BACKGROUND
flavonoids are a diverse group of plant secondary metabolites with various biological functions that play important roles during plant development.
they are involved in plant defenses against insects, pathogens and microbes, in absorption of free radicals and uv light, and in attraction of beneficial symbionts and pollinators  <cit> .
proanthocyanidins  are components of metabolites synthesized through the general flavonoid pathway.
their main known function is to provide protection against microbial pathogens and invasions of insects and herbivores through multiple mechanisms  <cit> .
pas have metal chelating activity that results in severe limitation of bacterial growth; pas can also associate with and irreversibly precipitate proteins, which is responsible for the astringent taste that repels herbivores.
furthermore, pas can be oxidized to quinones which not only are powerful antibiotics themselves, but also can initiate cross-linking of cell walls to increase the strength of this physical barrier to pathogens  <cit> .
in addition to these functional roles in plants, pas, especially those from cacao, have recently been suggested to be beneficial to humans by improving cardiovascular health through activation of nitric oxide synthase, by providing cancer chemopreventative effects, and also through neuroprotective activities  <cit> .
the biosynthetic pathways that lead to the production of flavan-3-ols -catechin and -epicatechin), the building blocks of pas, have been well studied in the model plant species maize  and arabidopsis as summarized in figure  <dig> <cit> .
biosynthesis of flavan-3-ols involves three principal enzymes: leucoanthocyanidin reductase , anthocyanidin synthase , and anthocyanidin reductase .
the synthesis of pas and anthocyanins share common steps leading to flavan- <dig> -diols , which can be converted to catechin  by lar  <cit>  or to anthocyanidin by ans  <cit> .
anthocyanidin then either serves as the substrate for the synthesis of epicatechin  by anr  <cit> , or can otherwise be converted to anthocyanin by glycosylation  <cit> .
both catechin and epicatechin act as the initiators for pa polymerization, with intermediates derived from leucoanthocyanidin, catechin or epicatechin added sequentially as extension units  <cit> .
however, the details of the polymerization process are unclear and it is not known whether this is a spontaneous or an enzyme-catalyzed reaction.
recent work has identified two new enzymes downstream of flavan-3-ols that are involved in key steps of pa polymer biosynthesis, an epicatechin 3’-o-glucosyltransferase in medicago truncatula <cit>  and mate transporters from arabidopsis and medicago truncatula <cit>  that transport epicatechin 3’-o-glucoside to the vacuole in which it is likely that pa polymerization occurs  <cit> .
ans and anr genes have been biochemically and genetically characterized in arabidopsis  <cit> , medicago truncatula <cit>  and vitis vinifera   <cit> .
the arabidopsis ans  mutant exhibits a deficiency in both anthocyanin accumulation in hypocotyls and pa deposition in seeds that results in a transparent testa phenotype  <cit> .
seeds of the arabidopsis banyuls  mutant exhibit a lack of pas and a hyper-accumulation of anthocyanins, resulting in a dark red color reminiscent of the famous banyuls wine produced in southern france  <cit> .
over-expression of anr genes from medicago and grape in tobacco results in a loss of anthocyanin pigments in flower petals and elevated levels of pas  <cit> .
antisense down-regulation of ans in medicago results in reduction of both anthocyanins in leaves and pas in seeds  <cit> .
lar genes have been isolated from various plant species including desmodium uncinatum <cit> , vitis vinifera <cit> , lotus corniculatus <cit>  and medicago truncatula <cit>  and their corresponding protein functions have been characterized by in vitro recombinant enzyme assays.
however, the genetic evidence for lar function is rather indirect and less convincing than it is for anr and ans, as discussed by pang et al.
<cit> .
it appears that the genomic sequence of arabidopsis thaliana does not contain an intact lar orthologue, and correspondingly, catechin is not detected in arabidopsis seed extracts  <cit> .
lar genes are expressed in other plant species that accumulate not only epicatechin but also catechin  <cit> .
for example, grape and lotus express both lar and anr genes and synthesize pas consisting of both catechin and epicatechin  <cit> .
in medicago, although both lar and anr are expressed, the pas are composed almost entirely of epicatechin  <cit> .
in arabidopsis and medicago, pa accumulation and gene expression is quantitatively and spatially limited to seed coats, making it remarkably difficult for biochemical analysis  <cit> .
in contrast, theobroma cacao  produces significant amounts of pas in various tissues including leaves and beans; up to 12% of dry weight in leaves  <cit>  and approximately 10% in mature beans  <cit> .
furthermore, large amounts of catechin and epicatechin monomers as well as their related polymers of different lengths have been detected in cocoa powder  <cit> .
considering the wide range of health benefits suggested for pas and its significance for plant resistance, we targeted this pathway for molecular-genetic analysis.
this manuscript describes the isolation and expression of the tcanr, tcans, and tclar genes encoding the key enzymes in proanthocyanidin biosynthesis.
we measured pa content in different cacao tissues and performed functional characterization of the tcanr, tcans, and tclar gene products through in vivo tests.
the results presented here provide background and genetic tools that will be useful in the development of new cacao varieties with altered pa profiles.
RESULTS
molecular cloning and sequence analysis of tcanr, tcans and tclar genes
the general pathway for the synthesis of proanthocyanidin and anthocyanins indicates the key enzymes anr, ans and lar that carry out the biochemical steps at a critical metabolic branch point .
to explore the genetic control of this important pathway in cacao, putative tcanr, tcans and tclar cdna sequences were identified in a collection of theobroma cacao expressed sequence tags   <cit>  by querying the cacao esttik database  with protein sequences of arabidopsis banyuls , arabidopsis ldox  and desmodium lar .
ests similar to each gene were assembled into contigs to determine consensus full-length open reading frames  by alignment with cdnas of homologous genes from other species and predictions from the orf finder program .
full-length cdnas of each gene were amplified by reverse transcription-pcr  using rna isolated from young leaves of cacao .
the tcanr cdna  contained a  <dig> -bp open reading frame  encoding a protein of  <dig> amino acids that showed a 63% identity with the arabidopsis banyuls gene at the amino acid level.
the tcans cdna  contained an orf of  <dig> -bp, which encodes a protein of  <dig> amino acids with 82% amino acid identity with the arabidopsis ldox gene.
the tclar cdna  contained a  <dig> -bp orf encoding a protein of  <dig> amino acids with 61% amino acid identity with the desmodium lar protein.
to determine the genomic sequences of the putative cacao ans, anr and lar genes, the cdnas were used to screen a cacao bac library by hybridization.
a portion of each hybridizing bac clone was sequenced using primers designed from the corresponding cdnas.
the genomic structure of each gene was established by alignment with its cdna sequence .
we also retrieved gene models of the ans and anr gene from arabidopsis  and lar from medicago and grape  and compared them with the corresponding cacao genes.
the coding region of tcanr  consisted of  <dig> exons and  <dig> introns distributed over  <dig> -bp.
the coding region of tcans  was shorter, having only  <dig> -bp and consisting of  <dig> exons and  <dig> intron.
the genomic organizations of these two cacao genes were nearly identical to the corresponding arabidopsis genes, which had the same exon and intron numbers.
moreover, the lengths of the exons were very similar between arabidopsis and cacao: for anr, the numbers of nucleotides were precisely the same for the exons  <dig>   <dig>   <dig>  and  <dig>  although the lengths of introns were more variable.
since lar does not have an orthologue in arabidopsis, we compared the genomic organization of tclar with mtlar and vvlar .
the genomic organization of the coding region of tclar  was similar to both mtlar and vvlar, consisting of  <dig> exons and  <dig> introns.
as observed with the anr gene, the middle three exons  of lar from all three species had identical lengths.
tclar exhibited an extremely long third intron of  <dig>  bp; similarly, vvlar also featured a long third intron of  <dig>  bp, while mtlar contained two long introns  that are 812-bp and  <dig> -bp respectively.
the analysis of the complete cacao genome sequence of the criollo variety was consistent with these results and allowed us to localize the position of each gene relative to the molecular-genetic map  <cit> .
the anr gene was located on chromosome  <dig>  and was  <dig>  identical to the cdna described above.
similarly, a single ans gene was located on chromosome  <dig>  and shared  <dig> % identity to the cdna described above.
the lar gene was also located on chromosome  <dig> tc03_g <dig>  genbank: gu <dig> ) and shares  <dig> % identity to its corresponding cdna.
however, two more distantly related genes were identified in the cacao genome assembly that were not identified in the cacao est collection.
one gene  encodes a protein that shares only 37% identity and 57% similarity with the tcans protein and a second  shares 63% identity and 76% similarity with the tclar protein.
for this study, we focused our further work on the three genes of highest similarity to the well-characterized corresponding arabidopsis and desmodium genes.
lar and anr proteins belong to the reductase-epimerase-dehydrogenase  superfamily, although their relationships are rather distant.
ans belongs to a different protein superfamily, the 2-oxoglutarate-dependent dioxygenase  superfamily, although it shares the same substrate with lar .
a phylogenetic tree was constructed using the neighbor-joining method with the sequences of functionally-tested proteins of lar, anr, ans and dfr from various plant species.
the tree construction also included all the ifr-like proteins from arabidopsis that are most closely related to lar proteins .
the tree was noticeably bifurcated into two clades: all red proteins  constituting one clade, and all ans proteins constituting the other.
within the red superfamily clade, the ifr, lar, anr and dfr proteins are clearly divided into four distinct groups with ifr and lar forming a subgroup that is distantly related to the subgroup formed by dfr and anr groups.
the cacao anr, ans and lar proteins used in the current research all clustered within their own groups.
expression profiles of tcanr, tcans and tclar genes
to assess the involvement of tcanr, tcans and tclar in pa biosynthesis in cacao, transcript levels were investigated in various tissues including leaves, flowers and pod tissues .
gene transcripts levels were assessed by semi-quantitative rt-pcr.
tcactin was chosen as a reference to normalize gene expression because both cacao microarray analyses  and data from this study suggested a relatively constant spatial and temporal expression of this gene for all cacao tissues examined.
pa synthesis genes, tcanr, tcans and tclar were expressed in all tissues examined, with relatively higher expression levels in pod exocarp and seeds and lower expression in leaves and flowers.
moreover, their expression levels were relatively similar within each tissue except seeds, in which the expression level of tclar was much lower than tcanr and tcans.
pa levels in various cacao tissues
to determine the concentrations of pas in different cacao tissues, samples were first extracted to obtain a soluble pa fraction.
the residues left after soluble pa extraction were assayed using butanol-hcl  to measure the amount of insoluble pas represented as larger polymers.
because insoluble pa polymers will crystallize and bind to proteins and cell wall components, this interference may reduce the extraction efficiency of the insoluble pas  <cit> .
as a result, comparing the relative amount of these two fractions within the same tissue is difficult.
however, the accumulation pattern of each fraction is comparable among different tissues.
high levels of both soluble and insoluble pas were detected in all tissues examined, ranging from approximately 40 mg/g dw in seeds to more than 170 mg/g dw in exocarp for soluble pas  and ranging from approximately  <dig>  mg/g dw in seeds to 8 mg/g dw in exocarps for insoluble pas .
the accumulation of both soluble and insoluble pas were in good correlation with the expression patterns of pa synthesis genes, both of which showed highest levels in fruit exocarp.
however, both soluble and insoluble pa levels were much lower in seeds than in other tissues, which correlated with tclar but not tcanr and tcans.
to determine the composition of monomer pas in cacao, soluble pas extracts prepared from all four tissues were further separated and quantified by hplc.
the data are presented for catechin and epicatechin concentrations as μg of catechin or epicatechin per g of dry tissue .
pas consisted almost entirely of epicatechin with less than 2% catechin.
in all tissues examined, the accumulation of both catechin and epicatechin were well correlated with the expression of tclar and tcanr.
in exocarp, where both tclar and tcanr had the highest expression among all tissues tested, both catechin and epicatechin were also at maximal levels.
in leaves and flowers, where tcanr expression was much lower, epicatechin was found at correspondingly lower levels.
likewise in seeds, which exhibited a much lower expression of tclar, catechin levels were also much lower.
functional analysis of the tcanr gene
while the sequences and expression patterns of the candidate cacao pa biosynthesis genes were consistent with their candidate functions relative to enzymes from other species, we conducted in vivo functional analysis of each gene in transgenic plants to gain direct evidence for their functions.
to investigate the in vivo function of tcanr, a genetic complementation experiment was performed by transferring the tcanr coding sequence under the control of an enhanced expression promoter  into the arabidopsis banyuls  mutant, which is defective in the gene encoding anr  <cit> .
ban mutant seeds are pale yellow due to a lack of pas, and there is more purple anthocyanin pigment deposited in hypocotyls compared to arabidopsis  control plants due to increased metabolic flux into this branch of the pathway.
from eight independent transgenic tcanr-overexpressing lines tested, five lines showed white hypocotyls, and  <dig> lines showed reduced pigmentation in hypocotyls as compared to banyuls mutants.
the lines with white hypocotyls also produced seeds exhibiting the wild-type phenotype that stained blue with dmaca reagent, suggesting the deposition of pas in the seed coat .
after pa extraction and quantification, all lines showed significantly increased levels of pas .
rt-pcr analysis confirmed expression of tcanr, and the expression levels positively correlated with pa accumulation .
functional analysis of tcans gene
to investigate the in vivo function of tcans, two model plants, tobacco and arabidopsis were utilized.
we used an arabidopsis ans  mutant to perform tests of transgenic complementation.
we used tobacco as a model system for metabolic flux analysis of the equilibrium between pa and anthocyanin synthesis.
introduction of functional ans genes into tobacco can result in flower petal color changes reflecting alterations in ans activity.
ans is involved in both pa synthesis and anthocyanin synthesis, as cyanidin, an anthocyanin precursor, can also be reduced to epicatechin by anr .
this was demonstrated in a recent study describing that down regulation of mtans in medicago resulted in decreased levels of both pas and anthocyanins  <cit> .
based on these results, we reasoned that if the putative cacao ans protein is truly a functional ans, over-expression of tcans in tobacco should result in increased accumulation of both anthocyanins and pas.
the orf of tcans, driven by the e12Ω promoter, was introduced into wild-type tobacco  for constitutive ectopic expression.
twelve independent hygromycin-resistant lines were generated, of which nine showed a visible increase in pink color intensity in flower petals.
the two lines displaying the greatest increase in petal color were chosen for further analysis.
rt-pcr analysis confirmed high tcans transcript levels in these two tobacco transgenic lines, which positively correlated with the color of the petals .
amplification of the tobacco ribosomal rna gene ntrrna, which served as an internal control, showed a relatively similar expression level in both wild-type control and transgenic tobacco plants.
as predicted, anthocyanin levels increased in the two transgenic lines .
the levels of pas in the petals of transgenic lines, quantified by dmaca assays, were also significantly higher as compared to untransformed samsun plants .
on average, a two-fold increase of pa was observed in the two lines compared to wild type.
the 35s-tcans transgene was also introduced into the arabidopsis ans  t-dna mutant, which produces hypocotyls that appear white to light green due to lack of anthocyanins, and seeds that appear light yellow due to lack of pas.
eighteen independent hygromycin resistant transgenic t <dig> seedlings were selected.
from these,  <dig> lines developed wild-type purple colored hypocotyls  and produced wild-type brown-colored seeds that stained blue after reacting with the dmaca reagent.
the color suggested deposition of pas in the seed coat, however the color intensity was lower than in wild type.
rt-pcr using rna extracted from t <dig> seedlings confirmed expression of tcans genes .
functional analysis of the tclar gene
because the lar gene is not present in arabidopsis  <cit> , we cannot obtain direct evidence of the in vivo function of tclar through genetic complementation analysis in arabidopsis.
therefore, to functionally characterize tclar, we used tobacco as a model system.
as over-expression of the anr gene can divert the metabolic flow from anthocyanin synthesis to pa synthesis  <cit> , we predicted that over-expression of the cacao lar gene in tobacco would result in a decrease in anthocyanin pigment and an increase in pa accumulation in flower petals.
transgenic tobacco plants were generated that constitutively expressed the orf of tclar under the control of the e12Ω promoter.
twenty-two independent transgenic lines that are resistant to hygromycin were generated.
nine of these exhibited a decrease in intensity of the visible pink color of petals.
two lines  exhibited virtually white petals .
rt-pcr showed that both lines expressed high levels of tclar transcripts .
quantification of soluble pa and anthocyanin levels indicated that anthocyanin levels in these two lines were about half those of wt controls, and that total pas accumulated to an approximately 5-fold higher level than in controls .
moreover, the levels of pas in transgenic tobacco petals were inversely proportional to the concentrations of anthocyanin, indicating diversion of metabolic flow from anthocyanin to pa synthesis.
insoluble pa levels were also estimated, but the levels were insignificant .
to confirm the increased accumulation of catechin, as it is the predicted product of tclar, we separated and quantified the pa extracts using hplc.
the hplc analysis showed that there is indeed an approximately 2-fold increase of catechin levels in both transgenic lines.
surprisingly, there is also a significant increase of epicatechin levels .
we also took advantage of the arabidopsis ans  t-dna mutant to examine lar function.
because arabidopsis lacks an lar gene  <cit>  and the ldox mutant is deficient in cyanidin , the ldox mutant exhibits a significant decrease of epicatechin and pa synthesis.
we reasoned that since the ldox mutant accumulates leucoanthocyanidin which can provide the substrate for a heterologous lar protein, the ldox mutant was potentially a good in vivo model to analyze lar function.
we predicted that over-expression of tclar should result in the synthesis of catechin in developing siliques of the ldox mutant arabidopsis, even if pas were not produced due to lack of epicatechin synthesis.
hplc separation and quantification of pa extracts from arabidopsis transgenic lines over-expressing tclar gene in the ldox mutant background revealed not only a significant increase of catechin, but also surprisingly, a modest increase of epicatechin .
quantification of total pas extracted from mature seeds also revealed significant pas increases in all transgenic lines compared to the ldox mutant .
these unexpected results prompted us to check the possibility that tclar may carry a dual enzymatic activity, capable of converting leucoanthocyanidin to both catechin and epicatechin.
thus, recombinant tclar protein was expressed in e. coli, purified and assayed using 3h -labeled leucocyanidin as substrate in the presence of nadph, followed by analysis of products by hplc-uv and radioactivity detection.
the negative control reaction  showed no product formation , whereas a peak with the same retention time and uv-spectrum as that of the pure -catechin standard  was detected when tclar protein was incubated with 3h -labeled leucocyanidin .
however, no epicatechin product was detected , suggesting that tclar does not exhibit duel functionality in this in vitro assay.
discussion
the distribution of cacao pa accumulation correlates with expression of the pa biosynthesis genes
the localization of pas in plant tissues has been well studied in the model plants arabidopsis, the legume medicago truncatula, and in the fruit species grape .
in arabidopsis, the expression of the atanr  gene and pa accumulation is limited to the seed coat  <cit> .
similarly in medicago the major localization of pas is also in the seed coat with very small amounts present in flowers, leaves, roots and stems  <cit> .
although theobroma cacao and arabidopsis are phylogenetically closely related, the pa accumulation profiles are quite different.
our results demonstrated that in cacao leaves, flowers and fruits, pas accumulate at high levels and this is correlated with expression of genes encoding the key enzymes involved in pa synthesis.
this is similar to vitis, in which both pa accumulation and genes involved in pa synthesis are found in leaves, fruit skins and seeds  <cit> .
in cacao, the expression of ans, anr, and lar were co-regulated and their expression correlated well with pa accumulation in most of the tissues, suggesting significant roles in pa synthesis for both anr and lar.
the only exception is in seeds, in which the expression level of tcans is higher than tcanr and tclar is much lower.
the high expression level of tcans may contribute to anthocyanin synthesis, because at the time when seed tissues were harvested , anthocyanins are actively synthesized  <cit> .
the pa level in seeds is also much lower than in other tissues, well correlated with the tclar but not tcanr.
the lar enzyme seems to be a rate-limiting factor of the pa synthesis.
to further explore the individual contributions of these proteins to catechin and epicatechin synthesis, flavan-3-ol monomer composition was examined by hplc analysis.
cacao flavan-3-ol monomers were composed almost entirely of epicatechin units with catechin units comprising less than 2% in all tissues examined.
this result is consistent with aprevious study of pa synthesis in cacao beans  <cit> , in which pa extension units were reported to be composed exclusively of epicatechin, and terminal units were mostly epicatechin with about 1% catechin.
similarly, medicago pas consist almost entirely of epicatechin units  <cit> .
in grape, both anr  and lars  contribute to pa synthesis in the fruits, in which pas consist of both catechin and epicatechin.
however, in grape leaves, although catechin is still present, lar genes are expressed at very low levels.
functional characterization of cacao pa biosynthetic genes ans, anr and lar
in vivo genetic analysis of tcans and tcanr verified their roles in pa biosynthesis.
over-expression of tcans in tobacco resulted in elevated levels of both anthocyanins and pas in flower petals.
it also restored anthocyanin synthesis in hypocotyls as well as pa accumulation in seeds of the arabidopsis ans  mutant.
similarly, over-expression of tcanr in the arabidopsis banyuls  mutant restored pa synthesis in seeds.
moreover, ectopic over-expression of tcanr in hypocotyls diverted the anthocyanin synthesis branch and resulted in decreased anthocyanin pigments related to gene expression levels.
the catalytic activities of lar proteins from grapevine, medicago and desmodium have been verified by in vitro assays of recombinant proteins  <cit> .
lotus lar showed weak activity in a recombinant dfr protein coupled assay  <cit> .
however, none of the enzyme assays was able to recover sufficient flavan-3-ol products for stereo chemical analysis to allow specific determination of catechin and epicatechin.
the in vivo activities of medicago and desmodium lar proteins was also tested in tobacco and white clover  <cit> .
in the former study  <cit> , the authors found that transgenic plants expressing the dular protein showed slightly elevated lar enzymatic activity compared to extracts from untransformed plants, but they did not demonstrate changes in metabolite synthesis.
in a more recent study of mtlar overexpresing transgenic tobacco plants, there were no detectable changes in anthocyanin and pa levels  <cit> .
the results presented in this work shows that a cacao tclar gene is co-regulated with tcanr in all tissues examined, providing a simple model to investigate lar enzyme functions.
in our results, tclar showed clear in vivo activities in both transgenic tobacco and arabidopsis plants by converting metabolic flow from anthocyanin synthesis to pa synthesis.
this data provides a direct genetic evidence for a clear role of lar in pa biosynthesis.
the metabolic flow divergence resulting from tclar over-expression could be due to perturbations of a hypothesized metabolic channeling mechanism that suggests that multiple enzymes in each subsequent step of the synthesis pathway interact as a means to increase the efficiency and throughput of the pathway  <cit> .
to our surprise, ectopic expression of tclar resulted in elevated levels of both catechin and epicatechin units in tobacco flowers  in contrast to the prediction that only catechin should be formed.
based on the sequence similarity between lar and anr, it is possible that lar could function redundantly to anr and convert cyanidin to epicatechin.
alternatively, lar may perform as a dual functional enzyme and convert leucoanthocyanidin to both catechin and epicatechin.
to test the possibility that lar performs the same activity as anr and uses cyanidin as a substrate to form epicatechin, we took advantage of the arabidopsis ldox  mutant.
since arabidopsis does not have an lar gene and synthesizes only epicatechin through ans and anr pathway, the ldox mutants have a significantly reduced pa  level due to lack of a supply of cyanidin, the epicatechin precursor.
thus, the ldox mutant provides a system that has leucoanthocyanidin but not cyanidin.
over-expression of tclar in the ldox mutant resulted in synthesis of catechin confirming its predicted enzymatic function.
however, hplc quantification showed that there was only a slight elevation of epicatechin levels compared to the significant elevation of catechin, suggesting that lar was less likely to perform dual function and convert leucoanthocyanidin to epicatechin.
when recombinant tclar was assayed in vitro, while we observed the production of catechin from  <dig> -trans- <dig> -cis-leucoanthocyanidin, we did not detect even trace amounts of epicatechin.
the failure to detect epicatechin in assays with the cacao lar enzyme suggests that it functions solely in converting leucoanthocyanidin to catechin.
however, it is possible that lar enzyme can synthesize epicatechin from  <dig> -cis- <dig> -trans-leucocyanidin as suggested by  <cit> ; but we could not test this possibility in the recombinant tclar enzyme assay because  <dig> -cis- <dig> -trans-leucocyanidin is not commercially available.
the increase in epicatechin in tclar over-expressing arabidopsis and tobacco plants could be due to the possible existence of a gene encoding a catechin-epicatechin epimerase.
one possible candidate gene that could be involved is anr.
vitis vinifera anr protein expressed and purified from e.coli showed epimerase activities in vitro and converted cyanidin to a mixture of catechin and epicatechin  <cit> .
similarly, two recombinant anr proteins cloned from camellia sinensis also showed epimerase activities in vitro <cit> .
however, we regard this as a remote possibility because arabidopsis itself does not synthesize catechin and thus it would be surprising if a catechin-epicatechin-specific epimerase were expressed in the absence of its substrate.
nevertheless, we cannot exclude the possible existence of such an epimerase that may also serve other functions important in plant development.
a second alternative hypothesis to explain epicatechin formation would be racemization of catechins by polymerization to proanthocyanidins followed by nonstereo-specific depolymerization.
an earlier report lends some support to this idea through the analysis of transgenic apple lines in which the mdans gene was silenced.
the transgenic lines showed a drastic reduction in anthocyanins together with significant increase of both catechin and epicatechin, concomitant with an increase of catechin derived pa polymers and a decrease in epicatechin derived polymers  <cit> .
based on these observations, the authors of this study suggested that the increased epicatechins were derived from non-stereospecific depolymerization of proanthocyanidins.
a third possible explanation for our results is the hypothesis that increased production of catechin in the transgenic plants alters the metabolic equilibrium in such a way as to increase flux through the epicatechin branch as a feedback mechanism to balance the relative amounts of catechin and epicatechin.
it is known that dular activity can be inhibited by catechin with an ic <dig> as low as 12 μm  <cit> .
feedback and feedforward mechanisms also exist at a transcriptional level in the flavonoid pathway.
for example, a bean chalcone synthase  promoter can be stimulated by low concentrations of trans-cinnamic acid , the first intermediate of the phenylpropanoid pathway while its activity can be repressed by high concentrations of ca  <cit> .
on the other hand, high concentrations of trans-p-coumaric acid, the second intermediate of the phenylpropanoid pathway, can still stimulate the chs promoter  <cit> .
it seem likely that a complex web of homeostatic mechanisms function to control the flux through this entire pathway and elucidation of these mechanisms remains a major objective of this field for the future.
CONCLUSIONS
we successfully isolated three genes encoding key pa synthesis enzymes in theobroma cacao, tcans, tcanr, and tclar.
in vivo genetic analysis of tcans and tcanr in tobacco and complementation in arabidopsis verified their roles in pa biosynthesis.
in vitro enzyme assays of tclar recombinant protein verified its predicted function.
moreover, in vivo overexpressing tclar in tobacco and arabidopsis ldox mutants successfully diverted metabolic flow to pa syntheis, which provide direct evidence for a clear role of the tclar gene in pa biosynthesis.
our results provide new knowledge and genetic tools for development of cacao varieties with novel pa profiles through conventional breeding or genetic approaches.

 flavonoids are a diverse group of plant secondary metabolites with various biological functions that play important roles during plant development. they are involved in plant defenses against insects, pathogens and microbes, in absorption of free radicals and uv light, and in attraction of beneficial symbionts and pollinators. proanthocyanidins are components of metabolites synthesized through the general flavonoid pathway. their main known function is to provide protection against microbial pathogens and invasions of insects and herbivores through multiple mechanisms. pas have metal chelating activity that results in severe limitation of bacterial growth; pas can also associate with and irreversibly precipitate proteins, which is responsible for the astringent taste that repels herbivores. furthermore, pas can be oxidized to quinones which not only are powerful antibiotics themselves, but also can initiate cross-linking of cell walls to increase the strength of this physical barrier to pathogens. in addition to these functional roles in plants, pas, especially those from cacao, have recently been suggested to be beneficial to humans by improving cardiovascular health through activation of nitric oxide synthase, by providing cancer chemopreventative effects, and also through neuroprotective activities. the biosynthetic pathways that lead to the production of flavan-3-ols -catechin and -epicatechin), the building blocks of pas, have been well studied in the model plant species maize and arabidopsis as summarized in figure . biosynthesis of flavan-3-ols involves three principal enzymes: leucoanthocyanidin reductase, anthocyanidin synthase, and anthocyanidin reductase. the synthesis of pas and anthocyanins share common steps leading to flavan- -diols, which can be converted to catechin by lar or to anthocyanidin by ans. anthocyanidin then either serves as the substrate for the synthesis of epicatechin by anr, or can otherwise be converted to anthocyanin by glycosylation. both catechin and epicatechin act as the initiators for pa polymerization, with intermediates derived from leucoanthocyanidin, catechin or epicatechin added sequentially as extension units. however, the details of the polymerization process are unclear and it is not known whether this is a spontaneous or an enzyme-catalyzed reaction. recent work has identified two new enzymes downstream of flavan-3-ols that are involved in key steps of pa polymer biosynthesis, an epicatechin 3’-o-glucosyltransferase in medicago truncatula and mate transporters from arabidopsis and medicago truncatula that transport epicatechin 3’-o-glucoside to the vacuole in which it is likely that pa polymerization occurs. ans and anr genes have been biochemically and genetically characterized in arabidopsis, medicago truncatula and vitis vinifera. the arabidopsis ans mutant exhibits a deficiency in both anthocyanin accumulation in hypocotyls and pa deposition in seeds that results in a transparent testa phenotype. seeds of the arabidopsis banyuls mutant exhibit a lack of pas and a hyper-accumulation of anthocyanins, resulting in a dark red color reminiscent of the famous banyuls wine produced in southern france. over-expression of anr genes from medicago and grape in tobacco results in a loss of anthocyanin pigments in flower petals and elevated levels of pas. antisense down-regulation of ans in medicago results in reduction of both anthocyanins in leaves and pas in seeds. lar genes have been isolated from various plant species including desmodium uncinatum, vitis vinifera, lotus corniculatus and medicago truncatula and their corresponding protein functions have been characterized by in vitro recombinant enzyme assays. however, the genetic evidence for lar function is rather indirect and less convincing than it is for anr and ans, as discussed by pang et al.. it appears that the genomic sequence of arabidopsis thaliana does not contain an intact lar orthologue, and correspondingly, catechin is not detected in arabidopsis seed extracts. lar genes are expressed in other plant species that accumulate not only epicatechin but also catechin. for example, grape and lotus express both lar and anr genes and synthesize pas consisting of both catechin and epicatechin. in medicago, although both lar and anr are expressed, the pas are composed almost entirely of epicatechin. in arabidopsis and medicago, pa accumulation and gene expression is quantitatively and spatially limited to seed coats, making it remarkably difficult for biochemical analysis. in contrast, theobroma cacao produces significant amounts of pas in various tissues including leaves and beans; up to 12% of dry weight in leaves and approximately 10% in mature beans. furthermore, large amounts of catechin and epicatechin monomers as well as their related polymers of different lengths have been detected in cocoa powder. considering the wide range of health benefits suggested for pas and its significance for plant resistance, we targeted this pathway for molecular-genetic analysis. this manuscript describes the isolation and expression of the tcanr, tcans, and tclar genes encoding the key enzymes in proanthocyanidin biosynthesis. we measured pa content in different cacao tissues and performed functional characterization of the tcanr, tcans, and tclar gene products through in vivo tests. the results presented here provide background and genetic tools that will be useful in the development of new cacao varieties with altered pa profiles.  molecular cloning and sequence analysis of tcanr, tcans and tclar genes the general pathway for the synthesis of proanthocyanidin and anthocyanins indicates the key enzymes anr, ans and lar that carry out the biochemical steps at a critical metabolic branch point. to explore the genetic control of this important pathway in cacao, putative tcanr, tcans and tclar cdna sequences were identified in a collection of theobroma cacao expressed sequence tags by querying the cacao esttik database with protein sequences of arabidopsis banyuls, arabidopsis ldox and desmodium lar. ests similar to each gene were assembled into contigs to determine consensus full-length open reading frames by alignment with cdnas of homologous genes from other species and predictions from the orf finder program. full-length cdnas of each gene were amplified by reverse transcription-pcr using rna isolated from young leaves of cacao. the tcanr cdna contained a -bp open reading frame encoding a protein of amino acids that showed a 63% identity with the arabidopsis banyuls gene at the amino acid level. the tcans cdna contained an orf of -bp, which encodes a protein of amino acids with 82% amino acid identity with the arabidopsis ldox gene. the tclar cdna contained a -bp orf encoding a protein of amino acids with 61% amino acid identity with the desmodium lar protein. to determine the genomic sequences of the putative cacao ans, anr and lar genes, the cdnas were used to screen a cacao bac library by hybridization. a portion of each hybridizing bac clone was sequenced using primers designed from the corresponding cdnas. the genomic structure of each gene was established by alignment with its cdna sequence. we also retrieved gene models of the ans and anr gene from arabidopsis and lar from medicago and grape and compared them with the corresponding cacao genes. the coding region of tcanr consisted of exons and introns distributed over -bp. the coding region of tcans was shorter, having only -bp and consisting of exons and intron. the genomic organizations of these two cacao genes were nearly identical to the corresponding arabidopsis genes, which had the same exon and intron numbers. moreover, the lengths of the exons were very similar between arabidopsis and cacao: for anr, the numbers of nucleotides were precisely the same for the exons and although the lengths of introns were more variable. since lar does not have an orthologue in arabidopsis, we compared the genomic organization of tclar with mtlar and vvlar. the genomic organization of the coding region of tclar was similar to both mtlar and vvlar, consisting of exons and introns. as observed with the anr gene, the middle three exons of lar from all three species had identical lengths. tclar exhibited an extremely long third intron of  bp; similarly, vvlar also featured a long third intron of  bp, while mtlar contained two long introns that are 812-bp and -bp respectively. the analysis of the complete cacao genome sequence of the criollo variety was consistent with these results and allowed us to localize the position of each gene relative to the molecular-genetic map. the anr gene was located on chromosome and was identical to the cdna described above. similarly, a single ans gene was located on chromosome and shared % identity to the cdna described above. the lar gene was also located on chromosome tc03_g genbank: gu ) and shares % identity to its corresponding cdna. however, two more distantly related genes were identified in the cacao genome assembly that were not identified in the cacao est collection. one gene encodes a protein that shares only 37% identity and 57% similarity with the tcans protein and a second shares 63% identity and 76% similarity with the tclar protein. for this study, we focused our further work on the three genes of highest similarity to the well-characterized corresponding arabidopsis and desmodium genes. lar and anr proteins belong to the reductase-epimerase-dehydrogenase superfamily, although their relationships are rather distant. ans belongs to a different protein superfamily, the 2-oxoglutarate-dependent dioxygenase superfamily, although it shares the same substrate with lar. a phylogenetic tree was constructed using the neighbor-joining method with the sequences of functionally-tested proteins of lar, anr, ans and dfr from various plant species. the tree construction also included all the ifr-like proteins from arabidopsis that are most closely related to lar proteins. the tree was noticeably bifurcated into two clades: all red proteins constituting one clade, and all ans proteins constituting the other. within the red superfamily clade, the ifr, lar, anr and dfr proteins are clearly divided into four distinct groups with ifr and lar forming a subgroup that is distantly related to the subgroup formed by dfr and anr groups. the cacao anr, ans and lar proteins used in the current research all clustered within their own groups. expression profiles of tcanr, tcans and tclar genes to assess the involvement of tcanr, tcans and tclar in pa biosynthesis in cacao, transcript levels were investigated in various tissues including leaves, flowers and pod tissues. gene transcripts levels were assessed by semi-quantitative rt-pcr. tcactin was chosen as a reference to normalize gene expression because both cacao microarray analyses and data from this study suggested a relatively constant spatial and temporal expression of this gene for all cacao tissues examined. pa synthesis genes, tcanr, tcans and tclar were expressed in all tissues examined, with relatively higher expression levels in pod exocarp and seeds and lower expression in leaves and flowers. moreover, their expression levels were relatively similar within each tissue except seeds, in which the expression level of tclar was much lower than tcanr and tcans. pa levels in various cacao tissues to determine the concentrations of pas in different cacao tissues, samples were first extracted to obtain a soluble pa fraction. the residues left after soluble pa extraction were assayed using butanol-hcl to measure the amount of insoluble pas represented as larger polymers. because insoluble pa polymers will crystallize and bind to proteins and cell wall components, this interference may reduce the extraction efficiency of the insoluble pas. as a result, comparing the relative amount of these two fractions within the same tissue is difficult. however, the accumulation pattern of each fraction is comparable among different tissues. high levels of both soluble and insoluble pas were detected in all tissues examined, ranging from approximately 40 mg/g dw in seeds to more than 170 mg/g dw in exocarp for soluble pas and ranging from approximately  mg/g dw in seeds to 8 mg/g dw in exocarps for insoluble pas. the accumulation of both soluble and insoluble pas were in good correlation with the expression patterns of pa synthesis genes, both of which showed highest levels in fruit exocarp. however, both soluble and insoluble pa levels were much lower in seeds than in other tissues, which correlated with tclar but not tcanr and tcans. to determine the composition of monomer pas in cacao, soluble pas extracts prepared from all four tissues were further separated and quantified by hplc. the data are presented for catechin and epicatechin concentrations as μg of catechin or epicatechin per g of dry tissue. pas consisted almost entirely of epicatechin with less than 2% catechin. in all tissues examined, the accumulation of both catechin and epicatechin were well correlated with the expression of tclar and tcanr. in exocarp, where both tclar and tcanr had the highest expression among all tissues tested, both catechin and epicatechin were also at maximal levels. in leaves and flowers, where tcanr expression was much lower, epicatechin was found at correspondingly lower levels. likewise in seeds, which exhibited a much lower expression of tclar, catechin levels were also much lower. functional analysis of the tcanr gene while the sequences and expression patterns of the candidate cacao pa biosynthesis genes were consistent with their candidate functions relative to enzymes from other species, we conducted in vivo functional analysis of each gene in transgenic plants to gain direct evidence for their functions. to investigate the in vivo function of tcanr, a genetic complementation experiment was performed by transferring the tcanr coding sequence under the control of an enhanced expression promoter into the arabidopsis banyuls mutant, which is defective in the gene encoding anr. ban mutant seeds are pale yellow due to a lack of pas, and there is more purple anthocyanin pigment deposited in hypocotyls compared to arabidopsis control plants due to increased metabolic flux into this branch of the pathway. from eight independent transgenic tcanr-overexpressing lines tested, five lines showed white hypocotyls, and lines showed reduced pigmentation in hypocotyls as compared to banyuls mutants. the lines with white hypocotyls also produced seeds exhibiting the wild-type phenotype that stained blue with dmaca reagent, suggesting the deposition of pas in the seed coat. after pa extraction and quantification, all lines showed significantly increased levels of pas. rt-pcr analysis confirmed expression of tcanr, and the expression levels positively correlated with pa accumulation. functional analysis of tcans gene to investigate the in vivo function of tcans, two model plants, tobacco and arabidopsis were utilized. we used an arabidopsis ans mutant to perform tests of transgenic complementation. we used tobacco as a model system for metabolic flux analysis of the equilibrium between pa and anthocyanin synthesis. introduction of functional ans genes into tobacco can result in flower petal color changes reflecting alterations in ans activity. ans is involved in both pa synthesis and anthocyanin synthesis, as cyanidin, an anthocyanin precursor, can also be reduced to epicatechin by anr. this was demonstrated in a recent study describing that down regulation of mtans in medicago resulted in decreased levels of both pas and anthocyanins. based on these results, we reasoned that if the putative cacao ans protein is truly a functional ans, over-expression of tcans in tobacco should result in increased accumulation of both anthocyanins and pas. the orf of tcans, driven by the e12Ω promoter, was introduced into wild-type tobacco for constitutive ectopic expression. twelve independent hygromycin-resistant lines were generated, of which nine showed a visible increase in pink color intensity in flower petals. the two lines displaying the greatest increase in petal color were chosen for further analysis. rt-pcr analysis confirmed high tcans transcript levels in these two tobacco transgenic lines, which positively correlated with the color of the petals. amplification of the tobacco ribosomal rna gene ntrrna, which served as an internal control, showed a relatively similar expression level in both wild-type control and transgenic tobacco plants. as predicted, anthocyanin levels increased in the two transgenic lines. the levels of pas in the petals of transgenic lines, quantified by dmaca assays, were also significantly higher as compared to untransformed samsun plants. on average, a two-fold increase of pa was observed in the two lines compared to wild type. the 35s-tcans transgene was also introduced into the arabidopsis ans t-dna mutant, which produces hypocotyls that appear white to light green due to lack of anthocyanins, and seeds that appear light yellow due to lack of pas. eighteen independent hygromycin resistant transgenic t seedlings were selected. from these, lines developed wild-type purple colored hypocotyls and produced wild-type brown-colored seeds that stained blue after reacting with the dmaca reagent. the color suggested deposition of pas in the seed coat, however the color intensity was lower than in wild type. rt-pcr using rna extracted from t seedlings confirmed expression of tcans genes. functional analysis of the tclar gene because the lar gene is not present in arabidopsis, we cannot obtain direct evidence of the in vivo function of tclar through genetic complementation analysis in arabidopsis. therefore, to functionally characterize tclar, we used tobacco as a model system. as over-expression of the anr gene can divert the metabolic flow from anthocyanin synthesis to pa synthesis, we predicted that over-expression of the cacao lar gene in tobacco would result in a decrease in anthocyanin pigment and an increase in pa accumulation in flower petals. transgenic tobacco plants were generated that constitutively expressed the orf of tclar under the control of the e12Ω promoter. twenty-two independent transgenic lines that are resistant to hygromycin were generated. nine of these exhibited a decrease in intensity of the visible pink color of petals. two lines exhibited virtually white petals. rt-pcr showed that both lines expressed high levels of tclar transcripts. quantification of soluble pa and anthocyanin levels indicated that anthocyanin levels in these two lines were about half those of wt controls, and that total pas accumulated to an approximately 5-fold higher level than in controls. moreover, the levels of pas in transgenic tobacco petals were inversely proportional to the concentrations of anthocyanin, indicating diversion of metabolic flow from anthocyanin to pa synthesis. insoluble pa levels were also estimated, but the levels were insignificant. to confirm the increased accumulation of catechin, as it is the predicted product of tclar, we separated and quantified the pa extracts using hplc. the hplc analysis showed that there is indeed an approximately 2-fold increase of catechin levels in both transgenic lines. surprisingly, there is also a significant increase of epicatechin levels. we also took advantage of the arabidopsis ans t-dna mutant to examine lar function. because arabidopsis lacks an lar gene and the ldox mutant is deficient in cyanidin, the ldox mutant exhibits a significant decrease of epicatechin and pa synthesis. we reasoned that since the ldox mutant accumulates leucoanthocyanidin which can provide the substrate for a heterologous lar protein, the ldox mutant was potentially a good in vivo model to analyze lar function. we predicted that over-expression of tclar should result in the synthesis of catechin in developing siliques of the ldox mutant arabidopsis, even if pas were not produced due to lack of epicatechin synthesis. hplc separation and quantification of pa extracts from arabidopsis transgenic lines over-expressing tclar gene in the ldox mutant background revealed not only a significant increase of catechin, but also surprisingly, a modest increase of epicatechin. quantification of total pas extracted from mature seeds also revealed significant pas increases in all transgenic lines compared to the ldox mutant. these unexpected results prompted us to check the possibility that tclar may carry a dual enzymatic activity, capable of converting leucoanthocyanidin to both catechin and epicatechin. thus, recombinant tclar protein was expressed in e. coli, purified and assayed using 3h -labeled leucocyanidin as substrate in the presence of nadph, followed by analysis of products by hplc-uv and radioactivity detection. the negative control reaction showed no product formation, whereas a peak with the same retention time and uv-spectrum as that of the pure -catechin standard was detected when tclar protein was incubated with 3h -labeled leucocyanidin. however, no epicatechin product was detected, suggesting that tclar does not exhibit duel functionality in this in vitro assay. discussion the distribution of cacao pa accumulation correlates with expression of the pa biosynthesis genes the localization of pas in plant tissues has been well studied in the model plants arabidopsis, the legume medicago truncatula, and in the fruit species grape. in arabidopsis, the expression of the atanr gene and pa accumulation is limited to the seed coat. similarly in medicago the major localization of pas is also in the seed coat with very small amounts present in flowers, leaves, roots and stems. although theobroma cacao and arabidopsis are phylogenetically closely related, the pa accumulation profiles are quite different. our results demonstrated that in cacao leaves, flowers and fruits, pas accumulate at high levels and this is correlated with expression of genes encoding the key enzymes involved in pa synthesis. this is similar to vitis, in which both pa accumulation and genes involved in pa synthesis are found in leaves, fruit skins and seeds. in cacao, the expression of ans, anr, and lar were co-regulated and their expression correlated well with pa accumulation in most of the tissues, suggesting significant roles in pa synthesis for both anr and lar. the only exception is in seeds, in which the expression level of tcans is higher than tcanr and tclar is much lower. the high expression level of tcans may contribute to anthocyanin synthesis, because at the time when seed tissues were harvested, anthocyanins are actively synthesized. the pa level in seeds is also much lower than in other tissues, well correlated with the tclar but not tcanr. the lar enzyme seems to be a rate-limiting factor of the pa synthesis. to further explore the individual contributions of these proteins to catechin and epicatechin synthesis, flavan-3-ol monomer composition was examined by hplc analysis. cacao flavan-3-ol monomers were composed almost entirely of epicatechin units with catechin units comprising less than 2% in all tissues examined. this result is consistent with aprevious study of pa synthesis in cacao beans, in which pa extension units were reported to be composed exclusively of epicatechin, and terminal units were mostly epicatechin with about 1% catechin. similarly, medicago pas consist almost entirely of epicatechin units. in grape, both anr and lars contribute to pa synthesis in the fruits, in which pas consist of both catechin and epicatechin. however, in grape leaves, although catechin is still present, lar genes are expressed at very low levels. functional characterization of cacao pa biosynthetic genes ans, anr and lar in vivo genetic analysis of tcans and tcanr verified their roles in pa biosynthesis. over-expression of tcans in tobacco resulted in elevated levels of both anthocyanins and pas in flower petals. it also restored anthocyanin synthesis in hypocotyls as well as pa accumulation in seeds of the arabidopsis ans mutant. similarly, over-expression of tcanr in the arabidopsis banyuls mutant restored pa synthesis in seeds. moreover, ectopic over-expression of tcanr in hypocotyls diverted the anthocyanin synthesis branch and resulted in decreased anthocyanin pigments related to gene expression levels. the catalytic activities of lar proteins from grapevine, medicago and desmodium have been verified by in vitro assays of recombinant proteins. lotus lar showed weak activity in a recombinant dfr protein coupled assay. however, none of the enzyme assays was able to recover sufficient flavan-3-ol products for stereo chemical analysis to allow specific determination of catechin and epicatechin. the in vivo activities of medicago and desmodium lar proteins was also tested in tobacco and white clover. in the former study, the authors found that transgenic plants expressing the dular protein showed slightly elevated lar enzymatic activity compared to extracts from untransformed plants, but they did not demonstrate changes in metabolite synthesis. in a more recent study of mtlar overexpresing transgenic tobacco plants, there were no detectable changes in anthocyanin and pa levels. the results presented in this work shows that a cacao tclar gene is co-regulated with tcanr in all tissues examined, providing a simple model to investigate lar enzyme functions. in our results, tclar showed clear in vivo activities in both transgenic tobacco and arabidopsis plants by converting metabolic flow from anthocyanin synthesis to pa synthesis. this data provides a direct genetic evidence for a clear role of lar in pa biosynthesis. the metabolic flow divergence resulting from tclar over-expression could be due to perturbations of a hypothesized metabolic channeling mechanism that suggests that multiple enzymes in each subsequent step of the synthesis pathway interact as a means to increase the efficiency and throughput of the pathway. to our surprise, ectopic expression of tclar resulted in elevated levels of both catechin and epicatechin units in tobacco flowers in contrast to the prediction that only catechin should be formed. based on the sequence similarity between lar and anr, it is possible that lar could function redundantly to anr and convert cyanidin to epicatechin. alternatively, lar may perform as a dual functional enzyme and convert leucoanthocyanidin to both catechin and epicatechin. to test the possibility that lar performs the same activity as anr and uses cyanidin as a substrate to form epicatechin, we took advantage of the arabidopsis ldox mutant. since arabidopsis does not have an lar gene and synthesizes only epicatechin through ans and anr pathway, the ldox mutants have a significantly reduced pa level due to lack of a supply of cyanidin, the epicatechin precursor. thus, the ldox mutant provides a system that has leucoanthocyanidin but not cyanidin. over-expression of tclar in the ldox mutant resulted in synthesis of catechin confirming its predicted enzymatic function. however, hplc quantification showed that there was only a slight elevation of epicatechin levels compared to the significant elevation of catechin, suggesting that lar was less likely to perform dual function and convert leucoanthocyanidin to epicatechin. when recombinant tclar was assayed in vitro, while we observed the production of catechin from -trans- -cis-leucoanthocyanidin, we did not detect even trace amounts of epicatechin. the failure to detect epicatechin in assays with the cacao lar enzyme suggests that it functions solely in converting leucoanthocyanidin to catechin. however, it is possible that lar enzyme can synthesize epicatechin from -cis- -trans-leucocyanidin as suggested by; but we could not test this possibility in the recombinant tclar enzyme assay because -cis- -trans-leucocyanidin is not commercially available. the increase in epicatechin in tclar over-expressing arabidopsis and tobacco plants could be due to the possible existence of a gene encoding a catechin-epicatechin epimerase. one possible candidate gene that could be involved is anr. vitis vinifera anr protein expressed and purified from e.coli showed epimerase activities in vitro and converted cyanidin to a mixture of catechin and epicatechin. similarly, two recombinant anr proteins cloned from camellia sinensis also showed epimerase activities in vitro. however, we regard this as a remote possibility because arabidopsis itself does not synthesize catechin and thus it would be surprising if a catechin-epicatechin-specific epimerase were expressed in the absence of its substrate. nevertheless, we cannot exclude the possible existence of such an epimerase that may also serve other functions important in plant development. a second alternative hypothesis to explain epicatechin formation would be racemization of catechins by polymerization to proanthocyanidins followed by nonstereo-specific depolymerization. an earlier report lends some support to this idea through the analysis of transgenic apple lines in which the mdans gene was silenced. the transgenic lines showed a drastic reduction in anthocyanins together with significant increase of both catechin and epicatechin, concomitant with an increase of catechin derived pa polymers and a decrease in epicatechin derived polymers. based on these observations, the authors of this study suggested that the increased epicatechins were derived from non-stereospecific depolymerization of proanthocyanidins. a third possible explanation for our results is the hypothesis that increased production of catechin in the transgenic plants alters the metabolic equilibrium in such a way as to increase flux through the epicatechin branch as a feedback mechanism to balance the relative amounts of catechin and epicatechin. it is known that dular activity can be inhibited by catechin with an ic as low as 12 μm. feedback and feedforward mechanisms also exist at a transcriptional level in the flavonoid pathway. for example, a bean chalcone synthase promoter can be stimulated by low concentrations of trans-cinnamic acid, the first intermediate of the phenylpropanoid pathway while its activity can be repressed by high concentrations of ca. on the other hand, high concentrations of trans-p-coumaric acid, the second intermediate of the phenylpropanoid pathway, can still stimulate the chs promoter. it seem likely that a complex web of homeostatic mechanisms function to control the flux through this entire pathway and elucidation of these mechanisms remains a major objective of this field for the future.  we successfully isolated three genes encoding key pa synthesis enzymes in theobroma cacao, tcans, tcanr, and tclar. in vivo genetic analysis of tcans and tcanr in tobacco and complementation in arabidopsis verified their roles in pa biosynthesis. in vitro enzyme assays of tclar recombinant protein verified its predicted function. moreover, in vivo overexpressing tclar in tobacco and arabidopsis ldox mutants successfully diverted metabolic flow to pa syntheis, which provide direct evidence for a clear role of the tclar gene in pa biosynthesis. our results provide new knowledge and genetic tools for development of cacao varieties with novel pa profiles through conventional breeding or genetic approaches. 
to dissect the genetic basis of pa biosynthetic pathway in cacao, we have isolated three genes encoding key pa synthesis enzymes, anthocyanidin synthase, anthocyanidin reductase and leucoanthocyanidin reductase. we measured the expression levels of tcanr, tcans and tclar and pa content in cacao leaves, flowers, pod exocarp and seeds. our results confirm the in vivo function of cacao ans and anr predicted based on sequence homology to previously characterized enzymes from other species. in addition, our results provide a clear functional analysis of a lar gene in vivo. overexpression of tcans in an arabidopsis ldox mutant complemented its pa deficient phenotype in seeds. overexpressing tclar in arabidopsis ldox mutant also resulted in elevated synthesis of not only catechin but also epicatechin. overexpression of tcans in tobacco resulted in increased content of both anthocyanidins and pas in flower petals. pas have been associated with human health benefits and also play important roles in pest and disease defense throughout the plant. recombinant tclar protein converted leucoanthocyanidin to catechin in vitro. transgenic tobacco overexpressing tclar had decreased amounts of anthocyanidins and increased pas. in all tissues examined, all three genes were abundantly expressed and well correlated with pa accumulation levels, suggesting their active roles in pa synthesis.
 the proanthocyanidins, a subgroup of flavonoids, accumulate to levels of approximately 10% total dry weight of cacao seeds. pas have been associated with human health benefits and also play important roles in pest and disease defense throughout the plant.  to dissect the genetic basis of pa biosynthetic pathway in cacao, we have isolated three genes encoding key pa synthesis enzymes, anthocyanidin synthase, anthocyanidin reductase and leucoanthocyanidin reductase. we measured the expression levels of tcanr, tcans and tclar and pa content in cacao leaves, flowers, pod exocarp and seeds. in all tissues examined, all three genes were abundantly expressed and well correlated with pa accumulation levels, suggesting their active roles in pa synthesis. overexpression of tcanr in an arabidopsis ban mutant complemented the pa deficient phenotype in seeds and resulted in reduced anthocyanidin levels in hypocotyls. overexpression of tcans in tobacco resulted in increased content of both anthocyanidins and pas in flower petals. overexpression of tcans in an arabidopsis ldox mutant complemented its pa deficient phenotype in seeds. recombinant tclar protein converted leucoanthocyanidin to catechin in vitro. transgenic tobacco overexpressing tclar had decreased amounts of anthocyanidins and increased pas. overexpressing tclar in arabidopsis ldox mutant also resulted in elevated synthesis of not only catechin but also epicatechin.  our results confirm the in vivo function of cacao ans and anr predicted based on sequence homology to previously characterized enzymes from other species. in addition, our results provide a clear functional analysis of a lar gene in vivo. 