Optimising the DPPH Assay for Cell-Free Marine Microorganism Supernatants

Antioxidants prevent ageing and are usually quantified and screened using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. However, this assay cannot be used for salt-containing samples, such as the cell-free supernatants of marine microorganisms that are aggregated under these conditions. Herein, the DPPH solvent (methanol or ethanol) and its water content were optimized to enable the analysis of salt-containing samples, aggregation was observed for alcohol contents of >70%. The water content of methanol influenced the activities of standard antioxidants but did not significantly affect that of the samples. Based on solution stability considerations, 70% aqueous methanol was chosen as the optimal DPPH solvent. The developed method was successfully applied to the cell-free supernatants of marine bacteria (Pseudoalteromonas rubra and Pseudoalteromonas xiamenensis), revealing their high antioxidant activities. Furthermore, it was concluded that this method would be useful for the screening of marine microorganism–derived antioxidants, which also has numerous potential applications, such as salt-fermented foods.     

Spray Drying of Chitosan Acid Salts: Process Development,Scaling Up and Physicochemical Material Characterization

We investigated a spray drying process for preparing water-soluble salts of high molecular weight chitosan (CH) intended for pharmaceutical excipient applications. CH was derived from chitin of marine lobster origin (Panulirus argus). The effects of organic acid (acetic or lactic acid) and the ratio (difference) of inlet/outlet air temperature (140/90 °C or 160/100 °C) on spray drying were studied. The yield of spray-dried CH salt powders ranged from 50% to 99% in laboratory and industrial-scale processes. The spray-dried dry powder of CH salts consisted of spherical agglomerated particles with an average diameter of 36.2 ± 7.0 µm (CH acetate) and 108.6 ± 11.5 µm (CH lactate). After dispersing the spray-dried CH salt powder samples in purified water, the mean particle sizes obtained for the CH acetate salts were 31.4 nm (batch A001), 33.0 nm (A002) and 44.2 nm (A003), and for the CH lactate salts 100.8 nm (batch L001), 103.2 nm (L002) and 121.8 nm (L003). The optimum process conditions for spray drying were found: an inlet air temperature of 160 ± 5 °C, an outlet temperature of 100 ± 5 °C and an atomizer disk rotational speed of 18,200 min⁻¹. The X-ray powder diffraction (XRPD) and differential scanning calorimetry (DSC) results confirmed the amorphous state of the CH salts. The ¹H nuclear magnetic resonance (NMR) and Fourier transform infrared (FT-IR) spectra of CH acetate and lactate salts verified that the spray drying process does not affect the polymer backbone. In conclusion, both laboratory and industrial-scale spray drying methods for preparing water-soluble acid salts of CH are reproducible, and the physicochemical properties of the corresponding CH acid salts are uniform.     

Salt effects on growth and mineral contents of Diplachne fusca (Kallar grass)

Effects of NaCl, KC1, and CaCl₂ alone, and in combinations of NaCl/KCl and NaCl/CaCl₂ on growth and ion uptake by ‘Kallar'grass in soil and in solution cultures were studied. In soil up to 150 mM NaCl and KCl had little effect on growth but CaCl₂ depressed growth strongly. Dry weights in 150 mM NaCl/KCl decreased but remained little affected in NaCl/CaCl₂. Plant response to Na⁺ was little affected by Ca⁺⁺ or K⁺ and no interactions between Na⁺ and Ca⁺⁺ or Na⁺ and K⁺ were observed. Plant tissue exhibited a strong selectivity for K⁺ over Na⁺ Concentrations of Na and Cl in the tops exceeded those in the roots. Leaf transections are presented providing some information on the tissue anatomy.     

Leaf Responses to Reduced Iron Availability in Two Tomato Genotypes: T3238FER (Iron Efficient) and T3238fer (Iron Inefficient)

Abstract

The present work is aimed at evaluating some effects induced by different levels of iron availability in the growth medium for two different tomato (Lycopersicon esculentum Mill.) genotypes, the T3238fer (Tfer), unable to activate mechanisms for iron mobilization and uptake known as “strategy I,” and its correspondent wild‐type T3238FER (TFER). By using different iron concentration in the growth solution, the most suitable iron level to induce phenotypic differences between the two genotypes without being lethal for the mutant was found to be 40 µM Fe‐Na‐EDTA. The analyses were carried out also on plants grown with 80 µM Fe‐Na‐EDTA, an iron concentration at which the two genotypes showed no phenotypic differences. A significant decrease in total leaf iron and chlorophyll content was detected in both genotypes following reduction of iron concentration in the nutrient solution, and was particularly evident in Tfer40, which showed symptoms of chlorosis. The photo‐electron transport rate of the whole chain was significantly affected by growth conditions as well as by genotype, the lowest activity being detected in Tfer40 plants. Chlorophyll a fluorescence analysis revealed an increase in non‐photochemical quenching (q _NP) of Tfer plants grown at both iron concentrations, indicating the activation of photoprotective mechanisms, which, however, were not sufficient to prevent photoinhibition when plants were grown at 40 µM iron, as indicated by significant reduction in PSII photochemistry (F _v/F _m) and photochemical quenching (q _P). The actual quantum yield of PSII (Φ_PSII) and the intrinsic PSII efficiency (Φ_EXC) showed the same behavior of q _P and F _v/F _m ratio. A significant effect of mutation and iron supply on all the pigments was detected, and was particularly evident in the mutant grown at 40 µM iron. A different behavior was shown by the three pigments involved in the xantophyll cycle, violaxanthin being less affected than chlorophylls and the other carotenoids, and zeaxanthin even increasing, due to the xanthophyll cycle activation. In conclusion, the interaction between iron deprivation and fer mutation induced functional alterations to the photosynthetic apparatus. Anyway, as far as concerns the photo‐electron transport activity, the influence of fer mutation seemed to occur independently from iron supply.     

Response to salinity of three grain legumes for potential cultivation in arid areas

Abstract

The material balance of all the chemical species associated with the phosphate sorption reaction by two amorphous clays, silica-alumina gel and synthetic goethite was investigated adjusting the initial pH to 4.0, 5.0, 6.0, and 7.0. Phosphate sorption was found to decrease with increasing initial pH. During phosphate sorption, the equilibrium pH rose, adsorption of Cl^- decreased to a state of negative adsorption, adsorption of Na⁺ increased markedly, silicate was released, and Al in the solution was precipitated. With increasing initial pH, the amount of apparent released OH^- increased, the amount of Na⁺ adsorption increased, and the amount of released silicate decreased. The overall relationship of the material balance is explained in terms of the charge balance associated with phosphate sorption.     

Iron transport in plants: Future research in view of a plant nutritionist and a molecular biologist

Iron is attractive to plant physiologists since J. Sachs has proven in 1868 the essentiality and the possible leaf uptake of Fe. It lasted about 100 years before the principal processes for Fe mobilization in the rhizosphere were discovered and classified as two distinct strategies for Fe acquisition. During the 80's and 90's of the last century the uptake of Fe²⁺ and Fe^III-phytosiderophores by specific transporters in strategy I- and strategy II-plants, respectively, were postulated without any application of the new approaching molecular techniques. In the following decade, the various transporters for Fe uptake by roots, such as AtIRT1 in Arabidopsis or ZmYS1 in maize and their possible regulation were characterized. In the following years with fast developing molecular approaches further Fe trans ortsrs were genetically described with often only vague physiological functions. In view of a plant nutritionist, besides uptake processes by roots, the following transport processes within the respective target tissue have to be considered by molecular biologists in more detail: 1) radial transfer of Fe from the root cortex through the endodermis, 2) xylem loading in roots, 3) transfer of Fe from xylem to phloem via transfer cells, 4) phloem loading with Fe in source leaves and retranslocation to sink organs, and 5) remobilization and retranslocation via the phloem during senescence of perennial plants. The importance of these various specific transport processes for a well-regulated Fe homeostasis in plants and new strategies to identify and characterize proteins involved in Fe transport and homeostasis will be discussed.     

Regulation and function of AtNRAMP4 metal transporter protein

The NRAMP gene family encodes integral membrane proteins mediating the transport of a broad range of transition metals in bacteria, fungi, plants, and animals. We studied the regulation of AtNRAMP4 in Arabidopsis. In a previous study, we showed that AtNRAMP3 and AtNRAMP4 transport manganese (Mn), iron (Fe), and cadmium (Cd). In this study, we show that, in contrast to AtNRAMP3, AtNRAMP4 complements the growth phenotype of the zrt1zrt2 Zn uptake deficient yeast mutant. In a previous study, we have shown that, under Fe starvation, AtNRAMP4 mRNA levels are up-regulated in Arabidopsis. To analyze the regulation of AtNRAMP4 at the protein level, we generated specific antibodies against AtNRAMP4 protein. The antiserum was able to recognize a tagged version of AtNRAMP4 expressed in yeast. The antibody did not reveal any change in AtNRAMP4 protein level upon Fe starvation in Arabidopsis thaliana ecotype Columbia plants. In AtNRAMP4 overexpressing plants, high levels of AtNRAMP4 protein could be detected. AtNRAMP4 overexpressing plants display cadmium hypersensitivity in a medium containing 50 μm FeEDTA as Fe source. However, despite the constitutive accumulation of AtNRAMP4 protein, AtNRAMP4 over-expressing plants did not display Cd hypersensitivity under high Fe supply (100 μm FeHBED). AtNRAMP4 over-expressing lines displayed the same sensitivity to Zn as controls under all conditions tested. Our results suggest a translational level for the regulation of AtNRAMP4. Over-expression of AtNRAMP4 in Arabidopsis thaliana confers a slight hypersensitivity to Cd but not to Zn.     

Rice phenolics efflux transporter 2 (PEZ2) plays an important role in solubilizing apoplasmic iron

Iron (Fe) is an essential micronutrient for plants; however, despite its abundance in soils it is not readily available in neutral to alkaline soils. Plants secrete phenolics, such as protocatechuic acid (PCA) and caffeic acid (CA), to absorb and utilize precipitated apoplasmic Fe from root surfaces. However, the synthesis and secretion of phenolics have not been well characterized in plants. We have identified and characterized a rice (Oryza sativa L.) mutant with reduced amounts of PCA and CA in xylem sap and root exudates; hence we named it phenolics efflux zero 2 (pez2). PEZ2 localized to the plasma membrane in onion (Allium cepa L.) epidermal cells and transported PCA when expressed in Xenopus laevis oocytes. PEZ2 expression was observed in whole root near root tips. Similarly, strong expression was observed in leaves. In line with reduced amounts of PCA and CA in xylem sap, the xylem Fe concentration was also low in pez2 plants. These results suggest that PEZ2 is involved in solubilization of apoplasmic Fe in rice.     

Physiological responses and adaptive strategies of wheat seedlings to salt and alkali stresses

Abstract

Effects of salt (NaCl?:?Na₂SO₄) and alkali (NaHCO₃?:?Na₂CO₃) stresses on the contents of inorganic ions and organic solutes in wheat shoots were compared to explore the physiological responses and adaptive strategies of wheat to these stresses. Wheat significantly accumulated Na⁺ and simultaneously accumulated Cl^?, soluble sugars and proline to maintain osmotic and ionic balance under salt stress. Compared with salt stress, the high pH from alkali stress enhanced Na⁺ accumulation and affected the absorption of inorganic anions. To maintain ionic and osmotic balance, wheat accumulated organic acids, soluble sugars and proline. The accumulation of Cl^? and organic acids was the main difference in the physiological responses and adaptive mechanisms to salt and alkali stresses, respectively.     

Effect of Mixed-Salt Salinity on Growth and Ion Relations of a Barnyardgrass Species

ABSTRACT

Recent investigations of the plant genome have revealed a large degree of similarity among cereal crops (specifically within the family Triticeae) and other related grass species. Recognition of the close genetic relationship among the grasses indicates that more exotic species, such as weedy grasses, may be exploited by plant breeders to enhance biotic/abiotic stress tolerance in cereal crops. Echinochloa crus-galli (L.) ^{1 1} Mention of company names or products is for the benefit of the reader and does not imply endorsement, guarantee, or preferential treatment by the USDA or its agents. Beauv. (barnyardgrass), a wide-spread, persistent C₄ weed species of agricultural importance, is reported to tolerate high levels of salinity. Growth and ion relations were studied in barnyardgrass using mixed-salt salinity (with final electrical conductivities (ECi) of 3 (control), 7, 11, and 19 dS m^?1 in the irrigation water) to determine whether the mechanism(s) for its salt tolerance are similar to those previously reported for other cereal crops. Such a finding would indicate that barnyardgrass may possess valuable genetic traits. Ion accumulation and ion selectivity ratios in the various treatments were determined from oven-dried shoot tissue. Significant (P < 0.01) linear regressions were obtained between either fresh weight or plant height and ECi. The point of 50% reduction (C₅₀) in fresh weight was predicted to occur at about 13.9 dS m^?1; the C₅₀ value for plant height was at about 22.9 dS m^?1. Statistical analysis of calculated Gapon constants for potassium (K)/sodium (Na) indicated that ion selectivity was not affected until after 11 dS m^?1. Calcium (Ca)/Na selectivity was not significantly affected by salinity. We concluded that, similar to some cereal crops, barnyardgrass maintains growth under saline conditions by maintaining high K/Na ratios, possibly because of better membrane integrity due to high Ca selectivity.