Biocatalytic regio- and stereoselective access to ω-3 endocannabinoid epoxides with peroxygenase from oat flour.

The biocatalytic epoxidation of ethanolamides of ω-3 fatty acids EPA and DHA, regarded as biologically active ω-3 endocannabinoids, in the presence of a peroxygenase-containing preparation from oat flour was investigated. Good regio- and steroselectivity toward the formation of the epoxide on the terminal double bond in the chain was observed with both these fatty acid derivatives and chiral monoepoxides 1 or 2 in 74% optical purity and 51-53% yields were isolated and spectroscopically characterized. The use of acetone as cosolvent in the reaction medium allowed to increase the concentration of starting substrates up to 40 mM and to further improve the selectivity in the epoxidation of DHA-EA. Due to the easy availability of the enzymatic preparation, the method offers a valuable strategy for the access to oxyfunctionalized derivatives of fatty acids.

Sprouted oat as a potential gluten-free ingredient with enhanced nutritional and bioactive properties.

This study is aimed to produce and characterize a novel gluten-free ingredient from oat through sprouting at 18 °C for 96 h. The nutritional and bioactive properties as well as key enzymatic activities were studied in sprouted oat powder and compared with those of oat grain powder (control). Sprouted oat powder was an excellent source of protein (10.7%), ß-glucan (2.1%), thiamine (687.1 µg/100 g), riboflavin (218.4 µg/100 g), and minerals (P, K, Mg and Ca), and presented better amino acid and fatty acid compositions and levels of γ-aminobutyric acid (54.9 mg/100 g), free phenolics (507.4 mg GA/100 g) and antioxidant capacity (1744.3 mg TE/100 g) than control. Enhanced protease and α-amylase and reduced lipase activities were observed in sprouted oat powder, which are promising features to improve its nutritional, sensorial and health-promoting properties. These results support the use of sprouted oat powder as a promising gluten-free functional ingredient.

Extreme drought alters progeny dispersal unit properties of winter wild oat (Avena sterilis L.).

MAIN CONCLUSION: The dead husk is a vital component of the dispersal unit whose biochemical properties can be modified following exposure to drought. This might affect seed performance and fate, soil properties and consequently plant biodiversity. We investigated the effects of extreme drought on the dispersal unit (DU) properties of winter wild oat (Avena sterilis L.) in the Mediterranean ecosystems focusing on a commonly ignored component of the DU, namely the dead floral bracts (husk). DUs were collected from a climate change experimental research station in the Judean Hills, Israel, simulating extreme drought and from two additional sites differing in the rainfall amounts. Our results showed that drought conditions significantly affected A. sterilis reproductive traits displaying reduced DUs and caryopses weights. The husk contributes profoundly to seed performance showing that germination from the intact DUs or the intact florets 1 was higher, faster and more homogenous compared to naked caryopses; no effect of drought on germination properties was observed. The husk stored hundreds of proteins that retain enzymatic activity and multiple metabolites including phytohormones. Changes in rainfall amounts affected the composition and levels of proteins and other metabolites accumulated in the husk, with a notable effect on abscisic acid (ABA). The husk of both control and drought plants released upon hydration substances that selectively inhibited other species seed germination as well as substances that promoted microbial growth. Our data showed that the dead husk represents a functional component of the DU that have been evolved to nurture the embryo and to ensure its success in its unique habitat. Furthermore, drought conditions can modify husk biochemical properties, which in turn might affect seed performance and fate, soil microbiota and soil fertility and consequently plant species diversity.

Dynamic changes in the phenolic composition and antioxidant activity of oats during simultaneous hydrolysis and fermentation.

Solid-state fermentation (SSF) is the preferred method of enhancing the phenolic content of oats, while scientific optimization for improving specific phenolic compounds is limited. In this study, sequential targeting of phenolic conversion in simultaneous hydrolysis and fermentation (SHF) of oats was investigated. The results revealed that SHF with adding cellulase at 0, 6 and 12 days could increase the total phenolic content by 4.4%, 67.8% and 59.1%, respectively, over that of SSF. The α-amylase and CMCase activity were highly correlated with the soluble and insoluble phenolic contents in SHF (-6 and -12) systems (r > 0.8, p < 0.05). Interestingly, the content of phenolic fraction, such as ferulic acid, was up-regulated, whereas sinapic acid was down-regulated. These results indicated that the phenolic conversion occurred in SHF, resulting in variation in DPPH and ABTS+ radical scavenging abilities. This research provided metabolic understanding of the optimization of phenolic compounds to increase the functional ingredient of oats.

A chitinase with antifungal activity from naked oat (Avena chinensis) seeds.

A chitinase was purified from naked oat (Avena chinensis) seeds using simple chromatographic techniques. Its molecular weight and isoelectric point were determined as 35 kDa and 8.9, respectively. The purified chitinase exhibited specific activity of 3.6 U/mg and 15.6% yield using colloidal chitin as substrate. Partial amino acid sequence analysis and homology search indicated that it probably belonged to Class I plant chitinase, glycosyl hydrolase family 19. With chitin as substrate, the optimum pH and temperature of the chitinase were pH 7.0 and 40°C, respectively. The chitinase was remarkably stable from 30°C up to 50°C, but was inactivated at high temperatures above 85°C. Antifungal activity in vitro tests demonstrated this purified chitinase had potent, dose-dependent inhibitory activity against the fungi Panus conchatus and Trichoderma reesei. PRACTICAL APPLICATIONS: Chitinase has broad applications in many fields including the food industry and is recognized as one of the antifungal substances with potential use in plant disease resistance or biological control in agriculture. This study developed cost-effective purification methods for producing chitinase from naked oat (Avena chinensis) seeds, which may favor large-scale production of the enzyme. The remarkable stability of the chitinase at moderate temperatures (30°C-50°C), makes it a potentially useful enzyme in bioprocessing to produce chitooligosaccharides for various applications in the food, health, and agriculture sectors.

New insights into phospholipases in oat (Avena sativa) from bioinformatic analysis.

Phospholipases from plants in particular from oat (Avena sativa) could not be purified to homogeneity due to their association with other proteins. Interestingly, bioinformatics is a useful tool for the identification of such new sequences of enzymes. The Avena sativa phospholipases could be identified by functional proteomics and bioinformatics analysis with the aid of database searches. Based upon Transcriptome Shotgun Assembly (TSA) sequences, predicted genes were identified for Avena sativa PLD, PLA, and PLC, and assigned as AsPLD1, AsPLA2_1, and AsPiPLC1, respectively. Insights into the structural characterization of the oat predicted enzymes were analyzed using in silico approaches. Our results on sequence analysis of the oat phospholipases provide a detail view of the main residues' characteristics of such biocatalysts.

Prediction and analysis of GH14 family ß-amylases in oat seedling extract: Structure and function insights using in silico approaches.

Oat (Avena sativa L.) seedling extract exhibited a high degree of catalytic activities. Bioinformatics were used to identify ß-amylases as abundant enzymes in the oat seedling extract. These identified oat enzymes are a member of the GH14 family. Proteins in the Avena sativa seedling extract were separated by SDS-PAGE and 2 major protein bands with an apparent molecular weights of 53 and 42 kDa were the subject of this study. These materials were digested with trypsin and the amino acid sequences of the tryptic peptides were determined by LC/ESI/MS/MS and database searches. These sequences were used to identify cDNAs from expressed sequence tags (EST) and Transcriptome Shotgun Assembly (TSA) of Avena sativa. Based upon EST and TSA sequences, at least 6 predicted different sequences were identified and assigned as ß-amylases. Insights into structural characterization of the oat predicted ß-amylases were analyzed using in silico approaches. The identified ß-amylases conserved the two Glu residues assigned as the "putative" catalytic residues, which would act as an acid and base pair in the catalytic process. A similar core (ß/α)8-barrel architecture was found in the predicted oat ß-amylases with a specific location of the active site in a pocket-like cavity structure made at one end of this core (ß/α)8-barrel domain. This suggests an accessibility of the non-reducing end of the substrate towards the oat ß-amylases and thus confirming that are exo-acting hydrolases. The results provide a detailed view of the main residues involved in catalysis in this kind of enzyme.

Towards take-all control: a C-21ß oxidase required for acylation of triterpene defence compounds in oat.

Oats produce avenacins, antifungal triterpenes that are synthesized in the roots and provide protection against take-all and other soilborne diseases. Avenacins are acylated at the carbon-21 position of the triterpene scaffold, a modification critical for antifungal activity. We have previously characterized several steps in the avenacin pathway, including those required for acylation. However, transfer of the acyl group to the scaffold requires the C-21ß position to be oxidized first, by an as yet uncharacterized enzyme. We mined oat transcriptome data to identify candidate cytochrome P450 enzymes that may catalyse C-21ß oxidation. Candidates were screened for activity by transient expression in Nicotiana benthamiana. We identified a cytochrome P450 enzyme AsCYP72A475 as a triterpene C-21ß hydroxylase, and showed that expression of this enzyme together with early pathway steps yields C-21ß oxidized avenacin intermediates. We further demonstrate that AsCYP72A475 is synonymous with Sad6, a previously uncharacterized locus required for avenacin biosynthesis. sad6 mutants are compromised in avenacin acylation and have enhanced disease susceptibility. The discovery of AsCYP72A475 represents an important advance in the understanding of triterpene biosynthesis and paves the way for engineering the avenacin pathway into wheat and other cereals for control of take-all and other diseases.

PK-profiling method for identifying the expression of resistance-associated genes in partially resistant oats to crown rust.

BACKGROUND: Protein kinases play a key role in plant cell homeostasis and the activation of defense mechanisms. Partial resistance to fungi in plants is interesting because of its durability. However, the variable number of minor loci associated with this type of resistance hampers the reliable identification of the full range of genes involved. The present work reports the technique of protein kinase (PK)-profiling for the identification of the PK genes induced in the partially resistant oats line MN841801-1 following exposure to the fungus Puccinia coronata. This is the first time this technique has been used with cDNA (complementary DNA) from a suppression subtractive hybridization library obtained after the hybridization of cDNAs from inoculated and mock-inoculated plants. RESULTS: Six degenerate primers based on the conserved domains of protein kinases were used in a PK-profiling assay including cDNA from mock-inoculated leaves and subtracted cDNA. Of the 75.7% of sequences cloned and sequenced that showed significant similarity to resistance genes, 76% were found to code for PKs. Translation and ClustalW2 alignment of each sequence cloned with the complete sequences of the most similar B. distachyon PKs allowed those of the partially resistant oat line to be deduced and characterized. Further, a phylogenetic study carried out after alignment of these B. distachyon PK sequences with the most similar protein sequences of related species also allowed to deduce different functions for the PK cloned. RT-qPCR (Reverse Transcription-quantitative PCR) was analyzed on nine representative sequences to validate the reliability of the employed PK-profiling method as a tool for identifying the expression of resistance-associated genes. CONCLUSIONS: PK-profiling would appear to be a useful tool for the identification of the PKs expressed in oats after challenge by P. coronata, and perhaps other pathogens. Most of the PKs studied are related to receptor-like protein kinases expressed shortly after infection. This is in agreement with previous studies indicating a close relationship between partial resistance and the first layer of defense against pathogen used by plants.

Enzymatic action mechanism of phenolic mobilization in oats (Avena sativa L.) during solid-state fermentation with Monascus anka.

This work aims to investigate the effects of carbohydrate-hydrolysing enzymes on the release of phenolics in oat fermentation with Monascus anka. There were good correlations between phenolic content and α-amylase, xylanase and FPase activities. A high level of α-amylase activity (141.07 U/g) was observed, while xylanase (2.40 U/g), total cellulase (0.52 U/g) and ß-glucosidase activities (0.028 U/g) were relatively low in the fermentation system. The phenolic content of oat powder treated with crude enzyme from fermented oats significantly increased, especially that of the ferulic acid in the insoluble fraction and the vanillic acid in the soluble fraction. The surface SEM morphology of the oats showed that the cell wall structure was damaged by the crude enzyme treatment, which led to the release of phenolics. This study could provide metabolic understanding for optimization of phenolic compounds which could more efficiently increase the nutrition of oat intended for functional food ingredients.

Lipid-modifying enzymes in oat and faba bean.

The aim was to study lipase, lipoxygenase (LOX) and peroxygenase (POX) activities in oat and faba bean samples to be able to evaluate their potential in formation of lipid-derived off-flavours. Lipase and LOX activities were measured by spectroscopy, and POX activities via the formation of epoxides. An ultra-high performance liquid chromatography method was developed to study the formation of fatty acid epoxides. The epoxides of esters were measured by gas chromatography. Mass spectroscopy was used to verify the identity of the epoxides. Both oat and faba bean possessed high lipase activities. In faba bean, LOX catalysed the formation of hydroperoxides, whose break-down products are the likely cause of off-flavours. Since oat had low LOX activity, autoxidation is needed to initiate lipid oxidation. Oat had high POX activity, which is able to convert hydroperoxides to epoxy and hydroxy fatty acids that could contribute significantly to off-flavours. POX activity in the faba bean was low. Thus, in faba bean volatile lipid oxidation products could rapidly be formed by LOX, whereas in oat reactions are slower due to the need of autoxidation prior to further reactions.

A translational synthetic biology platform for rapid access to gram-scale quantities of novel drug-like molecules.

Plants are an excellent source of drug leads. However availability is limited by access to source species, low abundance and recalcitrance to chemical synthesis. Although plant genomics is yielding a wealth of genes for natural product biosynthesis, the translation of this genetic information into small molecules for evaluation as drug leads represents a major bottleneck. For example, the yeast platform for artemisinic acid production is estimated to have taken >150 person years to develop. Here we demonstrate the power of plant transient transfection technology for rapid, scalable biosynthesis and isolation of triterpenes, one of the largest and most structurally diverse families of plant natural products. Using pathway engineering and improved agro-infiltration methodology we are able to generate gram-scale quantities of purified triterpene in just a few weeks. In contrast to heterologous expression in microbes, this system does not depend on re-engineering of the host. We next exploit agro-infection for quick and easy combinatorial biosynthesis without the need for generation of multi-gene constructs, so affording an easy entrée to suites of molecules, some new-to-nature, that are recalcitrant to chemical synthesis. We use this platform to purify a suite of bespoke triterpene analogs and demonstrate differences in anti-proliferative and anti-inflammatory activity in bioassays, providing proof of concept of this system for accessing and evaluating medicinally important bioactives. Together with new genome mining algorithms for plant pathway discovery and advances in plant synthetic biology, this advance provides new routes to synthesize and access previously inaccessible natural products and analogs and has the potential to reinvigorate drug discovery pipelines.

Identification and functional analysis of new peroxygenases in oat.

MAIN CONCLUSION: Two new peroxygenases for the biosynthesis of epoxy fatty acids in oat were identified and functionally analyzed by heterologous expression along with rationally designed site-directed mutagenesis. Oat (Avena sativa L.) contains a large family of peroxygenases, a group of heme-containing monooxygenases catalyzing hydroperoxide-dependent epoxidation of unsaturated fatty acids. Here, we report identification and functional analysis of two new peroxygenases AsPXG2 and AsPXG3 from oat. The open reading frame (ORF) of AsPXG2 contains 702 bps encoding a polypeptide of 233 amino acids, while the ORF of AsPXG3 is 627 bps coding for 208 amino acids. Both AsPXG2 and AsPXG3 comprise a single transmembrane domain, conserved histidines for heme binding and a conserved EF-hand motif for calcium binding, but they only share about 50% amino acid sequence identity with each other. When expressed in Escherichia coli and Pichia pastoris, AsPXG3 showed high epoxidation activity, while AsPXG2 exhibited no activity in E. coli and low activity in P. pastoris. AsPXG3 could effectively epoxidize both mono- and polyunsaturated fatty acids with linolenic acid being the most preferred substrate. Site-directed mutagenesis was employed to investigate the structure-function relationship of oat peroxygenase on 12 conserved residues of AsPXG3. Replacement of two conserved histidines, the ligands to the prosthetic heme group of the peroxygenase, by alanine resulted in complete loss of activity. Substitution of three conserved residues surrounding the two histidines resulted in reduction of the enzymatic activity by more than 80%. These results imply that these conserved residues might be located in or near the catalytic pocket, where the two histidine residues coordinate the heme group and the surrounding residues define the shape and size of the pocket for interaction with the heme as well as two substrates.

Efecto del consumo de harina de avena (avena sativa) y frijoles negros (phaseolus vulgaris) sobre la actividad de las disacaridasas intestinales en ratas / Effect of the consumption of flour of oats (avena sativa) and black beans (phaseolus vulgaris) on the activity of intestinal disaccharidases in rats

Objetivo: En este trabajo se estudió el efecto de la ingesta crónica de harina de caraotas negras y harina de avena sobre la actividad de las disacaridasas intestinales en ratas. Metodología: 15 ratas Sprague Dawley, con un peso inicial promedio de 85g se dividieron en tres grupos, un grupo control sin fibra, un grupo alimentado con harina de caraotas negras y un grupo con harina de avena por 21 días. Los animales fueron sacrificados, el intestino delgado se dividió en tres porciones (proximal, media, y distal), y se obtuvo un homogenato por raspado de la mucosa. Resultados: La actividad de las disacaridasas se estimó mediante la determinación de glucosa por un método enzimático con peroxidasa. El orden de actividad enzimática fue Maltasa > Sacarasa > Lactasa, obteniéndose una mayor actividad en la región intestinal media para cada disacaridasa. El consumo de harina de caraotas produjo un incremento significativo en la actividad de la maltasa y sacarasa, en esta última, de más de un 100% en relación con el control. La suplementación de las dietas con avena produjo una disminución de la actividad de la sacarasa de un 40% y no produjo ningún efecto sobre la actividad de la maltasa y lactasa. Conclusiones: Estos resultados señalan que el efecto que produce la ingesta crónica de fibra dietética sobre la actividad de las disacaridasas intestinales no se puede generalizar, cada enzima reacciona de manera particular frente a cada tipo de fibra, y puede variar además, según el segmento del intestino delgado estudiado (AU)

Objective: In this work was studied the effect of chronic intake of black beans flour and oat flour on the activity of the intestinal disaccharidases in rats. Methodology: 15 Sprague Dawley rats, with an initial average weight of 85g were distributed, into three groups, a fiber free control group, a group fed with black beans flour and a group with oat flour for 21 days. Then, the rats were sacrificed and the small intestine was divided into three sections (proximal, medial, and distal). The homogenate was obtained by scraping the mucosa of each section. Results: The activity of the disaccharidases was estimated by the glucose determination using peroxidase enzymatic method. The order of enzyme activity was maltase> Sucrase > lactase, obtaining a greater activity in the middle intestinal region for each disaccharidase. The consumption of beans flour produced a significant increase in the activity of maltase and sucrase, the latter, with more than 100% in relation to control. Diets with oats supplementation resulted in a decrease of 40% sucrase activity and did not produce any effect on the activity of maltase and lactase. Conclusions: These results indicate that the effect of chronic dietary fiber intake on the activity of intestinal disaccharidases cannot be generalized, each enzyme reacts in a particular way to each type of fiber and may also vary according to the segment of the small intestine (AU)

Engineering and Application of LOV2-Based Photoswitches.

Cellular optogenetic switches, a novel class of biological tools, have improved our understanding of biological phenomena that were previously intractable. While the design and engineering of these proteins has historically varied, they are all based on borrowed elements from plant and bacterial photoreceptors. In general terms, each of the optogenetic switches designed to date exploits the endogenous light-induced change in photoreceptor conformation while repurposing its effect to target a different biological phenomenon. We focus on the well-characterized light-oxygen-voltage 2 (LOV2) domain from Avena sativa phototropin 1 as our cornerstone for design. While the function of the LOV2 domain in the context of the phototropin protein is not fully elucidated, its thorough biophysical characterization as an isolated domain has created a strong foundation for engineering of photoswitches. In this chapter, we examine the biophysical characteristics of the LOV2 domain that may be exploited to produce an optogenetic switch and summarize previous design efforts to provide guidelines for an effective design. Furthermore, we provide protocols for assays including fluorescence polarization, phage display, and microscopy that are optimized for validating, improving, and using newly designed photoswitches.

A conserved amino acid residue critical for product and substrate specificity in plant triterpene synthases.

Triterpenes are structurally complex plant natural products with numerous medicinal applications. They are synthesized through an origami-like process that involves cyclization of the linear 30 carbon precursor 2,3-oxidosqualene into different triterpene scaffolds. Here, through a forward genetic screen in planta, we identify a conserved amino acid residue that determines product specificity in triterpene synthases from diverse plant species. Mutation of this residue results in a major change in triterpene cyclization, with production of tetracyclic rather than pentacyclic products. The mutated enzymes also use the more highly oxygenated substrate dioxidosqualene in preference to 2,3-oxidosqualene when expressed in yeast. Our discoveries provide new insights into triterpene cyclization, revealing hidden functional diversity within triterpene synthases. They further open up opportunities to engineer novel oxygenated triterpene scaffolds by manipulating the precursor supply.

Identification of a new oat ß-amylase by functional proteomics.

Oat (Avena sativa L.) seed extracts exhibited a high degree of catalytic activity including amylase activities. Proteins in the oat seed extracts were optimized for their amylolytic activities. Oat extract with amylolytic activity was separated by SDS-PAGE and a major protein band with an apparent molecular mass of 53 kDa was subjected to tryptic digestion. The generated amino acid sequences were analyzed by liquid chromatography­tandem mass spectrometry (LC/ESI/MS/MS) and database searches. These sequences were used to identify a partial cDNA from expressed sequence tags (ESTs) of A. sativa L. Based upon EST sequences, a predicted full-length gene was identified, with an open reading frame of 1464 bp encoding a protein of 488 amino acid residues (AsBAMY), with a theoretical molecular mass of 55 kDa identified as a ß-amylase belonging to the plant ß-amylase family. Primary structure of oat ß-amylase (AsBAMY) protein indicated high similarity with other ß-amylase from other cereals such as wheat (Triticum aestivum), barley (Hordeum vulgare), and rye (Secale cereale) with two conserved Glu residues (E184 and E378) assigned as the "putative" catalytic residues which would act as an acid and base pair in the catalytic process. In addition, a 3D-model of AsBAMY was built from known X-ray structures and sequence alignments. A similar core (ß/α)8-barrel architecture was found in AsBAMY like the other cereal ß-amylases with a specific location of the active site in a pocket-like cavity structure made at one end of this core (ß/α)8-barrel domain suggesting an accessibility of the non-reducing end of the substrate and thus confirming the results of AsBAMY exo-acting hydrolase.

Proteomic analysis of salt-responsive proteins in oat roots (Avena sativa L.).

BACKGROUND: Oat is considered as a moderately salt-tolerant crop that could be used to improve saline and alkaline soil. Previous studies have focused on short-term salt stress exposure (0.5-48 h), while molecular mechanisms of salt tolerance in oat remain unclear. RESULTS: Long-term salt stress (16 days) increased the levels of superoxide dismutase activity, peroxidase activity, malondialdehyde content, putrescine content, spermidine content and soluble sugar content and reduced catalase activity in oat roots. The stress also caused changes in protein profiles in the roots. At least 1400 reproducible protein spots were identified in a two-dimensional electrophoresis gel, among which 23 were differentially expressed between treated vs control plants and 13 were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. CONCLUSION: These differentially expressed proteins are involved in five types of biological process: (1) two fructose-bisphosphate aldolases, four alcohol dehydrogenases, an enolase, a UDP-glucuronic acid decarboxylase and an F1-ATPase alpha subunit related to carbohydrate and energy metabolism; (2) a choline monooxygenase related to stress and defense; (3) a lipase related to fat metabolism; (4) a polyubiquitin related to protein degradation; (5) a 14-3-3 protein related to signaling. © 2015 Society of Chemical Industry.

Evaluation of post-germinative lipid peroxidation and enzymatic antioxidant potential in lead absorbing oat (Avena sativa) seedlings.

The objective of the present research was to study the impact of lead (Pb) on growth, metal uptake and antioxidative potential of oat seeds under metal stress. To achieve these objectives, few experiments were conducted to assess the effect of this particular metal on various anti-oxidative enzymes, during initial metabolism after germination, in presence of lead. Pb is not an oxido-reducing metal, the oxidative stress induced by Pb in growing oat seedlings appears to be an indirect effect of Pb toxicity, leading to production of ROS with simultaneous decrease in tissue levels of superoxide dismutase and catalase. Content of free radical like superoxide anion and metabolite such as H2O2 were found to be more in plumule as compared to radical and endosperm of oat seedling. In response to various concentrations of lead ranging from 25-400 ppm, activities of peroxidase, glutathione peroxidase and ascorbate peroxidase were induced in plumule, radical and cotyledon on the 3rd, 6th and 9th days after germination. Growth parameters like length, fresh weight and dry weight were substantially affected in addition to reduced germination upto 49% only. The results indicated that even at the lowest concentration tested, a low inhibition of growth was obtained.

Eugenol-inhibited root growth in Avena fatua involves ROS-mediated oxidative damage.

Plant essential oils and their constituent monoterpenes are widely known plant growth retardants but their mechanism of action is not well understood. We explored the mechanism of phytotoxicity of eugenol, a monoterpenoid alcohol, proposed as a natural herbicide. Eugenol (100-1000 µM) retarded the germination of Avena fatua and strongly inhibited its root growth compared to the coleoptile growth. We further investigated the underlying physiological and biochemical alterations leading to the root growth inhibition. Eugenol induced the generation of reactive oxygen species (ROS) leading to oxidative stress and membrane damage in the root tissue. ROS generation measured in terms of hydrogen peroxide, superoxide anion and hydroxyl radical content increased significantly in the range of 24 to 144, 21 to 91, 46 to 173% over the control at 100 to 1000 µM eugenol, respectively. The disruption in membrane integrity was indicated by 25 to 125% increase in malondialdehyde (lipid peroxidation byproduct), and decreased conjugated diene content (~10 to 41%). The electrolyte leakage suggesting membrane damage increased both under light as well as dark conditions measured over a period from 0 to 30 h. In defense to the oxidative damage due to eugenol, a significant upregulation in the ROS-scavenging antioxidant enzyme machinery was observed. The activities of superoxide dismutases, catalases, ascorbate peroxidases, guaiacol peroxidases and glutathione reductases were elevated by ~1.5 to 2.8, 2 to 4.3, 1.9 to 5.0, 1.4 to 3.9, 2.5 to 5.5 times, respectively, in response to 100 to 1000 µM eugenol. The study concludes that eugenol inhibits early root growth through ROS-mediated oxidative damage, despite an activation of the antioxidant enzyme machinery.