BACKGROUND
flavonoids are polyphenol antioxidants found naturally in plants, which possess key pharmacological activities, including antioxidant, antimutagenic, anticarcinogenic, and antibacterial properties  <cit> .
flavonoids in most higher plants can be divided into six major subgroups: chalcones, flavones, flavonols, flavan-3-ols , anthocyanins, and proanthocyanins   <cit> .
the structure of the flavonoid b ring is the primary determinant of the antioxidant activity of flavonoids  <cit> , and flavonoids can be divided into three subclasses according to the hydroxylation pattern of their b-ring, including b-ring 4′-hydroxylated, 3′4′-dihydroxylated, and 3′4′5′-trihydroxylated compounds.
the number of hydroxyl groups on the b-ring affects the capacity to inhibit lipid peroxidation  <cit> .
for instance, liu and yang reported that the antioxidant activity of epigallocatechin-3-gallate  is greater than that over epigallocatechin  at concentrations of up to 100 mg · l− <dig>  <cit> .
in the flavonoid biosynthesis pathway, the hydroxylation pattern of the b-ring is determined by two cytochrome p450-dependent monooxygenases : flavonoid 3′-hydroxylase  and flavonoid 3′,5′-hydroxylase .
hydroxylation of the 5′-position by f3′5′h is a particularly important step, which determines the b-ring tri-hydroxyl flavonoid end-product  formed in plants, as illustrated in figure  <dig> figure  <dig>
biosynthesis pathway and end-product accumulation of flavonoids in
camellia sinensis
.
biosynthesis pathway of flavonoids.
chs, chalcone synthase; chi, chalcone isomerase; f3h, flavanone 3-hydroxylase; f3′h, flavonoid 3′-hydroxylase; f3′5′h, flavonoid 3′,5′-hydroxylase; dfr, dihydroflavonol 4-reductase; fls, flavonol synthase; lar, leucoanthocyanidin reductase; ans, anthocyanidin synthase; anr, anthocyanidin reductase; ufgt, udp-glycose flavonoid glycosyltransferase; uggt, udp-glucose galloyl-1-o-β-d-glucosyltransferase; ecgt, epicatechins: 1-o-galloyl-β-d-glucose o-galloyltransferase;  relative quantity of different flavonoid compounds.
the data for relative quantity of different flavonoid compounds were quoted from jiang .
f3′5′hs have been previously cloned and functionally analyzed from multiple plants, including grape   <cit> , petunia , snapdragon   <cit> , cineraria   <cit> , tomato   <cit> , big leaf periwinkle   <cit> , and potato   <cit> .
through heterologous expression in transgenic plants and yeasts, f3′5′hs were shown to hydroxylate a broad range of flavonoid substrates, including naringenin , dihydrokaempferol , kaempferol  and apigenin  <cit> .
however, optimum substrates for these enzymes remain to be determined.
tea  is an important commercial crop, the leaves of which can be processed into popular nonalcoholic beverages.
because of the high flavonoid content, epidemiological and pathological studies have suggested that tea consumption may potentially be protective against human cancers  <cit>  and high blood pressure  <cit> , and contribute to weight reduction  <cit> .
the total concentration of flavonoid compounds is around 12–24% of tea leaf dry mass  <cit> .
we have previously shown that catechins are among the most abundant flavonoids in tea leaves, followed by proanthocyanidins , flavonols, flavones and anthocyanins   <cit> .
in recent years, some of the flavonoid structural and regulatory genes have been cloned, and functions of these genes have been investigated  <cit> .
while 4′-hydroxylated catechins are very rare or undetectable in tea leaves  <cit> , 3′4′5′-trihydroxylated catechins , egc, and egcg), are the most abundant flavonoids in young leaves and the stem, with significantly higher concentrations than 3′4′-dihydroxylated catechins , epicatechin  and ecg) .
therefore, characterizing the pattern of b-ring hydroxylation is clearly a valuable contribution to the understanding of flavonoid biosynthesis in tea plants.
however, it has not yet been possible to prepare active membrane-bound f3′5′h enzymes from camellia sinensis, and it is still unclear whether b-ring hydroxylation occurs at the level of flavanones and/or dihydroflavonols, in vivo.
aiming to analyze the in vivo expression pattern of csf3′5′h and to characterize the function of this gene in vitro, we isolated the csf3′5′h gene from tea cdna library.
we found that csf3′5′h was highly expressed in the bud, but little or no csf3′5′h was detected in the root.
csf3′5′h expression was enhanced by light and sucrose treatment, and over-expression of csf3′5′h resulted in production of delphinidin derivatives, producing redder flowers in transgenic tobacco plants, in comparison to with wild type.
heterologous expression of modified csf3′5′h in yeast revealed that 4′-hydroxylated flavanone  is the optimum substrate for csf3′5′h, and the ratio of 3′4′5′- to 3′4′-hydroxylated products in the modified csf3′5′h transgenic cells was significantly higher than in vvf3′5′h cells.
RESULTS
isolation and characterization of the csf3′5′h gene
the csf3′5′h gene  was successfully cloned from the cdna library of the 3rd tea leaf, and encoded  <dig> amino acid residues.
a blast search  performed with the coding sequence revealed  <dig>   <dig> and 81% identity with cyclamen persicum , cyclamen graecum  and vitis vinifera  genes, respectively.
the phylogenetic tree  was generated using protein sequences from several plant f3′5′h and f3′h enzymes retrieved from the ncbi database.
the tree demonstrated that f3′hs and f3′5′hs were grouped in cyp75b and cyp75a clusters, respectively.
csf3′5′h was grouped into the cyp75a subfamilies, and most closely related to the f3′5′h enzymes of cyclamen persicum, cyclamen graecum and vitis vinifera.figure  <dig>
phylogenetic tree for a selection of f3′5′h protein.
phylogenetic tree based on amino acid sequences of f3′hs and f3′5′hs in various plant species from the ncbi web page.
accession numbers are displayed in the figure.
bootstrap values  are shown at nodes.
expression pattern of csf3′5′h in tea
the expression pattern of csf3′5′h in tea was detected by qrt-pcr.
the gadph gene , expected to show a constitutive expression pattern, was used as control  <cit> .
csf3′5′h expression was tissue specific, expressed highly in leaves and stem , with transcripts peaking in the buds.
we also assessed substrate specificity of crude extracts from tea leaves, measuring hydroxylation of n and dihydroquercetin , which yielded eriodictyol  and dihydromyricetin , respectively .
the enzyme activities of these crude extracts were  <dig>  and  <dig>  pcat · g− <dig> protein, respectively.
surprisingly hydroxylation of n did not yield, 3′4′5′-hydroxylated product .figure  <dig>
expression of csf3′5′h in different tea tissues.
relative expression of csf3′5′h in different tea tissues analyzed by qrt-pcr and semi-quantitative rt pcr for csf3′5′h and gapdh in different tea tissues.
relative expression of csf3′5′h in different light and sucrose conditions analyzed by qrt-pcr and semi-quantitative rt pcr for gapdh and csf3′5′h in different light and sucrose conditions.
the data represent the mean ± sd from three independent measurements.
the different letters  and *indicated the significant level at p <  <dig> .
hplc chromatograms of flavanones or dihydroflavonols formation in csf3′5′h assays with tea leaf enzyme.
reaction assay with substrate n of heat-denatured protein in the control treatment ;  reaction assay with substrate n of the crude enzyme extract from the leave of tea;  reaction assay with substrate dhq of heat-denatured protein in the control treatment;  reaction assay with substrate dhq of the crude enzyme extract from the leave of tea.
interestingly, csf3′5′h transcripts were barely detected in the root, and the monomer and polymer of 3′ 4′ -dihydroxylated catechins , but no 3′ 4′ 5′-trihydroxylated catechins, accumulated in camellia sinensis roots  <cit> , indicating that extremely low csf3′5′h expression might directly lead to absence of b-ring tri-hydroxyl catechins in the root.
we used tissue culture seedlings, developed from the embryo of tea-seeds, to assess the direct influence of light and sucrose on csf3′5′h expression.
csf3′5′h expression levels in light-exposed and sucrose-induced seedlings were significantly increased by  <dig>  and  <dig> -fold, respectively , indicating that csf3′5′h expression can be efficiently induced by light and sucrose.
functional analysis of the csf3′5′h gene in nicotiana tabacum
the vector for constitutive expression of the 35s:csf3′5′h gene was introduced into tobacco ‘g28’ , which lacks f3′5′h genes and has pink flowers  <cit> .
about  <dig> independent transgenic tobacco plants were obtained.
most flowers from the transgenic plants exhibited a clear color change from pale pink of the host to magenta .figure  <dig>
flower color after overexpression of csf3′5′h and qrt-pcr of transgenic tobacco plants.
tobacco flowers of wild-type  and csf3′5′h transgenes .
tobacco flowers of wild-type , csf3′5′h transgenes and qrt-pcr for csf3′5′h in flowers from ck and transgenic lines.
hplc chromatograms of anthocyanidins  and flavonol glacosides  in tobacco flowers from ck and line  <dig> .
concentration of anthocyanidins in tobacco flowers from ck, csf3′5′h transgenes  and vector control.
the data represent the mean ± sd from three independent measurements.
qrt-pcr for flavonoid-related genes in tobacco flowers from ck, csf3′5′h transgenes  and vector control.
*indicated the significant level at p <  <dig> .
#indicated the significant level compared between every detected lines versus ck .
the expression of csf3′5′h in several transgenic lines with magenta flowers was detected by qrt-pcr, with β-actin  used as reference gene , and we found varying levels of csf3′5′h gene expression in glyphosate-resistant transgenic tobaccos.
to investigate whether the flavonoid biosynthesis pathway was affected by over-expression of csf3′5′h, the flavonoid pathway genes , chi , f3h , f3′h , dfr , fls , ans , anr , ufgt ) from nicotiana tabacum were examined by qrt-pcr in wild type  and transgenic lines.
the expression levels of chs, f3h, ans, anr, ufgt genes in transgenic lines significantly increased in comparison to the wild type and vector control , suggesting that expression of these genes was stimulated by the over-expression of csf3′5′h in transgenic lines.
the level of glycosylated flavonoids in flowers was assessed by reverse phase hplc and lc-ms. 3′,5′-hydroxylated flavonol glacoside  was detected in the petals of the transgenic lines, but not in wild-type tobaccos .
however, the concentration of myr in the flowers was too low to quantify .
petal pigments were extracted and chemically converted to anthocyanidins, for anglicizing the anthocyanin components by reverse phase hplc.
petals expressing the csf3′5′h gene contained a novel 3′,5′-hydroxylated anthocyanidin  and increased cyaniding  derivative content.
the ratio of delphinin to total anthocyanin compounds in transgenic tobacco plants reached a maximum of  <dig> % , and the average anthocyanin concentration in the petals of transgenic tobaccos was  <dig> -fold higher than in wild-type plants, suggesting that csf3′5′h encodes a protein with b-ring 3′, 5′-hydroxylation function, and that anthocyanin synthesis can be stimulated by csf3′5′h over-expression in transgenic lines.
heterologous csf3′5′h expression in yeast
the yeast strain saccharomyces cerevisiae wat <dig>  engineered to over-express the arabidopsis thaliana p <dig> reductase  <cit> , is a suitable heterologous host for p <dig> expression  <cit> .
a pyes-dest 52a csf3′5′h vector was transformed into wat <dig>  however, these transgenic cells did not produce functional f3′5h protein , so the codon optimized yeast csf3′5′h sequence  was designed and transformed into wat <dig>  resulting in only minimal activity of approximately  <dig>  pkat · l− <dig> culture, with n as substrate.figure  <dig>
optimization of
csf3′5′h
expression in
saccharomyces cerevisiae
‘wat11’.
comparison of amino acid sequences encoded by csf3′5′h and vvf3′5′h proteins.
the boxed regions represent fusion between csf3′5′h and vvf3′5′h.
primary structure schemes of expressed csf3′5′h sequence variants and resulting expression strength expressed as csf3′5′h activity · l− <dig> culture.
enzyme activity was expressed as pkat · l− <dig> culture.
the data represent the mean ± sd from three independent measurements.
transgenic cells, harboring the vitis vinifera f3′5′h  gene, achieved a high overall f3′5′h activity of  <dig>  pkat · l− <dig> culture with n as substrate.
with the predicted signal peptide, both f3′5′hs were translated into precursor proteins and delivered to the er.
we hypothesized that imperfect recognition of the camellia sinensis signal peptide might account for low expression levels detected in saccharomyces cerevisiae cells, and tested this hypothesis by fusing csf3′5′h with vvf3′5′h at three different points of the sequence based on amino acid sequence homology .
the 5′-sequences of ycsf3′5′h were replaced by vvf3′5′h at  <dig> aa ,  <dig> aa ,  <dig> aa  respectively.
vitis vinifera sequences were fused to ycsf3′5′h and cloned into the plasmid pyes-dest 52a for transformation of wat <dig> cells.
the cells containing fsi, replaced at signal and leader peptide region, led to high f3′5′h activity in the range of  <dig>  pkat · l− <dig> culture, a significant increase in comparison with the reference construct  .
these results indicated that csf3′5′h signal peptide might be imperfectly recognized in saccharomyces cerevisiae cells.
the cells transformed with fsii also resulted in f3′5′h activity, albeit significantly less .
generally, overall activities of chimeras are often low, e.g.
most chimeras between limonene 3-hydroxylase and limonene 6-hydroxylase achieve no, or less than 5% of that of wild type  <cit> .
unexpectedly, f3′5′h activity was undetected in cells transformed with fsiii.
in comparison to vvf3′5′h, the fsiii fusion gene was only altered at the 3′-terminal sequence.
finally, we assessed the substrate specificity of cells expressing fsi and vvf3′5′h.
based on previous findings and other intermediate compounds in the catechin synthesis pathway, we assessed catalysis of n, e, k, quercetin , dhk, dhq, pelargonidin , cya and c .
wat <dig> cells transformed with pyes-dest 52a vector were used as controls.
as observed with vvf3′5′h, fsi preferred b-ring 4′-hydroxylated compounds  to 3′, 4′-hydroxylated compounds .
no activity was detected with pel, cya and c as substrates, in both transgenic cells.
both proteins displayed highest activities with n and significant activities with k and dhk, yielding 3′4′- and 3′4′5′-forms as products.
interestingly, for fsi with n as substrate, the ratio of 3′4′5′- to 3′4′-hydroxylated products  was significantly higher than for vvf3′5′h .figure  <dig>
hplc chromatograms of products from pyes-dest52-
fs
and pyes-dest52-
vvf3′5′h
with flavanones, flavonols and dihydroflavonols as substrates.
hplc chromatograms of products from pyes-dest52- fsi with n , e , k , q , dhk  and dhq  as substrates; hplc chromatograms of products from pyes-dest52-vvf3′5′h with n , e , k , q , dhk  and dhq  as substrates; hplc chromatograms of products from control treatment with n , e , k , q , dhk  and dhq  as substrates.
accepted substrates and enzyme activity units for f3′5′h
substrate
modified-csf3′5′h
vvf3′5′h
class
3′-hydroxyla-tion product
5′-hydroxylat-ion product
3′-hydroxylat-ion product
5′-hydroxylat-ion product
enzyme activity was expressed as pkat · l− <dig> culture.
the data represent mean ± sd from three independent measurements.
— indicates results below the detection limit.
microsomes from wat <dig> cells transformed by pyes-dest 52a -fsi and -vvf3′5′h were assayed for nadph-dependent flavonoid 3′, 5′-hydroxylation with n, k and dhk as substrates.
no activity was detected with microsomes from the control, pyes-dest 52a-transformed cells.
in contrast the km values of the microsome extracted from fsi-transformed cells, with n, k, and dhk as substrates, were  <dig> ,  <dig> , and  <dig>  μm, respectively , indicating that n might be the optimum substrate for the csf3′5′h enzyme.
fsi achieved significantly higher km values than vvf3′5′h with k and dhk as substrates, but lower km values with n. however, the max reaction rates  for fsi and vvf3′5′h with n as substrate were significantly lower than the values with k and dhk as substrates.table  <dig>
comparison of steady-state kinetic parameters for cinnamate 4-hydroxylation in yeast microsomes
substrate
modified-csf3′5′h
vvf3′5′h
class
km
vmax
km
vmax
experiments were carried out in 50 mm phosphate buffer ph  <dig>  at 28°c for 30 min.
protein concentration of f3′5′h -transformed yeast microsomes was  <dig>  mg/ml in the reaction system.
the data represent the mean ± sd from three independent measurements.
concentration dependence of f3′5′h observed in yeast microsomes.
f3′5′h-containing microsomes originating from transformed pyes-dest52-fsi and -vvf3′5′h cells were incubated in 50 mm phosphate buffer ph  <dig>  at 28°c.
the solid line represents the result of a multi-iterative fitting of experimental data using the michaelis-menten equation.
insert: michaelis-menten double-reciprocal plot.
the data represent the mean ± sd from three independent measurements.
discussion
the role of csf3′5′h in catechin formation in tea leaves
all flavonoids are hydroxylated at the 4′ position of the b-ring.
b-ring hydroxylation patterns determie the color of anthocyanins and thus have been extensively investigated in ornamental plants for color engineering.
the f3′5′h gene is commonly known as the blue gene  <cit>  and previous studies have shown that f3′5′h catalyzes the hydroxylation at the 3′ and 5′ positions of flavonoids to determine the hydroxylation pattern of the b-ring  <cit> .
flavonoids are important secondary metabolites in tea and account for  <dig> to 36% of the dry weight of fresh leaves and tender stem.
3′,4′,5′-trihydroxylated flavan-3-ols  are the most abundant flavonoids in tea leaves, present at significantly higher levels than 3′ 4′-dihydroxylated catechins.
catechins anabolic and catabolic processes are dynamic and subject to complex regulatory control, but the link between f3′5′h gene activity and relative catechin content is not well understood, due to the lack of easily assessable reporters.
herein, we demonstrated that the csf3′5′h gene is highly expressed in the leaves and stem, but expressed at extremely low levels in the root, as previously reported  <cit> .
we have previously shown that the tea plant root lacks tri-hydroxyl groups in b-ring flavonols and flavan-3-ols, indicating that csf3′5′h participates in the control of tri-hydroxyl flavan-3-ols synthesis in tea plant.
we found that csf3′5′h gene transcripts peaked in the bud, and we were unable to explain the role of csf3′5′h in the accumulation of leaf end-products.
the content of most flavonoids such as galloylated catechins, pas, and anthocyanidin were highest in the bud or first leaf and declined gradually with the leaf development  <cit> .
these findings indicated that f3′5′h expression was closely associated with the accumulation of end-products of flavonoids in tea leaves.
csf3′5′h expression was significantly increased after seven days of treatment with light or sucrose, indicated that csf3′5′h expression can be efficiently induced by light and sucrose.
cloning analysis revealed that the csf3′5′h gene promoter contains several light-responsive promoter elements , further indicating that light might be a key factor in the control of csf3′5′h transcription.
anthocyanidin accumulation in csf3′5′h transgenic tobacco
the main anthocyanin in the wild-type tobacco corolla is cyanidin  <cit> .
as shown above, most transgenic plant flower petals contained delphinins.
interestingly, the cyanidin and delphinin content was significantly higher in transgenic tobacco plants than wild type plants, indicating that csf3′5′h performs both 3′,5′- and 3′ -hydroxylation in vivo, in agreement with results of heterologous expression of f3′5′hs in pericallis × hybrida  <cit> , senecio cruentus  <cit> , antirrhinum kelloggii  <cit> , and solanum lycopersicum  <cit> .
however, the hydroxylation pattern of the b-ring cannot be elucidated by tobacco transgenic experiments.
flavonoid pathway, which is a complex metabolic network in plants, starts with general phenylpropanoid metabolism and leads to a myriad of end-products.
the enzymes of flavonoid biosynthesis are likely to function as multienzyme complexes, which facilitate the direct transfer, or channeling of active sites  <cit> .
therefore, the overall concentrations of the intermediates, including free flavanones and flavanols, are extremely low in vivo  <cit> .
csf3′5′h transgenic tobacco plants produced deeper and redder flowers than wild-type plants.
the qrt-pcr results indicated that the flavonoid pathway genes, including chs, f3h, ans, anr, ufgt, could be stimulated by csf3′5′h over-expression in transgenic lines.
f3′5′h, a crucial microsomal cytochrome p <dig> enzyme in these pathways, may serve to anchor the complexes to the microsme membrane  <cit> .
therefore, our results indicate that over-expression of csf3′5′h may stimulate metabolic flux toward anthocyanin products in tobacco petals by formatting more enzyme complexes.
the transgenic lines, however, did not produce blue flowers in this study.
these findings demonstrated that blue flowers are not necessarily generated only by controlling the anthocyanin content  <cit> .
indeed, previous studies have reported that anthocyanidin content, co-pigments, metal ion type and concentration, ph of vacuoles, anthocyanin localization and shapes of surface cells all contribute to the final flower color  <cit> .
however, our findings do reveal a clear impact of csf3′5′h gene on flower phenotype.
this gene might therefore be applied to molecular design of flower color in ornamental plants.
heterologous expression of csf3′5′h in yeast
heterologous expressions of csf3′5′h in yeast were carried out to further confirm the catalytic position of csf3′5′h enzyme in flavonoid pathways.
to our knowledge, the camellia sinensis f3′5′h gene has not been previously successfully expressed in yeast.
for effective expression of csf3′5′h in yeast, a codon optimized yeast csf3′5′h sequence  was designed, but only minor activity was detected.
generally, the presence of an n-terminal signal peptide can translocate p <dig> proteins into the endoplasmic reticulum .
we further optimized ycsf3′5′h by replacing the n-terminal sequence with a signal peptide from the vvf3′5′h gene.
fortunately, transgenic cells expressing the fusion f3′5′h gene exhibited high f3′5′h activity, indicating that the signal peptide of csf3′5′h might be imperfectly recognized in s. cerevisiae cells.
unexpectedly, another fusion gene , only altered at the 3′-terminal sequence in comparison to vvf3′5′h, not achieving detectable f3′5′h activity.
these results suggested that the region of f3′5′h conferring enzymatic activity might be located at the c-terminal of f3′5′h.
indeed, previous reports have suggested that the functional difference between f3′h and f3′5′h is determined by the c-terminal end  <cit> .
f3′5′hs have been shown to hydroxylate a broad range of flavonoid substrates, including n, dhk, k and apigenin, possibly allowing the formation of 3′,4′- and 3′,4′,5′-hydroxylated flavonoids.
however, the optimum substrate for the f3′5′h enzymes needs to be further defined, in vivo and in vitro.
f3′5′h enzymes from catharanthus roseus and petunia x hybrida have achieved highest activities with naringenin and apigenin  <cit> , and n and dhk are equally hydroxylated by osteospermum hybrida f3′5′h, whereas f3′h from gerbera hybrida exhibits a clear substrate preference for n  <cit> .
in contrast, the f3′5′h gene from tomato  has a preference for naringenin, with a km value of  <dig>  μm  <cit> .
to assess substrate specificity of the modified csf3′5′h , flavanones , flavonols , dihydroflavonols , anthocyanins  and catechin  were selected as substrates.
4′- hydroxylated flavanone  was the optimum substrate for the csf3′5′h enzyme, and was effectively converted to both 3′4′- and 3′4′5′-forms.
interestingly, with n as substrate in fsi transgenic cells, the ratio of 3′4′5′- to 3′4′-hydroxylated products was significantly higher than in vvf3′5′h cells.
modified csf3′5′h genes could thus tailor flavonoid metabolism, enhancing the yields of specific b-ring tri-hydroxyl products.
the broad substrate acceptance is consistent with the possibility that multiple paths lead to the same intermediates, and that competition could occur in vivo.
the substrates used in vivo are mostly not yet precisely identified  <cit> .
we also detected the b-ring hydroxyl reaction patterns of total enzyme extract from tea leaves.
interesting, with n as a substrate, the 3′4′5′-hydroxylated flavanone product  was undetected and only the 3′4′-hydroxylated product  was detected.
it is not known whether the product p might be efficiently transformed into other end-products or the product e synthesized by another p <dig> gene .
therefore, further analysis should be carried out to further describe the mechanism underlying b-ring hydroxylation in vivo.
CONCLUSIONS
in this study, the csf3′5′h gene was isolated from the tea cdna library.
phylogenetic analyses revealed that the camellia sinensis f3′5′h gene belongs to the cyp75a subfamily.
qrt-pcr analysis indicated csf3′5′h is highly expressed in the bud, but very little is expressed in the root.
over-expression of csf3′5′h resulted in production of new delphinidin derivatives in the corollas of transgenic tobacco plants, increased the content of cyanidin derivatives and produced deeper and redder flowers in transgenic plants.
heterologous expressions of csf3′5′h in yeast were carried out to demonstrate the function of csf3′5′h enzyme in vitro.
heterologous expression of the modified ycsf3′5′h  in yeast revealed the 4′-hydroxylated flavanone naringenin to be the optimum substrate for the csf3′5′h enzyme; naringenin was effectively converted into both 3′4′- and 3′4′5′-forms.
importantly, the ratio of 3′4′5′- to 3′4′-hydroxylated products was significantly higher in modified ycsf3′5′h transgenic cells than in vvf3′5′h cells.
the findings reported here provide a basis for better understanding of the role of camellia sinensis f3′5′h in b-ring hydroxylation of flavonoids, in vitro and in vivo.

 flavonoids are polyphenol antioxidants found naturally in plants, which possess key pharmacological activities, including antioxidant, antimutagenic, anticarcinogenic, and antibacterial properties. flavonoids in most higher plants can be divided into six major subgroups: chalcones, flavones, flavonols, flavan-3-ols, anthocyanins, and proanthocyanins. the structure of the flavonoid b ring is the primary determinant of the antioxidant activity of flavonoids, and flavonoids can be divided into three subclasses according to the hydroxylation pattern of their b-ring, including b-ring 4′-hydroxylated, 3′4′-dihydroxylated, and 3′4′5′-trihydroxylated compounds. the number of hydroxyl groups on the b-ring affects the capacity to inhibit lipid peroxidation. for instance, liu and yang reported that the antioxidant activity of epigallocatechin-3-gallate is greater than that over epigallocatechin at concentrations of up to 100 mg · l−. in the flavonoid biosynthesis pathway, the hydroxylation pattern of the b-ring is determined by two cytochrome p450-dependent monooxygenases: flavonoid 3′-hydroxylase and flavonoid 3′,5′-hydroxylase. hydroxylation of the 5′-position by f3′5′h is a particularly important step, which determines the b-ring tri-hydroxyl flavonoid end-product formed in plants, as illustrated in figure  figure biosynthesis pathway and end-product accumulation of flavonoids in camellia sinensis. biosynthesis pathway of flavonoids. chs, chalcone synthase; chi, chalcone isomerase; f3h, flavanone 3-hydroxylase; f3′h, flavonoid 3′-hydroxylase; f3′5′h, flavonoid 3′,5′-hydroxylase; dfr, dihydroflavonol 4-reductase; fls, flavonol synthase; lar, leucoanthocyanidin reductase; ans, anthocyanidin synthase; anr, anthocyanidin reductase; ufgt, udp-glycose flavonoid glycosyltransferase; uggt, udp-glucose galloyl-1-o-β-d-glucosyltransferase; ecgt, epicatechins: 1-o-galloyl-β-d-glucose o-galloyltransferase; relative quantity of different flavonoid compounds. the data for relative quantity of different flavonoid compounds were quoted from jiang. f3′5′hs have been previously cloned and functionally analyzed from multiple plants, including grape, petunia, snapdragon, cineraria, tomato, big leaf periwinkle, and potato. through heterologous expression in transgenic plants and yeasts, f3′5′hs were shown to hydroxylate a broad range of flavonoid substrates, including naringenin, dihydrokaempferol, kaempferol and apigenin. however, optimum substrates for these enzymes remain to be determined. tea is an important commercial crop, the leaves of which can be processed into popular nonalcoholic beverages. because of the high flavonoid content, epidemiological and pathological studies have suggested that tea consumption may potentially be protective against human cancers and high blood pressure, and contribute to weight reduction. the total concentration of flavonoid compounds is around 12–24% of tea leaf dry mass. we have previously shown that catechins are among the most abundant flavonoids in tea leaves, followed by proanthocyanidins, flavonols, flavones and anthocyanins. in recent years, some of the flavonoid structural and regulatory genes have been cloned, and functions of these genes have been investigated. while 4′-hydroxylated catechins are very rare or undetectable in tea leaves, 3′4′5′-trihydroxylated catechins, egc, and egcg), are the most abundant flavonoids in young leaves and the stem, with significantly higher concentrations than 3′4′-dihydroxylated catechins, epicatechin and ecg). therefore, characterizing the pattern of b-ring hydroxylation is clearly a valuable contribution to the understanding of flavonoid biosynthesis in tea plants. however, it has not yet been possible to prepare active membrane-bound f3′5′h enzymes from camellia sinensis, and it is still unclear whether b-ring hydroxylation occurs at the level of flavanones and/or dihydroflavonols, in vivo. aiming to analyze the in vivo expression pattern of csf3′5′h and to characterize the function of this gene in vitro, we isolated the csf3′5′h gene from tea cdna library. we found that csf3′5′h was highly expressed in the bud, but little or no csf3′5′h was detected in the root. csf3′5′h expression was enhanced by light and sucrose treatment, and over-expression of csf3′5′h resulted in production of delphinidin derivatives, producing redder flowers in transgenic tobacco plants, in comparison to with wild type. heterologous expression of modified csf3′5′h in yeast revealed that 4′-hydroxylated flavanone is the optimum substrate for csf3′5′h, and the ratio of 3′4′5′- to 3′4′-hydroxylated products in the modified csf3′5′h transgenic cells was significantly higher than in vvf3′5′h cells.  isolation and characterization of the csf3′5′h gene the csf3′5′h gene was successfully cloned from the cdna library of the 3rd tea leaf, and encoded amino acid residues. a blast search performed with the coding sequence revealed and 81% identity with cyclamen persicum, cyclamen graecum and vitis vinifera genes, respectively. the phylogenetic tree was generated using protein sequences from several plant f3′5′h and f3′h enzymes retrieved from the ncbi database. the tree demonstrated that f3′hs and f3′5′hs were grouped in cyp75b and cyp75a clusters, respectively. csf3′5′h was grouped into the cyp75a subfamilies, and most closely related to the f3′5′h enzymes of cyclamen persicum, cyclamen graecum and vitis vinifera.figure phylogenetic tree for a selection of f3′5′h protein. phylogenetic tree based on amino acid sequences of f3′hs and f3′5′hs in various plant species from the ncbi web page. accession numbers are displayed in the figure. bootstrap values are shown at nodes. expression pattern of csf3′5′h in tea the expression pattern of csf3′5′h in tea was detected by qrt-pcr. the gadph gene, expected to show a constitutive expression pattern, was used as control. csf3′5′h expression was tissue specific, expressed highly in leaves and stem, with transcripts peaking in the buds. we also assessed substrate specificity of crude extracts from tea leaves, measuring hydroxylation of n and dihydroquercetin, which yielded eriodictyol and dihydromyricetin, respectively. the enzyme activities of these crude extracts were and pcat · g− protein, respectively. surprisingly hydroxylation of n did not yield, 3′4′5′-hydroxylated product.figure expression of csf3′5′h in different tea tissues. relative expression of csf3′5′h in different tea tissues analyzed by qrt-pcr and semi-quantitative rt pcr for csf3′5′h and gapdh in different tea tissues. relative expression of csf3′5′h in different light and sucrose conditions analyzed by qrt-pcr and semi-quantitative rt pcr for gapdh and csf3′5′h in different light and sucrose conditions. the data represent the mean ± sd from three independent measurements. the different letters and *indicated the significant level at p < . hplc chromatograms of flavanones or dihydroflavonols formation in csf3′5′h assays with tea leaf enzyme. reaction assay with substrate n of heat-denatured protein in the control treatment; reaction assay with substrate n of the crude enzyme extract from the leave of tea; reaction assay with substrate dhq of heat-denatured protein in the control treatment; reaction assay with substrate dhq of the crude enzyme extract from the leave of tea. interestingly, csf3′5′h transcripts were barely detected in the root, and the monomer and polymer of 3′ 4′ -dihydroxylated catechins, but no 3′ 4′ 5′-trihydroxylated catechins, accumulated in camellia sinensis roots, indicating that extremely low csf3′5′h expression might directly lead to absence of b-ring tri-hydroxyl catechins in the root. we used tissue culture seedlings, developed from the embryo of tea-seeds, to assess the direct influence of light and sucrose on csf3′5′h expression. csf3′5′h expression levels in light-exposed and sucrose-induced seedlings were significantly increased by and -fold, respectively, indicating that csf3′5′h expression can be efficiently induced by light and sucrose. functional analysis of the csf3′5′h gene in nicotiana tabacum the vector for constitutive expression of the 35s:csf3′5′h gene was introduced into tobacco ‘g28’, which lacks f3′5′h genes and has pink flowers. about independent transgenic tobacco plants were obtained. most flowers from the transgenic plants exhibited a clear color change from pale pink of the host to magenta.figure flower color after overexpression of csf3′5′h and qrt-pcr of transgenic tobacco plants. tobacco flowers of wild-type and csf3′5′h transgenes. tobacco flowers of wild-type, csf3′5′h transgenes and qrt-pcr for csf3′5′h in flowers from ck and transgenic lines. hplc chromatograms of anthocyanidins and flavonol glacosides in tobacco flowers from ck and line. concentration of anthocyanidins in tobacco flowers from ck, csf3′5′h transgenes and vector control. the data represent the mean ± sd from three independent measurements. qrt-pcr for flavonoid-related genes in tobacco flowers from ck, csf3′5′h transgenes and vector control. *indicated the significant level at p < . #indicated the significant level compared between every detected lines versus ck. the expression of csf3′5′h in several transgenic lines with magenta flowers was detected by qrt-pcr, with β-actin used as reference gene, and we found varying levels of csf3′5′h gene expression in glyphosate-resistant transgenic tobaccos. to investigate whether the flavonoid biosynthesis pathway was affected by over-expression of csf3′5′h, the flavonoid pathway genes, chi, f3h, f3′h, dfr, fls, ans, anr, ufgt ) from nicotiana tabacum were examined by qrt-pcr in wild type and transgenic lines. the expression levels of chs, f3h, ans, anr, ufgt genes in transgenic lines significantly increased in comparison to the wild type and vector control, suggesting that expression of these genes was stimulated by the over-expression of csf3′5′h in transgenic lines. the level of glycosylated flavonoids in flowers was assessed by reverse phase hplc and lc-ms. 3′,5′-hydroxylated flavonol glacoside was detected in the petals of the transgenic lines, but not in wild-type tobaccos. however, the concentration of myr in the flowers was too low to quantify. petal pigments were extracted and chemically converted to anthocyanidins, for anglicizing the anthocyanin components by reverse phase hplc. petals expressing the csf3′5′h gene contained a novel 3′,5′-hydroxylated anthocyanidin and increased cyaniding derivative content. the ratio of delphinin to total anthocyanin compounds in transgenic tobacco plants reached a maximum of %, and the average anthocyanin concentration in the petals of transgenic tobaccos was -fold higher than in wild-type plants, suggesting that csf3′5′h encodes a protein with b-ring 3′, 5′-hydroxylation function, and that anthocyanin synthesis can be stimulated by csf3′5′h over-expression in transgenic lines. heterologous csf3′5′h expression in yeast the yeast strain saccharomyces cerevisiae wat engineered to over-express the arabidopsis thaliana p reductase, is a suitable heterologous host for p expression. a pyes-dest 52a csf3′5′h vector was transformed into wat however, these transgenic cells did not produce functional f3′5h protein, so the codon optimized yeast csf3′5′h sequence was designed and transformed into wat resulting in only minimal activity of approximately pkat · l− culture, with n as substrate.figure optimization of csf3′5′h expression in saccharomyces cerevisiae ‘wat11’. comparison of amino acid sequences encoded by csf3′5′h and vvf3′5′h proteins. the boxed regions represent fusion between csf3′5′h and vvf3′5′h. primary structure schemes of expressed csf3′5′h sequence variants and resulting expression strength expressed as csf3′5′h activity · l− culture. enzyme activity was expressed as pkat · l− culture. the data represent the mean ± sd from three independent measurements. transgenic cells, harboring the vitis vinifera f3′5′h gene, achieved a high overall f3′5′h activity of pkat · l− culture with n as substrate. with the predicted signal peptide, both f3′5′hs were translated into precursor proteins and delivered to the er. we hypothesized that imperfect recognition of the camellia sinensis signal peptide might account for low expression levels detected in saccharomyces cerevisiae cells, and tested this hypothesis by fusing csf3′5′h with vvf3′5′h at three different points of the sequence based on amino acid sequence homology. the 5′-sequences of ycsf3′5′h were replaced by vvf3′5′h at aa, aa, aa respectively. vitis vinifera sequences were fused to ycsf3′5′h and cloned into the plasmid pyes-dest 52a for transformation of wat cells. the cells containing fsi, replaced at signal and leader peptide region, led to high f3′5′h activity in the range of pkat · l− culture, a significant increase in comparison with the reference construct. these results indicated that csf3′5′h signal peptide might be imperfectly recognized in saccharomyces cerevisiae cells. the cells transformed with fsii also resulted in f3′5′h activity, albeit significantly less. generally, overall activities of chimeras are often low, e.g. most chimeras between limonene 3-hydroxylase and limonene 6-hydroxylase achieve no, or less than 5% of that of wild type. unexpectedly, f3′5′h activity was undetected in cells transformed with fsiii. in comparison to vvf3′5′h, the fsiii fusion gene was only altered at the 3′-terminal sequence. finally, we assessed the substrate specificity of cells expressing fsi and vvf3′5′h. based on previous findings and other intermediate compounds in the catechin synthesis pathway, we assessed catalysis of n, e, k, quercetin, dhk, dhq, pelargonidin, cya and c. wat cells transformed with pyes-dest 52a vector were used as controls. as observed with vvf3′5′h, fsi preferred b-ring 4′-hydroxylated compounds to 3′, 4′-hydroxylated compounds. no activity was detected with pel, cya and c as substrates, in both transgenic cells. both proteins displayed highest activities with n and significant activities with k and dhk, yielding 3′4′- and 3′4′5′-forms as products. interestingly, for fsi with n as substrate, the ratio of 3′4′5′- to 3′4′-hydroxylated products was significantly higher than for vvf3′5′h.figure hplc chromatograms of products from pyes-dest52- fs and pyes-dest52- vvf3′5′h with flavanones, flavonols and dihydroflavonols as substrates. hplc chromatograms of products from pyes-dest52- fsi with n, e, k, q, dhk and dhq as substrates; hplc chromatograms of products from pyes-dest52-vvf3′5′h with n, e, k, q, dhk and dhq as substrates; hplc chromatograms of products from control treatment with n, e, k, q, dhk and dhq as substrates. accepted substrates and enzyme activity units for f3′5′h substrate modified-csf3′5′h vvf3′5′h class 3′-hydroxyla-tion product 5′-hydroxylat-ion product 3′-hydroxylat-ion product 5′-hydroxylat-ion product enzyme activity was expressed as pkat · l− culture. the data represent mean ± sd from three independent measurements. — indicates results below the detection limit. microsomes from wat cells transformed by pyes-dest 52a -fsi and -vvf3′5′h were assayed for nadph-dependent flavonoid 3′, 5′-hydroxylation with n, k and dhk as substrates. no activity was detected with microsomes from the control, pyes-dest 52a-transformed cells. in contrast the km values of the microsome extracted from fsi-transformed cells, with n, k, and dhk as substrates, were,, and  μm, respectively, indicating that n might be the optimum substrate for the csf3′5′h enzyme. fsi achieved significantly higher km values than vvf3′5′h with k and dhk as substrates, but lower km values with n. however, the max reaction rates for fsi and vvf3′5′h with n as substrate were significantly lower than the values with k and dhk as substrates.table comparison of steady-state kinetic parameters for cinnamate 4-hydroxylation in yeast microsomes substrate modified-csf3′5′h vvf3′5′h class km vmax km vmax experiments were carried out in 50 mm phosphate buffer ph  at 28°c for 30 min. protein concentration of f3′5′h -transformed yeast microsomes was  mg/ml in the reaction system. the data represent the mean ± sd from three independent measurements. concentration dependence of f3′5′h observed in yeast microsomes. f3′5′h-containing microsomes originating from transformed pyes-dest52-fsi and -vvf3′5′h cells were incubated in 50 mm phosphate buffer ph  at 28°c. the solid line represents the result of a multi-iterative fitting of experimental data using the michaelis-menten equation. insert: michaelis-menten double-reciprocal plot. the data represent the mean ± sd from three independent measurements. discussion the role of csf3′5′h in catechin formation in tea leaves all flavonoids are hydroxylated at the 4′ position of the b-ring. b-ring hydroxylation patterns determie the color of anthocyanins and thus have been extensively investigated in ornamental plants for color engineering. the f3′5′h gene is commonly known as the blue gene and previous studies have shown that f3′5′h catalyzes the hydroxylation at the 3′ and 5′ positions of flavonoids to determine the hydroxylation pattern of the b-ring. flavonoids are important secondary metabolites in tea and account for to 36% of the dry weight of fresh leaves and tender stem. 3′,4′,5′-trihydroxylated flavan-3-ols are the most abundant flavonoids in tea leaves, present at significantly higher levels than 3′ 4′-dihydroxylated catechins. catechins anabolic and catabolic processes are dynamic and subject to complex regulatory control, but the link between f3′5′h gene activity and relative catechin content is not well understood, due to the lack of easily assessable reporters. herein, we demonstrated that the csf3′5′h gene is highly expressed in the leaves and stem, but expressed at extremely low levels in the root, as previously reported. we have previously shown that the tea plant root lacks tri-hydroxyl groups in b-ring flavonols and flavan-3-ols, indicating that csf3′5′h participates in the control of tri-hydroxyl flavan-3-ols synthesis in tea plant. we found that csf3′5′h gene transcripts peaked in the bud, and we were unable to explain the role of csf3′5′h in the accumulation of leaf end-products. the content of most flavonoids such as galloylated catechins, pas, and anthocyanidin were highest in the bud or first leaf and declined gradually with the leaf development. these findings indicated that f3′5′h expression was closely associated with the accumulation of end-products of flavonoids in tea leaves. csf3′5′h expression was significantly increased after seven days of treatment with light or sucrose, indicated that csf3′5′h expression can be efficiently induced by light and sucrose. cloning analysis revealed that the csf3′5′h gene promoter contains several light-responsive promoter elements, further indicating that light might be a key factor in the control of csf3′5′h transcription. anthocyanidin accumulation in csf3′5′h transgenic tobacco the main anthocyanin in the wild-type tobacco corolla is cyanidin. as shown above, most transgenic plant flower petals contained delphinins. interestingly, the cyanidin and delphinin content was significantly higher in transgenic tobacco plants than wild type plants, indicating that csf3′5′h performs both 3′,5′- and 3′ -hydroxylation in vivo, in agreement with results of heterologous expression of f3′5′hs in pericallis × hybrida, senecio cruentus, antirrhinum kelloggii, and solanum lycopersicum. however, the hydroxylation pattern of the b-ring cannot be elucidated by tobacco transgenic experiments. flavonoid pathway, which is a complex metabolic network in plants, starts with general phenylpropanoid metabolism and leads to a myriad of end-products. the enzymes of flavonoid biosynthesis are likely to function as multienzyme complexes, which facilitate the direct transfer, or channeling of active sites. therefore, the overall concentrations of the intermediates, including free flavanones and flavanols, are extremely low in vivo. csf3′5′h transgenic tobacco plants produced deeper and redder flowers than wild-type plants. the qrt-pcr results indicated that the flavonoid pathway genes, including chs, f3h, ans, anr, ufgt, could be stimulated by csf3′5′h over-expression in transgenic lines. f3′5′h, a crucial microsomal cytochrome p enzyme in these pathways, may serve to anchor the complexes to the microsme membrane. therefore, our results indicate that over-expression of csf3′5′h may stimulate metabolic flux toward anthocyanin products in tobacco petals by formatting more enzyme complexes. the transgenic lines, however, did not produce blue flowers in this study. these findings demonstrated that blue flowers are not necessarily generated only by controlling the anthocyanin content. indeed, previous studies have reported that anthocyanidin content, co-pigments, metal ion type and concentration, ph of vacuoles, anthocyanin localization and shapes of surface cells all contribute to the final flower color. however, our findings do reveal a clear impact of csf3′5′h gene on flower phenotype. this gene might therefore be applied to molecular design of flower color in ornamental plants. heterologous expression of csf3′5′h in yeast heterologous expressions of csf3′5′h in yeast were carried out to further confirm the catalytic position of csf3′5′h enzyme in flavonoid pathways. to our knowledge, the camellia sinensis f3′5′h gene has not been previously successfully expressed in yeast. for effective expression of csf3′5′h in yeast, a codon optimized yeast csf3′5′h sequence was designed, but only minor activity was detected. generally, the presence of an n-terminal signal peptide can translocate p proteins into the endoplasmic reticulum. we further optimized ycsf3′5′h by replacing the n-terminal sequence with a signal peptide from the vvf3′5′h gene. fortunately, transgenic cells expressing the fusion f3′5′h gene exhibited high f3′5′h activity, indicating that the signal peptide of csf3′5′h might be imperfectly recognized in s. cerevisiae cells. unexpectedly, another fusion gene, only altered at the 3′-terminal sequence in comparison to vvf3′5′h, not achieving detectable f3′5′h activity. these results suggested that the region of f3′5′h conferring enzymatic activity might be located at the c-terminal of f3′5′h. indeed, previous reports have suggested that the functional difference between f3′h and f3′5′h is determined by the c-terminal end. f3′5′hs have been shown to hydroxylate a broad range of flavonoid substrates, including n, dhk, k and apigenin, possibly allowing the formation of 3′,4′- and 3′,4′,5′-hydroxylated flavonoids. however, the optimum substrate for the f3′5′h enzymes needs to be further defined, in vivo and in vitro. f3′5′h enzymes from catharanthus roseus and petunia x hybrida have achieved highest activities with naringenin and apigenin, and n and dhk are equally hydroxylated by osteospermum hybrida f3′5′h, whereas f3′h from gerbera hybrida exhibits a clear substrate preference for n. in contrast, the f3′5′h gene from tomato has a preference for naringenin, with a km value of  μm. to assess substrate specificity of the modified csf3′5′h, flavanones, flavonols, dihydroflavonols, anthocyanins and catechin were selected as substrates. 4′- hydroxylated flavanone was the optimum substrate for the csf3′5′h enzyme, and was effectively converted to both 3′4′- and 3′4′5′-forms. interestingly, with n as substrate in fsi transgenic cells, the ratio of 3′4′5′- to 3′4′-hydroxylated products was significantly higher than in vvf3′5′h cells. modified csf3′5′h genes could thus tailor flavonoid metabolism, enhancing the yields of specific b-ring tri-hydroxyl products. the broad substrate acceptance is consistent with the possibility that multiple paths lead to the same intermediates, and that competition could occur in vivo. the substrates used in vivo are mostly not yet precisely identified. we also detected the b-ring hydroxyl reaction patterns of total enzyme extract from tea leaves. interesting, with n as a substrate, the 3′4′5′-hydroxylated flavanone product was undetected and only the 3′4′-hydroxylated product was detected. it is not known whether the product p might be efficiently transformed into other end-products or the product e synthesized by another p gene. therefore, further analysis should be carried out to further describe the mechanism underlying b-ring hydroxylation in vivo.  in this study, the csf3′5′h gene was isolated from the tea cdna library. phylogenetic analyses revealed that the camellia sinensis f3′5′h gene belongs to the cyp75a subfamily. qrt-pcr analysis indicated csf3′5′h is highly expressed in the bud, but very little is expressed in the root. over-expression of csf3′5′h resulted in production of new delphinidin derivatives in the corollas of transgenic tobacco plants, increased the content of cyanidin derivatives and produced deeper and redder flowers in transgenic plants. heterologous expressions of csf3′5′h in yeast were carried out to demonstrate the function of csf3′5′h enzyme in vitro. heterologous expression of the modified ycsf3′5′h in yeast revealed the 4′-hydroxylated flavanone naringenin to be the optimum substrate for the csf3′5′h enzyme; naringenin was effectively converted into both 3′4′- and 3′4′5′-forms. importantly, the ratio of 3′4′5′- to 3′4′-hydroxylated products was significantly higher in modified ycsf3′5′h transgenic cells than in vvf3′5′h cells. the findings reported here provide a basis for better understanding of the role of camellia sinensis f3′5′h in b-ring hydroxylation of flavonoids, in vitro and in vivo. 
these findings provide animportant basis for further studies of flavonoid biosynthesis in tea plants. flavonoid 3′,5′-hydroxylase, an important branch point enzyme in tea plant flavan-3-ol synthesis, belongs to the cyp75a subfamily and catalyzes the conversion of flavones, flavanones, dihydroflavonols and flavonols into 3′,4′,5′-hydroxylated derivatives. csf3′5′h is a key controller of tri-hydroxyl flavan-3-ol synthesis in tea plants, which can effectively convert 4′-hydroxylated flavanone into 3′4′5′- and/or 3′4′-hydroxylated products. heterologous expression of the modified csf3′5′h revealed that 4′-hydroxylated flavanone is the optimum substrate for csf3′5′h, and was efficiently converted into both 3′4′- and 3′4′5′-forms. expression pattern analysis revealed that csf3′5′h expression was tissue specific, very high in the buds and extremely low in the roots. csf3′5′h expression was enhanced by light and sucrose. over-expression of csf3′5′h produced new-delphinidin derivatives, and increased the cyanidin derivative content of corollas of transgenic tobacco plants, resulting in the deeper transgenic plant flower color. heterologous expressions of csf3′5′h in yeast were carried out to demonstrate the function of csf3′5′h enzyme in vitro. the camellia sinensis f3′5′h gene was isolated from tea cdna library. however, whether b-ring hydroxylation occurs at the level of flavanones and/or dihydroflavonols, in vivo remains unknown. camellia sinensisflavonoid 3′5′-hydroxylasefunctional analysisheterologous expressioncatechinsissue-copyright-statement© the author 2014 the ratio of 3′4′5′- to 3′4′-hydroxylated products in fsi transgenic cells was significantly higher than vvf3′5′h cells.
 flavonoid 3′,5′-hydroxylase, an important branch point enzyme in tea plant flavan-3-ol synthesis, belongs to the cyp75a subfamily and catalyzes the conversion of flavones, flavanones, dihydroflavonols and flavonols into 3′,4′,5′-hydroxylated derivatives. however, whether b-ring hydroxylation occurs at the level of flavanones and/or dihydroflavonols, in vivo remains unknown.  the camellia sinensis f3′5′h gene was isolated from tea cdna library. expression pattern analysis revealed that csf3′5′h expression was tissue specific, very high in the buds and extremely low in the roots. csf3′5′h expression was enhanced by light and sucrose. over-expression of csf3′5′h produced new-delphinidin derivatives, and increased the cyanidin derivative content of corollas of transgenic tobacco plants, resulting in the deeper transgenic plant flower color. heterologous expressions of csf3′5′h in yeast were carried out to demonstrate the function of csf3′5′h enzyme in vitro. heterologous expression of the modified csf3′5′h revealed that 4′-hydroxylated flavanone is the optimum substrate for csf3′5′h, and was efficiently converted into both 3′4′- and 3′4′5′-forms. the ratio of 3′4′5′- to 3′4′-hydroxylated products in fsi transgenic cells was significantly higher than vvf3′5′h cells.  csf3′5′h is a key controller of tri-hydroxyl flavan-3-ol synthesis in tea plants, which can effectively convert 4′-hydroxylated flavanone into 3′4′5′- and/or 3′4′-hydroxylated products. these findings provide animportant basis for further studies of flavonoid biosynthesis in tea plants. such studies would help accelerate flavonoid metabolic engineering in order to increase b-ring tri-hydroxyl product yields. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords camellia sinensisflavonoid 3′5′-hydroxylasefunctional analysisheterologous expressioncatechinsissue-copyright-statement© the author 2014 