[Show abstract][Hide abstract] ABSTRACT: Green leaf volatiles (GLVs) are C6-aliphatic aldehydes/alcohols/acetates, and biosynthesized from the central precursor fatty acid 13-hydroperoxides by 13-hydroperoxide lyases (HPLs) in various plant species. While GLVs have been implicated as defense compounds in plants, GLVs give characteristic grassy note to a bouquet of aroma in green tea, which is manufactured from young leaves of Camellia sinensis. Here we identify three HPL-related genes from C. sinensis via RNA-Sequencing (RNA-Seq) in silico, and functionally characterized a candidate gene, CYP74B24, as a gene encoding tea HPL. Recombinant CYP74B24 protein heterologously expressed in Escherichia coli specifically produced (Z)-3-hexenal from 13-HPOT with the optimal pH 6.0 in vitro. CYP74B24 gene was expressed throughout the aerial organs in a rather constitutive manner and further induced by mechanical wounding. Constitutive expression of CYP74B24 gene in intact tea leaves might account for low but substantial and constitutive formation of a subset of GLVs, some of which are stored as glycosides. Our results not only provide novel insights into the biological roles that GLVs play in tea plants, but also serve as basis for the improvement of aroma quality in tea manufacturing processes.
Full-text · Article · Nov 2015 · Plant Physiology and Biochemistry
[Show abstract][Hide abstract] ABSTRACT: Main conclusion:
Allene oxide synthases (AOSs) were isolated from liverworts and charophytes. These AOSs exhibited enzymatic properties similar to those of angiosperms but formed a distinct phylogenetic clade. Allene oxide synthase (AOS) and hydroperoxide lyase (HPL) mediate the formation of precursors of jasmonates and carbon-six volatiles, respectively. AOS and HPL utilize fatty acid hydroperoxides and belong to the plant cytochrome P450 74 (CYP74) family that mediates plant defense against herbivores, pathogens, or abiotic stresses. Although members of the CYP74 family have been reported in mosses and other species, the evolution and function of multiple CYP74 genes in plants remain elusive. Here, we show that the liverwort Marchantia polymorpha belongs to a basal group in the evolution of land plants; has two closely related proteins (59 % identity), MpAOS1 and MpAOS2, that are similar to moss PpAOS1 (49 and 47 % identity, respectively); and exhibits AOS activity but not HPL activity. We also found that the green microalgae Klebsormidium flaccidum, consist of multicellular and non-branching filaments, contains an enzyme, KfAOS, that is similar to PpAOS1 (37 % identity), and converts 13-hydroperoxide of linolenic acid to 12-oxo-phytodienoic acid in a coupled reaction with allene oxide cyclase. Phylogenetic analysis showed two evolutionarily distinct clusters. One cluster comprised AOS and HPL from charophytic algae, liverworts, and mosses, including MpAOSs and KfAOS. The other cluster was formed by angiosperm CYP74. Our results suggest that plant CYP74 enzymes with AOS, HPL, and divinyl ether synthase activities have arisen multiple times and in the two different clades, which occurred prior to the divergence of the flowering plant lineage.
[Show abstract][Hide abstract] ABSTRACT: The hydroperoxide lyase (HPL) pathway for six carbon (C6) volatiles and the allene oxide synthase (AOS) pathway for jasmonates (JAs) share the first part of the pathway. To avoid competition, a separate localization of HPL and AOS might be important. A fusion protein comprising Arabidopsis HPL and green fluorescent protein was transported into chloroplasts, where AOS was located. Arabidopsis harboring β-glucuronidase (GUS) gene downstream of Arabidopsis HPL promoter (pAtHPL::GUS) showed different GUS activity in floral organs compared with that from pAtAOS::GUS. With pAtHPL::GUS, wounding enhanced GUS activity at the periphery of cotyledons; while with pAtAOS::GUS, GUS activity was high in the vasculature. The distribution of the ability to form C6 volatiles correlated with the profile of HPL promoter activity; however, this ability unchanged after wounding. Inconsistency between the AOS promoter activity and JA levels was also evident. Thus, an additional factor should also control the ability to form C6 volatiles and JAs.
Full-text · Article · Jan 2015 · Journal of Plant Interactions
[Show abstract][Hide abstract] ABSTRACT: Plant specialized metabolites play important roles in human life. These metabolites, however, are often produced in small amounts in particular plant species. Moreover, some of these species are endangered in their natural habitats, thus further limiting the availability of some plant specialized metabolites. Microbial production of these compounds may circumvent this problem. Considerable progress has been made in the microbial production of various plant specialized metabolites over the past decade. Now, the microbial production of these compounds is becoming robust, fine-tuned, and commercially relevant systems using the methods of synthetic biology. This review describes the progress of microbial production of plant specialized metabolites, including phenylpropanoids, flavonoids, stilbenoids, diarylheptanoids, phenylbutanoids, terpenoids, and alkaloids, and discusses future challenges in this field.
Full-text · Article · Dec 2014 · Plant Biotechnology
[Show abstract][Hide abstract] ABSTRACT: Fresh ginger (Zingiber officinale) rhizome is characterized by a pleasant citrus aroma and pungent flavor. The majority of the aroma-contributing volatiles are monoterpenoids, especially geraniol derivatives such as geranial, geranyl acetate, geraniol and citronellol. In this study, we investigated the interconversion of geraniol derivatives by incorporation experiments using deuterium-labeled geraniol and geranyl acetate. GC-MS analysis revealed that the incorporated geraniol and geranyl acetate were transformed and detected as geranial, geraniol, geranyl acetate and citronellol; however, nerol and neral were hardly detected. Next, we isolated and characterized a cDNA encoding geraniol dehydrogenase (ZoGeDH) by expressed sequence tag database mining. Phylogenic analysis of ZoGeDH resulted in its categorization into the cinnamyl alcohol dehydrogenase (CAD) group, along with the previously reported GeDHs of sweet basil (Ocimum basilicum) and wild perilla (P. setoyensis, P. citriodora, and P. frutescens). The recombinant ZoGeDH catalyzed the NADP-dependent oxidation from geraniol to citral. Furthermore, its substrate specificity was the highest for geraniol and nerol, while that for cinnamyl alcohol was 32% of the activity observed for geraniol. The expression levels of ZoGeDH in various ginger plant tissues were in accordance with the accumulation of geranial, except in old rhizome.
Preview · Article · Dec 2014 · Plant Biotechnology
[Show abstract][Hide abstract] ABSTRACT: Phenylpropenes such as eugenol, isoeugenol, chavicol, and anethole are C6-C3 volatile compounds derived from phenylalanine by modification of its benzene ring and reduction of its propyl side chain, with the final reduction step catalyzed by a product-specific phenylpropene synthase. Recent advances in the biochemical and molecular analysis of phenylpropene synthases have improved our understanding of their evolution, structural properties, and reaction mechanism, providing insights into how plants produce and regulate the many types of phenylpropene volatiles. Since phenylpropenes are important in determining the flavor of foods and the quality of essential oils in cosmetics, the identification of the genes and enzymes responsible for the biosynthesis of phenylpropene volatiles has also provided us with tools to meet the challenge of improving plant aromas through genetic engineering.
Preview · Article · Dec 2014 · Plant Biotechnology
[Show abstract][Hide abstract] ABSTRACT: Flavonoids are phenolic secondary metabolites commonly occurring in plants. In particular, prenylated flavonoids exhibit a wide range of biological activities, including antitumor, antibacterial and antioxidant activities. In this study, we attempted to produce prenylated flavonoids in tomato as a host plant by means of metabolic engineering through the introduction of both the naringenin 8-dimethylallyltransferase (N8DT) gene, encoding a prenyltransferase from Sophora flavescens, and the chalcone isomerase (CHI) gene from Nicotiana tabacum cv. Samsun-NN. Liquid chromatography-tandem mass spectrometry analysis revealed the production of 8-dimethylallyl naringenin in the double transformants, while the production level was lower than N8DT single transformants. In addition, tomato fruits over-expressing both N8DT and CHI genes accumulated high levels of rutin compared with wild-type tomato. A possible endogenous regulation of the synthesis of flavonoid derivatives is discussed.
Preview · Article · Dec 2014 · Plant Biotechnology
[Show abstract][Hide abstract] ABSTRACT: Prenyl residues confer divergent biological activities such as anti-pathogenic and anti-herbivorous activities on phenolic compounds, including flavonoids, coumarins, and xanthons. To date, about 1,000 prenylated phenolics have been isolated, with these compounds containing various prenyl residues. However, all currently described plant prenyltransferases (PTs) have been shown specific for dimethylallyl diphosphate (DMADP) as the prenyl donor, while most of the cDNAs encoding these genes have been isolated from the Leguminosae. In this study we describe the identification of a novel PT gene from Citrus limon, Cl-PT1, belonging to the homogentisate PT family. This gene encodes a PT that differs from other known PTs, including flavonoid-specific PTs, in polypeptide sequence. This membrane-bound enzyme was specific for geranyl diphosphate (GDP) as the prenyl donor and coumarin as the prenyl acceptor. Moreover, the gene product was targeted to plastid in plant cells. To our knowledge, this is the novel aromatic PT specific to GDP from citrus species.
[Show abstract][Hide abstract] ABSTRACT: Furanocoumarins constitute a subfamily of coumarin compounds with important defense properties against pathogens and insects, as well as allelopathic functions in plants. Furanocoumarins are divided into two subgroups according to the alignment of the furan ring with the lactone structure; linear psoralen and angular angelicin derivatives. Determination of furanocoumarin type is based on the prenylation position of the common precursor to all furanocoumarins, umbelliferone, at C6 or C8, which gives rise to psoralen or angelicin derivatives, respectively. Here, we identified a membrane-bound prenyltransferase PcPT from parsley (Petroselinum crispum), and characterized the properties of the gene product. PcPT expression in different parsley tissues is increased by UV irradiation with a concomitant increase in furanocoumarin production. This enzyme has strict substrate specificity towards umbelliferone and dimethylallyl diphosphate, and a strong preference for the C6-position of the prenylated product (demethylsuberosin), leading to linear furanocoumarins. The C8-prenylated derivative (osthenol) is also formed, but to a much lesser extent. PcPT protein is targeted to the plastids in planta. Introduction of this PcPT in a coumarin-producing plant Ruta graveolens showed an increased consumption of endogenous umbelliferone. Expression of PcPT and a 4-coumaroyl-CoA 2'-hydroxylase gene in Nicotiana benthamiana, which does not produce furanocoumarins, resulted in demethylsuberosin formation, indicating that furanocoumarin production may be reconstructed in a metabolic engineering approach. These results demonstrate that a single prenyltransferase, such as PcPT, opens the path to linear furanocoumarins in parsley, but might also catalyse the synthesis of osthenol, the first intermediate committed to the angular furanocoumarin pathway, in other plants. This article is protected by copyright. All rights reserved.
Full-text · Article · Dec 2013 · The Plant Journal
[Show abstract][Hide abstract] ABSTRACT: A variety of volatile phenylpropenes, C6 -C3 compounds are widely distributed in the plant kingdom, whereas prenylated phenylpropenes are limited to a few plant species. In this study, we analysed the volatile profiles from Illicium anisatum leaves and identified two O-prenylated phenylpropenes, 4-allyl-2-methoxy-1-[(3-methylbut-2-en-1-yl)oxy]benzene [O-dimethylallyleugenol (9)] and 5-allyl-1,3-dimethoxy-2-(3-methylbut-2-en-1-yl)oxy]benzene [O-dimethylallyl-6-methoxyeugenol (11)] as major constituents. The structure-activity relationship of a series of eugenol derivatives showed that specific phenylpropenes, including eugenol (1), isoeugenol (2) and 6-methoxyeugenol (6), with a phenolic hydroxy group had antifungal activity for a fungal pathogen, whereas guaiacol, a simple phenolic compound, and allylbenzene had no such activity. The eugenol derivatives that exhibited antifungal activity, in turn, had no significant toxicant property for mite oviposition. Interestingly, O-dimethylallyleugenol (9) in which the phenolic oxygen was masked with a dimethylallyl group exhibited a specific, potent oviposition deterrent activity for mites. The sharp contrast in structural requirements of phenylpropenes suggested distinct mechanisms underlying the two biological activities and the importance of a phenolic hydroxy group and its dimethylallylation for the structure-based design of new functional properties of phenylpropenes.
[Show abstract][Hide abstract] ABSTRACT: Eugenol, a volatile phenylpropene found in many plant species, exhibits antibacterial and acaricidal activities. This study attempted to modify the production of eugenol and its glycosides by introducing petunia coniferyl alcohol acetyltransferase (PhCFAT) and eugenol synthase (PhEGS) into hybrid aspen. Gas chromatography analyses revealed that wild-type hybrid aspen produced small amount of eugenol in leaves. The heterologous overexpression of PhCFAT alone resulted in up to 7-fold higher eugenol levels and up to 22-fold eugenol glycoside levels in leaves of transgenic aspen plants. The overexpression of PhEGS alone resulted in a subtle increase in either eugenol or eugenol glycosides, and the overexpression of both PhCFAT and PhEGS resulted in significant increases in the levels of both eugenol and eugenol glycosides which were nonetheless lower than the increases seen with overexpression of PhCFAT alone. On the other hand, overexpression of PhCFAT in transgenic Arabidopsis and tobacco did not cause any synthesis of eugenol. These results indicate that aspen leaves, but not Arabidopsis and tobacco leaves, have a partially active pathway to eugenol that is limited by the level of CFAT activity and thus the flux of this pathway can be increased by the introduction of a single heterologous gene.
No preview · Article · Jun 2013 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: Coumarins, a large group of polyphenols, play important roles in the defense mechanisms of plants, and they also exhibit various biological activities beneficial to human health, often enhanced by prenylation. Despite the high abundance of prenylated coumarins in citrus fruits, there has been no report on coumarin-specific prenyltransferase activity in citrus. In this study, we detected both O- and C-prenyltransferase activities of coumarin substrates in a microsome fraction prepared from lemon (Citrus limon) peel, where large amounts of prenylated coumarins accumulate. Bergaptol was the most preferred substrate out of various coumarin derivatives tested, and geranyl diphosphate (GPP) was accepted exclusively as prenyl donor substrate. Further enzymatic characterization of bergaptol 5-O-geranyltransferase activity revealed its unique properties: apparent K(m) values for GPP (9 µM) and bergaptol (140 µM) and a broad divalent cation requirement. These findings provide information towards the discovery of a yet unidentified coumarin-specific prenyltransferase gene.
[Show abstract][Hide abstract] ABSTRACT: Phenylbutanone raspberry ketone, accumulating in the mature fruits of raspberry (Rubus idaeus), imparts the characteristic aroma to the fruits. Here we describe the isolation and characterization of raspberry ketone/zingerone synthase 1 (RZS1), which catalyzed the NADPH-dependent reduction of 4-hydroxybenzalacetone and 3-methoxy-4-hydroxybenzalacetone to raspberry ketone and zingerone (the latter not found in raspberry), respectively. Its apparent K(m) values for 4-hydroxybenzalacetone and NADPH were 88 μM and 202 μM, respectively. RZS1 preferred 4-hydroxybenzalacetone to 3-methoxy-4-hydroxybenzalacetone as a substrate by a factor of 1.7, and showed a 6-fold preference for 4-hydroxybenzalacetone over p-coumaraldehyde, and no activity for coniferaldehyde. Expression analysis of the RZS1 gene throughout the plant revealed that its transcript level was highest in mature fruits. We conclude that RZS1 is responsible for hydrogenation of the α,β-unsaturated double bond of phenylbutenones, the final step of the raspberry ketone biosynthesis, in the raspberry fruits.
No preview · Article · Aug 2011 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: Flavonoids are natural compounds found in many plants, including the important fruit crop, tomato. Prenylated flavonoids consist of a large group of compounds, which often exhibit antitumour, antibacterial and/or anti-androgen activities. In this study, we engineered the biosynthesis of prenylated flavonoids using a Streptomyces prenyltransferase HypSc (SCO7190) possessing broad-range substrate specificity, in tomato as a host plant. LC/MS/MS analysis demonstrated the generation of 3'-dimethylallyl naringenin in tomato fruits when recombinant HypSc protein was targeted to the plastids, whereas the recombinant protein hardly produced this compound in vitro. This is the first report confirming the accumulation of a prenylated flavonoid using a bacterial prenyltransferase in transgenic plants, and our results suggest that the product specificities of prenyltransferases can be significantly influenced by the host plant.
[Show abstract][Hide abstract] ABSTRACT: Floral scent has been extensively investigated in plants of the South American genus Petunia. Flowers of Petunia integrifolia emit mostly benzaldehyde, while flowers of Petunia axillaris subsp. axillaris emit a mixture of volatile benzenoid and phenylpropanoid compounds that include isoeugenol and eugenol. Flowers of the artificial hybrid Petunia hybrida, a cross between P. integrifolia and P. axillaris, emit a similar spectrum of volatiles as P. axillaris subsp. axillaris. However, the flowers of P. axillaris subsp. parodii emit neither isoeugenol nor eugenol but contain high levels of dihydroconiferyl acetate in the petals, the main scent-synthesizing and scent-emitting organs. We recently showed that both isoeugenol and eugenol in P. hybrida are biosynthesized from coniferyl acetate in reactions catalyzed by isoeugenol synthase (PhIGS1) and eugenol synthase (PhEGS1), respectively, via a quinone methide-like intermediate. Here we show that P. axillaris subsp. parodii has a functional EGS gene that is expressed in flowers, but its IGS gene contains a frame-shift mutation that renders it inactive. Despite the presence of active EGS enzyme in P. axillaris subsp. parodii, in the absence of IGS activity the coniferyl acetate substrate is converted by an as yet unknown enzyme to dihydroconiferyl acetate. By contrast, suppressing the expression of PhIGS1 in P. hybrida by RNA interference also leads to a decrease in isoeugenol biosynthesis, but instead of the accumulation of dihydroconiferyl acetate, the flowers synthesize higher levels of eugenol.
Full-text · Article · Mar 2009 · The Plant Journal
[Show abstract][Hide abstract] ABSTRACT: The phenylpropene t-anethole imparts the characteristic sweet aroma of anise (Pimpinella anisum, family Apiaceae) seeds and leaves. Here we report that the aerial parts of the anise plant accumulate t-anethole as the plant matures, with the highest levels of t-anethole found in fruits. Although the anise plant is covered with trichomes, t-anethole accumulates inside the leaves and not in the trichomes or the epidermal cell layer. We have obtained anise cDNA encoding t-anol/isoeugenol synthase 1 (AIS1), an NADPH-dependent enzyme that can biosynthesize t-anol and isoeugenol (the latter not found in anise) from coumaryl acetate and coniferyl acetate, respectively. In addition, we have obtained a cDNA encoding S-[methyl-14C]adenosyl-l-methionine:t-anol/isoeugenol O-methyltransferase 1 (AIMT1), an enzyme that can convert t-anol or isoeugenol to t-anethole or methylisoeugenol, respectively, via methylation of the para-OH group. The genes encoding AIS1 and AIMT1 were expressed throughout the plant and their transcript levels were highest in developing fruits. The AIS1 protein is 59% identical to petunia (Petunia hybrida) isoeugenol synthase 1 and displays apparent Km values of 145 microm for coumaryl acetate and 230 microm for coniferyl acetate. AIMT1 prefers isoeugenol to t-anol by a factor of 2, with Km values of 19.3 microm for isoeugenol and 54.5 microm for S-[methyl-14C]adenosyl-l-methionine. The AIMT1 protein sequence is approximately 40% identical to basil (Ocimum basilicum) and Clarkia breweri phenylpropene O-methyltransferases, but unlike these enzymes, which do not show large discrimination between substrates with isomeric propenyl side chains, AIMT1 shows a 10-fold preference for t-anol over chavicol and for isoeugenol over eugenol.
[Show abstract][Hide abstract] ABSTRACT: Many plants synthesize the volatile phenylpropene compounds eugenol and isoeugenol to serve in defense against herbivores and pathogens and to attract pollinators. Clarkia breweri flowers emit a mixture of eugenol and isoeugenol, while Petunia hybrida flowers emit mostly isoeugenol with small amounts of eugenol. We recently reported the identification of a petunia enzyme, isoeugenol synthase 1 (PhIGS1) that catalyzes the formation of isoeugenol, and an Ocimum basilicum (basil) enzyme, eugenol synthase 1 (ObEGS1), that produces eugenol. ObEGS1 and PhIGS1 both utilize coniferyl acetate, are 52% sequence identical, and belong to a family of NADPH-dependent reductases involved in secondary metabolism. Here we show that C. breweri flowers have two closely related proteins (96% identity), CbIGS1 and CbEGS1, that are similar to ObEGS1 (58% and 59% identity, respectively) and catalyze the formation of isoeugenol and eugenol, respectively. In vitro mutagenesis experiments demonstrate that substitution of only a single residue can substantially affect the product specificity of these enzymes. A third C. breweri enzyme identified, CbEGS2, also catalyzes the formation of eugenol from coniferyl acetate and is only 46% identical to CbIGS1 and CbEGS1 but more similar (>70%) to other types of reductases. We also found that petunia flowers contain an enzyme, PhEGS1, that is highly similar to CbEGS2 (82% identity) and that converts coniferyl acetate to eugenol. Our results indicate that plant enzymes with EGS and IGS activities have arisen multiple times and in different protein lineages.
Full-text · Article · Jun 2008 · The Plant Journal
[Show abstract][Hide abstract] ABSTRACT: Oxylipin metabolism represents one of the important hormonal and defensive mechanisms employed by plants, algae, or animals. It begins mostly with the reaction of lipoxygenases (LOXs), which catalyze the oxygenation of polyunsaturated fatty acids to form the corresponding hydroperoxides. At present, little information about LOXs in cyanobacteria has been reported. Herein, we report the first isolation of two LOX genes (NpLOX1 and NpLOX2) from a cyanobacterium, Nostoc punctiforme ATCC29133. Incubations of recombinant NpLOX1 and NpLOX2 proteins expressed in Eschelichia coli with linoleic acid resulted in the predominant formation of linoleic acid 13-S-hydroperoxide. Other C18 and C20 fatty acids could also be substrates for NpLOX enzymes. Phylogenetic analysis of NpLOX sequences showed that the NpLOX enzymes shared a high homology with LOX sequence of a bacterial pathogen, Pseudomonas aeruginosa, and these bacterial LOXs formed a subfamily distinct from those of plants, algae, and mammals.
Full-text · Article · May 2007 · Current Microbiology