Flexibility modulates the catalytic activity of a thermostable enzyme: key information from optical spectroscopy and molecular dynamics simulation.

Enzymes are dynamical macromolecules and their conformation can be altered via local fluctuations of side chains, large scale loop and even domain motions which are intimately linked to their function. Herein, we have addressed the role of dynamic flexibility in the catalytic activity of a thermostable enzyme almond beta-glucosidase (BGL). Optical spectroscopy and classical molecular dynamics (MD) simulation were employed to study the thermal stability, catalytic activity and dynamical flexibility of the enzyme. An enzyme assay reveals high thermal stability and optimum catalytic activity at 333 K. Polarization-gated fluorescence anisotropy measurements employing 8-anilino-1-napthelenesulfonic acid (ANS) have indicated increasing flexibility of the enzyme with an increase in temperature. A study of the atomic 3D structure of the enzyme shows the presence of four loop regions (LRs) strategically placed over the catalytic barrel as a lid. MD simulations have indicated that the flexibility of BGL increases concurrently with temperature through different fluctuating characteristics of the enzyme's LRs. Principal Component Analysis (PCA) and the Steered Molecular Dynamics (SMD) simulation manifest the gatekeeper role of the four LRs through their dynamic fluctuations surrounding the active site which controls the catalytic activity of BGL.

Reconstruction of the Evolutionary Histories of UGT Gene Superfamily in Legumes Clarifies the Functional Divergence of Duplicates in Specialized Metabolism.

Plant uridine 5'-diphosphate glycosyltransferases (UGTs) influence the physiochemical properties of several classes of specialized metabolites including triterpenoids via glycosylation. To uncover the evolutionary past of UGTs of soyasaponins (a group of beneficial triterpene glycosides widespread among Leguminosae), the UGT gene superfamily in Medicago truncatula, Glycine max, Phaseolus vulgaris, Lotus japonicus, and Trifolium pratense genomes were systematically mined. A total of 834 nonredundant UGTs were identified and categorized into 98 putative orthologous loci (POLs) using tree-based and graph-based methods. Major key findings in this study were of, (i) 17 POLs represent potential catalysts for triterpene glycosylation in legumes, (ii) UGTs responsible for the addition of second (UGT73P2: galactosyltransferase and UGT73P10: arabinosyltransferase) and third (UGT91H4: rhamnosyltransferase and UGT91H9: glucosyltransferase) sugars of the C-3 sugar chain of soyasaponins were resulted from duplication events occurred before and after the hologalegina-millettoid split, respectively, and followed neofunctionalization in species-/ lineage-specific manner, and (iii) UGTs responsible for the C-22-O glycosylation of group A (arabinosyltransferase) and DDMP saponins (DDMPtransferase) and the second sugar of C-22 sugar chain of group A saponins (UGT73F2: glucosyltransferase) may all share a common ancestor. Our findings showed a way to trace the evolutionary history of UGTs involved in specialized metabolism.

Structures and kinetics for plant nucleoside triphosphate diphosphohydrolases support a domain motion catalytic mechanism.

Extracellular nucleoside triphosphate diphosphohydrolases (NTPDases) are enzymes that hydrolyze extracellular nucleotides to the respective monophosphate nucleotides. In the past 20 years, NTPDases belonging to mammalian, parasitic and prokaryotic domains of life have been discovered, cloned and characterized. We reveal the first structures of NTPDases from the legume plant species Trifolium repens (7WC) and Vigna unguiculata subsp. cylindrica (DbLNP). Four crystal structures of 7WC and DbLNP were determined at resolutions between 1.9 and 2.6 Å. For 7WC, structures were determined for an -apo form (1.89 Å) and with the product AMP (2.15 Å) and adenine and phosphate (1.76 Å) bound. For DbLNP, a structure was solved with phosphate and manganese bound (2.60 Å). Thorough kinetic data and analysis is presented. The structure of 7WC and DbLNP reveals that these NTPDases can adopt two conformations depending on the molecule and co-factor bound in the active site. A central hinge region creates a "butterfly-like" motion of the domains that reduces the width of the inter-domain active site cleft upon molecule binding. This phenomenon has been previously described in Rattus norvegicus and Legionella pneumophila NTPDaseI and Toxoplasma gondii NTPDaseIII suggesting a common catalytic mechanism across the domains of life.

The effects of PPO activity on the proteome of ingested red clover and implications for improving the nutrition of grazing cattle.

UNLABELLED: Increasing the rumen-stable protein content of feed would lead to improved nitrogen utilisation in cattle, and less nitrogenous waste. Red clover (Trifolium pratense L.) is a high protein ruminant feed containing high polyphenol oxidase (PPO) activity. PPO mediated protein-quinone binding has been linked to protecting plant proteins from proteolysis. To explore the mechanism underlying the effect of PPO on protein protection in fresh forage feeds, proteomic components of feed down-boli produced from wild-type red clover and a low PPO mutant, at point of ingestion and after 4h in vitro incubation with rumen inoculum were analysed. Significant differences in proteomic profiles between wild-type and mutant red clover were determined after 4h incubation, with over 50% less spots in mutant than wild-type proteomes, indicating decreased proteolysis in the latter. Protein identifications revealed preferentially retained proteins localised within the chloroplast, suggesting that PPO mediated protection in the wild-type operates due to the proximity of target proteins to the enzyme and substrates, either diffusing into this compartment from the vacuole or are present in the chloroplast. This increased understanding of protein targets of PPO indicates that wider exploitation of the trait could contribute to increased protein use efficiency in grazing cattle. BIOLOGICAL SIGNIFICANCE: One of the main challenges for sustainable livestock farming is improving capture of dietary nitrogen by ruminants. Typically up to 70% of ingested protein-N is excreted representing a loss of productivity potential and a serious environmental problem in terms of nitrogenous pollution of lands and water. Identification of key characteristics of rumen-protected protein will deliver target traits for selection in forage breeding programmes. The chloroplastic enzyme PPO catalyzes the oxidation of phenols to quinones, which react with protein. Little is currently known about the intracellular protein targets of the products of PPO activity or the mechanism underlying protein complexing, including whether there is any specificity to the reaction. Here we have determined significant differences in the proteomes of freshly ingested down boli corresponding to the presence or absence of active PPO. These results show that in the presence of PPO the forage protein is less amenable to proteolysis and provide the novel information that the protected proteins are putatively chloroplastically located. These data also contribute to a growing evidence base that a chloroplastic PPO substrate exists in red clover in addition to the currently known vacuolar substrates.

NO is involved in spermidine-induced drought tolerance in white clover via activation of antioxidant enzymes and genes.

Nitric oxide (NO), a key signaling molecule, can be induced by polyamines (PAs), which play an important role in improving drought tolerance in plants. This study was to further investigate the role of NO in spermidine (Spd)-induced drought tolerance associated with antioxidant defense in leaves of white clover (Trifolium repens) under drought stress induced by -0.3 MPa polyethylene glycol (PEG-6000) solution. A hydroponic growth method was used for cultivating plants in a controlled growth chamber for 30-33 days until the second leaves were fully expanded. Two relative independent experiments were carried out in our study. One is that exogenous application of Spd or an NO donor (sodium nitroprusside (SNP)) significantly improved drought tolerance in whole plants, as demonstrated by better phenotypic appearance, increased relative water content (RWC), and decreased electrolyte leakage (EL) and malondialdehyde (MDA) content in leaves as compared to untreated plants. For another detached leaf experiment, PEG induced an increase in the generation of NO in cells and significantly improved activities of nitrate reductase (NR) and nitric oxide synthase (NOS). These responses could be blocked by pre-treatment with a Spd biosynthetic inhibitor, dicyclohexyl amine (DCHA), and then reversed by application of exogenous Spd. Meanwhile, PEG induced up-regulation of activities and gene transcript levels of corresponding antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX) to varying degrees, while these effects were partially blocked by pre-treatment with DCHA, the scavenger of NO, the inhibitors of NR or NOS. In addition, Spd-induced antioxidant enzyme activities and gene expression also could be effectively inhibited by an NO scavenger as well as inhibitors of NR and NOS. These findings suggest that both Spd and NO can enhance drought tolerance. Spd was involved in drought stress-activated NR and NOS pathways associated with NO release, which mediated antioxidant defense and thus contributed to drought tolerance in white clover.

High expression of SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE coincides with initiation of various developmental pathways in in vitro culture of Trifolium nigrescens.

The aim of this study was to identify and examine the expression pattern of the ortholog of SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE gene from Trifolium nigrescens (TnSERK) in embryogenic and non-regenerative cultures of immature cotyledonary-stage zygotic embryos (CsZEs). In the presence of 1-naphthaleneacetic acid and N(6)-[2-isopentenyl]-adenine, the CsZE regenerated embryoids directly and in a lengthy culture produced callus which was embryogenic or remained non-regenerative. As revealed by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), the TnSERK was expressed in both embryogenic and non-regenerative cultures, but the expression level was significantly higher in embryogenic ones. An in situ RNA hybridization assay revealed that the expression of TnSERK preceded the induction of cell division in explants, and then, it was maintained exclusively in actively dividing cells from which embryoids, embryo-like structures (ELSs), callus or tracheary elements were produced. However, the cells involved in different morphogenic events differed in intensity of hybridization signal which was the highest in embryogenic cells. The TnSERK was up-regulated during the development of embryoids, but in cotyledonary embryos, it was preferentially expressed in the regions of the apical meristems. The occurrence of morphological and anatomical abnormalities in embryoid development was preceded by a decline in TnSERK expression, and this coincided with the parenchymatization of the ground tissue in developing ELSs. TnSERK was also down-regulated during the maturation of parenchyma and xylem elements in CsZE and callus. Altogether, these data suggest the involvement of TnSERK in the induction of various developmental programs related to differentiation/transdifferentiation and totipotent state of cell(s).

Combined effect of Cd and Pb spiked field soils on bioaccumulation, DNA damage, and peroxidase activities in Trifolium repens.

The present study was designed to investigate the combined effects of Cd and Pb on accumulation and genotoxic potential in white clover (Trifolium repens). For this purpose, T. repens was exposed to contaminated soils (2.5-20 mg kg(-1) cadmium (Cd), 250-2000 mg kg(-1) lead (Pb) and a mixture of these two heavy metals) for 3, 10 and 56 days. The resulting bioaccumulation of Cd and Pb, DNA damage (comet assay) and peroxidase activities (APOX and GPOX) were determined. The exposure time is a determinant factor in experiments designed to measure the influence of heavy metal contamination. The accumulation of Cd or Pb resulting from exposure to the two-metal mixture does not appear to depend significantly on whether the white clover is exposed to soil containing one heavy metal or both. However, when T. repens is exposed to a Cd/Pb mixture, the percentage of DNA damage is lower than when the plant is exposed to monometallic Cd. DNA damage is close to that observed in the case of monometallic Pb exposure. Peroxidase activity cannot be associated with DNA damage under these experimental conditions.

Polyphenol oxidase-mediated protection against oxidative stress is not associated with enhanced photosynthetic efficiency.

BACKGROUND AND AIMS: Polyphenol oxidases (PPOs) catalyse the oxidation of monophenols and/or o-diphenols to highly reactive o-quinones, which in turn interact with oxygen and proteins to form reactive oxygen species (ROS) and typical brown-pigmented complexes. Hence PPOs can affect local levels of oxygen and ROS. Although the currently known substrates are located in the vacuole, the enzyme is targeted to the thylakoid lumen, suggesting a role for PPOs in photosynthesis. The current study was designed to investigate the potential involvement of PPOs in the photosynthetic response to oxidative stress. METHODS: Photosynthesis (A, Fv/Fm, ΦPSII, qN, qP, NPQ) was measured in leaves of a wild-type and a low-PPO mutant of red clover (Trifolium pratense 'Milvus') under control conditions and under a stress treatment designed to induce photooxidative stress: cold/high light (2 °C/580 µmol m(2 )s(-1)) or 0-10 µm methyl viologen. Foliar protein content and oxidation state were also determined. KEY RESULTS: Photosynthetic performance, and chlorophyll and protein content during 4 d of cold/high light stress and 3 d of subsequent recovery under control growth conditions showed similar susceptibility to stress in both lines. However, more extensive oxidative damage to protein in mutants than wild-types was observed after treatment of attached leaves with methyl viologen. In addition, PPO activity could be associated with an increased capacity to dissipate excess energy, but only at relatively low methyl viologen doses. CONCLUSIONS: The presence of PPO activity in leaves did not correspond to a direct role for the enzyme in the regulation or protection of photosynthesis under cold stress. However, an indication that PPO could be involved in cellular protection against low-level oxidative stress requires further investigation.

Exogenous NO depletes Cd-induced toxicity by eliminating oxidative damage, re-establishing ATPase activity, and maintaining stress-related hormone equilibrium in white clover plants.

Various nitric oxide (NO) regulators [including the NO donor sodium nitroprusside (SNP), the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), the NO-synthase inhibitor N (G)-nitro-L-Arg-methyl ester (L-NAME), and the SNP analogues sodium nitrite/nitrate and sodium ferrocyanide] were investigated to elucidate the role of NO in white clover (Trifolium repens L.) plants after long-term (5 days) exposure to cadmium (Cd). A dose of 100 µM Cd stress significantly restrained plant growth and decreased the concentrations of chlorophyll and NO in vivo, whereas it disrupted the balance of stress-related hormones and enhanced the accumulation of Cd, thereby inducing reactive oxygen species (ROS) burst. However, the inhibition of plant growth was relieved by 50 µM SNP through its stimulation of ROS-scavenging compounds (ascorbic acid, ascorbate peroxidase, catalase, glutathione reductase, non-protein thiol, superoxide dismutase, and total glutathione), regulation of H(+)-ATPase activity of proton pumps, and increasing jasmonic acid and proline but decreasing ethylene in plant tissues. Even so, the alleviating effect of SNP on plant growth was counteracted by cPTIO and L-NAME and was not observed with SNP analogues, suggesting that the protective roles of SNP are related to the induction of NO. These results suggest that NO may improve the Cd tolerance of white clover plants by eliminating oxidative damage, re-establishing ATPase activity, and maintaining hormone equilibrium. Improving our understanding of the role of NO in white clover plants is key to expanding the plantations to various regions and the recovery of pasture species in the future.

Protection of polyunsaturated oils against ruminal biohydrogenation and oxidation during storage using a polyphenol oxidase containing extract from red clover.

Polyunsaturated fatty acid (PUFA) are to a large extent subject to biohydrogenation in a ruminal environment, which results to the healthy value of these PUFA being lost upon dietary addition to ruminants. PUFA are also prone to lipid oxidation upon storage. Therefore, it was tested whether emulsions could be protected against in vitro ruminal biohydrogenation and oxidation during storage by using protein extracts rich in polyphenol oxidase, an enzyme responsible for browning of plant tissues. PUFA rich emulsions were made with a protein extract from red clover (Trifolium pratense L.) before adding a synthetic diphenol (4-methylcatechol) to induce protection. Results after in vitro incubation confirmed the hypothesis and indicated the potential to prevent PUFA in linseed or fish oil from ruminal biohydrogenation and oxidation during storage through addition of 4-methylcatechol to the emulsions. Protection depended on the amount of oil present and protein concentrations in the emulsions. Protection efficiency increased with increasing the amounts of diphenol present in the emulsion per unit interfacial surface area. It is suggested that protection is caused by an effective encapsulation by cross-linking of the protein layer at the emulsion interface. For the first time, a method is described to protect PUFA using an enzyme abundantly available in nature, polyphenol oxidase, in combination with 4-methylcatechol.

Antioxidant responses of Annelids, Brassicaceae and Fabaceae to pollutants: a review.

Pollutants, such as Metal Trace Elements (MTEs) and organic compounds (polycyclic aromatic hydrocarbons, pesticides), can impact DNA structure of living organisms and thus generate damage. For instance, cadmium is a well-known genotoxic and mechanisms explaining its clastogenicity are mainly indirect: inhibition of DNA repair mechanisms and/or induction of Reactive Oxygen Species (ROS). Animal or vegetal cells use antioxidant defense systems to protect themselves against ROS produced during oxidative stress. Because tolerance of organisms depends, at least partially, on their ability to cope with ROS, the mechanisms of production and management of ROS were investigated a lot in Ecotoxicology as markers of biotic and abiotic stress. This was mainly done through the measurement of enzyme activities The present Review focuses on 3 test species living in close contact with soil that are often used in soil ecotoxicology: the worm Eisenia fetida, and two plant species, Trifolium repens (white clover) and Brassica oleracea (cabbage). E. fetida is a soil-dwelling organism commonly used for biomonitoring. T. repens is a symbiotic plant species which forms root nodule with soil bacteria, while B. oleracea is a non-symbiotic plant. In literature, some oxidative stress enzyme activities have already been measured in those species but such analyses do not allow distinction between individual enzyme involvements in oxidative stress. Gene expression studies would allow this distinction at the transcriptomic level. A literature review and a data search in molecular database were carried out on the basis of keywords in Scopus, in PubMed and in Genbank™ for each species. Molecular data regarding E. fetida were already available in databases, but a lack of data regarding oxidative stress related genes was observed for T. repens and B. oleracea. By exploiting the conservation observed between species and using molecular biology techniques, we partially cloned missing candidates involved in oxidative stress and in metal detoxification in E. fetida, T. repens and B. oleracea.

Design and application of an in vivo reporter assay for phenylalanine ammonia-lyase.

Phenylalanine ammonia-lyase (PAL) is an important enzyme that links primary metabolism to secondary metabolism. Its efficiency is often a critical factor that affects the overall flux of a related metabolic pathway, the titer of the final products, and the efficacy of PAL-based therapies. Thus, PAL is a common target for metabolic engineering, and it is of significant interest to screen efficient PALs for industrial and medical applications. In this study, a novel and efficient visible reporter assay for screening of PAL efficiency in Escherichia coli was established based on a plant type III polyketide biosynthetic pathway. The candidate PALs were co-expressed with a 4-coumarate:CoA ligase 4CL1 from Arabidopsis thaliana and curcuminoid synthase (CUS) from Oryza sativa in E. coli BL21(DE3) to form a dicinnamoylmethane biosynthetic pathway. Taking advantage of the yellow color of the product, a microplate-based assay was designed to measure the titer of dicinnamoylmethane, which was validated by HPLC analysis. The different titers of the product reflect the overall performance (expression level and enzymatic activity) of the individual PALs in E. coli. Using this system, we have screened three PALs (PAL1, PAL3, and PAL4) from Trifolium pratense, among which PAL1 showed the best performance in E. coli. The engineered E. coli strain containing PAL1, 4CL1, and CUS led to the production of dicinnamoylmethane at a high level of 0.36 g/l. Supplement of 2-fluoro-phenylalanine yielded two fluorinated dicinnamoylmethane derivatives, 6,6'-difluoro-dicinnamoylmethane and 6-fluoro-dicinnamoylmethane, of which the latter is a new curcuminoid.

Searching for the bull's eye: agents and targets of selection vary among geographically disparate cyanogenesis clines in white clover (Trifolium repens L.).

The recurrent evolution of adaptive clines within a species can be used to elucidate the selective factors and genetic responses that underlie adaptation. White clover is polymorphic for cyanogenesis (HCN release with tissue damage), and climate-associated cyanogenesis clines have evolved throughout the native and introduced species range. This polymorphism arises through two independently segregating Mendelian polymorphisms for the presence/absence of two required components: cyanogenic glucosides and their hydrolyzing enzyme linamarase. Cyanogenesis is commonly thought to function in herbivore defense; however, the individual cyanogenic components may also serve other physiological functions. To test whether cyanogenesis clines have evolved in response to the same selective pressures acting on the same genetic targets, we examined cyanogenesis cline shape and its environmental correlates in three world regions: southern New Zealand, the central United States and the US Pacific Northwest. For some regional comparisons, cline shapes are remarkably similar despite large differences in the spatial scales over which clines occur (40-1600 km). However, we also find evidence for major differences in both the agents and targets of selection among the sampled clines. Variation in cyanogenesis frequency is best predicted using a combination of minimum winter temperature and aridity variables. Together, our results provide evidence that recurrent adaptive clines do not necessarily reflect shared adaptive processes.

Gene expression patterns, localization, and substrates of polyphenol oxidase in red clover ( Trifolium pratense L.).

Polyphenol oxidase (PPO) genes and their corresponding enzyme activities occur in many plants; natural PPO substrates and enzyme/substrate localization are less well characterized. Leaf and root PPO activities in Arabidopsis and five legumes were compared with those of high-PPO red clover ( Trifolium pratense L.). Red clover PPO enzyme activity decreased leaves > stem > nodules > peduncle = petiole > embryo; PPO1 and PPO4 genes were expressed early in leaf emergence, whereas PPO4 and PPO5 predominated in mature leaves. PPO1 was expressed in embryos and nodules. PPO substrates, phaselic acid and clovamide, were detected in leaves, and clovamide was detected in nodules. Phaselic acid and clovamide, along with caffeic and chlorogenic acids, were suitable substrates for PPO1, PPO4, and PPO5 genes expressed in alfalfa ( Medicago sativa L.) leaves. PPO enzyme presence and activity were colocalized in leaves and nodules by cytochemistry. Substrates and PPO activity were localized in developing squashed cell layer of nodules, suggesting PPO may have a developmental role in nodules.

Antioxidant and photosystem II responses contribute to explain the drought-heat contrasting tolerance of two forage legumes.

Identification of metabolic targets of environmental stress factors is critical to improve the stress tolerance of plants. Studying the biochemical and physiological responses of plants with different capacities to deal with stress is a valid approach to reach this objective. Lotus corniculatus (lotus) and Trifolium pratense (clover) are legumes with contrasting summer stress tolerances. In stress conditions, which are defined as drought, heat or a combination of both, we found that differential biochemical responses of leaves explain these behaviours. Lotus and clover showed differences in water loss control, proline accumulation and antioxidant enzymatic capacity. Drought and/or heat stress induced a large accumulation of proline in the tolerant species (lotus), whereas heat stress did not cause proline accumulation in the sensitive species (clover). In lotus, Mn-SOD and Fe-SOD were induced by drought, but in clover, the SOD-isoform profile was not affected by stress. Moreover, lotus has more SOD-isoforms and a higher total SOD activity than clover. The functionality and electrophoretic profile of photosystem II (PSII) proteins under stress also exhibited differences between the two species. In lotus, PSII activity was drastically affected by combined stress and, interestingly, was correlated with D2 protein degradation. Possible implications of this event as an adaption mechanism in tolerant species are discussed. We conclude that the stress-tolerant capability of lotus is related to its ability to respond to oxidative damage and adaption of the photosynthetic machinery. This reveals that these two aspects should be included in the evaluation of the tolerance of species to stress conditions.

The Tr-cp 14 cysteine protease in white clover (Trifolium repens) is localized to the endoplasmic reticulum and is associated with programmed cell death during development of tracheary elements.

Cysteine proteases are known to be associated with programmed cell death, developmental senescence and some types of pathogen and stress-induced responses. In the present study, we have characterized the cysteine protease Tr-cp 14 in white clover (Trifolium repens). Tr-cp 14 belongs to the C1A family of cysteine proteases with homology to XCP1 and XCP2 from Arabidopsis thaliana and p48h-17 from Zinnia elegans, which previously have been reported to be associated with tracheary element differentiation. The proform as well as the processed form of the protein was detected in petioles, flowers and leaves, but the processed form was more abundant in leaves and petioles than in flowers. The Tr-cp 14 protein was localized to differentiating tracheary elements within the xylem, indicating that the cysteine protease is involved in protein re-mobilization during tracheary element differentiation. Immunogold studies suggest that the protease prior to the burst of the vacuole was associated to the ER cisternae. After disruption of the tonoplast, it was found in the cytoplasm, and, in later stages, associated with disintegrating material dispersed throughout the cell.

Recurrent gene deletions and the evolution of adaptive cyanogenesis polymorphisms in white clover (Trifolium repens L.).

Understanding the molecular evolution of genes that underlie intraspecific polymorphisms can provide insights into the process of adaptive evolution. For adaptive polymorphisms characterized by gene presence/absence (P/A) variation, underlying loci commonly show signatures of long-term balancing selection, with gene-presence and gene-absence alleles maintained as two divergent lineages. We examined the molecular evolution of two unlinked P/A polymorphisms that underlie a well-documented adaptive polymorphism for cyanogenesis (hydrogen cyanide release with tissue damage) in white clover. Both cyanogenic and acyanogenic plants occur in this species, and the ecological forces that maintain this chemical defence polymorphism have been studied for several decades. Using a sample of 65 plants, we investigated the molecular evolution of sequences flanking the two underlying cyanogenesis genes: Ac/ac (controlling the presence/absence of cyanogenic glucosides) and Li/li (controlling the presence/absence of their hydrolysing enzyme, linamarase). A combination of genome walking, PCR assays, DNA sequence analysis and Southern blotting was used to test whether these adaptive P/A polymorphisms show evidence of long-term balancing selection, or whether gene-absence alleles have evolved repeatedly through independent deletion events. For both loci, we detect no signatures of balancing selection in the closest flanking genomic sequences. Instead, we find evidence for variation in the size of the deletions characterizing gene-absence alleles. These observations strongly suggest that both of these polymorphisms have been evolving through recurrent gene deletions over time. We discuss the genetic mechanisms that could account for this surprising pattern and the implications of these findings for mechanisms of rapid adaptive evolution in white clover.

Expression and functional characterization of a white clover isoflavone synthase in tobacco.

BACKGROUND AND AIMS: Trifolium repens (white clover) is a valuable component of pastures due to its ability to fix nitrogen. Productivity of T. repens is sometimes threatened by insect pests, and it has been suggested that phenylpropanoid-derived isoflavonoids such as formononetin can protect white clover from insect damage. The aim of this study was to isolate and functionally characterize an isoflavone synthase (IFS2_12) from T. repens by expressing it in Nicotiana tabacum (tobacco), a plant which does not naturally produce isoflavonoids. METHODS: To induce anthocyanin production and increase isoflavonoid precursors in tobacco, the tomato R2R3 MYB transcription factor ANT1 was expressed in tobacco (Nt-ANT1 plants). IFS2_12 was heterologously expressed in tobacco both transiently and stably, and isoflavonoids in leaf extracts were analysed by liquid chromatography (LC) coupled to mass spectrometry (MS(n)). As a positive control, a double construct of soybean IFS and alfalfa chalcone isomerase (IFS/CHI), which had been previously shown to induce isoflavonoid production in tobacco, was also expressed. Stable transformants expressing IFS2_12, soybean/alfalfa IFS/CHI and ANT1 were crossed and the resulting plants were analysed for isoflavonoid production. KEY RESULTS: Leaves of tobacco plants expressing ANT1 had a range of phenotypes from mainly green to uniformly bronze coloured. Both transient and stable expression of the IFS2_12 or IFS/CHI constructs resulted in the production of the isoflavonoid genistein and its conjugates. The highest levels (up to 19·2 mg g(-1) d. wt) accumulated in a progeny of a cross between a purple ANT1 and a IFS/ CHI transformant, while the second highest concentration was found in a plant derived from a selfed IFS2-12 transformant. CONCLUSIONS: It is concluded that the gene IFS2_12 isolated from T. repens encodes an isoflavone synthase. This study paves the way for engineering white clover plants with higher levels of isoflavonoids than naturally found in this species for sufficient insect protection.

Halloysite clay nanotubes and platinum nanoparticles dispersed in ionic liquid applied in the development of a catecholamine biosensor.

Halloysite clay nanotubes were used as a support for the immobilization of the enzyme peroxidase from clover sprouts (Trifolium), and employed together with platinum nanoparticles in 1-butyl-3-methylimidazolium hexafluorophosphate ionic liquid (Pt-BMI·PF(6)) in the development of a new biosensor for the determination of catecholamines by square-wave voltammetry. Under optimized conditions, the analytical curves showed detection limits of 0.05, 0.06, 0.07, 0.12 µM for dopamine, isoproterenol, dobutamine and epinephrine, respectively. The biosensor demonstrated high sensitivity, good repeatability and reproducibility, and long-term stability (18% decrease in response over 150 days). A recovery study of dopamine in pharmaceutical samples gave values from 97.5 to 101.4%. The proposed biosensor was successfully applied to the determination of dopamine in pharmaceutical samples, with a maximum relative error of ±1.0% in relation to the standard (spectrophotometric) method. The good analytical performance of the proposed method can be attributed to the efficient immobilization of the peroxidase in the nanoclay, and the facilitation of electron transfer between the protein and the electrode surface due to the presence of the Pt nanoparticles and ionic liquid.

Changes in 1-aminocyclopropane-1-carboxlate (ACC) oxidase expression and enzyme activity in response to excess manganese in white clover (Trifolium repens L.).

To examine the effect on Mn treatment on the ACO gene family of white clover [Trifolium repens (L.) cv. Grasslands Challenge], rooted stolon cuttings were maintained in modified Hoaglands medium, at pH 5.4, containing either 5.2 µM Mn or 100 µM Mn over a 9-day time course. A significant uptake of Mn was observed in leaf tissue of plants grown in the 100 µM Mn treatment after 24 h and the content increased in these plants to reach 334 mg/kg DW at the conclusion of the time course. The growth of plants, measured as the petiole extension rate (PER), was significantly less in the 100 µM Mn treatment by day 9, while significantly less accumulation of leaf biomass was observed by day 7. The activity of a cell wall-associated H(2)O(2)-generating NADH peroxidase was shown to be higher in the 100 µM Mn treatment after day 5 of the time course while no significant difference in a H(2)O(2)-consuming guaiacol peroxidase activity was observed between the two Mn treatments. The expression of two leaf-associated ACC oxidase (ACO) genes, TR-ACO2 and TR-ACO3 was examined over the 9-day course but no difference between the two treatments was observed. In contrast, TR-ACO2 enzyme activity was measured and shown to decrease in the 100 µM Mn treatment after day 5 of the time course, with a concomitant decrease in TR-ACO2 accumulation, as determined by western analysis. Using 2DE and western analysis, evidence for post-translational modification of TR-ACO2 was observed.