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2-Phenylethanol (2PE) is a representative aromatic aroma compound in tea (Camellia sinensis) leaves. However, its formation in tea remains unexplored. In our study, feeding experiments of [2H8]L-phenylalanine (Phe), [2H5]phenylpyruvic acid (PPA), or (E/Z)-phenylacetaldoxime (PAOx) showed that three biosynthesis pathways for 2PE derived from L-Phe occurred in tea leaves, namely, pathway I (via phenylacetaldehyde (PAld)), pathway II (via PPA and PAld), and pathway III (via (E/Z)-PAOx and PAld). Furthermore, increasing temperature resulted in increased flux into the pathway for 2PE from L-Phe via PPA and PAld. In addition, tomato fruits and petunia flowers also contained the 2PE biosynthetic pathway from L-Phe via PPA and PAld and increasing temperatures led to increased flux into this pathway, suggesting that such a phenomenon might be common among most plants containing 2PE. This represents a characteristic example of changes in flux into the biosynthesis pathways of volatile compounds in plants in response to stresses.

Fragrance is an important quality index of horticultural flowers. Floral volatile formation in flowers during plant growth has been widely studied, but less is known about floral volatile formation in cut flowers and its responses to postharvest conditions. In this study, cut rose (Rosa hybrida cv. Tineke) flowers subjected to 5, 15 and 30 °C for 36 h showed increased concentrations of volatile phenylpropanoids/benzenoids (VPBs) including 2-phenylethanol (2PE), phenylacetaldehyde, benzyl alcohol, benzaldehyde, and phenethyl acetate, but a reduced 3,5-dimethoxytoluene concentration, as temperatures increased. l -[2H8]Phenylalanine (Phe) tracing in vivo suggested that phenylpyruvic acid (PPA) was involved in the increase in 2PE in response to increasing temperature. Genes for two aromatic amino acid aminotransferases (AAATs) were isolated and functionally characterized. Transient expression analyses in Nicotiana benthamiana plants provided in vivo evidence that RhAAAT2 was able to convert l -Phe into PPA, and that it was localized in the cytoplasm. These results advance our understanding of floral aroma formation in flowers after harvest.

Correction for ‘Insects (Thrips hawaiiensis (Morgan)) change the stereochemical configuration of 1-phenylethanol emitted from tea (Camellia sinensis) flowers’ by Ying Zhou et al., RSC Adv., 2017, 7, 32336–32343. DOI: 10.1039/C7RA03219F.

Metabolite formation is a biochemical and physiological feature of plants developed as an environmental response during the evolutionary process. These metabolites help defend plants against environmental stresses, but are also important quality components in crops. Utilizing the stress response to improve natural quality components in plants has attracted increasing research interest. Tea, which is processed by the tender shoots or leaves of tea plant (Camellia sinensis (L.) O. Kuntze), is the second most popular beverage worldwide after water. Aroma is an important factor affecting tea character and quality. The defense responses of tea leaves against various stresses during preharvest (tea growth process) and postharvest (tea manufacturing) processing can result in aroma formation. Herein, we summarize recent investigations into the biosyntheses of several characteristic aroma compounds prevalent in teas and derived from volatile fatty acid derivatives, terpenes, and phenylpropanoids/benzenoids. Several key aroma synthetic genes from tea leaves have been isolated, cloned, sequenced, and functionally characterized. Biotic stress (such as tea green leafhopper attack) and abiotic stress (such as light, temperature, and wounding) could enhance the expression of aroma synthetic genes, resulting in the abundant accumulation of characteristic aroma compounds in tea leaves. Understanding the specific relationships between characteristic aroma compounds and stresses is key to improving tea quality safely and effectively.

Fine bubbles of hydrogen were employed as a new reaction medium for the autoclave-free gas–liquid–solid multiphase hydrogenation of nitro groups on a multigram scale. Furthermore, ultrafine bubbles were examined by nanoparticle-tracking analysis in organic solvents.

A series of imidazolium salts with various N-aryl groups were synthesized, and their catalytic activities were evaluated to investigate the contribution of the N-aryl groups to the catalytic activity in the synthesis of γ-butyrolactone through an a3→d3-umpolung addition. Imidazolylidenes with 2,6-diethylphenyl groups were effective catalysts, and several mechanistic studies, including a deuterium kinetic isotope effect study, revealed that both steric and kinetic effects were responsible for the enhanced catalytic activity.

2-Phenylethanol (2PE) is a representative aromatic aroma compound in tea (

Plants synthesize and emit a large variety of volatile organic compounds, which possess extremely important ecological functions. Rapid progress in the method and instruments available to separate and identify plant metabolites has led to intensive investigations of the biosynthetic pathways that form plant volatiles, which has resulted in insightful and useful discoveries. In this review, we summarize and present the successful techniques currently used for studying metabolites involved in the formation of the major classes of plant volatile compounds, including volatile fatty acid derivatives, volatile isoprenoids, volatile phenylpropanoids/benzenoids, carotenoid-derived aroma compounds, and glycosidically bound volatile compounds. We discuss the extraction, identification, and quantitative analysis of many of these metabolites, which are unstable and occur in plants at trace levels.

Insect attacks change the quantity of volatiles emitted from plants, but little is known about the effects on the volatiles' stereochemical configurations. 1-Phenylethanol (1PE) is a major internal volatile of tea (Camellia sinensis) flowers. In the present study, insect-damaged C. sinensis flowers in the field emitted a different (R)-/(S)-1PE pattern than that emitted from undamaged flowers. Laboratory experiments showed that changes in the (R)-/(S)-1PE ratio were caused by treatments with Thrips hawaiiensis (Morgan), a pest of cut flowers and vegetable crops. These treatments led to an increase in jasmonic acid (JA) through the activation of allene oxide synthase, which is involved in JA biosynthesis. Exposing flowers to exogenous JA induced an (R)-/(S)-1PE emission pattern that was similar to that induced by a T. hawaiiensis attack, suggesting that JA was involved in the changes in the stereochemical configuration of 1PE emitted from the flowers after insect treatments. This study provides new insights into the involvement of volatiles in interactions between plants and insects, showing that not only the quantity but also the stereochemical configuration of plant volatiles is affected by insects.

The self-assembling gold nanoparticle (GNP)-supported l -proline derivative, which was readily synthesized in four steps from 4-hydroxy- l -proline and chloroauric acid, was used for enamine-based aldol reactions. The modified Brust-Schiffrin (BS) synthesis was favored over the ligand exchange reaction in order to develop a new and simple synthetic pathway for nanoparticle-supported catalyst. Use of immobilized organocatalyst on nanoscale solid carrier led to excellent selectivities (up to 94:6 dr and 94% ee) in asymmetric reactions of ketones and benzaldehydes. GNPs operate under pseudo-homogeneous conditions which are easily recycled and reused at least five times without significant loss of weight, activity, diastereo- and enantioselectivities.

Catechins and theaflavins are important metabolites contributing to tea function and quality. Catechins are known to transform into theaflavins during the tea manufacturing process, but the same transformation in preharvest tea leaves is unknown. Herein, we determined that shade treatment (dark), an agronomic practise widely used in tea cultivation, reduced the contents of most catechins, but increased the theaflavin contents, in preharvest tea leaves (cv. Yinghong No.9). This was attributed to the activation of polyphenoloxidase (PPO) activity in darkness. Furthermore, CsPPO3 was highly expressed under darkness, and thus CsPPO3 had been cloned, sequenced, and characterization. The CsPPO3 recombinant protein exhibited PPO function. Furthermore, shade treatment also reduced the catechin contents and increased the theaflavin contents in Yabukita and Hoshinomidori, suggesting that this phenomenon might not be specific to certain tea cultivars. This information will aid in understanding of theaflavin formation and its response to environmental factors at the preharvest tea stage.

Metabolite formation is a biochemical and physiological feature of plants developed as an environmental response during the evolutionary process. These metabolites help defend plants against environmental stresses, but are also important quality components in crops. Utilizing the stress response to improve natural quality components in plants has attracted increasing research interest. Tea, which is processed by the tender shoots or leaves of tea plant (

Tea plants (Camellia sinensis) store volatile organic compounds (VOCs; monoterpene, aromatic, and aliphatic alcohols) in the leaves in the form of water-soluble diglycosides, primarily as β-primeverosides (6-O-β-d-xylopyranosyl-β-d-glucopyranosides). These VOCs play a critical role in plant defenses and tea aroma quality, yet little is known about their biosynthesis and physiological roles in planta. Here, we identified two UDP-glycosyltransferases (UGTs) from C. sinensis, UGT85K11 (CsGT1) and UGT94P1 (CsGT2), converting VOCs into β-primeverosides by sequential glucosylation and xylosylation, respectively. CsGT1 exhibits a broad substrate specificity toward monoterpene, aromatic, and aliphatic alcohols to produce the respective glucosides. On the other hand, CsGT2 specifically catalyzes the xylosylation of the 6′-hydroxy group of the sugar moiety of geranyl β-d-glucopyranoside, producing geranyl β-primeveroside. Homology modeling, followed by site-directed mutagenesis of CsGT2, identified a unique isoleucine-141 residue playing a crucial role in sugar donor specificity toward UDP-xylose. The transcripts of both CsGTs were mainly expressed in young leaves, along with β-PRIMEVEROSIDASE encoding a diglycoside-specific glycosidase. In conclusion, our findings reveal the mechanism of aroma β-primeveroside biosynthesis in C. sinensis. This information can be used to preserve tea aroma better during the manufacturing process and to investigate the mechanism of plant chemical defenses.

The natural xanthines caffeine, theobromine, and theophylline are of major commercial importance as flavor constituents in coffee, cocoa, tea, and a number of other beverages. However, their exploitation for authenticity, a requirement in these commodities that have a large origin-based price-range, by the standard method of isotope ratio monitoring by mass spectrometry (irm-MS) is limited. We have now developed a methodology that overcomes this deficit that exploits the power of isotopic quantitative (13)C nuclear magnetic resonance (NMR) spectrometry combined with chemical modification of the xanthines to enable the determination of positional intramolecular (13)C/(12)C ratios (δ(13)Ci) with high precision. However, only caffeine is amenable to analysis: theobromine and theophylline present substantial difficulties due to their poor solubility. However, their N-methylation to caffeine makes spectral acquisition feasible. The method is confirmed as robust, with good repeatability of the δ(13)Ci values in caffeine appropriate for isotope fractionation measurements at natural abundance. It is shown that there is negligible isotope fractionation during the chemical N-methylation procedure. Thus, the method preserves the original positional δ(13)Ci values. The method has been applied to measure the position-specific variation of the (13)C/(12)C distribution in caffeine. Not only is a clear difference between caffeine isolated from different sources observed, but theobromine from cocoa is found to show a (13)C pattern distinct from that of caffeine.

A previous study found that 1-phenylethanol (1PE) was a major endogenous volatile compound in tea (Camellia sinensis) flowers and can be transformed to glycosically conjugated 1PE (1PE-Gly). However, occurrences of 1PE-Gly in plants remain unknown. In this study, four 1PE-Glys have been isolated from tea flowers. Three of them were determined as (R)-1PE β-d-glucopyranoside ((R)-1PE-Glu), (S)-1PE-Glu, and (S)-1PE β-primeveroside ((S)-1PE-Pri), respectively, on the basis of NMR, MS, LC-MS, and GC-MS evidence. The other one was identified as (R)-1PE-Pri on the basis of LC-MS and GC-MS data. Moreover, these 1PE-Glys were chemically synthesized as the authentic standards to further confirm their occurrences in tea flowers. 1PE-Glu had a higher molar concentration than 1PE-Pri in each floral stage and organ. The ratio of (R) to (S) differed between 1PE-Glu and 1PE-Pri. In addition, a 1PE-Gly hydrolase β-primeverosidase recombinant protein produced in Escherichia coli exhibited high hydrolysis activity toward (R)-1PE-Pri. However, β-primeverosidase transcript level was not highly expressed in the anther part, which accumulated the highest contents of 1PE-Gly and 1PE. This suggests that 1PE-Gly may not be easily hydrolyzed to liberate 1PE in tea flowers. This study provides evidence of occurrences of 1PE-Glys in plants for the first time.

A flower-inducing compound, LDS1, was isolated from a free-floating aquatic plant, Lemna paucicostata . The chemical structure and the absolute stereochemistry of LDS1 were determined as (9 R ,13 R ,11 E ,15 Z )-9,13-dihydroxy-10-oxooctadeca-11,15-dienoic acid for its most abundant diastereomer. LDS1 was enzymatically produced when the plant was exposed to drought stress, and induced flowering at a concentration of 10 nM.

Camellia sinensis synthesizes and emits a large variety of volatile phenylpropanoids and benzenoids (VPB). To investigate the enzymes involved in the formation of these VPB compounds, a new C. sinensis short-chain dehydrogenase/reductase (CsSDR) was isolated, cloned, sequenced, and functionally characterized. The complete open reading frame of CsSDR contains 996 nucleotides with a calculated protein molecular mass of 34.5 kDa. The CsSDR recombinant protein produced in Escherichia coli exhibited dehydrogenase-reductase activity towards several major VPB compounds in C. sinensis flowers with a strong preference for NADP/NADPH co-factors, and showed affinity for (R)/(S)-1-phenylethanol (1PE), phenylacetaldehyde, benzaldehyde, and benzyl alcohol, and no affinity for acetophenone (AP) and 2-phenylethanol. CsSDR showed the highest catalytic efficiency towards (R)/(S)-1PE. Furthermore, the transient expression analysis in Nicotiana benthamiana plants validated that CsSDR could convert 1PE to AP in plants. CsSDR transcript level was not significantly affected by floral development and some jasmonic acid-related environmental stress, and CsSDR transcript accumulation was detected in most floral tissues such as receptacle and anther, which were main storage locations of VPB compounds. Our results indicate that CsSDR is expressed in C. sinensis flowers and is likely to contribute to a number of floral VPB compounds including the 1PE derivative AP.

We produced low temperature (15 degrees C) processed green tea (LTPGT) with higher aroma contents than normal green tea (Sencha). Normal temperature processed green tea (NTPGT), involved storing at 25 degrees C, and Sencha had no storing process. Sensory evaluation showed LTPGT had higher levels of floral and sweet odorants than NTPGT and Sencha. Aroma extract dilution analysis and gas chromatography-mass spectrometry-olfactometry indicated LTPGT had 12 aroma compounds with high factor dilution values (FD). Amongst LTPGT's 12 compounds, indole, jasmine lactone, cis-jasmone, coumarin, and methyl epijasmonate contributed to floral, fruity and sweet characters. In particular, indole increased initially, peaking at 16 h, then gradually decreased; Feeding experiments suggested [N-15]indole and [N-15]oxygenated indoles (OX-indoles) were produced from [N-15]anthranilic acid. We proposed the increase in indole was due to transformation of anthranilic acid during the 16 h storage and the subsequent decline in indole level was due to its conversion to OX-indoles.

Tea plant (Camellia sinensis) biosynthesizes a wide variety of specialized metabolites, including phenolic compounds such as catechins. Flavonol, one of the major flavonoid subclasses, in C. sinensis is present in the O-glycoside form, such as quercetin 3-O-β-D-glucopyranoside, kaempferol 3-O-β-D-glucopyranoside, and rutin (quercetin 3-O-β- glucopyranosyl-6-O-α-rhamnoside). These flavonol glycosides are highly accumulated, constituting up to 2-3% (w/w dry weight) of tea leaves; however, their biosynthetic machinery in C. sinensis remains elusive. Using high-throughput RNA sequencing from the fresh leaves of a cultivar (C. sinensis var sinensis cv Yabukita) and rapid amplification of cDNA ends (RACE) cloning with degenerate oligonucleotide primers, we identified a full-length cDNA of UDP-glycosyltransferase, designated as UGT73A17, and characterized the biochemical and molecular functions of UGT73A17. Recombinant UGT73A17 protein catalyzed 3-O-glucosylation of quercetin, yielding quercetin 3-O-β-D-glucopyranoside in vitro. The preferential expression of UGT73A17 gene in the mature, relative to young leaves, stems and roots, is roughly consistent with the accumulation pattern of flavonol glycosides in C. sinensis, suggesting that UGT73A17, in part, participates in the biosynthesis of flavonol glycosides in planta .

A method for carbon isotope ratio (δ(13)C) analysis was developed for compound-specific isotope analysis of tea volatiles, and the values were compared with the δ(13)C value from bulk isotope analyses. The δ(13)C value of 2-phenylethanol liberated via enzymatic hydrolysis of the 2-phenylethyl β-primeveroside standard was examined first. Isotope fractionations for 2-phenylethyl β-primeveroside from preparative high-performance liquid chromatography (HPLC) were also analyzed. The enzymatic treatment and the preparative HPLC process did not cause carbon isotope fractionations, substantiating the strategies available for δ(13)C analysis of volatile compounds. On the basis of the gas chromatography-combustion-isotope ratio mass spectrometry data from 2-phenylethanol, it was possible to derive the conditions for enzyme treatment and preparative HPLC of the glycoconjugates of 2-phenylethanol, (Z)-3-hexenol, and benzyl alcohol isolated from green tea leaves. Larger variations in δ(13)C were found for individual volatile compounds compared with bulk analytical data from the leaves, indicating the potential to utilize this strategy in assigning the geographical origin of green tea.

An autoclave-free, gas–liquid–solid multiphase hydrogenation of carbon–carbon unsaturated bonds using hydrogen micro and nanobubbles (MNBs) is developed. The process allows the liquid phase of the reaction mixture to maintain a high concentration of hydrogen gas.

Tea aroma is one of the most important factors affecting the character and quality of tea. Recent advances in methods and instruments for separating and identifying volatile compounds have led to intensive investigations of volatile compounds in tea. These studies have resulted in a number of insightful and useful discoveries. Here we summarize the recent investigations into tea volatile compounds: the volatile compounds in tea products; the metabolic pathways of volatile formation in tea plants and the glycosidically-bound volatile compounds in tea; and the techniques used for studying such compounds. Finally, we discuss practical applications for the improvement of aroma and flavor quality in teas.

The α-ketol oxylipin (12Z,15Z)-9-hydroxy-10-oxooctadeca-12,15-dienoic acid (1) reacts with epinephrine (2) to afford unique cycloadducts, KE1 (3) and KE2 (4), both of which strongly induce flowering in Lemna paucicostata. Here we isolate compound 5 from the reaction mixture and identify it as a Michael-type adduct of 1 with adrenochrome (6), an oxidized form of 2. Compound 5 was gradually converted into KEs in aqueous solution, suggesting that the compound is an intermediate of KEs. From these results, we newly propose the reaction mechanism of the cycloaddition between 1 and 2.

Changes of endogenous 9, 10-ketol-octadecadienoic acid (KODA) concentrations, which is synthesized from linolenic acid by 9-lipoxygenase, were analyzed in apple [Malus ×sylvestris (L.) Mill. var. domestica (Borkh.) Mansf.] buds. In addition, the effects of 15, 16-chloro, hydroxy-9-hydroxy-10-oxo-12(Z)-octadecenoic acid (CKODA) application, which is an analog of KODA, on flower bud formation and the expression of MdTFL1 (terminal flower 1) and MdFT1 (flowering locus t 1) genes in apple buds were investigated in heavy-crop treatment (HCT) and under shade. An increase of endogenous KODA in the buds in the fruit-thinning treatment, which resulted in a higher proportion of flower bud formation than in HCT, was observed at 63 days after full bloom, but no such increase was found in HCT. In the shade-treated and heavy-crop trees, the expression of MdTFL1 in the buds to which CKODA was applied was lower than that in untreated buds. In contrast, under shade, the expression of MdFT1 in the CKODA-treated buds was higher than that in untreated buds. These results suggest that endogenous KODA may be associated with flower bud formation, and its application may be effective at improving the proportion of flower bud formation through its effect on MdTFL1 and MdFT1.

1-Phenylethanol (1PE) can be used as a fragrance in food flavoring and cosmetic industries and as an intermediate in the pharmaceutical industry. 1PE can be synthesized from acetophenone, and the cost of 1PE is higher than the cost of acetophenone. Therefore, it is important to establish an effective and low-cost approach for producing 1PE. Our previous studies found that tea (Camellia sinensis) flowers, which are an abundant and waste resource, contained enzymes that could transform acetophenone to 1PE. In the present study, we extracted crude enzymes from tea flowers and optimized the production conditions of 1PE using response surface methodology. The optimized conditions were an extraction pH of 7.0, a reaction pH of 5.3, a reaction temperature of 55 °C, a reaction time of 100 min, a coenzyme NADPH concentration of 3.75 μmol/mL in the reaction assay, and a substrate acetophenone concentration of 1.25 μmol/mL in the reaction assay. The results provide essential information for future industrial 1PE production using plant-derived enzymes.

The cyanoglycoside, prunasin, was isolated for the first time as an aroma precursor of benzaldehyde from fresh tea leaves (Camellia sinensis var. sinensis cv. Yabukita). Prunasin was readily hydrolyzed by a crude enzyme prepared from the fresh tea leaves to liberate benzaldehyde. The isomerization of prunasin under neutral conditions was also recognized.

The self-assembling gold nanoparticle (GNP)-supported l -proline derivative, which was readily synthesized in four steps from 4-hydroxy- l -proline and chloroauric acid, was used for enamine-based aldol reactions. The modified Brust-Schiffrin (BS) synthesis was favored over the ligand exchange reaction in order to develop a new and simple synthetic pathway for nanoparticle-supported catalyst. Use of immobilized organocatalyst on nanoscale solid carrier led to excellent selectivities (up to 94:6 dr and 94% ee) in asymmetric reactions of ketones and benzaldehydes. GNPs operate under pseudo-homogeneous conditions which are easily recycled and reused at least five times without significant loss of weight, activity, diastereo- and enantioselectivities.

Plants emit chemically diverse volatile compounds for attracting pollinators or putting up a chemical defense against herbivores. 2-Phenylethanol (2PE) is one of the abundantly emitted scent compounds in rose flowers. Feeding experiments with l-[2H8]phenylalanine into rose flowers and subsequent analysis using gas chromatography–mass spectrometry analysis revealed the hypothetical biosynthetic intermediates to [2H8]-2PE, and the biochemical and genetic analyses elucidated the principal pathway to [2H8]-2PE. We recently found season-specific 2PE pathway producing [2H7]-2PE from l-[2H8]phenylalanine. This is a unique example where the dominant pathway to a specific compound changes with the seasons. This review focuses on the biosynthesis of floral volatiles and their regulation to adapt to the changes in the environment.

Indole is a characteristic volatile constituent in oolong tea. Our previous study indicated that indole was mostly accumulated at the turn over stage of oolong tea manufacturing process. However, formation of indole in tea leaves remains unknown. In this study, one tryptophan synthase α-subunit (TSA) and three tryptophan synthase β-subunits (TSBs) from tea leaves were isolated, cloned, sequenced, and functionally characterized. Combination of CsTSA and CsTSB2 recombinant protein produced in Escherichia coli exhibited the ability of transformation from indole-3-glycerol phosphate to indole. CsTSB2 was highly expressed during the turn over process of oolong tea. Continuous mechanical damage, simulating the turn over process, significantly enhanced the expression level of CsTSB2 and amount of indole. These suggested that accumulation of indole in oolong tea was due to the activation of CsTSB2 by continuous wounding stress from the turn over process. Black teas contain much less indole, although wounding stress is also involved in the manufacturing process. Stable isotope labeling indicated that tea leaf cell disruption from the rolling process of black tea did not lead to the conversion of indole, but terminated the synthesis of indole. Our study provided evidence concerning formation of indole in tea leaves for the first time.

Floral volatile phenylpropanoids and benzenoids (VPBs) play important ecological functions and have potential economic applications. Little is known about how multi-factors in integration regulate the formation and emission of floral VPBs. In the present study, we investigated effects of multi factors including endogenous circadian clock, light, and temperature on the formation and emission of VPBs, which are major volatiles in flowers of Petunia× hybrida cv. 'Mitchell Diploid'. Endogenous circadian clock was proposed as the most important factor regulating rhythmic emission of VPBs and expressions of structural genes involved in the upstream biosynthetic pathway of VPBs, but did not affect expression levels of structural genes involved in the downstream pathway and VPBs-related regulators. In contrast to light, temperature was a more constant factor affecting emission of VPBs. VPBs emission could be inhibited within a short time by increasing temperature. The information will contribute to our understanding of emission mechanism of floral volatiles.

Ecological adaptations to seasonal changes are often observed in the phenotypic traits of plants and animals and these adaptations are usually expressed through the production of different biochemical end products. In this study, ecological adaptations are observed in a biochemical pathway without alteration of the end products. We present an alternative principal pathway to the characteristic floral scent compound 2-phenylethanol (2PE) in roses. The new pathway is seasonally induced in summer as a heat adaptation that uses rose phenylpyruvate decarboxylase (RyPPDC) as a novel enzyme. RyPPDC transcript levels and the resulting production of 2PE are increased time-dependently under high temperatures. The novel summer pathway produces levels of 2PE that are several orders of magnitude higher than those produced by the previously known pathway. Our results indicate that the alternative principal pathway identified here is a seasonal adaptation for managing the weakened volatility of summer roses.

BACKGROUND Carotenoids are a major class of plant pigments and fulfill many functions in different organisms that either produce or consume them. Although the color of the stamina of tea (Camellia sinensis) flowers is clearly due to the presence of carotenoids, the carotenoid profile and content remain to be discovered. RESULTS We investigated the carotenoid profile of tea flowers and determined changes in concentrations over the floral development. The flowers contained oxygenated xanthophylls such as neoxanthin, lutein and zeaxanthin, as well as the hydrocarbons β-carotene and α-carotene. Flowers of the tea plant contain to vegetables comparable amounts of carotenoids. The content of 9′-cis-epoxycarotenoids, which serve as abscisic acid precursors, as well as changes in concentration of abscisic acid were studied. The concentrations of carotenoids decreased whereas the abscisic acid content increased over the floral development. Exogenously applied S-abscisic acid affected water uptake, flower opening and carotenoid accumulation. CONCLUSION In summary, this paper reports, for the first time, the carotenoid profile and content of tea flowers. The study revealed that carotenoids in tea flowers are an interesting target in respect of possible applications of tea flower extracts as well as biological functions of abscisic acid during floral development. © 2012 Society of Chemical Industry

We investigated the effect of 9,10-ketol-octadecadienoic acid (KODA), which is synthesized from linolenic acid by 9-lipoxygenase, on 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase gene expression, their enzyme activities, and ethylene production in apple fruit [Malus sylvestris (L.) Mill. Var. domestica (Borkh.) Mansf.]. The fruit was treated with C-KODA, which is a derivative of KODA, at 63 or 119 d after full bloom (DAFB). Endogenous KODA in the skin of the fruit did not change significantly from 48 to 119 DAFB on the tree, and during storage after harvest at 119 DAFB. However, softening of the fruit treated with C-KODA at 63 DAFB was inhibited. ACC synthase and oxidase activities, ACC and MACC concentrations and ethylene production were lower in the C-KODA-treated fruit than in the untreated control at 119 DAFB. In general, mRNA transcript levels of MdACS1 and MdACO1 in the skin of the C-KODA-treated fruit were lower than those in the untreated control fruit. In contrast, softening was promoted in the fruit stored after C-KODA treatment at 119 DAFB. ACC synthase activity, ACC and MACC concentration, ethylene production and the mRNA transcript levels of MdACS1 and MdACO1 in the skin were also increased in the C-KODA-treated skin. These results suggest that C-KODA may influence ethylene production via the expression of MdACS1 and MdACO1 in apple fruit, but that the effect is different depending on the time of treatment.

Plant oxylipins are an important class of signaling molecules in plants. The cyclic adducts of epinephrine or norepinephrine with the naturally occurring oxylipin (12Z,15Z)-9-hydroxy-10-oxo-octadeca-12,15-dienoic acid (KODA, 1) or its synthetic analogues (2-6) have been reported to possess flower-inducing activity toward Lemna paucicostata. By in vivo and in vitro experiments with seedlings of Pharbitis nil cv. Violet carbonyl groups of the alpha-ketols (1 and 3) and the ketones (7 and 9) were enantio-selectively reduced to give their corresponding vicinal diols (2 and 4) and alcohols (8 and 10). The stereochemistry at the oxymethine carbon was determined based on the long range C H coupling constants and the modified Mosher's method. Orientation of the adjacent hydroxyl group in (1 and 3) did not affect the enantio-selectivity, whereas the conversion was slightly affected and higher yields were obtained with the R-enantiomers of the alpha-ketols.

As a defense response to attacks by herbivores such as the smaller tea tortrix ( Adoxophyes honmai Yasuda), tea ( Camellia sinensis ) leaves emit numerous volatiles such as (Z)-3-hexen-1-ol, linalool, α-farnesene, benzyl nitrile, indole, nerolidol, and ocimenes in higher concentration. Attack of Kanzawa spider mites ( Tetranychus kanzawai Kishida), another major pest insect of tea crops, induced the emission of α-farnesene and ocimenes from tea leaves. The exogenous application of jasmonic acid to tea leaves induced a volatile blend that was similar, although not identical, to that induced by the smaller tea tortrix. Most of these herbivore-induced plant volatiles (HIPV) were not stored in the tea leaves but emitted after the herbivore attack. Both the adaxial and abaxial epidermal layers of tea leaves emitted blends of similar composition. Furthermore, HIPV such as α-farnesene were emitted mostly from damaged but not from undamaged leaf regions. A principal component analysis of metabolites (m/z 70-1000) in undamaged tea leaves exposed or not to HIPV suggests that external signaling via HIPV may lead to more drastic changes in the metabolite spectrum of tea leaves than internal signaling via vascular connections, although total catechin contents were slightly but not significantly increased in the external signaling via HIPV.

2-Phenylethanol (2PE) is a prominent scent compound released from flowers of Damask roses (Rosa×damascena) and some hybrid roses (Rosa 'Hoh-Jun' and Rosa 'Yves Piaget'). 2PE is biosynthesized from l-phenylalanine (l-Phe) via the intermediate phenylacetaldehyde (PAld) by two key enzymes, aromatic amino acid decarboxylase (AADC) and phenylacetaldehyde reductase (PAR). Here we describe substrate specificity and cofactor preference in addition to molecular characterization of rose-PAR and recombinant PAR from R.×damascena. The deduced amino acid sequence of the full-length cDNA encoded a protein exhibiting 77% and 75% identity with Solanum lycopersicum PAR1 and 2, respectively. The transcripts of PAR were higher in petals than calyxes and leaves and peaking at the unfurling stage 4. Recombinant PAR and rose-PAR catalyzed reduction of PAld to 2PE using NADPH as the preferred cofactor. Reductase activity of rose-PAR and recombinant PAR were higher for aromatic and aliphatic aldehydes than for keto-carbonyl groups. Both PARs showed that (S)-[4-(2)H] NADPH was preferentially used over the (R)-[4-(2)H] isomer to give [1-(2)H]-2PE from PAld, indicating that PAR can be classified as short-chain dehydrogenase reductase (SDR).

Fluorogenic aromatic aldehydes bearing arylethynyl groups were developed. They were used for monitoring the reaction progress of organocatalytic aldol reactions in DMSO through an increase in the fluorescence intensity based on the formation of the florescent aldol product. The ratios of the fluorescence intensities of the aldols to the aldehydes were more than 300. These results suggest that the fluorescence assay system using the aldehyde is useful for the rapid identification of superior aldol catalysts and reaction conditions.

Damascenone is well-known for its potent flavour with an extremely low odour threshold. Several glycosidically bound precursors of damascenone have been isolated from several plants, but little is known about their occurrences in green tea infusions. In this work, three major glycosidic precursors of damascenone, 9-O-β- d -glucopyranosyl-megastigma-6,7-dien-3,5,9-triol (1a), 9-O-β- d -glucopyranosyl-3-hydroxy-7,8-didehydro-β-ionol (2a), and 3-O-β- d -glucopyranosyl-3-hydroxy-7,8-didehydro-β-ionol (2b) were isolated and identified in green tea infusions, and the stereochemistries at C-3 and C-9 positions of aglycone parts of the three glycosidic precursors were determined as (3S, 9R)-1a, (3R, 9R)-2a, and (3R, 9R)-2b, respectively. Compounds 1a, 2a, and 2b as well as 3-O-β- d -glucopyranosyl-megastigma-6,7-dien-3,5,9-triol (1b) were hydrolysed to form damascenone in a model system with strong acidic conditions (pH 2.0) and at high temperature (90 °C). In contrast to hydrolysis of 2a and 2b, more damascenone was transformed from 1a and 1b. Furthermore, the β- d -glucosyl moiety at the C-3 position gave a higher dehydration rate from megastigma-6,7-dien-3,5,9-triol to 3-hydroxy-7,8-didehydro-β-ionol than compound 1a carrying the sugar residue at C-9 position. Interestingly, the four glycosidic precursors of damascenone were not hydrolysed to give damascenone under slightly acidic conditions (pH 5.4 and 120 °C for 10 min), but they could be transformed to damascenone in the presence of green tea infusions even under the equal conditions.

Purple-fleshed sweet potatoes (Ipomoea batatas L.) contain a very complex anthocyanin profile due to the presence of several non-, mono-, and diacylated glucosides of cyanidin and peonidin. In this study, the anthocyanin composition of four Japanese purple sweet potato cultivars (Chiran Murasaki, Tanegashima Murasaki, Naka Murasaki, and Purple Sweet) were investigated by HPLC-DAD and ESI-MSn analyses. The HPLC chromatograms of the different cultivars show a remarkable variation of the two major pigments, cyanidin-3-(6''-caffeoylsophoroside)-5-glucoside and peonidin-3-(6''-caffeoylsophoroside)-5-glucoside, respectively. According to this, they can be categorized into two groups on the basis of the peonidin/cyanidin ratio: the cultivars Chiran Murasaki and Purple Sweet showed a high content of peonidin derivatives (peonidin type), whereas the varieties Tanegashima Murasaki and Naka Murasaki were classified as cyanidin types. By means of high-speed countercurrent chromatography (HSCCC) the nonacylated 3-sophoroside-5-glucoside of cyanidin was isolated on a preparative scale. Furthermore, it was possible to isolate the monoacylated cyanidin-3-(6''-caffeoylsophoroside)-5-glucoside as well as three diacylated major pigments, cyanidin-3-(6'',6'''-dicaffeoylsophoroside)-5-glucoside, cyanidin-3-(6''-caffeoyl-6'''-p-hydroxy-benzoylsophoroside)-5-glucoside, and peonidin-3-(6''-caffeoyl-6'''-p-hydroxybenzoyl-sophoroside)-5-glucoside. The purity and identity of the so-obtained pigments were confirmed by NMR measurements.

Oxylipin glucosides (2–4) were isolated from Lemna paucicostata with their structures and absolute configurations elucidated by spectroscopic and chemical methods. Compounds 2–4 were glucosides of C14 oxylipin which were synthesized from α-linolenic acid via the 9-lipoxygenase pathway.

While beneficial health properties of tea leaves have been extensively studied, less attention has been given to that of flowers of the tea ( Camellia sinensis ) plant. In this work, the ethanolic extract and its ethyl acetate-soluble fraction (EEA) from the tea flowers were found to possess the potent antioxidant activity using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical-scavenging assay. The compounds present in EEA had comparatively strong DPPH scavenging activity and strongly contributed to the antioxidant activity of the tea flowers. From EEA, besides eight catechins, five flavonol glycosides were isolated and their structures were elucidated on the basis of mass spectrometry and nuclear magnetic resonance spectroscopy as myricetin 3- O -β- d -galactopyranoside, quercetin 3- O -β- d -galactopyranoside, kaempferol 3- O -β- d -galactopyranoside, kaempferol 3- O -β- d -glucopyranoside, and kaempferol 3- O -[α- l -rhamnopyranosyl-(1-6)-β- d -glucopyranoside]. In addition, epigallocatechin gallate and epicatechin gallate were found as the major active components responsible for the antioxidant activity of tea flowers through the use of a combination of preparative liquid chromatography separation and DPPH assay.

Conventionally, tea flavor is analyzed through the use of a combination of gas chromatography–mass spectrometry and human taste panel. These methods present time-consuming or inaccurate factor. In this work, a rapid, accurate and nondestructive approach was put forward to identify coumarin-enriched Japanese green teas and evaluate their particular flavor using electronic nose (E-nose) technique. The multivariable analyses including principal component analysis and cluster analysis were applied to distinguish the tea samples and evaluate the particular (coumarin-like) flavor of coumarin-enriched tea under different infusion conditions. A correct classification was achieved for the seven tea samples with different content of coumarin. The E-nose successfully characterized the drying temperature-dependently trend of coumarin content during the manufacture process of coumarin-enriched green tea. It also revealed that the comparatively low-infusion temperature and long-infusion time were favorable for the emission of coumarin-like flavor of the tea infusion. In addition, a comparatively newly developed “absolute value expression” (AVE) method was employed to divide the tea flavors into quality and express them numerically. Using AVE, the role of coumarin in the total flavor of coumarin-enriched green tea was elucidated. These results suggest that E-nose could be employed to identify the green teas with particular flavor and evaluate the tea flavor.

The isolated protoplasts of rose flowers were used to investigate the metabolic pathway in rose flower leading from shikimic acid or L-phenylalanine (L-Phe) to 2-phenylethanol (2PE), a dominant volatile compound in hybrid roses such as Rosa damascena Mill., R. 'Hoh-Jun', and R. 'Yves Piaget'. Deuterium-labeled L-Phe ([2H8]L-Phe) was supplied to the protoplasts isolated from R. 'Yves Piaget' petals. The volatile end products ([2Hn]-2PE, n=6-8) and their related intermediates ([2Hn]phenylacetaldehyde, n=6-8) were detected in the protoplasts by gas chromatography-mass spectrometry (GC-MS). In addition, we chemically synthesized [2,3,4,5,6-13C5]shikimic acid, a new stable isotopomer, to investigate the formation of 2PE from shikimic acid by GC-MS and nuclear magnetic resonance. We proposed the hypothetical biochemical pathway of 2PE from shikimic acid via chorismic acid, L-Phe, and phenylacetaldehyde. This protoplast system facilitates findings of metabolic intermediates and simplifies the complex branching biosynthetic pathways of floral scents to distinct individual events.

(12Z,15Z)-9-Hydroxy-10-oxooctadeca-12,15-dienoic acid (1) and norepinephrine (2) undergo cycloaddition to afford FN1 (3) and FN2 (4), both of which induce flowering in Lemna paucicostata. Although the derivatives of 1 were suggested to also yield FN-like compounds after reacting with 2, their structures have not been elucidated. In this report, we investigated the structure and stereochemistry of seven novel FN analogs. These analogs were shown to be formed in the same regio- and stereocontrolled manner as FNs. Moreover, the activity of FN analogs on flowering induction was investigated, and we determined that all analogs, except for compound 8, were effective flowering inducers for L. paucicostata.