BACKGROUND
flavonoids represent a class of plant secondary metabolites that have evolved a variety of physiological functions including pigmentation, pathogen defense, and uv protection  <cit> .
additionally, metabolic engineering of flavonoids has become an important target for plant biotechnology, as flavonoids provide health benefits to foods, favorable agronomic traits to crops, and may be used in the future to color commercial transgenic materials such as grains to facilitate their identification and monitoring  <cit> .
commercial soybean  merr.)
has a yellow grain.
however rare spontaneous mutants exist that have black  or brown  seed coat  color phenotypes.
black  and brown  soybean seed coats contain proanthocyanidins  but differ in the presence/absence of anthocyanins  <cit> .
a goal for biosafety is to engineer a novel red seed coat color as a marker for transgenic soybean grains to facilitate their identification  <cit> , and could potentially be achieved by the suppression of anthocyanin-specific genes that are overexpressed in the black soybean seed coat.
however the genes have not yet been identified.
six genetic loci   <cit>  identified by classical genetics control flavonoid-based seed coat color in soybean.
the i locus controls the presence or absence and spatial distribution of flavonoid pigments and has four alleles ; i gives completely non-pigmented seed coat, ii restricts pigment to the hilum and ik to a saddle-shaped region, whereas the i allele results in a fully pigmented seed coat  <cit> .
the recessive i allele results from spontaneous deletion of chs <dig> or chs <dig> promoter sequences and results in the abolishment of a posttranscriptional rna silencing mechanism that results in the increased accumulation of chalcone synthase  transcripts in the seed coat  <cit> .
the t locus is a pleoiotropic locus that controls the type and abundance of flavonoid pigments in the seed coat in addition to other traits such as seed coat cracking and trichome pigmentation  <cit> .
genetic polymorphisms that affect the expression or function of the flavonoid 3'-hydroxylase gene  have been shown to co-segregate with recessive t alleles  <cit> .
the w <dig> locus controls flower color and affects seed color only in an irt background; where w <dig> and w <dig> alleles give imperfect black and buff seed coat colors, respectively  <cit> .
the w <dig> allele for purple flower color was shown to code flavonoid 3',5'-hydroxylase , as a 65-bp insertion in the gene  co-segregated with white flower color   <cit> .
the wp locus was suggested to code the flavonone 3-hydroxylase gene  by microarray analysis as high levels of f3h <dig> transcripts co-segregated with purple flower color , and low levels with pink  flowers  <cit> .
in the seed coat, recessive wp resulted in a change from black  to a lighter grayish  color  <cit> .
the o locus affects the color of brown  seed coats, with the recessive o allele giving a red-brown  phenotype  <cit> .
the o locus has been suggested to code the proanthocyanidin  biosynthesis gene anthocyanidin reductase , as the gene was located between markers that flank the o locus on the soybean physical map  <cit> .
however molecular genetics analyses have not yet demonstrated the identity of the o locus gene.
finally, the r locus controls the presence  or absence  of anthocyanins in black  or brown  seed coats, respectively  <cit> .
despite the identification of several pigment genes from soybean, only transcripts for anthocyanidin synthase  genes  have been shown to be overexpressed in the black  seed coat compared to the brown  nearly-isogenic line by northern blot  <cit> .
the upregulation of several ans genes suggests the r locus to code a regulatory factor, and raises the possibility that other isogenes for anthocyanin biosynthesis may be upregulated.
recently, a cdna coding the udp-glycose:flavonoid-3-o-glycosyltransferase  gene  was isolated from the black  seed coat and shown to function in anthocyanin biosynthesis in vitro and by complementation of a gene mutation in arabidopsis  <cit> .
however ugt78k <dig> expressions have not been investigated in relation to seed coat color.
the soybean genome sequence glyma <dig> was predicted to code  <dig>  protein-coding genes with nearly 75% of the genes present in multiple copies  <cit> .
this may suggest a relatively high frequency of functional redundancy and increased difficulty in identifying soybean genes by traditional approaches.
however, using transcriptome analysis tools such as the soybean genechip equipped with  <dig>  probe sets in combination with broad-coverage metabolite analysis methodologies such as lc-ms/ms, gene functions could potentially be efficiently predicted.
the combined analysis of transcriptome and metabolite data has been shown to be a powerful approach for the functional identification of unknown genes  <cit> .
metabolite differences caused by the overexpression of a transcription factor, the exposure to a nutritional stress, and by species differences have been correlated with differences in transcriptome profiles to successfully predict the functions of unknown genes in flavonoid, glucosinolate, and alkaloid biosyntheses  <cit> .
in the present study we have employed targeted and non-targeted metabolite analysis methodologies and have demonstrated that black  and brown  nearly-isogenic soybean seed coats do not just differ in the presence/absence of anthocyanins, and have extensive differences in procyanidin, flavonoid and phenylpropanoid compositions.
the underlying differences in gene expressions were then identified by microarray analysis, and the putative functions of  <dig> unknown genes were assigned by comparison to metabolite data.
from the set of differentially regulated genes, two putative late-stage anthocyanin genes were selected and the functions of their coded enzymes were validated in vitro.
in addition, a set of gene candidates potentially coded by the r locus have been provided.
RESULTS
qrt-pcr indicates differential expressions of anthocyanin and proanthocyanidin genes in nearly-isogenic black  and brown  soybean seed coats
among the flavonoid genes identified from soybean seed coats, only ans transcripts were found to be overexpressed in the seed coat of black  soybean compared to the brown  isoline, however no rflp difference was observed when probing ans genes  <cit> .
this suggests that ans overexpression is not the only molecular difference between black  and brown  seed coats.
to examine whether other anthocyanin genes are upregulated, transcript profiles of the recently identified ugt78k <dig>  <cit>  and the dfr <dig> gene  were measured from seed coat cdna by quantitative rt-pcr  using ans2/ans <dig> transcripts  as a positive control.
phosphenol pyruvate carboxylase   was used as an endogenous reference for normalization of the qrt-pcr measurements of target genes across seed coat samples, as pepc is expressed at similar levels in a wide range of soybean tissues and has been used previously as a reference for gene expressions during soybean seed coat development  <cit> .
qrt-pcr demonstrated ugt78k <dig> to be significantly overexpressed in the black  seed coat at the transition stage  and early maturation stage  of seed development compared to the brown  isoline, and to be almost undetectable at earlier stages .
stereomicroscopy and photospectroscopic measurements confirmed these developmental stages to coincide with the stages of anthocyanin biosynthesis in the black  seed coat .
in contrast to ugt78k <dig>  dfr <dig> was not differentially expressed at the stages of anthocyanin biosynthesis .
ans2/ans <dig> mrnas were confirmed to be overexpressed in the black  seed coat at most stages  as shown previously  <cit> .
the seed coat palisade cells of black  soybean are unusual compared to those of other model plants used for the study of anthocyanin biosynthesis  because black  soybean palisade cells synthesize both anthocyanins and proanthocyanidins .
anr and lar enzymes have been identified to catalyze the conversion of cyanidin to epicatechin and leucocyanidin to catechin, respectively, which are the putative initial biochemical steps of pa biosynthesis  <cit> .
to investigate how a seed tissue may coordinate the biosynthesis of these parallel pigment pathways, the expressions of putative pa genes anr <dig>  and lar <dig>  were examined in relation to anthocyanin gene expressions.
qrt-pcr demonstrated anr <dig> expressions to decrease throughout seed coat development in both genotypes and to be almost undetectable by the stages of ugt78k <dig> overexpressions and anthocyanin biosynthesis .
by contrast, lar <dig> expressions were downregulated at the stages of anthocyanin biosynthesis in the black  seed coat .
in conclusion, these results show that the recently identified ugt78k <dig> is upregulated in the black  seed coat, in addition to ans2/ans <dig>  and that there is a simultaneous downregulation of the pa gene lar <dig> at the stages of anthocyanin biosynthesis.
these results demonstrate that expression differences between black  and brown  seed coats are not limited to anthocyanin genes, and may suggest extensive differences in gene expressions and phenolic compositions.
combined analysis of targeted and non-targeted methodologies indicate overaccumulation of anthocyanins, altered procyanidin, and reduced flavonol, benzoic acid, and isoflavone compositions in the seed coat of black  soybean
the structures of anthocyanins from only a few  black soybean varieties have been described  <cit>  and information regarding other phenolic compositions may be limited to proanthocyanidins , where acid-catalyzed hydrolysis of black  and brown  seed coats showed the pas to be 3',4'-hydroxylated  <cit> .
a more extensive analysis of phenolic composition differences between black  and brown  seed coats could greatly improve our understanding of seed pigmentation, and may suggest gene activities that function in pigment biosynthesis.
for these reasons, black  and brown  clark seed coat metabolites were extracted at the transition stage  of seed development with multiple solvents and analyzed by targeted high performance liquid chromatography-diode array detection  and non-targeted hplc-tandem mass spectrometry .
to ensure seed coats were at the same stage of development, the days post anthesis, pod length, pod color, embryo morphology, and transcript levels of the developmental marker gene gm-r1083- <dig>  a putative cutin biosynthesis gene  <cit> , and the metabolism gene dfr <dig> were ensured to be equivalent between black  and brown  tissues .
for a targeted analysis of anthocyanins, black  and brown  clark seed coats were extracted with acidified aqueous methanol and metabolites were identified using hplc-dad by measuring the absorbance at  <dig> nm in comparison to authentic standards.
ten cyanic pigments were identified, the most abundant were the anthocyanins cyanidin-3-o-glucoside , cyanidin-3-o-galactoside  and petunidin-3-o-glucoside  with lesser amounts of delphinidin-, pelargonidin-, and peonidin-3-o-glucosides , the aglycone cyanidin , the 3-deoxyanthocyanidin diosmetinidin , and the two unidentified anthocyanin-like compounds  .
to our knowledge this is the first report of a 3-deoxyanthocyanidin from the soybean seed coat.
the structures of all identified anthocyanins are represented in figure  <dig>  by contrast, cyanic pigments were not detected from the brown  seed coat at any stage of seed development .
*for chemical structures see fig.
<dig>
to compare the structural features of pas, black  and brown  seed coats were extracted with 70% acetone and subjected to phloroglucinol cleavage analysis  <cit> .
pa free monomers, polymer subunit compositions, and mean degree of polymerization  did not differ significantly between nearly-isogenic black  and brown  seed coats.
the soluble pa mdp was  <dig>  and the subunit and monomer compositions consisted mainly of epicatechin with minor amounts of unidentified compounds .
reaction with dmaca reagent demonstrated similar amounts of soluble pas from both black  and brown  seed coats  .
further extraction of seed coat residues at 50°c in the presence of acid and phloroglucinol yielded only low levels of epicatechin-phloroglucinol adduct, epicatechin terminal units, and several unidentified compounds , and acid catalyzed cleavage of seed coat residues at 95°c identified relatively high, but equivalent amounts of 3',4'-hydroxylated solvent insoluble pas .
for a non-targeted analysis of soluble phenolic metabolites, nearly isogenic black  and brown  clark seed coats were extracted with acidified aqueous methanol and analyzed by hplc-ms/ms over a mass range of  <dig> amu to  <dig> amu.
identities were assigned to peaks by comparison of retention times and mass fragmentation patterns to authentic standards.
peaks with no corresponding authentic standards were identified by mass fragmentation patterns in comparison to the literature.
a total of  <dig> peaks were detected, of which  <dig> peaks were assigned to  <dig> compounds .
twenty-eight compounds were detected from the black  seed coat ; consisting mainly of unidentified compounds  and anthocyanins , followed by procyanidins .
by contrast,  <dig> compounds were detected from the brown  seed coat ; consisting mainly of unidentified compounds , followed by flavonols  and procyanidins .
the majority of compounds  were exclusive to the black  or brown  seed coat, suggesting extensively different biochemical specializations of these tissues.
the distribution of compound classes from black  and brown  clark seed coats is illustrated in figure  <dig>
*based on co-chromatographic and ms fragamentation comparison to an authentic standard.
ablack soybean , brown soybean , both .
btrace amounts in irt.
the  <dig> compounds exclusive to the black  seed coat were anthocyanins , procyanidins , and unknown compounds  .
hplc-ms/ms  confirmed the identity of anthocyanins found by hplc-dad .
procyanidin tetramers and larger oligomers were not detected as they exceeded the mass range  analyzed by the hplc-ms/ms experiments, however  <dig> pa trimers  and the a-type procyanidin dimer were exclusive to the black  seed coat .
the  <dig> compounds exclusive to the brown  seed coat consisted of  <dig> unknown compounds ,  <dig> flavonols ,  <dig> benzoic acids , and the isoflavone daidzein o-hexoside malonylated .
taken together, our metabolite analysis experiments show that nearly-isogenic black  and brown  clark seed coats have compositional differences not limited to the presence or absence of anthocyanins; with specific flavonol, isoflavone, and benzoic acids exclusive to the brown  seed coat and anthocyanins and unique procyanidin oligomers exclusive to the black  seed coat.
these results may suggest extensive differences in the underlying gene expressions.
microarray analysis indicates concomitant upregulation of specific phenylpropanoid, flavonoid, and anthocyanin isogenes and downregulation of other flavonoid, benzoic acid, and isoflavone isogenes in the black  seed coat transcriptome
nearly 75% of genes in the soybean genome are present in multiple copies  <cit> , and previously only transcripts for ans isogenes were found to be overexpressed in the black  seed coat compared to the nearly-isogenic brown  tissue  <cit> .
for a global analysis of isogene expressions, black  and brown  clark seed coats were dissected at the transition stage of seed development  for microarray analysis using the affymetrix soybean genechip.
seed coats were ensured to be at the same stage of development as described above for the metabolite analyses .
of the  <dig>  probe sets present on the soybean genechip,  <dig> were found to be upregulated more than 2-fold in black  soybean .
classification using the gene-bins ontology tool  demonstrated the majority of upregulated genes to be "unclassified without homology"  or "unclassified with homology" , followed by "biosynthesis of secondary metabolites"  .
this latter class consisted exclusively of putative  phenylpropanoid, flavonoid, and anthocyanin isogenes.
interestingly, putative isogenes of the phenylpropanoid pathway , the flavonoid pathway , and the late-stage anthocyanin pathway  were all upregulated more than 2-fold in the black  seed coat, more than 234-fold in the case of the gst <dig> probe set .
aexpression values were obtained using rma .
bsee table s <dig> for the complete list of upregulated genes.
*previously characterized flavonoid biosynthesis genes.
a blastp search of the soybean genome sequence glyma <dig>  using the conserved plant secondary product glycosyltransferase signature sequence   <cit>  identified  <dig> annotated family  <dig> glycosyltransferases .
of the  <dig> ugts, only  <dig> were found to be upregulated more than 2-fold from the black  seed coat; the recently identified uf3gt gene ugt78k <dig>  <cit>  was upregulated more than  <dig> -fold and a second uncharacterized glycosyltransferase, which we named ugt78k <dig> in accordance with ugt nomenclature  <cit> , was upregulated more than 3-fold .
a search of the soybean genome sequence with the conserved omt-domain sequence  and the ontology 'o-methyltransferase', identified  <dig> annotated omts .
of the  <dig> omt genes, only probe sets for omt-like and omt <dig> were upregulated in the black  seed coat, more than  <dig> -fold and  <dig> -fold, respectively .
a total of  <dig> probe sets were downregulated more than 2-fold in the black  seed coat ; among these,  <dig> probe sets by more than 10-fold,  <dig> probe sets by more than 3-fold, with the remainder between 2- and 3-fold .
classification of down-regulated genes using the gene-bins ontology tool demonstrated the majority to be "unclassified without homology"  or "unclassified with homology" , followed by "biosynthesis of secondary metabolites"  .
the latter class consisted primarily of characterized and putative flavonoid isogenes.
isogenes for common flavonoid biosynthesis , pa biosynthesis , and isoflavone biosynthesis  were all down-regulated more than 2-fold in the black  seed coat, more than 35-fold in the case of the chs <dig> .
the flavonoid genes chs <dig> and f3'h were down-regulated more than 21-fold and  <dig> -fold, respectively, the putative isoflavone gene ifr1-like by more than  <dig> -fold, and the putative pa gene lar <dig> by more than  <dig> -fold .
aexpression values were obtained using rma .
bsee table s <dig> for the complete list of up-regulated genes.
*previously characterized flavonoid biosynthesis genes.
of the differentially regulated probe sets,  <dig> represented transcription factor genes,  <dig> were upregulated and  <dig> downregulated , however none of these genes clustered with known flavonoid transcription factor orthologs by phylogenetic analysis .
a previous study mapped the r locus between markers a668_ <dig> and k387_ <dig> on mlg k   <cit> .
nineteen differentially-regulated probe sets were associated with genes located on chromosome  <dig> of the soybean genome sequence glyma <dig> .
interestingly, all genes were located in a  <dig>  mb region of the distal arm of chromosome  <dig>  and only the downregulated ap2/erf  and serine carboxypeptidase-like protein  and the upregulated polyamine oxidase  were located between markers  that flank the putative position of the r locus  .
to validate the microarray results,  <dig> differentially-regulated genes were selected and their relative expression profiles were measured by semi-quantitative rt-pcr  using black  and brown  seed coat cdna as templates.
phosphenol pyruvate carboxylase  was used as the endogenous control to ensure equal loading and the flavonoid genes chs7/chs <dig> and the putative pa gene anr <dig> were used as negative controls as they were not found to be differentially expressed in the microarray analysis.
semi-qrt-pcr confirmed the differential-expressions of all  <dig> genes and demonstrated similar expressions for chs7/chs <dig> and anr <dig> .
furthermore, as the upregulated probe set gmaaffx.
<dig> .s <dig> did not distinguish between chs <dig> and chs <dig> expressions, semi-qrt-pcr was performed with gene specific primers to show the chs <dig> gene, and not the chs <dig> gene to be upregulated in the black  seed coat .
in summary, these results show that numerous specific anthocyanin, flavonoid and phenylpropanoid isogenes are upregulated and other flavonoid, benzoic acid, and isoflavone isogenes are downregulated in the black  seed coat transcriptome relative to that of the nearly-isogenic brown  tissue.
putative assignment of function to genes differentially-regulated between black  and brown  seed coats
gene functions can be successfully predicted based on a combined analysis of transcriptome and metabolite data  <cit> .
we compared differences in gene expressions determined by microarray to differences in metabolite compositions to assign putative functions to genes from the black  and brown  seed coats .
in addition to  <dig> previously characterized soybean flavonoid genes, several uncharacterized genes belonging to large multi-gene families with possible roles in anthocyanin biosynthesis and transport were upregulated; including one glycosyltransferase , two o-methyltransferases  and two glutathione s-transferases  .
considering the accumulation of metabolites with specific molecular structures in the black  seed coat, putative functions of the differentially-expressed genes were assigned to specific structural modifications and transport functions .
anthocyanins in the seed coat of black  soybean are glycosylated at the 3-position of the c-ring .
the glycosyltransferase ugt78k <dig> is assigned to code a udp-glycose:flavonoid 3-o-glycosyltransferase  protein  and may have functional redundancies in anthocyanin biosynthesis with the previously characterized ugt78k <dig>  <cit> , which was also upregulated in the black  seed coat .
two anthocyanins  and one deoxyanthocyanidin  from the black  seed coat are methylated at the 3-position of the b-ring  .
the two upregulated o-methyltransferases were assigned to code anthocyanin 3'-o-methyltransferase  proteins  and may have redundant function in anthocyanin biosynthesis this tissue.
ugt78k <dig> and omt <dig> code udp-glycose:flavonoid 3-o-glycosyltransferase and anthocyanin 3'-o-methyltransferase proteins, respectively
reducing the expressions of late-stage anthocyanin genes by rna interference  in the seed coat of black soybean may be an effective strategy to engineer seed coat color while minimizing unintended effects on other flavonoid subpathways.
however, functional redundancies may require that multiple genes be silenced in order to reduce enzyme activity enough to reduce pigment levels.
two glycosyltransferases  were upregulated in the black  seed coat relative to the nearly-isogenic brown  tissue  and the upregulation paralleled the accumulation of anthocyanins with 3-o-glycosylated structures , suggesting the function of these genes as udp-glycose:flavonoid 3-o-glycosyltransferases .
we have characterized ugt78k <dig> previously and shown it to possess uf3gt activity towards anthocyanidins in vitro and in vivo  <cit> .
to determine the catalytic function of ugt78k <dig> , the orf was cloned into an n-terminal hexahistidine fusion tag vector and expressed in e. coli for analysis of the coded recombinant protein in vitro.
the recombinant protein  was purified by ion-metal-affinity chromatography  had an apparent mass of  <dig>  kda that matched well with the calculated mass of the native ugt78k <dig> protein  in addition to the  <dig>  kda n-terminal hexahistidine fusion tag encoded by the pet-14b vector .
in assays containing udp-glucose as the sugar donor and cyanidin as the acceptor, the recombinant enzyme transferred glucose to the 3-position of cyanidin to form cyanidin-3-o-glucoside , whereas the boiled enzyme and bacteria expressing the corresponding empty vector could not catalyze this reaction .
similarly, in assays containing udp-galactose as the sugar donor and cyanidin as the acceptor, the recombinant enzyme transferred galactose to the 3-position of cyanidin to form cyanidin-3-o-galactoside .
cyanidin-3-o-glucoside and cyanidin-3-o-galactoside from recombinant enzyme assays were identified by hplc-dad in comparison to the authentic standards, and confirmed by hplc-ms/ms with the characteristic parent ions  <dig>  + <dig> and the ms/ms fragment  <dig>  + <dig> corresponding to the loss of glucose .
as we have performed the kinetics analysis for recombinant ugt78k <dig>  we were able to employ the same experimental conditions to test the kinetics of ugt78k <dig>  interestingly, ugt78k <dig> converted cyanidin-3-o-glucoside with greater velocity than ugt78k <dig> and higher concentrations of cyanidin were required to inhibit its activity  despite the enzymes having 93% amino acid similarity .
methylation of the b-ring of anthocyanins has a reddening effect on flower color  and thus engineering the expression of 3'-o-methyltransferases could change the redness of seed color.
however, despite the identification of several flavonoid omts from soybean, none have been shown to accept anthocyanins as substrates.
our microarray analysis identified two o-methyltransferases  to be upregulated in the black  seed coat  and the upregulation paralleled the accumulation of 3'-o-methylated anthocyanins in this tissue , suggesting the function of these genes as anthocyanin 3'-o-methyltransferases .
following the approach described above, the orf of omt <dig>  was cloned for expression and functional characterization of the corresponding recombinant protein in vitro.
the purified recombinant protein had an apparent mass of  <dig>  kda that matched well with the calculated mass  in addition to the  <dig>  kda n-terminal hexahistidine fusion tag encoded by the pet-14b vector .
in assays containing s-adenosyl-l-methionine  as the methyl donor and cyanidin-3-o-glucoside as the acceptor, the recombinant enzyme transferred a methyl group to the 3'-position of the acceptor to form peonidin-3-o-glucoside , whereas the boiled enzyme and bacteria expressing the corresponding empty vector could not catalyze this reaction .
peonidin-3-o-glucoside from recombinant enzyme assays was identified by hplc-dad in comparison to the authentic standard, and confirmed by hplc-ms/ms with the characteristic parent ion  <dig>  + <dig> and the ms/ms fragment  <dig>  + <dig> corresponding to the loss of glucose .
to further investigate the function these genes, ugt78k <dig> and omt <dig> expression profiles were measured from seed coat cdna throughout black  and brown  soybean seed development by quantitative rt-pcr   and expressions were compared to anthocyanin accumulation profiles throughout seed development .
similar to ugt78k <dig>  ugt78k <dig> and omt <dig> expressions peaked at the transition and early maturation stages of seed development  in the black  seed coat , the stages at which anthocyanins accumulate , and were low or almost undetectable at early stages of seed development in the black  seed coat and at all stages in the brown  seed coat.
together the qrt-pcr and recombinant enzyme assays suggest that ugt78k <dig> and omt <dig> code uf3gt and aomt proteins, respectively, that function in anthocyanin pigment biosynthesis in the seed of black  soybean.
discussion
the seed coat compositions of black  and brown  soybean  were known to differ only by the presence/absence of anthocyanins, however our metabolite analysis demonstrated these nearly-isogenic tissues to have extensive differences in anthocyanin, proanthocyanidin , flavonoid, and phenylpropanoid content .
of the  <dig> compounds detected,  <dig> compounds  were exclusive to either the black  or the brown  seed coat.
analysis of the distributions of compound classes between seed coats demonstrated the black  to a have a greater proportion of anthocyanins and pas, and the brown  to have a greater proportion of flavonol, benzoic acid, isoflavone, and unknown metabolite compositions .
metabolites exclusive to the black  seed coat included pa trimers  and an a-type procyanidin dimer in addition to various anthocyanins .
the anthocyanins identified from variety clark were mainly similar to those reported previously from various korean varieties  <cit>  however clark was the first shown to contain a 3-deoxyanthocyanidin  in the seed coat .
3-deoxyanthocyanidins are relatively rare intensely coloured pigments found in sorghum bicolour in response to fungal attack  <cit> , and from the cotyledons of yellow soybean in response to uv-c treatment  <cit> .
the biosynthesis of 3-deoxyanthocyanidins from flavonones has been shown to be a minor activity of a dfr isogene in sorghum  <cit> .
it would be interesting to determine whether the dfr gene that was highly overexpressed in the black clark seed coat  had similar catalytic activity, and whether the korean varieties contain genetic alterations related to dfr <dig>
analysis of the black  seed coat transcriptome compared to that of the nearly-isogenic brown  tissue identified  <dig> probe sets to be upregulated .
this is almost double the number of probe sets upregulated by overexpression of the myb transcription factor pap <dig> in arabidopsis   <cit> , similar to the number upregulated by overexpression of the anthocyanin factor lap <dig> in m. truncatula , and less than half the number upregulated by overexpression of lap <dig> in m. sativa   <cit> .
the higher numbers of genes upregulated in anthocyanin-overaccumulating tissues of black soybean, m. truncatula and m. sativa likely reflect the fact that legumes have undergone genome duplication and that genes with multiple copies are frequently retained  <cit> .
our data supported this hypothesis as duplicate copies of several genes were upregulated in the black  seed coat .
functional redundancies can complicate gene identification by traditional approaches.
previous to the present study, genes for only  <dig> flavonoid enzymes  were identified to be involved in pigment biosynthesis in the soybean seed coat  <cit> .
in the present study,  <dig> isogenes for  <dig> proteins were identified to have putative roles in flavonoid biosynthesis in black  or brown  seed coats using a combined analysis of microarray and metabolite data .
transcriptome analysis has previously demonstrated a critical role of chs <dig> and chs <dig> genes for isoflavonoid biosynthesis in developing embryos of soybean  <cit> .
however, our microarray and semi-quantitative rt-pcr data demonstrated that chs <dig> and chs <dig> were not differentially regulated in black  and brown  seed coats .
interestingly, it was chs <dig> that was upregulated and chs <dig> and chs <dig> that were downregulatedin the black  seed coat .
as chs <dig> is upregulated with anthocyanin genes, it likely has a role in anthocyanin biosynthesis in this tissue.
consistent with this, spontaneous mutations of the dominant ii allele to the recessive i allele have been shown to affect the promoter region of the chs <dig> gene  <cit> .
by contrast, the downregulation of chs <dig> and chs <dig> may suggest that they have a role in the parallel downregulated biochemical pathways, such as flavonol and isoflavonoid biosynthesis, as these metabolites, and transcript levels of genes for their biosynthesis, were reduced or not present in the black  seed coat .
these results emphasize the complexity of isogene expressions that exist to achieve a single enzyme function in soybean.
microarray analysis identified differentially regulated probe sets for only  <dig> gene functions  that have been previously shown to be involved in pa biosynthesis and transport.
recombinant lar enzymes from several legume species have been shown to convert leucocyanidin to -catechin in vitro  <cit> .
however, the in vivo role of lar in pa biosynthesis remains questionable as endogenous expression has been shown not to correlate with pa levels in m. truncatula and heterologous expression has failed to increase pa accumulations  <cit> .
similarly, our metabolite data showed that soybean pas lack catechin subunits  and that lar <dig> expressions  do not parallel pa accumulations during seed development .
interestingly,  <dig> pas were found to be exclusive to the black  seed coat  however the total subunit compositions, the mean degree of polymerization , and the total amounts of soluble and solvent-insoluble pas were not different compared to the brown  seed coat .
these results suggest the pa oligomers of black  and brown  seed coats may differ only in their subunit linkages.
however, the potential influence of lar or other pa genes on pa subunit linkages  remains to be determined.
from the set of differentially regulated genes identified by microarray, two putative late-stage anthocyanin genes were selected and the functions of their coded enzymes were determined in vitro.
ugt78k <dig> was found to code a uf3gt enzyme for anthocyanin biosynthesis .
a uf3gt cdna  has recently been isolated from the black seed coat and functionally characterized  <cit> .
ugt78k <dig> was 93% similar to ugt78k <dig> but possessed increased catalytic activity towards cyanidin .
the identification gene redundancy has important implications for seed coat color engineering by rna interference, as both copies of the gene may have to be silenced to achieve a significant reduction in uf3gt activity.
similarly, two omt genes  were found to be overexpressed in the black seed coat .
omt <dig> was cloned and found to code an aomt enzyme in vitro .
uf3gt and omt genes may be ideal candidates for seed coat color engineering as suppression of uf3gt genes  may result in a reduction in anthocyanin levels similar to that caused by a t-dna insertion in the arabidopsis uf3gt  <cit>  and upregulation of an aomt may result in a more red seed coat, as methylation of the b-ring of anthocyanins has a reddening effect on flower color .
however, it remains to be determined whether altering the expressions of these genes could be use to modify seed coat color without causing unintended effects on alternative biochemical pathways.
prior to the present study, currently known soybean genes did not map between markers a668_ <dig> and k387_ <dig> on chromosome  <dig> of the soybean physical genomic map, where the r locus is located, leaving no obvious candidates for the r locus gene  <cit> .
in our study,  <dig> genes identified to be differentially regulated in the black  soybean seed coat transcriptome were found to be located on chromosome  <dig> of the soybean genome sequence glyma <dig> .
among the gene families represented, only wd <dig> and myb have known roles in flavonoid regulation.
however the genes demonstrated did not cluster with known flavonoid regulators by phylogenetic analysis .
a mob1/phocein gene was commonly upregulated in the black  soybean seed coat and arabidopsis seedlings overexpressing pap <dig>  <cit> .
other regulatory gene families represented on chromosome  <dig> were ap2/erf, serine/threonine protein kinase, calcium/calmodulin-dependent protein kinase, and ethylene-responsive element-binding protein.
interestingly, all upregulated and downregulated genes on chromosome  <dig> were located in  <dig>  mb region that includes the nearest sequence markers to the r locus.
only the downregulated ap2/erf transcription factor, the serine carboxypeptidase gene, and the gene moderately similar to the a. thaliana polyamine oxidase atpao <dig> were located between these sequence markers .
as a result our study provides a short list of gene candidates that may serve as the focus of future efforts to identify the r locus gene.
CONCLUSIONS
metabolite composition and gene expression differences between black  and brown  seed coats are far more extensive than previously thought.
putative anthocyanin, proanthocyanidin, flavonoid, and phenylpropanoid isogenes were differentially-expressed between black  and brown  seed coats, and ugt78k <dig> and omt <dig> were validated to code udp-glycose:flavonoid-3-o-glycosyltransferase and anthocyanin 3'-o-methyltransferase proteins in vitro, respectively.
duplicate gene copies for several enzymes were overexpressed in the black  seed coat suggesting more than one isogene may have to be silenced to engineer seed coat color using rna interference.

 flavonoids represent a class of plant secondary metabolites that have evolved a variety of physiological functions including pigmentation, pathogen defense, and uv protection. additionally, metabolic engineering of flavonoids has become an important target for plant biotechnology, as flavonoids provide health benefits to foods, favorable agronomic traits to crops, and may be used in the future to color commercial transgenic materials such as grains to facilitate their identification and monitoring. commercial soybean merr.) has a yellow grain. however rare spontaneous mutants exist that have black or brown seed coat color phenotypes. black and brown soybean seed coats contain proanthocyanidins but differ in the presence/absence of anthocyanins. a goal for biosafety is to engineer a novel red seed coat color as a marker for transgenic soybean grains to facilitate their identification, and could potentially be achieved by the suppression of anthocyanin-specific genes that are overexpressed in the black soybean seed coat. however the genes have not yet been identified. six genetic loci identified by classical genetics control flavonoid-based seed coat color in soybean. the i locus controls the presence or absence and spatial distribution of flavonoid pigments and has four alleles; i gives completely non-pigmented seed coat, ii restricts pigment to the hilum and ik to a saddle-shaped region, whereas the i allele results in a fully pigmented seed coat. the recessive i allele results from spontaneous deletion of chs or chs promoter sequences and results in the abolishment of a posttranscriptional rna silencing mechanism that results in the increased accumulation of chalcone synthase transcripts in the seed coat. the t locus is a pleoiotropic locus that controls the type and abundance of flavonoid pigments in the seed coat in addition to other traits such as seed coat cracking and trichome pigmentation. genetic polymorphisms that affect the expression or function of the flavonoid 3'-hydroxylase gene have been shown to co-segregate with recessive t alleles. the w locus controls flower color and affects seed color only in an irt background; where w and w alleles give imperfect black and buff seed coat colors, respectively. the w allele for purple flower color was shown to code flavonoid 3',5'-hydroxylase, as a 65-bp insertion in the gene co-segregated with white flower color. the wp locus was suggested to code the flavonone 3-hydroxylase gene by microarray analysis as high levels of f3h transcripts co-segregated with purple flower color, and low levels with pink flowers. in the seed coat, recessive wp resulted in a change from black to a lighter grayish color. the o locus affects the color of brown seed coats, with the recessive o allele giving a red-brown phenotype. the o locus has been suggested to code the proanthocyanidin biosynthesis gene anthocyanidin reductase, as the gene was located between markers that flank the o locus on the soybean physical map. however molecular genetics analyses have not yet demonstrated the identity of the o locus gene. finally, the r locus controls the presence or absence of anthocyanins in black or brown seed coats, respectively. despite the identification of several pigment genes from soybean, only transcripts for anthocyanidin synthase genes have been shown to be overexpressed in the black seed coat compared to the brown nearly-isogenic line by northern blot. the upregulation of several ans genes suggests the r locus to code a regulatory factor, and raises the possibility that other isogenes for anthocyanin biosynthesis may be upregulated. recently, a cdna coding the udp-glycose:flavonoid-3-o-glycosyltransferase gene was isolated from the black seed coat and shown to function in anthocyanin biosynthesis in vitro and by complementation of a gene mutation in arabidopsis. however ugt78k expressions have not been investigated in relation to seed coat color. the soybean genome sequence glyma was predicted to code protein-coding genes with nearly 75% of the genes present in multiple copies. this may suggest a relatively high frequency of functional redundancy and increased difficulty in identifying soybean genes by traditional approaches. however, using transcriptome analysis tools such as the soybean genechip equipped with probe sets in combination with broad-coverage metabolite analysis methodologies such as lc-ms/ms, gene functions could potentially be efficiently predicted. the combined analysis of transcriptome and metabolite data has been shown to be a powerful approach for the functional identification of unknown genes. metabolite differences caused by the overexpression of a transcription factor, the exposure to a nutritional stress, and by species differences have been correlated with differences in transcriptome profiles to successfully predict the functions of unknown genes in flavonoid, glucosinolate, and alkaloid biosyntheses. in the present study we have employed targeted and non-targeted metabolite analysis methodologies and have demonstrated that black and brown nearly-isogenic soybean seed coats do not just differ in the presence/absence of anthocyanins, and have extensive differences in procyanidin, flavonoid and phenylpropanoid compositions. the underlying differences in gene expressions were then identified by microarray analysis, and the putative functions of unknown genes were assigned by comparison to metabolite data. from the set of differentially regulated genes, two putative late-stage anthocyanin genes were selected and the functions of their coded enzymes were validated in vitro. in addition, a set of gene candidates potentially coded by the r locus have been provided.  qrt-pcr indicates differential expressions of anthocyanin and proanthocyanidin genes in nearly-isogenic black and brown soybean seed coats among the flavonoid genes identified from soybean seed coats, only ans transcripts were found to be overexpressed in the seed coat of black soybean compared to the brown isoline, however no rflp difference was observed when probing ans genes. this suggests that ans overexpression is not the only molecular difference between black and brown seed coats. to examine whether other anthocyanin genes are upregulated, transcript profiles of the recently identified ugt78k and the dfr gene were measured from seed coat cdna by quantitative rt-pcr using ans2/ans transcripts as a positive control. phosphenol pyruvate carboxylase was used as an endogenous reference for normalization of the qrt-pcr measurements of target genes across seed coat samples, as pepc is expressed at similar levels in a wide range of soybean tissues and has been used previously as a reference for gene expressions during soybean seed coat development. qrt-pcr demonstrated ugt78k to be significantly overexpressed in the black seed coat at the transition stage and early maturation stage of seed development compared to the brown isoline, and to be almost undetectable at earlier stages. stereomicroscopy and photospectroscopic measurements confirmed these developmental stages to coincide with the stages of anthocyanin biosynthesis in the black seed coat. in contrast to ugt78k dfr was not differentially expressed at the stages of anthocyanin biosynthesis. ans2/ans mrnas were confirmed to be overexpressed in the black seed coat at most stages as shown previously. the seed coat palisade cells of black soybean are unusual compared to those of other model plants used for the study of anthocyanin biosynthesis because black soybean palisade cells synthesize both anthocyanins and proanthocyanidins. anr and lar enzymes have been identified to catalyze the conversion of cyanidin to epicatechin and leucocyanidin to catechin, respectively, which are the putative initial biochemical steps of pa biosynthesis. to investigate how a seed tissue may coordinate the biosynthesis of these parallel pigment pathways, the expressions of putative pa genes anr and lar were examined in relation to anthocyanin gene expressions. qrt-pcr demonstrated anr expressions to decrease throughout seed coat development in both genotypes and to be almost undetectable by the stages of ugt78k overexpressions and anthocyanin biosynthesis. by contrast, lar expressions were downregulated at the stages of anthocyanin biosynthesis in the black seed coat. in conclusion, these results show that the recently identified ugt78k is upregulated in the black seed coat, in addition to ans2/ans and that there is a simultaneous downregulation of the pa gene lar at the stages of anthocyanin biosynthesis. these results demonstrate that expression differences between black and brown seed coats are not limited to anthocyanin genes, and may suggest extensive differences in gene expressions and phenolic compositions. combined analysis of targeted and non-targeted methodologies indicate overaccumulation of anthocyanins, altered procyanidin, and reduced flavonol, benzoic acid, and isoflavone compositions in the seed coat of black soybean the structures of anthocyanins from only a few black soybean varieties have been described and information regarding other phenolic compositions may be limited to proanthocyanidins, where acid-catalyzed hydrolysis of black and brown seed coats showed the pas to be 3',4'-hydroxylated. a more extensive analysis of phenolic composition differences between black and brown seed coats could greatly improve our understanding of seed pigmentation, and may suggest gene activities that function in pigment biosynthesis. for these reasons, black and brown clark seed coat metabolites were extracted at the transition stage of seed development with multiple solvents and analyzed by targeted high performance liquid chromatography-diode array detection and non-targeted hplc-tandem mass spectrometry. to ensure seed coats were at the same stage of development, the days post anthesis, pod length, pod color, embryo morphology, and transcript levels of the developmental marker gene gm-r1083- a putative cutin biosynthesis gene, and the metabolism gene dfr were ensured to be equivalent between black and brown tissues. for a targeted analysis of anthocyanins, black and brown clark seed coats were extracted with acidified aqueous methanol and metabolites were identified using hplc-dad by measuring the absorbance at nm in comparison to authentic standards. ten cyanic pigments were identified, the most abundant were the anthocyanins cyanidin-3-o-glucoside, cyanidin-3-o-galactoside and petunidin-3-o-glucoside with lesser amounts of delphinidin-, pelargonidin-, and peonidin-3-o-glucosides, the aglycone cyanidin, the 3-deoxyanthocyanidin diosmetinidin, and the two unidentified anthocyanin-like compounds. to our knowledge this is the first report of a 3-deoxyanthocyanidin from the soybean seed coat. the structures of all identified anthocyanins are represented in figure by contrast, cyanic pigments were not detected from the brown seed coat at any stage of seed development. *for chemical structures see fig. to compare the structural features of pas, black and brown seed coats were extracted with 70% acetone and subjected to phloroglucinol cleavage analysis. pa free monomers, polymer subunit compositions, and mean degree of polymerization did not differ significantly between nearly-isogenic black and brown seed coats. the soluble pa mdp was and the subunit and monomer compositions consisted mainly of epicatechin with minor amounts of unidentified compounds. reaction with dmaca reagent demonstrated similar amounts of soluble pas from both black and brown seed coats. further extraction of seed coat residues at 50°c in the presence of acid and phloroglucinol yielded only low levels of epicatechin-phloroglucinol adduct, epicatechin terminal units, and several unidentified compounds, and acid catalyzed cleavage of seed coat residues at 95°c identified relatively high, but equivalent amounts of 3',4'-hydroxylated solvent insoluble pas. for a non-targeted analysis of soluble phenolic metabolites, nearly isogenic black and brown clark seed coats were extracted with acidified aqueous methanol and analyzed by hplc-ms/ms over a mass range of amu to amu. identities were assigned to peaks by comparison of retention times and mass fragmentation patterns to authentic standards. peaks with no corresponding authentic standards were identified by mass fragmentation patterns in comparison to the literature. a total of peaks were detected, of which peaks were assigned to compounds. twenty-eight compounds were detected from the black seed coat; consisting mainly of unidentified compounds and anthocyanins, followed by procyanidins. by contrast, compounds were detected from the brown seed coat; consisting mainly of unidentified compounds, followed by flavonols and procyanidins. the majority of compounds were exclusive to the black or brown seed coat, suggesting extensively different biochemical specializations of these tissues. the distribution of compound classes from black and brown clark seed coats is illustrated in figure *based on co-chromatographic and ms fragamentation comparison to an authentic standard. ablack soybean, brown soybean, both. btrace amounts in irt. the compounds exclusive to the black seed coat were anthocyanins, procyanidins, and unknown compounds. hplc-ms/ms confirmed the identity of anthocyanins found by hplc-dad. procyanidin tetramers and larger oligomers were not detected as they exceeded the mass range analyzed by the hplc-ms/ms experiments, however pa trimers and the a-type procyanidin dimer were exclusive to the black seed coat. the compounds exclusive to the brown seed coat consisted of unknown compounds, flavonols, benzoic acids, and the isoflavone daidzein o-hexoside malonylated. taken together, our metabolite analysis experiments show that nearly-isogenic black and brown clark seed coats have compositional differences not limited to the presence or absence of anthocyanins; with specific flavonol, isoflavone, and benzoic acids exclusive to the brown seed coat and anthocyanins and unique procyanidin oligomers exclusive to the black seed coat. these results may suggest extensive differences in the underlying gene expressions. microarray analysis indicates concomitant upregulation of specific phenylpropanoid, flavonoid, and anthocyanin isogenes and downregulation of other flavonoid, benzoic acid, and isoflavone isogenes in the black seed coat transcriptome nearly 75% of genes in the soybean genome are present in multiple copies, and previously only transcripts for ans isogenes were found to be overexpressed in the black seed coat compared to the nearly-isogenic brown tissue. for a global analysis of isogene expressions, black and brown clark seed coats were dissected at the transition stage of seed development for microarray analysis using the affymetrix soybean genechip. seed coats were ensured to be at the same stage of development as described above for the metabolite analyses. of the probe sets present on the soybean genechip, were found to be upregulated more than 2-fold in black soybean. classification using the gene-bins ontology tool demonstrated the majority of upregulated genes to be "unclassified without homology" or "unclassified with homology", followed by "biosynthesis of secondary metabolites". this latter class consisted exclusively of putative phenylpropanoid, flavonoid, and anthocyanin isogenes. interestingly, putative isogenes of the phenylpropanoid pathway, the flavonoid pathway, and the late-stage anthocyanin pathway were all upregulated more than 2-fold in the black seed coat, more than 234-fold in the case of the gst probe set. aexpression values were obtained using rma. bsee table s for the complete list of upregulated genes. *previously characterized flavonoid biosynthesis genes. a blastp search of the soybean genome sequence glyma using the conserved plant secondary product glycosyltransferase signature sequence identified annotated family glycosyltransferases. of the ugts, only were found to be upregulated more than 2-fold from the black seed coat; the recently identified uf3gt gene ugt78k was upregulated more than -fold and a second uncharacterized glycosyltransferase, which we named ugt78k in accordance with ugt nomenclature, was upregulated more than 3-fold. a search of the soybean genome sequence with the conserved omt-domain sequence and the ontology'o-methyltransferase', identified annotated omts. of the omt genes, only probe sets for omt-like and omt were upregulated in the black seed coat, more than -fold and -fold, respectively. a total of probe sets were downregulated more than 2-fold in the black seed coat; among these, probe sets by more than 10-fold, probe sets by more than 3-fold, with the remainder between 2- and 3-fold. classification of down-regulated genes using the gene-bins ontology tool demonstrated the majority to be "unclassified without homology" or "unclassified with homology", followed by "biosynthesis of secondary metabolites". the latter class consisted primarily of characterized and putative flavonoid isogenes. isogenes for common flavonoid biosynthesis, pa biosynthesis, and isoflavone biosynthesis were all down-regulated more than 2-fold in the black seed coat, more than 35-fold in the case of the chs. the flavonoid genes chs and f3'h were down-regulated more than 21-fold and -fold, respectively, the putative isoflavone gene ifr1-like by more than -fold, and the putative pa gene lar by more than -fold. aexpression values were obtained using rma. bsee table s for the complete list of up-regulated genes. *previously characterized flavonoid biosynthesis genes. of the differentially regulated probe sets, represented transcription factor genes, were upregulated and downregulated, however none of these genes clustered with known flavonoid transcription factor orthologs by phylogenetic analysis. a previous study mapped the r locus between markers a668_ and k387_ on mlg k. nineteen differentially-regulated probe sets were associated with genes located on chromosome of the soybean genome sequence glyma. interestingly, all genes were located in a mb region of the distal arm of chromosome and only the downregulated ap2/erf and serine carboxypeptidase-like protein and the upregulated polyamine oxidase were located between markers that flank the putative position of the r locus. to validate the microarray results, differentially-regulated genes were selected and their relative expression profiles were measured by semi-quantitative rt-pcr using black and brown seed coat cdna as templates. phosphenol pyruvate carboxylase was used as the endogenous control to ensure equal loading and the flavonoid genes chs7/chs and the putative pa gene anr were used as negative controls as they were not found to be differentially expressed in the microarray analysis. semi-qrt-pcr confirmed the differential-expressions of all genes and demonstrated similar expressions for chs7/chs and anr. furthermore, as the upregulated probe set gmaaffx..s did not distinguish between chs and chs expressions, semi-qrt-pcr was performed with gene specific primers to show the chs gene, and not the chs gene to be upregulated in the black seed coat. in summary, these results show that numerous specific anthocyanin, flavonoid and phenylpropanoid isogenes are upregulated and other flavonoid, benzoic acid, and isoflavone isogenes are downregulated in the black seed coat transcriptome relative to that of the nearly-isogenic brown tissue. putative assignment of function to genes differentially-regulated between black and brown seed coats gene functions can be successfully predicted based on a combined analysis of transcriptome and metabolite data. we compared differences in gene expressions determined by microarray to differences in metabolite compositions to assign putative functions to genes from the black and brown seed coats. in addition to previously characterized soybean flavonoid genes, several uncharacterized genes belonging to large multi-gene families with possible roles in anthocyanin biosynthesis and transport were upregulated; including one glycosyltransferase, two o-methyltransferases and two glutathione s-transferases. considering the accumulation of metabolites with specific molecular structures in the black seed coat, putative functions of the differentially-expressed genes were assigned to specific structural modifications and transport functions. anthocyanins in the seed coat of black soybean are glycosylated at the 3-position of the c-ring. the glycosyltransferase ugt78k is assigned to code a udp-glycose:flavonoid 3-o-glycosyltransferase protein and may have functional redundancies in anthocyanin biosynthesis with the previously characterized ugt78k, which was also upregulated in the black seed coat. two anthocyanins and one deoxyanthocyanidin from the black seed coat are methylated at the 3-position of the b-ring. the two upregulated o-methyltransferases were assigned to code anthocyanin 3'-o-methyltransferase proteins and may have redundant function in anthocyanin biosynthesis this tissue. ugt78k and omt code udp-glycose:flavonoid 3-o-glycosyltransferase and anthocyanin 3'-o-methyltransferase proteins, respectively reducing the expressions of late-stage anthocyanin genes by rna interference in the seed coat of black soybean may be an effective strategy to engineer seed coat color while minimizing unintended effects on other flavonoid subpathways. however, functional redundancies may require that multiple genes be silenced in order to reduce enzyme activity enough to reduce pigment levels. two glycosyltransferases were upregulated in the black seed coat relative to the nearly-isogenic brown tissue and the upregulation paralleled the accumulation of anthocyanins with 3-o-glycosylated structures, suggesting the function of these genes as udp-glycose:flavonoid 3-o-glycosyltransferases. we have characterized ugt78k previously and shown it to possess uf3gt activity towards anthocyanidins in vitro and in vivo. to determine the catalytic function of ugt78k, the orf was cloned into an n-terminal hexahistidine fusion tag vector and expressed in e. coli for analysis of the coded recombinant protein in vitro. the recombinant protein was purified by ion-metal-affinity chromatography had an apparent mass of kda that matched well with the calculated mass of the native ugt78k protein in addition to the kda n-terminal hexahistidine fusion tag encoded by the pet-14b vector. in assays containing udp-glucose as the sugar donor and cyanidin as the acceptor, the recombinant enzyme transferred glucose to the 3-position of cyanidin to form cyanidin-3-o-glucoside, whereas the boiled enzyme and bacteria expressing the corresponding empty vector could not catalyze this reaction. similarly, in assays containing udp-galactose as the sugar donor and cyanidin as the acceptor, the recombinant enzyme transferred galactose to the 3-position of cyanidin to form cyanidin-3-o-galactoside. cyanidin-3-o-glucoside and cyanidin-3-o-galactoside from recombinant enzyme assays were identified by hplc-dad in comparison to the authentic standards, and confirmed by hplc-ms/ms with the characteristic parent ions + and the ms/ms fragment + corresponding to the loss of glucose. as we have performed the kinetics analysis for recombinant ugt78k we were able to employ the same experimental conditions to test the kinetics of ugt78k interestingly, ugt78k converted cyanidin-3-o-glucoside with greater velocity than ugt78k and higher concentrations of cyanidin were required to inhibit its activity despite the enzymes having 93% amino acid similarity. methylation of the b-ring of anthocyanins has a reddening effect on flower color and thus engineering the expression of 3'-o-methyltransferases could change the redness of seed color. however, despite the identification of several flavonoid omts from soybean, none have been shown to accept anthocyanins as substrates. our microarray analysis identified two o-methyltransferases to be upregulated in the black seed coat and the upregulation paralleled the accumulation of 3'-o-methylated anthocyanins in this tissue, suggesting the function of these genes as anthocyanin 3'-o-methyltransferases. following the approach described above, the orf of omt was cloned for expression and functional characterization of the corresponding recombinant protein in vitro. the purified recombinant protein had an apparent mass of kda that matched well with the calculated mass in addition to the kda n-terminal hexahistidine fusion tag encoded by the pet-14b vector. in assays containing s-adenosyl-l-methionine as the methyl donor and cyanidin-3-o-glucoside as the acceptor, the recombinant enzyme transferred a methyl group to the 3'-position of the acceptor to form peonidin-3-o-glucoside, whereas the boiled enzyme and bacteria expressing the corresponding empty vector could not catalyze this reaction. peonidin-3-o-glucoside from recombinant enzyme assays was identified by hplc-dad in comparison to the authentic standard, and confirmed by hplc-ms/ms with the characteristic parent ion + and the ms/ms fragment + corresponding to the loss of glucose. to further investigate the function these genes, ugt78k and omt expression profiles were measured from seed coat cdna throughout black and brown soybean seed development by quantitative rt-pcr and expressions were compared to anthocyanin accumulation profiles throughout seed development. similar to ugt78k ugt78k and omt expressions peaked at the transition and early maturation stages of seed development in the black seed coat, the stages at which anthocyanins accumulate, and were low or almost undetectable at early stages of seed development in the black seed coat and at all stages in the brown seed coat. together the qrt-pcr and recombinant enzyme assays suggest that ugt78k and omt code uf3gt and aomt proteins, respectively, that function in anthocyanin pigment biosynthesis in the seed of black soybean. discussion the seed coat compositions of black and brown soybean were known to differ only by the presence/absence of anthocyanins, however our metabolite analysis demonstrated these nearly-isogenic tissues to have extensive differences in anthocyanin, proanthocyanidin, flavonoid, and phenylpropanoid content. of the compounds detected, compounds were exclusive to either the black or the brown seed coat. analysis of the distributions of compound classes between seed coats demonstrated the black to a have a greater proportion of anthocyanins and pas, and the brown to have a greater proportion of flavonol, benzoic acid, isoflavone, and unknown metabolite compositions. metabolites exclusive to the black seed coat included pa trimers and an a-type procyanidin dimer in addition to various anthocyanins. the anthocyanins identified from variety clark were mainly similar to those reported previously from various korean varieties however clark was the first shown to contain a 3-deoxyanthocyanidin in the seed coat. 3-deoxyanthocyanidins are relatively rare intensely coloured pigments found in sorghum bicolour in response to fungal attack, and from the cotyledons of yellow soybean in response to uv-c treatment. the biosynthesis of 3-deoxyanthocyanidins from flavonones has been shown to be a minor activity of a dfr isogene in sorghum. it would be interesting to determine whether the dfr gene that was highly overexpressed in the black clark seed coat had similar catalytic activity, and whether the korean varieties contain genetic alterations related to dfr analysis of the black seed coat transcriptome compared to that of the nearly-isogenic brown tissue identified probe sets to be upregulated. this is almost double the number of probe sets upregulated by overexpression of the myb transcription factor pap in arabidopsis, similar to the number upregulated by overexpression of the anthocyanin factor lap in m. truncatula, and less than half the number upregulated by overexpression of lap in m. sativa. the higher numbers of genes upregulated in anthocyanin-overaccumulating tissues of black soybean, m. truncatula and m. sativa likely reflect the fact that legumes have undergone genome duplication and that genes with multiple copies are frequently retained. our data supported this hypothesis as duplicate copies of several genes were upregulated in the black seed coat. functional redundancies can complicate gene identification by traditional approaches. previous to the present study, genes for only flavonoid enzymes were identified to be involved in pigment biosynthesis in the soybean seed coat. in the present study, isogenes for proteins were identified to have putative roles in flavonoid biosynthesis in black or brown seed coats using a combined analysis of microarray and metabolite data. transcriptome analysis has previously demonstrated a critical role of chs and chs genes for isoflavonoid biosynthesis in developing embryos of soybean. however, our microarray and semi-quantitative rt-pcr data demonstrated that chs and chs were not differentially regulated in black and brown seed coats. interestingly, it was chs that was upregulated and chs and chs that were downregulatedin the black seed coat. as chs is upregulated with anthocyanin genes, it likely has a role in anthocyanin biosynthesis in this tissue. consistent with this, spontaneous mutations of the dominant ii allele to the recessive i allele have been shown to affect the promoter region of the chs gene. by contrast, the downregulation of chs and chs may suggest that they have a role in the parallel downregulated biochemical pathways, such as flavonol and isoflavonoid biosynthesis, as these metabolites, and transcript levels of genes for their biosynthesis, were reduced or not present in the black seed coat. these results emphasize the complexity of isogene expressions that exist to achieve a single enzyme function in soybean. microarray analysis identified differentially regulated probe sets for only gene functions that have been previously shown to be involved in pa biosynthesis and transport. recombinant lar enzymes from several legume species have been shown to convert leucocyanidin to -catechin in vitro. however, the in vivo role of lar in pa biosynthesis remains questionable as endogenous expression has been shown not to correlate with pa levels in m. truncatula and heterologous expression has failed to increase pa accumulations. similarly, our metabolite data showed that soybean pas lack catechin subunits and that lar expressions do not parallel pa accumulations during seed development. interestingly, pas were found to be exclusive to the black seed coat however the total subunit compositions, the mean degree of polymerization, and the total amounts of soluble and solvent-insoluble pas were not different compared to the brown seed coat. these results suggest the pa oligomers of black and brown seed coats may differ only in their subunit linkages. however, the potential influence of lar or other pa genes on pa subunit linkages remains to be determined. from the set of differentially regulated genes identified by microarray, two putative late-stage anthocyanin genes were selected and the functions of their coded enzymes were determined in vitro. ugt78k was found to code a uf3gt enzyme for anthocyanin biosynthesis. a uf3gt cdna has recently been isolated from the black seed coat and functionally characterized. ugt78k was 93% similar to ugt78k but possessed increased catalytic activity towards cyanidin. the identification gene redundancy has important implications for seed coat color engineering by rna interference, as both copies of the gene may have to be silenced to achieve a significant reduction in uf3gt activity. similarly, two omt genes were found to be overexpressed in the black seed coat. omt was cloned and found to code an aomt enzyme in vitro. uf3gt and omt genes may be ideal candidates for seed coat color engineering as suppression of uf3gt genes may result in a reduction in anthocyanin levels similar to that caused by a t-dna insertion in the arabidopsis uf3gt and upregulation of an aomt may result in a more red seed coat, as methylation of the b-ring of anthocyanins has a reddening effect on flower color. however, it remains to be determined whether altering the expressions of these genes could be use to modify seed coat color without causing unintended effects on alternative biochemical pathways. prior to the present study, currently known soybean genes did not map between markers a668_ and k387_ on chromosome of the soybean physical genomic map, where the r locus is located, leaving no obvious candidates for the r locus gene. in our study, genes identified to be differentially regulated in the black soybean seed coat transcriptome were found to be located on chromosome of the soybean genome sequence glyma. among the gene families represented, only wd and myb have known roles in flavonoid regulation. however the genes demonstrated did not cluster with known flavonoid regulators by phylogenetic analysis. a mob1/phocein gene was commonly upregulated in the black soybean seed coat and arabidopsis seedlings overexpressing pap. other regulatory gene families represented on chromosome were ap2/erf, serine/threonine protein kinase, calcium/calmodulin-dependent protein kinase, and ethylene-responsive element-binding protein. interestingly, all upregulated and downregulated genes on chromosome were located in mb region that includes the nearest sequence markers to the r locus. only the downregulated ap2/erf transcription factor, the serine carboxypeptidase gene, and the gene moderately similar to the a. thaliana polyamine oxidase atpao were located between these sequence markers. as a result our study provides a short list of gene candidates that may serve as the focus of future efforts to identify the r locus gene.  metabolite composition and gene expression differences between black and brown seed coats are far more extensive than previously thought. putative anthocyanin, proanthocyanidin, flavonoid, and phenylpropanoid isogenes were differentially-expressed between black and brown seed coats, and ugt78k and omt were validated to code udp-glycose:flavonoid-3-o-glycosyltransferase and anthocyanin 3'-o-methyltransferase proteins in vitro, respectively. duplicate gene copies for several enzymes were overexpressed in the black seed coat suggesting more than one isogene may have to be silenced to engineer seed coat color using rna interference. 
we recently identified and characterized a udp-glycose:flavonoid-3-o-glycosyltransferase from the seed coat of black soybean with the aim to engineer seed coat color by suppression of an anthocyanin-specific gene. duplicate gene copies for several enzymes were overexpressed in the black seed coat suggesting more than one isogene may have to be silenced to engineer seed coat color using rna interference. metabolite composition and gene expression differences between black and brown seed coats are far more extensive than previously thought. putative anthocyanin, proanthocyanidin, flavonoid, and phenylpropanoid isogenes were differentially-expressed between black and brown seed coats, and ugt78k and omt were validated to code udp-glycose:flavonoid-3-o-glycosyltransferase and anthocyanin 3'-o-methyltransferase proteins in vitro, respectively. a combined analysis of metabolite and gene expression data enabled the assignment of putative functions to biosynthesis and transport isogenes. however, it remained to be investigated whether ugt78k was overexpressed with anthocyanin biosynthesis in the black seed coat compared to the nearly-isogenic brown tissue. two differentially expressed late-stage anthocyanin biosynthesis isogenes were further characterized, as they may serve as useful targets for the manipulation of soybean grain color while minimizing the potential for unintended effects on the plant system. the recombinant enzymes of two genes were validated to catalyze late-stage steps in anthocyanin biosynthesis in vitro and expression profiles of the corresponding genes were shown to parallel anthocyanin biosynthesis during black seed coat development. the seed coats of nearly-isogenic black and brown soybean were known to differ by the presence or absence of anthocyanins, respectively, with genes for only a single enzyme found to be differentially expressed between isolines. the r locus controls the color of pigmented soybean seeds. in this study, we performed a combined analysis of transcriptome and metabolite data to elucidate the control of the r locus over seed coat biochemistry and to identify pigment biosynthesis genes. however information about its control over seed coat biochemistry and gene expressions remains limited. a global analysis of gene expressions identified ugt78k and other anthocyanin, flavonoid, and phenylpropanoid isogenes to be differentially expressed between isolines. metabolite composition differences were found to not be limited to anthocyanins, with specific proanthocyanidins, isoflavones, and phenylpropanoids present exclusively in the black or the brown seed coat.
 the r locus controls the color of pigmented soybean seeds. however information about its control over seed coat biochemistry and gene expressions remains limited. the seed coats of nearly-isogenic black and brown soybean were known to differ by the presence or absence of anthocyanins, respectively, with genes for only a single enzyme found to be differentially expressed between isolines. we recently identified and characterized a udp-glycose:flavonoid-3-o-glycosyltransferase from the seed coat of black soybean with the aim to engineer seed coat color by suppression of an anthocyanin-specific gene. however, it remained to be investigated whether ugt78k was overexpressed with anthocyanin biosynthesis in the black seed coat compared to the nearly-isogenic brown tissue. in this study, we performed a combined analysis of transcriptome and metabolite data to elucidate the control of the r locus over seed coat biochemistry and to identify pigment biosynthesis genes. two differentially expressed late-stage anthocyanin biosynthesis isogenes were further characterized, as they may serve as useful targets for the manipulation of soybean grain color while minimizing the potential for unintended effects on the plant system.  metabolite composition differences were found to not be limited to anthocyanins, with specific proanthocyanidins, isoflavones, and phenylpropanoids present exclusively in the black or the brown seed coat. a global analysis of gene expressions identified ugt78k and other anthocyanin, flavonoid, and phenylpropanoid isogenes to be differentially expressed between isolines. a combined analysis of metabolite and gene expression data enabled the assignment of putative functions to biosynthesis and transport isogenes. the recombinant enzymes of two genes were validated to catalyze late-stage steps in anthocyanin biosynthesis in vitro and expression profiles of the corresponding genes were shown to parallel anthocyanin biosynthesis during black seed coat development.  metabolite composition and gene expression differences between black and brown seed coats are far more extensive than previously thought. putative anthocyanin, proanthocyanidin, flavonoid, and phenylpropanoid isogenes were differentially-expressed between black and brown seed coats, and ugt78k and omt were validated to code udp-glycose:flavonoid-3-o-glycosyltransferase and anthocyanin 3'-o-methyltransferase proteins in vitro, respectively. duplicate gene copies for several enzymes were overexpressed in the black seed coat suggesting more than one isogene may have to be silenced to engineer seed coat color using rna interference. 