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NPC Natural Product Communications

EDITOR-IN-CHIEF

DR. PAWAN K AGRAWAL Natural Product Inc. 7963, Anderson Park Lane, Westerville, Ohio 43081, USA agrawal@naturalproduct.us

EDITORS

PROFESSOR ALEJANDRO F. BARRERO Department of Organic Chemistry, University of Granada, Campus de Fuente Nueva, s/n, 18071, Granada, Spain afbarre@ugr.es

PROFESSOR ALESSANDRA BRACA Dipartimento di Chimica Bioorganicae Biofarmacia, Universita di Pisa, via Bonanno 33, 56126 Pisa, Italy braca@farm.unipi.it

PROFESSOR DE-AN GUO State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100083, China gda5958@163.com

PROFESSOR YOSHIHIRO MIMAKI School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Horinouchi 1432-1, Hachioji, Tokyo 192-0392, Japan mimakiy@ps.toyaku.ac.jp

PROFESSOR STEPHEN G. PYNE Department of Chemistry University of Wollongong Wollongong, New South Wales, 2522, Australia spyne@uow.edu.au

PROFESSOR MANFRED G. REINECKE Department of Chemistry, Texas Christian University, Forts Worth, TX 76129, USA m.reinecke@tcu.edu

PROFESSOR WILLIAM N. SETZER Department of Chemistry The University of Alabama in Huntsville Huntsville, AL 35809, USA wsetzer@chemistry.uah.edu

PROFESSOR YASUHIRO TEZUKA Faculty of Pharmaceutical Sciences Hokuriku University Ho-3 Kanagawa-machi, Kanazawa 920-1181, Japan y-tezuka@hokuriku-u.ac.jp

PROFESSOR DAVID E. THURSTON Department of Pharmacy and Forensic Science, King’s College London, Britannia House, 7 Trinity Street, London SE1 1DB, UK. david.thurston@kcl.ac.uk

ADVISORY BOARD

Prof. Viqar Uddin Ahmad Karachi, Pakistan

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Prof. Yoshinori Asakawa Tokushima, Japan

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Prof. Anna R. Bilia Florence, Italy

Prof. Maurizio Bruno Palermo, Italy

Prof. César A. N. Catalán Tucumán, Argentina

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Prof. Cristina Gracia-Viguera Murcia, Spain

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Prof. Niel A. Koorbanally Durban, South Africa

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Prof. Stefano Serra Milano, Italy

Prof. Monique Simmonds Richmond, UK

Dr. Bikram Singh Palampur, India

Prof. John L. Sorensen Manitoba, Canada

Prof. Johannes van Staden Scottsville, South Africa

Prof. Valentin Stonik Vladivostok, Russia

Prof. Winston F. Tinto Barbados, West Indies

Prof. Sylvia Urban Melbourne, Australia

Prof. Karen Valant-Vetschera Vienna, Austria

HONORARY EDITOR

PROFESSOR GERALD BLUNDEN The School of Pharmacy & Biomedical Sciences,

University of Portsmouth, Portsmouth, PO1 2DT U.K.

axuf64@dsl.pipex.com

Low Caffeine Content in Novel Grafted Tea with Camellia sinensis as Scions and Camellia oleifera as Stocks Wei-Wei Deng, Min Li, Chen-Chen Gu, Da-Xiang Li, Lin-Long Ma, Yang Jin and Xiao-Chun Wan* State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, 130 Changjiang West Road, Hefei 230036, Anhui, China xcwan@ahau.edu.cn

Received: January 15th, 2015; Accepted: March 30th, 2015

Caffeine, a purine alkaloid, is a major secondary metabolite in tea leaves. The demand for low caffeine tea is increasing in recent years, especially for health reasons. We report a novel grafted tea material with low caffeine content. The grafted tea plant had Camellia sinensis as scions and C. oleifera as stocks. The content of purine alkaloids was determined in the leaves of one-year-old grafted tea plants by HPLC. We also characterized caffeine synthase (CS), a key enzyme involved in caffeine biosynthesis in tea plants, at the expression level. The expression patterns of CS were examined in grafted and control leaves by Western blot, using a self-prepared polyclonal antibody with high specificity and sensitivity. The expression of related genes (TCS1, tea caffeine synthase gene, GenBank accession No. AB031280; sAMS, SAM synthetase gene, AJ277206; TIDH, IMP dehydrogenase gene, EU106658) in the caffeine biosynthetic pathway was investigated by qRT-PCR. HPLC showed that the caffeine content was only 38% as compared with the non-grafted tea leaves. Immunoblotting analysis showed that CS protein decreased by half in the leaves of grafted tea plants. qRT-PCR revealed no significant changes in the expression of two genes in the upstream pathway (sAMS and TIDH), while the expression of TCS1 was greatly decreased (50%). Taken together, these data revealed that the low caffeine content in the grafted tea leaves is due to low TCS1 expression and CS protein accumulation. Keywords: Grafted tea, HPLC, Western blot, qRT-PCR, Low caffeine. Tea (Camellia sinensis) is one of the most popular drinks in the world, due to its unique flavor and health benefits [1-5]. Caffeine is a major secondary metabolite in tea, which imparts taste, and also makes it refreshing and exciting. However, caffeine is not good for children and pregnant women. Trying to decrease caffeine content in tea became a research hotspot up to now. Biosynthesis of caffeine involves four steps: xanthosine → 7-methyxanthosine → 7-methylxanthine→theobromine→caffeine [6]. In the caffeine biosynthesis pathway, 7-methylxanthine and theobromine could be converted to caffeine by caffeine synthase (CS, EC 2.1.1.160). C. oleifera is a unique woody edible oil tree in China and one of the four major woody oil crops in the world. Its fruit and oil have high nutritional and medicinal values [7]. Here, we report a new grafted tea of low caffeine content with C. sinensis as scions and C. oleifera as stocks. The contents of purine alkaloids in the leaves of one-year-old grafted tea plants were determined by HPLC. The expression of CS was examined in grafted and control tea leaves by Western blot, using a self-prepared polyclonal antibody with high specificity and sensitivity. The expression patterns of the genes related to caffeine biosynthetic pathway were also investigated by qRT-PCR. The concentrations of caffeine, theobromine and 7-methylxanthine were determined in leaves of seedlings of grafted tea, C. sinensis and C. oleifera (Figure 1). Although all three methylxanthines found in C. oleifera were present in low amounts, caffeine was the main purine alkaloid in the three seedlings. The biggest concentration of caffeine was found in the leaves of C. sinensis seedlings (40.6 ± 2.3 μmol·g-1 fresh weight). In grafted tea, the content of caffeine was significantly lower - 25.3 μmol·g-1 fresh weight - than in C. sinensis. The concentrations of the other two purine alkaloids, theobromine and 7-methylxanthine, were also decreased in leaves of grafted seedlings. A small amount of caffeine and very little theobromine and 7-methylxanthine were also detected in C. oleifera. The expression patterns of CS in leaves of grafted tea, C. sinensis and C. oleifera, were also examined by

Figure 1: Purine alkaloid contents in the leaves of Camellia sinensis, Camellia oleifera and grafted seedlings.

Figure 2: Western blot analysis of CS in leaves of seedlings of C. sinensis (1), grafted tea (2), and C. oleifera (3).

immunoblotting (Figure 2). In grafted tea seedlings, the expression of CS was much lower than in C. sinensis. We did not find any

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790 Natural Product Communications Vol. 10 (5) 2015 Deng et al.

Figure 3: Caffeine biosynthesis pathway in tea plants. sAMS (SAM synthetase), TIDH (IMP dehydrogenase), and TCS1 (caffeine synthase) studied in this work are shown. IMP, inosine-5’-monophosphate; XMP, xanthosine-5’-monophosphate; SAM, S-adenosyl-L-methionine; SAH, S-adenosyl-L-homocysteine.

Figure 4: Expression of sAMS (SAM synthase), TIDH (IMP dehydrogenase), and TCS1 (caffeine synthase) in the leaves of Camellia sinenesis, C. oleifera and grafted tea. GAPDH was used as reference gene. The expression level of the genes in C. sinenesis was set as “1.0”. Data represent mean of three biological samples. The experiments were repeated three times with similar results.

expression of CS in the leaves of C. oleifera using a CS polyclonal antibody with high specificity and sensitivity prepared from tea leaves. The expression patterns of CS matched with the concentration changes of purine alkaloids in the grafted tea seedlings (Figure 1). To some extent, the low caffeine content in leaves of grafted tea seedlings is due to the low CS expression. Caffeine biosynthesis starts with the methylation of xanthosine and S-adenosylmethionine (SAM) is the methyl donor [8, 9]. Subsequently there are four major steps from xanthosine to caffeine (Figure 3, revised according to [10]). We investigated here the expression of TCS1, TIDH and sAMS, which encode caffeine synthase, inosine-5'-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) and S-adenosylmethionine synthetase (MAT, EC 2.5.1.6). The expression of TCS1 was highest in the C. sinensis leaves, but significantly decreased in the grafted tea leaves. However, different expression patterns were found with sAMS and TIDH. The highest expression of sAMS was found in grafted tea leaves, and was similar in C. oleifera and C. sinensis leaves. TIDH

expression was similar in C. sinensis and grafted tea leaves, but low in C. oleifera (Figure 4). TCS1 regulates the biosynthesis of theobromine and caffeine, and is specific for caffeine biosynthesis. However, sAMS catalyzes the biosynthesis of SAM, the universal methyl donor, from methionine and ATP. It is also very important in regulation of plant development, abiotic and biotic stresses. Besides, SAM is an intermediate compound for the biosynthesis of polyamines, nicotianamine, biotin and ethylene [11-13]. TIDH encodes IMPDH, which is an important enzyme of de novo guanine nucleotide biosynthesis. In most organisms, the reaction of IMP to XMP is the first step in a branched pathway that leads to the biosynthesis of GMP. It has also been reported that IMPDH participates in ureide biosynthesis in nodules of tropical legumes and in caffeine biosynthesis [14]. The decreased content of caffeine in leaves of grafted seedlings was in accordance with the low expression of TCS1, which is specific for caffeine biosynthesis. However, the expression of sAMS and TIDH was not directly related to caffeine biosynthesis, probably because of their participation in many other metabolic pathways. In this study, we found that the content of caffeine was greatly decreased in leaves of grafted tea seedlings and that CS protein and TCS1 expression in leaves of grafted tea seedlings were also reduced. Thus it is very plausible that the decreased purine alkaloid concentration in grafted seedlings is, in part, a result of low TCS1 expression and CS protein accumulation. This grafted seedling may provide a new low caffeine tea in the future market. Experimental

Plant materials and graft treatment: The grafted tea plants with C. sinensis as scions and C. oleifera as stocks were grown at a plantation in Shucheng, Liu’an city, Anhui Province, China. The leaves of one-year-old grafted plants, C. sinensis and C. oleifera seedlings were collected, immediately frozen in liquid nitrogen and stored at -80°C until use (Figure 5).

Low caffeine in grafted tea material Natural Product Communications Vol. 10 (5) 2015 791

Figure 5: Samples used for alkaloids determination and caffeine synthase analysis. 1 - one-year-old grafted seedlings; 2 - leaves of C. sinenesis seedlings; 3 - leaves of grafted seedlings; 4 - leaves of C. oleifera seedlings.

Determination of purine alkaloid content by HPLC: Tea samples were homogenized in liquid nitrogen, and freeze-dried overnight. Samples (100 mg) were extracted in 2 mL boiling water for 20 min. After centrifugation at 6,000 x g for 15 min the supernatant was recovered and the debris was re-extracted twice as above. The pooled extracts (5 mL) were filtered through 0.45 μm nylon filters. Purine alkaloids were analyzed in a Waters 600 HPLC equipped with a 2489 UV-VIS detector. A reverse-phase C18 column (Gemini 5μm, 250 mm × 4.60 mm, Phenomenex, USA) was used to separate the alkaloids with a flow rate of 1.0 mL/min. The column oven temperature was set at 25°C. The mobile phase was as previously described [15], with a slight modification as follows: The solvents were 0.2%, v/v, acetic acid (A) and 100% acetonitrile (B) and the gradient was A 92% from 0 to 25 min, to 83%, v/v, at 30 min, to 10% at 32 min, to 92% at 40 min. HPLC grade acetic acid, methanol and acetonitrile were obtained from Tedia Co., Ltd (Fairfield, USA). Caffeine, 7-methylxanthine and theobromine standards were obtained from Sigma (St. Louis, USA). Western blot analysis: Preparation of the CS antibody was as previously described [16]. The open reading frame (ORF) of TCS1 gene from tea leaves was amplified, and ligated into the pGEX-4T-2 expression vector. The recombinant protein was expressed by induction with IPTG. In vitro enzyme activity was tested, and the recombinant protein was subsequently purified by affinity chromatography. The CS antibody was further refined by immunizing white rabbits with the purified protein. The antibody titer and specificity of the polyclonal antibody were confirmed by ELISA and Western blotting. The method for extraction of total soluble protein was similar to that of a previous report [17]. SDS-PAGE and Western blot analysis was performed as previously described [17, 18]. Briefly, samples (about 1 g) were ground in liquid nitrogen. An extraction buffer [50 mM Tris (pH 7.5), 20 mM KCl, 13 mM DTT] was added in a 1:5 ratio (plant tissue: buffer). After homogenization, samples were ground with 100 μL phenylmethanesulfonyl fluoride (PMSF, 0.1 mol.L-1) and 100 μL nonylphenoxypoly (ethyleneoxy) ethanol (NP-40). After centrifugation (16,000 x g, 30 min), the supernatant was collected and precipitated with 15 mL of 10%, w/v, trichloroacetic acid in

cold (-20°C) acetone for 4-12 h. The precipitate was washed 2-3 times with 0.07% DTT, w/v, in cold (-20°C) 80% acetone. Proteins were dried under vacuum, solubilized in rehydration buffer {7 M urea, 2 M thiourea, 0.4%, w/v, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane (CHAPS), 60 mM DTT, 0.4%, w/v, PMSF}, kept at room temperature for 10 min, and then stored at -80°C until use. The total amount of protein was measured with a Bradford protein assay kit (Bio-Rad) using bovine serum albumin as standard. Proteins were separated by SDS-PAGE (12% resolving gel, 5% stacking gel), and transferred to polyvinylidene fluoride membranes using a semidry electroblotter. After blocking for 2 h in a TBS buffer (Boster Biological Technology Co., Ltd) with 5%, w/v, non-fat dried milk at room temperature, the membranes were incubated with CS antibody for 1 h. After washing 3 times with TBST buffer, the membranes were incubated with anti-rat (IgG) (Sigma, St. Louis, USA) secondary antibody, and the complexes were visualized using a detection kit (Thermo, Rockford, USA). qRT-PCR analysis: Total RNA was extracted from tea leaves using the RNAprep Pure Plant Kit (Tiangen, China) following the manufacturer instruction. First strand cDNA was produced from 0.5 μg of total RNA using a PrimeScript RT Reagent Kit (Takara). qRT-PCR was performed using 2 μL of cDNA and 0.4 μM of each primer in a 25 μL reaction volume with SYBR Premix Ex TaqTM II (Perfect Real Time; Takara) on a Mini-Opticon real-time PCR system (Bio-Rad, Hercules, USA). GAPDH was used as normalization gene. The specific primers for qRT-PCR analysis were designed based on the 3’ or 5’-untranslated regions (UTR) of individual genes using the Primer Premier 5 software (http://www.premierbiosoft.com/primerdesign/). The gene specific primers were prepared from the tea database. The primer sequences were as follows: TCS1 (AB031280), 5’-AGCAAAGCTACCGAAGACCA-3’(forward) and 5’-TCCACACAAGAGCAAAATGC-3’(reverse); sAMS (AJ277206), 5’-CTTACGCCATTGGTGTTCCT-3’(forward) and 5’-GGCAGCAGTCTTCAAGAACC-3’(reverse); TIDH (EU106658), 5’-TGCTGAAAGAAGTGGTGTGC-3’(forward) and 5’-TCCCTTTTGTCATTGCTTCC-3’(reverse); GAPDH (GE651107), 5’-TTGGCATCGTTGAGGGTCT-3’(forward) and 5’-CAGTGGGAACACGGAAAGC-3’(reverse). Three replicates of each PCR were carried out using the following program steps: 95°C for 30 s, followed by 40 cycles (95°C for 5 s and 60°C for 30 s). The amplification efficiency of all tested genes was 95-110%. Data were analyzed according to the threshold cycle (Ct). The relative changes in gene expression were quantified using the 2-Ct method [19]. Differences shown in Figure 4 were calculated using GraphPad Prism 5.01 software. Acknowledgments - This study was supported by the National Natural Science Foundation of China (NSFC) projects (31300576, 31170283), Science and Technology Projects in Anhui Province (13Z03012) and Anhui Major Demonstration Project for Leading Talent Team on Tea Chemistry and Health. We would like to thank Professor Paulo Mazzafera, State University of Campinas, for his critical reading of the manuscript.

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Natural Product Communications Vol. 10 (5) 2015 Published online (www.naturalproduct.us)

A Stepwise Protocol for Induction and Selection of Prominent Coniferous Cell Cultures for the Production of -Thujaplicin Shinjiro Ogita, Masahito Shichiken, Chizuru Ito, Toshiyuki Yamashita, Taiji Nomura and Yasuo Kato 783

Low Caffeine Content in Novel Grafted Tea with Camellia sinensis as Scions and Camellia oleifera as Stocks Wei-Wei Deng, Min Li, Chen-Chen Gu, Da-Xiang Li, Lin-Long Ma, Yang Jin and Xiao-Chun Wan 789

Accounts/Reivews

Revisiting Caffeine Biosynthesis – Speculations about the Proximate Source of its Purine Ring Thomas W. Baumann 793

Biosynthesis of Caffeine Underlying the Diversity of Motif Bʹ Methyltransferase Fumiyo Nakayama, Kouichi Mizuno and Misako Kato 799

Occurrence, Biosynthesis and Metabolism of Theanine (γ-Glutamyl-L-ethylamide) in Plants: A Comprehensive Review Hiroshi Ashihara 803

Involvement of Allelopathy in the Formation of Monospecific Colonies of Ferns Hisashi Kato-Noguchi 811

Plant Cell, Tissue and Organ Culture: the Most Flexible Foundations for Plant Metabolic Engineering Applications Shinjiro Ogita 815

Natural Product Communications 2015

Volume 10, Number 5

Contents

Gerald Blunden Award (2014) Page

Stereocontrolled Total Synthesis of Tetrodotoxin from myo-Inositol and D-Glucose by Three Routes: Aspects for Constructing Complex Multi-Functionalized Cyclitols with Branched-Chain Structures Ken-ichi Sato, Shoji Akai and Juji Yoshimura 691

Metabolism and Biological Function of Natural Products in Plants (Guest editor: Hiroshi Ashihara)

Original Paper

(a) Biosynthesis

Occurrence and De novo Biosynthesis of Caffeine and Theanine in Seedlings of Tea (Camellia sinensis) Wei-Wei Deng and Hiroshi Ashihara 703

Metabolism of Purine Alkaloids and Xanthine in Leaves of Maté (Ilex paraguariensis) Yuling Yin, Riko Katahira and Hiroshi Ashihara 707

Comparative Analysis of Two DOPA Dioxygenases from Phytolacca americana Kana Takahashi, Kazuko Yoshida and Masaaki Sakuta 713 Biochemical Analysis of Phytolacca DOPA Dioxygenase Kana Takahashi, Kazuko Yoshida, Kei Yura, Hiroshi Ashihara and Masaaki Sakuta 717

Unraveling the Biosynthesis of Pilocarpine in Pilocarpus microphyllus Alexandra Christine Helena Frankland Sawaya, Yanna Dias Costa and Paulo Mazzafera 721

(b) Allelopathy

Transcriptomic Evaluation of Plant Growth Inhibitory Activity of Goniothalamin from the Malaysian Medicinal Plant Goniothalamus andersonii Naoya Wasano, Tomoko Takemura, Raihan Ismil, Baki Bakar and Yoshiharu Fujii 725

Momilactone Sensitive Proteins in Arabidopsis thaliana Hisashi Kato-Noguchi and Shinya Kitajima 729

Effect of Caffeine on the Expression Pattern of Water-Soluble Proteins in Rice (Oryza sativa) Seedlings Wei-Wei Deng, Hamako Sasamoto and Hiroshi Ashihara 733

Short Term Effect of Caffeine on Purine, Pyrimidine and Pyridine Metabolism in Rice (Oryza sativa) Seedlings Wei-Wei Deng, Riko Katahira and Hiroshi Ashihara 737

Cyanamide Phytotoxicity in Soybean (Glycine max) Seedlings involves Aldehyde Dehydrogenase Inhibition and Oxidative Stress John S. Maninang, Shin Okazaki and Yoshiharu Fujii 743

Allelopathy in a Leguminous Mangrove Plant, Derris indica: Protoplast Co-culture Bioassay and Rotenone Effect Aya Inoue, Daisuke Mori, Reiko Minagawa, Yoshiharu Fujii and Hamako Sasamoto 747

Effect of Purine Alkaloids on the Proliferation of Lettuce Cells Derived from Protoplasts Hamako Sasamoto, Yoshiharu Fujii and Hiroshi Ashihara 751

A Protocol for Axenic Liquid Cell Cultures of a Woody Leguminous Mangrove, Caesalpinia crista, and their Amino Acids Profiling Aya Inoue, Shinjiro Ogita, Shinpei Tsuchiya, Reiko Minagawa and Hamako Sasamoto 755

Phytotoxic Substance with Allelopathic Activity in Brachiaria decumbens Ai Kobayashi and Hisashi Kato-Noguchi 761

Isolation and Identification of an Allelopathic Substance from Hibiscus sabdariffa Prapaipit Suwitchayanon, Piyatida Pukclai, Osamu Ohno, Kiyotake Suenaga and Hisashi Kato-Noguchi 765

Angelicin as the Principal Allelochemical in Heracleum sosnowskyi Fruit Maryia Mishyna, Nikolai Laman, Valery Prokhorov and Yoshiharu Fujii 767

Identification of Octanal as Plant Growth Inhibitory Volatile Compound Released from Heracleum sosnowskyi Fruit Maryia Mishyna, Nikolai Laman, Valery Prokhorov, John Solomon Maninang and Yoshiharu Fujii 771

Identification of Safranal as the Main Allelochemical from Saffron (Crocus sativus) Hossein Mardani, Takayuki Sekine, Majid Azizi, Maryia Mishyna and Yoshiharu Fujii 775 (c) Biotechnology

The Biosynthetic Activities of Primary and Secondary Metabolites in Suspension Cultures of Aquilaria microcarpa Shinjiro Ogita, Jung-Bum Lee, Fumiya Kurosaki and Yasuo Kato 779

Continued inside backcover