Cytochrome P450-dependent Monooxygenase Research Articles

In silico Analysis and Characterization of the F5H Gene Family in Sorghum bicolor and Its Role in Lignin Production

Ferulate 5-hydroxylase (F5H), a cytochrome P450-dependent monooxygenase, catalyzes the hydroxylation of coniferaldehyde, a crucial step in the formation of syringyl lignin monomer (S). However, evolutionary divergence, expression patterns under abiotic stress conditions (ABA, PEG and NaOH) and lignin content-related features of the F5H gene family in Sorghum bicolor have not been explored. This study envisaged mining of Sorghum genomic data leading to the identification of 61 SbF5H genes. Bioinformatics analysis revealed the phylogenetic evolutionary relations, gene structures, conserved motifs, physicochemical properties, and promoter cis-acting elements related to these genes and their encoded proteins. Based on the gene structural and phylogenetic features, these 61 SbF5Hs were grouped into 4 subclasses. The in silico expression analysis revealed higher accumulation of SbF5H1 transcripts in embryo and in root under stress conditions. Similarly, Other SbF5H genes have shown expression in stem and root, thus indicating SbF5H genes involvement in Sorghum lignin biosynthesis. By exploring into the functional aspects of the F5H gene, our study sought to shed light on its significance in influencing not only the chemical makeup of lignin but also the resultant plant phenotypes. This insight into the molecular mechanisms governing lignin biosynthesis can have implications for bioenergy production and crop improvement.

Long-term impact of rearing substrates on susceptibility to insecticides and metabolic enzyme activities in the house fly Musca domestica.

Musca domestica Linnaeus is a devastating insect pest of medical and veterinary importance with reports of resistance development to commonly used insecticides worldwide. Rearing substrates usually play a crucial role in determining susceptibility to insecticides and control of insect pests. The aim of the present study was to investigate the effect of five rearing substrates of M. domestica on its susceptibility to different insecticides and activities of metabolic enzymes. After 30 generations of rearing, susceptibility of M. domestica to tested insecticides, viz., malathion, pirimiphos-methyl, alpha-cypermethrin, deltamethrin, methomyl, propoxur, spinetoram, and chlorfenapyr had evident differences. Musca domestica reared on hen liver exhibited reduced susceptibility to all insecticides followed by the strain reared on poultry manure. However, M. domestica reared on milk-based diet showed the highest susceptibility to tested insecticides followed by the strain reared on manures of buffalo and horse. In addition, M. domestica reared on different substrates exhibited significant differences (p < 0.01) in the activities of glutathione S-transferase (GST), cytochrome P450-dependent monooxygenase, and carboxylesterase (CarE). Overall, hen liver and poultry manure strains exhibited higher activities of metabolic enzymes than those of the milk-based diet, buffalo, and horse manure strains. In conclusion, the data of the present study exhibited a significant effect of rearing substrates on the susceptibility to insecticides and activities of metabolic enzymes in M. domestica. These results could be helpful for the sustainable management of M. domestica on different hosts by selecting appropriate insecticides.

Relationship between insecticide resistance profiles in Anopheles gambiae sensu lato and agricultural practices in Côte d’Ivoire

BackgroundInsecticide-based malaria vector control is increasingly undermined due to the development of insecticide resistance in mosquitoes. Insecticide resistance may partially be related to the use of pesticides in agriculture, while the level and mechanisms of resistance might differ between agricultural practices. The current study aimed to assess whether phenotypic insecticide resistance and associated molecular resistance mechanisms in Anopheles gambiae sensu lato differ between agricultural practices.MethodsWe collected An. gambiae s.l. larvae in six sites with three different agricultural practices, including rice, vegetable and cocoa cultivation. We then exposed the emerging adult females to discriminating concentrations of bendiocarb (0.1%), deltamethrin (0.05%), DDT (4%) and malathion (5%) using the standard World Health Organization insecticide susceptibility test. To investigate underlying molecular mechanisms of resistance, we used multiplex TaqMan qPCR assays. We determined the frequency of target-site mutations, including Vgsc-L995F/S and Vgsc-N1570Y, and Ace1-G280S. In addition, we measured the expression levels of genes previously associated with insecticide resistance in An. gambiae s.l., including the cytochrome P450-dependent monooxygenases CYP4G16, CYP6M2, CYP6P1, CYP6P3, CYP6P4, CYP6Z1 and CYP9K1, and the glutathione S-transferase GSTe2.ResultsThe An. gambiae s.l. populations from all six agricultural sites were resistant to bendiocarb, deltamethrin and DDT, while the populations from the two vegetable cultivation sites were additionally resistant to malathion. Most tested mosquitoes carried at least one mutant Vgsc-L995F allele that is associated with pyrethroid and DDT resistance. In the cocoa cultivation sites, we observed the highest 995F frequencies (80–87%), including a majority of homozygous mutants and several in co-occurrence with the Vgsc-N1570Y mutation. We detected the Ace1 mutation most frequently in vegetable-growing sites (51–60%), at a moderate frequency in rice (20–22%) and rarely in cocoa-growing sites (3–4%). In contrast, CYP6M2, CYP6P3, CYP6P4, CYP6Z1 and CYP9K1, previously associated with metabolic insecticide resistance, showed the highest expression levels in the populations from rice-growing sites compared to the susceptible Kisumu reference strain.ConclusionIn our study, we observed intriguing associations between the type of agricultural practices and certain insecticide resistance profiles in the malaria vector An. gambiae s.l. which might arise from the use of pesticides deployed for protecting crops.Graphical

Acaricide resistance in predatory mites of the genus Euseius (Acari: Phytoseiidae) and predation capacity on Brevipalpus yothersi (Acari: Tenuipalpidae)

One of the main citrus diseases in Brazil is citrus leprosis, transmitted by the mite Brevipalpus yothersi Baker (Acari: Tenuipalpidae). The predatory mites Euseius citrifolius Denmark & Muma and Euseius concordis (Chant) (Acari: Phytoseiidae) are important natural enemies for the pest mite. The objective of this work was to evaluate the susceptibility of different populations of E. concordis and E. citrifolius, from citrus groves in the state of São Paulo (SP), to the acaricides abamectin, fenpropathrin and chlorfenapyr; characterize the abamectin resistance in E. citrifolius, including studies on cross-resistance and biochemical mechanisms of resistance; and evaluate the predation capacity of an abamectin resistant population of E. citrifolius on B. yothersi. Significant differences in susceptibility to acaricides were observed among populations of E. citrifolius. Resistance ratios of 15.5 and 3.1 (times) were observed for abamectin and chlorfenapyr, respectively, comparing populations of E. citrifolius collected in the municipalities of Taquaral-SP and Bofete-SP. Studies with the synergist piperonyl butoxide indicated the involvement of cytochrome P450-dependent monooxygenases in the resistance of E. citrifolius to abamectin. Populations of E. concordis and E. citrifolius collected in Taquaral-SP showed low susceptibility to fenpropathrin, with LC50 values at least 95 times higher than the recommended concentration for the control of Brevipalpus mites in citrus in Brazil. The population of E. citrifolius from Taquaral showed a high capacity for predation of B. yothersi larvae, with consumption of up to 49.4 larvae per adult female per day.

Genome-Wide Identification and Expression Pattern Analysis of the F5H Gene Family in Flax (Linum usitatissimum L.)

Ferulate 5-hydroxylase (F5H) is a cytochrome P450-dependent monooxygenase that plays a key role in the biosynthesis of syringyl (S) lignin. In this study, mining of flax (Linum usitatissimum) genomic data enabled the identification of nine LuF5H genes. Bioinformatics analysis revealed the physicochemical properties, gene structures, conserved motifs, phylogenetic evolutionary features and promoter cis-acting elements related to these genes and their encoded proteins. Based on the gene structural and phylogenetic features, the nine LuF5Hs were assigned to two subclasses. The expression levels of the nine LuF5Hs was analyzed by the RNA-seq technique, and the RNA-seq data were verified by qRT-PCR. The results of the RNA-seq analysis showed that LuF5H genes belonging to the same subclass exhibited similar expression patterns. Notably, the transcripts of the LuF5H3 and LuF5H7 subclass 1 genes accumulated at high levels in stem tissues, thus indicating that LuF5H3 and LuF5H7 are the main LuF5H genes involved in flax lignin biosynthesis. Furthermore, the expression levels of LuF5H1, LuF5H3, LuF5H4 and LuF5H7 were upregulated 1.2–1.9-fold under drought, NaCl stress and brassinosteroid treatment conditions. This first comprehensive study of the flax F5H gene family provides valuable data for use in gene function analysis toward improving flax fiber quality and reducing flax manufacturing costs and associated environmental pollution.

Perfluoroethylcyclohexane sulphonate, an emerging perfluoroalkyl substance, disrupts mitochondrial membranes and the expression of key molecular targets in vitro

Perfluoroethylcyclohexane sulphonate (PFECHS) is an emerging, replacement perfluoroalkyl substance (PFAS) with little information available on the toxic effects or potencies with which to characterize its potential impacts on aquatic environments. This study aimed to characterize effects of PFECHS using in vitro systems, including rainbow trout liver cells (RTL-W1 cell line) and lymphocytes separated from whole blood. It was determined that exposure to PFECHS caused minor acute toxic effects for most endpoints and that little PFECHS was concentrated into cells with a mean in vitro bioconcentration factor of 81 ± 25 L/kg. However, PFECHS was observed to affect the mitochondrial membrane and key molecular receptors, such as the peroxisome proliferator receptor, cytochrome p450-dependent monooxygenases, and receptors involved in oxidative stress. Also, glutathione-S-transferase was significantly down-regulated at a near environmentally relevant exposure concentration of 400 ng/L. These results are the first to report bioconcentration of PFECHS, as well as its effects on the peroxisome proliferator and glutathione-S-transferase receptors, suggesting that even with little bioconcentration, PFECHS has potential to cause adverse effects.

Predicting the bioremediation potential of earthworms of different ecotypes through a multi-biomarker approach

Earthworms are attracting the attention of bioremediation research because of their short-term impact on pollutant fate. However, earthworm-assisted bioremediation largely depends on the earthworm sensitivity to target pollutants and its metabolic capacity to break down contaminants. The most studied species in soil bioremediation has been Eisenia fetida, which inhabits the soil surface feeding on decomposing organic residues. Therefore, its bioremediation potential may be limited to organic matter-rich topsoil. We compared the detoxification potential against organophosphate (OP) pesticides of three earthworm species representative of the main ecotypes: epigeic, anecic, and endogeic. Selected biomarkers of pesticide detoxification (esterases, cytochrome P450-dependent monooxygenase, and glutathione S-transferase) and oxidative homeostasis (total antioxidant capacity, glutathione levels, and glutathione reductase [GR] and catalase activities) were measured in the muscle wall and gastrointestinal tract of E. fetida (epigeic), Lumbricus terrestris (anecic) and Aporrectodea caliginosa (endogeic). Our results show that L. terrestris was the most suitable species to bioremediate OP-contaminated soil for the following reasons: 1) Gut carboxylesterase (CbE) activity of L. terrestris was higher than that of E. fetida, whereas muscle CbE activity was more sensitivity to OP inhibition than that of E. fetida, which means a high capacity to inactivate the toxic oxon metabolites of OPs. 2) Muscle and gut phosphotriesterase activities were significantly higher in L. terrestris than in the other species. 3) Enzymatic (catalase and GR) and molecular mechanisms of free radical inactivation (glutathione) were 3- to 4-fold higher in L. terrestris concerning E. fetida and A. caliginosa, which reveals a higher potential to keep the cellular oxidative homeostasis against reactive metabolites formed during OP metabolism. Together with biological and ecological traits, these toxicological traits suggest L. terrestris a better candidate for soil bioremediation than epigeic earthworms.

Xenobiotic-sensing nuclear receptors as targets for phthalates-induced lung injury and antagonism of lycopene

Phthalates are extensively used in the production of plastics products and have been verified to induce lung injury. Lycopene (LYC) has proved an effective preventive and can be utilized to prevent phthalates-induced toxicity. However, the role of phthalate in pathogenesis of lung injury remain poorly researched, and little work has been devoted whether LYC could alleviate phthalate-induced lung toxicity via modulating nuclear xenobiotic receptors (NXRs) response. Here, di (2-ethylhexyl) phthalate (DEHP) is used as the representative of phthalates for further studies on toxicity of phthalates and the antagonistic role of LYC in phthalates-induced lung injury. We found that DEHP exposure caused alveoli destruction and alveolar epithelial cells type II damage. Mechanistically, DEHP exposure increased nuclear accumulation of aryl hydrocarbon receptor (AHR) and its downstream genes level, including cytochrome P450-dependent monooxygenase (CYP) 1A1 and CYP1B1. Constitutive androstane receptor (CAR) and their downstream gene level, including CYP2E1 are also increased after phthalates exposure. Significantly, LYC supplementation relieves lung injury from DEHP exposure by inhibiting the activation of NXRs. We confirm that NXRs plays a key role in phthalates-induced lung injury. Our study showed that LYC may have a positive role in alleviating the toxicity effects of phthalates, which provides an effective strategy for revising phthalates-induced injury.

Biochemical and insecticidal effects of plant essential oils on insecticide resistant and susceptible populations of Musca domestica L. point to a potential cross-resistance risk

Essential oils (EOs) can provide important alternatives to chemical insecticides in the control of pests. In this study, 12 EOs of native plant species from Iran were evaluated for their adulticidal activity against the house fly. In addition, we examined the insecticidal activity of Zataria multiflora and Rosmarinus officinalis EOs on adult female house flies from pyrethroid and organophosphate resistant and susceptible populations, using both fumigant and topical bioassays. The involvement of detoxification enzymes in susceptibility was investigated with synergism experiments in vivo, while the inhibitory effects of R. officinalis and Zataria multiflora EOs on the activities of cytochrome P450-dependent monooxygenases (P450s), carboxylesterases (CarEs) and glutathione S-transferases (GSTs) were determined by enzymatic inhibition assays in vitro. The EOs of Z. multiflora, Mentha pulegium, R. officinalis and Thymus vulgaris were the most effective against adults in contact topical assays, while oils extracted from Eucalyptus cinerea, Z. multiflora, Citrus sinensis, R. officinalis, Pinus eldarica and Lavandula angustifolia where the most effective in fumigant assays. Rosmarinus officinalis and Z. multiflora EOs were selected for further investigation and showed higher toxicity against a susceptible population, compared to two insecticide-resistant populations. Correlation analysis suggested cross-resistance between these EOs and pyrethroids in the resistant populations. The toxicity of both EOs on the resistant populations was synergized by three detoxification enzyme inhibitors. Further, in vitro inhibition studies showed that R. officinalis and Z. multiflora EOs more effectively inhibited the activities of the detoxification enzymes from flies of the susceptible population compared to those of the pyrethroid resistant populations. Synergistic and enzymatic assays further revealed that increased activities of P450s, GSTs, and CarEs are possibly involved in the cross-resistance between EOs and pyrethroids. Investigating the molecular mechanisms of P450s, GSTs, and CarEs in the resistance to EOs should be subject to further studies.

Methoxyfenozide tolerance in Chrysoperla carnea: Inheritance, dominance and preliminary detoxification mechanisms.

Lacewings exist in insecticide-dominant cropping systems. They are prime biological control agents due to outstanding ability of insecticide resistance development. This study examines occurrence of methoxyfenozide resistance and its subsequent effects on cross-resistance to other insecticides, inheritance and mechanism of resistance in C. carnea. Methoxy-SEL strain of C. carnea selected for 15 generations developed 3531.67-fold resistance to methoxyfenozide. Overlapping fiducial limits of LC50s of F1 and F1’ (reciprocal crosses) suggested an autosomal and incompletely dominant mode of inheritance. Resistance to methoxyfenozide was polygenic and its realized heritability value was high (h2 = 0.62). Both PBO and DEF significantly changed LC50s indicating cytochrome P450-dependent monooxygenases and esterases detoxifying the resistance in Methoxy-SEL strain. Resistance to all tested insecticide was unstable but decrease rate was very negligible. These results have implications forpreservation of biological control and effective use in insecticide-dominant cropping systems.

Multi-insecticide resistant malaria vectors in the field remain susceptible to malathion, despite the presence of Ace1 point mutations.

Insecticide resistance in Anopheles mosquitoes is seriously threatening the success of insecticide-based malaria vector control. Surveillance of insecticide resistance in mosquito populations and identifying the underlying mechanisms enables optimisation of vector control strategies. Here, we investigated the molecular mechanisms of insecticide resistance in three Anopheles coluzzii field populations from southern Côte d'Ivoire, including Agboville, Dabou and Tiassalé. All three populations were resistant to bendiocarb, deltamethrin and DDT, but not or only very weakly resistant to malathion. The absence of malathion resistance is an unexpected result because we found the acetylcholinesterase mutation Ace1-G280S at high frequencies, which would typically confer cross-resistance to carbamates and organophosphates, including malathion. Notably, Tiassalé was the most susceptible population to malathion while being the most resistant one to the pyrethroid deltamethrin. The resistance ratio to deltamethrin between Tiassalé and the laboratory reference colony was 1,800 fold. By sequencing the transcriptome of individual mosquitoes, we found numerous cytochrome P450-dependent monooxygenases - including CYP6M2, CYP6P2, CYP6P3, CYP6P4 and CYP6P5 - overexpressed in all three field populations. This could be an indication for negative cross-resistance caused by overexpression of pyrethroid-detoxifying cytochrome P450s that may activate pro-insecticides, thereby increasing malathion susceptibility. In addition to the P450s, we found several overexpressed carboxylesterases, glutathione S-transferases and other candidates putatively involved in insecticide resistance.

Features of the Bioconversion of Pentacyclic Triterpenoid Oleanolic Acid Using Rhodococcus Actinobacteria

The ability of actinobacteria of the genus Rhodococcus to transform oleanolic acid (OA), a plant pentacyclic triterpenoid, was shown for the first time using bioresources of the Regional Specialized Collection of AlkanotrophicMicroorganisms (IEGM; WDCM #768;www.iegmcol.ru). The most promising strains (R.opacus IEGM 488 and R.rhodochrousIEGM 285) were selected, and these catalyzed80% bioconversion of OA (0.5 g/L) in the presence of n-hexadecane (0.1% v/v) for seven days. The process of OA bioconversion was accompanied by a gradual decrease in the culture medium pH. Adaptive responses of bacterial cells to the OA effects included the formation of compact cellular aggregates, a marked change in the surface-to-volume ratio of cells, and a significant increase in the Zeta potential values. The results demonstrated that the process of OA bioconversion was catalyzed by membrane-bound enzyme complexes. Participation of cytochrome P450-dependent monooxygenases in the oxidation of the OA moleculewas confirmedusing specific inhibitors. The obtained data expand our knowledge on the catalytic activity of actinobacteria of the genus Rhodococcus and their possible use as biocatalysts for the bioconversion of complex hydrophobic compounds. The results can also be used inthe searchfor promising OA derivatives to be used in the synthesis of biologically active agents.&#x0D; Keywords: bioconversion, oleanolic acid, Rhodococcus, biologically active compounds

Lycopene Preventing DEHP-Induced Renal Cell Damage Is Targeted by Aryl Hydrocarbon Receptor.

Di (2-ethylhexyl) phthalate (DEHP) is an environmentally persistent and bioaccumulative plasticizer. Accumulation of DEHP in the body can eventually cause kidney damage. As a type of natural carotenoid, lycopene (LYC) has a potential protective effect on renal cells, but the protective mechanism has not yet been elucidated. The major goal of this study was to see how effective LYC was at treating DEHP-induced nephrotoxicity in mice. ICR mice were treated with DEHP (500 mg/kg BW/day or 1000 mg/kg BW/day) or LYC (5 mg/kg BW/day) for 28 days. Through histopathology and ultrastructure, we found that LYC attenuated DEHP-induced renal tubular cell and glomerular damage. LYC relieved DEHP-induced kidney injury evidenced by lower levels of blood urea nitrogen (Bun), creatinine (Cre), and uric acid (Uric). Meanwhile, the reduced expression of kidney injury molecule-1 (Kim-1) also supported it. Notably, LYC can alleviate the activity or content of cytochrome P450 system (CYP450s) interfered with by DEHP. In addition, LYC treatment reduced nuclear accumulation of DEHP-induced aromatic hydrocarbon receptor (AhR) and AhR nuclear transporter (Arnt), and its downstream target genes such as cytochrome P450-dependent monooxygenase (CYP) 1A1, 1A2, and 1B1 expression significantly decreased to normal in the LYC treatment group. In summary, LYC can mediate the AhR/Arnt signaling system to prevent kidney toxicity in mice caused by DEHP exposure.

Level of Pyrethroid-Resistance Associated with Cytochrome P450 Expression in German Cockroach Blattella germanica (Blattodea: Ectobiidae) in the Field Collected Strains.

Background:Cytochrome P450-dependent monooxygenases are a very important metabolic system involve in insecticide resistance. This study was conducted to find the association between the expression level of cytochrome P450 (CYP450) and permethrin-resistance level among four strains of the German cockroach Blattella germanica (L) (Blattodea: Ectobiidae).Methods:Three field strains of German cockroach with different frequency of exposure to pesticides, and a laboratory susceptible strain were used in the present study. Insecticide susceptibility bioassays were carried out to detect resistance to permethrin. The concentration of CYP450 in each strain was determined using ion-exchange HPLC chromatography. Biochemical assays was performed to analyse CYP450 activities.Results:The resistance ratios (RR) to permethrin among three field strains were 3.29, 4.10 and 6.17-fold comping with the susceptible stain. The CYP450 activity of three field strains was 1.6, 2.4 and 2.7 times higher than in the susceptible stain. The amount of CYP450 per mg of protein was significantly different between the susceptible and the three resistant strains. The resistant cockroaches showed a relatively high expression of CYP450 enzymes. A strong correlation was found between permethrin resistance level and total concentration of CYP450 enzymes.Conclusion:The results of current study show that more frequent usage of a pyrethroid insecticide cause the metabolic insecticide resistance to rise in German cockroach. Therefore, there is a ratio correlation between resistance level and monooxygenases activities in insect. Thus, the control program must be managed according to history of pesticide usage.

De Novo Biosynthesis of the Oleanane-Type Triterpenoids of Tunicosaponins in Yeast.

Tunicosaponins are natural products extracted from Psammosilene tunicoides, which is an important ingredient of Yunnan Baiyao Powder, an ancient and famous Asian herbal medicine. The representative aglycones of tunicosaponins are the oleanane-type triterpenoids of gypsogenin and quillaic acid, which were found to manipulate a broad range of virus-host fusion via wrapping the heptad repeat-2 (HR2) domain prevalent in viral envelopes. However, the unknown biosynthetic pathway and difficulty in chemical synthesis hinder the therapeutic use of tunicosaponins. Here, two novel cytochrome P450-dependent monooxygenases that take part in the biosynthesis of tunicosaponins, CYP716A262 (CYP091) and CYP72A567 (CYP099), were identified from P. tunicoides. In addition, the whole biosynthesis pathway of the tunicosaponin aglycones was reconstituted in yeast by transforming the platform strain BY-bAS with the CYP716A262 and CYP716A567 genes, the resulting strain could produce 146.84 and 314.01 mg/L of gypsogenin and quillaic acid, respectively. This synthetic biology platform for complicated metabolic pathways elucidation and microbial cell factories construction can provide alternative sources of important natural products, helping conserve natural plant resources.

SmJRB1 positively regulates the accumulation of phenolic acid in Salvia miltiorrhiza

Phenolic acids rank high among the major active metabolites produced in the Chinese herb, Salvia miltiorrhiza, which has a rapidly escalating medicinal demand. Phenolic acid biosynthesis can be induced by methyl jasmonate (MeJA), although the underlying molecular mechanism remains obscure. A jasmonic acid-responsive basic helix-loop-helix (bHLH) transcriptional factor, SmJRB1, was identified in this study from the MeJA-induced transcriptome library of S. miltiorrhiza. The SmJRB1 is localized in the nuclei illustrated by the transient-transformation of epidermal cells in tobacco leaves. Overexpression of the SmJRB1 significantly promoted the accumulation of phenolic acids and upregulated the genes that encode the principal enzymes such as rosmarinic acid synthase I (RAS1) and cytochrome P450-dependent monooxygenase (CYP98A14), which are known to promote the phenolic acid biosynthetic pathway. Further investigation revealed that SmJRB1 triggered the activation of RAS1 transcription, according to the Dual-Luciferase (Dual-LUC) and yeast one-hybrid (Y1H) assays. Furthermore, yeast two-hybrid (Y2H) and biomolecular fluorescence complementation (BiFC) assays demonstrated that SmJRB1 interacted with the Jasmonate ZIM-domain proteins (JAZ) SmJAZ3 and SmJAZ9, and was most likely responsible for the co-regulation of the biosynthesis of phenolic acids. Overall, these results demonstrate that the SmJRB1 is an activator that enhances the phenolic acid accumulation in S. miltiorrhiza, and the study provides insights into the phenolic acid accumulation-related regulatory mechanisms in S. miltiorrhiza, as well as on the metabolic manipulation of the bioactive substances present.

Spatio-temporal control of phenylpropanoid biosynthesis by inducible complementation of a cinnamate 4-hydroxylase mutant.

Cinnamate 4-hydroxylase (C4H) is a cytochrome P450-dependent monooxygenase that catalyzes the second step of the general phenylpropanoid pathway. Arabidopsis reduced epidermal fluorescence 3 (ref3) mutants, which carry hypomorphic mutations in C4H, exhibit global alterations in phenylpropanoid biosynthesis and have developmental abnormalities including dwarfing. Here we report the characterization of a conditional Arabidopsis C4H line (ref3-2pOpC4H), in which wild-type C4H is expressed in the ref3-2 background. Expression of C4H in plants with well-developed primary inflorescence stems resulted in restoration of fertility and the production of substantial amounts of lignin, revealing that the developmental window for lignification is remarkably plastic. Following induction of C4H expression in ref3-2pOpC4H, we observed rapid and significant reductions in the levels of numerous metabolites, including several benzoyl and cinnamoyl esters and amino acid conjugates. These atypical conjugates were quickly replaced with their sinapoylated equivalents, suggesting that phenolic esters are subjected to substantial amounts of turnover in wild-type plants. Furthermore, using localized application of dexamethasone to ref3-2pOpC4H, we show that phenylpropanoids are not transported appreciably from their site of synthesis. Finally, we identified a defective Casparian strip diffusion barrier in the ref3-2 mutant root endodermis, which is restored by induction of C4H expression.

High resistance to insect growth disruptors and control failure likelihood in Brazilian populations of the tomato pinworm Tuta absoluta

This study aimed to compare the susceptibility of tomato pinworm, Tuta absoluta, from four Brazilian regions to insect growth disruptor (IGD) insecticides by tomato leaf-dip bioassays. Larval mortality was assessed seven days after exposure to insecticide-treated leaves. The control failure likelihood (CFL) was estimated after larval bioassays with IGDs recommended label rate. Mortality data were subjected to Probit and variance analysis. All populations of tomato pinworm showed significant resistance to one or more insecticides. The LC50 values ranged from 0.34 to 0.63 g L−1 (chlorfluazuron), 0.17 to 0.92 g L−1 (teflubenzuron), 0.13 to 1.28 g L−1 (novaluron), 0.29 to 0.46 g L−1 (lufenuron), and 0.71 to 4.60 g L−1 (methoxyfenozide). The resistance ratios varied from 1.2 to 1.8-fold (chlorfluazuron), 1.4 to 5.5-fold (teflubenzuron), 1.2 to 9.9-fold (novaluron), 1.0 to 1.6-fold (lufenuron), and 1.5 to 6.5-fold (methoxyfenozide). Despite the low resistance ratios ( 54%, indicating likely control failure at the IGD label rates. We detected significant variations among populations of T. absoluta for activity of enzymes glutathione S-transferases (GST), cytochrome P450-dependent monooxygenases (MFO) and α-esterase (EST), however there was no significant difference between the populations for activity of β-esterase (EST). The evolution of resistance in T. absoluta populations to IGDs observed in this study is likely due to high selection pressure, demanding insecticide resistance management practices and environmentally sustainable tactics to obtain a more effective control of this pest.

The methyl jasmonate-responsive transcription factor SmMYB1 promotes phenolic acid biosynthesis in Salvia miltiorrhiza

Water-soluble phenolic acids are major bioactive compounds in the medicinal plant species Salvia miltiorrhiza. Phenolic acid biosynthesis is induced by methyl jasmonate (MeJA) in this important Chinese herb. Here, we investigated the mechanism underlying this induction by analyzing a transcriptome library of S. miltiorrhiza in response to MeJA. Global transcriptome analysis identified the MeJA-responsive R2R3-MYB transcription factor-encoding gene SmMYB1. Overexpressing SmMYB1 significantly promoted phenolic acid accumulation and upregulated the expression of genes encoding key enzymes in the phenolic acid biosynthesis pathway, including cytochrome P450-dependent monooxygenase (CYP98A14). Dual-luciferase (dual-LUC) assays and/or an electrophoretic mobility shift assays (EMSAs) indicated that SmMYB1 activated the expression of CYP98A14, as well as the expression of genes encoding anthocyanin biosynthesis pathway enzymes, including chalcone isomerase (CHI) and anthocyanidin synthase (ANS). In addition, SmMYB1 was shown to interact with SmMYC2 to additively promote CYP98A14 expression compared to the action of SmMYB1 alone. Taken together, these results demonstrate that SmMYB1 is an activator that improves the accumulation of phenolic acids and anthocyanins in S. miltiorrhiza. These findings lay the foundation for in-depth studies of the molecular mechanism underlying MeJA-mediated phenolic acid biosynthesis and for the metabolic engineering of bioactive ingredients in S. miltiorrhiza.

Saposin D acting on macrophage bacteriostatic function in experimental tuberculosis infection

The protection against tuberculosis infection is largely determined by the ability of host tissue macrophages to limit the growth and spread of mycobacteria. Able to multiply within the host macrophages, mycobacteria have developed a number of protective mechanisms preventing phagosome-lysosome fusion, thereby evading damaging effects of lysosomal enzymes. Saposins are small, acidic, thermostable, non-enzymatic glycoproteins that participate as co-fac-tors in degradation of short oligosaccharide head group glycosphingolipids. Saposins A, B, C and D are formed in acidic endosomes due to cleavage of initial prosaposin molecule. The effect of saposins on human immune response is mediated by their involvement in presenting mycobacterial antigens on CD1 molecules. Preliminary studies with electron microscopy allowed to uncover saposin D-bound damaging effect on Mycobacterium tuberculosis in acidic environment. These data allowed us to suggest that saposin D is an important protective component fighting against TB infection. The aim of the study was to explore how saposin D deficiency might affect formation of anti-tuberculosis immune response and ability of macrophages to inhibit M. tuberculosis growth. Materials and methods. Interstitial pulmonary macrophages and peritoneal macrophages were isolated from wild type C57BL/6 strain and saposin D deficient C57BL/6-SapD-/- mouse strains. Results. It was found that as compared to macrophages from mice, macrophages from wild type strain significantly better controlled mycobacteria growth in vitro. To study an opportunity of compensating for deficient saposin D in peritoneal macrophages from C57BL/6-SapD-/- mice, a saposin D gene-bearing lentiviral vector was created. Transfection of SAPD-deficient peritoneal macrophages with expression vector compensated for saposin D deficiency in such cells and restored bactericidal function. The mechanisms of action for current anti-TB drugs are mediated by various metabolic pathways in mycobacteria (inhibited biosynthesis of fatty acids, arabinogalactan, peptidoglycan and protein; inhibition of DNA-dependent processes, proton pumps and cytochrome P450-dependent monooxygenases). Conclusion. It was shown that saposin D deficiency affects activation of macrophage bactericidal function in vitro. Our study data may be a prerequisite for biologically substantiated potential of using a vector construct bearing natural human protein gene such as saposin D, as a new anti-tuberculosis drug.