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2 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 HONORARY EDITOR PROFESSOR GERALD BLUNDEN The School of Pharmacy & Biomedical Sciences, University of Portsmouth, Portsmouth, PO1 2DT U.K. axuf64@dsl.pipex.com 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, 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 , China gda5958@163.com PROFESSOR YOSHIHIRO MIMAKI School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Horinouchi , Hachioji, Tokyo , 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 , 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 Prof. Giovanni Appendino Novara, Italy Prof. Yoshinori Asakawa Tokushima, Japan Prof. Roberto G. S. Berlinck São Carlos, Brazil Prof. Anna R. Bilia Florence, Italy Prof. Maurizio Bruno Palermo, Italy Prof. César A. N. 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3 NPC Natural Product Communications Low Caffeine Content in Novel Grafted Tea with Camellia sinensis as Scions and Camellia oleifera as Stocks 2015 Vol. 10 No 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 , Anhui, China xcwan@ahau.edu.cn Received: January 15 th, 2015; Accepted: March 30 th, 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 ). 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-yearold 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 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

4 790 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. expression was similar in C. sinensis and grafted tea leaves, but low in C. oleifera (Figure 4). 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 ) and S-adenosylmethionine synthetase (MAT, EC ). 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 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).

5 Low caffeine in grafted tea material Natural Product Communications Vol. 10 (5) 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 ( 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 %. 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 ( , ), 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. References [1] Cabrera C, Artacho R, Giménez R. (2006) Beneficial effects of green tea-a review. Journal of the American College of Nutrition, 25, [2] Kris-Etherton PM, Keen CL. (2002) Evidence that the antioxidant flavonoids in tea and cocoa are beneficial for cardiovascular health. Current Opinion in Lipidology, 13, [3] Peng L, Song X, Shi X, Li J, Ye C. (2008) An improved HPLC method for simultaneous determination of phenolic compounds, purine alkaloids and theanine in Camellia species. Journal of Food Composition and Analysis, 21, [4] Sueoka N, Suganuma M, Sueoka E, Okabe S, Matsuyama S, Imai K, Nakachi K, Fujiki H. (2001) A new function of green tea: Prevention of lifestyle-related diseases. Annals of the New York Academy of Sciences, 928, [5] Yang CS, Maliakal P, Meng X. (2002) Inhibition of carcinogenesis by tea. Annual Review of Pharmacology and Toxicology, 42,

6 792 Natural Product Communications Vol. 10 (5) 2015 Deng et al. [6] Ashihara H, Sano H, Crozier A. (2008) Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering. Phytochemistry, 69, [7] Li YF, Hu L, Wang LH. (2009) Current status and prospect of researches and utilizations on Camellia oleifera resources (in Chinese). Guangxi Agricultural Sciences, 40, [8] Ashihara H, Crozier A. (1999) Biosynthesis and metabolism of caffeine and related purine alkaloids in plants. Advances in Botanical Research Incorporating Advances in Plant Pathology, 30, [9] Ashihara H., Crozier A. (2001) Caffeine: a well known but little mentioned compound in plant science. Trends in Plant Science. 6, [10] Li Y, Ogita S, Keya CA, Ashihara H. (2008) Expression of caffeine biosynthesis genes in tea (Camellia sinensis). Zeitschrift für Naturforschung. C, Journal of Biosciences, 63, [11] Roeder S, Dreschler K, Wirtz M, Cristescu SM, van Harren FJ, Hell R, Piechulla B. (2009) SAM levels, gene expression of SAM synthetase, methionine synthase and ACC oxidase, and ethylene emission from N. suaveolens flowers. Plant Molecular Biology, 70, [12] Gong B, Li X, Van den Langenberg KM, Wen D, Sun S, Wei M, Li Y, Yang F, Shi Q, Wang X. (2014) Overexpression of S-adenosyl-L-methionine synthetase increased tomato tolerance to alkali stress through polyamine metabolism. Plant Biotechnology Journal, 12, [13] Kim SH, Kim SH, Palaniyandi SA, Yang SH, Suh JW. (2015) Expression of potato S-adenosyl-L-methionine synthase (SbSAMS) gene altered developmental characteristics and stress responses in transgenic Arabidopsis plants. Plant Physiology and Biochemistry, 87, [14] Nishimura K, Ashihara H. (1993) IMP dehydrogenase from tea leaves and suspension-cultured Catharanthus roseus cells. Phytochemistry, 34, [15] Xu W, Song Q, Li D, Wan X. (2012) Discrimination of the production season of Chinese green tea by chemical analysis in combination with supervised pattern recognition. Journal of Agricultural and Food Chemistry, 28, [16] Jin Y, Li M, Ma LL, Deng WW, Zhang ZZ. (2015) Prokaryotic expression of caffeine synthase (TCS1), its polyclonal antibody preparation and identification. Bulletin of Botanical Research, in press [17] Deng WW, Wang S, Chen Q, Zhang ZZ, Hu XY. (2012) Effect of salt treatment on theanine biosynthesis in Camellia sinensis seedlings. Plant Physiology and Biochemistry, 56, [18] Tian L, Kong WF, Pan QH, Zhan JC, Wen PF, Chen JY, Wan SB, Huang WD. (2006) Expression of the chalcone synthase gene from grape and preparation of an anti-chs antibody. Protein Expression and Purification, 50, [19] Schmittgen TD, Livak KJ. (2008) Analyzing real-time PCR data by the comparative C(T) method. Nature Protocols, 3,

7 Natural Product Communications Vol. 10 (5) 2015 Published online ( 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

8 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 Original Paper (a) Biosynthesis Metabolism and Biological Function of Natural Products in Plants (Guest editor: Hiroshi Ashihara) 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