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Herbicides are the extensively used class of pesticides, which beside the active ingredient, in their formulation accompanying substances such as emulsifiers, surfactants and others is needed. The potential toxicity of these synthetic chemicals could pose serious risks to the human health, nontarget organism and environment. In this work we developed biodegradable lignin nanoparticles (LNPs) as environmentally friendly and controlled release carriers of 2,4-dichlorophenoxyacetic acid 2,4-D and 2-methyl-4-chlorophenoxyacetic acid MCPA. LNPs were synthesized via solvent-free nanoprecipitation, achieving high entrapment efficiencies of 90.7% (2,4-D) and 97.4% (MCPA), confirmed by ultraviolet–visible spectroscopy. In vitro release studies revealed sustained herbicide release in buffer solutions (pH 5.5–7.5), with 68–74% release over 72 h, compared to rapid release from commercial formulations. Bioactivity assays of on Descurainia sophia showed that LNP-encapsulated formulation of herbicides reduced weed dry weight by 62.31% and density by 56.09% compared to untreated controls, statistically matching the weed control efficacy of commercial formulations. Field trials further validated these results. LNP-encapsulated 2,4-D + MCPA reduced Amaranthus blitoides dry weight by 91.10% and density by 65.09%, while this new formulation decreased Chenopodium album dry weight and density by 96.01% and by 66.75%, respectively. Notably, lignin’s inherent biodegradability and non-toxic nature provide a sustainable alternative to conventional synthetic adjuvants, significantly reducing the risks of environmental contamination. Our study highlights the potential of lignin-based nanoencapsulation to preserve weed control efficacy while promoting environmentally friendly and safer herbicide formulations.

Resistance to auxinic herbicides is increasing in a range of dicotyledonous weed species, but in most cases the biochemical mechanism of resistance is unknown. Using 14C-labelled herbicide, the mechanism of resistance to 2,4-dichlorophenoxyacetic acid (2,4-D) in two wild radish (Raphanus raphanistrum L.) populations was identified as an inability to translocate 2,4-D out of the treated leaf. Although 2,4-D was metabolized in wild radish, and in a different manner to the well-characterized crop species wheat and bean, there was no difference in metabolism between the susceptible and resistant populations. Reduced translocation of 2,4-D in the latter was also not due to sequestration of the herbicide, or to reduced uptake by the leaf epidermis or mesophyll cells. Application of auxin efflux or ABCB transporter inhibitors to 2,4-D-susceptible plants caused a mimicking of the reduced-translocation resistance phenotype, suggesting that 2,4-D resistance in the populations under investigation could be due to an alteration in the activity of a plasma membrane ABCB-type auxin transporter responsible for facilitating long-distance transport of 2,4-D.

The study reports the response to herbicide of the 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading fungal strain Umbelopsis isabellina. A comparative analysis covered 41 free amino acids as well as 140 lipid species of fatty acids, phospholipids, acylglycerols, sphingolipids, and sterols. 2,4-D presence led to a decrease in fungal catalase activity, associated with a higher amount of thiobarbituric acid-reactive substances (TBARS). Damage to cells treated with the herbicide resulted in increased membrane permeability and decreased membrane fluidity. Detailed lipidomic profiling showed changes in the fatty acids composition such as an increase in the level of linoleic acid (C18:2). Moreover, an increase in the phosphatidylethanolamine/phosphatidylcholine ratio was observed. Analysis of fungal lipid profiles revealed that the presence of 2,4-D was accompanied by the accumulation of triacylglycerols, a decrease in ergosterol content, and a considerable rise in the level of sphingolipid ceramides. In the exponential phase of growth, increased levels of leucine, glycine, serine, asparagine, and hydroxyproline were found. The results obtained in our study confirmed that in the cultures of U. isabellina oxidative stress was caused by 2,4-D. The herbicide itself forced changes not only to membrane lipids but also to neutral lipids and amino acids, as the difference of tested compounds profiles between 2,4-D-containing and control samples was consequently lower as the pesticide degradation progressed. The presented findings may have a significant impact on the basic understanding of 2,4-D biodegradation and may be applied for process optimization on metabolomic and lipidomic levels.

Synthetic auxins are used in agriculture as herbicides worldwide, which leads to localized pollution and their potential entry into food crops during early developmental stages. Triticum aestivum L. is a major agricultural crop, and for this reason, understanding the mechanisms by which herbicides affect photosynthetic and lipid metabolic processes in wheat is crucial for assessing yield reduction risks. This study aimed to evaluate the toxic effects of three synthetic auxins, 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and clopyralid (CLD) on growth parameters, membrane permeability, lipid peroxidation (LPO) product content, fatty acid (FA) profiles, and photosynthetic pigment levels in both etiolated and green spring wheat seedlings. FA content was assessed using gas chromatography-mass spectrometry. The results revealed that NAA and 2,4-D exerted the most pronounced inhibitory effects on seedling growth, whereas 2,4-D and CLD increased membrane permeability. In etiolated seedlings exposed to synthetic auxins, there was an elevation in FA content noted. Conversely, in green seedlings, exposure to all tested synthetic auxins led to a reduction in FA content, particularly affecting polyunsaturated fatty acids (PUFAs), as well as declines in chlorophyll and carotenoid levels. CLD reduced odd-chain fatty acid content (OCFAs) and very long-chain fatty acid content (VLCFAs) to undetectable levels. The increase in LPO products under the action of 2,4-D and CLD indicates oxidative stress as a possible cause of the decrease in PUFA content in green seedlings. These findings suggest that synthetic auxins have detrimental impacts on the photosynthetic apparatus of wheat, which in turn may have negative consequences for its productivity.

Peroxisomes, single-membrane-bounded organelles with essentially oxidative metabolism, are key in plant responses to abiotic and biotic stresses. Recently, the presence of nitric oxide (NO) described in peroxisomes opened the possibility of new cellular functions, as NO regulates diverse biological processes by directly modifying proteins. However, this mechanism has not yet been analysed in peroxisomes. This study assessed the presence of S-nitrosylation in pea-leaf peroxisomes, purified S-nitrosylated peroxisome proteins by immunoprecipitation, and identified the purified proteins by two different mass-spectrometry techniques (matrix-assisted laser desorption/ionization tandem time-of-flight and two-dimensional nano-liquid chromatography coupled to ion-trap tandem mass spectrometry). Six peroxisomal proteins were identified as putative targets of S-nitrosylation involved in photorespiration, β-oxidation, and reactive oxygen species detoxification. The activity of three of these proteins (catalase, glycolate oxidase, and malate dehydrogenase) is inhibited by NO donors. NO metabolism/S-nitrosylation and peroxisomes were analysed under two different types of abiotic stress, i.e. cadmium and 2,4-dichlorophenoxy acetic acid (2,4-D). Both types of stress reduced NO production in pea plants, and an increase in S-nitrosylation was observed in pea extracts under 2,4-D treatment while no total changes were observed in peroxisomes. However, the S-nitrosylation levels of catalase and glycolate oxidase changed under cadmium and 2,4-D treatments, suggesting that this post-translational modification could be involved in the regulation of H₂O₂ level under abiotic stress.

Enzymes secreted by white rot fungi (WRF), such as laccase, offer a promising approach for the treatment of hazardous xenobiotic compounds. This study conducted a comprehensive analysis of the impact of the pesticides 2,4-dichlorophenoxyacetic acid (2,4-D) and chlorpyrifos on the laccase of Phlebia brevispora BAFC 633 through in vitro and bioinformatics analyses. The fungal strain was shown to be tolerant to both pesticides, with notable morphological and ultrastructural alterations in the mycelium. Laccase activity and two isoenzymes (53 and 70 kDa) were detected in all initial treatments. The laccase was concentrated for subsequent catalytic evaluation in the presence of both pesticides, showing high stability at a pH of 3.6 and a temperature range of 50–60 °C. The lacI gene, corresponding to this laccase, was modeled, and its structure revealed a defined catalytic pocket validated with a drug score of 0.61. Molecular docking estimated affinity energies of −5.06 and −9.41 Kcal mol−1 for 2,4-D and chlorpyrifos, respectively. Molecular Mechanics Poisson–Boltzmann Surface Area (MM/PBSA) analysis through 250 ns of molecular dynamics revealed stronger hydrophobic interactions of laccase with chlorpyrifos and highlighted the importance of residue His460 in stabilizing both complexes. Understanding the impact of these agrochemicals on the catalytic function of laccase is crucial for developing future biotechnological strategies involving this enzyme.

The effects of auxins 2,4-D (2,4-dichlorophenoxyacetic acid), NAA (1-naphthaleneacetic acid) or picloram (4-amino-3,5,6-trichloropicolinic acid; 9 µM) and cytokinin BA (benzyloadenine; 4.5 µM) applied in the early stages of somatic embryogenesis (SE) on specific stages of SE in Picea abies and P. omorika were investigated. The highest SE initiation frequency was obtained after 2,4-D application in P. omorika (22.00%) and picloram application in P. abies (10.48%). NAA treatment significantly promoted embryogenic tissue (ET) proliferation in P. abies, while 2,4-D treatment reduced it. This reduction was related to the oxidative stress level, which was lower with the presence of NAA in the proliferation medium and higher with the presence of 2,4-D. The reduced oxidative stress level after NAA treatment suggests that hydrogen peroxide (H2O2) acts as a signalling molecule and promotes ET proliferation. NAA and picloram in the proliferation medium decreased the further production and maturation of P. omorika somatic embryos compared with that under 2,4-D. The quality of the germinated P. abies embryos and their development into plantlets depended on the auxin type and were the highest in NAA-originated embryos. These results show that different auxin types can generate different physiological responses in plant materials during SE in both spruce species.

The microRNA miR393 has been shown to play a role in plant development and in the stress response by targeting mRNAs that code for the auxin receptors in Arabidopsis. In this study, we verified that two rice auxin receptor gene homologs (OsTIR1 and OsAFB2) could be targeted by OsmiR393 (Os for Oryza sativa). Two new phenotypes (increased tillers and early flowering) and two previously observed phenotypes (reduced tolerance to salt and drought and hyposensitivity to auxin) were observed in the OsmiR393-overexpressing rice plants. The OsmiR393-overexpressing rice demonstrated hyposensitivity to synthetic auxin-analog treatments. These data indicated that the phenotypes of OsmiR393-overexpressing rice may be caused through hyposensitivity to the auxin signal by reduced expression of two auxin receptor genes (OsTIR1 and OsAFB2). The expression of an auxin transporter (OsAUX1) and a tillering inhibitor (OsTB1) were downregulated by overexpression of OsmiR393, which suggested that a gene chain from OsmiR393 to rice tillering may be from OsTIR1 and OsAFB2 to OsAUX1, which affected the transportation of auxin, then to OsTB1, which finally controlled tillering. The positive phenotypes (increased tillers and early flowering) and negative phenotypes (reduced tolerance to salt and hyposensitivity to auxin) of OsmiR393-overexpressing rice present a dilemma for molecular breeding.

Callogenesis is one of the most powerful biotechnological approaches for in vitro secondary metabolite production and indirect organogenesis in Passiflora caerulea . Comprehensive knowledge of callogenesis and optimized protocol can be obtained by the application of a combination of machine learning (ML) and optimization algorithms. In the present investigation, the callogenesis responses (i.e., callogenesis rate and callus fresh weight) of P . caerulea were predicted based on different types and concentrations of plant growth regulators (PGRs) (i.e., 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP), 1-naphthaleneacetic acid (NAA), and indole-3-Butyric Acid (IBA)) as well as explant types (i.e., leaf, node, and internode) using multilayer perceptron (MLP). Moreover, the developed models were integrated into the genetic algorithm (GA) to optimize the concentration of PGRs and explant types for maximizing callogenesis responses. Furthermore, sensitivity analysis was conducted to assess the importance of each input variable on the callogenesis responses. The results showed that MLP had high predictive accuracy (R 2 > 0.81) in both training and testing sets for modeling all studied parameters. Based on the results of the optimization process, the highest callogenesis rate (100%) would be obtained from the leaf explant cultured in the medium supplemented with 0.52 mg/L IBA plus 0.43 mg/L NAA plus 1.4 mg/L 2,4-D plus 0.2 mg/L BAP. The results of the sensitivity analysis showed the explant-dependent impact of the exogenous application of PGRs on callogenesis. Generally, the results showed that a combination of MLP and GA can display a forward-thinking aid to optimize and predict in vitro culture systems and consequentially cope with several challenges faced currently in Passiflora tissue culture.

Abstract Background and Aims Resistance to synthetic auxin herbicides such as 2,4-dichlorophenoxyacetic acid (2,4-D) is increasing in weed populations worldwide, which is of concern given the recent introduction of synthetic auxin-resistant transgenic crops. Due to the complex mode of action of the auxinic herbicides, the mechanisms of evolved resistance remain largely uncharacterized. The aims of this study were to assess the level of diversity in resistance mechanisms in 11 populations of the problem weed Raphanus raphanistrum, and to use a high-throughput, whole-genome transcriptomic analysis on one resistant and one susceptible population to identify important changes in gene expression in response to 2,4-D. Methods Levels of 2,4-D and dicamba (3,6-dichloro-2-methoxybenzoic acid) resistance were quantified in a dose–response study and the populations were further screened for auxin selectivity, 2,4-D translocation and metabolism, expression of key 2,4-D-responsive genes and activation of the mitogen-activated proein kinase (MAPK) pathway. Potential links between resistance levels and mechanisms were assessed using correlation analysis. Key Results The transcriptomic study revealed early deployment of the plant defence response in the 2,4-D-treated resistant population, and there was a corresponding positive relationship between auxinic herbicide resistance and constitutive MAPK phosphorylation across all populations. Populations with shoot-wide translocation of 2,4-D had similar resistance levels to those with restricted translocation, suggesting that reduced translocation may not be as strong a resistance mechanism as originally thought. Differences in auxin selectivity between populations point to the likelihood of different resistance-conferring alterations in auxin signalling and/or perception in the different populations. Conclusions 2,4-D resistance in wild radish appears to result from subtly different auxin signalling alterations in different populations, supplemented by an enhanced defence response and, in some cases, reduced 2,4-D translocation. This study highlights the dangers of applying knowledge generated from a few populations of a weed species to the species as a whole.