Mechanism Actions of Coniferyl Alcohol in Improving Cardiac Dysfunction in Renovascular Hypertension Studied by Experimental Verification and Network Pharmacology.

Renovascular hypertension (RH), a secondary hypertension, can significantly impact heart health, resulting in heart damage and dysfunction, thereby elevating the risk of cardiovascular diseases. Coniferol (CA), which has vascular relaxation properties, is expected to be able to treat hypertension-related diseases. However, its potential effects on cardiac function after RH remain unclear. In this study, in combination with network pharmacology, the antihypertensive and cardioprotective effects of CA in a two-kidney, one-clip (2K1C) mice model and its ability to mitigate angiotensin II (Ang II)-induced hypertrophy in H9C2 cells were investigated. The findings revealed that CA effectively reduced blood pressure, myocardial tissue damage, and inflammation after RH. The possible targets of CA for RH treatment were screened by network pharmacology. The interleukin-17 (IL-17) and tumor necrosis factor (TNF) signaling pathways were identified using a Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. The inflammatory response was identified using a Gene Ontology (GO) enrichment analysis. Western blot analysis confirmed that CA reduced the expression of IL-17, matrix metallopeptidase 9 (MMP9), cyclooxygenase 2 (COX2), and TNF α in heart tissues and the H9C2 cells. In summary, CA inhibited cardiac inflammation and fibrohypertrophy following RH. This effect was closely linked to the expression of MMP9/COX2/TNF α/IL-17. This study sheds light on the therapeutic potential of CA for treating RH-induced myocardial hypertrophy and provides insights into its underlying mechanisms, positioning CA as a promising candidate for future drug development.

Production of phenylpropanoids and flavonolignans from glycerol by metabolically engineered Escherichia coli.

Phenylpropanoids are a group of plant natural products with medicinal importance derived from aromatic amino acids. Here, we report the production of two representative phenylpropanoids-coniferyl alcohol (CA) and dihydroquercetin (DHQ)-from glycerol by engineered Escherichia coli. First, an E. coli strain capable of producing 187.7 mg/L of CA from glycerol was constructed by the introduction of hpaBC from E. coli and OMT1, 4CL4, and CCR1 from Arabidopsis thaliana to the p-coumaric acid producer. Next, an E. coli strain capable of producing 239.4 mg/L of DHQ from glycerol was constructed by the introduction of F3H, TT7, and CPR from A. thaliana to the naringenin producer, followed by engineering the signal peptide of a cytochrome P450 TT7. Furthermore, to demonstrate the production of flavonolignans, a group of heterodimeric phenylpropanoids, from glycerol, ascorbate peroxidase 1 from Silybum marianum was employed and engineered to produce 0.04 µg/L of silybin and 1.29 µg/L of isosilybin from glycerol by stepwise culture. Finally, a single strain harboring all the 16 necessary genes was constructed, resulting in 0.12 µg/L of isosilybin production directly from glycerol. The strategies described here will be useful for the production of pharmaceutically important yet complex natural products.

Vanillic acid and methoxyhydroquinone production from guaiacyl units and related aromatic compounds using Aspergillus niger cell factories.

BACKGROUND: The aromatic compounds vanillin and vanillic acid are important fragrances used in the food, beverage, cosmetic and pharmaceutical industries. Currently, most aromatic compounds used in products are chemically synthesized, while only a small percentage is extracted from natural sources. The metabolism of vanillin and vanillic acid has been studied for decades in microorganisms and many studies have been conducted that showed that both can be produced from ferulic acid using bacteria. In contrast, the degradation of vanillin and vanillic acid by fungi is poorly studied and no genes involved in this metabolic pathway have been identified. In this study, we aimed to clarify this metabolic pathway in Aspergillus niger and identify the genes involved. RESULTS: Using whole-genome transcriptome data, four genes involved in vanillin and vanillic acid metabolism were identified. These include vanillin dehydrogenase (vdhA), vanillic acid hydroxylase (vhyA), and two genes encoding novel enzymes, which function as methoxyhydroquinone 1,2-dioxygenase (mhdA) and 4-oxo-monomethyl adipate esterase (omeA). Deletion of these genes in A. niger confirmed their role in aromatic metabolism and the enzymatic activities of these enzymes were verified. In addition, we demonstrated that mhdA and vhyA deletion mutants can be used as fungal cell factories for the accumulation of vanillic acid and methoxyhydroquinone from guaiacyl lignin units and related aromatic compounds. CONCLUSIONS: This study provides new insights into the fungal aromatic metabolic pathways involved in the degradation of guaiacyl units and related aromatic compounds. The identification of the involved genes unlocks new potential for engineering aromatic compound-producing fungal cell factories.

Green production of silybin and isosilybin by merging metabolic engineering approaches and enzymatic catalysis.

Silymarin extracted from milk thistle seeds, is used for treating hepatic diseases. Silybin and isosilybin are its main components, and synthesized from coupling of taxifolin and coniferyl alcohol. Here, the biosynthetic pathways of taxifolin and coniferyl alcohol were reconstructed in Saccharomyces cerevisiae for the first time. To alleviate substantial burden caused by a great deal of genetic manipulation, expression of the enzymes (e.g. ZWF1, TYR1 and ARO8) playing multiple roles in the relevant biosynthetic pathways was selectively optimized. The strain YT1035 overexpressing seven heterologous enzymes and five native enzymes and the strain YC1053 overexpressing seven heterologous enzymes and four native enzymes, respectively produce 336.8 mg/L taxifolin and 201.1 mg/L coniferyl alcohol. Silybin and isosilybin are synthesized from taxifolin and coniferyl alcohol under catalysis of APX1t (the truncated milk thistle peroxidase), with a yield of 62.5%. This study demonstrates an approach for producing silybin and isosilybin from glucose for the first time.

Characterization of a sweet basil acyltransferase involved in eugenol biosynthesis.

Sweet basil (Ocimum basilicum) plants produce its characteristic phenylpropene-rich essential oil in specialized structures known as peltate glandular trichomes (PGTs). Eugenol and chavicol are the major phenylpropenes produced by sweet basil varieties whose synthetic pathways are not fully elucidated. Eugenol is derived from coniferyl acetate by a reaction catalysed by eugenol synthase. An acyltransferase is proposed to convert coniferyl alcohol to coniferyl acetate which is the first committed step towards eugenol synthesis. Here, we perform a comparative next-generation transcriptome sequencing of different tissues of sweet basil, namely PGT, leaf, leaf stripped of PGTs (leaf-PGT), and roots, to identify differentially expressed transcripts specific to PGT. From these data, we identified a PGT-enriched BAHD acyltransferase gene ObCAAT1 and functionally characterized it. In vitro coupled reaction of ObCAAT1 with eugenol synthase in the presence of coniferyl alcohol resulted in eugenol production. Analysis of ObCAAT1-RNAi transgenic lines showed decreased levels of eugenol and accumulation of coniferyl alcohol and its derivatives. Coniferyl alcohol acts as a common substrate for phenylpropene and lignin biosynthesis. No differences were found in total lignin content of PGTs and leaves of transgenic lines, indicating that phenylpropene biosynthesis is not coupled to lignification in sweet basil.

Characterization of human S-adenosyl-homocysteine hydrolase in vitro and identification of its potential inhibitors.

Human S-adenosyl-homocysteine hydrolase (SAHH, E.C.3.3.1.1) has been considered to be an attractive target for the design of medicines to treat human disease, because of its important role in regulating biological methylation reactions to catalyse the reversible hydrolysis of S-adenosylhomocysteine (SAH) to adenosine (Ado) and l-homocysteine (Hcy). In this study, SAHH protein was successfully cloned and purified with optimized, Pichia pastoris (P. pastoris) expression system. The biological activity results revealed that, among the tested compounds screened by ChemMapper and SciFinder Scholar, 4-(3-hydroxyprop-1-en-1-yl)-2-methoxyphenol (coniferyl alcohol, CAS: 458-35-5, ZINC: 12359045) exhibited the highest inhibition against rSAHH (IC50= 34 nM). Molecular docking studies showed that coniferyl alcohol was well docked into the active cavity of SAHH. And several H-bonds formed between them, which stabilized coniferyl alcohol in the active site of rSAHH with a proper conformation.

Histology and cell wall biochemistry of stone cells in the physical defence of conifers against insects.

Conifers possess an array of physical and chemical defences against stem-boring insects. Stone cells provide a physical defence associated with resistance against bark beetles and weevils. In Sitka spruce (Picea sitchensis), abundance of stone cells in the cortex of apical shoots is positively correlated with resistance to white pine weevil (Pissodes strobi). We identified histological, biochemical and molecular differences in the stone cell phenotype of weevil resistant (R) or susceptible (S) Sitka spruce genotypes. R trees displayed significantly higher quantities of cortical stone cells near the apical shoot node, the primary site for weevil feeding. Lignin, cellulose, xylan and mannan were the most abundant components of stone cell secondary walls, respectively. Lignin composition of stone cells isolated from R trees contained a higher percentage of G-lignin compared with S trees. Transcript profiling revealed higher transcript abundance in the R genotype of coumarate 3-hydroxylase, a key monolignol biosynthetic gene. Developing stone cells in current year apical shoots incorporated fluorescent-tagged monolignol into the secondary cell wall, while mature stone cells of previous year apical shoots did not. Stone cell development is an ephemeral process, and fortification of shoot tips in R trees is an effective strategy against insect feeding.

Three-steps in one-pot: whole-cell biocatalytic synthesis of enantiopure (+)- and (-)-pinoresinol via kinetic resolution.

BACKGROUND: Pinoresinol is a high-value plant-derived lignan with multiple health supporting effects. Enantiomerically pure pinoresinol can be isolated from natural sources, but with low efficiency. Most chemical and biocatalytic approaches that have been described for the synthesis of pinoresinol furnish the racemic mixture. In this study we devised a three-step biocatalytic cascade for the production of enantiomerically pure pinoresinol from the cheap compound eugenol. Two consecutive oxidations of eugenol through vanillyl-alcohol oxidase and laccase are followed by kinetic resolution of racemic pinoresinol by enantiospecific pinoresinol reductases. RESULTS: The addition of the enantiospecific pinoresinol reductase from Arabidopsis thaliana for kinetic resolution of (±)-pinoresinol to an in vitro cascade involving the vanillyl-alcohol oxidase from Penicillium simplicissimum and the bacterial laccase CgL1 from Corynebacterium glutamicum resulted in increasing ee values for (+)-pinoresinol; however, an ee value of 34% was achieved in the best case. The ee value could be increased up to ≥ 99% by applying Escherichia coli-based whole-cell biocatalysts. The optimized process operated in a one-pot "two-cell" sequential mode and yielded 876 µM (+)-pinoresinol with an ee value of 98%. Switching the reductase to the enantiospecific pinoresinol lariciresinol reductase from Forsythia intermedia enabled the production of 610 µM (-)-pinoresinol with an ee value of 97%. CONCLUSION: A new approach for the synthesis of enantiomerically pure (+)- and (-)-pinoresinol is described that combines three biotransformation steps in one pot. By switching the reductase in the last step, the whole-cell biocatalysts can be directed to produce either (+)- or (-)-pinoresinol. The products of the reductases' activity, (-)-lariciresinol and (-)-secoisolariciresinol, are valuable precursors that can also be applied for the synthesis of further lignans.

Norway spruce (Picea abies) laccases: characterization of a laccase in a lignin-forming tissue culture.

Secondarily thickened cell walls of water-conducting vessels and tracheids and support-giving sclerenchyma cells contain lignin that makes the cell walls water impermeable and strong. To what extent laccases and peroxidases contribute to lignin biosynthesis in muro is under active evaluation. We performed an in silico study of Norway spruce (Picea abies (L.) Karst.) laccases utilizing available genomic data. As many as 292 laccase encoding sequences (genes, gene fragments, and pseudogenes) were detected in the spruce genome. Out of the 112 genes annotated as laccases, 79 are expressed at some level. We isolated five full-length laccase cDNAs from developing xylem and an extracellular lignin-forming cell culture of spruce. In addition, we purified and biochemically characterized one culture medium laccase from the lignin-forming cell culture. This laccase has an acidic pH optimum (pH 3.8-4.2) for coniferyl alcohol oxidation. It has a high affinity to coniferyl alcohol with an apparent Km value of 3.5 µM; however, the laccase has a lower catalytic efficiency (V(max)/K(m)) for coniferyl alcohol oxidation compared with some purified culture medium peroxidases. The properties are discussed in the context of the information already known about laccases/coniferyl alcohol oxidases of coniferous plants.

Lignans: Advances in Biosynthesis, Bioavailability, and Pharmacological Activity.

Lignans, a group of naturally occurring compounds abundant in various plant-based foods, are becoming increasingly popular due to their potential health benefits. The literature suggests that these bioactive substances can reduce the risk of certain types of cancer, such as postmenopausal colon and breast cancer. Moreover, the significance of lignans for improving cardiovascular health has been recognized, as studies have revealed a potential correlation between the intake of lignans and a decreased risk of cardiovascular disease. These complex molecules possess diverse bioactive capabilities, rendering them potential alternatives for preventing chronic diseases. Further research is needed to examine the mechanisms responsible for their beneficial outcomes. Recent research has emphasized the pharmacological properties of lignans as effective substances for human health. Incorporating foods rich in lignans into the diet may be a practical approach to enhancing protection against life-threatening ailments, such as cardiovascular diseases and malignancies.

Identification, Molecular Cloning, and Functional Characterization of a Coniferyl Alcohol Acyltransferase Involved in the Biosynthesis of Dibenzocyclooctadiene Lignans in Schisandra chinensis.

Schisandra chinensis owes its therapeutic efficacy to the dibenzocyclooctadiene lignans, which are limited to the Schisandraceae family and whose biosynthetic pathway has not been elucidated. Coniferyl alcohol is the synthetic precursor of various types of lignans and can be acetylated to form coniferyl acetate by coniferyl alcohol acyltransferase (CFAT), which belongs to the BAHD acyltransferase family. This catalytic reaction is important because it is the first committed step of the hypothetical biosynthetic pathway in which coniferyl alcohol gives rise to dibenzocyclooctadiene lignans. However, the gene encoding CFAT in S. chinensis has not been identified. In this study, firstly we identified 37 ScBAHD genes from the transcriptome datasets of S. chinensis. According to bioinformatics, phylogenetic, and expression profile analyses, 1 BAHD gene, named ScBAHD1, was cloned from S. chinensis. The heterologous expression in Escherichia coli and in vitro activity assays revealed that the recombinant enzyme of ScBAHD1 exhibits acetyltransferase activity with coniferyl alcohol and some other alcohol substrates by using acetyl-CoA as the acetyl donor, which indicates ScBAHD1 functions as ScCFAT. Subcellular localization analysis showed that ScCFAT is mainly located in the cytoplasm. In addition, we generated a three-dimensional (3D) structure of ScCFAT by homology modeling and explored the conformational interaction between protein and ligands by molecular docking simulations. Overall, this study identified the first enzyme with catalytic activity from the Schisandraceae family and laid foundations for future investigations to complete the biosynthetic pathway of dibenzocyclooctadiene lignans.

Assessment of attractancy and safeness of (E)-coniferyl alcohol for management of female adults of Oriental fruit fly, Bactrocera dorsalis (Hendel).

BACKGROUND: Bactrocera dorsalis is a devastating pest on fruits and vegetables because the adult female is the key factor that determines the population density of offspring and the degree of host damage. Unfortunately, there is still a lack of effective female attractants for behavioral control. Males of B. dorsalis fed on methyl eugenol (ME) were shown to be more sexually attracted to females and, therefore, were more successful in mating over ME-deprived males. RESULTS: In the current study, we demonstrated that (E)-coniferyl alcohol (E-CF), one of the ME metabolites in males, was highly attractive to sexually-mature females in laboratory bioassays. During the dusk courtship period, mature females showed the highest response to E-CF. However, there were no significant differences in olfactory responses to E-CF between virgin and mated mature females. Moreover, no obvious signs and symptoms of toxicity or death were observed in mice during a 14-day acute oral toxicity test. Toxicologically, no significant changes were observed in body weight, water intake, food consumption and absolute and relative organ weights between control and treated groups of healthy-looking mice, implying that E-CF could be regarded as non-toxic. Furthermore, cytotoxicity assessment revealed that E-CF was non-toxic against human fetal lung fibroblast 1 (HFL1), human breast cancer (MDA-MB-231), mouse embryonic hepatocytes (BNL-CL.2) and Spodoptera frugiperda ovary (SF-9) cell lines. CONCLUSIONS: E-CF proved to be an effective, promising and eco-friendly lure to B. dorsalis females. Therefore, this study may facilitate the development of novel control strategies against B. dorsalis in the field.

Phylogeny and functional characterization of the cinnamyl alcohol dehydrogenase gene family in Phryma leptostachya.

Phryma leptostachya has attracted increasing attention because it is rich in furofuran lignans with a wide range of biological activities. Biosynthesis of furofuran lignans begins with the dimerization of coniferyl alcohol, one of the monolignol. Cinnamyl alcohol dehydrogenase (CAD) catalyzes the final step of monolignol biosynthesis, reducing cinnamyl aldehydes to cinnamyl alcohol. As it is in the terminal position of monolignol biosynthesis, its type and activity can cause significant changes in the total amount and composition of lignans. Herein, combined with bioinformatics analysis and in vitro enzyme assays, we clarified that CAD in P. leptostachya belonged to a multigene family, and identified nearly the entire CAD gene family. Our in-depth characterization about the functions and structures of two major CAD isoforms, PlCAD2 and PlCAD3, showed that PlCAD2 exhibited the highest catalytic activity, and coniferyl aldehyde was its preferred substrate, followed by PlCAD3, and sinapyl aldehyde was its preferred substrate. Considering the accumulation patterns of furofuran lignans and expression patterns of PlCADs, we speculated that PlCAD2 was the predominant CAD isoform responsible for furofuran lignans biosynthesis in P. leptostachya. Moreover, these CADs found here can also provide effective biological parts for lignans and lignins biosynthesis.

Dual Mechanisms of Coniferyl Alcohol in Phenylpropanoid Pathway Regulation.

Lignin is a complex phenolic polymer that imparts cell wall strength, facilitates water transport and functions as a physical barrier to pathogens in all vascular plants. Lignin biosynthesis is a carbon-consuming, non-reversible process, which requires tight regulation. Here, we report that a major monomer unit of the lignin polymer can function as a signal molecule to trigger proteolysis of the enzyme L-phenylalanine ammonia-lyase, the entry point into the lignin biosynthetic pathway, and feedback regulate the expression levels of lignin biosynthetic genes. These findings highlight the highly complex regulation of lignin biosynthesis and shed light on the biological importance of monolignols as signaling molecules.

Density functional theory study on the coupling and reactions of diferuloylputrescine as a lignin monomer.

Diferuloylputrescine has been found in a variety of plant species, and recent work has provided evidence of its covalent bonding into lignin. Results from nuclear magnetic resonance spectroscopy revealed the presence of bonding patterns consistent with homo-coupling of diferuloylputrescine and the possibility of cross-coupling with lignin. In the present work, density functional theory calculations have been applied to assess the energetics associated with radical coupling, rearomatization, and dehydrogenation for possible homo-coupled dimers of diferuloylputrescine and cross-coupled dimers of diferuloylputrescine and coniferyl alcohol. The values obtained for these reaction energetics are consistent with those reported for monolignols and other novel lignin monomers. As such, this study shows that there would be no thermodynamic impediment to the incorporation of diferuloylputrescine into the lignin polymer and its addition to the growing list of non-canonical lignin monomers.

Metabolic Changes of Cholangiocarcinoma Cells in Response to Coniferyl Alcohol Treatment.

Cholangiocarcinoma (CCA) is a major cause of mortality in Northeast Thailand with about 14,000 deaths each year. There is an urgent necessity for novel drug discovery to increase effective treatment possibilities. A recent study reported that lignin derived from Scoparia dulcis can cause CCA cell inhibition. However, there is no evidence on the inhibitory effect of coniferyl alcohol (CA), which is recognized as a major monolignol-monomer forming a very complex structure of lignin. Therefore, we aimed to investigate the effect of CA on CCA cell apoptosis. We demonstrated that a half-inhibitory concentration of CA on KKU-100 cells at 48 h and 72 h was 361.87 ± 30.58 and 268.27 ± 18.61 µg/mL, respectively, and on KKU-213 cells 184.37 ± 11.15 and 151.03 ± 24.99 µg/mL, respectively. Furthermore, CA induced CCA cell apoptosis as demonstrated by annexin V/PI staining in correspondence with an increase in the BAX/Bcl-2 ratio. A metabonomic study indicated that CA significantly decreased the intracellular concentrations of glutathione and succinate in KKU-213 cells and increased dihydrogen acetone phosphate levels in KKU-100 cells treated with 200 µg/mL of CA compared to the control group. In conclusion, CA induced cellular metabolic changes which are involved in the antioxidant defense mechanism, glycerophospholipid metabolism and the tricarboxylic acid cycle. CA may serve as a potent anticancer agent for CCA treatment by inducing CCA cellular apoptosis.

Antifungal Activity against Botryosphaeriaceae Fungi of the Hydro-Methanolic Extract of Silybum marianum Capitula Conjugated with Stevioside.

Silybum marianum (L.) Gaertn, viz. milk thistle, has been the focus of research efforts in the past few years, albeit almost exclusively restricted to the medicinal properties of its fruits (achenes). Given that other milk thistle plant organs and tissues have been scarcely investigated for the presence of bioactive compounds, in this study, we present a phytochemical analysis of the extracts of S. marianum capitula during the flowering phenological stage (stage 67). Gas chromatography-mass spectroscopy results evidenced the presence of high contents of coniferyl alcohol (47.4%), and secondarily of ferulic acid ester, opening a new valorization strategy of this plant based on the former high-added-value component. Moreover, the application of the hydro-methanolic extracts as an antifungal agent has been also explored. Specifically, their activity against three fungal species responsible for the so-called Botryosphaeria dieback of grapevine (Neofusicoccum parvum, Dothiorella viticola and Diplodia seriata) has been assayed both in vitro and in vivo. From the mycelial growth inhibition assays, the best results (EC90 values of 303, 366, and 355 µg·mL-1 for N. parvum, D. viticola, and D. seriata, respectively) were not obtained for the hydroalcoholic extract alone, but after its conjugation with stevioside, which resulted in a strong synergistic behavior. Greenhouse experiments confirmed the efficacy of the conjugated complexes, pointing to the potential of the combination of milk thistle extracts with stevioside as a promising plant protection product in organic Viticulture.

Pulsed-ozonolysis assisted oxidative treatment of forestry biomass for lignin fractionation.

Lignocellulosic biomass has been used to produce biomolecules of industrial interest through thermochemical, biological, and chemical transformation. However, few works have been developed over lignin fractionation to obtain monolignols with commercial potentials, such as sinapyl, coniferyl, and p-coumaryl alcohols. This study is focused on developing a thermochemical method to delignify biomass. Additionally, an oxidative treatment with ozone was studied to increase the release of monolignol compounds. The results showed that with 30 sec of ozonation in liquid samples from softwood sawdust a total concentration of 368.50 ± 0.73 mg/kg of monolignols was released after microwave-assisted extraction (256.5 ± 0.51 mg/kg of sinapyl alcohol and 112 ± 0.22 mg/kg of coniferyl alcohol) and 629.20 ± 0.21 mg/kg was released after thermal treatment (453.70 ± 0.15 mg/kg of sinapyl alcohol and 175.5 ± 0.06 mg/kg of coniferyl alcohol). For p-coumaryl alcohol, 16.32 mg/kg was obtained only in hardwood samples. The results of the present study showed that ozonolysis improves monolignols release from forestry residues.

Kinetic study on the heterogeneous degradation of coniferyl alcohol by OH radicals.

Coniferyl alcohol derived from lignin pyrolysis, is a potential tracer for wood burning emissions, but its atmospheric stability toward OH radicals is not well known. In this work, the degradation kinetics of coniferyl alcohol by OH radicals was studied using a flow reactor at different OH concentrations, temperatures, and relative humidity (RH). The results showed that coniferyl alcohol could be degraded effectively by OH radials, and the average second-order rate constant (k2) was (11.6 ±â€¯0.5) × 10-12 cm3 molecule-1 s-1 at the temperature and RH of 25 °C and 40%, respectively. Additionally, increasing temperature facilitated the degradation of coniferyl alcohol and the Arrhenius equation could be expressed as k2 = (1.7 ±â€¯0.3) × 10-9exp [-(1480.2 ±â€¯55.6)/T] at 40% RH. Meanwhile, increasing RH had a negative impact on the degradation of coniferyl alcohol. According to the k2 obtained under different conditions, the atmospheric lifetime of coniferyl alcohol was in the range of 13.5 ±â€¯0.4 h to 22.9 ±â€¯1.4 h. The results suggested that the atmosphere lifetime of coniferyl alcohol was predominantly controlled by OH radicals.

Fraxinus mandshurica 4-coumarate-CoA ligase 2 enhances drought and osmotic stress tolerance of tobacco by increasing coniferyl alcohol content.

4-Coumarate-CoA ligase (4CL) is an important branch point in the phenylpropane pathway and plays important roles in plant growth and development. In this study, the 4CL2 gene from Fraxinus mandshurica (designated Fm4CL2) was identified and isolated. Sequence analysis revealed that Fm4CL2 is a type I 4CL gene involved in lignin biosynthesis. Analysis of cell wall components revealed that Fm4CL2-overexpressing (OE-Fm4CL2) tobacco showed increased lignin content (by 58.9%) and decreased hemicellulose content (by 41.2%). Detection of small-molecule metabolites in the lignin pathway revealed that coumaric acid content decreased by 48% and coniferyl alcohol content increased by 250% compared with the control values. Compared with wild type, OE-Fm4CL2 tobacco showed increased xylem cell layer number (by 120%) and cell wall thickness (by 54.5%). Under osmotic stress, transgenic tobacco showed higher growth than wild-type tobacco. The germination rate of transgenic tobacco was higher than that of wild type. Reactive oxygen species accumulation and malondialdehyde content were significantly lower in transgenic tobacco than in wild type. Under drought, the expression of stress-related genes was higher in 35S-Fm4CL2-infected Fraxinus mandshurica plants than in control plants. These results indicate that Fm4CL2 overexpression can enhance drought and osmotic stress tolerance of plants.