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The canonical arsRBC genes of the ars1 operon in Pseudomonas putida KT2440, which confer tolerance to arsenate and arsenite, are followed by a series of additional ORFs culminating in phoN1. The phoN1 gene encodes an acetyltransferase that imparts resistance to the glutamine synthetase inhibitor herbicide phosphinothricin (PPT). The co‐expression of phoN1 and ars genes in response to environmental arsenic, along with the physiological effects, was analysed through transcriptomics of cells exposed to the oxyanion and phenotypic characterization of P. putida strains deficient in different components of the bifan motif governing arsenic resistance in this bacterium. Genetic separation of arsRBC and phoN1 revealed that their associated phenotypes operate independently, indicating that their natural co‐regulation is not functionally required for simultaneous response to the same signal. The data suggest a scenario of associative evolution, akin to Pavlovian conditioning, where two unrelated but frequently co‐occurring signals result in one regulating the other's response – even if there is no functional link between the signal and the response. Such surrogate regulatory events may provide an efficient solution to complex regulatory challenges and serve as a genetic patch to address transient gaps in evolving regulatory networks. [ABSTRACT FROM AUTHOR]

The fast spread of bacteria that are resistant to many classes of antibiotics (multidrug resistant) is a global threat to human and animal health with a worrisome scenario ahead. Novel therapeutical strategies are of crucial importance to combat this phenomenon. For this purpose, we investigated the antimicrobial properties of the naturally occurring tripeptide Bialaphos and a dipeptide L-leucyl-L-phosphinoithricin, the synthesis and diastereomers separation of which are herein described. We demonstrate that these compounds are effective on clinical isolates of the human pathogen Klebsiella pneumoniae, causing hospital-acquired and community-acquired infections. The tested isolates were remarkable for their resistance to more than 20 commercial antibiotics of different classes. Based on previous literature data and our experiments consisting of glutamine supplementation, we suggest that both compounds release phosphinothricin—a well-known nanomolar inhibitor of glutamine synthetase—after their penetration in the bacterial cells; and, in this way, exert their antibacterial effect by negatively affecting nitrogen assimilation in this pathogen. [ABSTRACT FROM AUTHOR]

Glufosinate is a key herbicide to manage glyphosate‐resistant weeds mainly because it is a broad‐spectrum herbicide, and transgenic glufosinate‐resistant crops are available. Although glufosinate use has increased exponentially over the past decade, the treated area with this herbicide is far less than that with glyphosate. This is because glufosinate often provides inconsistent performance in the field, which is attributed to several factors including environmental conditions, application technology, and weed species. Glufosinate is also highly hydrophilic and does not translocate well in plants, generally providing poor control of grasses and perennial species. In the soil, glufosinate is rapidly degraded by microorganisms, leaving no residual activity. While there have been concerns regarding glufosinate toxicology, its proper use can be considered safe. Glufosinate is a fast‐acting herbicide that was first discovered as a natural product, and is the only herbicide presently targeting glutamine synthetase. The mode of action of glufosinate has been controversial, and the causes for the rapid phytotoxicity have often been attributed to ammonia accumulation. Recent studies indicate that the contact activity of glufosinate results from the accumulation of reactive oxygen species and subsequent lipid peroxidation. Glufosinate disrupts both photorespiration and the light reactions of photosynthesis, leading to photoreduction of molecular oxygen, which generates reactive oxygen species. The new understanding of the mode of action provided new ideas to improve the herbicidal activity of glufosinate. Finally, a very few weed species have evolved glufosinate resistance in the field, and the resistance mechanisms are generally not well understood requiring further investigation. © 2020 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Active ingredient of the commercial herbicide BASTA (B), phosphinothricin, acts as an inhibitor of glutamine synthetase (GS), a key enzyme in ammonium assimilation. The treatment with BASTA leads to an elevation of ammonium levels in plants and further to various physiological alterations, ammonium toxicity and lethality. Results of the present study emphasize the complexity underlying control mechanisms that determine BASTA interaction with essential oil (EO) from Nepeta rtanjensis (Nr EO), bioherbicide inducing oxidative stress in target plants. Simultaneous application of Nr EO and BASTA, two agents showing differential mode of action, suspends BASTA-induced ammonium toxicity in Arabidopsis thaliana plants. This is achieved through maintaining GS activity, which sustains a sub-toxic and/or sub-lethal ammonium concentration in tissues. As revealed by the present study, regulation of GS activity, as influenced by BASTA and Nr EO, occurs at transcriptional, posttranscriptional, and/or posttranslational levels. Two genes encoding cytosolic GS, GLN1;1 and GLN1;3 , are highlighted as the main isozymes in Arabidopsis shoots contributing to Nr EO-induced overcoming of BASTA-generated ammonium toxicity. The effects of Nr EO might be ascribed to its major component nepetalactone, but the contribution of minor EO components should not be neglected. Although of fundamental significance, the results of the present study suggest possible low efficiency of BASTA in plantations of medicinal/aromatic plants such as Nepeta species. Furthermore, these results highlight the possibility of using Nr EO as a bioherbicide in BASTA-treated crop fields to mitigate the effect of BASTA residues in contaminated soils. [ABSTRACT FROM AUTHOR]

Main conclusion: Glufosinate is primarily toxic to plants due to a massive light-dependent generation of reactive oxygen species rather than ammonia accumulation or carbon assimilation inhibition. Glutamine synthetase (GS) plays a key role in plant nitrogen metabolism and photorespiration. Glufosinate (C5H12NO4P) targets GS and causes catastrophic consequences leading to rapid plant cell death, and the causes for phytoxicity have been attributed to ammonia accumulation and carbon assimilation restriction. This study aimed to examine the biochemical and physiological consequences of GS inhibition to identify the actual cause for rapid phytotoxicity. Monocotyledonous and dicotyledonous species with different forms of carbon assimilation (C3 versus C4) were selected as model plants. Glufosinate sensitivity was proportional to the uptake of herbicide between species. Herbicide uptake also correlated with the level of GS inhibition and ammonia accumulation in planta even with all species having the same levels of enzyme sensitivity in vitro. Depletion of both glutamine and glutamate occurred in glufosinate-treated leaves; however, amino acid starvation would be expected to cause a slow plant response. Ammonia accumulation in response to GS inhibition, often reported as the driver of glufosinate phytotoxicity, occurred in all species, but did not correlate with either reductions in carbon assimilation or cell death. This is supported by the fact that plants can accumulate high levels of ammonia but show low inhibition of carbon assimilation and absence of phytotoxicity. Glufosinate-treated plants showed a massive light-dependent generation of reactive oxygen species, followed by malondialdehyde accumulation. Consequently, we propose that glufosinate is toxic to plants not because of ammonia accumulation nor carbon assimilation inhibition, but the production of reactive oxygen species driving the catastrophic lipid peroxidation of the cell membranes and rapid cell death. [ABSTRACT FROM AUTHOR]

l-Phosphinothricin (glufosinate or 2-amino-4-((hydroxy(methyl) phosphinyl) butyric acid ammonium salt (AHPB)), which is a structural analog of glutamate, is a recognized herbicide that acts on weeds through inhibition of glutamine synthetase. Due to the structural similarity between phosphinothricin and some bisphosphonates (BPs), this study focuses on investigating the possibility of repurposing phosphinothricin as a bisphosphonate analogue, particularly in two medicine-related activities: image probing and as an anti-cancer drug. As BP is a competitive inhibitor of human farnesyl pyrophosphate synthase (HFPPS), in silico molecular docking and dynamic simulations studies were established to evaluate the binding and stability of phosphinothricin with HFPPS, while the results showed good binding and stability in the active site of the enzyme in relation to alendronate. For the purpose of inspecting bone-tissue accumulation of phosphinothricin, a technetium (99mTc)–phosphinothricin complex was developed and its stability and tissue distribution were scrutinized. The radioactive complex showed rapid, high and sustained uptake into bone tissues. Finally, the cytotoxic activity of phosphinothricin was tested against breast and lung cancer cells, with the results indicating cytotoxic activity in relation to alendronate. All the above results provide support for the use of phosphinothricin as a potential anti-cancer drug and of its technetium complex as an imaging probe. [ABSTRACT FROM AUTHOR]

Phosphinothricin (PPT) is a widely used and non-selective herbicide. PPT-resistance genes, especially PPT N -acetyltransferase genes, have been used in the development of transgenic PPT-resistant crops. However, there are only a limited number of available PPT-resistance genes for use in plant biotechnology. In this study, we found that Enterobacter LSJC7 is highly resistant to PPT and can acetylate PPT to N -acetyl phosphinothricin (Ac-PPT). Furthermore, a novel PPT N -acetyltransferase gene, named LsarsN , was identified from LSJC7. When LsarsN was expressed in E. coli AW3110, it confered resistance to PPT. Ac-PPT was detected in both the culture medium and cells of AW3110 expressing the LsarsN- pET22b plasmid. The purified LsArsN protein also showed strong N -acetylation ability in vitro , and its enzymatic kinetic curve was fitted with the Michaelis-Mentan equation. Compared with wild-type LsArsN, both R72A and R74A mutants showed significantly lower PPT N -acetylation ability. In summary, our results systematically characterized LsArsN with strong ability for PPT N -acetylation, which lays the groundwork for future research into the use of this novel gene, LsarsN , to create PPT-resistant crops. [Display omitted] • Enterobacter LSJC7 is highly resistant to phosphinothricin. • LSJC7 acetylates phosphinothricin to N -acetyl phosphinothricin. • E. coli expressing LsarsN confers resistance to phosphinothricin. • LsArsN acetylates phosphinothricin in vitro. [ABSTRACT FROM AUTHOR]

Objectives: To develop a host-vector system for heterologous protein co-expression and purification in the Trichoderma reesei (teleomorph Hypocrea jecorina) industrial strain RUT-C30. Results: The co-expression and purification system is based on: (i) an efficient and reliable selectable marker using bar (phosphinothricin acetyltransferase gene); (ii) a compact hygromycin B resistance marker; and (iii) a versatile integration plasmid for gene expression and knockout. Fluorescent protein genes were introduced into T. reesei and the corresponding proteins were purified from fermentation broth. The host-vector system was used in a proof-of-principle approach to achieve the co-expression of an alkaline endoglucanase and an alkaline cellobiohydrolase. Conclusions: This protocol can be used at an industrial scale to produce large amounts of proteins in T. reesei. [ABSTRACT FROM AUTHOR]

We compared the transformation frequency of a positive selection system, which employs an Escherichia coli-derived pmi (phosphomannose isomerase) gene as the selectable marker and mannose as the selective agent, to commonly used negative selection systems of bar (phosphinothricin acetyltransferase) /phosphinothricin (PPT), npt II (neomycin phosphotransferase) /G418 (geneticin), and hpt (hygromycin phosphotransferase) /Hygromycin. Transgenic sugarcane plants were obtained following biolistic transformation of callus with four different selectable marker genes. Under different selective agents, transformed callus was selectively proliferated on MS medium containing 13.57 μM of 2,4-D, regenerated on MS medium with 4.44 μM of BA, 9.29 μM of KT, and 0.89 μM of NAA, and rooted on half of MS medium with 13.38 μM of NAA and 0.44 μM of 6-BA. The results demonstrated that the transformation frequency of 4.2% scored in the pmi/mannose positive system was significantly higher than those in the negative selection systems (0.47% for bar/PPT, 1.38% for npt II/G418, and 0.63% for hpt/Hygromycin, respectively). Transgenic sugarcane plants using the pmi/mannose system were confirmed by polymerase chain reaction (PCR) analysis and chlorophenol red (CPR) assay. The CPR assay was found to efficiently and qualitatively detect the transgenic sugarcane from the nontransformed plants because medium color changes in the CPR assay were highly responsive to pmi gene expression and enzyme activity. Thus, a pmi/mannose positive selection system for sugarcane transformation was developed to obviate the use of negative selection systems based on herbicides and antibiotics. [ABSTRACT FROM AUTHOR]

Highlights: [•] A novel phosphinothricin N-acetyltransferase gene, repat, from the marine bacterium was isolated. [•] RePAT can utilize both l-phosphinothricin and l-methionine sulfoximine. [•] RePAT is a cold-adaptive enzyme. [Copyright &y& Elsevier]

We have developed marker-free transgenic wheat using a transcription factor, AtDREB1A cloned from Arabidopsis. Southern hybridization confirmed a transgenic event with a single copy insertion. PCR analysis of the T plants showed four were positive only for AtDREB1A. A T1 plant (HRCB3#17-37) was marker-free and had good expression of drought tolerance in comparison with untransformed plants. The leaf relative water content of this T transgenic plant was 12-15 % higher than that of the wild type during stress with an 8 % higher yield under water deficit conditions compared with wild type plants. [ABSTRACT FROM AUTHOR]

Highlights: [•] Phosphinothricin (PPT) was completely acetylated into N-AcPt with THF and Ac2O. [•] N-AcPt, nontoxic to WT rice, showed negative selection effect on argE transgenic rice. [•] ⩾0.1mgml−1 of N-AcPt induced self-destruction of leaf discs of argE transgenic rice. [•] Also, seed germination of them was completely restricted in ⩾0.5mgml−1 of N-AcPt. [•] This system will be useful for developing selectable marker free transgenic rice. [ABSTRACT FROM AUTHOR]

Herbicide phosphinothricin (PPT) inhibits glutamine synthetase (GS), a key enzyme in nitrogen assimilation, thus causing ammonia accumulation, glutamine depletion and eventually plant death. However, the growth response of Lotus corniculatus L. plants immersed in solutions with a broad range of PPT concentrations is biphasic, with pronounced stimulating effect on biomass production at concentrations ≤50 μM and growth inhibition at higher concentrations. The growth stimulation at low PPT concentrations is a result of activation of chloroplastic isoform GS2, while the growth suppression is caused by inhibition of both cytosolic GS1 and GS2 at higher PPT concentrations. Since the results are obtained in cell-free system (e.g. protein extracts), to which the principles of homeostasis are not applicable, this PPT effect is an unambiguous example of direct stimulation hormesis. A detailed molecular mechanism of concentration-dependent interaction of both PPT and a related GS inhibitor, methionine sulfoximine, with GS holoenzymes is proposed. The mechanism is in concurrence with all experimental and literature data. [ABSTRACT FROM AUTHOR]

l-Methionine sulfoximine (MSO) and dl-Phosphinothricin (PPT), two non-proteinogenic amino acids known as inhibitors of Glutamine Synthetase, cause a dose-dependent increase in the phosphorylation of the mTOR substrate S6 kinase 1. The effect is particularly evident in glutamine-depleted cells, where mTOR activity is very low, but is detectable for PPT also in the presence of glutamine. The stimulation of mTOR activity by either MSO or PPT is strongly synergized by essential amino acids. Thus, the non-proteinogenic amino acids MSO and PPT are mTOR activators. [ABSTRACT FROM AUTHOR]

Human tissue-plasminogen activator (t-PA) is a thrombolytic protein that plays an active role in dissolving fibrin clots by fibrinolysis and in activating the transition from plasminogen to plasmin in blood vessels. The recombinant tissue-plasminogen activator (rTPA) produced in plants is a soluble protein derived from human t-PA. In order to produce rTPA protein in plants, a DNA fragment encoding the t-PA protein was cloned into a plant binary plasmid that included the cauliflower mosaic virus 35S promoter, a tobacco etch virus (TEV) leader sequence, an N-terminal signal peptide of the alfala glucose-regulated endoplasmic reticular protein, and an 35S terminator. The insertion of the rTPA gene in genomic DNA of phospinothricin-resistant tobacco plants was confirmed by polymerase chain reaction (PCR). The presence of the rTPA-specific transcript in the total RNAs of transgenic plants leaves was verified by reverse transcription (RT)-PCR. Immunoblot analysis of the transformed tobacco leaves revealed single bands of 68-kDa rTPAs. Enzyme-linked immunosorbent assay experiments demonstrated that the highest level of rTPA expression was 0.0014% of the total soluble protein in tobacco plants. Fibrinolysis of rTPA was confirmed by fibrin plate assay and zymography. These studies demonstrate that plants can be employed as bioreactor systems for the production of an enzymatically active t-PA with properties similar to the recombinant t-PA produced by mammalian cells. [ABSTRACT FROM AUTHOR]

The toxicity of three antibiotics commonly used to eliminate Agrobacterium tumefaciens from plant tissue during transformation were tested to determine their effect on Pinus pinea L. morphogenesis. Cefotaxime and vancomycin at a dose of 250 µg ml−1, as well as ticarcillin at 300 µg ml−1, were essentially nontoxic to the culture and significantly enhanced regeneration and shoot development. Kanamycin, a widely used selection agent for plant transformation, strongly inhibited regeneration even at very low doses. On the contrary, phosphinothricin and its ammonium salt glufosinate in the commercial formulation Finale® proved to be the best selection agents because they were less toxic to stone pine cotyledons. Thus, they could be applied immediately after transformation to avoid escapes. Other schemes for selection and recovery of transgenic stone pine trees are discussed. [ABSTRACT FROM AUTHOR]

In a study to optimize the use of the bar gene as a selectable marker gene in plant transformation, the selection mechanism of phosphinothricin in vitro was studied. It was found that tissues with a high anabolic activity were more sensitive to the toxicity of ammonium than tissues with a low anabolic activity. However, tissues with a low anabolic and high catabolic activity were more sensitive to glutamine deprivation. The metabolic (anabolic and catabolic) activity of callusing hypocotyl explants of Brassica napus L. was influenced by the carbon source of the medium and the light condition. The expression of the bar gene regulated by the light-inducible promoter derived from the gene coding for the small subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase was also influenced by both light condition and carbon source. Finally, a bioassay to screen for, and to quantify the resistance of expiants to, phosphinothricin is presented. [ABSTRACT FROM AUTHOR]

Phosphinothricin (glufosinate), an irreversible inhibitor of glutamine synthetase, causes an inhibition of photosynthesis in C ( Sinapis alba) and C ( Zea mays) plants under atmospheric conditions (400 ppm CO, 21% O). This photosynthesis inhibition is proceeding slower in C leaves. Under non-photorespiratory conditions (1000 ppm CO, 2% O) there is no inhibition of photosynthesis. The inhibition of glutamine synthetase by phosphinothricin results in an accumulation of NH. The NH-accumulation is lower in C plants than in C plants. The inhibition of glutamine synthetase through phosphinothricin in mustard leaves results in a decrease in glutamine, glutamate, aspartate, asparagine, serine, and glycine. In contrast to this, a considerable increase in leucine and valine following phosphinothricin treatment is measured. With the addition of either glutamine, glutamate, aspartate, glycine or serine, photosynthesis inhibition by phosphinothricin can be reduced, although the NH-accumulation is greatly increased. This indicates that NH-accumulation cannot be the primary cause for photosynthesis inhibition by phosphinothricin. The investigations demonstrate the inhibition of transmination of glyoxylate to glycine in photorespiration through the total lack of amino donors. This could result in a glyoxylate accumulation inhibiting ribulose-1,5-bisphosphate-carboxylase and consequently CO-fixation. [ABSTRACT FROM AUTHOR]

Sugarcane being the major contributor of sugar and potential source of biofuel around the globe, occupies significant commercial importance. Red rot is the most devastating disease of sugarcane, severely affecting its quality as well as yield. Here we report the overexpression of SUGARWIN1 and SUGARWIN2 genes in any field crop for the first time. For this purpose, SUGAWIN1 and SUGARWIN2 were cloned downstream of maize ubiquitin (Ubi-1) promoter to construct two independent expression cassettes. The bar gene conferring resistance against phosphinothricin was used as selectable marker. Embryogenic calli of sugarcane were bombarded with both expression cassettes and selected on regeneration medium supplemented with phosphinothricin. The phosphinothricin-resistant shoots were rooted and then, analyzed using molecular tools at the genomic as well as transcriptomic levels. The transcriptomic analysis, using real time qPCR, showed that expression of SUGARWIN1 (SWO) and SUGARWIN2 (SWT) was higher in transgenic plants as compared to untransformed plants. Our results further demonstrated that over expression of these genes under maize ubiquitin (Ubi-1) promoter causes significant restriction in proliferation of red rot causal agent, Colletotrichum falcatum in sugarcane transgenic plants, under in vitro conditions. This report may open up exciting possibilities to extend this technology to other monocots for the development of crops with better ability to withstand fungal pathogens. [ABSTRACT FROM AUTHOR]

Phosphinothricin (PPT) is one of the most widely used herbicides. PTT targets glutamine synthetase (GS) activity in plants, and its phytotoxicity is ascribed to ammonium accumulation and reactive oxygen species bursts, which drives rapid lipid peroxidation of cell membranes. In agricultural fields, PPT is extensively sprayed on plant foliage; however, a portion of the herbicide reaches the soil. According to the present study, PPT absorbed via roots can be phytotoxic to Arabidopsis, inducing more adverse effects in roots than in shoots. Alterations in plant physiology caused by 10 days exposure to herbicide via roots are reflected through growth suppression, reduced chlorophyll content, perturbations in the sugar and organic acid metabolism, modifications in the activities and abundances of GS, catalase, peroxidase, and superoxide dismutase. Antagonistic interaction of Nepeta rtanjensis essential oil (NrEO) and PPT, emphasizes the existence of complex control mechanisms at the transcriptional and posttranslational level, which result in the mitigation of PPT-induced ammonium toxicity and in providing more efficient antioxidant defense of plants. Simultaneous application of the two agents in the field cannot be recommended; however, NrEO might be considered as the PPT post-treatment for reducing harmful effects of herbicide residues in the soil on non-target plants. [ABSTRACT FROM AUTHOR]