the apc subunits is tpr, which consists of 3- <dig> tandem repeats of  <dig> amino-acid residues.
however, individual tpr domains can be dispersed in the protein sequence  <cit> .
tprs appear to act as versatile protein-protein interaction domains and it has been hypothesized that the tpr-containing proteins in the apc form a scaffold on which the other subunits assemble  <cit> .
five apc subunits contain tpr domains: cdc <dig>  apc <dig>  cdc <dig>  apc <dig> and cdc <dig>  tpr-containing sequences of primitive red algae cyanidioschyzon merolae; green algae volvox carteri, chlorella sp., micromonas sp and ostreococcus sp; the bryophyte physcomitrella patens; the licophyte selaginella moellendorffii; the land plants vitis vinifera and sorghum bicolor; and the poplar, rice and arabidopsis sequences were used to gain insight into the evolutionary relationship between land plants and algae.
a phylogenetic tree was constructed with the mega <dig> neighbor-joining method, employing multiple alignments of  <dig> cdc <dig>   <dig> apc <dig>   <dig> cdc <dig>   <dig> apc <dig> and  <dig> cdc <dig> genes, with bootstrap analysis of  <dig>  replicates to ensure statistical reliability .
subsequently, the  <dig> apc subunits were divided into five phylogenetic groups of ortholog genes.
the cladogram obtained shows the oscdc <dig> genes in the same branch, located in the same group as s. bicolor.
grapevine cdc <dig> genes are in the same group as poplar.
the cladogram also shows that the poplar and grapevine genes are in the same branches in every group, and this could reveal slow evolutionary rates in woody when compared to herbaceous plants.
on the other hand, while arabidopsis cdc27a is in the same group of cdc27b, it is in a separate branch, and sequence divergence and evolution may reflect the accelerated life cycle of this plant.
interestingly, the apc <dig> and cdc <dig> clades do not contain duplicated genes.
we have not identified an apc <dig> sequence in red algae c. merolae; however, this subunit sequence is not so well conserved among different organisms, and it is possible that the similarity is too low to be identified by blast comparison.
the apc <dig> gene is absent in green algae chlorella sp and v. carteri, as well as in the red algae c. merolae, but it is present in green algae ostreococcus sp and micromonas sp.
chlorophytes  and trebouxiophytes  have apparently lost the apc <dig> gene, but the prasinophytes  and land plants retained this tpr subunit.
these data also suggest that the primitive ancestor of chlorophyceae and trebouxiophyceae classes lost apc <dig> gene during green algae evolution .
the apc <dig> gene is also not found in the yeasts, and it could mean that it is not required for apc function in unicellular organisms and it could be a recent duplication of another tpr subunit.
the arabidopsis genome codes for six cdc <dig> genes and three ccs <dig> genes.
in addition, ccs <dig> genes have been divided into two types  on the basis of functional and sequence analysis  <cit> .
to gain an understanding of the evolutionary relationship between activators from land plants and algae, phylogenetic analysis was performed for activators from plants and algae genomes .
we used est sequences from zea mays, medicago truncatula and saccharum officinarum to better estimate divergence between monocot and dicot sequences.
the obtained phylogenetic tree reveals three major class groups.
three genes, cmcdc <dig>  cmccs <dig> and smcdc20_ <dig>  are isolated from the others and thus need to be treated as nonconserved sequences.
one clade groups all cdc <dig> sequences , while two other clades included the ccs52a and ccs52b sequences .
the cdc <dig> clade is divided into two subfamilies: one with s. moellendorffii and p. patens and another with algae and land plants.
the number of cdc <dig> copies varies according to species.
arabidopsis has six copies, rice three, poplar five, grapevine four, sorghum three, s. moellendorffii three and p. patens four.
the ccs52a clade contains more duplications than the ccs52b clade.
all algae ccs <dig> genes, two copies of ptccs52a, atccs52a and smccs <dig>  and three copies of ppccs <dig> genes are grouped into the ccs52a branch.
curiously, only one copy of the cdc <dig> and ccs <dig>  genes was found in algae genomes.
the absence of ccs52b type genes in algae, p. patens and s. moellendorffii is interesting because these species are at the base of plant evolution where divergence into a-type and b-type has not occurred yet.
like arabidopsis, poplar has two copies of ccs52a; however poplar has an additional copy of ccs52b.
analyses of poplar gene evolution must take into account the most significant event in the recent evolution of the genus: a genome-wide duplication event at approximately 65mya and is still detectable over approximately 92% of the genome  <cit> .
based on the age estimates of duplicate genes and homology microsynteny analyses, cdc <dig> and ccs <dig> gene pairs are represented within segmental duplication regions associated with the recent salicoid duplication event.
comparison of arabidopsis, rice and poplar tprs proteins and determination of orthologous relationships
progress in comparative genomics allows assessing the impact of gene and genome evolution on the appearance of novel biological functions and their effect on organismal complexity  <cit> .
the importance of gene duplication in supplying raw genetic material for biological evolution has been recognized for decades and is still extensively studied.
gene duplication generates functional redundancy, which allows two identical genes resulting from the duplication to accumulate mutations with relaxed selection pressure  <cit> .
differentiated or novel function may be achieved after a period of evolution.
segmental duplication  is the most prevalent way to generate redundant genes  <cit> .
segmental duplication can also happen on a smaller scale, resulting in duplicated exons, rather than an entire gene  <cit> .
these segmental duplication blocks can create protein fragments  that have structure and function  <cit> .
the tpr domain consists of a 34-residue repeat that adopts a helix-turn-helix conformation, which is associated with protein-protein interactions  <cit> .
the subunits cdc <dig>  apc <dig>  cdc <dig>  apc <dig> and cdc <dig> have tpr domains, but the total number of tpr repeats and the position in each sequence is variable.
exon shuffling may be responsible for internal duplications in repeats and we decided to investigate the exon-intron organization of tpr genes in arabidopsis, rice and poplar   <cit> .
the number and position of exons and introns in the genomic sequences are remarkably conserved in some of the apc subunit genes.
the cdc <dig> gene has two homologs in arabidopsis and poplar, and only one in rice.
interestingly, the cdc <dig> gene maintains  <dig> exons in monocot and dicots.
the apc <dig> and apc <dig> genes have one copy in all genomes and also maintain  <dig> and  <dig> exons respectively, in the three genomes.
on the other hand, the cdc <dig> gene has two homologs in rice and only one in arabidopsis and poplar, but the most significant information is the lower number of exons when compared with other tpr-containing genes.
the oscdc23_ <dig> has  <dig> exons  and atcdc <dig>  ptcdc <dig> and oscdc23_ <dig> have  <dig> exons.
examining the cdc <dig> gene we identified  <dig> exons in poplar,  <dig> exons in arabidopsis and  <dig> exons in rice.
we next compared the exon organization of all tpr genes from arabidopsis using domain prediction.
nine tpr repeat sequences were identified in atcdc27a and atcdc <dig> sequences, two in atapc <dig> and ten in the atapc <dig> and atcdc <dig> sequences .
atcdc27b has the same number of tpr domains as atcdc27a.
tandem arrangement of tpr domains occurs predominantly at the c-terminal of atcdc27a, atcdc27b, atcdc <dig>  atapc <dig> and atcdc <dig> sequences.
atapc <dig> has only two tpr domains, one in the middle of the sequence and another at the c-terminal.
the results show that tpr tandem repeat topology on protein sequences is at least partially related with the exon organization of tpr subunit genes.
in the apc subunits, the tandem arrangement of tpr domains suggests that the elements evolved by duplication followed by primary sequence divergence.
because tpr are conserved structure repeat elements which are often divergent at the primary sequence level, tpr domain sequences can diverge a great deal over time and have poor consensus motifs.
an approach to investigate the evolution of tpr domains in the subunits is to identify patterns of tpr duplication from the alignments.
distinct patterns of repetition can often be distinguished, and we tested whether the arabidopsis tpr proteins have defined internal repetition blocks.
the sequences of tpr proteins were aligned to each other or in pairs using the methodology described in  <cit> .
the pairwise sequence similarities between all repeating domains in a protein were examined using the smith-waterman algorithm to identify patterns of duplication from the alignments  <cit> .
the alignment scores between the domains were displayed in a matrix .
comparing the proteins to themselves, there are similarities among domains 4- <dig> in atcdc27; and among domains 5- <dig> in atcdc <dig>  atapc <dig> has a pattern for domains 1- <dig>  atcdc <dig> has patterns of similarities for domains 6-8; and no pattern was found to for atapc <dig>  comparing the proteins in pairs, we can observe a diagonal match between atcdc <dig> domains 2- <dig> and atcdc <dig> domains 3- <dig>  there are also indications of similarities for the pairs atcdc <dig> and atcdc <dig>  atcdc <dig> and atcdc <dig>  although there are no unambiguous duplication patterns for any of the proteins, the results suggest that tpr domains in the apc genes have evolved by duplication of the central elements, or that higher constraints are imposed to changes in the primary sequence of these repeats.
expression analysis of apc subunits and activators genes
gene duplication can be a source of innovation for the increased developmental complexity of plants.
expression patterns can provide important clues for gene function under specific conditions.
we examined the expression of the apc subunit and activators genes in 5-day old rice roots and shoots, and in sheath and blade of mature tissues .
as expected, there are higher levels of gene expression in tissues with high proliferation rates, although there is more mrna in shoots than in roots.
however, a complex pattern is observed when their expression is examined in mature blade.
in general, there is a general decrease in expression for several genes, as expected.
however, the degree of reduction is variable.
the mrna levels of osapc <dig>  osapc <dig>  osapc <dig>  osapc <dig>  oscdc <dig>  oscdc23_ <dig>  osapc <dig>  osapc11_ <dig>  oscdc <dig>  and osapc <dig> are reduced in both sheath and blade compared to aerial part.
on the other hand, osapc11_ <dig> and oscdc <dig> mrna levels are reduced only in the sheath, but not in the blade.
finally, there is no reduction of osapc <dig> mrna levels in both sheath and blade.
in contrast, expression levels of activator genes are markedly reduced in mature leaves.
however, although ccs52a mrna levels are lower in both sheath and blade, the reduction is less pronounced.
in medicago, the ccs52a gene has been implicated in control of endoreduplication in nodules  <cit> .
recently, it has been shown that the ccs52a gene is important for maintenance of meristem activity in roots of arabidopsis  <cit> .
therefore, it is possible that the ccs52a could form a specialized complex with some of the apc subunits and assume a unique function in mature leaves.
growth response to dark is part of an integrated developmental change throughout all the plant organs  <cit> .
in arabidopsis, hypocotyl cells undergo up to two rounds of endoreduplication in light-grown seedlings, whereas an additional round can be observed specifically during dark-grown development  <cit> .
the endoreduplication regulatory mechanism is thought to be common to the g1-s transition of the mitotic cell cycle  <cit> .
g1-s transition is important for cdk inactivation by cyclin proteolysis, mediated by apccdh <dig>  which then maintains cyclin instability in g <dig> and enables a new round of dna replication by allowing the assembly of pre-replication complex   <cit> .
almost all rice apc subunits have similar expression patterns, both in roots and shoots .
however, osapc11_ <dig> and oscdc <dig> mrna levels increased in darkness.
interestingly, the two oscdc <dig> genes are differentially expressed in etiolated roots.
while oscdc23_ <dig> mrna levels did not change in roots grown in the dark or light, oscdc23_ <dig> mrna levels in roots were much lower when rice plants were grown in the dark.
similarly, in etiolated roots osapc11_ <dig> mrna levels increased while osapc11_ <dig> levels decreased.
these results could indicate that, following gene duplication, the apc <dig> and cdc <dig> genes are assuming new functions in rice.
the activators osccs52a and osccs52b exhibited high levels of mrna in darkness and oscdc20_ <dig> showed a lower level.
one hypothesis for the high mrna levels of ccs <dig> genes is that the apcccs <dig> acts as a negative regulator of cyca2;3/cdka; <dig> complex in arabidopsis  <cit> .
the cyca2;3/cdka;1complex acts as a negative regulator of endocycle and the apc activation by ccs <dig> may increase cyca2; <dig> degradation and consequently the progression of endocycle in darkness  <cit> .
to investigate the expression of the apc genes and activators during rice seed development, we examined the expression of the apc subunits and activators in endosperm, pericarp and inflorescence tissues .
expression levels were measured in the inflorescence at reproductive stage  <dig> , pericarp and endosperm at reproductive stage  <dig> .
levels of mrna from all apc subunits were higher in the endosperm, except osapc <dig> and osapc <dig>  which were present in higher amounts in the inflorescence.
the duplicated oscdc <dig> genes displayed similar expression profiles in the seed tissues examined.
however, the osapc <dig> gene seems to be differentially expressed; osapc11_ <dig> showed reduced amounts of mrna in the inflorescence when compared to the pericarp and endosperm.
using the genevestigator analysis tool, we compared the expression levels of the apc genes with qrt-pcr results   <cit> .
although only the genes osapc <dig>  osapc <dig>  osapc <dig> and osapc <dig> are present in the microarray slides, their expression is similar to what we observed in this work.
the expression patterns of the various apc activator genes were analyzed.
the osccs52b transcript was expressed at low levels in the inflorescence and oscdc20_ <dig> was barely detected in the same tissue.
interestingly, transcripts for oscdc20_ <dig> and oscdc20_ <dig> were detected in inflorescence, a tissue with a high rate of proliferation, suggesting their involvement in mitotic activities  <cit> .
the osccs52a had high levels in the endosperm and this may be explained by the fact that the transition from a mitotic to an endocycle in endosperm cells involves a spike in activity of the master cell cycle regulators belonging to the cdk family and apc complex, which subsequently decreases as endocycle progresses  <cit> .
these contrasting expression patterns for osccs <dig> and oscdc <dig> could suggest distinct functions for these apc activators during seed development.
based on the direct relationship between the increase of auxin and cellular proliferation we treated rice seedlings with naphthaleneacetic acid .
after treatment for  <dig> and  <dig> hours, all apc and activator genes increased their expression.
this result indicates that the rice apc subunits and activators might be involved in the regulation of the cell division events triggered by auxin in the seedlings.
we analyzed the expression in leaves of ptapc <dig>  ptcdc27_ <dig>  ptcdc20_ <dig>  ptccs52a1_ <dig> and ptccs52b genes by qrt-pcr .
interestingly, ptccs52b exhibited lower mrna levels when compared to ptccs52a1_ <dig>  recent data suggest that the mtccs52b may have specific roles in m-phase progression, alternative or complementary to those of mtccs52a  <cit> .
thus, ccs52a might have major roles in post-mitotic, differentiating cells, in which degradation of specific apc targets could contribute to differentiation of given cell types, tissues, or organs  <cit> .
CONCLUSIONS
through genome-wide bioinformatics analyses of the complete genomes of arabidopsis, rice and poplar, we identified ortholog genes of apc subunits and activators.
in addition, their occurrence was demonstrated in algae, bryophyte and other monocotyledonous and dicotyledonous species as well.
a distinctive feature of these genomes is the presence of characteristic duplication patterns .
in arabidopsis, only the cdc <dig> gene is duplicated.
rice and poplar have two duplicated subunits, cdc23-apc <dig> and cdc27-apc <dig> respectively.
among other plant genomes, grape and p. patens have two copies of the cdc <dig> and cdc <dig> genes, respectively, and grape has two copies of the apc <dig> gene.
the two isoforms of cdc <dig> and apc <dig> genes in rice are differentially expressed in light-dark grown plants and one of apc <dig> subunits is also more expressed in the inflorescence than the other.
in arabidopsis, the duplicated copies of the cdc <dig> gene exhibit significant sequence differences, and seem to have assumed specialized functions, indicative of the occurrence of apc subcomplexes  <cit> .
while the coding sequences of the rice genes are quite conserved, it is possible that their controlling elements have diverged and that they are assuming new functions in the apc during plant development.
paralogs usually display different functions, whereas orthologs may retain the same function  <cit> .
we tested the frequency of ka/ks for cdc <dig>  cdc <dig>  apc <dig> and activators orthologous genes and found interesting values .
the cdc <dig> and cdc <dig> tpr genes had the highest values  and the activators and apc <dig> subunit lower.
these results reveal the importance of maintenance of the amino acids conserved in ring finger proteins and the adapters.
analyses of the promoters of the rice subunit genes revealed different regulatory sites for duplicated genes, suggesting different regulation of their expression .
as expected, the most common cis-acting elements in all genes analyzed have functions related to cell division.
strikingly, oscdc <dig> and oscdc23_ <dig> and oscdc23_ <dig> do not show any cis element in common and only two cis-regulatory elements are found in both osapc11_ <dig> and osapc11_ <dig>  however, the osapc <dig> and oscdc <dig> gene sequences present very little variation across taxa, possibly due to strong purifying selection.
as in yeasts, apc <dig> is not present in the green algae chlamydomonas reinhardtii, v. carteri and chlorella spp.
the absence of this subunit at the root of animal and land-plant evolution suggests that this subunit may have appeared after a gene duplication event of a tpr-containing subunit gene, and it may not be essential for apc activity in unicellular organisms.
curiously, it might be the case also in later metazoans, once drosophila apc <dig> knockout mutant flies and null mutants are viable and fertile  <cit> .
the activators cdc <dig> and ccs <dig> have many copies in plants.
this is in sharp contrast with the situation in metazoans, where only one copy of each regulator is present per genome.
due to the duplications, arabidopsis contains three ccs <dig> activators; ccs52a, ccs52a <dig> and ccs52b.
in addition, six cdc <dig> genes were found, but atcdc20_ <dig> is probably a pseudogene because the important motif ken-box is absent  <cit> .
in poplar, the ccs52a and ccs52b genes are also duplicated.
five cdc <dig> genes are present in poplar, the same number found in arabidopsis.
interestingly, rice contains one copy of the ccs52a and ccs52b genes; and three copies of cdc <dig> genes.
this suggests that the duplication of the ccs <dig> genes occurred in dicotyledonous plants after the separation from monocotyledons.
recent duplication of the cdc <dig> genes has occurred also in arabidopsis and poplar.
plants can adopt dramatically different alternative developmental pathways and must integrate cell-cycle progression, growth and development in response to environmental cues.
it is believed that multiple members of a specific gene family of a particular organism are the natural products generated from the long evolutionary history that the organism experienced  <cit> .
the number of members of a gene family reflects a succession of genomic rearrangements and expansions due to extensive duplication and diversification that occurred in the course of evolution.
phylogenetic analysis of the apc subunits and activator proteins is very illuminating.
the fact that all subunit types except apc <dig>  were found in all plant genomes analyzed, including red algae, green algae and other primitive organisms, suggests that the complete set of genes encoding the apc subunits was already present in the common ancestor of plants and animals.
the activators descend from a common ancestor and correspond to well-conserved structures.
in all algae species analyzed, there is only one copy of cdc <dig> and ccs <dig> genes.
land plants have amplified the number of activators and this fact may be associated with substrate specificity and or with the complexity of their developmental programs.
by comparing the similarity between different tpr domains within one protein it might be possible to trace the evolutionary history of the internal duplications.
the alignment scores between domains showed limited similarities among tpr subunits of atcdc <dig>  atcdc <dig>  atapc <dig>  atcdc <dig> and atapc <dig>  the sequence of tpr subunits can diverge a great deal over time, limiting their consensus motifs and making it difficult to detect unambiguous duplication patterns for any of the proteins.
nevertheless, pairwise comparisons indicated greater similarity for the central domains, especially between cdc <dig> and cdc <dig>  cdc <dig> and cdc <dig>  and cdc <dig> and cdc <dig>  in all cases, higher similarity scores between tpr subunits  <dig> in cdc <dig> and cdc <dig>  and tpr subunit  <dig> in cdc <dig> were detected, suggesting that the tpr tandem evolution most likely started from the middle domains.
because the primary amino acid sequence of tpr subunits tends to diverge, the higher degree of similarity among the central subunits may also indicate that stronger evolutionary constraints are applied to these regions.
previously, we have shown that the arabidopsis subunit genes are differentially expressed according to whether the plants are grown in the dark or under light.
when arabidopsis plants were grown in the dark, only atapc <dig>  atapc <dig> and atcdc <dig> increased mrna levels  <cit> .
however, qrt-pcr expression profiles of rice apc and activator genes showed spatial modulation of gene expression of the duplicated subunit genes in different tissues  of plants grown under different light treatment.
osapc11_ <dig> mrna accumulates to higher levels in etiolated roots when compared with osapc11_ <dig> mrna, and oscdc23_ <dig> mrna is present in etiolated roots at almost twice the levels of oscdc23_ <dig>  likewise, the rice osapc <dig> genes are differentially expressed in the inflorescence, with activators that have very complex expression patterns.
altogether, these data suggest that the duplicated rice apc subunits and activators could participate in different apc subcomplexes and that they may have assumed new specialized functions as they diverged during evolution.
most e <dig> ligases are composed by one or two polypeptides  <cit> .
in contrast, the cell cycle regulators scf and apc are formed by a larger number of subunits.
however, for both complexes, biochemical activity can be achieved by only two subunits, a cullin related protein together with a ring finger protein.
in both cases, substrate specificity is defined by the adaptor subunit.
the human genome contains  <dig> f-box proteins - the scf activator - and two apc activators, cdh <dig> and cdc <dig>  <cit> .
in plants, scf-related complexes are involved in many physiological processes and approximately  <dig> f-box proteins have been identified in arabidopsis  <cit> .
the number of apc activators have also suffered an expansion in the higher plant lineages, though a more modest one, the number varying from nine in poplar and arabidopsis to six in rice.
we and others have shown that differential expression of apc subunits could be a source of complexity in regulation of the apc  <cit> .
the results presented here suggest that gene duplication, followed by sequence divergence and differential expression, could be another tier of regulation of ubiquitin-mediated proteolysis mediated by specific apc complexes composed by particular subunit isoforms.
future studies employing functional genomics approaches will be required to define the impact of duplications or splicing variants on cell-cycle progression at the cellular level and the associated plant developmental processes at the whole-organism level.

 cell proliferation is controlled by an universally conserved molecular machinery in which the key players are cyclin-dependent kinases and cyclins. eukaryotes have therefore evolved elaborate mechanisms for cdk regulation. an irreversible mechanism of cdk down-regulation is destruction of cyclin subunits. at the g1- to s-phase and metaphase to anaphase transitions, cdks are irreversibly inactivated by ubiquitin-mediated proteolysis of cognate cyclins. degradation of protein substrates through the ubiquitin-proteasome pathway involves the activity of different e ligases, among them the anaphase-promoting complex. the apc was first identified based on its role in facilitating the multiubiquitination and targeting of a- and b-type cyclins for proteasome-mediated destruction during mitosis. apc is a multiprotein complex conserved from plants to man and contains at least core subunits. the apc is regulated in part by two associated proteins, cdc and ccs52/cdh that can both activate the apc with proper timing and provide substrate specificity. the apc is activated at metaphase/anaphase transition by the cdc protein and later in telophase and g by the cdh protein. substrates that have a destruction box, ken-box or a-box motifs are recognized and ubiquitinated by the apc. the apc has important functions in mitosis, meiosis, g1-s-phase and in post-mitotic differentiated cells. the mitosis-specific activator cdc is itself an apc substrate. other targets of apc/c degradation are: cyclins a and b; protein kinases plk cdc aurora a and b; regulators of dna replication geminin, cdc6; and the anaphase inhibitor securin. the proteolytic events triggered by the apc are required to release sister chromatides cohesion during anaphase, to control the exit from mitosis and to prevent premature entry into s-phase. although the role of the apc in controlling cell-cycle progression has been extensively investigated, has recent work shown the unexpected presence of apc subunits in differentiated tissues, in particular in the nervous system of vertebrates. in plants, the expression of several apc subunits has been detected in differentiated tissues of arabidopsis. in addition, while both cdc27a and cdc27b genes are essential during gametogenesis, the cdc27b subunit has been implicated also in post-embryogenic differentiation at meristem levels. besides, it has been shown that reduced levels of apc and apc subunits in arabidopsis lead to plants with several defects in vascular development. these results indicate that, while the overall structure of the apc is conserved among eukaryotes, this e ligase may have assumed specialized functions in the diverse kingdoms. gene duplication and retention in plants has been extensive and gene families are generally larger in plants than in animals. nevertheless, almost all studies on the apc function in plants have been carried out in the model plant arabidopsis thaliana. comparative genomic analyses can provide valuable insights into the organization of cell cycle machinery and the evolution of these protein complexes. in this article, we describe thorough searches for the predicted sequences of the apc subunits and the activators cdc20/ccs in plant sequence databases. our results indicate that land plants and green algae orthologs have an ancient evolutionary origin. we present phylogenetic analysis of tpr subunits and activators, and their orthologs from other plants. an evolutionary analysis of arabidopsis tpr domain suggests that tandem regions could have been created from duplication of internal sequence domains. overall, our data support the proposal that apc subunits and activators have been conserved in the course of evolution. however, while other eukaryotes like fungi and metazoans have only one copy of each apc subunit and its regulators, gene duplication of different subunits have occurred in arabidopsis, rice and poplar, and in other plant genomes as well. an attractive proposal is that gene duplication of apc subunits may result in the formation of apc subcomplexes that evolved to assume restricted specialized roles during plant development. indeed, in arabidopsis, duplication of the cdc subunit led to substantial sequence divergence and specialization. here we show that the two copies of rice cdc and apc subunits and the activators genes are differentially expressed in plant tissues or when grown under either dark or light-mediated developmental programs, suggesting the duplicated genes could be assuming new functions in the plant.  identification of apc subunits and activator genes in order to carry out the cross-species comparison of the apc and activators, we have searched for homologous sequences in the genomic research rice genome database and doe join genome institute poplar genome database. the sequences of the arabidopsis apc subunits have been published. even after exhaustive data mining, some rice and poplar sequences were only found in the est database gene index dfci. subsequently, the smart motif identification tool was used to identify predicted domains in all candidate proteins. using this strategy, we report the apc/activators in the dicotyledonous poplar and the monocotyledon rice plants. all apc subunits and activators were found, and they contained the predicted conserved domains, providing one of many examples for the evolutionary conservation of the eukaryotic cell-cycle machinery. agene index project access number http://compbio.dfci.harvard.edu/tgi/ two cdc homologs have been identified in arabidopsis atcdc27a and atcdc27b. in poplar, two homologs of cdc were also identified: ptcdc27_ and ptcdc27_ however, a careful inspection of the ptcdc27_ deposited sequence revealed a stop codon tag at nucleotide position 1471- this fragment was amplified and sequenced and the presence of the stop codon was discarded. the expression of both genes in leaves was confirmed by quantitative pcr. we also found two homologs of cdc in the bryophyte physcomitrella patens genome. however, only one copy of cdc was found in the rice genome as well as other monocot plants. on the other hand, the rice genome has two oscdc and osapc subunits. we examined other complete genomes and found two cdc and apc genes in grapevine. two homologs of ptapc were found in the poplar and grapevine genomes, a feature that has not been reported in any other eukaryotic species so far. the structures of many predicted genes were considerably misannotated. comparing est databases from plant species and using bioinformatics tools, it was possible to identify the correct gene sequences. the genomic sequence of osapc was not found in the tigr database, but a partial cds was identified in the gene index database. most likely, the largest subunit of the apc had come apart by misannotation. diverse exon-intron structures needed correction in the genomics sequences. osapc may contain an intron in the genomic orf and oscdc23_ may have a fragment of an exon involved in the formation of tpr domain erroneously annotated as an intron. the 3'region of predicted ptapc and the 5'-3'regions of ptapc were incomplete. however, we found fragments from 3'regions of both genes and fragments from the 5'region of ptapc in the est database. interestingly, osapc oscdc and osapc11_ have a divergent 5'region compared to arabidopsis and poplar genes. the first methionine of oscdc and osapc11_ are upstream from a consensus start codon in cdc and apc genes, but only for cdc there is est support. the opposite occurs with osapc where the first methionine is downstream from the consensus start codon. the apc genes atcdc oscdc ptcdc and osapc were identified in est databases. arabidopsis contains three ccs genes, atccs52a atccs52a and atccs52b; and five cdc genes. in addition, we found another cdc homolog in the arabidopsis genome. this atcdc20_ showed major differences from conserved structures of other cdc20; the cds does not have the c-box sequence element ipxr) that was first identified in the n-terminal region of cdc although it is conserved in all known apc/c co-activators, and therefore atcdc20_ could be a pseudogene. the rice genome has three predicted cdc genes compared to five in poplar and arabidopsis. rice has two ccs genes compared to four and three genes in poplar and arabidopsis, respectively. exon-intron organization of activators was analyzed and we found a number of annotation errors. atcdc20_ ptcdc20_ ptccs52a and ptccs52a1_ have errors in the 5' region. we found that only ptcdc20_ is without a c-box. oscdc20_ atcdc20_ ptcdc20_ ptcdc20_ and ptcdc20_ had mistakes in the 3' region. this region contains the ir-tail sequence element that occupies the c terminus of apc activators and the apc subunit apc comparing with est databases, all sequences were deduced and the ir-tail was identified. in ptccs52a1_ and oscdc20_ genes, introns were incorrectly included. the opposite happened in the oscdc20_ gene, where one exon was absent. alternative splicing variants were found in the arabidopsis, rice and poplar apc/activator genes and could represent another layer of complexity in the organization and function of the apc. we selected only a single variant for further analysis. duplication of cell-cycle machinery components is rare in metazoans but it is a widespread phenomenon in plants.it has been proposed that duplication, followed by sequence divergence of promoter and/or coding regions, leads to novel and specialized functions unique to the plant kingdom. still, additional file shows that duplications of individual subunits of the apc are not present in all plants; on the contrary, they may be restricted to one or to a small group of phylogenetically related plant species and may be involved in the organization of developmental events unique to this group. chromosomal location of genes for apc subunits and activator the close similarity and presence of putative subunits and paralogs prompted us to investigate the genome distribution of plant apc/activators. apc genes were found in the five arabidopsis chromosomes, of the rice chromosomes and of the poplar chromosomes. only one apc subunit is duplicated in the arabidopsis genome, atcdc27a and atcdc27b, and they are located on different chromosomes, and respectively; they share 47% identity and 64% protein similarity. a duplication event is predicted to have occurred in this case according to the tool paralogons in arabidopsis. arabidopsis cdc and ccs genes are located on chromosomes and atccs52a and atccs52a appear to be the result of a recent duplication and atcdc20_ and atcdc20_ are present in tandem on chromosome. the rice apc and activator genes are distributed among all chromosomes, except chromosomes and based on the sequence consensus, rice genome, segmental duplication information from the tigr database was used to identify paralog genes. each pair of paralogs located in the corresponding segmental duplication regions share high sequence similarity. two subunits - cdc and apc - are duplicated. oscdc23_ and oscdc23_ are located on chromosomes and respectively and share 92% identity. osapc11_ and osapc11_ are located on chromosome and respectively and share 97% identify. no evidences for recent duplication events in rice activators were found. in-silico chromosome mapping revealed that poplar apc/activators are scattered throughout the genome. paralogs are also located on different chromosomes or scaffolds. it is possible, however, that some of the apparently closely related genes are in fact alleles from unassembled haplotypes, which are potential artifacts from shotgun assembly of this highly heterozygous genome. however, the apparent co-orthologs are divergent at the nucleotide level, as well as in the flanking gene order, and they are identified in the syntenic blocks which argue against the classification of the scaffold as a haplotype. duplications of two apc subunits were identified; cdc and apc ptcdc27_ is located on linkage group viii and ptcdc27_ on scaffold_ both are very similar to each other - 88% identity and 85% similarity in their amino-acid sequences, respectively. ptapc13_ is 95% identical to ptapc13_ and they are located on linkage groups iv and xi, respectively. poplar ccs genes are closely related, suggesting a recent genomic duplication event. linkage groups iii and i and viii and x share large megabase-blocks in complete colinearity. ptcdc20_ and ptcdc20_ are located in chromosomal regions that might represent paralog segments - linkage groups xiii and xix. other cdc genes are located on linkage group xvi and scaffold_ the scattered distribution of apc/activators is in good agreement with previous studies that described large-scale duplication events in poplar. phylogenetic analysis of apc tprs subunits and activators proteins an important question is why the apc is composed of many different subunits, while most e ligases are composed of one to three subunits. most of the apc subunits are conserved in all eukaryotes and remain tightly associated throughout the cell cycle. it seems probable that the complexity of multiprotein domains is related to the intricate topology of apc. one important domain in the apc subunits is tpr, which consists of 3- tandem repeats of amino-acid residues. however, individual tpr domains can be dispersed in the protein sequence. tprs appear to act as versatile protein-protein interaction domains and it has been hypothesized that the tpr-containing proteins in the apc form a scaffold on which the other subunits assemble. five apc subunits contain tpr domains: cdc apc cdc apc and cdc tpr-containing sequences of primitive red algae cyanidioschyzon merolae; green algae volvox carteri, chlorella sp., micromonas sp and ostreococcus sp; the bryophyte physcomitrella patens; the licophyte selaginella moellendorffii; the land plants vitis vinifera and sorghum bicolor; and the poplar, rice and arabidopsis sequences were used to gain insight into the evolutionary relationship between land plants and algae. a phylogenetic tree was constructed with the mega neighbor-joining method, employing multiple alignments of cdc apc cdc apc and cdc genes, with bootstrap analysis of replicates to ensure statistical reliability. subsequently, the apc subunits were divided into five phylogenetic groups of ortholog genes. the cladogram obtained shows the oscdc genes in the same branch, located in the same group as s. bicolor. grapevine cdc genes are in the same group as poplar. the cladogram also shows that the poplar and grapevine genes are in the same branches in every group, and this could reveal slow evolutionary rates in woody when compared to herbaceous plants. on the other hand, while arabidopsis cdc27a is in the same group of cdc27b, it is in a separate branch, and sequence divergence and evolution may reflect the accelerated life cycle of this plant. interestingly, the apc and cdc clades do not contain duplicated genes. we have not identified an apc sequence in red algae c. merolae; however, this subunit sequence is not so well conserved among different organisms, and it is possible that the similarity is too low to be identified by blast comparison. the apc gene is absent in green algae chlorella sp and v. carteri, as well as in the red algae c. merolae, but it is present in green algae ostreococcus sp and micromonas sp. chlorophytes and trebouxiophytes have apparently lost the apc gene, but the prasinophytes and land plants retained this tpr subunit. these data also suggest that the primitive ancestor of chlorophyceae and trebouxiophyceae classes lost apc gene during green algae evolution. the apc gene is also not found in the yeasts, and it could mean that it is not required for apc function in unicellular organisms and it could be a recent duplication of another tpr subunit. the arabidopsis genome codes for six cdc genes and three ccs genes. in addition, ccs genes have been divided into two types on the basis of functional and sequence analysis. to gain an understanding of the evolutionary relationship between activators from land plants and algae, phylogenetic analysis was performed for activators from plants and algae genomes. we used est sequences from zea mays, medicago truncatula and saccharum officinarum to better estimate divergence between monocot and dicot sequences. the obtained phylogenetic tree reveals three major class groups. three genes, cmcdc cmccs and smcdc20_ are isolated from the others and thus need to be treated as nonconserved sequences. one clade groups all cdc sequences, while two other clades included the ccs52a and ccs52b sequences. the cdc clade is divided into two subfamilies: one with s. moellendorffii and p. patens and another with algae and land plants. the number of cdc copies varies according to species. arabidopsis has six copies, rice three, poplar five, grapevine four, sorghum three, s. moellendorffii three and p. patens four. the ccs52a clade contains more duplications than the ccs52b clade. all algae ccs genes, two copies of ptccs52a, atccs52a and smccs and three copies of ppccs genes are grouped into the ccs52a branch. curiously, only one copy of the cdc and ccs genes was found in algae genomes. the absence of ccs52b type genes in algae, p. patens and s. moellendorffii is interesting because these species are at the base of plant evolution where divergence into a-type and b-type has not occurred yet. like arabidopsis, poplar has two copies of ccs52a; however poplar has an additional copy of ccs52b. analyses of poplar gene evolution must take into account the most significant event in the recent evolution of the genus: a genome-wide duplication event at approximately 65mya and is still detectable over approximately 92% of the genome. based on the age estimates of duplicate genes and homology microsynteny analyses, cdc and ccs gene pairs are represented within segmental duplication regions associated with the recent salicoid duplication event. comparison of arabidopsis, rice and poplar tprs proteins and determination of orthologous relationships progress in comparative genomics allows assessing the impact of gene and genome evolution on the appearance of novel biological functions and their effect on organismal complexity. the importance of gene duplication in supplying raw genetic material for biological evolution has been recognized for decades and is still extensively studied. gene duplication generates functional redundancy, which allows two identical genes resulting from the duplication to accumulate mutations with relaxed selection pressure. differentiated or novel function may be achieved after a period of evolution. segmental duplication is the most prevalent way to generate redundant genes. segmental duplication can also happen on a smaller scale, resulting in duplicated exons, rather than an entire gene. these segmental duplication blocks can create protein fragments that have structure and function. the tpr domain consists of a 34-residue repeat that adopts a helix-turn-helix conformation, which is associated with protein-protein interactions. the subunits cdc apc cdc apc and cdc have tpr domains, but the total number of tpr repeats and the position in each sequence is variable. exon shuffling may be responsible for internal duplications in repeats and we decided to investigate the exon-intron organization of tpr genes in arabidopsis, rice and poplar. the number and position of exons and introns in the genomic sequences are remarkably conserved in some of the apc subunit genes. the cdc gene has two homologs in arabidopsis and poplar, and only one in rice. interestingly, the cdc gene maintains exons in monocot and dicots. the apc and apc genes have one copy in all genomes and also maintain and exons respectively, in the three genomes. on the other hand, the cdc gene has two homologs in rice and only one in arabidopsis and poplar, but the most significant information is the lower number of exons when compared with other tpr-containing genes. the oscdc23_ has exons and atcdc ptcdc and oscdc23_ have exons. examining the cdc gene we identified exons in poplar, exons in arabidopsis and exons in rice. we next compared the exon organization of all tpr genes from arabidopsis using domain prediction. nine tpr repeat sequences were identified in atcdc27a and atcdc sequences, two in atapc and ten in the atapc and atcdc sequences. atcdc27b has the same number of tpr domains as atcdc27a. tandem arrangement of tpr domains occurs predominantly at the c-terminal of atcdc27a, atcdc27b, atcdc atapc and atcdc sequences. atapc has only two tpr domains, one in the middle of the sequence and another at the c-terminal. the results show that tpr tandem repeat topology on protein sequences is at least partially related with the exon organization of tpr subunit genes. in the apc subunits, the tandem arrangement of tpr domains suggests that the elements evolved by duplication followed by primary sequence divergence. because tpr are conserved structure repeat elements which are often divergent at the primary sequence level, tpr domain sequences can diverge a great deal over time and have poor consensus motifs. an approach to investigate the evolution of tpr domains in the subunits is to identify patterns of tpr duplication from the alignments. distinct patterns of repetition can often be distinguished, and we tested whether the arabidopsis tpr proteins have defined internal repetition blocks. the sequences of tpr proteins were aligned to each other or in pairs using the methodology described in. the pairwise sequence similarities between all repeating domains in a protein were examined using the smith-waterman algorithm to identify patterns of duplication from the alignments. the alignment scores between the domains were displayed in a matrix. comparing the proteins to themselves, there are similarities among domains 4- in atcdc27; and among domains 5- in atcdc atapc has a pattern for domains 1- atcdc has patterns of similarities for domains 6-8; and no pattern was found to for atapc comparing the proteins in pairs, we can observe a diagonal match between atcdc domains 2- and atcdc domains 3- there are also indications of similarities for the pairs atcdc and atcdc atcdc and atcdc although there are no unambiguous duplication patterns for any of the proteins, the results suggest that tpr domains in the apc genes have evolved by duplication of the central elements, or that higher constraints are imposed to changes in the primary sequence of these repeats. expression analysis of apc subunits and activators genes gene duplication can be a source of innovation for the increased developmental complexity of plants. expression patterns can provide important clues for gene function under specific conditions. we examined the expression of the apc subunit and activators genes in 5-day old rice roots and shoots, and in sheath and blade of mature tissues. as expected, there are higher levels of gene expression in tissues with high proliferation rates, although there is more mrna in shoots than in roots. however, a complex pattern is observed when their expression is examined in mature blade. in general, there is a general decrease in expression for several genes, as expected. however, the degree of reduction is variable. the mrna levels of osapc osapc osapc osapc oscdc oscdc23_ osapc osapc11_ oscdc and osapc are reduced in both sheath and blade compared to aerial part. on the other hand, osapc11_ and oscdc mrna levels are reduced only in the sheath, but not in the blade. finally, there is no reduction of osapc mrna levels in both sheath and blade. in contrast, expression levels of activator genes are markedly reduced in mature leaves. however, although ccs52a mrna levels are lower in both sheath and blade, the reduction is less pronounced. in medicago, the ccs52a gene has been implicated in control of endoreduplication in nodules. recently, it has been shown that the ccs52a gene is important for maintenance of meristem activity in roots of arabidopsis. therefore, it is possible that the ccs52a could form a specialized complex with some of the apc subunits and assume a unique function in mature leaves. growth response to dark is part of an integrated developmental change throughout all the plant organs. in arabidopsis, hypocotyl cells undergo up to two rounds of endoreduplication in light-grown seedlings, whereas an additional round can be observed specifically during dark-grown development. the endoreduplication regulatory mechanism is thought to be common to the g1-s transition of the mitotic cell cycle. g1-s transition is important for cdk inactivation by cyclin proteolysis, mediated by apccdh which then maintains cyclin instability in g and enables a new round of dna replication by allowing the assembly of pre-replication complex. almost all rice apc subunits have similar expression patterns, both in roots and shoots. however, osapc11_ and oscdc mrna levels increased in darkness. interestingly, the two oscdc genes are differentially expressed in etiolated roots. while oscdc23_ mrna levels did not change in roots grown in the dark or light, oscdc23_ mrna levels in roots were much lower when rice plants were grown in the dark. similarly, in etiolated roots osapc11_ mrna levels increased while osapc11_ levels decreased. these results could indicate that, following gene duplication, the apc and cdc genes are assuming new functions in rice. the activators osccs52a and osccs52b exhibited high levels of mrna in darkness and oscdc20_ showed a lower level. one hypothesis for the high mrna levels of ccs genes is that the apcccs acts as a negative regulator of cyca2;3/cdka; complex in arabidopsis. the cyca2;3/cdka;1complex acts as a negative regulator of endocycle and the apc activation by ccs may increase cyca2; degradation and consequently the progression of endocycle in darkness. to investigate the expression of the apc genes and activators during rice seed development, we examined the expression of the apc subunits and activators in endosperm, pericarp and inflorescence tissues. expression levels were measured in the inflorescence at reproductive stage, pericarp and endosperm at reproductive stage. levels of mrna from all apc subunits were higher in the endosperm, except osapc and osapc which were present in higher amounts in the inflorescence. the duplicated oscdc genes displayed similar expression profiles in the seed tissues examined. however, the osapc gene seems to be differentially expressed; osapc11_ showed reduced amounts of mrna in the inflorescence when compared to the pericarp and endosperm. using the genevestigator analysis tool, we compared the expression levels of the apc genes with qrt-pcr results. although only the genes osapc osapc osapc and osapc are present in the microarray slides, their expression is similar to what we observed in this work. the expression patterns of the various apc activator genes were analyzed. the osccs52b transcript was expressed at low levels in the inflorescence and oscdc20_ was barely detected in the same tissue. interestingly, transcripts for oscdc20_ and oscdc20_ were detected in inflorescence, a tissue with a high rate of proliferation, suggesting their involvement in mitotic activities. the osccs52a had high levels in the endosperm and this may be explained by the fact that the transition from a mitotic to an endocycle in endosperm cells involves a spike in activity of the master cell cycle regulators belonging to the cdk family and apc complex, which subsequently decreases as endocycle progresses. these contrasting expression patterns for osccs and oscdc could suggest distinct functions for these apc activators during seed development. based on the direct relationship between the increase of auxin and cellular proliferation we treated rice seedlings with naphthaleneacetic acid. after treatment for and hours, all apc and activator genes increased their expression. this result indicates that the rice apc subunits and activators might be involved in the regulation of the cell division events triggered by auxin in the seedlings. we analyzed the expression in leaves of ptapc ptcdc27_ ptcdc20_ ptccs52a1_ and ptccs52b genes by qrt-pcr. interestingly, ptccs52b exhibited lower mrna levels when compared to ptccs52a1_ recent data suggest that the mtccs52b may have specific roles in m-phase progression, alternative or complementary to those of mtccs52a. thus, ccs52a might have major roles in post-mitotic, differentiating cells, in which degradation of specific apc targets could contribute to differentiation of given cell types, tissues, or organs.  through genome-wide bioinformatics analyses of the complete genomes of arabidopsis, rice and poplar, we identified ortholog genes of apc subunits and activators. in addition, their occurrence was demonstrated in algae, bryophyte and other monocotyledonous and dicotyledonous species as well. a distinctive feature of these genomes is the presence of characteristic duplication patterns. in arabidopsis, only the cdc gene is duplicated. rice and poplar have two duplicated subunits, cdc23-apc and cdc27-apc respectively. among other plant genomes, grape and p. patens have two copies of the cdc and cdc genes, respectively, and grape has two copies of the apc gene. the two isoforms of cdc and apc genes in rice are differentially expressed in light-dark grown plants and one of apc subunits is also more expressed in the inflorescence than the other. in arabidopsis, the duplicated copies of the cdc gene exhibit significant sequence differences, and seem to have assumed specialized functions, indicative of the occurrence of apc subcomplexes. while the coding sequences of the rice genes are quite conserved, it is possible that their controlling elements have diverged and that they are assuming new functions in the apc during plant development. paralogs usually display different functions, whereas orthologs may retain the same function. we tested the frequency of ka/ks for cdc cdc apc and activators orthologous genes and found interesting values. the cdc and cdc tpr genes had the highest values and the activators and apc subunit lower. these results reveal the importance of maintenance of the amino acids conserved in ring finger proteins and the adapters. analyses of the promoters of the rice subunit genes revealed different regulatory sites for duplicated genes, suggesting different regulation of their expression. as expected, the most common cis-acting elements in all genes analyzed have functions related to cell division. strikingly, oscdc and oscdc23_ and oscdc23_ do not show any cis element in common and only two cis-regulatory elements are found in both osapc11_ and osapc11_ however, the osapc and oscdc gene sequences present very little variation across taxa, possibly due to strong purifying selection. as in yeasts, apc is not present in the green algae chlamydomonas reinhardtii, v. carteri and chlorella spp. the absence of this subunit at the root of animal and land-plant evolution suggests that this subunit may have appeared after a gene duplication event of a tpr-containing subunit gene, and it may not be essential for apc activity in unicellular organisms. curiously, it might be the case also in later metazoans, once drosophila apc knockout mutant flies and null mutants are viable and fertile. the activators cdc and ccs have many copies in plants. this is in sharp contrast with the situation in metazoans, where only one copy of each regulator is present per genome. due to the duplications, arabidopsis contains three ccs activators; ccs52a, ccs52a and ccs52b. in addition, six cdc genes were found, but atcdc20_ is probably a pseudogene because the important motif ken-box is absent. in poplar, the ccs52a and ccs52b genes are also duplicated. five cdc genes are present in poplar, the same number found in arabidopsis. interestingly, rice contains one copy of the ccs52a and ccs52b genes; and three copies of cdc genes. this suggests that the duplication of the ccs genes occurred in dicotyledonous plants after the separation from monocotyledons. recent duplication of the cdc genes has occurred also in arabidopsis and poplar. plants can adopt dramatically different alternative developmental pathways and must integrate cell-cycle progression, growth and development in response to environmental cues. it is believed that multiple members of a specific gene family of a particular organism are the natural products generated from the long evolutionary history that the organism experienced. the number of members of a gene family reflects a succession of genomic rearrangements and expansions due to extensive duplication and diversification that occurred in the course of evolution. phylogenetic analysis of the apc subunits and activator proteins is very illuminating. the fact that all subunit types except apc were found in all plant genomes analyzed, including red algae, green algae and other primitive organisms, suggests that the complete set of genes encoding the apc subunits was already present in the common ancestor of plants and animals. the activators descend from a common ancestor and correspond to well-conserved structures. in all algae species analyzed, there is only one copy of cdc and ccs genes. land plants have amplified the number of activators and this fact may be associated with substrate specificity and or with the complexity of their developmental programs. by comparing the similarity between different tpr domains within one protein it might be possible to trace the evolutionary history of the internal duplications. the alignment scores between domains showed limited similarities among tpr subunits of atcdc atcdc atapc atcdc and atapc the sequence of tpr subunits can diverge a great deal over time, limiting their consensus motifs and making it difficult to detect unambiguous duplication patterns for any of the proteins. nevertheless, pairwise comparisons indicated greater similarity for the central domains, especially between cdc and cdc cdc and cdc and cdc and cdc in all cases, higher similarity scores between tpr subunits in cdc and cdc and tpr subunit in cdc were detected, suggesting that the tpr tandem evolution most likely started from the middle domains. because the primary amino acid sequence of tpr subunits tends to diverge, the higher degree of similarity among the central subunits may also indicate that stronger evolutionary constraints are applied to these regions. previously, we have shown that the arabidopsis subunit genes are differentially expressed according to whether the plants are grown in the dark or under light. when arabidopsis plants were grown in the dark, only atapc atapc and atcdc increased mrna levels. however, qrt-pcr expression profiles of rice apc and activator genes showed spatial modulation of gene expression of the duplicated subunit genes in different tissues of plants grown under different light treatment. osapc11_ mrna accumulates to higher levels in etiolated roots when compared with osapc11_ mrna, and oscdc23_ mrna is present in etiolated roots at almost twice the levels of oscdc23_ likewise, the rice osapc genes are differentially expressed in the inflorescence, with activators that have very complex expression patterns. altogether, these data suggest that the duplicated rice apc subunits and activators could participate in different apc subcomplexes and that they may have assumed new specialized functions as they diverged during evolution. most e ligases are composed by one or two polypeptides. in contrast, the cell cycle regulators scf and apc are formed by a larger number of subunits. however, for both complexes, biochemical activity can be achieved by only two subunits, a cullin related protein together with a ring finger protein. in both cases, substrate specificity is defined by the adaptor subunit. the human genome contains f-box proteins - the scf activator - and two apc activators, cdh and cdc. in plants, scf-related complexes are involved in many physiological processes and approximately f-box proteins have been identified in arabidopsis. the number of apc activators have also suffered an expansion in the higher plant lineages, though a more modest one, the number varying from nine in poplar and arabidopsis to six in rice. we and others have shown that differential expression of apc subunits could be a source of complexity in regulation of the apc. the results presented here suggest that gene duplication, followed by sequence divergence and differential expression, could be another tier of regulation of ubiquitin-mediated proteolysis mediated by specific apc complexes composed by particular subunit isoforms. future studies employing functional genomics approaches will be required to define the impact of duplications or splicing variants on cell-cycle progression at the cellular level and the associated plant developmental processes at the whole-organism level. 
expression patterns of paralog subunits and activators in rice could indicate that this duplication, rather than complete redundancy, could reflect initial specialization steps. here, we describe an in-depth search for apc subunits and activator genes in the arabidopsis, rice and poplar genomes. phylogenetic analyses indicate that some apc subunits and activator genes have experienced gene duplication events in plants, in contrast to animals. the absence of subunit apc from the genome of some green algae species and as well as from early metazoan lineages, could mean that apc is not required for apc function in unicellular organisms and it may be a result of duplication of another tetratricopeptide subunit. analyses of tpr evolution suggest that duplications of subunits started from the central domains. the increased complexity of the apc gene structure, tied to the diversification of expression paths, suggests that land plants developed sophisticated mechanisms of apc regulation to cope with the sedentary life style and its associated environmental exposures.
 the orderly progression through mitosis is regulated by the anaphase-promoting complex, a large multiprotein e ubiquitin ligase that targets key cell-cycle regulators for destruction by the s proteasome. the apc is composed of at least subunits and associates with additional regulatory activators during mitosis and interphase cycles. despite extensive research on apc and activator functions in the cell cycle, only a few components have been functionally characterized in plants.  here, we describe an in-depth search for apc subunits and activator genes in the arabidopsis, rice and poplar genomes. also, searches in other genomes that are not completely sequenced were performed. phylogenetic analyses indicate that some apc subunits and activator genes have experienced gene duplication events in plants, in contrast to animals. expression patterns of paralog subunits and activators in rice could indicate that this duplication, rather than complete redundancy, could reflect initial specialization steps. the absence of subunit apc from the genome of some green algae species and as well as from early metazoan lineages, could mean that apc is not required for apc function in unicellular organisms and it may be a result of duplication of another tetratricopeptide subunit. analyses of tpr evolution suggest that duplications of subunits started from the central domains.  the increased complexity of the apc gene structure, tied to the diversification of expression paths, suggests that land plants developed sophisticated mechanisms of apc regulation to cope with the sedentary life style and its associated environmental exposures. 