Assessing phytotoxicity and tolerance levels of ZnO nanoparticles on Raphanus sativus: implications for widespread adoptions.

The escalating release of zinc oxide nanoparticles (ZnO NPs) into the environment poses a substantial threat, potentially leading to increased concentrations of zinc (Zn) in the soil and subsequent phytotoxic effects. This study aimed to assess the effects of ZnO NPs on Raphanus sativus (R. sativus) concerning its tolerance levels, toxicity, and accumulation. ZnO NPs were synthesized by the wet chemical method and characterized by powder X-ray diffraction (PXRD), Fourier-transform infrared (FTIR) spectroscopy, ultraviolet-visible (UV-vis) spectroscopy, dynamic light scattering (DLS), and scanning electron microscopy (SEM). The effect of ZnO NPs (70 nm) on R. sativus grown in coir was evaluated. The application of 1,000 mg/L of ZnO NPs resulted in a significant increase (p < 0.05) in soluble protein content, carbohydrates, chlorophyll a (Chl-a), chlorophyll b (Chl-b), total chlorophylls, carotenoids, and antioxidants by 24.7%, 58.5%, 38.0%, 42.2%, 39.9%, 11.2%, and 7.7%, respectively. Interestingly, this dose had no impact on the indole acetic acid (IAA) content. Conversely, the use of 2,000 mg/L of ZnO NPs in the same medium led to a significant reduction (p < 0.05) in soluble protein content by 23.1%, accompanied by a notable increase in IAA by 31.1%, indicating potential toxicity. The use of atomic absorption spectroscopy confirmed the internalization of zinc in seedlings, with a statistically significant increase (p < 0.05). In control plants without ZnO NPs, Zn concentration was 0.36 mg/g, while at the highest ZnO NPs tested dose of 10,000 mg/L, it significantly rose to 1.76 mg/g, causing leaf chlorosis and stunted seedling growth. This suggests potential health risks related to Zn toxicity for consumers. Given the adverse effects on R. sativus at concentrations above 1000 mg/L, caution is advised in the application and release of ZnO NPs, highlighting the importance of responsible practices to mitigate harm to plant life and consumer health. The study demonstrated the tolerance of R. sativus to high Zn levels, classifying it as a Zn-tolerant species.

Antibiotic Residues and Zinc Concentrations in the Livers and Kidneys of Portuguese Piglets-Relationship to Antibiotic and Zinc Resistance in Intestinal Escherichia coli.

Metal ions such as zinc and copper have been used as alternatives to antibiotics, to improve animal health and growth rates in pig farming. This study aims to determine antibiotic residues and Zn concentration in piglets' livers (n = 56) and kidneys (n = 60); and to examine the correlation between the use of Zn and antibiotics, and resistance to Zn and antibiotics of Escherichia coli isolated from piglets' faeces (n = 60). Samples were collected from randomly selected healthy piglets (n = 60); antibiotic residues were quantified by ultra-high-performance-liquid-chromatography time-of-flight mass spectrometry (UHPLC-ToF-MS); Zn was quantified using flame atomic absorption spectrometry (FAAS); microbiological methods were used for E. coli isolation, antibiotic susceptibility, and Zn minimal inhibitory concentration; and Real-Time PCR was used for gene detection. The presence of antibiotic residues and Zn concentrations in the liver was found to be negatively correlated, whilst no significant difference was observed in the kidney. In E. coli isolated from piglet faeces considered to be susceptible or multi-drug-resistant, no significant difference was found between Zn concentrations in the liver and in the kidney, which appears to indicate that Zn accumulated in the liver and in the kidney does not promote resistance to antibiotics in E. coli. The isolates showed tolerance to Zn which would suggest that antibiotic resistance and phenotypic tolerance to Zn in these isolates are not related. The genes zitB and zntA associated to Zn tolerance, were predominantly found in the more resistant Zn isolates. The findings provide insights on how Zn use in pig production maintains antibiotic resistance and metal tolerance in bacteria, with implications for One Health.

Assessment of Drought and Zinc Stress Tolerance of Novel Miscanthus Hybrids and Arundo donax Clones Using Physiological, Biochemical, and Morphological Traits.

High-yield potential perennial crops, such as Miscanthus spp. and Arundo donax are amongst the most promising sources of sustainable biomass for bioproducts and bioenergy. Although several studies assessed the agronomic performance of these species on diverse marginal lands, research to date on drought and zinc (Zn) resistance is scarce. Thus, the objective of this study was to investigate the drought and Zn stress tolerance of seven novel Miscanthus hybrids and seven Arundo clones originating from different parts of Italy. We subjected both species to severe drought (less than 30%), and Zn stress (400 mg/kg-1 of ZnSO4) separately, after one month of growth. All plants were harvested after 28 days of stress, and the relative drought and Zn stress tolerance were determined by using a set of morpho-physio-biochemical and biomass attributes in relation to stress tolerance indices (STI). Principal component analysis (PCA), hierarchical clustering analysis (HCA) and stress tolerance indices (STI) were performed for each morpho-physio-biochemical and biomass parameters and showed significant relative differences among the seven genotypes of both crops. Heatmaps of these indices showed how the different genotypes clustered into four groups. Considering PCA ranking value, Miscanthus hybrid GRC10 (8.11) and Arundo clone PC1 (11.34) had the highest-ranking value under both stresses indicating these hybrids and clones are the most tolerant to drought and Zn stress. In contrast, hybrid GRC3 (-3.33 lowest ranking value) and clone CT2 (-5.84) were found to be the most sensitive to both drought and Zn stress.

Response and Tolerance of Macleaya cordata to Excess Zinc Based on Transcriptome and Proteome Patterns.

Macleaya cordata is a dominant plant of mine tailings and a zinc (Zn) accumulator with high Zn tolerance. In this study, M. cordata seedlings cultured in Hoagland solution were treated with 200 µmol·L-1 of Zn for 1 day or 7 days, and then, their leaves were taken for a comparative analysis of the transcriptomes and proteomes between the leaves of the control and Zn treatments. Differentially expressed genes included those that were iron (Fe)-deficiency-induced, such as vacuolar iron transporter VIT, ABC transporter ABCI17 and ferric reduction oxidase FRO. Those genes were significantly upregulated by Zn and could be responsible for Zn transport in the leaves of M. cordata. Differentially expressed proteins, such as chlorophyll a/b-binding proteins, ATP-dependent protease, and vacuolar-type ATPase located on the tonoplast, were significantly upregulated by Zn and, thus, could be important in chlorophyll biosynthesis and cytoplasm pH stabilization. Moreover, the changes in Zn accumulation, the production of hydrogen peroxide, and the numbers of mesophyll cells in the leaves of M. cordata were consistent with the expression of the genes and proteins. Thus, the proteins involved in the homeostasis of Zn and Fe are hypothesized to be the keys to the tolerance and accumulation of Zn in M. cordata. Such mechanisms in M. cordata can suggest novel approaches to genetically engineering and biofortifying crops.

Improving zinc phytoremediation characteristics in Salix pedicellata with a new acclimation approach.

Some species of Salix sp. (willows) are a potential phytoremediator that can accumulate substantial contents of mineral elements and, therefore, to detoxify soils contaminated with pollutants and heavy metals such as the zinc (Zn). However, high concentrations of Zn inhibit plant growth and reduce biomass production in plants. In an attempt to overcome this inconvenience and to enhance plant tolerance to Zn toxicity, we tested a new tolerance induction approach by acclimation in two clones of Salix pedicellata, named SPK-12 and SP-K20. The approach comprises two successive phases. The first is a "tolerance induction phase" consisting of gradual exposure of plants to low concentrations of Zn sulfate (ZnSO4) at regular intervals until reaching DI100 (ZnSO4 inhibitory concentration). And, the second is a "tolerance maintenance phase" to uphold the acquired tolerance to Zn toxicity. The SP-K20 clone was acclimated to DI100 threshold over 33 days without noticeable symptoms of chlorosis or growth inhibition. Compared to controls, the SP-K20 clone was able to accumulate high concentrations of Zn, suggesting that phytoremediation abilities of S. pedicellata have been improved throughout the applied approach. Acclimated Salix plants might thus improve metal phytoextraction in heavily polluted soils without biomass growth inhibition.

PtHSFA4a gene play critical roles in the adaptation of Arabidopsis thaliana plants to high-Zinc stress.

Heat shock transcription factors (HSFs) play pivotal roles in various abiotic stresses. However, only one of the studies on HSFs that participated in excess zinc (Zn) stress in our previous study in Populus ussuriensis. Here, overexpression of P. trichocarpa PtHSFA4a gene in Arabidopsis thaliana significantly improved excess Zn tolerance. It was found that PtHSFA4a-OE lines have higher seed germination rate than wild type (WT) when exposed to excess Zn. Also, PtHSFA4a-OE lines exhibit high viability and stronger root growth than WT in soil. PtHSFA4a reduced the intracellular concentration of free zinc ion of roots when overexpressed in A. thaliana. Our data indicate PtHSFA4a is the candidate gene to act as positive regulators in the resistance to excess Zn, extending our knowledge of excess Zn tolerance transcription factors.

The tonoplast-localized transporter OsHMA3 plays an important role in maintaining Zn homeostasis in rice.

In order to respond to fluctuating zinc (Zn) in the environment, plants must have a system to control Zn homeostasis. However, how plants maintain an appropriate level of Zn during their growth and development is still poorly understood. In this study, we found that OsHMA3, a tonoplast-localized transporter for Zn/Cd, plays an important role in Zn homeostasis in rice. Accessions with the functional allele of OsHMA3 showed greater tolerance to high Zn than those with the non-functional allele based on root elongation test. A 67Zn-labeling experiment showed that accessions with loss of function of OsHMA3 had lower Zn accumulation in the roots but similar concentrations in the shoots compared with functional OsHMA3 accessions. When exposed to Zn-free growing medium, the concentration in the root cell sap was rapidly decreased in accessions with functional OsHMA3, but less dramatic changes were observed in non-functional accessions. A mobility experiment showed that more Zn in the roots was translocated to the shoots in accessions with functional OsHMA3. Higher expression levels of OsZIP4, OsZIP5, OsZIP8, and OsZIP10 were found in the roots of accessions with functional OsHMA3 in response to Zn deficiency. Taken together, our results indicate that OsHMA3 plays an important role in rice roots in both Zn detoxification and storage by sequestration into the vacuoles, depending on Zn concentration in the environment.

Isolation of a fungus Pencicillium sp. with zinc tolerance and its mechanism of resistance.

A zinc (Zn)-tolerant fungus, designated BC109-2, was isolated from rhizosphere soil and was identified as Penicillium janthinellum BC109-2 based on ITS sequence analysis. To understand its Zn tolerance mechanisms, a series of studies was carried out addressing the subcellular distribution of Zn, its chemical forms, and the antioxidant system (superoxide dismutase, catalase, peroxidase, glutathione reductase, glutathione S-transferase, reduced glutathione, oxidized glutathione and malondialdehyde) of the fungus. The maximum level of resistance to Zn for strain BC109-2 is 2100 mg L-1. The Zn contents and percentages of cell wall and soluble fraction increased with increasing Zn concentration in the medium, which indicated extracellular accumulation/precipitation and vacuolar compartmentation mechanism might play significant role in the detoxificating process. The proportion of inactive forms of Zn was higher in the fungus, which indicated that BC109-2 mainly formed inactive Zn and stored it in the cell walls and vacuoles to decrease Zn toxicity. Furthermore, changes in antioxidant enzyme activities at various concentrations of Zn showed that the addition of Zn could cause oxidative stress in the fungal cells and that antioxidant enzymes in fungi played important roles in resistance to Zn toxicity. Moreover, the high level of lipid peroxidation showed that the protective effects of the antioxidant system were not sufficient at the high concentrations of Zn even though the antioxidant enzyme activity levels were very high. The purpose of this work is to figure out the heavy metal tolerance mechanisms of microorganisms in soil and the microbial isolate could be potentially used in bioremediation of Zn-contaminated environments.

The MTP1 promoters from Arabidopsis halleri reveal cis-regulating elements for the evolution of metal tolerance.

In the hyperaccumulator Arabidopsis halleri, the zinc (Zn) vacuolar transporter MTP1 is a key component of hypertolerance. Because protein sequences and functions are highly conserved between A. halleri and Arabidopsis thaliana, Zn tolerance in A. halleri may reflect the constitutively higher MTP1 expression compared with A. thaliana, based on copy number expansion and different cis regulation. Three MTP1 promoters were characterized in A. halleri ecotype I16. The comparison with the A. thaliana MTP1 promoter revealed different expression profiles correlated with specific cis-acting regulatory elements. The MTP1 5' untranslated region, highly conserved among A. thaliana, Arabidopsis lyrata and A. halleri, contains a dimer of MYB-binding motifs in the A. halleri promoters absent in the A. thaliana and A. lyrata sequences. Site-directed mutagenesis of these motifs revealed their role for expression in trichomes. A. thaliana mtp1 transgenic lines expressing AtMTP1 controlled by the native A. halleri promoter were more Zn-tolerant than lines carrying mutations on MYB-binding motifs. Differences in Zn tolerance were associated with different distribution of Zn among plant organs and in trichomes. The different cis-acting elements in the MTP1 promoters of A. halleri, particularly the MYB-binding sites, are probably involved in the evolution of Zn tolerance.

Control of Zn uptake in Arabidopsis halleri: a balance between Zn and Fe.

Zinc (Zn) is an essential plant micronutrient but is toxic in excess. To cope with excess Zn, plant species possess a strict metal homeostasis mechanism. The Zn hyperaccumulator Arabidopsis halleri has developed various adaptive mechanisms involving uptake, chelation, translocation and sequestration of Zn. In this mini review, we broadly discuss the different Zn tolerance mechanisms and then focus on controlled Zn uptake in A. halleri. Members of the ZRT/IRT-like protein (ZIP) family of metal transporters are mainly regulated by Zn and are involved in Zn uptake. A few members of the ZIP family, such as IRT1 and IRT2, are regulated by iron (Fe) and can transport multi-metals, including Zn, Fe, Mn, Cd, and Co. This mini-review also discusses the differential expression of multiple metal ZIP transporters in A. halleri and A. thaliana, a non-hyperaccumulator, with Zn exposure as well as Fe deficiency and their role in controlled Zn uptake and tolerance.

A mutation in the Arabidopsis thaliana cell wall biosynthesis gene pectin methylesterase 3 as well as its aberrant expression cause hypersensitivity specifically to Zn.

Defects in metal homeostasis factors are often accompanied by the loss of metal tolerance. Therefore, we screened for mutants with compromised growth in the presence of excess Zn(2+) in order to identify factors involved in Zn biology in plants. Here we report the isolation of six ozs (overly Zn sensitive) ethyl methanesulfonate Arabidopsis thaliana mutants with contrasting patterns of metal sensitivity, and the molecular characterization of two mutants hypersensitive specifically to Zn(2+) . Mutant ozs1 represents a non-functional allele of the vacuolar Zn transporter AtMTP1, providing additional genetic evidence for its major role in Zn(2+) tolerance in seedlings. Mutant ozs2 carries a semi-dominant mutation in the gene encoding pectin methylesterase 3 (AtPME3), an enzyme catalyzing demethylesterification of pectin. The mutation results in impaired proteolytic processing of AtPME3. Ectopic expression of AtPME3 causes strong Zn(2+) hypersensitivity that is tightly correlated with transcript abundance. Together these observations suggest detrimental effects on Golgi-localized processes. The ozs2 but not the ozs1 phenotype can be suppressed by extra Ca(2+) , indicating changes in apoplastic cation-binding capacity. However, we did not detect any changes in bulk metal-binding capacity, overall pectin methylesterification status or cell wall ultrastructure in ozs2, leading us to hypothesize that the ozs2 mutation causes hypersensitivity towards the specific interference of Zn ions with cell wall-controlled growth processes.

Plant Defensin type 1 (PDF1): protein promiscuity and expression variation within the Arabidopsis genus shed light on zinc tolerance acquisition in Arabidopsis halleri.

Plant defensins are recognized for their antifungal properties. However, a few type 1 defensins (PDF1s) were identified for their cellular zinc (Zn) tolerance properties after a study of the metal extremophile Arabidopsis halleri. In order to investigate whether different paralogues would display specialized functions, the A. halleri PDF1 family was characterized at the functional and genomic levels. Eleven PDF1s were isolated from A. halleri. Their ability to provide Zn tolerance in yeast cells, their activity against Fusarium oxysporum f. sp. melonii, and their level of expression in planta were compared with those of the seven A. thaliana PDF1s. The genomic organization of the PDF1 family was comparatively analysed within the Arabidopsis genus. AhPDF1s and AtPDF1s were able to confer Zn tolerance and AhPDF1s also displayed antifungal activity. PDF1 transcripts were constitutively more abundant in A. halleri than in A. thaliana. Within the Arabidopsis genus, the PDF1 family is evolutionarily dynamic, in terms of gain and loss of gene copy. Arabidopsis halleri PDF1s display no superior abilities to provide Zn tolerance. A constitutive increase in AhPDF1 transcript accumulation is proposed to be an evolutionary innovation co-opting the promiscuous PDF1 protein for its contribution to Zn tolerance in A. halleri.

A zinc-adapted fungus protects pines from zinc stress.

• Here we investigated zinc tolerance of ectomycorrhizal Scots pine (Pinus sylvestris) seedlings. An ectomycorrhizal genotype of Suillus bovinus, collected from a Zn-contaminated site and showing adaptive Zn tolerance in vitro, was compared with a nonadapted isolate from a nonpolluted area. • A dose-response experiment was performed. Dynamics of plant and fungal development, and phosphate and ammonium uptake capacity, were assessed under increasing Zn stress. Effects of Zn on transpiration, nutrient content and Zn accumulation were analysed. • Significant Zn-inoculation interaction effects were observed for several responses measured, including uptake rates of phosphate and ammonium; phosphorus, iron and Zn content in shoots; transpiration; biomass of external mycelia; and fungal biomass in roots. • The Zn-tolerant S. bovinus genotype was particularly efficient in protecting pines from Zn stress. The growth of a Zn-sensitive genotype from a normal wild-type population was inhibited at high Zn concentrations, and this isolate could not sustain the pines' acquisition of nutrients. This study shows that well adapted microbial root symbionts are a major component of the survival strategy of trees that colonize contaminated soils.