Research Note Low Expression of Flavonoid 3,5 -Hydroxylase (F3,5 H) Associated with Cyanidin-based Anthocyanins in Grape Leaf

Research Note Low Expression of Flavonoid 3,5 -Hydroxylase (F3,5 H) Associated with Cyanidin-based Anthocyanins in Grape Leaf Hironori Kobayashi, 1, 2 * Shunji Suzuki, 1 Fumiko Tanzawa, 2 and Tsutomu Takayanagi 1 Abstract: The aim of this study was to clarify anthocyanin composition and transcription profiles of anthocyanin-synthesis-related genes in grape (Vitis vinifera L.) leaf during development. Berry skin accumulated both cyanidin-based anthocyanins and delphinidin-based anthocyanins during development and showed up-regulation of flavonoid 3,5 -hydroxylase gene (F3,5 H) expression. Grape leaf, in contrast, accumulated a large amount of cyanidin-based anthocyanins during development. This result was supported by the low expression of F3,5 H in developing grape leaf. The low expression of F3,5 H and the low level of delphinidin-based anthocyanins in grape leaf were consistent in green-yellow-, purple-, and black-colored skin cultivars. Flavonoid 3 -hydroxylase gene (F3 H) expression increased in grape leaf during development. Since F3,5 H regulates the synthesis of delphinidin-based anthocyanins and F3 H regulates the synthesis of cyanidin-based anthocyanins in plants, it is possible that grape leaf accumulates large amount of cyanidin-based anthocyanins through the cyanidin synthesis pathway regulated by F3 H. Key words: flavonoid 3,5 -hydroxylase gene, flavonoid 3 -hydroxylase gene, cyanidin-based anthocyanin, delphinidin-based anthocyanin, grape The black/red color of grape berries is the result of anthocyanin accumulation in berry skins (Glories 1978). Anthocyanins are flavonoid compounds synthesized from phenylalanine (Boss et al. 1996) and are a key contributor to wine color (Castellarin et al. 2006, Castellarin and Gaspero 2007). In addition to their role in plant coloration, the anthocyanin formation pathway produces a wide range of compounds, such as flavonols, flavonones, and proanthocyanidins, in many plants and fruit, including grapes. Therefore, the composition and concentration of anthocyanins in grape skin affect wine taste (Glories 1988) in addition to wine color. Anthocyanins are classified into five groups based on the number of hydroxyl groups on the B-ring: cyanidin, peonidin, delphinidin, petunidin, and malvidin. Cyanidin and peonidin are referred to as cyanidin-based anthocyanins because they are synthesized from cyanidin. Delphinidin, petunidin, and malvidin are delphinidin-based anthocyanins because they are synthesized from delphinidin (Nyman and Kumpulainen 2001). Most anthocyanins are sensitive to ph, changing from red to blue as ph rises. In grape skin 1 Laboratory of Fruit Genetic Engineering, The Institute of Enology and Viticulture, University of Yamanashi, 1-13-1 Kofu, Yamanashi 400-0005, Japan, and 2 Laboratory of Enology & Viticulture, Mercian Corporation, 4-9-1 Jyonan, Fujisawa, Kanagawa 251-0057, Japan. *Corresponding author (email: kobayashi-hr@mercian.co.jp; tel.: +81-55-220-8394; fax: +81-55-220-8768) Manuscript submitted Feb 2009, revised Apr 2009, accepted May 2009. Publication costs of this article defrayed in part by page fees. Copyright 2009 by the American Society for Enology and Viticulture. All rights reserved. that generally has a ph of 4 or lower, cyanidin-based anthocyanins produce red color while delphinidin-based anthocyanins produce blue color. The composition of anthocyanins in berry skin differs among grape cultivars. The expression of flavonoid 3 -hydroxylase (F3 H) and flavonoid 3,5 -hydroxylase (F3,5 H) has a marked effect on anthocyanins composition in berry skin (Bogs et al. 2006, Castellarin et al. 2006, Joeng et al. 2006). Therefore, environmental effects including air temperature (Mori et al. 2007) and water deficit (Castellarin et al. 2007) affect anthocyanin composition via the expression level of F3 H and/or F3,5 H. Epidermal anthocyanins in grape leaf also act as light screening agents (Liakopoulos et al. 2006). The color change of aging leaves in autumn occurs due to the accumulation of anthocyanins prior to defoliation in both white and red/black cultivars. However, unlike berry skin, both anthocyanin composition and transcription profiles of anthocyanin-synthesis-related genes in grape leaf have not been clarified. In the present study, we determined anthocyanin composition and transcription profiles of anthocyaninsynthesis-related genes in grape leaf during development. Materials and Methods Plant materials. Light purple berried Vitis vinifera cv. Koshu, which is a grape cultivar indigenous to Japan, green-yellow berried Chardonnay, and black berried Merlot were used in this study. In the 2007 growing season, leaves and berries were collected four times: 14 weeks postflowering at the end of veraison, 17 weeks postflowering at early harvest, 19 weeks postflowering at harvest, and 22 weeks postflowering at the end of the ripening process. All plant materials were frozen immediately in liquid nitrogen and stored at -80 C until use. 362

Cyanidin-Based Anthocyanins in Grape Leaf 363 Determination of anthocyanin composition. To determine anthocyanin (cyanidin, peonidin, delphinidin, petunidin, and malvidin) composition in leaf and berry skin, each tissue was pulverized in liquid nitrogen. One gram of the powder was macerated in 20 ml 0.1% HCl-methanol for 4 hr at room temperature in the dark. The mixture was filtered through a 0.45-µm cellulose acetate filter (Advantec Toyo, Tokyo, Japan) and analyzed by reversed-phase high-performance liquid chromatography (LC-10Avp, Shimadzu, Kyoto, Japan) as described previously (Baranowski and Nagel 1981, Chang et al. 1993). In this study, the term cyanidin-based anthocyanins is used to indicate the gross weight of cyanidin-3-glucoside, cyanidin-3-acetylglucoside, cyanidin-3-coumarylglucoside, peonidin-3-glucoside, peonidin-3-acetylglucoside, and peonidin-3-coumarylglucoside. The term delphinidin-based anthocyanins is used to indicate the gross weight of delphinidin-3-glucoside, delphinidin-3-acetylglucoside, delphinidin-3-coumarylglucoside, petunidin-3-glucoside, petunidin-3-acetylglucoside, petunidin-3-coumarylglucoside, malvidin-3-glucoside, malvidin- 3-acetylglucoside, and malvidin-3-coumarylglucoside. RNA extraction. One gram of leaf or skin was pulverized in liquid nitrogen. The powder was added to 10 ml extraction buffer (0.1 M Tris-HCl [ph 9.5], 20 mm EDTA [ph 8.0], 1.4 M sodium chloride, 2% polyvinylpolypyrrolidone, 2% cetyl trimethyl ammonium bromide, and 0.2% β-mercaptoethanol) and the suspension was stirred rapidly for 10 min at 60 C. The suspension was extracted twice with an equal volume of chloroform:isoamyl alcohol (24:1, v/v). It was then centrifuged at 830 g for 30 min at room temperature. The supernatant was transferred to a new microtube and 2.5 ml 0.1 M lithium chloride was added. The microtube was incubated at 4 C for at least 16 hr and centrifuged at 13,200 g for 10 min at 4 C. After removal of the supernatant, the pellet was suspended in 1.0 ml TE buffer and total RNA was precipitated by adding 400 μl 0.1 M lithium chloride followed by incubation at 4 C for 16 hr and centrifugation at 13,200 g for 10 min at 4 C. Finally, total RNA was purified using an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) as described previously (Tesniere and Vayda 1991). Transcription analysis of anthocyanin-synthesisrelated genes. First-strand cdna was synthesized from 500 ng of total RNA using a PrimeScript RT Reagent Kit (TaKaRa-Bio Inc, Shiga, Japan) according to the manufacturer s instructions. Sequences of anthocyanin-synthesis-related genes were obtained from DFCI Grape Gene Index (http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/ gimain.pl?gudb=grape), and the nucleotide sequences of PCR primers used in this study were designed by Primer Express software version 3.0 (Applied Biosystems Japan Ltd., Tokyo, Japan): phenylalanine ammonia lyase (PAL) primers (5 -GCGCCACATATCCACTGATG-3 and 5 - CCTCGAATGCCCCTATTTTTT-3, corresponding to bases 1733-1752 and 1852-1832 of VvPAL, GenBank accession no. EF192469, respectively); cinnamate 4-hydroxylase (C4H) primers (5 -ATCCACCGCCACAACCAT-3 and 5 -GGC- CAGAATTATAGCGCAGAA-3, corresponding to bases 5015-5032 and 5090-5070 of Vv78X211789.9, GenBank accession AM468511, respectively); chalcone synthase (CHS) primers (5 -TCTGAGCGAGTATGGGAACATG-3 and 5 -CTGTGCTGGCTTTCCCTTCT-3, corresponding to bases 1469-1490 and 1563-1544 of VvCHS, GenBank accession AB015872, respectively); chalcone isomerase (CHI) primers (5 -GACGGGTCGCCAGTATTCAG-3 and 5 -GCTTTGGCTTCTGCGTCAGT-3, corresponding to bases 320-330 and 409-390 of VvCHI, GenBank accession X75963, respectively); F3 H primers (5 -TATGGGCT- GACCCTACAACGA-3 and 5 -CCTGGGCAAACAACCT- CATT-3, corresponding to bases 2566-2586 and 2664-2645 of VvF3 H4, GenBank accession AB213605, respectively); F3,5 H primers (5 -AGGGTCGGAGTCAAATGAGTTC-3 and 5 -CGCTGGATCCCTTGGATGT-3, corresponding to bases 639-660 and 758-740 of VvF3,5 H, GenBank accession AB213606, respectively); leucoanthocyanidin dioxygenase (LDOX) primers (5 -GCGATATGACCATCT- GGCCTAA-3 and 5 -ATCCCAACCCAAGCGATAGC-3 corresponding to bases 544-565 and 663-643 of VvLODX, GenBank accession X75966, respectively); UDP glucoseflavonoid 3-O-glucosyl transferase (UFGT) primers (5 -CT- TCTTCAGCACCAGCCAATC-3 and 5 -AGGCACAC- CGTCGGAGATAT-3, corresponding to bases 548-588 and 647-628 of VvUFGT, GenBank accession AB047099, respectively); and 18SrRNA primers (5 -CGAAAGCATTT- GCCAAGGAT-3 and 5 -CCTGGTCGGCATCGTTTATG-3, corresponding to bases 522-541 and 625-606 of Vv18S ribosomal RNA gene, GenBank accession AF207053, respectively). Real-time reverse transcription PCR (real-time RT- PCR) was performed using an ABI PRISM 7300 Real-Time PCR System (Applied Biosystems) with SYBR Green detection modules. Briefly, real-time RT-PCR was carried out in 50 µl reaction mixture containing 100 nm of each primer, 5 ng cdna, and 25 µl 2 x SYBR Green Master Mix Reagent (Applied Biosystems). Real-time RT-PCR conditions were as follows: 50 C for 2 min for RT reaction and 95 C for 10 min, then 40 cycles at 95 C for 15 sec and 60 C for 1 min for PCR amplification. Negative control and 18S rrna primers were used for background analysis and for normalization, respectively (Downey et al. 2003, Fujita et al. 2005). Dissociation curves for each sample were analyzed to verify the specificity of the amplification reaction. Data were analyzed using the 7300 system software SDS 1.3.0 (Applied Biosystems). Expression levels were determined as the number of amplification cycles needed to reach a fixed threshold, as described elsewhere (Pfaffl 2001, Reid et al. 2006). Triplicate experiments using three independent samples were performed in a 96-well reaction plate. Results and Discussion The biosynthetic pathway of five anthocyanins from phenylalanine in grape is shown (Figure 1). The concentrations of anthocyanins increased in both grape leaf and berry skin of light purple berried Koshu grape cultivar during development (Figure 2A, 2B). At the postflowering stage, berry

364 Kobayashi et al. skin accumulated more delphinidin-based anthocyanins than cyanidin-based anthocyanins (Figure 2A, week 14). During the course of berry development, cyanidin-based anthocyanins accumulated in berry skin, accounting for ~60% of the total anthocyanins accumulated in skin at the end of the ripening process (week 22). In contrast, grape leaf accumulated high levels of cyanidin-based anthocyanins during development, although delphinidin-based anthocyanins were also detected (Figure 2B). To determine why the composition of anthocyanins was entirely different between berry skin and grape leaf, we analyzed the transcription profiles of eight genes (PAL, C4H, CHS, CHI, F3 H, F3,5 H, LDOX, and UFGT) related to anthocyanin synthesis in both tissues during development (Figure 3). In berry skin, PAL and C4H expression decreased during berry development, while the expression of the other genes was up-regulated (Figure 3A). F3 H, which regulates the synthesis of cyanidin-based anthocyanins, and F3,5 H, which regulates the synthesis of delphinidin-based anthocyanins, were expressed at the same level during berry development. This result was supported by the observation that Koshu berry skin accumulated both cyanidin- and delphinidin-based anthocyanins (Figure 2A). Unexpectedly, the expression of the genes at harvest and at the end of ripening was more abundant in grape leaf than in berry skin (Figure 3, weeks 19 and 22). However, F3,5 H expression in grape leaf was not detected during development (Figure 3B). F3 H expression was increased in Figure 1 The flavonoid pathway leading to the synthesis of anthocyanins. Genes tested in the present study are enclosed in squares: PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; CHS, chalcone synthase; CHI, chalcone isomerase; CMT, caffeate-3-ometheyltransferase; F3 H, flavonoid 3 -hydroxylase; F3,5 H, flavonoid 3,5 -hydroxylase; LDOX, leucoanthocyanidin dioxygenase; UFGT, UDP glucose-flavonoid 3-O-glucosyl transferase; STS, stilbene synthase; 4CL, 4-coumarate ligase; C3H, coumarate-3-hydroxylase; F3H, flavonone-3- hydroxylase; DFR, dihydroflavonol 4-reductase. Figure 2 Anthocyanin composition in berry skin (A) and grape leaf (B) of purple berried Koshu during development: 14 weeks postflowering at the end of veraison, 17 weeks postflowering at early harvest, 19 weeks postflowering at harvest, 22 weeks postflowering at the end of the ripening process. Cyanidin-based anthocyanins include cyanidin and peonidin. Delphinidin-based anthocyanins include delphinidin, petunidin, and malvidin. Data were reproduced in triplicate experiments.

Cyanidin-Based Anthocyanins in Grape Leaf 365 grape leaf during development, suggesting that grape leaf actively synthesizes cyanidin-based but not delphinidinbased anthocyanins during development. This suggestion is based on results that show grape leaf accumulated a large amount of cyanidin-based anthocyanins during development, although delphinidin-based anthocyanins were also detected (Figure 2B). These results together indicate that grape leaf expresses F3,5 H at nondetectable levels and accumulates only cyanidin-based anthocyanins through the cyanidin synthesis pathway regulated by F3 H. Similar results to light purple berried Koshu were obtained in both green-yellow berried Chardonnay and black berried Merlot. Merlot, Koshu, and Chardonnay respectively accumulated 8976.6, 100.5, and 2.42 µg anthocyanins/g fresh weight of berry skin at harvest (Figure 4A). Merlot accumulated more delphinidin-based anthocyanins in berry Figure 3 Transcription profiles of anthocyanin-synthesis-related genes in Koshu berry skin (A) and grape leaf (B). 18S rrna was used as internal control. Data were calculated as gene expression relative to 18S rrna gene expression. Bars indicate means ± standard deviation of triplicate experiments.

366 Kobayashi et al. skin than cyanidin-based anthocyanins, while Chardonnay accumulated more cyanidin-based anthocyanins than delphinidin-based anthocyanins. F3 H and F3,5 H were transcribed in berry skins tested at harvest (Figure 4B), even if skin color appeared green-yellow. Considering that Chardonnay accumulated small amounts of anthocyanins in berry skin, the expression of F3 H and F3,5 H in berry skin may also be related to the synthesis of other flavonoids including flavonols and proanthocyanidins. In grape leaf, Merlot, Koshu, and Chardonnay respectively accumulated 22.1, 53.1, and 5.4 µg anthocyanins/g fresh weight of grape leaf at harvest (Figure 4C). Cyanidin-based anthocyanins were detected in grape leaves of all cultivars tested irrespective of skin color. Interestingly, F3,5 H was transcribed at a nondetectable level by RT-PCR in grape leaves tested at harvest, even in black berried Merlot (Figure 4D). UFGT plays an important role in anthocyanin synthesis in berry skin (Kobayashi et al. 2001) and is regulated by VvMYBA transcriptional factors (Kobayashi et al. 2002, Matus et al. 2009). The transcriptional induction of F3 H Figure 4 Anthocyanin population and F3 H and F3,5 H expression in berry skin and grape leaf of different cultivars. Cyanidin-based (cyanidin and peonidin) and delphinidin-based (delphinidin, petunidin, and malvidin) population in berry skin (A) and grape leaf (C) of Merlot (Me), Koshu (Ko), and Chardonnay (Ch) at 19 weeks postflowering at harvest. Values indicate total amount of anthocyanins in one gram of fresh weight of each tissue. F3 H and F3,5 H expression in berry skin (B) and grape leaf (D) determined using real-time RT-PCR. 18S rrna was used as internal control. Data were calculated as gene expression relative to 18S rrna gene expression. Gel images indicate F3 H and F3,5 H expression as analyzed by conventional RT-PCR. Bars indicate means ± standard deviation of triplicate experiments.

Cyanidin-Based Anthocyanins in Grape Leaf 367 and F3,5 H coincided with the beginning of anthocyanin biosynthesis in black-colored berry and green-yellow-colored berry (Castellarin et al. 2006). In addition, F3 H and F3,5 H expression was consistent with the chromatic evolution of ripening berries (Castellarin et al. 2006). However, other regulators might regulate the flavonoid pathway in grape leaf during development. It remains to be clarified why grape leaf does not express F3,5 H. Future studies of the proportion and quantification of other flavonoids, using genetic and epigenetic analyses of F3,5 H, would be useful to reveal the mechanisms of F3,5 H transcriptional regulation in grape leaf. Conclusion In the present study, we detected and analyzed the accumulation of cyanidin-based anthocyanins in grape leaf, which occurred irrespective of cultivar. 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