The Chemical Reactivity of Membrane Lipids.

It is well-known that aqueous dispersions of phospholipids spontaneously assemble into bilayer structures. These structures have numerous applications across chemistry and materials science and form the fundamental structural unit of the biological membrane. The particular environment of the lipid bilayer, with a water-poor low dielectric core surrounded by a more polar and better hydrated interfacial region, gives the membrane particular biophysical and physicochemical properties and presents a unique environment for chemical reactions to occur. Many different types of molecule spanning a range of sizes, from dissolved gases through small organics to proteins, are able to interact with membranes and promote chemical changes to lipids that subsequently affect the physicochemical properties of the bilayer. This Review describes the chemical reactivity exhibited by lipids in their membrane form, with an emphasis on conditions where the lipids are well hydrated in the form of bilayers. Key topics include the following: lytic reactions of glyceryl esters, including hydrolysis, aminolysis, and transesterification; oxidation reactions of alkenes in unsaturated fatty acids and sterols, including autoxidation and oxidation by singlet oxygen; reactivity of headgroups, particularly with reactive carbonyl species; and E/Z isomerization of alkenes. The consequences of reactivity for biological activity and biophysical properties are also discussed.

A novel soluble di-iron monooxygenase from the soil bacterium Solimonas soli.

Soluble di-iron monooxygenase (SDIMO) enzymes enable insertion of oxygen into diverse substrates and play significant roles in biogeochemistry, bioremediation and biocatalysis. An unusual SDIMO was detected in an earlier study in the genome of the soil organism Solimonas soli, but was not characterized. Here, we show that the S. soli SDIMO is part of a new clade, which we define as 'Group 7'; these share a conserved gene organization with alkene monooxygenases but have only low amino acid identity. The S. soli genes (named zmoABCD) could be functionally expressed in Pseudomonas putida KT2440 but not in Escherichia coli TOP10. The recombinants made epoxides from C2 C8 alkenes, preferring small linear alkenes (e.g. propene), but also epoxidating branched, carboxylated and chlorinated substrates. Enzymatic epoxidation of acrylic acid was observed for the first time. ZmoABCD oxidised the organochlorine pollutants vinyl chloride (VC) and cis-1,2-dichloroethene (cDCE), with the release of inorganic chloride from VC but not cDCE. The original host bacterium S. soli could not grow on any alkenes tested but grew well on phenol and n-octane. Further work is needed to link ZmoABCD and the other Group 7 SDIMOs to specific physiological and ecological roles.

Functional production and biochemical investigation of an integral membrane enzyme for olefin biosynthesis.

Integral membrane enzymes play essential roles in a plethora of biochemical processes. The fatty acid desaturases (FADS)-like superfamily is an important group of integral membrane enzymes that catalyze a wide array of reactions, including hydroxylation, desaturation, and cyclization; however, due to the membrane-bound nature, the majority of these enzymes have remained poorly understood. UndB is a member of the FADS-like superfamily, which catalyzes fatty acid decarboxylation, a chemically challenging reaction at the membrane interface. UndB reaction produces terminal olefins that are prominent biofuel candidates and building blocks of polymers with widespread industrial applications. Despite the great importance of UndB for several biotechnological applications, the enzyme has eluded comprehensive investigation. Here, we report details of the expression, solubilization, and purification of several constructs of UndB to achieve the optimally functional enzyme. We gained important insights into the biochemical, biophysical, and catalytic properties of UndB, including the thermal stability and factors influencing the enzyme activity. Additionally, we established the ability and kinetics of UndB to produce dienes by performing di-decarboxylation of diacids. We found that the reaction proceeds by forming a mono-carboxylic acid intermediate. Our findings shed light on the unexplored biochemical properties of the UndB and extend opportunities for its rigorous mechanistic and structural characterization.

Engineering cytochrome P450s for selective alkene to carbonyl oxidation.

The Wacker-Tsuji oxidation is an important aerobic oxidation process to synthesize ethanal from ethene and methyl ketones from 1-alkenes. Current challenges in aerobic alkene oxidation include selective carbonyl product formation beyond methyl ketones. This includes the regioselective oxidation of the terminal carbon atom of 1-alkenes, the regioselective ketone formation with internal alkenes as well as the enantioselective alkene to carbonyl oxidation. Recently, the potential of high-valent metal-oxo species for direct alkene to carbonyl oxidation was explored as carbonyl product formation is frequently reported as a side reaction of alkene epoxidation by cytochrome P450s. It was shown that such promiscuous P450s can be engineered via directed evolution to perform alkene to carbonyl oxidation reactions with high activity and selectivity. Here, we report a protocol to convert promiscuous P450s into efficient and selective enzymes for Wacker-type alkene oxidation. One round of directed evolution is described in detail, which includes the generation and handling of site-saturation libraries, recombinant protein expression, library screening in a 96-well plate format and the rescreening of variants with beneficial mutations. These protocols might be useful to engineer various P450s for selective alkene to carbonyl oxidation, and to engineer enzymes in general.

P450 fatty acid decarboxylase.

P450 fatty acid decarboxylases are able to utilize hydrogen peroxide as the sole cofactor to decarboxylate free fatty acids to produce α-olefins with abundant applications as drop-in biofuels and important chemical precursors. In this chapter, we review diverse approaches for discovery, characterization, engineering, and applications of P450 fatty acid decarboxylases. Information gained from structural data has been advancing our understandings of the unique mechanisms underlying alkene production, and providing important insights for exploring new activities. To build an efficient olefin-producing system, various engineering strategies have been proposed and applied to this unusual P450 catalytic system. Furthermore, we highlight a select number of applied examples of P450 fatty acid decarboxylases in enzyme cascades and metabolic engineering.

The 2-methylpropene degradation pathway in Mycobacteriaceae family strains.

Mycolicibacterium gadium IBE100 and Mycobacterium paragordonae IBE200 are aerobic, chemoorganoheterotrophic bacteria isolated from activated sludge from a wastewater treatment plant. They use 2-methylpropene (isobutene, 2-MP) as the sole source of carbon and energy. Here, we postulate a degradation pathway of 2-methylpropene derived from whole genome sequencing, differential expression analysis and peptide-mass fingerprinting. Key genes identified are coding for a 4-component soluble diiron monooxygenase with epoxidase activity, an epoxide hydrolase, and a 2-hydroxyisobutyryl-CoA mutase. In both strains, involved genes are arranged in clusters of 61.0 and 58.5 kbp, respectively, which also contain the genes coding for parts of the aerobic pathway of adenosylcobalamin synthesis. This vitamin is essential for the carbon rearrangement reaction catalysed by the mutase. These findings provide data for the identification of potential 2-methylpropene degraders.

Functional Characterization of a (E)-ß-Ocimene Synthase Gene Contributing to the Defense against Spodoptera litura.

Soybean is a worldwide crop that offers valuable proteins, fatty acids, and phytonutrients to humans but is always damaged by insect pests or pathogens. Plants have captured sophisticated defense mechanisms in resisting the attack of insects and pathogens. How to protect soybean in an environment- or human-friendly way or how to develop plant-based pest control is a hotpot. Herbivore-induced plant volatiles that are released by multiple plant species have been assessed in multi-systems against various insects, of which (E)-ß-ocimene has been reported to show anti-insect function in a variety of plants, including soybean. However, the responsible gene in soybean is unknown, and its mechanism of synthesis and anti-insect properties lacks comprehensive assessment. In this study, (E)-ß-ocimene was confirmed to be induced by Spodoptera litura treatment. A plastidic localized monoterpene synthase gene, designated as GmOCS, was identified to be responsible for the biosynthesis of (E)-ß-ocimene through genome-wide gene family screening and in vitro and in vivo assays. Results from transgenic soybean and tobacco confirmed that (E)-ß-ocimene catalyzed by GmOCS had pivotal roles in repelling a S. litura attack. This study advances the understanding of (E)-ß-ocimene synthesis and its function in crops, as well as provides a good candidate for further anti-insect soybean improvement.

Systematic In Vitro Metabolic Profiling of the OXIZID Synthetic Cannabinoids BZO-4en-POXIZID, BZO-POXIZID, 5F-BZO-POXIZID, BZO-HEXOXIZID and BZO-CHMOXIZID.

A new class of synthetic cannabinoids termed OXIZIDs has recently emerged on the recreational drug market. In order to continue the detection of new drugs in biological specimens, the identification of metabolites is essential. The aim of this study was to elucidate the metabolites of BZO-4en-POXIZID produced in human liver microsomes (HLMs) and human hepatocyte incubations and to compare the results with closely related analogs using the same experimental setup. Each drug was incubated for 1 h in HLM and BZO-4en-POXIZID was also incubated in human hepatocytes for up to 3 h. Subsequently, the incubates were analyzed by liquid chromatography-high-resolution mass spectrometry. BZO-4en-POXIZID metabolites were obtained in the incubation with HLMs and human hepatocytes, via the metabolic pathways of dihydrodiol formation, hydroxylation, reduction of the alkene bond and glucuronidation. The major metabolic pathway was found to be dihydrodiol formation at the pentenyl tail moiety. BZO-POXIZID, 5 F-BZO-POXIZID, BZO-HEXOXIZID and BZO-CHMOXIZID underwent similar metabolism to those reported in the literature, via the metabolic pathways of N-dealkylation, hydroxylation, ketone formation and oxidative defluorination (to alcohol or carboxylic acid). The results suggest that OXIZIDs are mainly metabolized at the N-alkyl moiety and the major metabolic pathways are hydroxylation when the N-alkyl moiety is a simple hydrocarbon, whereas functional-group-specific pathways (dihydrodiol formation and oxidative defluorination) are preferred when the moiety contains specific functional groups (alkene or fluoro), as has been observed for other synthetic cannabinoids. The major metabolites generated via these major metabolic pathways should serve as useful analytical targets for urine analysis. Furthermore, the higher abundance of glucuronidated metabolite suggests that enzymatic hydrolysis of glucuronides may be necessary for urine analysis to increase phase I metabolite concentration and improve detection.

D-π-A-Based Trisubstituted Alkenes as Environmentally Sensitive Fluorescent Probes to Detect Lewy Pathologies.

Lewy pathologies, which mainly consist of insoluble α-synuclein (α-syn) aggregates, are the hallmarks of Parkinson's disease and many other neurodegenerative diseases termed "synucleinopathies". Detection of Lewy pathologies with optical methods is of interest for preclinical studies, while the α-syn fluorescent probe is still in great demand. By rational design, we obtained a series of D-π-A-based trisubstituted alkenes with acceptable optical properties and high binding affinities to α-syn fibrils. Among these probes, FPQXN and TQXN-2 exhibited high binding affinities (6 and 8 nM, respectively), significant fluorescence enhancements (17.2- and 26.6-fold, respectively), and satisfying quantum yields (36.5% and 10.4%, respectively), which met the need for the in vitro neuropathological staining of Lewy pathologies in the PD brain sections. In addition, TQXN-2 showed great potential in fluorescent discrimination of Lewy pathologies and Aß plaques. Our research provides flexible tools for in vitro detection of α-syn aggregates and offers new structural frameworks for the further development of α-syn fluorescent probes.

Overexpression of SmLAC25 promotes lignin accumulation and decreases salvianolic acid content in Salvia miltiorrhiza.

Laccase (LAC) is a blue multicopper oxidase that contains four copper ions, which is involved in lignin polymerization and flavonoid biosynthesis in plants. Although dozens of LAC genes have been identified in Salvia miltiorrhiza Bunge (a model medicinal plant), most have not been functionally characterized. Here, we explored the expression patterns and the functionality of SmLAC25 in S. miltiorrhiza. SmLAC25 has a higher expression level in roots and responds to methyl jasmonate, auxin, abscisic acid, and gibberellin stimuli. The SmLAC25 protein is localized in the cytoplasm and chloroplasts. Recombinant SmLAC25 protein could oxidize coniferyl alcohol and sinapyl alcohol, two monomers of G-lignin and S-lignin. To investigate its function, we generated SmLAC25-overexpressed S. miltiorrhiza plantlets and hairy roots. The lignin content increased significantly in all SmLAC25-overexpressed plantlets and hairy roots, compared with the controls. However, the concentrations of rosmarinic acid and salvianolic acid B decreased significantly in all the SmLAC25-overexpressed lines. Further studies revealed that the transcription levels of some key enzyme genes in the lignin synthesis pathway (e.g., SmCCR and SmCOMT) were significantly improved in the SmLAC25-overexpressed lines, while the expression levels of multiple enzyme genes in the salvianolic acid biosynthesis pathway were inhibited. We speculated that the overexpression of SmLAC25 promoted the metabolic flux of lignin synthesis, which resulted in a decreased metabolic flux to the salvianolic acid biosynthesis pathway.

Genus Smenospongia: Untapped Treasure of Biometabolites-Biosynthesis, Synthesis, and Bioactivities.

Marine sponges continue to attract remarkable attention as one of the richest pools of bioactive metabolites in the marine environment. The genus Smenospongia (order Dictyoceratida, family Thorectidae) sponges can produce diverse classes of metabolites with unique and unusual chemical skeletons, including terpenoids (sesqui-, di-, and sesterterpenoids), indole alkaloids, aplysinopsins, bisspiroimidazolidinones, chromenes, γ-pyrones, phenyl alkenes, naphthoquinones, and polyketides that possessed diversified bioactivities. This review provided an overview of the reported metabolites from Smenospongia sponges, including their biosynthesis, synthesis, and bioactivities in the period from 1980 to June 2022. The structural characteristics and diverse bioactivities of these metabolites could attract a great deal of attention from natural-product chemists and pharmaceuticals seeking to develop these metabolites into medicine for the treatment and prevention of certain health concerns.

Synthetic metabolic pathways for conversion of CO2 into secreted short-to medium-chain hydrocarbons using cyanobacteria.

The objective of this study was to implement direct sunlight-driven conversion of CO2 into a naturally excreted ready-to-use fuel. We engineered four different synthetic metabolic modules for biosynthesis of short-to medium-chain length hydrocarbons in the model cyanobacterium Synechocystis sp. PCC 6803. In module 1, the combination of a truncated clostridial n-butanol pathway with over-expression of the native cyanobacterial aldehyde deformylating oxygenase resulted in small quantities of propane when cultured under closed conditions. Direct conversion of CO2 into propane was only observed in strains with CRISPRi-mediated repression of three native putative aldehyde reductases. In module 2, three different pathways towards pentane were evaluated based on the polyunsaturated fatty acid linoleic acid as an intermediate. Through combinatorial evaluation of reaction ingredients, it was concluded that linoleic acid undergoes a spontaneous non-enzymatic reaction to yield pentane and hexanal. When Synechocystis was added to the reaction, hexanal was converted into 1-hexanol, but there was no further stimulation of pentane biosynthesis even in the Synechocystis strains expressing GmLOX1. For modules 3 and 4, several different acyl-ACP thioesterases were evaluated in combination with two different decarboxylases. Small quantities of 1-heptene and 1-nonene were observed in strains expressing the desaturase-like enzyme UndB from Pseudomonas mendocina in combination with C8-C10 preferring thioesterases ('CaFatB3.5 and 'ChoFatB2.2). When UndB instead was combined with a C12-specific 'UcFatB1 thioesterase, this resulted in a ten-fold increase of alkene biosynthesis. When UndB was replaced with the light-dependent FAP decarboxylase, both undecane and tridecane accumulated, albeit with a 10-fold drop in productivity. Preliminary optimization of the RBS, promoter and gene order in some of the synthetic operons resulted in improved 1-alkene productivity, reaching a titer of 230 mg/L after 10 d with 15% carbon partitioning. In conclusion, the direct bioconversion of CO2 into secreted and ready-to-use hydrocarbon fuel was implemented with several different metabolic systems. Optimal productivity was observed with UndB and a C12 chain-length specific thioesterase, although further optimization of the entire biosynthetic system is still possible.

Insights into cyanobacterial alkane biosynthesis.

Alkanes are high-energy molecules that are compatible with enduring liquid fuel infrastructures, which make them highly suitable for being next-generation biofuels. Though biological production of alkanes has been reported in various microorganisms, the reports citing photosynthetic cyanobacteria as natural producers have been the most consistent for the long-chain alkanes and alkenes (C15-C19). However, the production of alkane in cyanobacteria is low, leading to its extraction being uneconomical for commercial purposes. In order to make alkane production economically feasible from cyanobacteria, the titre and yield need to be increased by several orders of magnitude. In the recent past, efforts have been made to enhance alkane production, although with a little gain in yield, leaving space for much improvement. Genetic manipulation in cyanobacteria is considered challenging, but recent advancements in genetic engineering tools may assist in manipulating the genome in order to enhance alkane production. Further, advancement in a basic understanding of metabolic pathways and gene functioning will guide future research for harvesting the potential of these tiny photosynthetically efficient factories. In this review, our focus would be to highlight the current knowledge available on cyanobacterial alkane production, and the potential aspects of developing cyanobacterium as an economical source of biofuel. Further insights into different metabolic pathways and hosts explored so far, and possible challenges in scaling up the production of alkanes will also be discussed.

Outer-sphere electron transfer does not underpin B12-dependent olefinic reductive dehalogenation in anaerobes.

Anaerobic microbial B12-dependent reductive dehalogenation may pave a way to remediate soil, sediment, and underground water contaminated with halogenated olefins. The chemical reaction is initiated by electron transfer (ET) from supernucleophilic cob(I)alamin (B12s). However, the inherent mechanism as outer-sphere or inner-sphere route is still under debate. To clarify the possibility of an outer-sphere pathway, we calculated free energy barriers of the initial steps of all outer-sphere ET routes by Marcus theory employing density functional theory (DFT). For 18 fluorinated, chlorinated, and brominated ethenes as representative olefins, 164 of 165 reactions with free energy barriers larger than 20 kcal mol-1 are not feasible under physiological dehalogenase conditions. Moreover, electronic structure analysis of perbromoethene with an outer-sphere free energy barrier of 18.2 kcal mol-1 reveals no ET initiation down to Co⋯Br and Co⋯C distances of 3.15 Å. The results demonstrate that the B12-catalyzed reductive dechlorination of olefins in microbes should proceed through an inner-sphere ET pathway.

Prevalence of toxigenic fungi and mycotoxins in Arabic coffee (Coffea arabica): Protective role of traditional coffee roasting, brewing and bacterial volatiles.

Fungal infection and synthesis of mycotoxins in coffee leads to significant economic losses. This study aimed to investigate the prevalence of toxigenic fungi, their metabolites, and the effect of traditional roasting and brewing on ochratoxin A (OTA) and aflatoxins (AFs) contents of naturally contaminated coffee samples. In addition, in vivo biocontrol assays were performed to explore the antagonistic activities of Bacillus simplex 350-3 (BS350-3) on the growth and mycotoxins synthesis of Aspergillus ochraceus and A. flavus. The relative density of A. niger, A. flavus, Penicillium verrucosum and A. carbonarius on green coffee bean was 60.82%, 7.21%, 3.09% and 1.03%, respectively. OTA contents were lowest in green coffee beans (2.15 µg/kg), followed by roasted (2.76 µg/kg) and soluble coffee (8.95 µg/kg). Likewise, AFs levels were highest in soluble coffee (90.58 µg/kg) followed by roasted (33.61 µg/kg) and green coffee (9.07 µg/kg). Roasting naturally contaminated coffee beans at three traditional methods; low, medium and high, followed by brewing resulted in reduction of 58.74% (3.50 µg/kg), 60.88% (3.72 µg/kg) and 64.70% (4.11 µg/kg) in OTA and 40.18% (34.65 µg/kg), 47.86% (41.17 µg/kg) and 62.38% (53.73 µg/kg) AFs contents, respectively. Significant inhibitions of AFs and OTA synthesis by A. flavus and A. carbonarius, respectively, on infected coffee beans were observed in presence of Bacillus simplex BS350-3 volatiles. Gas chromatography mass spectrochemistry (GC-MS/MS) analysis of head-space BS350-3 volatiles showed quinoline, benzenemethanamine and 1-Octadecene as bioactive antifungal molecules. These findings suggest that marketed coffee samples are generally contaminated with OTA and AFs, with a significant level of roasted and soluble coffee contaminated above EU permissible limits for OTA. Further, along with coffee roasting and brewing; microbial volatiles can be optimized to minimize the dietary exposure to mycotoxins.

Discovery, Biocatalytic Exploration and Structural Analysis of a 4-Ethylphenol Oxidase from Gulosibacter chungangensis.

The vanillyl-alcohol oxidase (VAO) family is a rich source of biocatalysts for the oxidative bioconversion of phenolic compounds. Through genome mining and sequence comparisons, we found that several family members lack a generally conserved catalytic aspartate. This finding led us to study a VAO-homolog featuring a glutamate residue in place of the common aspartate. This 4-ethylphenol oxidase from Gulosibacter chungangensis (Gc4EO) shares 42 % sequence identity with VAO from Penicillium simplicissimum, contains the same 8α-N3 -histidyl-bound FAD and uses oxygen as electron acceptor. However, Gc4EO features a distinct substrate scope and product specificity as it is primarily effective in the dehydrogenation of para-substituted phenols with little generation of hydroxylated products. The three-dimensional structure shows that the characteristic glutamate side chain creates a closely packed environment that may limit water accessibility and thereby protect from hydroxylation. With its high thermal stability, well defined structural properties and high expression yields, Gc4EO may become a catalyst of choice for the specific dehydrogenation of phenolic compounds bearing small substituents.

Exploring Marine-Derived Ascochlorins as Novel Human Dihydroorotate Dehydrogenase Inhibitors for Treatment of Triple-Negative Breast Cancer.

Human dihydroorotate dehydrogenase (hDHODH) is an attractive tumor target essential to de novo pyrimidine biosynthesis. Novel potent hDHODH inhibitors with low toxicity are urgently needed. Herein, we demonstrate the isolation of 25 ascochlorin (ASC) derivatives, including 13 new ones, from the coral-derived fungus Acremonium sclerotigenum, and several of them showed pronounced inhibitions against hDHODH and triple-negative breast cancer (TNBC) cell lines, MDA-MB-231/-468. Interestingly, we found that hDHODH is required for proliferation and survival of TNBC cells, and several ASCs significantly inhibited TNBC cell growth and induced their apoptosis via hDHODH inhibition. Furthermore, the novel and potent hDHODH inhibitors (1 and 21) efficiently suppressed tumor growth in patient-derived TNBC xenograft models without obvious body weight loss or overt toxicity in mice. Collectively, our findings offered a novel lead scaffold as the hDHODH inhibitor for further development of potent anticancer agents and a potential therapeutic strategy for TNBC.

Directed evolution of prenylated FMN-dependent Fdc supports efficient in vivo isobutene production.

Isobutene is a high value gaseous alkene used as fuel additive and a chemical building block. As an alternative to fossil fuel derived isobutene, we here develop a modified mevalonate pathway for the production of isobutene from glucose in vivo. The final step in the pathway consists of the decarboxylation of 3-methylcrotonic acid, catalysed by an evolved ferulic acid decarboxylase (Fdc) enzyme. Fdc belongs to the prFMN-dependent UbiD enzyme family that catalyses reversible decarboxylation of (hetero)aromatic acids or acrylic acids with extended conjugation. Following a screen of an Fdc library for inherent 3-methylcrotonic acid decarboxylase activity, directed evolution yields variants with up to an 80-fold increase in activity. Crystal structures of the evolved variants reveal that changes in the substrate binding pocket are responsible for increased selectivity. Solution and computational studies suggest that isobutene cycloelimination is rate limiting and strictly dependent on presence of the 3-methyl group.

Herbivore-induced volatiles influence moth preference by increasing the ß-Ocimene emission of neighbouring tea plants.

Herbivore-induced plant volatiles prime neighbouring plants to respond more strongly to subsequent attacks. However, the key volatiles that trigger this state and their priming mechanisms remain largely unknown. The tea geometrid Ectropis obliqua is one of the most devastating leaf-feeding pests of tea plants. Here, plant-plant communication experiments demonstrated that volatiles emitted from tea plants infested by E. obliqua larvae triggered neighbouring plants to release volatiles that repel E. obliqua adult, especially mated females. Volatile analyses revealed that the quantity of eight volatiles increased dramatically when plants were exposed to volatiles emitted by infested tea plants, including (Z)-3-hexenol, linalool, α-farnesene, ß-Ocimene and (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT). The results of behavioural bioassays demonstrated that ß-Ocimene strongly repelled mated E. obliqua females. Individual volatile compound exposure experiments revealed that (Z)-3-hexenol, linalool, α-farnesene and DMNT triggered the emission of ß-Ocimene from tea plants. Chemical inhibition experiments demonstrated that the emission of ß-Ocimene induced by (Z)-3-hexenol, linalool, α-farnesene and DMNT were dependent on Ca2+ and JA signalling. These findings help us to understand how E. obliqua moths respond to volatiles emitted from tea plants and provide new insight into volatile-mediated plant-plant interactions. They have potential significance for the development of novel insect and pest control strategies in crops.

Identification and Evaluation of Aromatic Volatile Compounds in 26 Cultivars and 8 Hybrids of Freesia hybrida.

Freesia hybrida is a group of cultivars in the genus Freesia with a strong floral scent composed of diverse volatile organic compounds (VOCs). In this study, the VOCs of 34 F. hybrida were extracted and analyzed by headspace solid phase microextraction and gas chromatography mass spectrometry (HS-SPME-GC-MS). A total of 164 VOCs whose relative contents were higher than 0.05% were detected. The numbers of VOCs in all germplasms differed between 11 to 38, and the relative contents ranged from 32.39% to 94.28%, in which most germplasms were higher than 80%. Terpenoids, especially monoterpenes, were the crucial type of VOCs in most germplasms, of which linalool and D-limonene were the most frequently occurring. Principal component analysis (PCA) clearly separated samples based on whether linalool was the main component, and hierarchical clustering analysis (HCA) clustered samples into 4 groups according to the preponderant compounds linalool and (E)-ß-ocimene. Comparison of parental species and hybrids showed heterosis in three hybrids, and the inherited and novel substances suggested that monoterpene played an important role in F. hybrida floral scent. This study established a foundation for the evaluation of Freesia genetic resources, breeding for the floral aroma and promoting commercial application.