GENE-37119; No.

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Gene xxx (2012) xxxxxx

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Gene
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Short Communication

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Wenjun Huang a, b, Wei Sun b, c, Ying Wang a,

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a r t i c l e

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a b s t r a c t

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Key Laboratory of Plant Germplasm Enhancement and Speciality Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan, Hubei, 430074, China Graduate University of the Chinese Academy of Sciences, Beijing, 100039, China Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, Guangdong, 510650, China

Isolation and molecular characterisation of avonoid 3-hydroxylase and avonoid 3, 5-hydroxylase genes from a traditional Chinese medicinal plant, Epimedium sagittatum

Article history: Accepted 15 November 2011 Available online xxxx Keywords: Anthocyanin CYP75 Epimedium Flavonoid biosynthesis

The epimedii herb, a traditional Chinese medicinal plant, has signicant pharmacological effects on human health. The bioactive components in the herb (Epimedium sagittatum (Sieb. et Zucc.) Maxim) are mainly prenylated avonol glycosides, which are end-products of the avonoid biosynthetic pathway. This has not been clearly elucidated until recently. The genes encoding avonoid 3-hydroxylase (F3H) and avonoid 3, 5-hydroxylase (F35H) involved in the avonoid biosynthetic pathway, designated as EsF3H and EsF3 5H, were isolated from E. sagittatum using a homology-based cloning method and deposited in the GenBank databases (GenBank ID: HM011054 and HM011055), respectively. EsF3H and EsF35H proteins shared high homology with other plant-specic avonoid hydroxylases and were clustered into the CYP75B and CYP75A group, respectively. In addition, four conserved cytochrome P450-featured motifs were found in the amino acid sequences of both genes. Transcription levels of both genes were detected in all tissues tested and were high in most of the pigmented tissues. Moreover, the expression levels of both EsF3H and EsF35H correlated positively with the anthocyanin accumulation pattern in leaves from E. sagittatum. The cloning and molecular characterisation of EsF3H and EsF35H genes will accelerate progress in the study of the avonoid biosynthetic pathway to elucidate the molecular mechanisms of the biosynthesis of the bioactive components in E. sagittatum. 2011 Elsevier B.V. All rights reserved.

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1. Introduction

Flavonoids represent a large class of secondary metabolites in plants that are best known as the characteristic red, blue, and purple anthocyanin pigments of plant tissues (Winkel-Shirley, 2001, 2002). They have a wide range of biological functions as they provide pigmentation to owers, fruits, and seeds in order to attract pollinators and seed dispersers, protect plants from UV radiation, defend against phytopathogens, act as signal molecules in plantmicrobe interactions, and are involved in auxin transport and pollen germination (Dixon and Paiva, 1995; Koes et al., 2005; Peer and Murphy, 2007). Flavonoids receive substantial public attention because of their signicant effects on human health. The antioxidant activity of avonoids plays a vital role in the prevention of neuronal and cardiovascular illnesses, cancer and diabetes (Harborne and Williams, 2000; Havsteen, 2002).

Abbreviations: CTAB, cetyl trimethyl ammonium bromide; CYP, cytochrome P450; RACE, rapid amplication of cDNA ends; qRT-PCR, quantitative reverse transcriptionpolymerase chain reaction; F3H, avonoid 3-hydroxylase; F35H, avonoid 3,5hydroxylase; FLS, avonol synthase; DFR, dihydroavonol 4-reductase. Corresponding author. Tel.: + 86 27 87510675; fax: + 86 27 87510331. E-mail address: yingwang@wbgcas.cn (Y. Wang). 0378-1119/$ see front matter 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2011.11.029

The genetics and biochemistry of the avonoid pathway have been characterised in several model plant species such as Arabidopsis, maize, petunia, and snapdragon, and the main structural and regulatory genes have been cloned (Holton and Cornish, 1995; Mol et al., 1998; Winkel-Shirley, 2001). The F3H and F35H genes, both of which belong to the cytochrome P450 superfamily, catalyse hydroxylation at the 3 and 3, 5 positions of the B-ring of the avonoids. This leads to the production of the red cyanidin-based pigments and the blue/violet delphinidin-based pigments, respectively (Ayabe and Akashi, 2006; Tanaka, 2006). In addition to the hydroxylation of anthocyanidins, both genes also catalyse the hydroxylation of avanones, avones and avonols. Since both the F3H and F35H genes were rst isolated from petunia (Brugliera et al., 1999; Holton et al., 1993), their homologues have been subsequently isolated from many plants such as the apple (Han et al., 2010), Arabidopsis (Schoenbohm et al., 2000), gentian (Tanaka et al., 1996), grape (Bogs et al., 2006) and tomato (Olsen et al., 2010). Of these two genes, F35H has evoked more interest from scientists and industries, because some important ornamental plants, such as roses, carnations and chrysanthemums lack F35H enzyme activity and cannot produce blue or violet owers (Tanaka, 2006). Both F3H and F35H play important roles in avonoid biosynthesis and regulation. They are two of the main structural genes encoding

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Please cite this article as: Huang, W., et al., Isolation and molecular characterisation of avonoid 3-hydroxylase and avonoid 3, 5-hydroxylase genes from a traditional Chinese medicinal plant, Epimedium sagittatum, Gene (2012), doi:10.1016/j.gene.2011.11.029

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enzymes for the avonoid biosynthesis and function as a regulatory factor in an important branch point between avonol and anthocyanin biosyntheses. It was demonstrated in the petunia, potato and tomato that F3H and F35H competed with avonol synthase (FLS) and dihydroavonol 4-reductase (DFR) for the same substrate to determine the branch ow (Olsen et al., 2010). In the grapevine, the transcription levels of VvF3H and VvF35H were consistent with the accumulation pattern of the anthocyanins (Castellarin et al., 2006). Recently, the expression pattern of apple F3H genes has also been reported to correspond to the accumulation patterns of avonoids in apple fruit (Han et al., 2010). Ectopic expression of F3H and F35H genes not only changed the anthocyanin composition and content but also altered the ower colour, such as ectopic expression of VvF3H and VvF35H in petunia (Bogs et al., 2006) and MdF3H in tobacco (Han et al., 2010). The epimedii herb (Yin-yang-huo, used by Chinese druggists) is one of the most popular traditional Chinese medicines, with more than two thousand years of history, and is collected from the dried aerial parts of the Epimedium species of Berberidaceae (Guo and Xiao, 2003; Li et al., 2005). Epimedium sagittatum (Sieb. et Zucc.) Maxim, together with four other Epimedium species, Epimedium brevicornu Maxim, Epimedium brevicornu pubescens Maxim, Epimedium wushanense T. S. Ying, and Epimedium koreanum Nakai, is recorded in the Chinese Pharmacopoeia (Pharmacopoeia Commission of PRC, 2005). Many studies have demonstrated that epimedii have extensive pharmacological efcacy for treating impotence, spermatorrhea, infertility, amenorrhea, rheumatic arthritis and chronic bronchitis (Lee et al., 1995; Meng et al., 2005; Wu et al., 2003). In addition, Epimedium species are used as ground covers and ornamental plants due to the abundance of colours and patterns in their leaves and owers. It has been reported that the main bioactive constituents of epimedii are prenylated avonol glycosides (Shen et al., 2007), which are endproducts of a branch of the avonoid biosynthetic pathway. To date, about 130 components have been identied (Wu et al., 2003), among which epimedin A, B, C and icariin are considered major bioactive components that comprise more than 52% of total avonoids in herba epimedii (Zhang et al., 2008). However, the biosynthetic pathways involved in producing these bioactive components are still unclear. Since the 1990s, the wild resources of medicinal Epimedium species have dramatically been reduced due to years of over-harvesting and curtailment of habitat, and recently, they have even become endangered (Xu et al., 2008). Considering the shortage of natural resources and the huge market demand, the creation of new cultivars is crucial for large scale cultivation. In order to manipulate the biosynthesis of the bioactive components, the rst step needed is to elucidate the avonoid biosynthetic pathway in E. sagittatum. The F3H and F35H genes not only encode enzymes involved in the avonoid biosynthetic pathway but also regulate the ow orientation of the anthocyanin and avonol pathways at an important branch point. Notably, a red-purple leaf colour has been discovered in some individuals of E. sagittatum, which contrasts with the green that is usually observed. Furthermore, the colour of leaves can change from redpurple to green as the developmental stage progresses. Therefore, it is possible that the EsF3H and EsF35H genes are responsible for this colour pattern by modulating the anthocyanin accumulation pattern. Here, we report the isolation, molecular characterisation and expression patterns of EsF3H and EsF35H genes in various tissues of E. sagittatum, particularly in green and red leaves that accumulate different anthocyanin contents, which shed light on the avonoid biosynthetic pathway in E. sagittatum.

introduced from Hunan province, China. Leaf, petiole, ower bud, ow- 141 er, and root were sampled and immediately frozen in liquid nitrogen 142 and kept at 70 C until required. 143 2.2. Total genomic DNA and RNA extraction Total genomic DNA from young leaves was extracted using the CTAB method (Doyle and Doyle, 1990). Total RNA was isolated from the various tissues collected above using the TRIzol reagent (Invitrogen, USA) except roots, from which total RNA was extracted with RNAiso Plus (Takara, Japan) combined with the RNAiso-mate for plant tissue (Takara, Japan). The RNA solution was digested with RQ RNase-free DNase I (Promega, USA) to remove any contaminating genomic DNA before the reverse transcription reaction.
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Based on the conserved domains of the F3H and F35H genes, two pairs of degenerate primers (F3H degF/R, F35H degF/R) were designed to amplify the corresponding sequences in E. sagittatum (Supplemental Table S1). The partial cDNA fragments of the putative F3H and F35H homologues were isolated by RT-PCR, using the total RNA extracted from the young red leaves and Superscript II reverse transcriptase (Invitrogen, USA). The 439 bp and 578 bp gene specic fragments of putative F3H and F35H, respectively, were amplied and ligated into the pMD18-T vector (Takara, Japan). Fifteen positive clones for each fragment were selected for DNA sequencing. The sequence results were BLASTed against the NR protein database to identify the putative EsF3H and EsF35H targets. To isolate the putative EsF3H and EsF35H full-length cDNA clones, 5 RACE and 3 RACE were carried out using the SMART RACE Amplication kit (Clontech, Japan). Four pairs of gene-specic primers (F3H. 5 GSP1/2 and F3H. 3GSP1/2, F35H. 5GSP1/2 and F35H. 3GSP1/2) were designed to amplify the 5 cDNA ends and 3 cDNA ends of EsF3 H and EsF35H clones, respectively (Supplemental Table S1). According to the manufacturer's instructions, the single fragments for each RACE reaction were amplied and ligated into the pMD18-T vector (Takara, Japan) and sequenced. Two pairs of gene-specic primers (F3H. F/R, F35H. F/R) were designed to isolate the full length cDNAs of EsF3H and EsF35H genes and the corresponding genomic sequences (Supplemental Table S1). One microliter of 5 cDNA-to-ready template and 1 L of genomic DNA template, combined with 1.25 units of PrimeSTAR HS DNA polymerase (Takara, Japan), were used in the kit-recommended 50 L PCR reaction. Cycling conditions were as follows: pre-denaturation at 98 C for 1 min, followed by 3032 cycles of amplication (98 C for 10 s, 55 C for 15 s and 72 C for 2 min), then succeeded by a nal extension at 72 C for 8 min. The amplied fragments were ligated into the pMD18-T vector (Takara, Japan) and sequenced. 2.4. Sequence and phylogenetic analysis The Blast program at the NCBI website was used to determine the putative F3H and F35H clones. The ORF nder program was used to search for open reading frames in the F3H and F35H nucleotide sequences. The Spidey program was used to analyse the exon and intron genomic structure. Conserved domains were searched for using the Conserved Domain program. Multi-alignment analysis was performed by the ClustalW program, and the neighbour-joining method by the MEGA 4.0 program was conducted to generate the phylogenetic tree from the ClustalW alignment results.

2. Materials and methods 2.1. Plant materials Plantlets of E. sagittatum were collected from individuals in the experimental eld of Wuhan Botanical Garden that were originally

Please cite this article as: Huang, W., et al., Isolation and molecular characterisation of avonoid 3-hydroxylase and avonoid 3, 5-hydroxylase genes from a traditional Chinese medicinal plant, Epimedium sagittatum, Gene (2012), doi:10.1016/j.gene.2011.11.029

2.5. Quantitative RT-PCR for transcription levels of EsF3H and EsF35H 196 To detect the transcription levels of EsF3H and EsF35H genes in 197 different tissues of E. sagittatum, quantitative RT-PCR was carried out. 198

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One microgram of each total RNA from leaf, petiole, ower bud, ower and root was used for the reverse transcription reaction with the Primescript RT reagent kit combined with gDNA eraser to remove any contaminated genomic DNA (Takara, Japan). Leaf tissues included three different samples: green old leaves, red old leaves and red young leaves, which were collected from a single plant at different developmental stages. qPCR system was recommended by the SYBR Premix Ex Taq II kit instructions (Takara, Japan) and run by Stepone Plus equipment (ABI, USA). Gene-specic primers (F3H.qF/R, F35H.qF/R) in the qPCR assay were designed, and the actin homologue (Actin.qF/R) was used as an internal control (Supplemental Table S1). The comparative Ct method was used to determine the relative expression, and the ower tissues were set as the calibrator sample (Livak and Schmittgen, 2001). 2.6. Determination of the anthocyanin content Anthocyanins were extracted and measured as described by Mancinelli (1990). Green and red mature leaves from the same E. sagittatum individual were harvested and frozen at 70 C until required. The samples (about 200300 mg) were ground into ne powder with liquid nitrogen. Anthocyanins were extracted with 1% HCl in methanol for 24 h at 4 C in darkness with occasional shaking. The extracts were centrifuged and decanted carefully, and their absorbance was measured at 530 nm (peak absorption of anthocyanins) and 657 nm (peak absorption of chlorophyll degradation products). The equation A530-0.25A657 was used to compensate for the absorption

of chlorophyll degradation products at 530 nm. Anthocyanin content was calculated as cyanidin-3-glucoside using 29,600 as the molecular extinction coefcient and 445 as the molecular weight because cyanidin-3-glucoside is the most common anthocyanin pigment in nature (Francis and Markakis, 1989). Three independent replicates were analysed for each sample. 3. Results and discussion 3.1. Isolation and sequence analysis of EsF3H and EsF35H clones To obtain the F3H and F35H clones from E. sagittatum, we employed a homology-based cloning method using degenerate primers designed in the conserved domains of the known F3H and F35H genes. Two fragments, 439 bp and 578 bp, were amplied and sequenced as the putative EsF3H and EsF35H targets, respectively. The deduced amino acid sequence of the partial fragment EsF3H and EsF35H gene showed 84% and 73% identities with Lobelia erinus F3H (GenBank accession no. BAF49324) and Vinca major F35H (GenBank accession no. ACZ63205), respectively. Only one representative candidate sequence of each gene was found to represent the EsF3H and EsF35H conserved domains from multiple clone sequencing results. This result indicates that only one copy of each gene exists in the genome of E. sagittatum. This was inconsistent with the reports about their reported copy numbers in grapes and petunias (Brugliera et al., 1999; Castellarin et al., 2006). Therefore, further DNA gel blot analysis is needed to

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Fig. 1. Comparison of the deduced amino acid sequences of E. sagittatum F3H and F35H. Numbers indicate position of amino acid from the N-terminus. (*) means identical, (:) means strong similar, (.) means weak similar. Identical amino acid residues are shaded in black, similar in grey. The P450-featured conserved motif, including the proline-rich hinge region, oxygen binding pocket motif, EXXR motif and heme binding domain are boxed.

Please cite this article as: Huang, W., et al., Isolation and molecular characterisation of avonoid 3-hydroxylase and avonoid 3, 5-hydroxylase genes from a traditional Chinese medicinal plant, Epimedium sagittatum, Gene (2012), doi:10.1016/j.gene.2011.11.029

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Fig. 2. Phylogenetic tree showing selected F3H and F35H genes from other plant species grouped into the CYP75A and CYP75B cytochrome P450 subfamily, respectively. The E. sagittatum F3H (ADE80941) and F35H (ADE80942) are marked in diamond. The protein sequences are retrieved from the GenBank database: F3H from Antirrhinum majus (ABB53383), Arabidopsis thaliana (Q9SD85), Brassica napus (ABC58723), Callistephus chinensis (AAG49298), Gentiana triora (BAD91808), Glycine max (ABW69386), Ipomoea purpurea (BAD00191), Malus x domestica (ACR14867), Matthiola incana (AAG49301), Petunia x hybrida (Q9SBQ9), Torenia hybrid cultivar (BAB87838), Vitis vinifera (ABH06586); F35H from Camellia sinensis (ABA40923), Campanula medium (BAA03440), Catharanthus roseus (CAA09850), Delphinium grandiorum (AAX51796), Eustoma grandiorum (BAA03439), Gentiana triora (BAA12735), Glycine max (AAM51564), Gossypium hirsutum (AAP31058), Petunia x hybrida hf1 (CAA80265), Petunia x hybrida hf2 (CAA80266), Verbena x hybrida (AAT34974), Vinca major (BAC97831), Vitis vinifera (CAI54277), Solanum melongena (CAA50155), Solanum lycopersicum (ADC80513), Solanum tuberosum (AAV85470). The tree was created from the ClustalW alignment using the neighbour joining method by the MEGA 4.0 program. The scale bar represents 0.05 amino acid substitutions per site, and the numbers next to the nodes are bootstrap values from 1000 replicates, and lower than 50 values are not indicated.

Please cite this article as: Huang, W., et al., Isolation and molecular characterisation of avonoid 3-hydroxylase and avonoid 3, 5-hydroxylase genes from a traditional Chinese medicinal plant, Epimedium sagittatum, Gene (2012), doi:10.1016/j.gene.2011.11.029

determine the copy numbers in E. sagittatum. In addition, the EsF35H degenerate primers amplied not only the target EsF35H fragment but also the off-target EsF3H fragment because fragment sequences from EsF35H clones also included the off-target EsF3H fragment sequences. The very high levels of identity between the conserved F3 H and F35H domains, belonging to the CYP75 family, complicate the degenerate primers design. To obtain the full-length cDNA sequences of the EsF3H and EsF35 H genes, 3-and 5-RACE methods were performed. The full-length cDNA of EsF3H was 1700 bp containing a 1545 bp open reading frame (ORF) encoding 514 amino acids (GenBank ID: HM011054). The full-length cDNA of EsF35H was 1633 bp containing a 1527 bp ORF encoding 508 amino acids (GenBank ID: HM011055). Remarkably, the EsF35H gene had a very short 3-untranslated region and no typical polyA signal sequence in the full-length cDNA sequence, which may indicate that there is another transcript that was not found. The deduced amino acid sequences of the EsF3H and EsF35H genes showed high homology to several avonoid hydroxylases from other species. The protein sequence encoded by EsF3H (GenBank ID: ADE80941) showed 74%, 74%, 70% identities and 86%, 85%, 82% positives with other plant F3H proteins from Malus x domestica (ACR14867), Vitis vinifera (BAE47004) and Petunia x hybrida (AAD56282), respectively, while the protein sequence encoded by EsF35H (GenBank ID: ADE80942) had a high identity of 74%, 73% and high positives of 84%, 83% with other plant F35H proteins from V. vinifera (BAE47007) and P. x hybrida (CAA80265), respectively. Pair-alignment analysis showed no signicant similarity between the full-length nucleotide sequences of EsF3H and EsF35H cDNA, but 49% identity and 71% positives between the deduced amino acid sequences (Fig. 1).

Phylogenetic analysis was carried out using the deduced amino acid sequences of the EsF3H and EsF35H genes with other known plant-specic avonoid hydroxylase proteins (Fig. 2). The phylogenetic tree was separated into two large groups, with all F3Hs and F35Hs genes clustered in the CYP75B and CYP75A clades, respectively (Fig. 2). The EsF3H and EsF35H genes were placed in the basal position of each clade (Fig. 2). The phylogenetic analysis suggested that P450 enzymes with the same function form a distinctive group. Against the conserved domain database, the P450 superfamily signature domain was found in both EsF3H and EsF35H sequences at N44K479 and P37V497 positions, respectively (data not shown). The proline-rich hinge region (P33PGPKPWP40 for EsF3H, P37PGPKGWP44 for EsF35H) is thought to act as a hinge that is required for optimal orientation of the P450 enzyme (Werck-Reichhart and Feyereisen, 2000). The motif (A/G)GX(D/E)T(T/S), which forms a binding pocket for oxygen molecules required for catalytic activity (Chapple, 1998), was present in both EsF3H and EsF35H. The absolutely conserved EXXR motif, used to stabilise the core structure, and the most characteristic P450 consensus sequence FxxGxRxCxG, which is located in the heme domain and responsible for carbon monoxide-binding ability, were also found in both EsF3H and EsF35 H genes (Werck-Reichhart and Feyereisen, 2000) (Fig. 1). Moreover, F3H-specic motifs VDVKG and VVVAAS (Boddu et al., 2004) have high similarity counterparts at M427DVKG431 and V76IVAAS81 of EsF3H gene, respectively, and the characteristic motif GGEK (Brugliera et al., 1999) distinguishing F3H from F35H was present at G421GKN424 in the EsF3H gene but not in the EsF35H gene. The genomic structure of the EsF3H and EsF35H genes was analysed by pairwise aligning between the full-length cDNA sequences and the corresponding genomic sequences. Fig. 3 showed that the EsF3H gene consisted of three exons and two introns, consistent

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Fig. 3. Genomic structure of F3H and F35H in E. sagittatum. Exons are shown by a grey box, and introns by a black line. Numbers indicate the length of exon and intron. Start codon and stop codon are shown for the entire open reading frame.

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with the genomic structure of F3H in the apple (Han et al., 2010), grape (Jeong et al., 2006) and morning glory (Hoshino et al., 2003). The EsF35H genomic sequence was split into two exons by one intron, which is identical to the F35H gene from V. major (Mori et al., 2004) and grape (Jeong et al., 2006) (Fig. 3). However, the F35H gene in several Solanaceae species, including the petunia, potato and tomato, have three exons and two introns (Olsen et al., 2010). 3.2. Expression patterns of EsF3H and EsF35H in various tissues To investigate the tissue-specic expression patterns of the EsF3H and EsF35H genes, different tissues, including green and red old leaves, red young leaves, petioles, ower buds, owers and roots from E. sagittatum, were used for quantitative RT-PCR analysis (Fig. 4a). The qPCR assay results indicated that both EsF3H and EsF35H genes were expressed in all tissues tested (Fig. 4b), which were in agreement

Fig. 4. Quantitative RT-PCR analysis of EsF3H and EsF35H transcription levels in various tissues of E. sagittatum. (a): Phenotype characteristics of seven samples used for qRT-PCR analysis. (b): Transcription levels of EsF3H and EsF35H genes in various tissues by qRT-PCR analysis. The comparative Ct method is used to determine the relative expression, and the ower is selected as the calibration sample. (c): Comparison of anthocyanin extraction colours in 1% HCl acidied methanol and anthocyanin contents from old green leaves (left) and old red leaves (right). (For interpretation of the references to colour in this gure legend, the reader is referred to the web version of this article.)

Please cite this article as: Huang, W., et al., Isolation and molecular characterisation of avonoid 3-hydroxylase and avonoid 3, 5-hydroxylase genes from a traditional Chinese medicinal plant, Epimedium sagittatum, Gene (2012), doi:10.1016/j.gene.2011.11.029

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with the expression patterns of grape VvF3H and VvF35H (Castellarin et al., 2006). EsF3H was strongly expressed in red-purple ower buds and red young leaves, moderately in green old leaves, owers and roots, and weakly in the red old leaf and petioles (Fig. 4b). An extremely high expression level of EsF35H was detected in the red young leaves and red-purple ower buds, and high expression was detected in red petioles, all of which may accumulate large amounts of anthocyanins, and weak expression levels were detected in green and red old leaves (Fig. 4b). Moreover, both EsF3H and EsF35H were abundantly expressed in pigmented tissues such as red young leaves, red-purple ower buds and red petioles, which all contain amounts of anthocyanins, except for the low expression level of EsF3H gene in red petioles. Therefore, it was hypothesised that the EsF3H and EsF35H genes corresponded to the accumulation pattern of anthocyanins in tissues from E. sagittatum, with the exception of the low transcription level of the EsF3H gene in pigmented petioles. Experiments investigating avonoid/anthocyanin contents and compositions in petioles may be needed to explain this exception in future because the mRNA levels and the ratio of F3H and F35H genes were shown to control avonoid composition in several grape organs (Jeong et al., 2006). Notably, both EsF3H and EsF35H genes were moderately expressed in the root tissues (Fig. 4b), where the bioactive components (prenylated avonol glycosides) of E. sagittatum also are abundantly deposited. This result indicated that the transcription levels of EsF3H and EsF35H maybe correlated with the accumulation pattern of the bioactive

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Q2 359
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388 389 390 391 392 393 394

Acknowledgments

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This work was supported by the National Natural Science Foundation of China (30800624), the CAS/CAFEA International Partnership Program for Creative Research Teams (0921101001), and the Hubei Natural Science Foundation (2009CDA073), and Knowledge Innovation Project of The Chinese Academy of Sciences (KSCX2-YW-N-043). Special thanks Professor Ling Yuan for critical review of our manuscript.

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In summary, we isolated the gene encoding avonoid 3-hydroxylase (F3H) and avonoid 3,5-hydroxylase (F35H) from E. sagittatum using a homology-based cloning method. Sequence analysis and molecular characterisation proved that EsF3H and EsF35H homologues were the real candidate genes, belonging to the CYP75B and CYP75A subfamilies, respectively. Transcription levels of the EsF3H and EsF35H genes were detected in all tissues tested, and they were highly expressed in most of the pigmented tissues. Moreover, the expression levels of both genes correlated positively with the anthocyanin accumulation pattern in leaves. The cloning and molecular characterisation of the EsF3H and EsF35H genes will accelerate studies about the avonoid biosynthetic pathway in E. sagittatum and further build an important foundation for genetic engineering to improve agricultural quality. Supplementary data to this article can be found online at doi:10. 1016/j.gene.2011.11.029.

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4. Conclusion

components, perhaps because both genes catalyse hydroxylation not only of anthocyanidins but also of avonols in E. sagittatum. The colour of leaves from a single plant can change from red to green as the developmental stage progresses, as the three different leaf samples collected for qPCR analysis showed (Fig. 4a). The red pigmented leaves accumulated more anthocyanins than the green leaves (Fig. 4c). The qPCR analysis results showed that the expression levels of both EsF3H and EsF35H genes were more highly expressed in young red leaves than in old green leaves. This was especially true for EsF35H expression, but both of the genes reduced their expression levels in the old red leaves, which were grown right before the turning stage for colour change; EsF35H expression levels had especially reduced dramatically (Fig). Biological physiology and gene expression could change sharply in fruits and vegetables at the turning stage of development. VvF3H and VvF35H expression patterns both increased to the max level in seed or skin at the vraison stage (Bogs et al., 2006; Castellarin et al., 2006). Anthocyanin contents also reduced signicantly when leaves turned from red to green (Fig. 4c). These partly contributed to the explanation for the dramatic decrease of EsF35H expression levels. The expression patterns of the EsF3H and EsF35H genes correlated positively with the anthocyanin accumulation patterns in leaves from E. sagittatum, which were consistent with previous reports in the apple and grape that MdF3H, VvF3H and VvF35H were expressed higher in red cultivars than in yellow or white cultivars (Bogs et al., 2006; Han et al., 2010).

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Please cite this article as: Huang, W., et al., Isolation and molecular characterisation of avonoid 3-hydroxylase and avonoid 3, 5-hydroxylase genes from a traditional Chinese medicinal plant, Epimedium sagittatum, Gene (2012), doi:10.1016/j.gene.2011.11.029

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