interest has grown in recent years for biodiesel fuel based on animal fat or plant oils due to its relative compatibility with automotive diesel engines . there are many plant species whose derived oils have been used for biodiesel production , such as sunflower ( pessoa et al . , 2010 ) , peanut ( perez et al . , 2010 ) , castor bean ( godoy et al . , 2011 ) , physic nut ( kumar and sharma , 2008 ) and soybean ( pessoa et al . , 2010 ) , among others . soybean ( glycine max ( l. ) merrill ) is one of the world s main protein and oil sources , representing about 30% of the plant oil used for food , feed and industry . plant oils used for such application are basically composed of triacylglycerides , glycerol esthers and fatty acids . the term mono- or diglyceride refers to the number of acids , and in soybean oil the predominant one is oleic acid ( pessoa et al . , 2010 ) . ethanol production may derive from degradation of cell wall associated with cellulolytic processes on biomass . the cell wall of higher plants is the first structure that produces signaling molecules in response to biotic or abiotic stress signals . they are composed of polysaccharide , proteins and lignin , the last found in specific cell types ( cassab et al . , 1988 ) . lignin , after cellulose , is the second most abundant land polymer , essential for the structural integrity of cell wall and protection against pathogen action . in vascular plants , lignin also accounts for mechanical resistance and transport of nutrients , water and metabolites ( baucher et al . , 2003 ) . lignin is formed by amorphous , highly complex molecules , whose polymer is made of mainly aromatic phenylpropane units ( rowell et al . , 2005 ) . soybean cultivated in association with other crops has been analyzed regarding inputs , outputs , requirement in non - renewable resources , higher energy use efficiency and cost / benefit ratio ; although presenting high bioenergy output rates , soybean has been considered one of the most energy - intensive crops ( mandal et al . , 2002 ) . one possible way , which has already been considered in several countries , is to increase soybean energy output through enhancing biofuel production from its cellulosic biomass ( comis , 2006 ; siqueira et al . , 2008 ) or from industrial postprocessed residues ( sensz and kaynar , 2006 ) . according to recently increasing demand for renewable biofuels world - wide , the viability of such an alternative to petroleum depends on the verification of net energy gain , environmental benefits , economical competitiveness and large - scale production without reducing food supplies ( hill et al . , 2006 ) . until recent increases in petroleum prices , high production costs made biofuels unprofitable without subsidies . transportation biofuels such as synfuel hydrocarbons or cellulosic ethanol , if produced from low - input biomass grown on agriculturally marginal land or from waste biomass , could provide much greater supplies and environmental benefits than food - based biofuels ( hill et al . , 2006 ) . the increasing demand for food combined with the relatively recent , but also growing , need for biofuel derived from legume species has justified scientific and technological efforts aimed at improving knowledge on legume biology , especially the symbiotic nitrogen fixation and assimilation which has been a specific target in fabales research . such relevance has proven evident after finishing ( or nearly completing ) and publishing the genome sequences of the legume species lotus japonicus ( sato et al . , 2008 ) , medicago truncatula ( barrel medic ) and soybean ( glycine max ; schmutz et al . , 2010 ) . also of note , soybean transcriptome databases have been launched as comprehensive tools for gene expression studies on distinct cultivars and several experimental treatments , allowing in silico investigation of a wide range of molecular and physiological processes including growth and development as well as responses to biotic or abiotic stress factors . for example , the soyxpress database ( cheng and strmvik , 2008 ) harbors 380,095 soybean ests ( expressed sequence tags ) from conventional and transgenic cultivars , with associated gene ontology terms , metabolic pathways , swissprot identifiers and affymetrix microarray gene expression data from several experiments . more recently , a transcriptional atlas of soybean was published , where 69,145 putative genes are predicted , 46,430 of which with high confidence ( libault et al . , 2010 ) ; this database comprises cdna - derived illumina solexa sequences from 14 conditions or tissues , and genes identified in silico with significant differential expression between libraries were experimentally validated via rt - qpcr . in brazil , landmark research has been carried out in the brazilian soybean genome consortium ( genosoja project ) , established in 2008 and involving several research groups in soybean genomics , with the aim to investigate structural and functional aspects of soybean gene expression , under relevant agricultural stresses , mainly asian rust , nematodes , drought and nitrogen fixation ( abdelnoor et al . , 2009 ) . the genosoja database includes public access data from the soybean genome ( schmutz et al . , 2010 ) , so it constitutes a comprehensive molecular genetics base for in silico and physiological analyses whose applications may reach basic research and also strategies to circumvent agronomical constrains in the brazilian soybean industry ( nascimento et al . , 2012 ) . brazils sugarcane bioethanol produced in 2008 represented ca 37.3% of the worldwide production of 65.6 billion liters ( renewable fuels association ) . although small in scale by brazil standards , production of bioethanol from biomass sources other than sugarcane is in agreement with worldwide interest in bioethanol as an energy source and , in this scenario , potentially useful candidates include agroindustry by - products , such as soybean molasses and residual field biomass ( siqueira et al . , 2008 ) . the brazilian production of soybean ranks second worldwide , representing about 28% of global production ( soystat , 2009 ) . molasses is a co - product of protein - concentrate meal obtained after de - oiled soybean processing ; quantitatively , one ton of soybean yields about 716 kg of de - oiled meal , from which are extracted 522 kg of protein concentrate ( usually for the animal feed industry ) and 190.8 kg molasses . this , if submitted to fermentation with specific saccharomyces strains , will result in 18.4 kg ethanol plus 533.6 kg vinasse ( 80.5% moisture ; siqueira et al . indeed , the relatively well - established production of biodiesel from soybean oil can be summed up by bioethanol obtained from the soybean industry waste and/or cellulosic biomass . in this direction , soybean molecular biology and functional genomics may be applied to aid in breeding programs aiming to increase not only oil content in seeds , but also other polymeric components that may support technological strategies towards producing biofuels or bioenergy , such as cell wall polysaccharides and lignin . thus , the use of the genosoja database for in silico predictions on soybean transcriptome related to enzymes and proteins involved in cell wall , lignin and fatty acids metabolism is expected to result in valuable information over the regulation of the coding gene expression in different cultivars , organs and also under stress factor treatments . in this work , gene and variety - specific transcriptional activity genosoja , the soybean genome project database ( nascimento et al . , 2012 ) was searched by keyword in order to identify sequences related to genes encoding products whose putative annotation is associated with fatty acids , cell wall polysaccharides ( mostly cellulose or pectin ) or lignin metabolism . lignin was considered separately , despite being a cell wall component , because its structural features are different from polysaccharides . to be included in the analysis platform , ests assembled in contigs and singlets ( unigenes ) , components of gene models also had to present gene ontology ( go ) terms associated with fatty acids , cell wall polysaccharides or lignin metabolism . annotation by blastx against public protein databases ( nr ) and go terms from the genosoja database were considered for in silico analyses . retrieved est sequences were assessed and organized considering the standard genosoja nomenclature for reads and cdna libraries , derived from buds , cotyledons , endosperm , epicotyls , flowers , leaves , roots , shoots , seeds , pods or stems ( nascimento et al . , 2012 ) . ests with extremely similar or identical putative annotations were counted , and their frequency was normalized for total number of ests in each corresponding library ( expressed as a percentage ) , or for total number of ests derived from the same cultivar . the normalized frequencies of contrasting libraries were compared to infer in silico gene expression variation among cdna libraries and varieties , according to the genosoja database ( nascimento et al . , 2012 ) . transcripts most directly annotated as involved in metabolism of fatty acids , lignin and cell wall main polysaccharides were separately analyzed for expression pattern by hierarchical clustering using the epclust / ebi expression profiler ( brazma and vilo , 2000 ) online tools , following standard parameters . fatty acids and lignin data matrices were directly used in correlation measure - based ( uncentered ) and upgma ( average ) distances , while cell wall - related data matrix was log2 normalized prior to clustering . statistical significance of pairwise comparisons was assessed , when necessary , from non - normalized transcript frequencies in different libraries , based on the audic and claverie ( 1997 ) test , with significant p - value  0.05 . a keyword - based search on the nr / go annotated genosoja est database ( ests + genemodels ) resulted in a total subset of 4,094 transcript reads strictly assigned to bioenergy gene products . among these , , 2,111 to cell wall polysaccharides , with 827 related to cellulose , 1,202 to pectin and 82 not directly associated with pectin or cellulose ( npc ) , and finally , 49 reads were associated with lignin terms . a normalized distribution of such ests in soybean cdna libraries is summarized in table s1 ( supplementary material ) , grouped by reproductive or vegetative organ - derived libraries and described according to tissue or treatment type . general detection of fatty acids or cell - wall related transcripts in different soybean organs is shown in figure 2 , for distinct reproductive and vegetative ones . the frequency of reads putatively associated with fatty acids and cell wall components identified in cdna libraries constructed from vegetative or reproductive soybean organs samples was comparatively analyzed , resulting in a more comprehensive picture of transcriptional response associated with different growth or developmental stages and stressing factors ( figure 3 ) . the transcript frequencies from specific genes , significantly annotated as coding for proteins directly involved in fatty acids , cell wall polysaccharides or lignin metabolism , were normalized as a percentage of the total number of reads in the library , and used for hierarchical clustering of such gene expression patterns ( figure 4 ) , in an attempt to identify possible co - regulation . in a similar investigative approach , hierarchical clustering of mostly the same genes was then performed considering the soybean commercial varieties used for cdna library construction ( figure 5 ) ; here , differences in the expression of these genes were potentially associated with genotypes . out of the total number of soybean ests putatively found as related to metabolism regarding biofuels / bioenergy , 47.2% are involved in fatty acid biosynthesis / catabolism ( fa ) , as expected for an oil - producing legume . the remaining ests found were assigned to cell wall components , with pectin - related ( cwp ) ones being are more abundant ( 29.3% ) than the cellulose - related ( cwc ) , these comprising 20.2% . lignin - associated ( cwl ) transcript reads accounted for only 1.2% , which is proportionally less than the amount of reads annotated as linked to other cell wall polysaccharide biopolymers ( npcl ) which accounted for 2.0% . a direct analysis of the distribution of the so - called  bioenergy  genes dataset across the cdna libraries revealed that transcription related to fa metabolism is most represented in cotyledon and pod libraries ( table s1 and figure 3 ) , as also expected for a lipid storage - specific organ in this legume . this was the case of libraries c04 , c08 and c03 , constructed from immature or very young cotyledons , where fa - related transcripts reached up to 2.8% of the total library ( figure 3b ) . within cell wall - related transcripts , the cwp fraction presented a higher frequency in epicotyls of 2-week - old seedling ( almost 3% ) and seed coat ( ca 0.7% ) libraries , respectively ep1 and s06 ( figure 3 ) . on the other hand , cwc associated reads were more frequent in stems ( 0.5% ) and root nodules ( 0.3% ) , corresponding to st2 and r01 libraries . the reads putatively assigned to npcl comprised on average no more than 0.2% of each library : observed maximum frequency for npcl reads was 0.22% ( in seed coats , s06 ) and 0.18% ( in immature leaves , l04 ) . the most frequently transcribed cwl sequences were found in stems ( 0.6% , st2 ) and pods ( 0.36% , s02 ) . such general and putative transcriptional picture indicates , although theoretical and validation - dependent , some insights concerning gene activity were directly associated with the main components of soybean biomass , other than lipids and proteins . for example , genes involved in pectin and other hemicelluloses metabolism seem to be more active in epicotyl and seed coat , which is of great interest since the hulls are a soybean industry residue potentially useful for novel strategies in biofuel production ( wu et al . , 2010 ) . in addition , leaves have been shown to be a main site for expression for npcl - related genes , which shall be considered when leaf material represents a higher proportion of plant biomass used in bioenergy production , but in its composition different hemicelluloses and other cell wall components are significant . stem - specific gene expression showed higher correlation to cellulose / lignin biosynthesis and rearrangement than in any other soybean organ , which is quite expected according to its structural support function , and very important to any further energetic usage of soybean stem - rich biomass . noteworthy , root nodules also had high transcription level associated with cwc , which may point to unknown molecular mechanisms underlying the activities of cellulose metabolism that may be essential to nodule formation , including structural changes in host root cell wall ( sherrier et al . , 2005 ) and proper functioning , with effects on nitrogen fixation and consequently in general plant / biomass growth . indeed , the observed transcriptional preference for cwl sequences in pods suggests that lignin may be even more structurally relevant to the fruit , perhaps in transpiration avoidance and water content maintenance ( anterola and lewis , 2002 ) . in more detail , the reproductive organs of soybean presented higher transcriptional activity associated with fa and npcl , while vegetative organs showed more frequent transcription of genes putatively annotated to cw(p / c / l ) ( figure 2a ) . among reproduction structures , the flowers , pods , seeds and cotyledons were the site of highest expression of fa genes , and a more intense expression of cw(p / c ) and cwl genes was observed in seeds and pods , respectively ( figure 2b ) , which is relatively expected for lipid - storage organs . regarding structures in developmental vegetation , fa gene expression did not presente marked differences in frequency levels compared to cell wall - related genes , except in shoots ( figure 2c ) . in fact , n most vegetative organs , fa gene transcription was in similar ranges as that observed for cw(p / c / l ) , and in stems the lignin - associated transcripts level were even higher than fa genes ( figure 2c ) . results from hierarchical clustering analyses provided clues to the transcriptional regulation of few but key - step genes whose products are essential in fa and cw metabolism . as a caveat one must note that the genosoja database is not fully comprehensive , that this work included just est / cdna data only , and that equivalent libraries were not constructed from all soybean genotypes used . nevertheless , comparative analysis of gene expression patterns may help to identify co - regulated genes concerning biomass / bioenergy - related gene expression , as well as to determine similarity levels among transcriptional profiles of libraries ( i.e. , organs , tissues , treatments ) , or even among soybean varieties . for cell wall associated transcripts , only cwl presented a relatively clear profile , possibly due to the fact that only 2 genes could be assessed , as these were the only matches in the keyword search . actually , they are considered to code for unknown soybean proteins putatively assigned to the o - methyltransferase domain , thus referring to the lignin biosynthetic pathway . from the genosoja database , their clustered expression pattern in cdna libraries was very distinct ( figure 4c ) , with acu22737.1  isoform  transcripts being found in more similar levels in distinct organs , whereas acu21012.1 expression was higher , although not statistically significant , in etiolated hypocotyls ( h05 ) and germinating shoots ( sh2 ) . nevertheless , acu22737.1 appears to be significantly more transcribed than acu21012.1 in roots ( p = 0.0069 , r06 ) , seeds with globular - stage embryo ( p = 0.0436 , s12 ) and seedlings ( p = 0.0078 , s10 ) . furthermore , it appears to be more , though not statistically significant , expressed in phytophthora sojae - infected hypocotyls ( h04 ) . hence , it is inferred that these lignin - associated soybean genes may have rather different regulation pathways triggered by light ( su et al . , 2005 ) , germination , organ - specific and defense - related cell wall component cross - linking . the other categories of cw genes did not present a highly informative clustering of expression patterns , mostly because of the generally low transcription level after normalization ( figure 4a , b ) . however , some patterns emerged , such as the higher and statistically significant expression level of the expansin coding gene in the seedling epicotyl ( ep1 ; 0.00027 < p < 0.03195 ) library compared to other libraries ( figure 4a ) . expansins are known to be essential to cell wall loosening in plant structures in elongation stages ( lee et al . , 2003 ) , especially in stems . herein , the expansin gene was detected to be significantly more expressed in etiolated seedling hypocotyls than in light - grown whole seedlings ( h05 vs. s10 , p = 0.00596 ) . in addition , it was proportionally more expressed when cotyledons were excluded from the seedling sample , since a significantly higher transcription was observed in seedlings minus cotyledons when compared to whole seedlings ( s11 vs. s10 , p = 0.03136 ) . grouping of the libraries based on expression profiles similarity was not as evident for cw genes as observed for fa genes ( figure 4b ) . here , the several soybean fatty acid desaturases ( fads ) were very similar with respect to frequency variation across the libraries , inferring co - regulation mechanisms for this group of key enzymes in fatty acid metabolism and lipid content manipulation ( pham et al . , 2010 ) . more specifically , transcripts for a putative microsomal 6-fad were represented in almost every library , being significantly higher ( p10 ) in those from cotyledons , seeds , roots , and floral meristem . it was the only fad - associated transcript detected in phytophthora sojae - infected hypocotyls ( h04 ) , indicating putative specific association to this patosystem . in seedlings ( s09 ) , it also was the most frequent significant fad - associated transcript differentially detected ( p = 0.0028 ) . in contrast , transcription associated with chloroplast 3-fad gene was higher in floral meristematic apices ( f04 , p10 ) , and expression levels detected for fad 8 were significantly and preferentially higher in seeds with globular - stage embryos ( s12 , p10 ) . the expression of the two putative lignin - related o - methyltransferases was profiled by hierarchical clustering among soybean varieties / cultivars used for library construction . this showed that acu22737.1 was more transcribed in almost all genotypes than acu21012.1 , reaching significance for differential expression only in asgrow a3237 ( p = 0.0436 ) . this did not apply to corolla , pi567374 , delsoy 5710 , ogden and minsoy x noir ri , since in these genotypes , expression levels were practically undetected for both genes . in williams genotypes both genes presented intermediate to low transcript levels ( figure 5b ) . varieties could be grouped following fa gene expression clustering . transcripts for the microsomal 6-fad were the most frequent ones in almost all genotypes , especially in l82 2024 ( figure 5a ) , except for as - grow a3237 , where they presented one of the lowest frequencies , and fad 8 transcripts were the most frequent among fad - coding genes ( p10 ) . concerning cw genes , expression clustering cellulose synthase - coding transcripts from cesa1 and cesa2 showed very distinct expression patterns in delsoy 5710 and jack genotypes , while in the corolla variety both genes seemed to be more intensely transcribed . the expression difference in these genes was observed to be significant for asgrow a3237 ( p = 0.0008 ) , clark ( p = 0.0175 ) , harosoy / harosoy 63 ( p = 0.0333 ) , williams / williams 82 ( p = 0.0020 and raiden ( p = 0.0001 ) . this type of information , despite being specific and not comprehensive for the several varieties tested so far , should be useful for soybean breeding programs focusing on new strategies , by conventional crosses / selection and by transgeny , to obtain varieties with application focus on biomass , bioenergy and cellulosic - based biofuels production ( pham et al . , 2010 ) . further studies on linking the expression data of these genes to fa and polysaccharide composition of selected parental varieties should prove to be very helpful . in conclusion , this work aimed at providing initial information from the genosoja database regarding gene expression directly associated with potential uses of soybean biomass and/or waste for bioenergy - producing technologies fa , cw polysaccharides and lignin metabolism and physiological properties in planta are ultimately governed by genomic features interacting with environmental factors , and are a first line of research focus towards improving soybean as feedstock for bioenergy , biodiesel , as well as for bioethanol and many other by - products and subproducts that could soon be commercially available due to intensive lignocellulosic chemistry and technologies . the soybean industry may soon benefit from investments in oil - protein - cellulose - lignin - biofuel from biomass and from conventional waste , not usually directed toward profitable and ecologically suitable destinations . finally , the highly relevant position of brazil as a soybean producer should further the development of this commodity as an additional bioenergy source in this country . the following online material is available for this article : table s1 - frequency distribution of reads ( ests ) from cdna libraries .
increasing efforts to preserve environmental resources have included the development of more efficient technologies to produce energy from renewable sources such as plant biomass , notably through biofuels and cellulosic residues . 
 the relevance of the soybean industry is due mostly to oil and protein production which , although interdependent , results from coordinated gene expression in primary metabolism . concerning biomass and biodiesel , a comprehensive analysis of gene regulation associated with cell wall components ( as polysaccharides and lignin ) and fatty acid metabolism may be very useful for finding new strategies in soybean breeding for the expanding bioenergy industry . searching the genosoja transcriptional database for enzymes and proteins directly involved in cell wall , lignin and fatty acid metabolism provides gene expression datasets with frequency distribution and specific regulation that is shared among several cultivars and organs , and also in response to different biotic / abiotic stress treatments 
 . these results may be useful as a starting point to depict the genosoja database regarding gene expression directly associated with potential applications of soybean biomass and/or residues for bioenergy - producing technologies .