Analysis of multiple biomarkers revealed the size matters of Cu particles for barley response under foliar exposure.

The impact of particle size of engineered nanoparticles (ENPs) on plant response has marginally been investigated under the foliar application so far. Concerning the significance of particle diameter for their properties and interaction with plants, the effect of size should be considered in the analysis of the effect of micronutrient-based ENPs on plants. It is of particular importance for ENPs containing Cu due to plants needing a relatively low amount of this element, thus there is a risk of overdosing during application as a fertilizer or pesticide. Here, we examined the biochemical and transcriptional response of barley (Hordeum vulgare L.) to Cu nanoparticles (nano-Cu) with different diameters (25 nm, 50 nm, 70 nm), microparticles (micro-Cu), and chelated Cu (EDTA-Cu). The plants suffering from Cu deficiency were foliar sprayed with Cu compounds at 1000 mg/L during the tillering stage. 1- and 7-day plants were analyzed in terms of biomass, Cu content, the activity of enzymes involved with antioxidant response, the content of low molecular weight compounds, and the expression of genes regulated metal homeostasis, aquaporins, and defense. The results showed that the Cu leaf level was differentiated over time and after 7 days it was higher under exposure to the smallest nano-Cu than other particulate Cu. Regardless of the duration of exposure, the Cu content was highest in plants treated with Cu-EDTA. The cluster analysis of all markers revealed a clear distinct response to the smallest nano-Cu and other particulate and ionic treatments. The bigger nano-Cu, depending on the markers, caused the medium effects between the nano-Cu 25 nm and micro-Cu and Cu-EDTA. The found size thresholds at the nanoscale will be useful for the fabrication of safe-by-design agrochemicals to provide crop security and attenuate environmental impact.

Transcriptional response of Cu-deficient barley (Hordeum vulgare L.) to foliar-applied nano-Cu: Molecular crosstalk between Cu loading into plants and changes in Cu homeostasis genes.

For safe and effective nutrient management, the cutting-edge approaches to plant fertilization are continuously developed. The aim of the study was to analyze the transcriptional response of barley suffering from Cu deficiency to foliar application of nanoparticulate Cu (nano-Cu) and its ionic form (CuSO4) at 100 and 1000 mg L-1 for the examination of their supplementing effect. The initial interactions of Cu-compounds with barley leaves were analyzed with spectroscopic (ICP-OES) and microscopic (SEM-EDS) methods. To determine Cu cellular status, the impact of Cu-compounds on the expression of genes involved in regulating Cu homeostasis (PAA1, PAA2, RAN1, COPT5), aquaporins (NIP2.1, PIP1.1, TIP1.1, TIP1.2) and antioxidant defense response (SOD CuZn, SOD Fe, SOD Mn, CAT) after 1 and 7 days of exposure was analyzed. Although Cu accumulation in plant leaves was detected overtime, the Cu content in leaves exposed to nano-Cu for 7 days was 44.5% lower than in CuSO4 at 100 mg L-1. However, nano-Cu aggregates remaining on the leaf surface indicated a potential difference between measured Cu content and the real Cu pool present in the plant. Our study revealed significant changes in the pattern of gene expression overtime depending on Cu-compound type and dose. Despite the initial puzzling patterns of gene expression, after 7 days all Cu transporters showed significant down-regulation under Cu-compounds exposure to prevent Cu excess in plant cells. Conversely, aquaporin gene expression was induced after 7 days, especially by nano-Cu and CuSO4 at 100 mg L-1 due to the stimulatory effect of low Cu doses. Our study revealed that the gradual release of Cu ions from nano-Cu at a lower rate provided a milder molecular response than CuSO4. It might indicate that nano-Cu maintained better metal balance in plants than the conventional compounds, thus may be considered as a long-term supplier of Cu.

The life cycle study revealed distinct impact of foliar-applied nano-Cu on antioxidant traits of barley grain comparing with conventional agents.

Despite that the applicability of Cu-based engineered nanoparticles (ENPs) as an antibacterial and antifungal agent for plant protection has been studied widely, little is known about their role in the improvement of crop yield and quality. Here, a full life study was performed to investigate the nutritional quality and bioactivity of barley grains under foliar application of nano-/microparticulate (nano-Cu, nano-CuO, micro-Cu) and ionic Cu compounds (CuSO4, CuEDTA). Hordeum vulgaris L. plants were sprayed with Cu compounds at 500 mg/L during the end of tillering and the beginning of heading. Yield, mineral composition, protein and dietary content, antioxidant (phenolic, anthocyanin, flavonoid, tannin, flavanol) content and antioxidant capacity of barley grain were evaluated. Grain yield was unaffected by all treatments. Only nano-Cu and ionic compounds enhanced Cu accumulation in grain: 2-fold increase was observed compared to the control (2.6 µg/kg). Nano-Cu also increased the dietary fiber content by 19.9 %, while no impact of the other treatments was determined. The content of phenolic compounds, the main group of antioxidants, remained unchanged after Cu supply. In general, for all Cu treatment, antiradical and reducing abilities were decreased or were at the similar level in relation to the control. On the other hand, chelating power in grain extracts was 2-4 times higher under nano-Cu/nano-CuO/micro-Cu than in the untreated sample, while the ionic compounds had no impact on the chelating indicator. Our results demonstrated that more favorable effects were triggered by nano-Cu than CuSO4 or CuEDTA on the tested indicators of barley grain, despite that both compounds resulted in similar superior Cu acquisition. It suggests that nano-Cu may be considered as an alternative agent to be used as economic and traditional fertilizers.

Diverse Partners of the Partitioning ParB Protein in Pseudomonas aeruginosa.

In the majority of bacterial species, the tripartite ParAB-parS system, composed of an ATPase (ParA), a DNA-binding protein (ParB), and its target parS sequence(s), assists in the chromosome partitioning. ParB forms large nucleoprotein complexes at parS(s), located in the vicinity of origin of chromosomal replication (oriC), which after replication are subsequently positioned by ParA in cell poles. Remarkably, ParA and ParB participate not only in the chromosome segregation but through interactions with various cellular partners they are also involved in other cell cycle-related processes, in a species-specific manner. In this work, we characterized Pseudomonas aeruginosa ParB interactions with the cognate ParA, showing that the N-terminal motif of ParB is required for these interactions, and demonstrated that ParAB-parS-mediated rapid segregation of newly replicated ori domains prevented structural maintenance of chromosome (SMC)-mediated cohesion of sister chromosomes. Furthermore, using proteome-wide techniques, we have identified other ParB partners in P. aeruginosa, which encompass a number of proteins, including the nucleoid-associated proteins NdpA(PA3849) and NdpA2, MinE (PA3245) of Min system, and transcriptional regulators and various enzymes, e.g., CTP synthetase (PA3637). Among them are also NTPases PA4465, PA5028, PA3481, and FleN (PA1454), three of them displaying polar localization in bacterial cells. Overall, this work presents the spectrum of P. aeruginosa ParB partners and implicates the role of this protein in the cross-talk between chromosome segregation and other cellular processes. IMPORTANCE In Pseudomonas aeruginosa, a Gram-negative pathogen causing life-threatening infections in immunocompromised patients, the ParAB-parS system is involved in the precise separation of newly replicated bacterial chromosomes. In this work, we identified and characterized proteins interacting with partitioning protein ParB. We mapped the domain of interactions with its cognate ParA partner and showed that ParB-ParA interactions are crucial for the chromosome segregation and for proper SMC action on DNA. We also demonstrated ParB interactions with other DNA binding proteins, metabolic enzymes, and NTPases displaying polar localization in the cells. Overall, this study uncovers novel players cooperating with the chromosome partition system in P. aeruginosa, supporting its important regulatory role in the bacterial cell cycle.

Unveiling of interactions between foliar-applied Cu nanoparticles and barley suffering from Cu deficiency.

The objective of this study was to evaluate nano-Cu-plant interactions under Cu deficiency. Nano-Cu at rates of 100 and 1000 mg L-1 was applied as foliar spray to Hordeum vulgare L. during increased demand for nutrients at tillering stage. Corresponding treatment with CuSO4 was used to exam the nano-specific effects. Cu compounds-plant leaves interactions were analyzed with spectroscopic and microscopic methods (ICP-OES, FTIR/ATR, SEM-EDS). Moreover, the effect of Cu compounds on plants in terms of biomass, pigments content, lipid peroxidation, antiradical properties, the activity of enzymes involved in plant defense against stress (SOD, CAT, POD, GR, PAL, PPO) and the content of non-enzymatic antioxidants (GSH, GSSG, TPC) was determined after 1 and 7 days of exposure. Cu loading to plant leaves increased over time, but the content of Cu under treatment with nano-Cu at 100 mg L-1 was lower by 76% than CuSO4 at 7th day of exposure. The changes induced by applied Cu compounds in biochemical traits were mostly observed after 1 day. Our data showed that CuSO4 exposure induce oxidative stress (increased MDA level and GSSG content) when compared to control and nano-Cu treated plants. Noteworthy, nano Cu at 100 mg L-1 demonstrated enhanced stress tolerance as indicated by boosted GSH content. After 7 days, the antioxidant response was almost same compared to control sample. However, based on other indicators (pigment content, chlorosis sign, biomass), it should be noted that CuSO4 caused serve oxidative burst of plant which may resulted in damage of defense system. Nano-Cu, especially at 100 mg L-1, showed promising effect on plant health, and obtained results may be useful for optimizing of nano-Cu application as fertilizer agent.

The antioxidant defense responses of Hordeum vulgare L. to polycyclic aromatic hydrocarbons and their derivatives in biochar-amended soil.

The recent studies indicated that the biochar (BC) may be a source of polycyclic aromatic hydrocarbons (PAHs) as well as their oxygen, nitrogen, or sulfur-containing derivatives that are considered as more toxic pollutants than their parent compounds. Here, the assessment of the impact of various biochars addition (1% wt.) to soil on barley Hordeum vulgare L. growth was presented. The concentrations of bioavailable PAHs and their derivatives in biochar were determined. PAHs increased reactive oxygen species generation resulting in oxidative stress in organisms. In this study, the response of soil-grown plants was examined in terms of the activity of the antioxidative enzymes (superoxide dismutase, catalase, peroxidase), lipid peroxidation, and the expression of genes related to oxidative stress. The results indicate that despite low content of a bioavailable fraction of parent compounds and their derivatives (up to 4.45 ± 0.24 ng gbiochar-1 and 0.83 ± 0.03 ng L-1, respectively) the biochemical response of plant was present, the activity of superoxide dismutase increased up to 2 times, but the activity of the other enzymes was lowered. The transcript level values support the studies on enzymatic activity. The presence of PAHs and their derivatives induced oxidative stress slightly but the plant was able to mitigate it.

Cross-examination of engineered nanomaterials in crop production: Application and related implications.

The review presents the current knowledge on the development and implementation of nanotechnology in crop production, giving particular attention to potential opportunities and challenges of the use of nano-sensors, nano-pesticides, and nano-fertilizers. Due to the size-dependent properties, e.g. high reactivity, targeted and controlled delivery of active ingredients, engineered nanomaterials (ENMs) are expected to be more efficient agrochemicals than conventional agents. Growing production and usage of ENMs result in the spread of ENMs in the environment. Because plants constitute an important component of the agri-ecosystem, they are subjected to the ENMs activity. A number of studies have confirmed the uptake and translocation of ENMs by plants as well as their positive/negative effects on plants. Here, these endpoints are briefly summarized to show the diversity of plant responses to ENMs. The review includes a detailed molecular analysis of ENMs-plant interactions. The transcriptomics, proteomics and metabolomics tools have been very recently employed to explore ENMs-induced effects in planta. The omics approach allows a comprehensive understanding of the specific machinery of ENMs occurring at the molecular level. The summary of data will be valuable in defining future studies on the ENMs-plant system, which is crucial for developing a suitable strategy for the ENMs usage.

Combined effect of nano-CuO and nano-ZnO in plant-related system: From bioavailability in soil to transcriptional regulation of metal homeostasis in barley.

The co-existence of engineered nanoparticles (ENPs) in the environment is an emerging issue remaining poorly investigated. The present study aimed at analyzing the fate of binary mixtures of CuO and ZnO ENPs in a soil-plant system. The ENPs were singly or jointly dosed into soil at 300 mg kg-1 and aged for 7 and 30 days. To evaluate nano-specific effects, individual and combined treatments of metal salts were also applied. Interactions between ENPs and soil-grown barley Hordeum vulgare were determined in terms of biomass, plant mineral composition as well as expression of genes regulating metal homeostasis (ZIP1,3,6,8,10,14, RAN1, PAA1,2, MTP1, COPT5) and detoxification (MT1-3). The bioavailability of Zn and Cu in bulk soil and in the rooting zone was determined using the 0.01 mol L-1 CaCl2 extraction. After combined treatment of ENPs, the extractable concentrations of Cu and Zn were lower than upon individual exposure in bulk soil. The opposite tendency was noted for metal salts. Genes related to metal uptake (ZIP) and cellular compartment (PAA2, RAN1) were mostly up-regulated by single rather than combined application of ENPs. The single and joint exposure to metals salts induced the down-regulation of these genes.

The chronic effects of CuO and ZnO nanoparticles on Eisenia fetida in relation to the bioavailability in aged soils.

The bioavailability and bioaccumulation of metal-based engineered nanoparticles (ENPs) in soils need to be evaluated in environmentally relevant scenarios. The aim of this study was an analysis of potentially available metal-component ENPs (nano-ZnO and nano-CuO) in soils. Earthworms (Eisenia fetida) were used to examine the bioaccumulation potential of ENPs. Micro-particles (micro-ZnO and micro-CuO) and metal salts (ZnCl2 and CuCl2) were used to evaluate the nano-effect and the activity of dissolved ions, respectively. Zn- and Cu-compounds were added to sandy loam and silt loam at a concentration of 10 mg kg-1. The bioavailable fractions of metals were extracted from soil using H2O, MgCl2 with CH3COONa or EDTA. EDTA was the most effective extractant of Zn and Cu (10.06-11.65 mg Zn kg-1 and 2.69-3.52 mg Cu kg-1), whereas the H2O-extractable metal concentration was at the lowest level (1.98-2.12 mg Zn kg-1 and 0.54-0.82 Cu mg kg-1). The bioavailable metal concentrations were significantly higher in silt loam than sandy loam soil, which was related to the higher pH value of silt. There were no significant differences between the Zn content in the earthworms incubated in the two soils, which may confirm the auto-regulation of the Zn content by earthworms. However, the bioaccumulation of Cu was strongly correlated with the extractable Cu concentrations. The juvenile earthworms accumulated Cu and Zn more than adults. Based on our results, aging neutralized the differences between the ionic and particulate effects of metal-compounds.

The effect of pH and ageing on the fate of CuO and ZnO nanoparticles in soils.

The objective of this study was to evaluate the fractionation of ZnO and CuO engineered nanoparticles (ENPs) in soils with a pH adjusted to 4.0, 6.5, and 9.0 after 1 day and 30 days of incubation. Based on the multi-stage extraction, 5 fractions of metals were determined. Moreover, the effect of ENPs on the activity of acid, neutral and alkaline phosphatase was determined. The results of the study revealed that pH had a dominant effect on the metal participation in soils. The levels of those fractions of metals differed between nano-ZnO and nano-CuO, which could have resulted from differences in the dissolution of the ENPs. After 1 day, the concentration of Zn2+ (0.02-7.4 mg L-1) was 10 times higher than that of Cu2+. The metal fractionation in soil treated with ENPs and metal salts may also confirm the role of ENP dissolution. The concentration of potentially bioavailable fraction of Zn increased with a drop in pH. At a 4 pH concentration of Zn in the treatment with nano-ZnO and ZnCl2 was at a similar level (42.1-45 mg kg-1), whereas the addition of nano-CuO resulted in a lower content of Cu (24.7 mg kg-1) than CuCl2 (36.5 mg kg-1). On the other hand, the concentration of fraction exchangeable of both metals in the alkaline soil did not exceed the level of 5.0 mg kg-1. Sample incubation time was especially important for metal participation in samples with a pH of 6.5. The greatest differentiation of metal fractionation between the soils was also noted at a pH of 6.5, which could also have been a result of other properties of the soils. The strong effect of pH on the lability of ENPs in soils confirmed a need to trace the fate of ENPs in extreme soil conditions as well as in changing environment.

Binding and spreading of ParB on DNA determine its biological function in Pseudomonas aeruginosa.

ParB protein of Pseudomonas aeruginosa belongs to a widely represented ParB family of chromosomally and plasmid-encoded partitioning type IA proteins. Ten putative parS sites are dispersed in the P. aeruginosa chromosome, with eight of them localizing in the oriC domain. After binding to parS, ParB spreads on the DNA, causing transcriptional silencing of nearby genes (A. A. Bartosik et al., J. Bacteriol. 186:6983-6998, 2004). We have studied ParB derivatives impaired in spreading either due to loss of DNA-binding ability or oligomerization. We defined specific determinants outside of the helix-turn-helix motif responsible for DNA binding. Analysis confirmed the localization of the main dimerization domain in the C terminus of ParB but also mapped another self-interactive domain in the N-terminal domain. Reverse genetics were used to introduce five parB alleles impaired in spreading into the P. aeruginosa chromosome. The single amino acid substitutions in ParB causing a defect in oligomerization but not in DNA binding caused a chromosome segregation defect, slowed the growth rate, and impaired motilities, similarly to the pleiotropic phenotype of parB-null mutants, indicating that the ability to spread is vital for ParB function in the cell. The toxicity of ParB overproduction in Pseudomonas spp. is not due to the spreading since several ParB derivatives defective in oligomerization were still toxic for P. aeruginosa when provided in excess.