cDNA cloning, expression, and antifungal activity of chitinase from Ficus microcarpa latex: difference in antifungal action of chitinase with and without chitin-binding domain.

MAIN CONCLUSION: A chitin-binding domain could contribute to the antifungal ability of chitinase through its affinity to the fungal lateral wall by hydrophobic interactions. Complementary DNA encoding the antifungal chitinase of gazyumaru (Ficus microcarpa), designated GlxChiB, was cloned and expressed in Escherichia coli cells. The results of cDNA cloning showed that the precursor of GlxChiB has an N-terminal endoplasmic reticulum targeting signal and C-terminal vacuolar targeting signal, whereas mature GlxChiB is composed of an N-terminal carbohydrate-binding module family-18 domain (CBM18) and a C-terminal glycoside hydrolase family-19 domain (GH19) with a short linker. To clarify the role of the CBM18 domain in the antifungal activity of chitinase, the recombinant GlxChiB (wild type) and its catalytic domain (CatD) were used in quantitative antifungal assays under different ionic strengths and microscopic observations against the fungus Trichoderma viride. The antifungal activity of the wild type was stronger than that of CatD under all ionic strength conditions used in this assay; however, the antifungal activity of CatD became weaker with increasing ionic strength, whereas that of the wild type was maintained. The results at high ionic strength further verified the contribution of the CBM18 domain to the antifungal ability of GlxChiB. The microscopic observations clearly showed that the wild type acted on both the tips and the lateral wall of fungal hyphae, while CatD acted only on the tips. These results suggest that the CBM18 domain could contribute to the antifungal ability of chitinase through its affinity to the fungal lateral wall by hydrophobic interactions.

Gene expression of PLAT and ATS3 proteins increases plant resistance to insects.

MAIN CONCLUSION: Genes of the PLAT protein family, including PLAT and ATS3 subfamilies of higher plants and homologs of liverwort, are involved in plant defense against insects. Laticifer cells in plants contain large amounts of anti-microbe or anti-insect proteins and are involved in plant defense against biotic stresses. We previously found that PLAT proteins accumulate in laticifers of fig tree (Ficus carica) at comparable levels to those of chitinases, and the transcript level of ATS3, another PLAT domain-containing protein, is highest in the transcriptome of laticifers of Euphorbia tirucalli. In this study, we investigated whether the PLAT domain-containing proteins are involved in defense against insects. Larvae of the lepidopteran Spodoptera litura showed retarded growth when fed with Nicotiana benthamiana leaves expressing F. carica PLAT or E. tirucalli ATS3 genes, introduced by agroinfiltration using expression vector pBYR2HS. Transcriptome analysis of these leaves indicated that ethylene and jasmonate signaling were activated, leading to increased expression of genes for PR-1, ß-1,3-glucanase, PR5 and trypsin inhibitors, suggesting an indirect mechanism of PLAT- and ATS3-induced resistance in the host plant. Direct cytotoxicity of PLAT and ATS3 to insects was also possible because heterologous expression of the corresponding genes in Drosophila melanogaster caused apoptosis-mediated cell death in this insect. Larval growth retardation of S. litura occurred when they were fed radish sprouts, a good host for agroinfiltration, expressing any of nine homologous genes of dicotyledon Arabidopsis thaliana, monocotyledon Brachypodium distachyon, conifer Picea sitchensis and liverwort Marchantia polymorpha. Of these nine genes, the heterologous expression of A. thaliana AT5G62200 and AT5G62210 caused significant increases in larval death. These results indicated that the PLAT protein family has largely conserved anti-insect activity in the plant kingdom (249 words).

A new P450 involved in the furanocoumarin pathway underlies a recent case of convergent evolution.

Furanocoumarins are phytoalexins often cited as an example to illustrate the arms race between plants and herbivorous insects. They are distributed in a limited number of phylogenetically distant plant lineages, but synthesized through a similar pathway, which raised the question of a unique or multiple emergence in higher plants. The furanocoumarin pathway was investigated in the fig tree (Ficus carica, Moraceae). Transcriptomic and metabolomic approaches led to the identification of CYP76F112, a cytochrome P450 catalyzing an original reaction. CYP76F112 emergence was inquired using phylogenetics combined with in silico modeling and site-directed mutagenesis. CYP76F112 was found to convert demethylsuberosin into marmesin with a very high affinity. This atypical cyclization reaction represents a key step within the polyphenol biosynthesis pathway. CYP76F112 evolutionary patterns suggests that the marmesin synthase activity appeared recently in the Moraceae family, through a lineage-specific expansion and diversification. The characterization of CYP76F112 as the first known marmesin synthase opens new prospects for the use of the furanocoumarin pathway. It also supports the multiple acquisition of furanocoumarin in angiosperms by convergent evolution, and opens new perspectives regarding the ability of cytochromes P450 to evolve new functions related to plant adaptation to their environment.

Convergent evolution of the UbiA prenyltransferase family underlies the independent acquisition of furanocoumarins in plants.

Furanocoumarins (FCs) are plant-specialized metabolites with potent allelochemical properties. The distribution of FCs is scattered with a chemotaxonomical tendency towards four distant families with highly similar FC pathways. The mechanism by which this pathway emerged and spread in plants has not been elucidated. Furanocoumarin biosynthesis was investigated in Ficus carica (fig, Moraceae), focusing on the first committed reaction catalysed by an umbelliferone dimethylallyltransferase (UDT). Comparative RNA-seq analysis among latexes of different fig organs led to the identification of a UDT. The phylogenetic relationship of this UDT to previously reported Apiaceae UDTs was evaluated. The expression pattern of F. carica prenyltransferase 1 (FcPT1) was related to the FC contents in different latexes. Enzymatic characterization demonstrated that one of the main functions of FcPT1 is UDT activity. Phylogenetic analysis suggested that FcPT1 and Apiaceae UDTs are derived from distinct ancestors, although they both belong to the UbiA superfamily. These findings are supported by significant differences in the related gene structures. This report describes the identification of FcPT1 involved in FC biosynthesis in fig and provides new insights into multiple origins of the FC pathway and, more broadly, into the adaptation of plants to their environments.

Comparative multi-omics analysis reveals diverse latex-based defense strategies against pests among latex-producing organs of the fig tree (Ficus carica).

MAIN CONCLUSION: Latexes in immature fruit, young petioles and lignified trunks of fig trees protect the plant using toxic proteins and metabolites in various organ-dependent ways. Latexes from plants contain high amounts of toxic proteins and metabolites, which attack microbes and herbivores after exudation at pest-induced wound sites. The protein and metabolite constituents of latexes are highly variable, depending on the plant species and organ. To determine the diversity of latex-based defense strategies in fig tree (Ficus carica) organs, we conducted comparative proteomic, transcriptomic and metabolomic analyses on latexes isolated from immature fruit, young petioles and lignified trunks of F. carica after constructing a unigene sequence library using RNA-seq data. Trypsin inhibitors were the most abundant proteins in petiole latex, while cysteine proteases ("ficins") were the most abundant in immature fruit and trunk latexes. Galloylglycerol, a possible defense-related metabolite, appeared to be highly accumulated in all three latexes. The expression levels of pathogenesis-related proteins were highest in the latex of trunk, suggesting that this latex had adapted a defensive role against microbe attacks. Although young petioles and immature fruit are both unlignified soft organs, and potential food for herbivorous insects, unigenes for the sesquiterpenoid pathway, which likely produces defense-associated volatiles, and the phenylpropanoid pathway, which produces toxic furanocoumarins, were expressed less in immature fruit latex. This difference may indicate that while petioles and fruit protect the plant from attack by herbivores, the fruit must also attract insect pollinators at younger stages and animals after ripening. We also suggest possible candidate transcription factors and signal transduction proteins that are involved in the differential expression of the unigenes.

Enzymatic hydrolysis of PET: functional roles of three Ca2+ ions bound to a cutinase-like enzyme, Cut190*, and its engineering for improved activity.

Cut190 from Saccharomonospora viridis AHK190 (Cut190) is the only cutinase that exhibits inactive (Ca2+-free) and active (Ca2+-bound) states, although other homologous cutinases always maintain the active states (Ca2+-free and bound). The X-ray crystallography of the S176A mutant of Cut190* (Cut190_S226P/R228S) showed that three Ca2+ ions were bound at sites 1-3 of the mutant. We analyzed the roles of three Ca2+ ions by mutation and concluded that they play different roles in Cut190* for activation (sites 1 and 3) and structural and thermal stabilization (sites 2 and 3). Based on these analyses, we elucidated the mechanism for the conformational change from the Ca2+-free inactive state to the Ca2+-bound active state, proposing the novel Ca2+ effect on structural dynamics of protein. The introduction of a disulfide bond at Asp250 and Glu296 in site 2 remarkably increased the melting temperatures of the mutant enzymes by more than 20-30 °C (while Ca2+-bound) and 4-14 °C (while Ca2+-free), indicating that a disulfide bond mimics the Ca2+ effect. Replacement of surface asparagine and glutamine with aspartic acid, glutamic acid, or histidine increased the melting temperatures. Engineered mutant enzymes were evaluated by an increase in melting temperatures and kinetic values, based on the hydrolysis of poly(butylene succinate-co-adipate) and microfiber polyethylene terephthalate (PET). A combined mutation, Q138A/D250C-E296C/Q123H/N202H, resulted in the highest thermostability, leading to the maximum degradation of PET film (more than 30%; approximately threefold at 70 °C, compared with that of Cut190* at 63 °C).

Acrolein-detoxifying isozymes of glutathione transferase in plants.

MAIN CONCLUSION: Acrolein is a lipid-derived highly reactive aldehyde, mediating oxidative signal and damage in plants. We found acrolein-scavenging glutathione transferase activity in plants and purified a low K M isozyme from spinach. Various environmental stressors on plants cause the generation of acrolein, a highly toxic aldehyde produced from lipid peroxides, via the promotion of the formation of reactive oxygen species, which oxidize membrane lipids. In mammals, acrolein is scavenged by glutathione transferase (GST; EC 2.5.1.18) isozymes of Alpha, Pi, and Mu classes, but plants lack these GST classes. We detected the acrolein-scavenging GST activity in four species of plants, and purified an isozyme showing this activity from spinach (Spinacia oleracea L.) leaves. The isozyme (GST-Acr), obtained after an affinity chromatography and two ion exchange chromatography steps, showed the K M value for acrolein 93 µM, the smallest value known for acrolein-detoxifying enzymes in plants. Peptide sequence homology search revealed that GST-Acr belongs to the GST Tau, a plant-specific class. The Arabidopsis thaliana GST Tau19, which has the closest sequence similar to spinach GST-Acr, also showed a high catalytic efficiency for acrolein. These results suggest that GST plays as a scavenger for acrolein in plants.

The novel heme-dependent inducible protein, SRRD regulates heme biosynthesis and circadian rhythms.

Heme plays a role in the regulation of the expression of genes related to circadian rhythms and heme metabolism. In order to identify new heme-regulated proteins, an RNA sequence analysis using mouse NIH3T3 cells treated without or with 5-aminolevulinic acid (ALA) was performed. Among the changes observed in the levels of various mRNAs including heme oxygenase-1 (HO-1) and ALA synthase-1 (ALAS1), a mouse homologue of the plant circadian-regulating protein SRR1, SRR1 domain containing (SRRD) was induced by the ALA treatment. The expression of SRRD was dependent on heme biosynthesis, and increased the production of heme. SRRD was expressed under circadian rhythms, and influenced the expression of clock genes including PER2, BMAL1, and CLOCK. The knockout of SRRD arrested the growth of cells, indicating that SRRD plays roles in heme-regulated circadian rhythms and cell proliferation.

Ferritin 2 domain-containing protein found in lacquer tree (Toxicodendron vernicifluum) sap has negative effects on laccase and peroxidase reactions.

Lacquer tree sap, a raw material of traditional paints in East Asia, is hardened through laccase-catalyzed oxidation and the following polymerization of phenolic compound urushiol. In the sap's water-insoluble fraction, we found two plantacyanins and a ferritin 2 domain-containing protein (TvFe2D, a homolog of Arabidopsis AT1G47980 and AT3G62730). The recombinant TvFe2D protein suppressed the accumulation of laccase-catalyzed oxidation products of a model substrate syringaldazine without decreasing oxygen consumption, the second substrate of laccase. The suppression was also observed when another substrate guaiacol or another oxidizing enzyme peroxidase was used. The functional domain of the suppression was the C-terminal half, downstream of the ferritin 2 domain. The results suggest that this protein may be involved in regulating the sap polymerization/hardening. We also discuss the possibility that homologous proteins of TvFe2D in other plants might be involved in the laccase- or peroxidase-mediated polymerization of phenolic compounds, such as lignin and flavonoids.

A simple and highly sensitive method of measuring heme oxygenase activity.

Heme oxygenase (HO) is a rate-limiting step of heme degradation, which catalyzes the conversion of heme into biliverdin, iron, and CO. HO has been characterized in microorganisms, insects, plants, and mammals. Previously used assays of HO activity were complicated and had low sensitivity. We found that the use of an eel bilirubin-bound fluorescent protein, UnaG, can achieve a highly sensitive and simple assay of HO activity. Using several enzyme sources including human culture cells, homogenates of plant tissues, and recombinant yeast HO, data were successfully obtained. The present method can facilitate the examination of HO in various organisms.

A regulatory cascade involving class II ETHYLENE RESPONSE FACTOR transcriptional repressors operates in the progression of leaf senescence.

Leaf senescence is the final process of leaf development that involves the mobilization of nutrients from old leaves to newly growing tissues. Despite the identification of several transcription factors involved in the regulation of this process, the mechanisms underlying the progression of leaf senescence are largely unknown. Herein, we describe the proteasome-mediated regulation of class II ETHYLENE RESPONSE FACTOR (ERF) transcriptional repressors and involvement of these factors in the progression of leaf senescence in Arabidopsis (Arabidopsis thaliana). Based on previous results showing that the tobacco (Nicotiana tabacum) ERF3 (NtERF3) specifically interacts with a ubiquitin-conjugating enzyme, we examined the stability of NtERF3 in vitro and confirmed its rapid degradation by plant protein extracts. Furthermore, NtERF3 accumulated in plants treated with a proteasome inhibitor. The Arabidopsis class II ERFs AtERF4 and AtERF8 were also regulated by the proteasome and increased with plant aging. Transgenic Arabidopsis plants with enhanced expression of NtERF3, AtERF4, or AtERF8 showed precocious leaf senescence. Our gene expression and chromatin immunoprecipitation analyses suggest that AtERF4 and AtERF8 targeted the EPITHIOSPECIFIER PROTEIN/EPITHIOSPECIFYING SENESCENCE REGULATOR gene and regulated the expression of many genes involved in the progression of leaf senescence. By contrast, an aterf4 aterf8 double mutant exhibited delayed leaf senescence. Our results provide insight into the important role of class II ERFs in the progression of leaf senescence.

Polyvinyl alcohol and polyethylene glycol form polymer bodies in the periplasm of Sphingomonads that are able to assimilate them.

Scanning electron microscopy (SEM) shows remarkable morphological surface changes in Sphingopyxis sp. 113P3 cells grown in polyvinyl alcohol (PVA) but not in Luria-Bertani medium (LB) (Hu et al. in Arch Microbiol 188: 235-241, 2007). However, transmission electron microscopy showed no surface changes in PVA-grown cells and revealed the presence of polymer bodies in the periplasm of PVA-grown cells, which were not observed in LB-grown cells. The presence of polymer bodies was supported by low-vacuum SEM observation of PVA- and LB-grown cells of strain 113P3, and the presence of similar polymer bodies was also found when Sphingopyxis macrogoltabida 103 and S. terrae were grown in polyethylene glycol (PEG). The extraction of PVA and PEG from the periplasmic fraction of cells using a modified Anraku and Heppel method and their analysis by MALDI-TOF mass spectrometry strongly suggested that the polymer bodies are composed of PVA and PEG, respectively, in Sphingopyxis sp. 113P3 (PVA degrader) and Sphingopyxis macrogoltabida 103 or S. terrae (PEG degraders). PEG-grown S. macrogoltabida 103 and S. terrae showed higher transport of (14)C-PEG 4000 than LB-grown cells. Recombinant PegB (TonB-dependent receptor-like protein consisting of a barrel structure) interacted with PEG 200, 4000 and 20000, suggesting that the barrel protein in the outer membrane contributes to the transport of PEG into the periplasm.

Comparative study of gene expression and major proteins' function of laticifers in lignified and unlignified organs of mulberry.

A laticifer is a cell involved in plant defense against biotic stresses such as herbivores and microorganisms; however, its gene expression is poorly understood. We compared protein accumulation and transcriptomes among laticifers of lignified and unlignified organs of mulberry (Morus alba), which has a non-articulated, branched type of laticifer. LA-a (equivalent to MLX56) and its homolog LA-b (insecticidal chitinase-like proteins containing two chitin-binding domains) were major proteins in laticifers of unlignified organs, and another protein (LA-c) was a major protein in laticifers of lignified organs. Purification, cDNA cloning, and bioassay of LA-c revealed that LA-c was an acidic class I chitinase having antifungal but not insecticidal activity. Comparative mRNA-Seq analysis using a GS-FLX revealed transcripts of other possible defense-related proteins. Jacalin-like lectin, galacturonase-inhibitor, and pathogenesis-related proteins were also abundant; however, the relative amounts differed among laticifers of lignified and unlignified organs. The results suggest a discontinuous laticifer network in planta and adaptation to different potential enemies among these organs.

Efficient depolymerization of polyethylene terephthalate (PET) and polyethylene furanoate by engineered PET hydrolase Cut190.

The enzymatic recycling of polyethylene terephthalate (PET) can be a promising approach to tackle the problem of plastic waste. The thermostability and activity of PET-hydrolyzing enzymes are still insufficient for practical application. Pretreatment of PET waste is needed for bio-recycling. Here, we analyzed the degradation of PET films, packages, and bottles using the newly engineered cutinase Cut190. Using gel permeation chromatography and high-performance liquid chromatography, the degradation of PET films by the Cut190 variant was shown to proceed via a repeating two-step hydrolysis process; initial endo-type scission of a surface polymer chain, followed by exo-type hydrolysis to produce mono/bis(2-hydroxyethyl) terephthalate and terephthalate from the ends of fragmented polymer molecules. Amorphous PET powders were degraded more than twofold higher than amorphous PET film with the same weight. Moreover, homogenization of post-consumer PET products, such as packages and bottles, increased their degradability, indicating the importance of surface area for the enzymatic hydrolysis of PET. In addition, it was required to maintain an alkaline pH to enable continuous enzymatic hydrolysis, by increasing the buffer concentration (HEPES, pH 9.0) depending on the level of the acidic products formed. The cationic surfactant dodecyltrimethylammonium chloride promoted PET degradation via adsorption on the PET surface and binding to the anionic surface of the Cut190 variant. The Cut190 variant also hydrolyzed polyethylene furanoate. Using the best performing Cut190 variant (L136F/Q138A/S226P/R228S/D250C-E296C/Q123H/N202H/K305del/L306del/N307del) and amorphous PET powders, more than 90 mM degradation products were obtained in 3 days and approximately 80 mM in 1 day.

The role of chaperone complex HSP90-SGT1-RAR1 as the associated machinery for hybrid inviability between Nicotiana gossei Domin and N. tabacum L.

Two cultured cell lines (GTH4 and GTH4S) of a Nicotiana interspecific F1 hybrid (N. gossei × N. tabacum) were comparatively analyzed to find genetic factors related to hybrid inviability. Both cell lines proliferated at 37 °C, but after shifting to 26 °C, GTH4 started to die similar to the F1 hybrid seedlings, whereas GTH4S survived. As cell death requires de novo expression of genes and proteins, we compared expressed protein profiles between the two cell lines, and found that NgSGT1, a cochaperone of the chaperone complex (HSP90-SGT1-RAR1), was expressed in GTH4 but not in GTH4S. Agrobacterium-mediated transient expression of NgSGT1, but not NtSGT1, induced cell death in leaves of N. tabacum, suggesting its possible role in hybrid inviability. Cell death in N. tabacum was also induced by transient expression of NgRAR1, but not NtRAR1. In contrast, transient expression of any parental combinations of three components revealed that NgRAR1 promoted cell death, whereas NtRAR1 suppressed it in N. tabacum. A specific inhibitor of HSP90, geldanamycin, inhibited the progression of hypersensitive response-like cell death in GTH4 and leaf tissue after agroinfiltration. The present study suggested that components of the chaperone complex are involved in the inviability of Nicotiana interspecific hybrid.

Two chitinase-like proteins abundantly accumulated in latex of mulberry show insecticidal activity.

BACKGROUND: Plant latex is the cytoplasm of highly specialized cells known as laticifers, and is thought to have a critical role in defense against herbivorous insects. Proteins abundantly accumulated in latex might therefore be involved in the defense system. RESULTS: We purified latex abundant protein a and b (LA-a and LA-b) from mulberry (Morus sp.) and analyzed their properties. LA-a and LA-b have molecular masses of approximately 50 and 46 kDa, respectively, and are abundant in the soluble fraction of latex. Western blotting analysis suggested that they share sequence similarity with each other. The sequences of LA-a and LA-b, as determined by Edman degradation, showed chitin-binding domains of plant chitinases at the N termini. These proteins showed small but significant chitinase and chitosanase activities. Lectin RCA120 indicated that, unlike common plant chitinases, LA-a and LA-b are glycosylated. LA-a and LA-b showed insecticidal activities when fed to larvae of the model insect Drosophila melanogaster. CONCLUSIONS: Our results suggest that the two LA proteins have a crucial role in defense against herbivorous insects, possibly by hydrolyzing their chitin.

Analysis of the eukaryotic community and metabolites found in clay wall material used in the construction of traditional Japanese buildings.

Clay wall (tsuchikabe in Japanese) material for Japanese traditional buildings is manufactured by fermenting a mixture of clay, sand, and rice straw. The aim of this study was to understand the fermentation process in order to gain insight into the ways waste biomass can be used to produce useful materials. In this study, in addition to Clostridium, we suggested that the family Nectriaceae and the Scutellinia sp. of fungi were important in degrading cell wall materials of rice straw, such as cellulose and/or lignin. The microorganisms in the clay wall material produced sulfur-containing inorganic compounds that may sulfurate minerals in clay particles, and polysaccharides that give viscosity to clay wall material, thus increasing workability for plastering, and possibly giving water-resistance to the dried clay wall.

Recombinant stromal APX defective in the unique loop region showed improved tolerance to hydrogen peroxide.

Ascorbate peroxidase (APX) of chloroplasts plays a pivotal role in detoxifying the hydrogen peroxide (H2O2) produced through photoexcitation of the photosystem, but it easily loses its catalytic activity under attack by excess H2O2. Here we report that the H2O2-tolerance of APX localized in the stroma of chloroplasts was improved by removing a unique 16-amino-acid-residue loop not found in H2O2-tolerant isoforms of cytosol and glyoxysome.

Radish sprouts as an efficient and rapidly available host for an agroinfiltration-based transient gene expression system.

Agroinfiltration, the infiltration of plants with Agrobacterium harboring a plasmid that contains a specific gene, is used to transiently express a gene in a heterologous organism. Using the "Tsukuba system", greater amounts of target protein accumulate compared with usual expression plasmids. Reported host plants, including Nicotiana benthamiana, a common plant for agroinfiltration, need several weeks after sowing to grow enough for infection. To shorten the culture period and, thereby, improve target protein production, we tested sprouts as host plants. Sprouts were grown in the dark to encourage elongation so that vacuum infiltration becomes easier, and this was followed by a few days of exposure to illumination before infection with pBYR2HS-EGFP, the EGFP expression plasmid of the Tsukuba system. Among six tested species of Fabaceae and Brassicaceae, radish showed the highest transient expression. Among six tested radish cultivars, Kaiware, Hakata, and Banryoku provided the best results. Culturing for 5 day, including 1 day of imbibition and 1 to 2 day of exposure to illumination resulted in EGFP fluorescence in 80% of the cotyledon area. Thus, a remarkable amount of EGFP was obtained only 8 day after seed imbibition. The EGFP amount in Kaiware cotyledons was comparable with Rubisco at ∼0.7 mg/g fresh weight. Kaiware sold in supermarkets could also be used, but resulted in lower expression levels.

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.