G-site residue S67 is involved in the fungicide-degrading activity of a tau class glutathione S-transferase from Carica papaya.

Thiram is a toxic fungicide extensively used for the management of pathogens in fruits. Although it is known that thiram degrades in plant tissues, the key enzymes involved in this process remain unexplored. In this study, we report that a tau class glutathione S-transferase (GST) from Carica papaya can degrade thiram. This enzyme was easily obtained by heterologous expression in Escherichia coli, showed low promiscuity toward other thiuram disulfides, and catalyzed thiram degradation under physiological reaction conditions. Site-directed mutagenesis indicated that G-site residue S67 shows a key influence for the enzymatic activity toward thiram, while mutation of residue S13, which reduced the GSH oxidase activity, did not significantly affect the thiram-degrading activity. The formation of dimethyl dithiocarbamate, which was subsequently converted into carbon disulfide, and dimethyl dithiocarbamoylsulfenic acid as the thiram degradation products suggested that thiram undergoes an alkaline hydrolysis that involves the rupture of the disulfide bond. Application of the GST selective inhibitor 4-chloro-7-nitro-2,1,3-benzoxadiazole reduced papaya peel thiram-degrading activity by 95%, indicating that this is the main degradation route of thiram in papaya. GST from Carica papaya also catalyzed the degradation of the fungicides chlorothalonil and thiabendazole, with residue S67 showing again a key influence for the enzymatic activity. These results fill an important knowledge gap in understanding the catalytic promiscuity of plant GSTs and reveal new insights into the fate and degradation products of thiram in fruits.

Integrative effects of microbial inoculation and amendments on improved crop safety in industrial soils co-contaminated with organic and inorganic pollutants.

Soils co-contaminated by organic and inorganic pollutants usually pose major ecological risks to soil ecosystems including plants. Thus, effective strategies are needed to alleviate the phytotoxicity caused by such co-contamination. In this study, microbial agents (a mixture of Bacillus subtilis, Sphingobacterium multivorum, and a commercial microbial product named OBT) and soil amendments (ß-cyclodextrin, rice husk, biochar, calcium magnesium phosphate fertilizer, and organic fertilizer) were evaluated to determine their applicability in alleviating toxicity to crops (maize and soybean) posed by polycyclic aromatic hydrocarbon (PAHs) and potentially toxic metals co-contaminated soils. The results showed that peroxidase, catalase, and superoxide dismutase activity levels in maize or soybean grown in severely or mildly contaminated soils were significantly enhanced by the integrative effects of amendments and microbial agents, compared with those in single plant treatments. The removal rates of Zn, Pb, and Cd in severely contaminated soils were 49 %, 47 %, and 51 % and 46 %, 45 %, and 48 %, for soybean and maize, respectively. The total contents of Cd, Pb, Zn, and PAHs in soil decreased by day 90. Soil organic matter content, levels of nutrient elements, and enzyme activity (catalase, urease, and dehydrogenase) increased after the amendments and application of microbial agents. Moreover, the amendments and microbial agents also increased the diversity and distribution of bacterial species in the soil. These results suggest that the amendments and microbial agents were beneficial for pollutant purification, improving the soil environment and enhancing both plant resistance to pollutants and immune systems of plants.

Amendments and bioaugmentation enhanced phytoremediation and micro-ecology for PAHs and heavy metals co-contaminated soils.

Co-existence of polycyclic aromatic hydrocarbons (PAHs) and multi-metals challenges the decontamination of large-scale contaminated sites. This study aims to comprehensively evaluate the remediation potential of intensified phytoremediation in coping with complex co-contaminated soils. Results showed that the removal of PAHs and heavy metals is time-dependent, pollution-relevant, and plant-specific. Removal of sixteen PAHs by Medicago sativa L. (37.3%) was significantly higher than that of Solanum nigrum L. (20.7%) after 30 days. S. nigrum L. removed higher amounts of Cd than Zn and Pb, while M. sativa L. uptake more Zn. Nevertheless, amendments and microbial agents significantly increased the phytoremediation efficiency of pollutants and shortened the gap between plants. Cd removal and PAHs dissipation reached up to 80% and 90% after 90 days for both plants. Heavy metal stability in soil was promoted after the intensified phytoremediation. Plant lipid peroxidation was alleviated, regulated by changed antioxidant defense systems (superoxide dismutase, peroxidase, catalase). Soil enzyme activities including dehydrogenase, urease, and catalase increased up to 5-fold. Soil bacterial diversity and structure were changed, being largely composed of Proteobacteria, Actinobacteria, Patescibacteria, Bacteroidetes, and Firmicutes. These findings provide a green and sustainable approach to decontaminating complex-polluted environments with comprehensive improvement of soil health.

Enhanced 2,4-D Metabolism in Two Resistant Papaver rhoeas Populations from Spain.

Corn poppy (Papaver rhoeas), the most problematic broadleaf weed in winter cereals in Southern Europe, has developed resistance to the widely-used herbicide, 2,4-D. The first reported resistance mechanism in this species to 2,4-D was reduced translocation from treated leaves to the rest of the plant. However, the presence of other non-target site resistance (NTSR) mechanisms has not been investigated up to date. Therefore, the main objective of this research was to reveal if enhanced 2,4-D metabolism is also present in two Spanish resistant (R) populations to synthetic auxins. With this aim, HPLC experiments at two 2,4-D rates (600 and 2,400 g ai ha-1) were conducted to identify and quantify the metabolites produced and evaluate possible differences in 2,4-D degradation between resistant (R) and susceptible (S) plants. Secondarily, to determine the role of cytochrome P450 in the resistance response, dose-response experiments were performed using malathion as its inhibitor. Three populations were used: S, only 2,4-D R (R-703) and multiple R to 2,4-D and ALS inhibitors (R-213). HPLC studies indicated the presence of two hydroxy metabolites in these R populations in shoots and roots, which were not detected in S plants, at both rates. Therefore, enhanced metabolism becomes a new NTSR mechanism in these two P. rhoeas populations from Spain. Results from the dose-response experiments also showed that pre-treatment of R plants with the cytochrome P450 (P450) inhibitor malathion reversed the phenotype to 2,4-D from resistant to susceptible in both R populations. Therefore, it could be hypothesized that a malathion inhibited P450 is responsible of the formation of the hydroxy metabolites detected in the metabolism studies. This and previous research indicate that two resistant mechanisms to 2,4-D could be present in populations R-703 and R-213: reduced translocation and enhanced metabolism. Future experiments are required to confirm these hypotheses, understand the role of P450, and the relationship between both NTSR mechanisms. On this basis, selection pressure with synthetic auxins bears the risk of promoting the evolution enhanced metabolism in Papaver rhoeas.