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Cyhalofop-butyl and pyribenzoxim are commonly used herbicides in rice-crayfish co-culture fields. In actual production, weed control in paddy fields is inseparable from cyhalofop-butyl and pyribenzoxim, while its risk to P. clarkii is still unclear. The present study investigated the risk of acute and subchronic toxicity of cyhalofop-butyl and pyribenzoxim to P. clarkii. The results showed that cyhalofop-butyl and pyribenzoxim exposure for 28 days could accumulate in P. clarkii muscle and inhibit P. clarkii growth. Further research found that the malondialdehyde (MDA) level and glutathione-S-transferase (GST) activity in muscle of P. clarkii were significantly increased after exposure to cyhalofop-butyl and pyribenzoxim (4 days and 28 days), and the superoxide dismutase (SOD) and catalase (CAT) activities were significantly altered. Histological results also confirmed cyhalofop-butyl and pyribenzoxim-induced muscle damage in P. clarkii. Additionally, after 28 days exposure to 1.02 mg/L cyhalofop-butyl and 10.4 mg/L pyribenzoxim, transcriptome analysis identified 2029 and 4246 differentially expressed genes (DEGs), respectively. Exposure to 1.02 mg/L cyhalofop-butyl significantly altered metabolism-related pathways, such as drug metabolism-other enzymes, glutathione metabolism, drug metabolism-cytochrome P450, fatty acid biosynthesis and fatty acid degradation. While the pathways related to antioxidant system and nutrient substances synthesis and metabolic were significantly enriched after exposure to 10.4 mg/L pyribenzoxim. This research has significant implications for scientific and rational use of herbicides under rice-crayfish co-culture and will contribute to the development of the highly productive agricultural model.

Background The photosystem II (PSII)-inhibiting herbicides are important for Australian farmers to control Lolium rigidum Gaud. and other weed species in trazine tolerant (TT)-canola fields. A L. rigidum population (R) collected from a TT-canola field from Western Australia showed multiple resistance to PSII, acetyl-coenzyme A carboxylase (ACCase) and acetolactate synthase (ALS) inhibitors. The mechanisms of multiple resistance in this R population were determined. Results The R population showed a low-level (about 3.0-fold) resistance to the PSII-inhibiting herbicides metribuzin and atrazine. Sequencing of the psbA gene revealed no differences between the R and susceptible (S) sequences. Furthermore, [14 C]-metribuzin experiments found no significant difference in metribuzin foliar uptake and translocation between the R and S plants. However, [14 C]-metribuzin metabolism in R plants was 2.3-fold greater than in S plants. The cytochrome P450 monooxygenase inhibitor piperonyl butoxide (PBO) enhanced plant mortality response to metribuzin and atrazine in both R and S populations. In addition, multiple resistance to ALS and ACCase inhibitors are due to known resistance mutations in ALS and ACCase genes. Conclusion The results demonstrate that enhanced metribuzin metabolism likely involving cytochrome P450 monooxygenase contributes to metribuzin resistance in Lolium rigidum. This is the first report of metabolic resistance to the PSII-inhibiting herbicide metribuzin in Australian Lolium rigidum. © 2020 Society of Chemical Industry.

Penoxsulam is an important herbicide for the control of Echinochloa crus-galli (L.) P. Beauv. Two resistant populations 17GA (R1) and 16NXB (R2) showed 17- and 3-fold resistance to penoxsulam, respectively. A known resistance mutation of Trp-574-Leu in ALS gene and enhanced rates of penoxsulam metabolism likely involving GST contribute to penoxsulam resistance in R1 population. This population had resistance to the ALS-inhibitors pyribenzoxim and bispyribac‑sodium and the auxin herbicide quinclorac, but was susceptible to ACCase-inhibitors quizalofop-p-ethyl and cyhalofop-butyl. No known mutations in the ALS gene conferring target site resistance to ALS-inhibiting herbicides were presented in R2 population. However, penoxsulam metabolism in R2 plants was about 4-fold greater than in susceptible population 14YC (S0) plants. The enzyme inhibitors piperonyl butoxide, malathion and 4-chloro-7-nitrobenzoxadiazole reversed penoxsulam resistance in this population. GST and P450 enzyme activities and the genes of GST1-1, GST1-2, GST1-3, CYP81A18, CYP81A12, CYP81A21 were increased significantly in R2 population. These results indicate that multiple resistance mechanisms had occurred in E. crus-galli populations in central China and resistance needs to be managed effectively by diverse chemical and non-chemical methods.

During bacterial conjugation, plasmid DNA is transferred from cell to cell. In Agrobacterium fabrum, conjugation is regulated by the phytochrome photoreceptors Agp1 and Agp2. Both contribute equally to this regulation. Agp1 and Agp2 are histidine kinases, but, for Agp2, we found no autophosphorylation activity. A clear autophosphorylation signal, however, was obtained with mutants in which the phosphoaccepting Asp of the C-terminal response regulator domain is replaced. Thus, the Agp2 histidine kinase differs from the classical transphosphorylation pattern. We performed size exclusion, photoconversion, dark reversion, autophosphorylation, chromophore assembly kinetics and fluorescence resonance energy transfer measurements on mixed Agp1/Agp2 samples. These assays pointed to an interaction between both proteins. This could partially explain the coaction of both phytochromes in the cell.

Prokaryotic (6-4) photolyases branch at the base of the evolution of cryptochromes and photolyases. Prototypical members contain an iron-sulphur cluster which was lost in the evolution of the other groups. In the Agrobacterium (6-4) photolyase PhrB, the repair of DNA lesions containing UV-induced (6-4) pyrimidine dimers is stimulated by Mg2+ . We propose that Mg2+ is required for efficient lesion binding and for charge stabilization after electron transfer from the FADH- chromophore to the DNA lesion. Furthermore, two highly conserved Asp residues close to the DNA-binding site are essential for the effect of Mg2+ . Simulations show that two Mg2+ bind to the region around these residues. On the other hand, DNA repair by eukaryotic (6-4) photolyases is not increased by Mg2+ . In these photolyases, structurally overlapping regions contain no Asp but positively charged Lys or Arg. During the evolution of photolyases, the role of Mg2+ in charge stabilization and enhancement of DNA binding was therefore taken over by a postiviely charged amino acid. Besides PhrB, another prokaryotic (6-4) photolyase from the marine cyanobacterium Prochlorococcus marinus, PromaPL, which contains no iron-sulphur cluster, was also investigated. This photolyase is stimulated by Mg2+ as well. The evolutionary loss of the iron-sulphur cluster due to limiting iron concentrations can occur in a marine environment as a result of iron deprivation. However, the evolutionary replacement of Mg2+ by a positively charged amino acid is unlikely to occur in a marine environment because the concentration of divalent cations in seawater is always sufficient. We therefore assume that this transition could have occurred in a freshwater environment.

The aim of the present study was to evaluate the enantioselective bioaccumulation, metabolism, and toxic effects of metolachlor and S-metolachlor in zebrafish. Five-month-old zebrafish were exposed to metolachlor and S-metolachlor for 28 days, then transferred to clean water and purified for 7 days. In the uptake phase, S-metolachlor was preferentially accumulated at low concentrations, while metolachlor was preferentially accumulated at high concentrations. The two chemicals were metabolized by >70% in zebrafish on the first day and showed same metabolic process. At the accumulation endpoint, S-metolachlor had no significant inhibitory effect on the enzymes activities of superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST) and developmental indicators of zebrafish. However, 300 μg/L metolachlor significantly inhibited the enzymes activities of SOD, CAT and GST and affected the liver development. The preferential enrichment of metolachlor at the high concentration may be the reason for its higher toxicity to zebrafish. Further research demonstrated that metolachlor significantly altered the expression of hypothalamic-pituitary-gonadal (HPG) axis-related genes, including gnrh2, gnrh3, lhβ, 17βhsd and cyp19a, thereby reducing the levels of testosterone (T) in females and sex hormones (estradiol and testosterone) in males. S-metolachlor increased the levels of estradiol (E2) in females by altering the expression of HPG axis-related genes such as fshβ, cyp17, 17βhsd and cyp19a. The mechanism of metolachlor and S-metolachlor on the endocrine disrupting effects of zebrafish is different, which may be sex-specific. 7 days after transferring the exposed zebrafish to clean water, most of the enzymes activities, sex hormone levels and related gene expression levels returned to normal, which may be related to the rapid metabolism of the two chemicals.

Photolyases are DNA-repairing flavoproteins that are represented in most phylogenetic taxa with the exception of placental mammals. These enzymes reduce the ultraviolet-induced DNA damage; thus, they have features that make them very attractive for dermatological or other medical uses, such as the prevention of human skin cancer and actinic keratosis. In this work, we identified a 50.8 kDa photolyase from the UVC-resistant Antarctic bacterium Hymenobacter sp. UV11. The enzyme was produced by recombinant DNA technology, purified using immobilized metal affinity chromatography and its activity was analyzed using different approaches: detection of cyclobutane pyrimidine dimers (CPDs) by immunochemistry, high-performance liquid chromatography and comet assays using Chinese Hamster Ovary (CHO) and immortalized nontumorigenic human epidermal (HaCat) cells. The information supports that the recombinant protein has the ability to repair the formation of CPDs, on both double- and single-stranded DNA. This CPD-photolyase was fully active on CHO and HaCat cell lines, suggesting that this enzyme could be used for medical or cosmetic purposes. Results also suggest that the UV11 CPD-photolyase uses MTHF as chromophore in the antenna domain. The potential use of this recombinant enzyme in the development of new inventions with pharmaceutical and cosmetic applications is discussed during this work.

PhrB from Agrobacterium fabrum is the first prokaryotic photolyase which repairs (6-4) UV DNA photoproducts. The protein harbors three cofactors: the enzymatically active FAD chromophore, a second chromophore, 6,7-dimethyl-8-ribityllumazine (DMRL) and a cubane-type Fe-S cluster. Tyr424 of PhrB is part of the DNA-binding site and could provide an electron link to the Fe-S cluster. The PhrBY424F mutant showed reduced binding of lesion DNA and loss of DNA repair. The mutant PhrBI51W is characterized by the loss of the DMRL chromophore, reduced photoreduction and reduced DNA repair capacity. We have determined the crystal structures of both mutants and found that both mutations only affect local protein environments, whereas the overall fold remained unchanged. The crystal structure of PhrBY424F revealed a water network extending to His366, which are part of the lesion-binding site. The crystal structure of PhrBI51W shows how the bulky Trp leads to structural rearrangements in the DMRL chromophore pocket. Spectral characterizations of PhrBI51W suggest that DMRL serves as an antenna chromophore for photoreduction and DNA repair in the wild type. The energy transfer from DMRL to FAD could represent a phylogenetically ancient process.

The ascomycete plant-pathogenic fungus Botrytis cinerea infects more than 1,400 plant species worldwide. Stimulatory effects of sublethal doses of fungicides on plant pathogens are of close relevance to disease management. In the present study, stimulatory effects of carbendazim on the virulence of B. cinerea to cucumber plants were investigated. Spraying carbendazim on cucumber plants at 3 to 200 μg/ml had stimulatory effects on the virulence of carbendazim-resistant isolates of B. cinerea and the maximum percent stimulations were 16.7 and 13.5% for isolates HBtom451 and HBstr491, respectively. Preconditioned mycelia (i.e., mycelia grown on potato dextrose agar [PDA] amended with carbendazim at concentrations of 10, 50, or 200 μg/ml) also showed increased virulence, and the maximum percent stimulations for isolates HBtom451 and HBstr491 were 7.9 and 9.5%, respectively. Compared with mycelia grown on PDA without carbendazim, virulence stimulation magnitudes of spraying carbendazim on leaves increased moderately but the concentrations of carbendazim that elicited the maximum stimulation increased 20- and 8-fold for preconditioned isolates HBtom451 and HBstr491, respectively. The time course of infection indicated that virulence stimulation was mediated by a direct stimulation mechanism. Studies of the physiological mechanism for stimulation demonstrated that carbendazim had no significant effects on tolerance to hydrogen peroxide, or on oxalic acid production in B. cinerea. These studies will deepen our understanding of quantitative features of hormetic effects of sublethal doses of fungicides on plant pathogens.

Prokaryotic (6-4) photolyases branch at the base of the evolution of cryptochromes and photolyases. Prototypical members contain an iron-sulphur cluster which was lost in the evolution of the other groups. In the Agrobacterium (6-4) photolyase PhrB, the repair of DNA lesions containing UV-induced (6-4) pyrimidine dimers is stimulated by Mg

Prokaryotic (6-4) photolyases branch at the base of the evolution of cryptochromes and photolyases. In theAgrobacterium(6-4) photolyase PhrB, the repair of DNA with UV-induced (6-4) pyrimidin dimers is stimulated by Mg2+. We show that Mg2+is required for efficient lesion binding and for charge stabilization after electron transfer from the FADH-chromophore to the DNA lesion. Two highly conserved Asp residues close to the DNA binding site are essential for the Mg2+effect. Simulations showed that two Mg2+bind to the region around these residues. DNA repair by eukaryotic (6-4) photolyases is not increased by Mg2+. Here, the structurally overlapping region contains no Asp but positively charged Lys or Arg. During evolution, charge stabilization and DNA binding by Mg2+was therefore replaced by a positive amino acid. We argue that this transition has evolved in a freshwater environment. Prokaryotic (6-4) photolyases usually contain an FeS cluster. DNA repair of a cyanobacterial member of this group which is missing the FeS cluster was also found to be stimulated by Mg2+.

Summary How the environment shapes the function and evolution of DNA repair systems is poorly understood. In a comparative study using zebrafish and the Somalian blind cavefish, Phreatichthys andruzzii, we reveal that during evolution for millions of years in continuous darkness, photoreactivation DNA repair function has been lost in P. andruzzii. We demonstrate that this loss results in part from loss-of-function mutations in pivotal DNA-repair genes. Specifically, C-terminal truncations in P. andruzzii DASH and 6-4 photolyase render these proteins predominantly cytoplasmic, with consequent loss in their functionality. In addition, we reveal a general absence of light-, UV-, and ROS-induced expression of P. andruzzii DNA-repair genes. This results from a loss of function of the D-box enhancer element, which coordinates and enhances DNA repair in response to sunlight. Our results point to P. andruzzii being the only species described, apart from placental mammals, that lacks the highly evolutionary conserved photoreactivation function. We predict that in the DNA repair systems of P. andruzzii, we may be witnessing the first stages in a process that previously occurred in the ancestors of placental mammals during the Mesozoic era.

FAD photoreduction mechanism by different aromatic residues in a phylogenetically ancient photolyase., Cryptochromes and photolyases form a flavoprotein family in which the FAD chromophore undergoes light induced changes of its redox state. During this process, termed photoreduction, electrons flow from the surface via conserved amino acid residues to FAD. The bacterial (6–4) photolyase PhrB belongs to a phylogenetically ancient group. Photoreduction of PhrB differs from the typical pattern because the amino acid of the electron cascade next to FAD is a tyrosine (Tyr391), whereas photolyases and cryptochromes of other groups have a tryptophan as direct electron donor of FAD. Mutagenesis studies have identified Trp342 and Trp390 as essential for charge transfer. Trp342 is located at the periphery of PhrB while Trp390 connects Trp342 and Tyr391. The role of Tyr391, which lies between Trp390 and FAD, is however unclear as its replacement by phenylalanine did not block photoreduction. Experiments reported here, which replace Tyr391 by Ala, show that photoreduction is blocked, underlining the relevance of Tyr/Phe at position 391 and indicating that charge transfer occurs via the triad 391-390-342. This raises the question, why PhrB positions a tyrosine at this location, having a less favourable ionisation potential than tryptophan, which occurs at this position in many proteins of the photolyase/cryptochrome family. Tunnelling matrix calculations show that tyrosine or phenylalanine can be involved in a productive bridged electron transfer between FAD and Trp390, in line with experimental findings. Since replacement of Tyr391 by Trp resulted in loss of FAD and DMRL chromophores, electron transfer cannot be studied experimentally in this mutant, but calculations on a mutant model suggest that Trp might participate in the electron transfer cascade. Charge transfer simulations reveal an unusual stabilization of the positive charge on site 391 compared to other photolyases or cryptochromes. Water molecules near Tyr391 offer a polar environment which stabilizes the positive charge on this site, thereby lowering the energetic barrier intrinsic to tyrosine. This opens a second charge transfer channel in addition to tunnelling through the tyrosine barrier, based on hopping and therefore transient oxidation of Tyr391, which enables a fast charge transfer similar to proteins utilizing a tryptophan-triad. Our results suggest that evolution of the first site of the redox chain has just been possible by tuning the protein structure and environment to manage a downhill hole transfer process from FAD to solvent.

Die (6-4) Photolyasen der bakteriellen Cryptochrom/Photolyase (BCP) -Gruppe gehoren zu einer Familie von Flavoproteinen, die als Reparaturenzyme fur UV-B-induzierte DNA-Lasionen (Photolyasen) oder als Blaulicht-Photorezeptoren (Cryptochrome) fungieren. Die (6-4) BCP-Proteine kommen ausschlieslich in Prokaryoten vor. In dieser Arbeit wurden 3 Mitglieder der (6-4) BCP-Gruppe, PhrB von Agrobacterium fabrum, CryB von Rhodobacter sphaeroides und Proma-PL von Prochlorococcus marinus mit einer eukaryotischen (6-4) Photolyase von Ostreococcus tauri OtCPF1 und einem Mitglied der Klasse III CPD Photolyasen PhrA von Agrobacterium fabrum verglichen. Es zeigte sich, dass die DNA-Reparatur-Effizienz von (6-4) BCP-Proteinen durch Mg2+ oder andere zweiwertige Kationen stimuliert wird, wahrend bei OtCPF1 und PhrA keine Wirkung von zweiwertigen Kationen beobachtet wurde. Der Einfluss zweiwertiger Kationen auf die Photoreduktion bei verschiedenen Photolyasen wurde ebenfalls untersucht. Die Photoreduktion von PhrB wurde durch Mg2+ negativ beeinflusst, wahrend bei PhrA Mg2+ einen stimulierenden Effekt hatte. Es stellte sich klar heraus, dass die Abhangigkeit von Mg2+ bei der DNA-Reparatur der (6-4) BCP und nicht bei der Photoreduktion zu suchen ist. Die veranlasste uns zu der Annahme, dass Mg2+ die DNA-Bindung und -Reparatur in (6-4) BCP-Proteinen beeinflusst. Zusammen mit den Strukturdaten und der Sequenzanalyse fanden wir eine vorgeschlagene Mg2+ Bindungsposition neben der DNA-Lasion und modifizierten die beiden betreffenden Aminosauren durch ortsgerichtete Mutagenese. Der Effekt von Mg2+ ging fur beide Mutanten verloren, wahrend die Basisreparaturaktivitat ohne Mg2+ von der Mutation nicht beeinflusst wurde. Vermutlich fordert Mg2+ die Bindung der (6-4) Lasion und erhoht die Elektronen-Affinitat des Substrats. Auserdem wird die Barriere fur Elektronentransfer verringert, wodurch und die Reaktion reibungsloser verlauft. Ich untersuchte auch die Reparatureffizienz fur verschiedene Langen einzelstrangiger und doppelstrangiger DNA. Je langer die DNA war, desto schneller war die Reparatur. Bei einer Lange der DNA von 12 nt oder mehr anderte sich allerdings mit Zunahme der Lange die Geschwindigkeit der Reparatur nicht mehr. In dieser Arbeit werden auch die Mutanten PhrB-Y424F und PhrB-I51W vorgestellt. Tyr424 von PhrB ist Teil der DNA-Bindungsstelle und konnte eine Elektronenverbindung zum Fe-S-Cluster bilden. Die PhrB-Y424F-Mutante zeigte eine starke Verringerung der Bindung von Lasions-DNA und DNA-Reparatur. Die Mutante PhrB-I51W ist durch den Verlust des DMRL-Chromophors, reduzierte Photoreduktion und reduzierte DNA-Reparaturkapazitat gekennzeichnet. Die Kristallstrukturen zeigen eine hohe Ubereinstimmung mit der Wildtyp-Struktur, somit beeinflussen Mutationen nur lokale Proteinumgebungen. Die Photoreduktion von PhrB sich unterscheidet von dem typischen Muster, da die dem FAD benachbarte Aminosaure der Elektronenkaskade ein Tyrosin (Tyr391) ist, wahrend Photolyasen und Cryptochrome anderer Gruppen ein Tryptophan als direkten Elektronendonor von FAD besitzen. In einer Mutante, in der Tyr391 durch Tryptophan ersetzt wurde, ging der Cofaktor-FAD verloren und die PhrB-Struktur war instabil. Trp342 und Trp390 sind fur den Ladungstransfer essentiell sind. Trp342 befindet sich an der Peripherie von PhrB. Die Rolle von Tyr391, die zwischen Trp390 und FAD liegt, war jedoch unklar, da der Ersatz durch Phenylalanin die Photoreduktion nicht blockierte. Bei der Substitution von Tyr391 durch Ala wurde die Photoreduktion blockiert, was darauf hindeutet, dass Tyr391 ein Teil der Elektronentransferkette ist und zeigt, dass der Ladungstransfer uber die Triade 342-391-390 erfolgt. Diese Ergebnisse deuten auf ein tunneling (Elektronentransfer ohne Ladungsanderung) zwischen Trp390 und FAD hin.