BACKGROUND
heterosis is a phenomenon that particular inbred lines can produce progenies with favourable phenotypes over their parents, such as stronger tolerance to stresses and higher yields.
two fundamental hypotheses for heterosis were defined in classical genetic studies, including genome-wide dominance complementation and locus-specific over-dominant effects  <cit> .
evidence showed that both play roles in heterosis with involvement of epistasis  <cit> .
another theory suggested that heterosis might result from the loss of control of metabolism among heterozygotes  <cit> .
along with the development of genome-wide expression technology such as est, sage, and microarrays, current technological advancements allow scientists to gain comprehensive information and knowledge on gene expression profiles of particular tissues from hybrids and their parental lines  <cit> .
as it is obvious that differentially-expressed genes  among hybrid triads may offer straightforward molecular clues to characterize phenotypic differences and genes responsible for favourable phenotypes, high-throughput gene expression profiling methods have been used for identifying degs in recent years.
various degs and their expression patterns in hybrids and their parental lines have been defined and analyzed, including over-dominance, high-parent dominance, additivity, low-parent dominance, and under-dominance  <cit> .
the distribution of degs varies among different samples, which not only reflects the complexity of rice transcriptomes but also points to the possibility where intricate molecular mechanisms may be involved in heterosis.
as both an important cereal crop and a model plant, the rice genome has been sequenced multiple times for its two major subspecies  <cit> .
we have been studying rice genome with a particular interest on the molecular mechanism of heterosis as one of the specific aims proposed for the super-hybrid rice genome project .
the project focuses on an elite super-hybrid  and its parental lines .
several datasets of degs as potential heterosis-associated genes as well as possible molecular mechanisms have been collected from major tissues, such as leaves, roots, and panicles  <cit> .
the mature embryo is an important developmental stage in the rice life cycle, as it is a synchronized, undeveloped miniature plant that consists of precursor tissues for root, leaf, and stem.
it was reported that its long-lived mrna reserved in dry plant seeds might, in general, contain a valuable molecular record of embryogenic development and primary biological process  <cit> .
a recent study suggested that the embryo of heterotic f <dig> possesses certain advantages at a very early developmental stage  <cit> .
therefore, it is important to know whether the mature embryo deposits heterotic potential for further development, as a genome-wide transcriptomic study for the mature rice embryo of a hybrid triad has not yet been reported to this date.
in this study, we constructed cdna libraries for mature embryos from 93- <dig>  pa64s and lyp <dig>  and randomly sequenced approximately  <dig>  ests from each library, resulting in  <dig>  high-quality ests and  <dig>  unigenes.
we analyzed these ests and identified  <dig> degs between lyp <dig> and its parental lines .
assigning the degs into functional categories and expression patterns, we realized that multiple modes are at work for heterosis.
all our est sequences were submitted to the ncbi dbest database under accession number fg <dig> to fg <dig>
RESULTS
est acquisition
we acquired approximately  <dig>  clones from each library, which yielded  <dig>  high-quality ests after removal of vector and low-quality sequences.
the collection was further refined by removing sequences that are not anchored in the oryza sativa gene index database  and predicted gene sequences of the 93- <dig> genome  <cit> , resulted in  <dig>  high-quality ests that include  <dig> ,  <dig> , and  <dig>  ests for 93- <dig>  pa64s, and lyp <dig>  respectively .
we assembled the ests into  <dig>  clusters, including  <dig>  contigs and  <dig>  singletons.
the three datasets contain  <dig>   <dig>  and  <dig> unigenes from 93- <dig>  pa64s, and lyp <dig>  respectively, with a similar cluster-size distribution .
only  <dig>  unigenes were found universally-expressed as they are shared by all three libraries; there are about a third of them are shared by two libraries and more than 50% unigenes in each library are unique .
since most of the unique genes were detected as single copies, we believe that the transcriptome of a mature rice embryo is rather complex and the current sampling is not deep enough to discover low-abundance genes as most of the genes should be shared among the cdna libraries rather than truly unique to each.
therefore, we focused our analysis on unigenes expressed in relatively high abundance.
functional annotation
we annotated  <dig>   out of a total of  <dig>  unigenes in our dataset based on sequence similarity to those in public databases .
among  <dig> un-annotated unigenes,  <dig> have significant similarity to the repeat sequence collection  and one to a non-coding rna sequence from the mouse genome .
the remaining  <dig> unannotated unigenes are rather short but have open reading frames more than  <dig> amino acids in length, and they may represent novel protein-coding genes or non-coding rnas as they are expressed in rice embryos.
alignments were performed by using blast , and  <dig>   unigenes were annotated.
we aligned the best hits to tigr ogi database with the gene index to go mapping database at tigr and assigned at least one go term to  <dig>   transcripts.
the go-annotated unigenes are  <dig>  ,  <dig>  , and  <dig>   for 93- <dig>  pa64s, and lyp <dig>  respectively.
we assigned most of the unigenes into several functional categories, including cellular process, metabolism process, multicellular organism development, stress tolerance, transport, cell, binding, catalytic activity, transcription regulator activity, translation regulator activity, and transporter activity .
we also annotated our datasets using kegg automatic annotation server  for pathway information .
we were able to offer  <dig>  unigenes ko numbers, including  <dig> ,  <dig> , and  <dig>  in the libraries of 93- <dig>  pa64s, and lyp <dig>  respectively.
the categories of metabolism, genetic information processing , environmental information processing , and cellular process involved  <dig> %,  <dig> %,  <dig> %, and  <dig> % of the total annotated unigenes, respectively.
as summarized in figure  <dig>  carbon , energy , and amino acid  metabolisms are major contributors among the subsets of metabolism.
in the category of gip, translation  and sorting  are the majority as opposed to transcription .
signal transduction  and membrane transport  constitute the majority of eip.
other than these major categories, the rest of cellular processes showed lower abundance although defence systems  were found more abundant among the low-abundance categories.
expression abundance
we calculated the expression abundance for each unigene using their cluster sizes  and sorted them into three abundance levels: high-abundance , medium-abundance , and low-abundance .
the top  <dig> bins of high-abundance genes constitute only  <dig> % of total unigenes in kind but  <dig> % of the total est counts .
the entire collection and its annotated details are listed in additional file 1: summary of unigenes.
their sequences are supplemented in additional file 2: a full list of unigene sequences.
alignments were performed by using blast , and  <dig>   unigenes were annotated.
atranscript per million,b stress-tolerance associated genes, and c development-associated genes.
to identify embryo-characteristic genes, we carried out a hierarchical clustering analysis based on est abundance, rice gene annotation in the unigene database at ncbi, and comparative analysis with representative libraries of other rice tissues selected from the digital differential display  database .
we also did a parallel analysis utilizing sage data of different tissues , which were generated from the same hybrid triad lines  <cit>  based on annotations in our database that contains predicted gene sequences from the 93- <dig> genome.
the results are rather consistent; not only is there a significant amount of high-abundance genes unique to the mature embryo  but also most of the medium-abundance genes are rather characteristic for the embryo .
in addition, we compared embryo-associated genes against all predicted genes from the parental genomes  based on go functional classification, and observed the enrichment of genes in several categories, such as response to stimulus and stress in the category biological processes .
data from this study are highlighted in bold and italic.
based on go functional classification, we found that  high-abundance genes are enriched in cellular and metabolic processes, response to stress, cell, binding, catalytic activity, and translation regulator activity;  medium-abundance genes are those for embryo development, response to external stimulus, transport, enzyme regulator activity, signal transducer activity, structural molecule activity, transcription regulator activity, and transporter activity; and  low-abundance genes are involved in anatomical structure morphogenesis, death, growth, reproduction, response to biotic stimulus, and motor activity .
we also analyzed the genes classified as different pathways.
we found that  high-abundance genes concentrate on carbohydrate metabolism, folding/sorting/degradation, membrane transport, signal transduction, and defence  systems, and  medium-abundance genes are mainly involved in the pathways of energy metabolism, lipid metabolism, amino acid metabolism, transcription and translation .
high-abundance genes were found in the pathways of glycolysis, pores ion channels, protein folding and associated processing, ubiquitin-mediated proteolysis, mapk signalling, progesterone-mediated oocyte maturation, and antigen processing/presentation; all appear performing important functions in keeping embryonic status and rapid growth recovery.
abundant embryo-characteristic gene families
we discovered four major gene families expressed in high-abundance in the rice embryo .
the most abundant gene family belongs to cytochrome p <dig> monooxygenases, making up 10% of total ests.
they are universally-expressed and senescence-associated in all eukaryotic species.
among plants, these enzymes are important for the biosynthesis of hormones, defensive compounds, and fatty acids.
the second group is composed of hsps , and they are multifunctional molecular chaperones and many show over-expression during embryonic development and are inducible by desiccation and other stress types.
we identified four low-molecular-weight  hsps in our dataset; they are hsp <dig> , hsp <dig> , hsp <dig>  and hsp <dig> as well as four classical hsps: hsp <dig>  hsp <dig>  hsp <dig>  and hsp <dig>  in numbers, there are 16% of the abundantly-expressed genes and  <dig> % of the total ests belonging to the hsp family.
the third protein family is the lea  proteins that are a group of proteins accumulating in high abundance during last stages of seed formation and periods of the water shortage in vegetative organs.
ample evidence bsuggested that lea proteins, especially its subgroup  <dig> members, are involved in desiccation resistance through a variety of machineries, including water retention, ion sequestration, and direct protein protection  <cit> .
the fourth group is the rab family, which are membrane-associated small gtp-binding proteins localized to discrete subcellular compartments and involved in signal transduction, cytoskeleton organization, and vesicle trafficking.
they are also stress-inducible.
we identified its four members whose ests make up  <dig> % of the total collection: rab <dig> , rab <dig>  rab <dig>  and rab <dig>
acytochrome p <dig> family, bheat shock protein, clate embryogenesis abundant protein, and d rab protein.
degs between lyp <dig> and its parents
based on p-values of  <dig>  and  <dig>  from general chi-squared test, we defined  <dig>  and  <dig>  degs, respectively, between lyp <dig> and its parental lines .
the full list of all degs is in additional file 4: degs and their expression patterns.
most of these degs belong to high- and medium-abundance genes,  <dig> % and  <dig> %, respectively.
we focus our discussion on the larger group  unless specified.
the degs cover almost all expression patterns with rather unequal distributions.
first, the majority of degs  exhibit either high- or low-parent dominances rather than deviate significantly from both .
second, of the  <dig> genes,  <dig> %  show differences between pa64s and lyp <dig>  whereas only  <dig> %  exhibit differences between 93- <dig> and lyp <dig>  this result indicated that the gene expression pattern of lyp <dig> is more similar to 93- <dig> than to pa64s at this developmental stage.
third, there are slightly less high-parent dominant genes  than low-parent dominant ones  in embryo both between 93- <dig> and lyp <dig>  and between pa64s and lyp <dig> , whereas over-dominant genes  are more than under-dominant genes .
finally, there are slightly more up-regulated genes than down-regulated genes in general.
when we used the more stringent p-value , most of the observed rules did not change except that the up-regulated genes between pa64s and lyp <dig> were found slightly more than down-regulated genes .
in general, the distribution pattern of degs in the embryo is similar to that of the panicle as compared to our previous sage study  <cit> .
when we displayed the fold change of degs /2]) in a two-dimensional plot , the consistency remains.
n, p, and l stand for paternal lines , maternal lines , and f <dig> hybrids , respectively.
integrated analysis with proteomic and sage data
we compared our degs to those identified from proteomic data generated from the same material .
the proteomic data were acquired from the same material as what we used in this study, mature embryo of 93- <dig>  pa64s, and lyp <dig>  over  <dig>  2-d gel spots  were analyzed and  <dig> differentially-expressed proteins were identified in the study.
we found that most of the high-abundance genes also highly expressed in the proteomic data, but the expression patterns were not always consistent.
there were only nine genes found consistent in both datasets, and three of them were not .
seven of the consistent degs were found expressed higher in lyp <dig> except early embryogenesis protein and glutelin; both are down-regulated.
most of the degs in the protein data fell into three general categories: additivity, over-dominance, and under-dominance, whereas most of the degs generated from the est data are in other groups: high- and low-parent dominance.
we also compared our est data to those of our sage experiments generated from the same hybrid triad.
the sage data were generated from nine sage libraries made from three tissues: root, leaf, and panicle of the same hybrid triad, 93- <dig>  pa64s, and lyp <dig>  roughly ten-thousand sage tags from each library were obtained and  <dig>  unique tags were annotated, and among them,  <dig>  degs  were detected  <cit> .
in this study, we identified  <dig> degs between the two datasets, which are shared by at least one tissue .
the expression patterns in both dataset were summarized in additional file 5: degs shared with sage data.
these genes are more likely to be associated with heterosis, especially when some of the degs have consistent expression pattern in all tissues.
alignments of degs to genome sequences
to understand the regulatory mechanism of degs, we traced their genomic sequences in 93- <dig> and pa64s, and identified the regulatory and transcript sequences among orthologous gene pairs.
we scrutinized the genomic sequences  from the genome sequences of 93- <dig> and pa64s for the  <dig> degs that are confirmed with our sage data.
we found that  <dig>  genes have almost identical sequences in all regions , and we believed that these genes might be regulated by distant trans-regulatory mechanisms other than cis-elements in their immediate promoters.
among the  <dig>  genes that showed significant sequence deviations,  <dig> ,  <dig> , and  <dig>  genes have differences in coding, 5'utr, and 3' utr regions, respectively.
we also analyzed well-defined regulatory sequence motifs and found that  <dig>  genes have obvious motif variations.
our results were summarized in table  <dig>
n stands for insignificant differences  and y stands for significant differences .
quantitative pcr-based experimental validation of degs
we chose eight stress-tolerance-associated and four development-associated genes–both expressed in high-abundance and functionally important to mature rice embryo–to validate their expression patterns, using quantitative real-time pcr or qrt-pcr.
the stress-tolerance-associated proteins are lea <dig>  hsp <dig> , hsp <dig>  tonoplast intrinsic protein, ubiquitin e <dig>  thaumatin-like protein, bowman-birk trypsin inhibitor, and cold-regulated protein .
the four development-associated genes included one embryo-specific protein and three metabolism-associated proteins .
almost all the results showed consistent trends with their corresponding est data .
we listed the primer sequences used in the experiment in additional file 6: primers for qrt-pcr.
discussion
genes in abundance: stress tolerance and development
high-abundance genes are known to characterize tissue- or organ-specificity since they are dominant in manifestation of major functions of the tissue or organ.
we found that the embryo shares  <dig> ,  <dig> , and  <dig>  genes with leaf, panicle, and root, respectively, and there are  <dig>  genes common to all tissues.
all high-abundance and most medium-abundance genes in our dataset are either specifically expressed or unique to their tissues of origin whereas only  <dig> genes in medium-abundance expressed universally at similar expression level, as compared to the sage data, distributing among the most basic go categories .
in an attempt to define functional characteristics of the mature rice embryo based on its high- and medium-abundance genes, we found that two major functional categories stood out: stress tolerance and development, which are composed of  <dig> % and  <dig> % of high-abundance genes, respectively.
furthermore, there are still large amount of genes in the universally expressed categories such as metabolism and cellular process which expressed specifically in high-abundance in mature embryo.
we identified a large number of genes that are in the category of anti-stress via various mechanisms, which include a complete water-deficit tolerance system and genes involved in resistance in oxidation damage and fungal pathogen infection .
water deficit is a major threatening factor to embryos, and a water-deficit tolerance system in plants was reported, including membrane proteins, chaperones , water channel proteins, protease inhibitors, proteases, and the ubiqutin system  <cit> .
the three high-abundance gene families  that we categorized in this study all have important functions in this system  <cit> .
our proteomic study with the same material also provided a footnote to this conclusion .
three gene families–em, ec, and germin proteins–have been suggested to regulate the development of rice embryo to seedling  <cit> .
the em and ec proteins are encoded by conserved mrnas stored in mature embryos but germin transcribes after growth recovery.
the em protein is involved in the process of seed desiccation, and the ec protein is the only bona fide zn metallothionein yet found in higher plants  <cit> ; both may play important roles in ion homeostasis during embryogenesis since em and ec proteins are expressed as the most abundantly expressed genes in our dataset with  <dig> and  <dig> ests, respectively.
in addition, we identified one embryogenic cell protein  <dig> in the high-abundance gene class as well as other development-associated proteins including embryo-specific protein, differentiation embryo protein  <dig>  and early embryogenesis protein in medium-abundance genes.
pathway analysis on the mrna repertoire provided a better glance on the basic physiology of rice mature embryos detected by pathway analysis, which showed that the embryo is well-prepared for rapid development.
first, genes in the pathways of metabolism, especially the subsets of carbon, energy, and amino acid metabolisms, are stored in high abundance.
material utilization and energy production, such as glycolysis , tca cycle, starch and sucrose metabolism, oxidative phosphorylation, and carbon fixation are of essence for the embryo.
second, genes participating in translation  and sorting  are in high abundance to ensure rapid translation of the reserved rna.
third, in the category of eip, signal transduction  and membrane transport  stood out obviously to guarantee rapid signal transduction in the recovery phase, especially the mapk signal pathway that is important in regulating cell cycle.
fourth, the defence  system is the highly-expressed gene category in our dataset for cellular processes, which ensures hormonal regulation in development and protection.
our detailed analyses indicated that several important pathways active in mature plant tissues are almost absent in embryos.
for example, we only identified nine ests involved in photosynthesis and none involved in respiration from our dataset.
although present at low level, transcription  and replication/repair  genes are important for growth recovery as compared to the complete absence of genes in the categories of signal molecules and interaction as well as the low level of cellular process genes in our dataset.
the reservoir of the reserved mrnas in the mature embryo appeared well-prepared for basic metabolism, response to environmental stimulus, and signal transduction to ensure rapid recovery.
in the contrary, secondary metabolism, cellular process, and transcription constitute a relatively small portion of the total, and most of these functions must depend upon new mrna syntheses.
possible biological mechanisms of heterosis
when displaying all degs into two-dimension space, we found a composite expression pattern that suggests multiple modes of action as they are classified into  <dig> patterns: over-dominance, high-parent dominance, additivity, low-parent dominance and under-dominance .
although up- and down-regulated degs are almost equal in numbers , up-regulated genes are dominant in the most abundant gene classes including cellular, metabolic, response to stress, embryo development, enzyme regulator activity, transcription regulator activity, and transporter activity .
strengthened stress-tolerance system in f1
the majority of stress-tolerance genes in our dataset exhibited differential expressions among the hybrid triad, and especially showed up-regulated expressions in lyp <dig> .
in the go term, five out of seven  degs are in the category of response to stress and up-regulated in f <dig>  in the gene families related to stress tolerance ,  <dig> % were differentially expressed whereas 89% of them are up-regulated together with  <dig> % down-regulated and  <dig> % additivity in f <dig>  and as shown in table  <dig>  among 65% of the degs, 77% of them are up-regulated  as opposed to 23% down-regulated .
functionally, stress-tolerance genes are classified into two groups: protective and injury-induced genes.
taking the water-deficit-tolerance related genes as examples, we know that protective genes, including chaperones , protease inhibitors, water-deficit induced membrane proteins, and water channel proteins, are counterparts of the protein-degrading mechanism, promoting survivability under water-deficit conditions.
injury-induced genes include proteases and ubiquitin system, which are involved in the degradation of proteins that are denatured during cellular water loss .
in lyp <dig>  most of the protective genes are up-regulated as compared to the down-regulated injury-induced genes.
protective genes that participate in other stress-related functions are also up-regulated in the f <dig>  proteomic data also added footnotes to this notion, where the most functionally important and representative genes, including lea <dig>  shsp, bowman-birk trypsin inhibitor, lipoprotein and lectin, thaumatin-like protein were found as up-regulated degs.
in addition, our sage data also pointed out a universal strengthening in the ability of stress-tolerance in the f <dig>  where heavy metal transport/detoxification protein, senescence-associated protein and rab <dig> protein were found up-regulated albeit in other tissues of lyp <dig>  we validated some of these results with qrt-pcr.
better preparation for growth recovery
deg analysis suggested that the f <dig> appears better prepared for growth recovery as compared to its parental lines.
first, genes participating in material and energy utilization are up-regulated and genes participating in anabolism are down-regulated .
in general, the up-regulated degs were found in carbon metabolism with an exception of the oxidative phosphorylation of a nadp-dependent malic enzyme in energy metabolism.
in contrast, genes in carbon, nitrogen fixation, and nucleotide metabolism are mostly down-regulated.
in amino acid metabolism, we identified genes both up-regulated and down-regulated, but all genes related to amino acid biosynthesis are down-regulated.
this indicated that lyp <dig> up-regulate material and energy generation and utilization to ensure its embryo to be active immediately when being re-hydrated as material anabolism is normally down-regulated.
second, in the category of gip, genes involved in protein folding and associated processing are up-regulated and genes of ubiquitin mediated proteolysis and proteasome are down-regulated.
genes involved in transcription are down-regulated but those involved in translation are not obviously biased, where both down- and up-regulated genes were found.
third, in eip, genes in membrane transport and mapk signal transduction are up-regulated but calcium signalling pathway and phosphatidylinositol signalling systems are down-regulated; the result suggested that mapk signalling pathway may be the major signal pathway triggered as development starts after re-hydration.
finally, degs involved in cellular process but not in the response  system appeared down-regulated.
some of these genes and their trends of expressions were found consistent in our sage and proteomic data.
for example, the up-regulation of an important enolase was confirmed in the protein data.
and the up-regulation of succinate dehydrogenase was validated based on the qrt-pcr test.
another instance is β-amylase that is a key enzyme for starch hydrolysis, which is not only found up-regulated in the embryo but also in the leaf and panicle of lyp <dig>
complementation and over-dominance
we found that embryo-characteristic genes among degs are primarily up-regulated and showed high-parent dominance  with the addition of over-dominance .
the high-parent dominance genes often occur in gene families.
for example, in the hsp family, the expression of lmw hsps has the tendency of pa64s, higher than that of 93- <dig>  or expressed in an over-dominance fashion.
in contrast, the expression level of classical hsps resembles that of 93- <dig>  which is higher than that of pa64s, exhibiting over-dominance.
in the lea and rab family, as well as other stress-tolerance genes, all the degs show high-parent dominance with expression levels similar to 93- <dig>  more than two third of the degs differ between pa64s and lyp <dig> , whereas less than one third show differences between 93- <dig> and lyp <dig> .
the result is consistent with the fact that lyp <dig> is more like 93- <dig> than pa64s in their phenotypic appearances .
therefore, it appears that the hybrid vigour of lyp <dig> may be contributed by a composite of beneficial alleles from both parents, complementing the relatively weaker alleles in a global fashion, and over-dominance is an essential strategy for stress tolerance and metabolism.
strengthening tissue-characteristic functions may contribute to heterosis
analyses on degs have indicated that the mode of action for heterosis is most likely multi-fold and at multiple levels.
if we have to point out a single mode of action, we believe that the strengthening of embryo-characteristic genes in lyp <dig> is most noticeable.
for instance, the f <dig>  lyp <dig>  is predicted to have a significantly better stress-tolerance system than its parental cultivars, supported by up-regulated protective genes and down-regulated injury-induced genes.
in addition, lyp <dig> keeps strong responses to environmental signals and potentially high-level activities after growth recovery.
therefore, when seeds re-hydrate, the f <dig> embryo may perform better than its parental lines.
this phenomenon can be cautiously generalized based on similar analyses among other tissues of the same hybrid line.
for example, in leaf, it was detected that up-regulated genes in hybrid are dominated by degs related to enhancing carbon assimilation whereas those related to decomposition of carbohydrates are down-regulated  <cit> , resulting in an increased efficiency of photosynthesis or energy production.
CONCLUSIONS
we reported a comparative transcriptomic analysis based on ests acquired from mature embryos of a rice hybrid and its parental lines.
our study not only provided an initial characteristic description of expression profiles of rice embryos but also pointed out the enrichment of genes involved in stress-tolerance and preparation-for-recover .
we also identified  <dig> degs between f <dig> and its parental lines.
functional analyses revealed clues for rice heterosis, such as multiple modes of actions and strengthening of embryo-characteristic function.
genes involved in stress-tolerance and preparation-for-recovery may play key roles for rice heterosis in mature embryos.

 heterosis is a phenomenon that particular inbred lines can produce progenies with favourable phenotypes over their parents, such as stronger tolerance to stresses and higher yields. two fundamental hypotheses for heterosis were defined in classical genetic studies, including genome-wide dominance complementation and locus-specific over-dominant effects. evidence showed that both play roles in heterosis with involvement of epistasis. another theory suggested that heterosis might result from the loss of control of metabolism among heterozygotes. along with the development of genome-wide expression technology such as est, sage, and microarrays, current technological advancements allow scientists to gain comprehensive information and knowledge on gene expression profiles of particular tissues from hybrids and their parental lines. as it is obvious that differentially-expressed genes among hybrid triads may offer straightforward molecular clues to characterize phenotypic differences and genes responsible for favourable phenotypes, high-throughput gene expression profiling methods have been used for identifying degs in recent years. various degs and their expression patterns in hybrids and their parental lines have been defined and analyzed, including over-dominance, high-parent dominance, additivity, low-parent dominance, and under-dominance. the distribution of degs varies among different samples, which not only reflects the complexity of rice transcriptomes but also points to the possibility where intricate molecular mechanisms may be involved in heterosis. as both an important cereal crop and a model plant, the rice genome has been sequenced multiple times for its two major subspecies. we have been studying rice genome with a particular interest on the molecular mechanism of heterosis as one of the specific aims proposed for the super-hybrid rice genome project. the project focuses on an elite super-hybrid and its parental lines. several datasets of degs as potential heterosis-associated genes as well as possible molecular mechanisms have been collected from major tissues, such as leaves, roots, and panicles. the mature embryo is an important developmental stage in the rice life cycle, as it is a synchronized, undeveloped miniature plant that consists of precursor tissues for root, leaf, and stem. it was reported that its long-lived mrna reserved in dry plant seeds might, in general, contain a valuable molecular record of embryogenic development and primary biological process. a recent study suggested that the embryo of heterotic f possesses certain advantages at a very early developmental stage. therefore, it is important to know whether the mature embryo deposits heterotic potential for further development, as a genome-wide transcriptomic study for the mature rice embryo of a hybrid triad has not yet been reported to this date. in this study, we constructed cdna libraries for mature embryos from 93- pa64s and lyp and randomly sequenced approximately ests from each library, resulting in high-quality ests and unigenes. we analyzed these ests and identified degs between lyp and its parental lines. assigning the degs into functional categories and expression patterns, we realized that multiple modes are at work for heterosis. all our est sequences were submitted to the ncbi dbest database under accession number fg to fg  est acquisition we acquired approximately clones from each library, which yielded high-quality ests after removal of vector and low-quality sequences. the collection was further refined by removing sequences that are not anchored in the oryza sativa gene index database and predicted gene sequences of the 93- genome, resulted in high-quality ests that include,, and ests for 93- pa64s, and lyp respectively. we assembled the ests into clusters, including contigs and singletons. the three datasets contain and unigenes from 93- pa64s, and lyp respectively, with a similar cluster-size distribution. only unigenes were found universally-expressed as they are shared by all three libraries; there are about a third of them are shared by two libraries and more than 50% unigenes in each library are unique. since most of the unique genes were detected as single copies, we believe that the transcriptome of a mature rice embryo is rather complex and the current sampling is not deep enough to discover low-abundance genes as most of the genes should be shared among the cdna libraries rather than truly unique to each. therefore, we focused our analysis on unigenes expressed in relatively high abundance. functional annotation we annotated out of a total of unigenes in our dataset based on sequence similarity to those in public databases. among un-annotated unigenes, have significant similarity to the repeat sequence collection and one to a non-coding rna sequence from the mouse genome. the remaining unannotated unigenes are rather short but have open reading frames more than amino acids in length, and they may represent novel protein-coding genes or non-coding rnas as they are expressed in rice embryos. alignments were performed by using blast, and unigenes were annotated. we aligned the best hits to tigr ogi database with the gene index to go mapping database at tigr and assigned at least one go term to transcripts. the go-annotated unigenes are,, and for 93- pa64s, and lyp respectively. we assigned most of the unigenes into several functional categories, including cellular process, metabolism process, multicellular organism development, stress tolerance, transport, cell, binding, catalytic activity, transcription regulator activity, translation regulator activity, and transporter activity. we also annotated our datasets using kegg automatic annotation server for pathway information. we were able to offer unigenes ko numbers, including,, and in the libraries of 93- pa64s, and lyp respectively. the categories of metabolism, genetic information processing, environmental information processing, and cellular process involved %, %, %, and % of the total annotated unigenes, respectively. as summarized in figure carbon, energy, and amino acid metabolisms are major contributors among the subsets of metabolism. in the category of gip, translation and sorting are the majority as opposed to transcription. signal transduction and membrane transport constitute the majority of eip. other than these major categories, the rest of cellular processes showed lower abundance although defence systems were found more abundant among the low-abundance categories. expression abundance we calculated the expression abundance for each unigene using their cluster sizes and sorted them into three abundance levels: high-abundance, medium-abundance, and low-abundance. the top bins of high-abundance genes constitute only % of total unigenes in kind but % of the total est counts. the entire collection and its annotated details are listed in additional file 1: summary of unigenes. their sequences are supplemented in additional file 2: a full list of unigene sequences. alignments were performed by using blast, and unigenes were annotated. atranscript per million,b stress-tolerance associated genes, and c development-associated genes. to identify embryo-characteristic genes, we carried out a hierarchical clustering analysis based on est abundance, rice gene annotation in the unigene database at ncbi, and comparative analysis with representative libraries of other rice tissues selected from the digital differential display database. we also did a parallel analysis utilizing sage data of different tissues, which were generated from the same hybrid triad lines based on annotations in our database that contains predicted gene sequences from the 93- genome. the results are rather consistent; not only is there a significant amount of high-abundance genes unique to the mature embryo but also most of the medium-abundance genes are rather characteristic for the embryo. in addition, we compared embryo-associated genes against all predicted genes from the parental genomes based on go functional classification, and observed the enrichment of genes in several categories, such as response to stimulus and stress in the category biological processes. data from this study are highlighted in bold and italic. based on go functional classification, we found that high-abundance genes are enriched in cellular and metabolic processes, response to stress, cell, binding, catalytic activity, and translation regulator activity; medium-abundance genes are those for embryo development, response to external stimulus, transport, enzyme regulator activity, signal transducer activity, structural molecule activity, transcription regulator activity, and transporter activity; and low-abundance genes are involved in anatomical structure morphogenesis, death, growth, reproduction, response to biotic stimulus, and motor activity. we also analyzed the genes classified as different pathways. we found that high-abundance genes concentrate on carbohydrate metabolism, folding/sorting/degradation, membrane transport, signal transduction, and defence systems, and medium-abundance genes are mainly involved in the pathways of energy metabolism, lipid metabolism, amino acid metabolism, transcription and translation. high-abundance genes were found in the pathways of glycolysis, pores ion channels, protein folding and associated processing, ubiquitin-mediated proteolysis, mapk signalling, progesterone-mediated oocyte maturation, and antigen processing/presentation; all appear performing important functions in keeping embryonic status and rapid growth recovery. abundant embryo-characteristic gene families we discovered four major gene families expressed in high-abundance in the rice embryo. the most abundant gene family belongs to cytochrome p monooxygenases, making up 10% of total ests. they are universally-expressed and senescence-associated in all eukaryotic species. among plants, these enzymes are important for the biosynthesis of hormones, defensive compounds, and fatty acids. the second group is composed of hsps, and they are multifunctional molecular chaperones and many show over-expression during embryonic development and are inducible by desiccation and other stress types. we identified four low-molecular-weight hsps in our dataset; they are hsp, hsp, hsp and hsp as well as four classical hsps: hsp hsp hsp and hsp in numbers, there are 16% of the abundantly-expressed genes and % of the total ests belonging to the hsp family. the third protein family is the lea proteins that are a group of proteins accumulating in high abundance during last stages of seed formation and periods of the water shortage in vegetative organs. ample evidence bsuggested that lea proteins, especially its subgroup members, are involved in desiccation resistance through a variety of machineries, including water retention, ion sequestration, and direct protein protection. the fourth group is the rab family, which are membrane-associated small gtp-binding proteins localized to discrete subcellular compartments and involved in signal transduction, cytoskeleton organization, and vesicle trafficking. they are also stress-inducible. we identified its four members whose ests make up % of the total collection: rab, rab rab and rab acytochrome p family, bheat shock protein, clate embryogenesis abundant protein, and d rab protein. degs between lyp and its parents based on p-values of and from general chi-squared test, we defined and degs, respectively, between lyp and its parental lines. the full list of all degs is in additional file 4: degs and their expression patterns. most of these degs belong to high- and medium-abundance genes, % and %, respectively. we focus our discussion on the larger group unless specified. the degs cover almost all expression patterns with rather unequal distributions. first, the majority of degs exhibit either high- or low-parent dominances rather than deviate significantly from both. second, of the genes, % show differences between pa64s and lyp whereas only % exhibit differences between 93- and lyp this result indicated that the gene expression pattern of lyp is more similar to 93- than to pa64s at this developmental stage. third, there are slightly less high-parent dominant genes than low-parent dominant ones in embryo both between 93- and lyp and between pa64s and lyp, whereas over-dominant genes are more than under-dominant genes. finally, there are slightly more up-regulated genes than down-regulated genes in general. when we used the more stringent p-value, most of the observed rules did not change except that the up-regulated genes between pa64s and lyp were found slightly more than down-regulated genes. in general, the distribution pattern of degs in the embryo is similar to that of the panicle as compared to our previous sage study. when we displayed the fold change of degs /2]) in a two-dimensional plot, the consistency remains. n, p, and l stand for paternal lines, maternal lines, and f hybrids, respectively. integrated analysis with proteomic and sage data we compared our degs to those identified from proteomic data generated from the same material. the proteomic data were acquired from the same material as what we used in this study, mature embryo of 93- pa64s, and lyp over 2-d gel spots were analyzed and differentially-expressed proteins were identified in the study. we found that most of the high-abundance genes also highly expressed in the proteomic data, but the expression patterns were not always consistent. there were only nine genes found consistent in both datasets, and three of them were not. seven of the consistent degs were found expressed higher in lyp except early embryogenesis protein and glutelin; both are down-regulated. most of the degs in the protein data fell into three general categories: additivity, over-dominance, and under-dominance, whereas most of the degs generated from the est data are in other groups: high- and low-parent dominance. we also compared our est data to those of our sage experiments generated from the same hybrid triad. the sage data were generated from nine sage libraries made from three tissues: root, leaf, and panicle of the same hybrid triad, 93- pa64s, and lyp roughly ten-thousand sage tags from each library were obtained and unique tags were annotated, and among them, degs were detected. in this study, we identified degs between the two datasets, which are shared by at least one tissue. the expression patterns in both dataset were summarized in additional file 5: degs shared with sage data. these genes are more likely to be associated with heterosis, especially when some of the degs have consistent expression pattern in all tissues. alignments of degs to genome sequences to understand the regulatory mechanism of degs, we traced their genomic sequences in 93- and pa64s, and identified the regulatory and transcript sequences among orthologous gene pairs. we scrutinized the genomic sequences from the genome sequences of 93- and pa64s for the degs that are confirmed with our sage data. we found that genes have almost identical sequences in all regions, and we believed that these genes might be regulated by distant trans-regulatory mechanisms other than cis-elements in their immediate promoters. among the genes that showed significant sequence deviations,,, and genes have differences in coding, 5'utr, and 3' utr regions, respectively. we also analyzed well-defined regulatory sequence motifs and found that genes have obvious motif variations. our results were summarized in table n stands for insignificant differences and y stands for significant differences. quantitative pcr-based experimental validation of degs we chose eight stress-tolerance-associated and four development-associated genes–both expressed in high-abundance and functionally important to mature rice embryo–to validate their expression patterns, using quantitative real-time pcr or qrt-pcr. the stress-tolerance-associated proteins are lea hsp, hsp tonoplast intrinsic protein, ubiquitin e thaumatin-like protein, bowman-birk trypsin inhibitor, and cold-regulated protein. the four development-associated genes included one embryo-specific protein and three metabolism-associated proteins. almost all the results showed consistent trends with their corresponding est data. we listed the primer sequences used in the experiment in additional file 6: primers for qrt-pcr. discussion genes in abundance: stress tolerance and development high-abundance genes are known to characterize tissue- or organ-specificity since they are dominant in manifestation of major functions of the tissue or organ. we found that the embryo shares,, and genes with leaf, panicle, and root, respectively, and there are genes common to all tissues. all high-abundance and most medium-abundance genes in our dataset are either specifically expressed or unique to their tissues of origin whereas only genes in medium-abundance expressed universally at similar expression level, as compared to the sage data, distributing among the most basic go categories. in an attempt to define functional characteristics of the mature rice embryo based on its high- and medium-abundance genes, we found that two major functional categories stood out: stress tolerance and development, which are composed of % and % of high-abundance genes, respectively. furthermore, there are still large amount of genes in the universally expressed categories such as metabolism and cellular process which expressed specifically in high-abundance in mature embryo. we identified a large number of genes that are in the category of anti-stress via various mechanisms, which include a complete water-deficit tolerance system and genes involved in resistance in oxidation damage and fungal pathogen infection. water deficit is a major threatening factor to embryos, and a water-deficit tolerance system in plants was reported, including membrane proteins, chaperones, water channel proteins, protease inhibitors, proteases, and the ubiqutin system. the three high-abundance gene families that we categorized in this study all have important functions in this system. our proteomic study with the same material also provided a footnote to this conclusion. three gene families–em, ec, and germin proteins–have been suggested to regulate the development of rice embryo to seedling. the em and ec proteins are encoded by conserved mrnas stored in mature embryos but germin transcribes after growth recovery. the em protein is involved in the process of seed desiccation, and the ec protein is the only bona fide zn metallothionein yet found in higher plants; both may play important roles in ion homeostasis during embryogenesis since em and ec proteins are expressed as the most abundantly expressed genes in our dataset with and ests, respectively. in addition, we identified one embryogenic cell protein in the high-abundance gene class as well as other development-associated proteins including embryo-specific protein, differentiation embryo protein and early embryogenesis protein in medium-abundance genes. pathway analysis on the mrna repertoire provided a better glance on the basic physiology of rice mature embryos detected by pathway analysis, which showed that the embryo is well-prepared for rapid development. first, genes in the pathways of metabolism, especially the subsets of carbon, energy, and amino acid metabolisms, are stored in high abundance. material utilization and energy production, such as glycolysis, tca cycle, starch and sucrose metabolism, oxidative phosphorylation, and carbon fixation are of essence for the embryo. second, genes participating in translation and sorting are in high abundance to ensure rapid translation of the reserved rna. third, in the category of eip, signal transduction and membrane transport stood out obviously to guarantee rapid signal transduction in the recovery phase, especially the mapk signal pathway that is important in regulating cell cycle. fourth, the defence system is the highly-expressed gene category in our dataset for cellular processes, which ensures hormonal regulation in development and protection. our detailed analyses indicated that several important pathways active in mature plant tissues are almost absent in embryos. for example, we only identified nine ests involved in photosynthesis and none involved in respiration from our dataset. although present at low level, transcription and replication/repair genes are important for growth recovery as compared to the complete absence of genes in the categories of signal molecules and interaction as well as the low level of cellular process genes in our dataset. the reservoir of the reserved mrnas in the mature embryo appeared well-prepared for basic metabolism, response to environmental stimulus, and signal transduction to ensure rapid recovery. in the contrary, secondary metabolism, cellular process, and transcription constitute a relatively small portion of the total, and most of these functions must depend upon new mrna syntheses. possible biological mechanisms of heterosis when displaying all degs into two-dimension space, we found a composite expression pattern that suggests multiple modes of action as they are classified into patterns: over-dominance, high-parent dominance, additivity, low-parent dominance and under-dominance. although up- and down-regulated degs are almost equal in numbers, up-regulated genes are dominant in the most abundant gene classes including cellular, metabolic, response to stress, embryo development, enzyme regulator activity, transcription regulator activity, and transporter activity. strengthened stress-tolerance system in f1 the majority of stress-tolerance genes in our dataset exhibited differential expressions among the hybrid triad, and especially showed up-regulated expressions in lyp. in the go term, five out of seven degs are in the category of response to stress and up-regulated in f in the gene families related to stress tolerance, % were differentially expressed whereas 89% of them are up-regulated together with % down-regulated and % additivity in f and as shown in table among 65% of the degs, 77% of them are up-regulated as opposed to 23% down-regulated. functionally, stress-tolerance genes are classified into two groups: protective and injury-induced genes. taking the water-deficit-tolerance related genes as examples, we know that protective genes, including chaperones, protease inhibitors, water-deficit induced membrane proteins, and water channel proteins, are counterparts of the protein-degrading mechanism, promoting survivability under water-deficit conditions. injury-induced genes include proteases and ubiquitin system, which are involved in the degradation of proteins that are denatured during cellular water loss. in lyp most of the protective genes are up-regulated as compared to the down-regulated injury-induced genes. protective genes that participate in other stress-related functions are also up-regulated in the f proteomic data also added footnotes to this notion, where the most functionally important and representative genes, including lea shsp, bowman-birk trypsin inhibitor, lipoprotein and lectin, thaumatin-like protein were found as up-regulated degs. in addition, our sage data also pointed out a universal strengthening in the ability of stress-tolerance in the f where heavy metal transport/detoxification protein, senescence-associated protein and rab protein were found up-regulated albeit in other tissues of lyp we validated some of these results with qrt-pcr. better preparation for growth recovery deg analysis suggested that the f appears better prepared for growth recovery as compared to its parental lines. first, genes participating in material and energy utilization are up-regulated and genes participating in anabolism are down-regulated. in general, the up-regulated degs were found in carbon metabolism with an exception of the oxidative phosphorylation of a nadp-dependent malic enzyme in energy metabolism. in contrast, genes in carbon, nitrogen fixation, and nucleotide metabolism are mostly down-regulated. in amino acid metabolism, we identified genes both up-regulated and down-regulated, but all genes related to amino acid biosynthesis are down-regulated. this indicated that lyp up-regulate material and energy generation and utilization to ensure its embryo to be active immediately when being re-hydrated as material anabolism is normally down-regulated. second, in the category of gip, genes involved in protein folding and associated processing are up-regulated and genes of ubiquitin mediated proteolysis and proteasome are down-regulated. genes involved in transcription are down-regulated but those involved in translation are not obviously biased, where both down- and up-regulated genes were found. third, in eip, genes in membrane transport and mapk signal transduction are up-regulated but calcium signalling pathway and phosphatidylinositol signalling systems are down-regulated; the result suggested that mapk signalling pathway may be the major signal pathway triggered as development starts after re-hydration. finally, degs involved in cellular process but not in the response system appeared down-regulated. some of these genes and their trends of expressions were found consistent in our sage and proteomic data. for example, the up-regulation of an important enolase was confirmed in the protein data. and the up-regulation of succinate dehydrogenase was validated based on the qrt-pcr test. another instance is β-amylase that is a key enzyme for starch hydrolysis, which is not only found up-regulated in the embryo but also in the leaf and panicle of lyp complementation and over-dominance we found that embryo-characteristic genes among degs are primarily up-regulated and showed high-parent dominance with the addition of over-dominance. the high-parent dominance genes often occur in gene families. for example, in the hsp family, the expression of lmw hsps has the tendency of pa64s, higher than that of 93- or expressed in an over-dominance fashion. in contrast, the expression level of classical hsps resembles that of 93- which is higher than that of pa64s, exhibiting over-dominance. in the lea and rab family, as well as other stress-tolerance genes, all the degs show high-parent dominance with expression levels similar to 93- more than two third of the degs differ between pa64s and lyp, whereas less than one third show differences between 93- and lyp. the result is consistent with the fact that lyp is more like 93- than pa64s in their phenotypic appearances. therefore, it appears that the hybrid vigour of lyp may be contributed by a composite of beneficial alleles from both parents, complementing the relatively weaker alleles in a global fashion, and over-dominance is an essential strategy for stress tolerance and metabolism. strengthening tissue-characteristic functions may contribute to heterosis analyses on degs have indicated that the mode of action for heterosis is most likely multi-fold and at multiple levels. if we have to point out a single mode of action, we believe that the strengthening of embryo-characteristic genes in lyp is most noticeable. for instance, the f lyp is predicted to have a significantly better stress-tolerance system than its parental cultivars, supported by up-regulated protective genes and down-regulated injury-induced genes. in addition, lyp keeps strong responses to environmental signals and potentially high-level activities after growth recovery. therefore, when seeds re-hydrate, the f embryo may perform better than its parental lines. this phenomenon can be cautiously generalized based on similar analyses among other tissues of the same hybrid line. for example, in leaf, it was detected that up-regulated genes in hybrid are dominated by degs related to enhancing carbon assimilation whereas those related to decomposition of carbohydrates are down-regulated, resulting in an increased efficiency of photosynthesis or energy production.  we reported a comparative transcriptomic analysis based on ests acquired from mature embryos of a rice hybrid and its parental lines. our study not only provided an initial characteristic description of expression profiles of rice embryos but also pointed out the enrichment of genes involved in stress-tolerance and preparation-for-recover. we also identified degs between f and its parental lines. functional analyses revealed clues for rice heterosis, such as multiple modes of actions and strengthening of embryo-characteristic function. genes involved in stress-tolerance and preparation-for-recovery may play key roles for rice heterosis in mature embryos. 
we carried out an est -based transcriptomic study, aiming at gaining molecular insights into embryonic development of a rice hybrid triad–an elite hybrid rice lyp and its parental lines –and possible relatedness to heterosis. we noticed that the high-abundance genes in mature rice embryos belong to two major functional categories, stress-tolerance and preparation-for-development, and we also identified differentially-expressed genes between lyp and its parental lines, representing typical expression patterns including over-dominance, high- and low-parent dominance, additivity, and under-dominance. our results suggested that massively strengthening tissue- characteristic functions may contribute to heterosis rather than a few simple mechanistic explanations at the individual gene level. in addition, the large collection of rice embryonic ests provides significant amount of data for future comparative analyses on plant development, especially for the important crops of the grass family. we generated high-quality ests from cdna libraries made from mature rice embryos. the mature embryo of rice is a synchronized and integrated tissue mass laying the foundation at molecular level for its growth, development, and differentiation toward a developing and ultimately a mature plant.
 the mature embryo of rice is a synchronized and integrated tissue mass laying the foundation at molecular level for its growth, development, and differentiation toward a developing and ultimately a mature plant. we carried out an est -based transcriptomic study, aiming at gaining molecular insights into embryonic development of a rice hybrid triad–an elite hybrid rice lyp and its parental lines –and possible relatedness to heterosis.  we generated high-quality ests from cdna libraries made from mature rice embryos. we classified these ests into unigenes and of them were annotated. we noticed that the high-abundance genes in mature rice embryos belong to two major functional categories, stress-tolerance and preparation-for-development, and we also identified differentially-expressed genes between lyp and its parental lines, representing typical expression patterns including over-dominance, high- and low-parent dominance, additivity, and under-dominance. in lyp the majority of embryo-associated genes were found not only abundantly and specifically enriched but also significantly up-regulated.  our results suggested that massively strengthening tissue- characteristic functions may contribute to heterosis rather than a few simple mechanistic explanations at the individual gene level. in addition, the large collection of rice embryonic ests provides significant amount of data for future comparative analyses on plant development, especially for the important crops of the grass family. 