Biosynthesis of dillapiole/apiole in dill (Anethum graveolens): characterization of regioselective phenylpropene O-methyltransferase.

The phenylpropene volatiles dillapiole and apiole impart one of the characteristic aromas of dill (Anethum graveolens) weeds. However, very few studies have been conducted to investigate the chemical composition of volatile compounds from different developmental stages and plant parts of A. graveolens. In this study, we examined the distribution of volatile phenylpropenes, including dillapiole, in dill plants at various developmental stages. We observed that young dill seedlings accumulate high levels of dillapiole and apiole, whereas a negligible proportion was found in the flowering plants and dry seeds. Based on transcriptomics and co-expression approaches with phenylpropene biosynthesis genes, we identified dill cDNA encoding S-adenosyl-L-methionine-dependent O-methyltransferase 1 (AgOMT1), an enzyme that can convert 6- and 2-hydroxymyristicin to dillapiole and apiole, respectively, via the methylation of the ortho-hydroxy group. The AgOMT1 protein shows an apparent Km value of 3.5 µm for 6-hydroxymyristicin and is 75% identical to the anise (Pimpinella anisum) O-methyltransferase (PaAIMT1) that can convert isoeugenol to methylisoeugenol via methylation of the hydroxy group at the para-position of the benzene ring. AgOMT1 showed a preference for 6-hydroxymyristicin, whereas PaAIMT1 displayed a large preference for isoeugenol. In vitro mutagenesis experiments demonstrated that substituting only a few residues can substantially affect the substrate specificity of these enzymes. Other plants belonging to the Apiaceae family contained homologous O-methyltransferase (OMT) proteins highly similar to AgOMT1, converting 6-hydroxymyristicin to dillapiole. Our results indicate that apiaceous phenylpropene OMTs with ortho-methylating activity evolved independently of phenylpropene OMTs of other plants and the enzymatic function of AgOMT1 and PaAIMT1 diverged recently.

CRISPR/Cas9-mediated disruption of ALLENE OXIDE SYNTHASE results in defective 12-oxo-phytodienoic acid accumulation and reduced defense against spider mite (Tetranychus urticae) in liverwort (Marchantia polymorpha).

Allene oxide synthase (AOS) is a key enzyme involved in the biosynthesis of 12-oxo-phytodienoic acid (OPDA) and jasmonic acid and plays an important role in plant defense against herbivore attacks. In the liverwort, Marchantia polymorpha, we previously identified cytosol-type MpAOS1 and chloroplast-type MpAOS2 that show AOS activities. However, there is no direct evidence to show the subcellular localization of MpAOSs and their contribution to plant defense via OPDA production in M. polymorpha. In this study, we generated M. polymorpha mutants, with the MpAOS1 and MpAOS2 genes disrupted via CRISPR/Cas9-mediated genome editing; the loss of OPDA production was analyzed in double-knockout mutants. On AOS mutants, the survival rate and oviposition of spider mites (Tetranychus urticae) increased relative to those on wild-type plants. Overall, these findings suggest that defense systems via OPDA-signaling pathways in response to spider mites have been established in M. polymorpha.

1-Octen-3-ol is formed from its primeveroside after mechanical wounding of soybean leaves.

KEY MESSAGE: Hydrolysis of 1-octen-3-yl ß-primeveroside implemented by a system with high structure-specificity is accountable for the rapid formation of 1-octen-3-ol from soybean leaves after mechanical wounding. 1-Octen-3-ol is a volatile compound ubiquitous in fungi; however, a subset of plant species also has the ability to form 1-octen-3-ol. Owing to its volatile nature, it has been anticipated that 1-octen-3-ol is associated with the effort of the emitter to control the behavior of the surrounding organisms; however, its ecological significance and the enzymes involved in its biosynthesis have not been fully elucidated, particularly in plants. We previously found that soybean (Glycine max) seeds contain 1-octen-3-yl ß-primeveroside (pri). To elucidate the physiological significance and the biosynthesis of 1-octen-3-ol in plants, changes in the amount of 1-octen-3-yl pri during development of soybean plants was examined. A high 1-octen-3-yl pri level was found in young developing green organs, such as young leaves and sepals. Treatment of soybean leaves with methyl jasmonates resulted in a significant increase in the amount of 1-octen-3-yl pri; suggesting its involvement in defense responses. Although 1-octen-3-ol was below the detection limit in intact soybean leaves, mechanical damage to the leaves caused rapid hydrolysis of almost all 1-octen-3-yl pri to liberate volatile 1-octen-3-ol. Under the same conditions, the other glycosides, including isoflavone glycoside and linalool diglycoside, were hardly hydrolyzed. Therefore, the enzyme system to liberate aglycone from glycosides in soybean leaves should have strict substrate specificity. 1-Octen-3-yl pri might function as a storage form of volatile 1-octen-3-ol for immediate response against stresses accompanying tissue wounding.

Green Leaf Volatile-Burst in Selaginella moellendorffii.

Green leaf volatiles (GLVs) consist of six-carbon volatile aldehydes, alcohols, and their esters. They are formed from polyunsaturated fatty acids and are involved in the defense of plants against herbivores and pathogens. GLVs generally have low concentrations in intact healthy plant tissues, but the biosynthetic pathway to form GLVs is quickly activated by mechanical damage to tissues, an event called the GLV-burst. Most seed plants have the ability to implement GLV-burst; however, this potential in non-seed plants has not been extensively researched. In this study, we examined the GLV-burst capacity of monilophytes, lycophytes, and bryophytes, and confirmed that monilophytes and lycophytes showed substantial GLV-burst ability, while bryophytes did not, with a few exceptions. When the genome sequence of a model lycophyte, Selaginella moellendorffii was reviewed, 10 genes were found that showed high similarity with the non-canonical cytochrome P450 enzymes, CYP74s, specialized in oxylipin formation. Recombinant proteins expressed with Escherichia coli showed that one of them had the ability to encode allene oxide synthase, and another encoded hydroperoxide lyase (HPL), preferring linolenic acid 13-hydroperoxide, and it was inferred that this gene was responsible for GLV-burst in S. moellendorffii. Based on the phylogenetic tree constructed with CYP74s of non-seed and seed plants, we hypothesized that HPL was acquired independently in the lycophyte and seed plants through diversification of CYP74 genes.

Production of raspberry ketone by redirecting the metabolic flux to the phenylpropanoid pathway in tobacco plants.

Raspberry ketone is one of the characteristic flavors of raspberry fruits, and it is an important and expensive ingredient in the flavor and fragrance industries. It is present at low levels in plant tissues, and its occurrence is limited to a few taxa. In this context, the stable production of nature-identical raspberry ketone using heterologous synthesis in plants hosts has recently garnered the attention of plant biochemists. In this study, we demonstrate the rational switching of the metabolic flow from anthocyanin pigments to volatile phenylbutanoid production via the phenylpropanoid pathway. This shift led to the efficient and stable production of raspberry ketone and its glycosides via heterologous expression of the biosynthetic enzymes benzalacetone synthase (BAS) and raspberry ketone/zingerone synthase 1 (RZS1) in the transgenic tobacco (Nicotiana tabacum 'Petit Havana SR-1'). Additionally, we achieved improved product titers by activating the phenylpropanoid pathway with the transcriptional factor, production of anthocyanin pigment 1 (PAP1), from Arabidopsis thaliana. We further demonstrated another metabolic-flow switching by RNA interference (RNAi)-mediated silencing of chalcone synthase (CHS) to increase pathway-intermediate p-coumaroyl-CoA in transgenic tobacco for raspberry-ketone production. The redirection of metabolic flux resulted in transgenic lines producing 0.45 µg/g of raspberry ketone and 4.5 µg/g, on the fresh weight basis, of its glycosides in the flowers. These results suggest that the intracellular enforcement of endogenous substrate supply is an important factor while engineering the phenylpropanoid pathway. This strategy might be useful for the production of other phenylpropanoids/polyketides that are produced via the pathway-intermediate p-coumaroyl-CoA, in tobacco plants.

Synthesis of deuterium-labeled cinnamic acids: Understanding the volatile benzenoid pathway in the flowers of the Japanese loquat Eriobotrya japonica.

Cinnamic acids are widely distributed in plants, including crops for human use, and exhibit a variety of activities that are beneficial to human health. They also occupy a pivotal position in the biosynthesis of phenylpropanoids such as lignins, anthocyanins, flavonoids, and coumarins. In this context, deuterium-labeled cinnamic acids have been used as tracers and internal standards in food and medicinal chemistry as well as plant biochemistry. Therefore, a concise synthesis of deuterium-labeled cinnamic acids would be highly desirable. In this study, we synthesized deuterium-labeled cinnamic acids using readily available deuterium sources. We also investigated a hydrogen-deuterium exchange reaction in an ethanol-d1 /Et3 N system. This method can introduce deuterium atoms at the ortho and para positions of the phenolic hydroxy groups as well as at the C-2 position of alkyl cinnamates and is applicable to various phenolic compounds. Using the synthesized labeled compounds, we demonstrated that the benzenoid volatiles, such as 4-methoxybenzaldehyde, in the scent of the flowers of the Japanese loquat Eriobotrya japonica are biosynthesized from phenylalanine via cinnamic and 4-coumaric acids. This study provides easy access to a variety of deuterium-labeled (poly)phenols, as well as to useful tools for studies of the metabolism of cinnamic acids in living systems.

Parallel evolution of UbiA superfamily proteins into aromatic O-prenyltransferases in plants.

Plants produce ∼300 aromatic compounds enzymatically linked to prenyl side chains via C-O bonds. These O-prenylated aromatic compounds have been found in taxonomically distant plant taxa, with some of them being beneficial or detrimental to human health. Although their O-prenyl moieties often play crucial roles in the biological activities of these compounds, no plant gene encoding an aromatic O-prenyltransferase (O-PT) has been isolated to date. This study describes the isolation of an aromatic O-PT gene, CpPT1, belonging to the UbiA superfamily, from grapefruit (Citrus × paradisi, Rutaceae). This gene was shown responsible for the biosynthesis of O-prenylated coumarin derivatives that alter drug pharmacokinetics in the human body. Another coumarin O-PT gene encoding a protein of the same family was identified in Angelica keiskei, an apiaceous medicinal plant containing pharmaceutically active O-prenylated coumarins. Phylogenetic analysis of these O-PTs suggested that aromatic O-prenylation activity evolved independently from the same ancestral gene in these distant plant taxa. These findings shed light on understanding the evolution of plant secondary (specialized) metabolites via the UbiA superfamily.

Fungal-Type Terpene Synthases in Marchantia polymorpha Are Involved in Sesquiterpene Biosynthesis in Oil Body Cells.

The liverwort Marchantia polymorpha possesses oil bodies in idioblastic oil body cells scattered in its thallus. Oil bodies are subcellular organelles in which specific sesquiterpenes and bisbibenzyls are accumulated. Therefore, a specialized system for the biosynthesis and accumulation of these defense compounds specifically in oil bodies has been implied. A recent study on M. polymorpha genome sequencing revealed 10 genes that shared high similarities with fungal-type terpene synthases (TPSs). Eight of these fungal-type TPS-like genes in M. polymorpha (MpFTPSL1-6, -9 and -10) are located within a 376-kb stretch on chromosome 6 and share similarities of over 94% at the nucleotide level. Therefore, these genes have likely originated from recent gene duplication events. The expression of a subset of MpFTPSLs was induced under non-axenic growth on vermiculite, which increased the amounts of sesquiterpenes and number of oil bodies. The tdTomato fluorescent protein-based in-fusion reporter assay with MpFTPSL2 promoter revealed fluorescent signals specifically in oil body cells of the thallus, indicating that MpFTPSL2 functions in oil body cells. Recombinant MpFTPSL2 expression in Escherichia coli led to sesquiterpene synthesis from farnesyl pyrophosphate. Moreover, suppression of a subset of MpFTPSLs through RNA interference reduced sesquiterpene accumulation in thalli grown on vermiculite. Taken together, these results suggest that at least a subset of MpFTPSLs is involved in sesquiterpene synthesis in oil body cells.

Biochemical characterization of the jasmonic acid methyltransferase gene from wasabi (Eutrema japonicum).

Methyl jasmonate and jasmonic acid play important roles as signaling molecules in regulating plant development and stress-related responses. Previous studies have shown that jasmonic acid carboxyl methyltransferase (JMT), which belongs to the SABATH methyltransferase gene family, catalyzes the transfer of methyl groups from S-adenosyl-L-methionine to the carboxyl groups of jasmonic acid. In the present study, we used RNA-seq analysis to identify a putative JMT gene, EujJMT, in wasabi (Eutrema japonicum). The EujJMT proteins showed the highest similarity (89% identity) to JMT proteins of Brassica rapa. Functional characterization of a recombinant EujJMT protein expressed in Escherichia coli showed the highest level of activity with jasmonic acid among the different carboxylic acids tested. The apparent Km value of EujJMT using jasmonic acid as substrate was 62.6 µM, which is comparable to the values of known JMTs. Phylogenetic analysis suggested that EujJMT shares a common ancestor with the JMTs of Arabidopsis and Brassica species and that the strict substrate specificity toward jasmonic acid is conserved among Brassicaceae JMTs.

Molecular cloning and characterization of UDP-glucose: Volatile benzenoid/phenylpropanoid glucosyltransferase in petunia flowers.

Volatile benzenoids/phenylpropanoids are characteristic scent compounds in petunia flowers and are reported to be stored as glycosides in the vacuoles of petal cells. Here, we used transcriptomics and co-expression approaches with volatile benzenoid/phenylpropanoid biosynthetic genes to identify three petunia genes (UGT85A96, UGT85A97, and UGT85A98) encoding UDP-glycosyltransferase. The analyses of spatiotemporal gene expression revealed that all UGT85 genes were highly expressed in floral tissues such as petals and pistils. Functional characterization of recombinant UGT85A96 and UGT85A98 proteins expressed in Escherichia coli showed that UGT85A98 could transfer a glucosyl moiety from UDP-glucose to the hydroxyl group of various substrates including volatile benzenoids/phenylpropanoids, terpene alcohol, flavonoids, and C6 alcohol, whereas UGT85A96 specifically catalyzes the glucosylation of 2-phenylethanol and benzyl alcohol. This report describes the first experimental evidence to identify UGT enzymes that catalyze the glycosylation of volatile benzenoids/phenylpropanoids in petunia flowers.

Suppressed Methionine γ-Lyase Expression Causes Hyperaccumulation of S-Methylmethionine in Soybean Seeds.

Several soybean (Glycine max) germplasms, such as Nishiyamahitashi 98-5 (NH), have an intense seaweed-like flavor after cooking because of their high seed S-methylmethionine (SMM) content. In this study, we compared the amounts of amino acids in the phloem sap, leaves, pods, and seeds between NH and the common soybean cultivar Fukuyutaka. This revealed a comparably higher SMM content alongside a higher free Met content in NH seeds, suggesting that the SMM-hyperaccumulation phenotype of NH soybean was related to Met metabolism in seeds. To investigate the molecular mechanism behind SMM hyperaccumulation, we examined the phenotype-associated gene locus in NH plants. Analyses of the quantitative trait loci in segregated offspring of the cross between NH and the common soybean cultivar Williams 82 indicated that one locus on chromosome 10 explains 71.4% of SMM hyperaccumulation. Subsequent fine-mapping revealed that a transposon insertion into the intron of a gene, Glyma.10g172700, is associated with the SMM-hyperaccumulation phenotype. The Glyma.10g172700-encoded recombinant protein showed Met-γ-lyase (MGL) activity in vitro, and the transposon-insertion mutation in NH efficiently suppressed Glyma.10g172700 expression in developing seeds. Exogenous administration of Met to sections of developing soybean seeds resulted in transient increases in Met levels, followed by continuous increases in SMM concentrations, which was likely caused by Met methyltransferase activity in the seeds. Accordingly, we propose that the SMM-hyperaccumulation phenotype is caused by suppressed MGL expression in developing soybean seeds, resulting in transient accumulation of Met, which is converted into SMM to avoid the harmful effects caused by excess free Met.

Molecular cloning and functional characterization of an O-methyltransferase catalyzing 4'-O-methylation of resveratrol in Acorus calamus.

Resveratrol and its methyl ethers, which belong to a class of natural polyphenol stilbenes, play important roles as biologically active compounds in plant defense as well as in human health. Although the biosynthetic pathway of resveratrol has been fully elucidated, the characterization of resveratrol-specific O-methyltransferases remains elusive. In this study, we used RNA-seq analysis to identify a putative aromatic O-methyltransferase gene, AcOMT1, in Acorus calamus. Recombinant AcOMT1 expressed in Escherichia coli showed high 4'-O-methylation activity toward resveratrol and its derivative, isorhapontigenin. We purified a reaction product enzymatically formed from resveratrol by AcOMT1 and confirmed it as 4'-O-methylresveratrol (deoxyrhapontigenin). Resveratrol and isorhapontigenin were the most preferred substrates with apparent Km values of 1.8 µM and 4.2 µM, respectively. Recombinant AcOMT1 exhibited reduced activity toward other resveratrol derivatives, piceatannol, oxyresveratrol, and pinostilbene. In contrast, recombinant AcOMT1 exhibited no activity toward pterostilbene or pinosylvin. These results indicate that AcOMT1 showed high 4'-O-methylation activity toward stilbenes with non-methylated phloroglucinol rings.

Transcriptional regulators involved in responses to volatile organic compounds in plants.

Field studies have shown that plants growing next to herbivore-infested plants acquire higher resistance to herbivore damage. This increased resistance is partly due to regulation of plant gene expression by volatile organic compounds (VOCs) released by plants that sense environmental challenges such as herbivores. The molecular basis for VOC sensing in plants, however, is poorly understood. Here, we report the identification of TOPLESS-like proteins (TPLs) that have VOC-binding activity and are involved in VOC sensing in tobacco. While screening for volatiles that induce stress-responsive gene expression in tobacco BY-2 cells and tobacco plants, we found that some sesquiterpenes induce the expression of stress-responsive genes. These results provided evidence that plants sense these VOCs and motivated us to analyze the mechanisms underlying volatile sensing using tobacco as a model system. Using a pulldown assay with caryophyllene derivative-linked beads, we identified TPLs as transcriptional co-repressors that bind volatile caryophyllene analogs. Overexpression of TPLs in cultured BY-2 cells or tobacco leaves reduced caryophyllene-induced gene expression, indicating that TPLs are involved in the responses to caryophyllene analogs in tobacco. We propose that unlike animals, which use membrane receptors for sensing odorants, a transcriptional co-repressor plays a role in sensing and mediating VOC signals in plant cells.

Silkworms suppress the release of green leaf volatiles by mulberry leaves with an enzyme from their spinnerets.

In response to herbivory, plants emit a blend of volatile organic compounds that includes green leaf volatiles (GLVs) and terpenoids. These volatiles are known to attract natural enemies of herbivores and are therefore considered to function as an indirect defense. Selection should favor herbivores that are able to suppress these volatile emissions, and thereby make themselves less conspicuous to natural enemies. We tested this possibility for silkworms, which were observed to leave secretions from their spinnerets while feeding on mulberry leaves. When we ablated the spinnerets of silkworms, no secretions were observed. Leaves infested by intact silkworms released smaller amounts of GLVs than leaves infested by ablated silkworms, indicating that the spinneret secretion suppressed GLV production. This difference in GLV emissions was also reflected in the behavioral response of Zenillia dolosa (Tachinidae), a parasitoid fly of silkworms. The flies laid fewer eggs when exposed to the volatiles from intact silkworm-infested leaves than when exposed to the volatiles from ablated silkworm-infested leaves. We identified a novel enzyme in the secretion from the spinneret that is responsible for the GLV suppression. The enzyme converted 13(S)-hydroperoxy-(9Z,11E,15Z)-octadecatrienoic acid, an intermediate in the biosynthetic pathway of GLVs, into its keto-derivative in a stereospecific manner. Taken together, this study shows that silkworms are able to feed on mulberry in a stealthy manner by suppressing GLV production with an enzyme in secretions of their spinnerets, which might be a countermeasure against induced indirect defense by mulberry plants.

Identification of a Hexenal Reductase That Modulates the Composition of Green Leaf Volatiles.

Green leaf volatiles (GLVs), including six-carbon (C6) aldehydes, alcohols, and esters, are formed when plant tissues are damaged. GLVs play roles in direct plant defense at wound sites, indirect plant defense via the attraction of herbivore predators, and plant-plant communication. GLV components provoke distinctive responses in their target recipients; therefore, the control of GLV composition is important for plants to appropriately manage stress responses. The reduction of C6-aldehydes into C6-alcohols is a key step in the control of GLV composition and also is important to avoid a toxic buildup of C6-aldehydes. However, the molecular mechanisms behind C6-aldehyde reduction remain poorly understood. In this study, we purified an Arabidopsis (Arabidopsis thaliana) NADPH-dependent cinnamaldehyde and hexenal reductase encoded by At4g37980, named here CINNAMALDEHYDE AND HEXENAL REDUCTASE (CHR). CHR T-DNA knockout mutant plants displayed a normal growth phenotype; however, we observed significant suppression of C6-alcohol production following partial mechanical wounding or herbivore infestation. Our data also showed that the parasitic wasp Cotesia vestalis was more attracted to GLVs emitted from herbivore-infested wild-type plants compared with GLVs emitted from chr plants, which corresponded with reduced C6-alcohol levels in the mutant. Moreover, chr plants were more susceptible to exogenous high-dose exposure to (Z)-3-hexenal, as indicated by their markedly lowered photosystem II activity. Our study shows that reductases play significant roles in changing GLV composition and, thus, are important in avoiding toxicity from volatile carbonyls and in the attraction of herbivore predators.

Transcriptome analysis of Petunia axillaris flowers reveals genes involved in morphological differentiation and metabolite transport.

The biosynthesis of plant secondary metabolites is associated with morphological and metabolic differentiation. As a consequence, gene expression profiles can change drastically, and primary and secondary metabolites, including intermediate and end-products, move dynamically within and between cells. However, little is known about the molecular mechanisms underlying differentiation and transport mechanisms. In this study, we performed a transcriptome analysis of Petunia axillaris subsp. parodii, which produces various volatiles in its corolla limbs and emits metabolites to attract pollinators. RNA-sequencing from leaves, buds, and limbs identified 53,243 unigenes. Analysis of differentially expressed genes, combined with gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses, showed that many biological processes were highly enriched in limbs. These included catabolic processes and signaling pathways of hormones, such as gibberellins, and metabolic pathways, including phenylpropanoids and fatty acids. Moreover, we identified five transporter genes that showed high expression in limbs, and we performed spatiotemporal expression analyses and homology searches to infer their putative functions. Our systematic analysis provides comprehensive transcriptomic information regarding morphological differentiation and metabolite transport in the Petunia flower and lays the foundation for establishing the specific mechanisms that control secondary metabolite biosynthesis in plants.

1-Octen-3-ol Is Formed from Its Glycoside during Processing of Soybean [ Glycine max (L.) Merr.] Seeds.

Soaking and maceration of dry soybean seeds induce the formation of aliphatic volatile compounds that impact the flavor properties of food products prepared from soybean. Most aliphatic volatile compounds are formed through oxygenation of unsaturated fatty acids by lipoxygenases; however, lipoxygenases are not responsible for the formation of 1-octen-3-ol. 1-Octen-3-ol in soybean products is in general an off-flavor compound; thus, a procedure to manage its formation is required. In this study, we show that the formation of 1-octen-3-ol after hydration of soybean seed powder is independent of oxygen, suggesting that 1-octen-3-ol is not formed de novo from unsaturated fatty acids but instead from its derivative. When crude methanol extract of soybean seeds was reacted with ß-glycosidases, 1-octen-3-ol was rather liberated from its glycoside. We purified the parent glycoside from soybean seeds and confirmed it as ( R)-1-octen-3-yl ß-primeveroside [( R)-1-octen-3-yl 6- O-ß-d-xylopyranosyl-ß-d-glucopyranoside]. Green immature soybean fruits (pericarp and seeds) contain a high amount of 1-octen-3-yl ß-primeveroside. Its amount decreases after hydration of dry soybean powder. The results indicate that management of 1-octen-3-ol levels in soybean products requires a different strategy than that applied to off-flavor compounds formed de novo.

Synthesis and inhibitory activity of mechanism-based 4-coumaroyl-CoA ligase inhibitors.

4-Coumaroyl-CoA ligase (4CL) is ubiquitous in the plant kingdom, and plays a central role in the biosynthesis of phenylpropanoids such as lignins, flavonoids, and coumarins. 4CL catalyzes the formation of the coenzyme A thioester of cinnamates such as 4-coumaric, caffeic, and ferulic acids, and the regulatory position of 4CL in the phenylpropanoid pathway renders the enzyme an attractive target that controls the composition of phenylpropanoids in plants. In this study, we designed and synthesized mechanism-based inhibitors for 4CL in order to develop useful tools for the investigation of physiological functions of 4CL and chemical agents that modulate plant growth with the ultimate goal to produce plant biomass that exhibits features that are beneficial to humans. The acylsulfamide backbone of the inhibitors in this study was adopted as a mimic of the acyladenylate intermediates in the catalytic reaction of 4CL. These acylsulfamide inhibitors and the important synthetic intermediates were fully characterized using two-dimensional NMR spectroscopy. Five 4CL proteins with distinct substrate specificity from four plant species, i.e., Arabidopsis thaliana, Glycine max (soybean), Populus trichocarpa (poplar), and Petunia hybrida (petunia), were used to evaluate the inhibitory activity, and the half-maximum inhibitory concentration (IC50) of each acylsulfamide in the presence of 4-coumaric acid (100 µM) was determined as an index of inhibitory activity. The synthetic acylsulfamides used in this study inhibited the 4CLs with IC50 values ranging from 0.10 to 722 µM, and the IC50 values of the most potent inhibitors for each 4CL were 0.10-2.4 µM. The structure-activity relationship observed in this study revealed that both the presence and the structure of the acyl group of the synthetic inhibitors strongly affect the inhibitory activity, and indicates that 4CL recognizes the acylsulfamide inhibitors as acyladenylate mimics.

Functional evolution of biosynthetic enzymes that produce plant volatiles.

Plants synthesize volatile compounds to attract pollinators. The volatiles emitted by flowers are often complex mixtures of organic compounds; pollinators are capable of distinctly recognizing different volatile compounds. Plants also produce volatile compounds to protect themselves against herbivores and pathogens. Some of the volatile compounds produced in floral and vegetative tissues are toxic to insects and microbes. To adapt changes in the environment, plants have evolved the ability to synthesize a unique set of volatiles. Intensive studies have identified and characterized the enzymes responsible for the formation of plant volatiles. In particular, many biosynthetic genes have been isolated and their enzymatic functions have been proposed. This review describes how plants have evolved the biosynthetic pathways leading to the formation of green leaf volatiles and phenylpropene volatiles.

n-Hexanal and (Z)-3-hexenal are generated from arachidonic acid and linolenic acid by a lipoxygenase in Marchantia polymorpha L.

Most terrestrial plants form green leaf volatiles (GLVs), which are mainly composed of six-carbon (C6) compounds. In our effort to study the distribution of the ability of lipoxygenase (LOX) to form GLVs, we found that a liverwort, Marchantia polymorpha, formed n-hexanal and (Z)-3-hexenal. Some LOXs execute a secondary reaction to form short chain volatiles. One of the LOXs from M. polymorpha (MpLOX7) oxygenized arachidonic and α-linolenic acids at almost equivalent efficiency and formed C6-aldehydes during its catalysis; these are likely formed from hydroperoxides of arachidonic and α-linolenic acids, with a cleavage of the bond between carbon at the base of the hydroperoxy group and carbon of double bond, which is energetically unfavorable. These lines of evidence suggest that one of the LOXs in liverwort employs an unprecedented reaction to form C6 aldehydes as by-products of its reaction with fatty acid substrates.