Inhibition of O-acetylserine (thiol) lyase as a promising new mechanism of action for herbicides.

Enzymes of the sulfur assimilation pathway of plants have been identified as potential targets for herbicide development, given their crucial role in synthesizing amino acids, coenzymes, and various sulfated compounds. In this pathway, O-acetylserine (thiol) lyase (OAS-TL; EC 2.5.1.47) catalyzes the synthesis of L-cysteine through the incorporation of sulfate into O-acetylserine (OAS). This study used an in silico approach to select seven inhibitors for OAS-TL. The in silico experiments revealed that S-benzyl-L-cysteine (SBC) had a better docking score (-7.0 kcal mol-1) than the substrate OAS (-6.6 kcal mol-1), indicating its suitable interaction with the active site of the enzyme. In vitro experiments showed that SBC is a non-competitive inhibitor of OAS-TL from Arabidopsis thaliana expressed heterologously in Escherichia coli, with a Kic of 4.29 mM and a Kiu of 5.12 mM. When added to the nutrient solution, SBC inhibited the growth of maize and morning glory weed plants due to the reduction of L-cysteine synthesis. Remarkably, morning glory was more sensitive than maize. As proof of its mechanism of action, L-cysteine supplementation to the nutrient solution mitigated the inhibitory effect of SBC on the growth of morning glory. Taken together, our data suggest that reduced L-cysteine synthesis is the primary cause of growth inhibition in maize and morning glory plants exposed to SBC. Furthermore, our findings indicate that inhibiting OAS-TL could potentially be a novel approach for herbicidal action.

The harmful acute effects of clomipramine in the rat liver: Impairments in mitochondrial bioenergetics.

Clomipramine, a tricyclic antidepressant used to treat depression and obsessive-compulsive disorder, has been linked to a few cases of acute hepatotoxicity. It is also recognized as a compound that hinders the functioning of mitochondria. Hence, the effects of clomipramine on mitochondria should endanger processes that are somewhat connected to energy metabolism in the liver. For this reason, the primary aim of this study was to examine how the effects of clomipramine on mitochondrial functions manifest in the intact liver. For this purpose, we used the isolated perfused rat liver, but also isolated hepatocytes and isolated mitochondria as experimental systems. According to the findings, clomipramine harmed metabolic processes and the cellular structure of the liver, especially the membrane structure. The considerable decrease in oxygen consumption in perfused livers strongly suggested that the mechanism of clomipramine toxicity involves the disruption of mitochondrial functions. Coherently, it could be observed that clomipramine inhibited both gluconeogenesis and ureagenesis, two processes that rely on ATP production within the mitochondria. Half-maximal inhibitory concentrations for gluconeogenesis and ureagenesis ranged from 36.87 µM to 59.64 µM. The levels of ATP as well as the ATP/ADP and ATP/AMP ratios were reduced, but distinctly, between the livers of fasted and fed rats. The results obtained from experiments conducted on isolated hepatocytes and isolated mitochondria unambiguously confirmed previous propositions about the effects of clomipramine on mitochondrial functions. These findings revealed at least three distinct mechanisms of action, including uncoupling of oxidative phosphorylation, inhibition of the FoF1-ATP synthase complex, and inhibition of mitochondrial electron flow. The elevation in activity of cytosolic and mitochondrial enzymes detected in the effluent perfusate from perfused livers, coupled with the increase in aminotransferase release and trypan blue uptake observed in isolated hepatocytes, provided further evidence of the hepatotoxicity of clomipramine. It can be concluded that impaired mitochondrial bioenergetics and cellular damage are important factors underlying the hepatotoxicity of clomipramine and that taking excessive amounts of clomipramine can lead to several risks including decreased ATP production, severe hypoglycemia, and potentially fatal outcomes.

Toluidine blue O directly and photodynamically impairs the bioenergetics of liver mitochondria: a potential mechanism of hepatotoxicity.

Toluidine blue O (TBO) is a phenothiazine dye that, due to its photochemical characteristics and high affinity for biomembranes, has been revealed as a new photosensitizer (PS) option for antimicrobial photodynamic therapy (PDT). This points to a possible association with membranous organelles like mitochondrion. Therefore, here we investigated its effects on mitochondrial bioenergetic functions both in the dark and under photostimulation. Two experimental systems were utilized: (a) isolated rat liver mitochondria and (b) isolated perfused rat liver. Our data revealed that, independently of photostimulation, TBO presented affinity for mitochondria. Under photostimulation, TBO increased the protein carbonylation and lipid peroxidation levels (up to 109.40 and 119.87%, respectively) and decreased the reduced glutathione levels (59.72%) in mitochondria. TBO also uncoupled oxidative phosphorylation and photoinactivated the respiratory chain complexes I, II, and IV, as well as the FoF1-ATP synthase complex. Without photostimulation, TBO caused uncoupling of oxidative phosphorylation and loss of inner mitochondrial membrane integrity and inhibited very strongly succinate oxidase activity. TBO's uncoupling effect was clearly seen in intact livers where it stimulated oxygen consumption at concentrations of 20 and 40 µM. Additionally, TBO (40 µM) reduced cellular ATP levels (52.46%) and ATP/ADP (45.98%) and ATP/AMP (74.17%) ratios. Consequently, TBO inhibited gluconeogenesis and ureagenesis whereas it stimulated glycogenolysis and glycolysis. In conclusion, we have revealed for the first time that the efficiency of TBO as a PS may be linked to its ability to photodynamically inhibit oxidative phosphorylation. In contrast, TBO is harmful to mitochondrial energy metabolism even without photostimulation, which may lead to adverse effects when used in PDT.

Reduction of Weed Growth under the Influence of Extracts and Metabolites Isolated from Miconia spp.

Weeds pose a problem, infesting areas and imposing competition and harvesting difficulties in agricultural systems. Studies that provide the use of alternative methods for weed control, in order to minimize negative impacts on the environment, have intensified. Native flora represents a source of unexplored metabolites with multiple applications, such as bioherbicides. Therefore, we aimed to carry out a preliminary phytochemical analysis of crude extracts and fractions of Miconia auricoma and M. ligustroides and to evaluate these and the isolated metabolites phytotoxicity on the growth of the target species. The growth bioassays were conducted with Petri dishes with lettuce, morning glory, and sourgrass seeds incubated in germination chambers. Phytochemical analysis revealed the presence of flavonoids, isolated myricetin, and a mixture of quercetin and myricetin. The results showed that seedling growth was affected in a dose-dependent manner, with the root most affected and the seedlings of the lettuce, morning glory, and sourgrass as the most sensitive species, respectively. Chloroform fractions and myricetin were the most inhibitory bioassays evaluated. The seedlings showed structural changes, such as yellowing, nonexpanded cotyledons, and less branched roots. These results indicate the phytotoxic potential of Miconia allelochemicals, since there was the appearance of abnormal seedlings and growth reduction.

The metabolic and toxic acute effects of phloretin in the rat liver.

The current study sought to evaluate the acute effects of phloretin (PH) on metabolic pathways involved in the maintenance of glycemia, specifically gluconeogenesis and glycogenolysis, in the perfused rat liver. The acute effects of PH on energy metabolism and toxicity parameters in isolated hepatocytes and mitochondria, as well as its effects on the activity of a few key enzymes, were also evaluated. PH inhibited gluconeogenesis from different substrates, stimulated glycogenolysis and glycolysis, and altered oxygen consumption. The citric acid cycle activity was inhibited by PH under gluconeogenic conditions. Similarly, PH reduced the cellular ATP/ADP and ATP/AMP ratios under gluconeogenic and glycogenolytic conditions. In isolated mitochondria, PH inhibited the electron transport chain and the FoF1-ATP synthase complex as well as acted as an uncoupler of oxidative phosphorylation, inhibiting the synthesis of ATP. PH also decreased the activities of malate dehydrogenase, glutamate dehydrogenase, glucose 6-phosphatase, and glucose 6-phosphate dehydrogenase. Part of the bioenergetic effects observed in isolated mitochondria was shown in isolated hepatocytes, in which PH inhibited mitochondrial respiration and decreased ATP levels. An aggravating aspect might be the finding that PH promotes the net oxidation of NADH, which contradicts the conventional belief that the compound operates as an antioxidant. Although trypan blue hepatocyte viability tests revealed substantial losses in cell viability over 120 min of incubation, PH did not promote extensive enzyme leakage from injured cells. In line with this effect, only after a lengthy period of infusion did PH considerably stimulate the release of enzymes into the effluent perfusate of livers. In conclusion, the increased glucose release caused by enhanced glycogenolysis, along with suppression of gluconeogenesis, is the opposite of what is predicted for antihyperglycemic agents. These effects were caused in part by disruption of mitochondrial bioenergetics, a result that should be considered when using PH for therapeutic purposes, particularly over long periods and in large doses.

Inhibiting tricin biosynthesis improves maize lignocellulose saccharification.

Lignin is a technological bottleneck to convert polysaccharides into fermentable sugars, and different strategies of genetic-based metabolic engineering have been applied to improve biomass saccharification. Using maize seedlings grown hydroponically for 24 h, we conducted a quick non-transgenic approach with five enzyme inhibitors of the lignin and tricin pathways. Two compounds [3,4-(methylenedioxy)cinnamic acid: MDCA and 2,4-pyridinedicarboxylic acid: PDCA] revealed interesting findings on root growth, lignin composition, and saccharification. By inhibiting hydroxycinnamoyl-CoA ligase, a key enzyme of phenylpropanoid pathway, MDCA decreased the lignin content and improved saccharification, but it decreased root growth. By inhibiting flavone synthase, a key enzyme of tricin biosynthesis, PDCA decreased total lignin content and improved saccharification without affecting root growth. PDCA was three-fold more effective than MDCA, suggesting that controlling lignin biosynthesis with enzymatic inhibitors may be an attractive strategy to improve biomass saccharification.

Cadmium uncouples mitochondrial oxidative phosphorylation and induces oxidative cellular stress in soybean roots.

Cadmium (Cd) inhibits soybean root growth, but its exact mode of action is still not completely understood. We evaluated the effects of Cd on growth, mitochondrial respiration, lipid peroxidation, total phenols, glutathione, and activities of lipoxygenase (LOX), superoxide dismutase (SOD), and catalase (CAT) in soybean roots. In primary roots, Cd stimulated KCN-insensitive respiration and KCN-SHAM-insensitive respiration, indicating the involvement of the alternative oxidase (AOX) pathway, while it decreased KCN-sensitive respiration, suggesting an inhibition of the cytochrome oxidase pathway (COX). In isolated mitochondria, Cd uncoupled the oxidative phosphorylation since it decreased state III respiration (coupled respiration) and ADP/O and respiratory control ratios, while it increased state IV respiration (depletion of exogenously added ADP). The uncoupling effect increased extramitochondrial LOX activity, lipid peroxidation, and oxidized and reduced glutathione, which induced an antioxidant response with enhanced SOD and CAT activities. In brief, our findings reveal that Cd acts as an uncoupler of the mitochondrial oxidative phosphorylation in soybean roots, disturbing cellular respiration and inducing oxidative cellular stress.

The photodynamic and intrinsic effects of Azure B on mitochondrial bioenergetics and the consequences of its intrinsic effects on hepatic energy metabolism.

BACKGROUND: The present study aimed to characterize the intrinsic and photodynamic effects of azure B (AB) on mitochondrial bioenergetics, as well as the consequences of its intrinsic effects on hepatic energy metabolism. METHODS: Two experimental systems were utilized: (a) isolated rat liver mitochondria and (b) isolated perfused rat liver. RESULTS: AB interacted with mitochondria regardless of photostimulation, but its binding degree was reduced by mitochondrial energization. Under photostimulation, AB caused lipid peroxidation and protein carbonylation and decreased the content of reduced glutathione (GSH) in mitochondria. AB impaired mitochondrial bioenergetics in at least three distinct ways: (1) uncoupling of oxidative phosphorylation; (2) photoinactivation of complexes I and II; and (3) photoinactivation of the FoF1-ATP synthase complex. Without photostimulation, AB also demonstrated mitochondrial toxicity, which was characterized by the induction of lipid peroxidation, loss of inner mitochondrial membrane integrity, and uncoupling of oxidative phosphorylation. The perfused rat liver experiments showed that mitochondria were one of the major targets of AB, even in intact cells. AB inhibited gluconeogenesis and ureagenesis, two biosynthetic pathways strictly dependent on intramitochondrially generated ATP. Contrariwise, AB stimulated glycogenolysis and glycolysis, which are required compensatory pathways for the inhibited oxidative phosphorylation. Similarly, AB reduced the cellular ATP content and the ATP/ADP and ATP/AMP ratios. CONCLUSIONS: Although the properties and severe photodynamic effects of AB on rat liver mitochondria might suggest its usefulness in PDT treatment of liver tumors, this possibility should be considered with precaution given the toxic intrinsic effects of AB on mitochondrial bioenergetics and energy-linked hepatic metabolism.

The photosensitiser azure A disrupts mitochondrial bioenergetics through intrinsic and photodynamic effects.

Azure A (AA) is a cationic molecule of the class of phenothiazines that has been applied in vitro as a photosensitising agent in photodynamic antimicrobial chemotherapy. It is a di-demethylated analogue of methylene blue (MB), which has been demonstrated to be intrinsically and photodynamically highly active on mitochondrial bioenergetics. However, as far as we know, there are no studies about the photodynamic effects of AA on mammalian mitochondria. Therefore, this investigation aimed to characterise the intrinsic and photodynamic acute effects of AA (0.540 µM) on isolated rat liver mitochondria, isolated hepatocytes, and isolated perfused rat liver. The effects of AA were assessed by evaluating several parameters of mitochondrial bioenergetics, oxidative stress, cell viability, and hepatic energy metabolism. The photodynamic effects of AA were assessed under simulated hypoxic conditions, a suitable way for mimicking the microenvironment of hypoxic solid tumour cells. AA interacted with the mitochondria and, upon photostimulation (10 min of light exposure), produced toxic amounts of reactive oxygen species (ROS), which damaged the organelle, as demonstrated by the high levels of lipid peroxidation and protein carbonylation. The photostimulated AA also depleted the GSH pool, which could compromise the mitochondrial antioxidant defence. Bioenergetically, AA photoinactivated the complexes I, II, and IV of the mitochondrial respiratory chain and the F1FO-ATP synthase complex, sharply inhibiting the oxidative phosphorylation. Upon photostimulation (10 min of light exposure), AA reduced the efficiency of mitochondrial energy transduction and oxidatively damaged lipids in isolated hepatocytes but did not decrease the viability of cells. Despite the useful photobiological properties, AA presented noticeable dark toxicity on mitochondrial bioenergetics, functioning predominantly as an uncoupler of oxidative phosphorylation. This harmful effect of AA was evidenced in isolated hepatocytes, in which AA diminished the cellular ATP content. In this case, the cells exhibited signs of cell viability reduction in the presence of high AA concentrations, but only after a long time of incubation (at least 90 min). The impairments on mitochondrial bioenergetics were also clearly manifested in intact perfused rat liver, in which AA diminished the cellular ATP content and stimulated the oxygen uptake. Consequently, gluconeogenesis and ureogenesis were strongly inhibited, whereas glycogenolysis and glycolysis were stimulated. AA also promoted the release of cytosolic and mitochondrial enzymes into the perfusate concomitantly with inhibition of oxygen consumption. In general, the intrinsic and photodynamic effects of AA were similar to those of MB, but AA caused some distinct effects such as the photoinactivation of the complex IV of the mitochondrial respiratory chain and a diminution of the ATP levels in the liver. It is evident that AA has the potential to be used in mitochondria-targeted photodynamic therapy, even under low oxygen concentrations. However, the fact that AA directly disrupts mitochondrial bioenergetics and affects several hepatic pathways that are linked to ATP metabolism, along with its ability to perturb cellular membranes and its little potential to reduce cell viability, could result in significant adverse effects especially in long-term treatments.

Aluminum oxide nanoparticles affect the cell wall structure and lignin composition slightly altering the soybean growth.

Aluminum oxide (Al2O3) nanoparticles (NPs) are among the nanoparticles most used industrially, but their impacts on living organisms are widely unknown. We evaluated the effects of 50-1000 mg L-1 Al2O3 NPs on the growth, metabolism of lignin and its monomeric composition in soybean plants. Al2O3 NPs did not affect the length of roots and stems. However, at the microscopic level, Al2O3 NPs altered the root surface inducing the formation of cracks near to root apexes and damage to the root cap. The results suggest that Al2O3 NPs were internalized and accumulated into the cytosol and cell wall of roots, probably interacting with organelles such as mitochondria. At the metabolic level, Al2O3 NPs increased soluble and cell wall-bound peroxidase activities in roots and stems but reduced phenylalanine ammonia-lyase activity in stems. Increased lignin contents were also detected in roots and stems. The Al2O3 NPs increased the p-hydroxyphenyl monomer levels in stems but reduced them in roots. The total phenolic content increased in roots and stems; cell wall-esterified p-coumaric and ferulic acids increased in roots, while the content of p-coumaric acid decreased in stems. In roots, the content of ionic aluminum (Al+3) was extremely low, corresponding to 0.0000252% of the aluminum applied in the nanoparticulate form. This finding suggests that all adverse effects observed were due to the Al2O3 NPs only. Altogether, these findings suggest that the structure and properties of the soybean cell wall were altered by the Al2O3 NPs, probably to reduce its uptake and phytotoxicity.

Suppression of a BAHD acyltransferase decreases p-coumaroyl on arabinoxylan and improves biomass digestibility in the model grass Setaria viridis.

Grass cell walls have hydroxycinnamic acids attached to arabinosyl residues of arabinoxylan (AX), and certain BAHD acyltransferases are involved in their addition. In this study, we characterized one of these BAHD genes in the cell wall of the model grass Setaria viridis. RNAi silenced lines of S. viridis (SvBAHD05) presented a decrease of up to 42% of ester-linked p-coumarate (pCA) and 50% of pCA-arabinofuranosyl, across three generations. Biomass from SvBAHD05 silenced plants exhibited up to 32% increase in biomass saccharification after acid pre-treatment, with no change in total lignin. Molecular dynamics simulations suggested that SvBAHD05 is a p-coumaroyl coenzyme A transferase (PAT) mainly involved in the addition of pCA to the arabinofuranosyl residues of AX in Setaria. Thus, our results provide evidence of p-coumaroylation of AX promoted by SvBAHD05 acyltransferase in the cell wall of the model grass S. viridis. Furthermore, SvBAHD05 is a promising biotechnological target to engineer crops for improved biomass digestibility for biofuels, biorefineries and animal feeding.

Feruloyl esterase activity and its role in regulating the feruloylation of maize cell walls.

Cell walls of grasses have ferulic acid (FA) ester-linked to the arabinosyl substitutions of arabinoxylan (AX). Feruloyl esterases (FAE) are carboxylic acid esterases that release FA from cell walls and synthetic substrates. Despite the importance of FA for cell wall recalcitrance and in response to biotic and abiotic stresses, the physiological function of plant FAEs remains unclear. Here, we developed a simple method for the determination of FAE activity (ZmFAE) in maize using the total protein extract and investigated its role in regulating the feruloylation of cell wall. The method includes a single protein extraction and enzymatic reaction with protein concentration as low as 65 µg at 35 °C for 30 min, using methyl ferulate as the substrate. The methodology allowed the determination of the apparent Km (392.82 µM) and Vmax (79.15 pkat mg-1 protein). We also found that ZmFAE activity was correlated (r = 0.829) with the levels of FA in seedling roots, plant roots and leaves of maize. Furthermore, the exposure to osmotic stress resulted in a 50% increase in ZmFAE activity in seedling roots. These data suggest that FAE-catalyzed reaction is important for cell wall feruloylation during plant development and in response to abiotic stress. We conclude proposing a model for the feruloylation and deferuloylation of AX, which explains the role of FAE in regulating the levels of ester-linked FA. Our model might orient further studies investigating the role of plant FAEs and assist strategies for genetic engineering of grasses to obtain plants with reduced biomass recalcitrance.

Cell wall remodeling under salt stress: Insights into changes in polysaccharides, feruloylation, lignification, and phenolic metabolism in maize.

Although cell wall polymers play important roles in the tolerance of plants to abiotic stress, the effects of salinity on cell wall composition and metabolism in grasses remain largely unexplored. Here, we conducted an in-depth study of changes in cell wall composition and phenolic metabolism induced upon salinity in maize seedlings and plants. Cell wall characterization revealed that salt stress modulated the deposition of cellulose, matrix polysaccharides and lignin in seedling roots, plant roots and stems. The extraction and analysis of arabinoxylans by size-exclusion chromatography, 2D-NMR spectroscopy and carbohydrate gel electrophoresis showed a reduction of arabinoxylan content in salt-stressed roots. Saponification and mild acid hydrolysis revealed that salinity also reduced the feruloylation of arabinoxylans in roots of seedlings and plants. Determination of lignin content and composition by nitrobenzene oxidation and 2D-NMR confirmed the increased incorporation of syringyl units in lignin of maize roots. Salt stress also induced the expression of genes and the activity of enzymes enrolled in phenylpropanoid biosynthesis. The UHPLC-MS-based metabolite profiling confirmed the modulation of phenolic profiling by salinity and the accumulation of ferulate and its derivatives 3- and 4-O-feruloyl quinate. In conclusion, we present a model for explaining cell wall remodeling in response to salinity.

Entacapone improves saccharification without affecting lignin and maize growth: An in silico, in vitro, and in vivo approach.

Caffeate 3-O-methyltransferase (COMT) catalyzes the methylation of the 3-hydroxyl group of caffeate to produce ferulate, an important precursor of the lignin biosynthesis. As a crucial drawback for biofuel production, lignin limits the enzymatic hydrolysis of polysaccharides to result in fermentable sugars. We hypothesized that a controlled inhibition of maize COMT can be an efficient approach to reduce ferulate and lignin, thus improving the saccharification process. First, we applied in silico techniques to prospect potential inhibitors of ZmaysCOMT, and the nitrocatechol entacapone was selected. Second, in vitro assays confirmed the inhibitory effect of entacapone on maize COMT. Finally, in vivo experiments revealed that entacapone reduced the contents of cell-wall-esterified hydroxycinnamates and increased saccharification of stems (18%) and leaves (70%), without negatively affecting maize growth and lignin biosynthesis. This non-genetically modified approach can be an alternative strategy to facilitate the enzymatic hydrolysis of biomass polysaccharides and increase saccharification for bioethanol production.

Chromatographic determination of shikimate for identification of conventional soybean and glyphosate resistant soybean / Determinação cromatográfica de shikimato para identificação de soja convencional e soja resistente ao glifosato

A sensitive and reliable process was established using high-performance liquid chromatography (HPLC) to distinguish conventional varieties of glyphosate-resistant genetically modified crops via shikimate detection in soybean (Glycine max L. Merril) seeds. Glyphosate has a well-defined mechanism of action. It is the only herbicide that specifically inhibits 5-enolpiruvilshikimate-3-phosphate synthase (EPSPS E.C. 2.5.1.19), which catalyzes the condensation of shikimate with phosphoenolpyruvate. This study is based on the concept that shikimate significantly accumulates in soybean plant tissues after EPSPS inhibition by glyphosate. In plants not subjected to glyphosate, shikimate is not easily detected because it quickly metabolizes into shikimate 3-phosphate and subsequently into 5-enolpiruvilshikimate 3-phosphate through the action of EPSPS. Conversely, in non-genetically modified plants subjected to glyphosate, shikimate metabolism is impaired, resulting in its accumulation. This metabolite can be detected in extremely low quantities (in the microgram range), through HPLC. In this study, six different contrasts were analyzed, each being formed by a transgenic cultivation and its parental strain, subject or not subject to the treatment of soaking with a 0.6% glyphosate solution. Chromatographic analyses indicated shikimate accumulation only in conventional cultivars with seeds previously soaked in a 0.6% glyphosate solution. Thus, this shikimate detection method can be used as a rapid and accurate means to distinguish soybeans with glyphosate-resistant qualities.

Este estudo estabelece um processo, sensível e confiável, com aplicação de cromatografia líquida de alta eficiência (HPLC) para distinguir variedades de soja convencionais de geneticamente modificadas, resistentes ao glifosato, por detecção de chiquimato nas sementes. O mecanismo de ação doglifosato é bem definido. É o único herbicida que inibe especificamente a enzima 5-enolpiruvilchiquimato-3-fosfato sintase (EPSPS, E.C. 2.5.1.19), que catalisa a condensação do chiquimato com fosfoenolpiruvato. O trabalho está baseado na concepção do chiquimato se acumular significativamente nos tecidos vegetais de soja convencional, após a inibição da EPSP sintase pelo glifosato. Em plantas não submetidas ao glifosato, o chiquimato não é facilmente detectado, pois rapidamente é metabolizado a chiquimato 3-fosfato e, a seguir, em 5-enolpiruvilchiquimato 3-fosfato, pela ação da EPSPS. Por outro lado, em plantas não geneticamente modificadas submetidas ao glifosato, a metabolização do chiquimato é prejudicada, resultando em seu acúmulo. Este metabólito pode ser detectado em quantidades extremamente baixas (na faixa de µg), por HPLC. Neste trabalho foram analisados seis contrastes diferentes, sendo cada contraste formado por uma cultivar transgênica e sua respectiva cultivar parental convencional, submetidas ou não a embebição com solução de glifosato 0,6%. As análises cromatográficas indicaram o acúmulo de chiquimato apenas em cultivares convencionais, nas quais as sementes foram previamente embebidas em solução de glifosato 0,6%. Os resultados demonstraram que adetecção de chiquimato pode ser utilizada como um método rápido e preciso na diferenciação de soja resistente ao glifosato de soja convencional.

Exogenous application of rosmarinic acid improves saccharification without affecting growth and lignification of maize.

Biomimetically incorporated into the lignin structure, rosmarinic acid improves in vitro maize cell wall saccharification; however, no in planta studies have been performed. We hypothesized that rosmarinic acid, itself, could inducer saccharification without disturbing plant growth. Its effects on growth, enzymes of the phenylpropanoid pathway, lignin, monomeric composition, and saccharification of maize were evaluated. In a short-term (24 h) exposure, rosmarinic acid caused deleterious effects on maize roots, inhibiting the first enzymes of the phenylpropanoid pathway, phenylalanine ammonia-lyase and tyrosine ammonia-lyase, altering lignin composition and slightly increasing saccharification. In a long-term (14 d) exposure, rosmarinic acid increased saccharification of maize stems by about 50% without any deleterious effects on plant growth, the phenylpropanoid pathway and lignin formation. This demonstrated that exogenous application of rosmarinic acid on maize plants improved saccharification, and represented an interesting approach in facilitating enzymatic hydrolysis of biomass polysaccharides and increasing bioethanol production.

Feruloyl esterases: Biocatalysts to overcome biomass recalcitrance and for the production of bioactive compounds.

Ferulic acid and its hydroxycinnamate derivatives represent one of the most abundant forms of low molecular weight phenolic compounds in plant biomass. Feruloyl esterases are part of a microorganism's plant cell wall-degrading enzymatic arsenal responsible for cleaving insoluble wall-bound hydroxycinnamates and soluble cytosolic conjugates. Stimulated by industrial requirements, accelerating scientific discoveries and knowledge transfer, continuous improvement efforts have been made to identify, create and repurposed biocatalysts dedicated to plant biomass conversion and biosynthesis of high-added value molecules. Here we review the basic knowledge and recent advances in biotechnological characteristics and the gene content encoding for feruloyl esterases. Information about several enzymes is systematically organized according to their function, biochemical properties, substrate specificity, and biotechnological applications. This review contributes to further structural, functional, and biotechnological R&D both for obtaining hydroxycinnamates from agricultural by-products as well as for lignocellulose biomass treatments aiming for production of bioethanol and other derivatives of industrial interest.

Trans-aconitic acid inhibits the growth and photosynthesis of Glycine max.

Grasses producing trans-aconitic acid, a geometric isomer of cis-aconitic acid, are often used in Glycine max rotation systems. However, the effects of trans-aconitic acid on Glycine max are unknown. We conducted a hydroponic experiment to evaluate the effects of 2.5-10 mM trans-aconitic acid on Glycine max growth and photosynthesis. The results revealed that the enhanced H2O2 production in the roots increased the membrane permeability and reduced the water uptake. These effects culminated with a reduced stomatal conductance (gs), which seems to be the main cause for a decreased photosynthetic rate (A). Due to low gs, the limited CO2 assimilation may have overexcited the photosystems, as indicated by the high production of H2O2 in leaves. After 96 h of incubation, and due to H2O2-induced damage to photosystems, a probable non-stomatal limitation for photosynthesis contributed to reducing A. This is corroborated by the significant decrease in the quantum yield of electron flow through photosystem II in vivo (ΦPSII) and the chlorophyll content. Taken together, the damage to the root system and photosynthetic apparatus caused by trans-aconitic acid significantly reduced the Glycine max plant growth.

Lignin-induced growth inhibition in soybean exposed to iron oxide nanoparticles.

Plants are occasionally exposed to environmental perturbations that limit their growth. One of these perturbations is the exposure to and interaction with various nanoparticles (NPs) that are discarded continuously into the environment. Hitherto, no study has been carried out evaluating the effects of iron oxide (γ-Fe2O3) NPs on soybean growth and lignin formation, as proposed herein. For comparative purposes, we also submitted soybean plants to non-nanoparticulate iron (FeCl3). Exposure of the plants to γ-Fe2O3 NPs increased cell wall-bound peroxidase (POD) activity but decreased phenylalanine ammonia lyase (PAL) activity due, probably, to the negative feedback of accumulated phenolic compounds. In contrast, FeCl3 decreased cell wall-bound POD activity. Both γ-Fe2O3 NPs and FeCl3 increased the lignin content of roots and stems. However, significant lignin-induced growth inhibition was noted only in stems after exposure to NPs, possibly due to changes in lignin monomer composition. In this case, γ-Fe2O3 NPs decreased the guaiacyl monomer content of roots but increased that of stems. The high levels of monomer guaiacyl in stems resulting from the action of γ-Fe2O3 NPs decreased syringyl/guaiacyl ratios, generating more highly cross-linked lignin followed by the stiffening of the cell wall and growth inhibition. In contrast, FeCl3 increased the contents of monomers p-hydroxyphenyl and syringyl in roots. The observed increase in the syringyl/guaiacyl ratio in plant roots submitted to FeCl3 agrees with the lack of effect on growth, due to the formation of a less condensed lignin. In brief, we here describe that γ-Fe2O3 NPs and FeCl3 act differently in soybean plants.