Phytosulfokine alpha enhances regeneration of transformed and untransformed protoplasts of Brassica oleracea.

Phytosulfokine-α (PSK-α) is a disulfated pentapeptide (YIYTQ) acting as an intercellular signal peptide and growth factor. It was originally isolated from conditioned medium of asparagus mesophyll cell cultures in 1996 and later characterized as a hormone-like signal molecule with important roles in numerous processes of in vivo plant growth and development. It is currently becoming a valuable mitogenic factor in plant breeding and biotechnology due to its stimulatory effect on in vitro cell elongation, proliferation and differentiation. The focus of our work was to review current knowledge about the roles of PSK-α in plant biotechnology and to evaluate its influence on the regeneration of protoplasts of four Brassica oleracea cultivars (two cauliflower and two cabbage) cultured under two distinctive protocols and with different protoplast densities. Protoplast regeneration was studied due to its high value for plant genome editing, which is generally limited by the inefficient regeneration of treated protoplasts of numerous important plant genotypes. Our hypothesis was that the stress related to PEG-mediated protoplast transformation and the following decrease in viable protoplast density in culture could be alleviated by the addition of PSK-α to the culture medium. We therefore tested whether PSK-α could increase cell division at the early stages of culture (5 and 15 days after protoplast isolation) and stimulate the formation of microcallus colonies up to the 30st day of culture and to evaluate its influence on callus organogenesis leading to shoot regeneration. The PSK-α showed a strong stimulatory effect on untransformed protoplast regeneration already during the first days of culture, accelerating cell division up to 5.3-fold and the formation of multicellular microcallus colonies up to 37.0-fold. The beneficial influence was retained at later stages of regeneration, when PSK improved shoot organogenesis even if it was present only during the first 10 days of culture. The highest numbers of shoots, however, were regenerated when PSK was present during the first days of culture and later in solid shoot regeneration medium. Finally, the addition of PSK-α to PEG-transformed protoplasts significantly enhanced their division rate and the formation of microcallus colonies in selection media, up to 44.0-fold.

Removal of toxic metals from sewage sludge by EDTA and hydrodynamic cavitation and use of the sludge as fertilizer.

Sewage sludge (SS) is rich in plant nutrients, including P, N, and organic C, but often contains toxic metals (TMs), which hinders its potential use in agriculture. The efficiency of removal of TMs by washing with ethylenediamine tetraacetate (EDTA), in combination with hydrodynamic cavitation (HC) and the usability of washed sewage sludge as fertilizer were investigated. The environmental risk was assessed. During 8 wash batches an average 35, 68, 47 and 45 % of Pb, Zn, Cd and Cu, respectively, as well as 22 and 5 % Mn and Fe were removed from the SS. The process solutions and EDTA were recycled at a pH gradient of 12.5-2, which was achieved by adding quicklime (CaO) and then acidification by H2SO4, so that no wastewater was produced, only solid waste (ReSoil® method). The quality of the recycled process solutions (they remained unsaturated with salts) and the efficiency of the washing process were maintained across all batches. On average, 46 % of the EDTA was lost during the process and was replenished. The initial leachability of EDTA-mobilized Pb, Zn, Cu, Cr and Fe remaining in the washed SS increased 6-, 17-, 3-, 11- and 11-fold, respectively, but not to hazardous levels except for Zn. After washing, P and K remained in the SS, plant-available P increased 3.3-fold, while total N and C were reduced by 20.28 and 2.44 %, respectively. Washed SS was used as fertilizer in the pot experiment. The yield of Brassica juncea did not improve, the uptake of TMs by the plants and the leaching of TMs from the soil were minimal. Our study highlighted the drawbacks and potential feasibility of the new SS washing method.

Apple (Malus domestica Borkh.) Cultivar 'Majda', a Naturally Non-Browning Cultivar: An Assessment of Its Qualities.

Browning of apple and apple products has been a topic of numerous research and there is a great number of methods available for browning prevention. However, one of the most efficient ways, and the one most acceptable for the consumers, is the selection of a non-browning cultivar. Cultivar 'Majda' is a Slovenian cultivar, a cross between 'Jonatan' and 'Golden Noble'. In this study, it was thoroughly examined and compared to the well-known cultivar 'Golden Delicious' with the aim to decipher the reason for non-browning. We have determined the content of sugars, organic acids, vitamin C, glutathione and phenolics in apple flesh, with the addition of phenolic content in apple peel and leaves. The change in color in halves and pomace was also measured and the activity of peroxidase (POX) and polyphenol oxidase (PPO) were determined. Additionally, the analyses of flesh were repeated post-storage. The most prominent results were high acidity (malic acid), low phenol content, especially hydroxycinnamic acid and flavan-3-ol content of cultivar 'Majda' in comparison to 'Golden Delicious', and no difference in PPO activity between cultivars. After the overview of the results, we believe that both low phenol content and high reduced glutathione content impact the non-browning characteristics of cultivar 'Majda'.

Demonstrational gardens with EDTA-washed soil. Part I: Remediation efficiency, effect on soil properties and toxicity hazards.

The viable chelator-based soil washing has yet to be demonstrated on a larger scale. Soil containing 1850, 3830 and 21 mg kg-1 Pb, Zn and Cd, respectively, was washed with 100 mmol EDTA kg-1 in a series of 16 batches (1 ton soil/batch) using the new ReSoil® technology. The ReSoil® recycled the process water and 85% of the EDTA, producing no wastewater and 14.4 kg ton-1 of waste. The soil washing removed 71, 28 and 53% of Pb, Zn and Cd, respectively, mainly from the carbonate fraction, saturated the soil with basic cations and increased the soil pH by up to 0.5 units. Raised beds (4 × 1 × 0.5 m) with original (contaminated) and remediated soil were constructed as lysmeters, and local produce was grown from July 2018 to November 2019. Throughout the gardening period, the concentration of Pb and Cd in the leachates from the remediated soil was lower and that of Zn was higher than in the original soil. Remediation decreased the concentration of plant-available and mobile toxic metals, as determined by CaCl2 and NH4NO3 extractions, and reduced the bioavailability of Pb, Zn, and Cd in the simulated human gastrointestinal phase by an average of 4.3, 1.7 and 2.7-fold, respectively. Revitalization with vermicompost, earthworms and rhizosphere soil, and spring fertilisation with compost and manure, had no significant effect on the mobility and accessibility of the toxic metals. The ReSoil® is a cost-effective technology (material cost = 18.27 € ton-1 soil) and showed the prospect of sustainable reuse of remediated soil.

Demonstrational gardens with EDTA-washed soil. Part II: Soil quality assessment using biological indicators.

In this study, we evaluated the impact of washing of Pb, Zn and Cd contaminated soil using EDTA-based technology (ReSoil®) on soil biological properties by measuring some of the most commonly used/sensitive biological indicators of soil perturbation. We estimated the temporal dynamics of the soil respiration, the activities of soil enzymes (dehydrogenase, ß-glucosidase, urease, acid and alkaline phosphatase), and the effect of the remediation process on arbuscular mycorrhizal (AM) fungi in original (Orig), remediated (Rem) and remediated vitalized (Rem+V) soils during a more than one-year garden experiment. ReSoil® technology initially affected the activity level of soil microbial respiration and all enzyme activities except urease and reduced AM fungal potential in the soil. However, after one year of vegetable cultivation and standard gardening practices, soil microbial respiration, acid and alkaline phosphatase in the Rem and Rem+V reached similar activities as in the Orig. Only the activities of dehydrogenase and ß-glucosidase remained lower in the remediated soil compared to the Orig. The frequency of arbuscular mycorrhiza in the root system, arbuscular density in the colonized root fragment, and the intensity of mycorrhizal colonization in the colonized root fragments in the remediated treatments increased with time; at the end of the experiment, no consistent differences in these parameters of mycorrhizal colonization were found among the treatments. Our results suggest a restored biological functioning of the remediated soil after one year of vegetable cultivation. In general, no differences were found between the Rem and Rem+V treatments, indicating that simple common garden practices are sufficient to restore soil functioning after remediation.

Demonstration gardens with EDTA-washed soil. Part III: Plant growth, soil physical properties and production of safe vegetables.

In previous reports large-scale EDTA-based soil washing using ReSoil® technology was demonstrated. In the current study, we established a vegetable garden with nine raised beds (4 × 1 × 0.5 m), three with original (contaminated) soil, three with remediated soil, and three with remediated soil vitalized by addition of vermicompost, earthworms, and rhizosphere inoculum. The garden was managed in 6 rotations between July 2018 and November 2019. Buckwheat was sown first as a green manure followed by spinach, lamb's lettuce, chicory, garlic, onion, leek, lettuce, carrot, kohlrabi and spinach again. Buckwheat growth on the remediated soil was reduced by half. Throughout the gardening process there were no remarkable differences in bulk density, hydraulic conductivity, available water capacity, and aggregate stability of the original and remediated soil. Biomass yield and plant performance, as measured by NDVI, also remained similar regardless of soil treatment. Remediation reduced Pb concentration in edible parts of vegetables from 76 (garlic) to 95% (kohlrabi), Zn concentration from 14 (lettuce) to 76% (first cutting of chicory), and Cd concentration from 33% (carrot) to 91% (leek and second cutting of chicory). The transfer of metals from soil to root and from root to shoot occurred in the order: Pb < Zn < Cd. The bioconcentration of toxic metals in edible plant parts was generally lower in the remediated soils. Application of ReSoil® technology and growing vegetables that exclude metals, especially Cd, has potential for safe food production on remediated soils. Vitalization had little effect on the properties of the remediated soil.

Hop Polyphenols in Relation to Verticillium Wilt Resistance and Their Antifungal Activity.

(1) Background: Verticillium wilt (VW) of hop is a devastating disease caused by the soil-borne fungi Verticillium nonalfalfae and Verticillium dahliae. As suggested by quantitative trait locus (QTL) mapping and RNA-Seq analyses, the underlying molecular mechanisms of resistance in hop are complex, consisting of preformed and induced defense responses, including the synthesis of various phenolic compounds. (2) Methods: We determined the total polyphenolic content at two phenological stages in roots and stems of 14 hop varieties differing in VW resistance, examined the changes in the total polyphenols of VW resistant variety Wye Target (WT) and susceptible Celeia (CE) on infection with V. nonalfalfae, and assessed the antifungal activity of six commercial phenolic compounds and total polyphenolic extracts from roots and stems of VW resistant WT and susceptible CE on the growth of two different V. nonalfalfae hop pathotypes. (3) Results: Generally, total polyphenols were higher in roots than stems and increased with maturation of the hop. Before flowering, the majority of VW resistant varieties had a significantly higher content of total polyphenols in stems than susceptible varieties. At the symptomatic stage of VW disease, total polyphenols decreased in VW resistant WT and susceptible CE plants in both roots and stems. The antifungal activity of total polyphenolic extracts against V. nonalfalfae was higher in hop extracts from stems than those from roots. Among the tested phenolic compounds, only p-coumaric acid and tyrosol markedly restricted fungal growth. (4) Conclusions: Although the correlation between VW resistance and total polyphenols content is not straightforward, higher levels of total polyphenols in the stems of the majority of VW resistant hop varieties at early phenological stages probably contribute to fast and efficient activation of signaling pathways, leading to successful defense against V. nonalfalfae infection.

Trace elements and APOE polymorphisms in pregnant women and their new-borns.

We investigated the relationship between lipid binding glycoprotein apolipoprotein E (apoE; gene APOE) polymorphisms (ε4 allele carriers versus no carriers = Îµ4+/ε4-) and trace elements (TEs) (e.g., (methyl)mercury, arsenic, lead, cadmium, selenium, manganese, copper, and zinc) in mothers (N = 223) and their new-borns (N = 213) exposed to potentially toxic metal(loid)s from seafood consumption. The apoE isoform encoded by the ε4 allele is believed to have beneficial effects in early life but represents a risk factor for age-associated diseases. Under certain conditions ε4 carriers are more susceptible to oxidative stress and metal(loid) toxicity. DNA from Croatian pregnant women (N = 223, third trimester) and their new-borns (N = 176), was genotyped for APOE by TaqMan® SNP assay - rs429358 and rs7412. Seafood intake data and TE levels in maternal urine, milk, hair, peripheral venous blood, mixed cord blood, and new-borns' urine were available from previous studies. We compared TEs between ε4+ and ε4- carriers using Mann-Whitney U tests and applied multiple linear regression models to analyse the TE's dependence on the presence of allele ε4 (genotypes ε3/ε4, ε4/ε4) in combination with other explanatory variables. We identified 17% (n = 37) and 20% (n = 35) ε4 allele carriers in mothers and new-borns, respectively. The Mann-Whitney U test showed that mothers with the ε4 allele had significantly higher mean levels of (methyl)mercury in peripheral venous blood, cord blood, and hair; arsenic in urine and cord blood; and selenium in peripheral venous blood and plasma. However, taking confounders into account, only the maternal plasma selenium remained statistically significant in the linear regression models (ε4 carriers vs non-carriers: 62.6 vs 54.9 ng/mL, p < 0.001). Literature suggestions of possible ε4 allele impact on Hg levels were not observed, while superior selenium status observed in healthy pregnant women carrying allele ε4 could be linked to the proposed APOE ε4 beneficial effects early in life.

Hop (Humulus lupulus L.) response mechanisms in drought stress: Proteomic analysis with physiology.

Drought is one of the major environmental devastating stressors that impair the growth and productivity of crop plants. Despite the relevance of drought stress, changes in physiology and resistance mechanisms are not completely understood for certain crops, including hop (Humulus lupulus L.). In this research the drought response of hop was studied using a conventional physiological approach (gas exchange techniques, fluorescence, relative water content measurements) and proteomic analysis (2D-DIGE). Plants of two cultivars (Aurora and Savinjski golding) were exposed to progressive drought in a pot experiment and analysed at different stress stages (mild, moderate and severe). Measurements of relative water content revealed a hydrostable water balance of hop. Photosynthesis was decreased due to stomatal and non-stomatal limitation to the same extent in both cultivars. Of 28 identified differentially abundant proteins, the majority were down regulated and included in photosynthetic (41%) and sugar metabolism (33%). Fifteen % of identified proteins were classified into the nitrogen metabolism, 4% were related to a ROS related pathway and 7% to other functions.

Potential environmental factors that influence the nitrogen concentration and δ(15)N values in the moss Hypnum cupressiforme collected inside and outside canopy drip lines.

Samples of the moss Hypnum cupressiforme were collected at 103 locations in forests of Slovenia. At each location, samples were taken at two types of sites: under tree canopies and in adjacent forest openings. The results show that the moss collected in the forest openings reflects the surrounding land-use characteristics and, consequently, the main N emission sources. For moss sampled under canopies, the characteristics of the forest at the moss-sampling locations are more important than the main emission sources outside the forest. A regression model was used to provide the nitrogen (N) concentration in moss from the forest openings in relation to the N concentration in moss under canopies and other environmental variables. The spatial distribution of the locations of the N concentrations and δ(15)N values in moss collected in the forest openings and under the canopies in relation to main N deposition sources is discussed.

Effect of Plum pox virus on chemical composition and fruit quality of plum.

The quality of fruit from PPV (Plum pox virus)-infected plum (Prunus domestica L.) trees was investigated during the last 3 weeks of ripening using healthy tree (T3), tree with short-term infection (T2), and tree with long-term infection (T1). The pomological variables (presence of necrosis on fruit, color of fruit, color of flesh, firmness, soluble solids content, and fruit weight) and composition of nutritive compounds (sugars and organic acids) and bioactive compounds (phenolics: anthocyanins, flavonols, and hydroxycinnamic acids) were evaluated. The results indicated that the PPV infection modified the ripening process (the most in T1) and the composition of nutritive and bioactive compounds in the healthy-looking part of the fruit. Long-term infected tree (T1) yielded fruit with the poorest pomological traits and with the most modified composition of nutritive and phenolic compounds. The short-term PPV infected tree also produced fruit with significantly altered phytochemicals composition, although the pomological traits had not changed significantly.

Influence of canopy drip on the indicative N, S and δ(15)N content in moss Hypnum cupressiforme.

Samples of Hypnum cupressiforme were collected at two types of site in forest areas: within the forest stand and within forest openings, and analyzed for N and S concentrations and δ(15)N. Mosses sampled within forest openings reflect the atmospheric N deposition; however, no influence of throughfall N deposition on the N in the moss that was sampled within the forest stand was found, nor was any influence of S deposition on the S in the moss found. For the N and S concentrations in the mosses sampled within forest openings, the within-site variability was comparable to the between-site variability, and for the δ(15)N, the within-site variability was lower than the between-site. The results showed that a short distance (<1 m) between the sampling location and the nearest tree canopy increases the N in the moss, and significantly higher values are found in mosses sampled in areas within the forest stand.

Soil physical characteristics after EDTA washing and amendment with inorganic and organic additives.

Soil washing has been established as suitable remediation technology, with most research focused on metal removing efficiency and toxic effect on plants, less on the influence on soil physical characteristics, which was the focus of this study. In soil column experiment highly contaminated soil and soil washed with EDTA, mixed with additives (gypsum, hydrogel, manure, peat) were tested. White clover was used as a soil cover. Yield, metal concentration in soil and plant, aggregate fractionation and stability, saturated hydraulic conductivity and soil water retention of the soil were measured. Soil washing decreased metal concentration in soil and plants, but yield of white clover on remediated soil was significantly lower compared to the original soil. Significant differences in water retention characteristics, aggregate fractionation and stability between original and remediated soil have been determined. Gypsum, hydrogel and peat increased plant available water, manure and peat increased yield on remediated soil.

Prediction of cadmium concentration in selected home-produced vegetables.

Soil contaminated with cadmium presents a potential hazard for humans, animals and plants. The latter play a major role in the transfer of cadmium to the food chain. The uptake of cadmium and its accumulation by plants is dependent on various soil, plants and environmental factors. In order to identify soil properties with statistically significant influence on cadmium concentration in vegetables and to reduce the collection of data, time and costs, regression models can be applied. The main objective of this research was to develop regression models to predict the concentration of cadmium in 9-vegetable species: zucchini, tomato, cabbage, onion, potato, carrot, red beet, endive and chicory, based on soil properties. Soil samples were collected from 123 home gardens of the Municipality of Celje and 59 of these gardens were also included in vegetable sampling. The concentration of elements (e.g. arsenic, cadmium, copper, lead, and zinc) in the samples was determined by Inductively Coupled Plasma Mass Spectrometry. Single (for cabbage, potato, red beet and chicory) and multiple (for tomato, onion, carrot and endive) linear regression models were developed. There was no statistically significant regression model for zucchini. The most significant parameter for the influencing the cadmium concentration in vegetables was the concentration of cadmium in soil. Other important soil properties were the content of organic matter, pH-value and the concentration of manganese. It was concluded that consuming carrots, red beets, endives, onions, potatoes and chicory which are grown in gardens with Cd concentrations (mgkg(-1) DW) above 2.4, 3.2, 6.3, 7.9, 8.3 and 10.9, respectively, might represent an important contribution to dietary Cd exposure.

Experimental evidence of false-positive Comet test results due to TiO2 particle--assay interactions.

We have studied the genotoxicity of TiO2 particles with a Comet assay on a unicellular organism, Tetrahymena thermophila. Exposure to bulk- or nano-TiO2 of free cells, cells embedded in gel or nuclei embedded in gel, all resulted in a positive Comet assay result but this outcome could not be confirmed by cytotoxicity measures such as lipid peroxidation, elevated reactive oxygen species or cell membrane composition. Published reports state that in the absence of cytotoxicity, nano- and bulk-TiO2 genotoxicity do not occur directly, and a possible explanation of our Comet assay results is that they are false positives resulting from post festum exposure interactions between particles and DNA. We suggest that before Comet assay is used for nanoparticle genotoxicity testing, evidence for the possibility of post festum exposure interactions should be considered. The acellular Comet test described in this report can be used for this purpose.

Phytochemicals in fruits of two Prunus domestica L. plum cultivars during ripening.

BACKGROUND: Plums are a rich source of nutritive and bioactive compounds. The objective was to evaluate the impact of fruit tissue (flesh and peel), ripening stage and cultivar on the composition of phytochemicals in plums (Prunus domestica L.) and to analyse the relation between total antioxidant activity (TAA) and the measured variables. RESULTS: The content of total sugars and total organic acids was higher in flesh but total phenolic content (TPC) and TAA was statistically higher in plum peel. The composition of sugars depended on fruit tissue, ripening stage and the cultivar. Ripening and fruit tissue affected the composition and concentrations of organic acids. TPC of 'Haganta' peel increased during ripening but there was no significant time trend for 'Jojo' peel and flesh of both cultivars. The composition of phenolics in peel depended on the cultivar, but in flesh on the cultivar and ripening. Ripening resulted in increased total anthocyanins and TAA in peel. TAA(peel) was positively correlated with TPC(peel) , total anthocyanins(peel), cyanidin 3-glucoside(peel) and peonidin 3-rutinoside(peel) in the cultivar 'Haganta'. CONCLUSION: The composition of phytochemicals in P. domestica fruit depends on fruit tissue, ripening stage and the cultivar. Peel contributes to the TAA on average 20 times more than flesh.

Electrochemical EDTA recycling after soil washing of Pb, Zn and Cd contaminated soil.

Recycling of chelant decreases the cost of EDTA-based soil washing. Current methods, however, are not effective when the spent soil washing solution contains more than one contaminating metal. In this study, we applied electrochemical treatment of the washing solution obtained after EDTA extraction of Pb, Zn and Cd contaminated soil. A sacrificial Al anode and stainless steel cathode in a conventional electrolytic cell at pH 10 efficiently removed Pb from the solution. The method efficiency, specific electricity and Al consumption were significantly higher for solutions with a higher initial metal concentration. Partial replacement of NaCl with KNO(3) as an electrolyte (aggressive Cl(-) are required to prevent passivisation of the Al anode) prevented EDTA degradation during the electrolysis. The addition of FeCl(3) to the acidified washing solution prior to electrolysis improved Zn removal. Using the novel method 98, 73 and 66% of Pb, Zn and Cd, respectively, were removed, while 88% of EDTA was preserved in the treated washing solution. The recycled EDTA retained 86, 84 and 85% of Pb, Zn and Cd extraction potential from contaminated soil, respectively.

Circadian rhythm of cholesterol synthesis in mouse liver: a statistical analysis of the post-squalene metabolites in wild-type and Crem-knock-out mice.

Circadian rhythms affect the total cholesterol levels in humans and animals, although their effect on cholesterol synthesis remain poorly understood. Here, we show for the first time that intermediates of the post-squalene portion of cholesterol synthesis also follow a circadian rhythm in the mouse liver. We used Crem-knock-out mice to investigate the effects of cAMP response element modulator (CREM) isoforms on cholesterol synthesis over time, as compared to wild-type mice. Multiple linear regression and cosinor statistical analysis were carried out on data obtained from 166 liver samples of mice, and the 24-h profiles were modelled across genotype, gender and zeitgeber time for lanosterol, 24,25-dihydrolanosterol, testis meiosis-activating sterol, and 7-dehydrocholesterol, along with cholesterol. The levels of these sterols were higher in female mice compared to males, although the genotype/gender factors showed no effects on the circadian oscillation of these sterols, except for 24,25-dihydrolanosterol. This study also highlights the importance of the statistical methods, where time, genotype and gender are the studied variables.

Uptake and biotransformation of arsenate in the lichen Hypogymnia physodes (L.) Nyl.

The uptake and metabolism of arsenate, As(V), as a function of time and concentration were examined in the lichen Hypogymnia physodes (L.) Nyl. Lichen thalli were exposed to As(V) in the form of a solution. Exponential uptake of As(V) from 4 microg mL(-1) As(V) solution was accompanied by constant arsenite, As(III), excretion back into the solution. Arsenate taken up into the lichens from 0, 0.1, 1, 10 microg mL(-1) As(V) solutions was partially transformed into As(III), dimethylarsinic acid (DMA) and (mono)methylarsonic acid (MA). 48 h after exposure, the main arsenic compound in the lichens was DMA in 0.1, As(III) in 1 and As(V) in 10 microg mL(-1) treatment. The proportion of methylated arsenic compounds decreased with increasing arsenate concentration in the exposure solution. These results suggest that at least two types of As(V) detoxification exist in lichens; arsenite excretion and methylation.