Nucleolytic activities and appearance of a new DNase in relation to nickel and manganese accumulation inAlyssum murale

Serpentine and non-serpentine plants of Alyssum murale, a nickel (Ni) accumulator plant, from North Greece, were studied in order to examine: (1) The ability of natural plants to accumulate metals; (2) the ability of their seedlings to tolerate increasing concentrations of Ni²⁺ or Mn²⁺ (0, 0.16, 0.32, 0.5 and 1 mmol/L), when grown in nutrient solution; (3) the activities and electrophoretic patterns of root and shoot DNases and RNases under the above conditions. Measurements of metal concentrations in serpentine and non-serpentine natural plants and the respective soils revealed: (1) Very low calcium (Ca)/magnesium (Mg) (0.16) ratio and high concentration of Ni in serpentine soil; (2) very high Ca/Mg (17) ratio and high concentration of manganese (Mn) in non-serpentine soil; (3) the ability of serpentine natural plants to accumulate Ni and the inability of plants of both serpentine and non-serpentine populations to accumulate Mn. A. murale plants grown in nutrient solution with increasing Ni²⁺ or Mn²⁺ concentrations showed a negative correlation between the Ni²⁺ or Mn²⁺ concentrations in the nutrient solution, and the chlorophyll concentration, shoot and especially root length. The accumulation of Ni²⁺ or Mn²⁺ in the plant showed a positive correlation with increasing Ni²⁺ or Mn²⁺ concentrations in the nutrient solution. Application of 0.5 mmol/L Ni²⁺ or Mn²⁺ resulted in the inhibition of DNase activities and the appearance of a new DNase form, in both root and shoot detected by electrophoresis in active ssDNA polyacrylamide gel. The new gel-extracted DNase showed nicking action against plasmid DNA and has been characterised as an endo-DNase. In contrast, electrophoretic patterns and RNase activities were unaffected. According to our studies on growth, both serpentine and non-serpentine plants of A. murale have a constitutive ability to tolerate and accumulate Ni²⁺ or Mn²⁺; they have similar DNase and RNase electrophoretic patterns and show a new DNase form under Ni²⁺ or Mn²⁺ stress. This is the first report on the response of nucleolytic enzymes under metallic elements hyperaccumulation.     

Foliar application of 24-epibrassinolide alleviates high-temperature-induced inhibition of photosynthesis in seedlings of two melon cultivars

Brassinosteroids (BRs), an important class of plant steroidal hormones, play a significant role in the amelioration of various biotic and abiotic stresses. 24-epibrassinolide (EBR), an active brassinosteroid, was applied exogenously in different concentrations to characterize a role of BRs in tolerance of melon (Cucumis melo L.) to high temperature (HT) stress and to investigate photosynthetic performance of HT-stressed, Honglvzaocui (HT-tolerant) and Baiyuxiang (HTsensitive), melon variety. Under HT, Honglvzaocui showed higher biomass accumulation and a lower index of heat injury compared with the Baiyuxiang. The exogenous application of 1.0 mg L^?1 EBR, the most effective concentration, alleviated dramatically the growth suppression caused by HT in both ecotypes. Similarly, EBR pretreatment of HTstressed plants attenuated the decrease in relative chlorophyll content, net photosynthetic rate, stomatal conductance, stomatal limitation, and water-use efficiency (WUE), as well as the maximal quantum yield of PSII photochemistry (F_v/F_m), the efficiency of excitation capture of open PSII center, the effective quantum yield of PSII photochemistry (Φ_PSII), photochemical quenching coefficient, and the photon activity distribution coefficients of PSI (α). EBR pretreatment further inhibited the increase in intracellular CO₂ concentration, leaf transpiration rate, minimal fluorescence of dark-adapted state, nonphotochemical quenching, thermal dissipation, and photon activity distribution coefficients of PSII. Results obtained here demonstrated that EBR could alleviate the detrimental effects of HT on the plant growth by improving photosynthesis in leaves, mainly reflected as up-regulation of photosynthetic pigment contents and photochemical activity associated with PSI.     

24-Epibrassinolide alleviates drought effects in young Carapa guianensis plants,improving the hydraulic safety margin,gas exchange and antioxidant defence

• Climate change is increasing the frequency of extreme events such as droughts, limiting plant growth and productivity. Exogenous application of plant growth regulators, such as 24-epibrassinolide (EBR), might be a solution as this molecule is organic, eco-friendly, and biodegradable. This is the first research to examine possible roles of EBR on the hydraulic safety margin, physiological behaviour, and metabolism in Carapa guianensis Aubl. (Meliaceae) exposed to drought. C. guianensis is a widely distributed tree in tropical forests of the Amazon.
• The objective was to determine whether EBR can improve tolerance to water deficit in young C. guianensis by measuring hydraulic traits, nutritional, biochemical and physiological responses, and biomass. The experiment had four randomized treatments: two water conditions (control and water deficit) and two concentrations of EBR (0 and 100 nM EBR).
• EBR increased the water potential and hydraulic safety margin, increased CO₂ fixation, and improved stomatal performance. EBR also stimulated antioxidant defences (SOD, CAT, APX, and POX).
• Overall, tretreatment with EBR improved drought tolerance of young C. guianensis plants.

     

Pretreatment with 24-Epibrassinolide Synergistically Protects Root Structures and Chloroplastic Pigments and Upregulates Antioxidant Enzymes and Biomass in Na+-Stressed Tomato Plants

Salt stress reduces plant growth by negatively interfering with the division rate and cellular expansion, limiting the growth and development of the roots, stems, and leaves. 24-Epibrassinolide (EBR) is a molecule extracted from plant tissues and is a plant growth regulator with a high capacity to modulate tolerance to abiotic stresses. The objective of this study was to verify the possible improvements promoted by pretreatment with EBR in salt-stressed tomato plants, evaluating the variables related to root anatomy, photosynthetic pigments, antioxidant system, and biomass accumulation. The experiment comprised four treatments: two salt conditions (0 and 150 mM NaCl, described as Na⁺ (?) and Na⁺ (?+), respectively) and two concentrations of 24-epibrassinolide (0 and 100 nM EBR, described as EBR (?) and EBR (?+), respectively). EBR modulated the protection and vascularization of root structures, as demonstrated by the increases in epidermis thickness (12%) and metaxilem diameter (119%), respectively. This steroid relieved oxidative damage, which was clearly linked to elevated activities of superoxide ascorbate peroxidase (24%) and guaiacol peroxidase (31%). EBR also benefited photosynthetic pigments, reducing the degradation of chlorophylls. In addition, pretreatment with EBR favoured a higher biomass, which was due to positive effects on leaf and root tissues, including better performance of photosynthetic machinery.

     

The role of Ca2+ ions in modulating changes induced in bean plants by an excess of Cu2+ ions. Chlorophyll fluorescence measurements

Ca²⁺-dependent influence of excess Cu²⁺ on the photosynthetic akpparatus monitored through chlorophyll fluorescence measurements was investigated in runner bean plants (Phaseolus coccineus L. cv. Pie kny Ja?) at three different growth stages. It was observed that the toxic effect of excess Cu²⁺ on plants depends both on their growth stages and the Ca²⁺ content in the medium. Increased Ca²⁺ content limits Cu²⁺ action on plants at their initial growth stage (I) through: stabilization of the PSII complex (increase of the ratio of variable to minimal fluorescence [F_v/F₀]), improved electron flow and reoxidative processes of the quinone primary electron acceptor of PSII (Q_A) (increase of quantum yield of PSII electron transport [φ_e] and photochemical quenching of fluorescence [q_P] values) and elimination of nonphotochemical energy dissipation (decrease of nonphotochemical fluorescence quenching from the Stern-Volmer equation [NPQ] and fraction of the absorbed light energy not used for photochemistry [LNU] values). At this growth stage excess Cu²⁺ decreases the rates of Q_A reduction as a result of decreased PSII activity at its donor side only at lower Ca²⁺ level. At the intermediate growth stage (II) the plants were less sensitive to Cu²⁺ treatment and also to changed Ca²⁺ content. A weakening of some photochemical processes by excess Cu²⁺ could be observed only at a higher Ca²⁺ dose. At the final growth stage of plants (III) Ca²⁺ ions exerted a decisively different effect on the mechanism of excess Cu²⁺ action on bean plants, visualized by decreased PSII stabilization and utilization of absorbed light energy at increased Ca²⁺ content in the medium.     

Influence of metal addition on ethanol production with <Emphasis Type="Italic">Pichia stipitis</Emphasis> ATCC 58784

Trace metals always act as cofactors or coenzymes in many cellular processes. Deficiency or excess of some metals will affect the fermentation of lignocellulosic hydrolysate. In order to make sure the deficient or excessive states of metals in culture medium, metal contents analysis was conducted in Pichia stipitis ATCC 58784 cells, synthetic medium, and diluted acid hydrolysate of rice straw. The results showed that Cu, Ni, and Co were deficient, and Al was a little excessive. So the influences of Cu²⁺, Al³⁺, Ni²⁺, and Co²⁺ additions on the growth and ethanol production of ATCC 58784 were further researched. Low concentration additions of Cu²⁺ and Al³⁺ (<0.24 mM and <0.23 mM, respectively) improved biomass growth of ATCC 58784 by 34 and 13%, respectively; however, higher concentrations decreased biomass growth. On the other hand, addition of Cu²⁺ (0.39 mM) did not affect volumetric ethanol production significantly (P = 0.05) and addition of Al³⁺ (0.38 mM) showed no influence on volumetric ethanol production (P = 0.68). Addition of 0.074 mM Co²⁺ inhibited biomass growth of ATCC 58784 by 13% and volumetric ethanol production by 10%. The biomass growth and volumetric ethanol production of ATCC 58784 was arrested by the addition of 0.33 mM of Ni²⁺ by 53 and 65%, respectively.     

Exogenous application of epibrassinolide attenuated Verticillium wilt in upland cotton by modulating the carbohydrates metabolism,plasma membrane ATPases and intracellular osmolytes

Verticillium dahliae toxin (Vd toxin) was employed as pathogen free model system to induce osmotic stress on upland cotton and its amelioration is investigated using epibrassinolide (EBR). In this study, we observed the physiological and biochemical differences among Vd toxin alone and EBR + Vd toxin treated plants using different levels of root (5, 10, 15 nM) and shoot (50, 100, 200 nM) applied EBR. Results revealed that in absence of EBR, Vd toxin caused 83 % plant wilting and the levels of glycine betaine and proline were 33 and 61 % higher than non treated control, respectively. However, the accumulation of these osmolytes was decreased in EBR treated plants with minimum values at 5 and 200 nM. Furthermore, the results depicted a remarkable decline in soluble sugars, photosynthesis, transpiration, chlorophyll content and chlorophyll florescence (Fv/Fm) in Vd toxin alone treated plants than EBR + Vd toxin. Besides, the activities of sucrose synthase, sucrose phosphate synthase and acid invertase were high in Vd + EBR treated plants with increased root and shoot biomass than Vd toxin alone treated plants. Moreover, EBR remarkably regulated the levels of plasma membrane ATPases and contents of total ATPase and Na⁺ K⁺-ATPase were elevated while contents of Ca²⁺ Mg²⁺-ATPase and H⁺ K⁺-ATPase were decreased as compared to Vd toxin alone treated plants. This study broadens our understanding of Verticillium wilt and demonstrates the potential role of EBR in mediating tolerance against Vd toxin induced stress of V. dahliae in cotton.     

Trace amounts of nickel in belowground rhizomes of Vaccinium myrtillus L. decrease anthocyanin concentrations in aerial shoots without water stress

Nickel (Ni) may impair plant water balance through detrimental effects on the belowground level. Bilberry (Vaccinium myrtillus L.) plants were grown in a mesic heath forest-type soil and subjected to Ni sulphate (NiSO₄·6H₂O) concentrations of 0, 10, 50, 100 and 500 mg m⁻² during an entire growing season in northern Finland (65°N). Biomass of belowground rhizomes, and tissue water content (TWC) and anthocyanin concentrations of aerial shoots were determined from mature plants in order to study rhizospheric Ni stress, and its possible long-distance effects on aerial shoots. As the major proportion of biomass of bilberry is invested in belowground parts, it was hypothesised that Ni-induced rhizospheric disturbance causes water stress in aerial shoots and increases their anthocyanin concentrations for osmotic regulation. Uptake of Ni from the soil to the rhizome and aerial shoots was measured with X-ray fluorescence spectrometry. Ni concentrations in the soil and rhizome exhibited a dose–response relationship, but the concentrations in the rhizome were about 10-fold lower (<3 mg Ni kg⁻¹) than those in the soil (<30 mg Ni kg⁻¹). Translocation of Ni from the rhizome to aerial shoots did not occur, as Ni concentrations in shoots remained at 1 mg Ni kg⁻¹. Although Ni concentrations in the rhizome were below the threshold values of Ni toxicity (i.e. 10–50 mg Ni kg⁻¹), Ni decreased the rhizome biomass. Anthocyanins decreased in aerial shoots along with the Ni accumulation in the rhizome, while TWC was unaffected. The result suggests that anthocyanins are not involved in osmotic regulation under Ni stress, since anthocyanins in aerial shoots responded to the Ni concentrations in the rhizome despite the lack of water stress.     

Translocation of nickel in xylem exudate of plants

Topped plants of tomato (Lycopersicon esculentum), cucumber (Cucumis sativus), corn (Zea mays), carrot (Daucus carota), and peanut (Arachis hypogaea) were treated with 0.5 to 50 micromolar Ni (containing ⁶³Ni) in nutrient solutions. Xylem exudate was collected for 10 hours or, in the case of corn, for 20 hours at 5-hour intervals. Electrophoresis of nutrient solution distributed all Ni cathodically as inorganic Ni²⁺. Low concentrations of Ni in tomato exudate migrated anodically, presumably bound to organic anion (carrier). However, this carrier became saturated at about 2 micromolar Ni in exudate, and excess Ni ran cathodically. Most of the Ni in cucumber, corn, carrot, and peanut exudate ran anodically, and its migration rate was identical for all exudates. Peanut root sap contained 14 to 735 micromolar Ni. The anodic Ni carriers in root sap and exudate appear identical. The carrier in root sap became saturated near 100 micromolar Ni, as shown by cathodic streaking of Ni exceeding that concentration. It appears that all five species translocate low concentrations of Ni in the same anionic form.     

24-Epibrasinolide Delays Chlorophyll Degradation and Stimulates the Photosynthetic Machinery in Magnesium-Stressed Soybean Plants

Adverse effects caused by inadequate magnesium (Mg) supply (deficiency or excess) often cause oxidative stress in chloroplasts and a decline in photosynthetic activity. However, 24-epibrassinolide (EBR) is a natural, biodegradable, and ecologically viable plant growth regulator with multiple roles in plant metabolism. This research aims to determine whether the foliar application of EBR (1) can delay chlorophyll degradation and/or (2) mitigate oxidative stress on the photosynthetic process in magnesium-stressed soybean plants. The experiment followed a completely randomized factorial design with two concentrations of 24-epibrassinolide (0 and 0.1 mM EBR, described as – EBR and?+?EBR, respectively) and three Mg supplies (0.0225, 2.25 and 225 mM Mg, described as low, control and high supply of Mg). Inadequate Mg supplies (deficiency and excess) negatively interfered with photosynthetic pigments, chlorophyll fluorescence and gas exchange. However, exogenous EBR sprayed in plants under high Mg maximized superoxide dismutase (37%), catalase (34%), ascorbate peroxidase (48%) and peroxidase (49%), protecting against oxidative stress and delaying chlorophyll degradation. Concomitantly, plants sprayed with this steroid had increases in Mg content, improving the photochemical efficiency and gas exchange because Mg plays an essential role during the light capture process.

     

Response to copper excess in Arabidopsis thaliana: Impact on the root system architecture,hormone distribution,lignin accumulation and mineral profile

Growth, in particular reorganization of the root system architecture, mineral homeostasis and root hormone distribution were studied in Arabidopsis thaliana upon copper excess. Five-week-old Arabidopsis plants growing in hydroponics were exposed to different Cu²⁺ concentrations (up to 5 μM). Root biomass was more severely inhibited than shoot biomass and Cu was mainly retained in roots. Cu²⁺ excess also induced important changes in the ionome. In roots, Mg, Ca, Fe and Zn concentrations increased, whereas K and S decreased. Shoot K, Ca, P, and Mn concentrations decreased upon Cu²⁺ exposure. Further, experiments with seedlings vertically grown on agar were carried out to investigate the root architecture changes. Increasing Cu²⁺ concentrations (up to 50 μM) reduced the primary root growth and increased the density of short lateral roots. Experiment of split-root system emphasized a local toxicity of Cu²⁺ on the root system. Observations of GUS reporter lines suggested changes in auxin and cytokinin accumulations and in mitotic activity within the primary and secondary root tips treated with Cu²⁺. At toxic Cu²⁺ concentrations (50 μM), these responses were accompanied by higher root apical meristem death. Contrary to previous reports, growth on high Cu²⁺ did not induce an ethylene production. Finally lignin deposition was detected in Cu²⁺-treated roots, probably impacting on the translocation of nutrients. The effects on mineral profile, hormonal status, mitotic activity, cell viability and lignin deposition changes on the Cu²⁺-induced reorganization of the root system architecture are discussed.     

24-epibrassionlide improves photosynthetic response of Rhododendron delavayi to drought

Rhododendron delavayi is an alpine evergreen ornamental plant with strong tolerance to drought stress. Brassinosteroids are promising agents for alleviating the negative effects of drought on plants, but the mechanism by which BRs induce plant resistance to drought is not well understood. The present study investigated the effects of exogenous spray of 24-epibrassionlide (EBR) at different concentrations (0~1 mg l⁻¹) on the physiological response of R. delavayi to drought caused by no watering for 10 days. With the increase in EBR concentration, net photosynthetic rate, stomatal conductance, transportation rate, light saturated photosynthetic rate, light compensation point, light saturation point, excitation energy capture efficiency of reaction center, actual photochemical efficiency of photosystem II (PSII), photochemical quenching and electron transport rate significantly increased, but there were no significant effects on photosynthetic pigment content. These results suggested that the EBR-induced improvement in CO₂ assimilation under drought was mainly related to stomatal and non-stomatal factors, and partially attributed to the increased photochemical efficiency of PSII. In addition, the leaf water potential increased with the increase in EBR concentration, while the malondialdehyde, superoxide dismutase, catalase, proline and soluble protein decreased. The results suggested EBR application partially alleviated the negative effect of drought on R. delavayi by improving water relations and decreasing lipid peroxidation and reactive oxygen species production. We concluded that exogenous application of EBR improved photosynthesis and alleviated the negative effects of drought-induced membrane peroxidation and severe oxidative stress.     

Enhanced sorption of Cu2+ and Ni2+ by acid-pretreated Chlorella vulgaris from single and binary metal solutions

The influence of HCl pretreatment (0.1 mM) on sorption ofCu²⁺ and Ni²⁺ by Chlorella vulgariswas tested using single and binary metal solutions. The optimal initial pH forsorption was 3.5 for Cu²⁺ and 5.5 for Ni²⁺. Second orderrate kinetics described well sorption by untreated and acid-pretreated cells.The kinetic constant q_e (metal sorption at equilibrium) for sorptionof test metals from single and binary metal solutions was increased afterpretreatment of the biomass with HCl. The Langmuir adsorption isotherm wasdeveloped for describing the various results for metal sorption. In single metalsolution, acid pretreatment enhanced q_max for Cu²⁺ andNi²⁺ sorption by approximately 70% and 65%, respectively.Cu²⁺ and Ni²⁺ mutually interfered with sorption of theother metal in the binary system. The combined presence of Cu²⁺ andNi²⁺ led to their decreased sorption by untreated biomass by 19% and88%, respectively. However, acid-pretreated biomass decreased Cu²⁺and Ni²⁺ sorption by 15 and 22%, respectively, when both the metalswere present in the solution. The results suggest a reduced mutual interferencein sorption of Cu²⁺ and Ni²⁺ from the binary metal systemdue to the acid pretreatment. Acid-pretreated cells sorbed twice the amount ofCu²⁺ and ten times that of Ni²⁺ than the untreated biomassfrom the binary metal system. Acid pretreatment more effectively enhanced thesorption of Ni²⁺ form the binary metal solution. The total metalsorption by untreated and acid-pretreated biomass depended on theCu²⁺ : Ni²⁺ ratio in the binary metal system. Acidpretreatment of C. vulgaris could be an effective andinexpensive strategy for enhancing Cu²⁺ and Ni²⁺ sorptionfrom single and binary metal solutions.     

Epibrassinolide Application Regulates Some Key Physio-biochemical Attributes As Well As Oxidative Defense System in Maize Plants Grown Under Saline Stress

It was aimed to investigate the ameliorative effect of exogenously applied 24-epibrassinolide (EBR) on some key growth parameters and mineral elements in two salt-stressed maize (PR 32T83 and PR 34N24) cultivars. A factorial experiment was designed with two electrical permeability (EC) levels (1.1 and 8.0 dS/m) and two levels (1.5 and 2.0 µM) of EBR supplied as a seed treatment, foliar spray, or both in combination. The foliar application of EBR was done once a week during the experiment. After 42 days of these treatments, the plants were harvested to assess growth, water relations, and oxidative and antioxidative systems. Salt stress markedly reduced plant fresh and dry weights, maximum fluorescence yield of PS-II, chlorophyll contents, leaf water potential, and leaf K and Ca, but it increased membrane permeability, the activities of superoxide dismutase (SOD; EC 1.15.1.1), peroxidase (POD; EC. 1.11.1.7), and catalase (CAT; EC. 1.11.1.6) enzymes, and the contents of proline and glycine betaine, leaf sap osmotic pressure, lipid peroxidation, hydrogen peroxide, and leaf Na and Cl. However, both seed treatment and foliar application of EBR to the maize plants exposed to saline conditions enhanced key growth attributes, water relations, and the activities of various antioxidant enzymes as well as the levels of proline, but they reduced electrolyte leakage, and H₂O₂ and MDA contents. Saline stress reduced leaf N, Ca²⁺, K⁺, and P contents as compared to those in the non-stressed plants. Both seed treatment and foliar application of EBR reduced Na⁺ and Cl^? concentrations, but increased those of N, Ca²⁺, K⁺, and P. Foliar application of EBR was more effective in increasing nutrient levels of plants grown at the high saline regime compared to the seed treatment of EBR. The study clearly indicates that both seed treatment and foliar application of EBR at the rate of 2.0 µM can overcome the detrimental effect of salinity stress on maize growth, which was found to be significantly linked to reduced concentrations of Na, Cl, MDA, and H₂O₂ as well as EL and increased activities of key antioxidant enzymes in the maize plants.     

Nickel hyperaccumulation in shoot cultures of Alyssum markgrafii

Shoot cultures of Alyssum markgrafii O.E. Shulz, endemic nickel hyperaccumulating species of central Balkan, were established and maintained on Murashige and Skoog medium supplemented with 0.2 mg dm^–3 benzyladenine (BA). Nickel in form of NiCl₂ . 6 H₂O was supplemented at 22 different concentrations ranging from 0.0001 to 15 mM but none of them was lethal to cultures. High Ni²⁺ concentrations (10 mM or more) arrested shoot growth which, upon transfer to Ni-free medium, commenced via axillary bud proliferation. Shoots that developed from axillary buds through the subculture manifested increased tolerance to Ni²⁺ expressed as shoot elongation. Shoot multiplication and dry biomass production decreased with increase of Ni²⁺ in medium. Only the accumulation of Ni²⁺ in tissues increased with Ni²⁺ content of the medium. Apart from shoot cultures, high Ni²⁺ accumulation was registered in undifferentiated callus cultured on medium with 0.5 mg dm^–3 BA and 0.5 mg dm^–3 naphthylacetic acid. Highest content of accumulated Ni was 2.37 g g^–1 (d.m.) in shoots and 2.65 g g^–1 (d.m.) in callus, both measured on medium with 15 mM Ni²⁺.     

The binding of Ni2+ to adenylyl-3',5'-adenosine and to poly(adenylic acid)

Studies of the binding of Ni²⁺ to adenylyl-3',5'-adenosine (ApA) at pH 6-0 by ultraviolet spectrophotometry indicate the formation of a 1:1 complex in the presence of a large excess of metal ion. At 25 °C. and ionic strength μ = 0.5 M, the stability constant of Ni(ApA) is evaluated to be K = 2.6 (±0.6) M^?1. The low stability is taken as evidence that the predominant complex species is one in which the ApA acts as a monodentate ligand, mainly through the adenine group. The rate constants for complex formation and dissociation, k_f = 1430 M^?1 s^?1 and k_b = 665 s^?1 (25°C. μ = 0.5M). determined by the temperature-jump relaxation technique, are consistent with this interpretation. The binding strength of Ni²⁺ to poly(adenylic acid) [poly(A)] has been studied at pH 7.0 using murexide as an indicator of the concentration of free Ni²⁺. Within the concentration range [Ni²⁺ = 1 × 10^?5 × 10^?3 M the data can be represented in the form of a linear Scatchard plot. i.e., the process can be described as the binding of Ni²⁺ to one class of independent binding sites. The number of binding sites per monomer is 0.26, and the stability constant K = 8.2×10³ M^?1 (25°C μ = 0.1 M). In kinetic studies of the reaction of Ni²⁺ with poly(A), two relaxation effects due to complex formation were detected, one with a concentration-independent time constant of about 0.4 ms, the other with a concentration-dependent time constant in the millisecond range. The concentration dependence of the longer relaxation time can be accounted for by a three-step mechanism which consists of a fast second-order association reaction followed by two first-order steps. There is evidence, however, that the overall process is more complicated than expressed by the three-step mechanism.     

Changes in morphological and anatomical structure of cabbage (Brassica oleracea L.) outer leaves and in ultrastructure of their chloroplasts caused by an in vitro excess of nickel

Morphological, anatomical and ultrastructural changes, the quantity and quality of stomata, and the chlorophyll (Chl) content in primary outer leaves of cabbage plants cv. Sława from Enkhouizen were examined. The plants were grown in agar with basic MS medium containing added nickel (as NiSO₄×7 H₂O) in concentrations of 0, 5, 10, and 20 g m^-3 (Ni₀, Ni₅, Ni₁₀, Ni₂₀). Reduction of leaf blade area, of succulence and of leaf density, and growth of specific leaf area were noticed in plants treated with all concentrations of Ni. In Ni-treated plants the total number of stomata and open stomata decreased, and the number of defective stomata in both adaxial and abaxial side of leaves was higher. In all Ni-treated samples the volume of spongy and palisade mesophyll cells was smaller in comparison to control, and it was decreasing when the Ni concentration was increasing; at the same time, the number of mesophyll cells on the same area of cross sections of leaves was increasing. In comparison to control, the intercellular spaces of mesophyll tissue decreased in Ni₁₀ and Ni₂₀ plants and increased in Ni₅ plants. In Ni5 plants the number of chloroplasts in mesophyll cells was higher than in the Ni₀ control. Reduction of grana size and increase of number of non-appressed lamellae, which often had central arrangement, were observed. In the Ni₁₀ and Ni₂₀ plants, the number and size of chloroplasts decreased, and their internal membranes (especially grana) were reduced and swollen. In Ni₅ plants the concentration of Chl in leaves was slightly higher than in the control; in Ni₁₀ and Ni₂₀ plants it was lower than in the control. This revised version was published online in June 2006 with corrections to the Cover Date.     

Changes in morphological and anatomical structure of cabbage (Brassica oleracea L.) outer leaves and in ultrastructure of their chloroplasts caused by an in vitro excess of nickel

Morphological, anatomical and ultrastructural changes, the quantity and quality of stomata, and the chlorophyll (Chl) content in primary outer leaves of cabbage plants cv. S?awa from Enkhouizen were examined. The plants were grown in agar with basic MS medium containing added nickel (as NiSO₄×7 H₂O) in concentrations of 0, 5, 10, and 20 g m^-3 (Ni₀, Ni₅, Ni₁₀, Ni₂₀). Reduction of leaf blade area, of succulence and of leaf density, and growth of specific leaf area were noticed in plants treated with all concentrations of Ni. In Ni-treated plants the total number of stomata and open stomata decreased, and the number of defective stomata in both adaxial and abaxial side of leaves was higher. In all Ni-treated samples the volume of spongy and palisade mesophyll cells was smaller in comparison to control, and it was decreasing when the Ni concentration was increasing; at the same time, the number of mesophyll cells on the same area of cross sections of leaves was increasing. In comparison to control, the intercellular spaces of mesophyll tissue decreased in Ni₁₀ and Ni₂₀ plants and increased in Ni₅ plants. In Ni5 plants the number of chloroplasts in mesophyll cells was higher than in the Ni₀ control. Reduction of grana size and increase of number of non-appressed lamellae, which often had central arrangement, were observed. In the Ni₁₀ and Ni₂₀ plants, the number and size of chloroplasts decreased, and their internal membranes (especially grana) were reduced and swollen. In Ni₅ plants the concentration of Chl in leaves was slightly higher than in the control; in Ni₁₀ and Ni₂₀ plants it was lower than in the control.

     

Ni2+-uptake inPseudomonas putida strain S4: a possible role of Mg2+-uptake pump

Essential metal ion homeostasis is based on regulated uptake of metal ions, both during its scarcity and abundance.Pseudomonas putida strain S4, a multimetal resistant bacterium, was employed to investigate Ni²⁺ entry into cells. It was observed that Mg²⁺ regulates the entry of Ni²⁺ and by this plays a protective role to minimize Ni²⁺ toxicity in this strain. This protection was evident in both growth as well as viability. Intracellular accumulation of Ni²⁺ varied in accordance with Mg²⁺ concentrations in the medium. It was hypothesized that Ni²⁺ enters the cell using a broad Mg²⁺ pump, i.e. the CorA system, as the CorA inhibitor, i.e. Co(III) Hex, also inhibits Ni²⁺ uptake. This led to the inference that Mg²⁺-based protection was basically due to competitive inhibition of Ni²⁺ uptake. We also show that Zn²⁺ can further regulate the entry of Ni²⁺     

Nickel transport systems in microorganisms

The transition metal Ni is an essential cofactor for a number of enzymatic reactions in both prokaryotes and eukaryotes. Molecular analyses have revealed the existence of two major types of high-affinity Ni²⁺ transporters in bacteria. The Nik system of Escherichia coli is a member of the ABC transporter family and provides Ni²⁺ ion for the anaerobic biosynthesis of hydrogenases. The periplasmic binding protein of the transporter, NikA, is likely to play a dual role. It acts as the primary binder in the uptake process and is also involved in negative chemotaxis to escape Ni overload. Expression of the nik operon is controlled by the Ni-responsive repressor NikR, which shows functional similarity to the ferric ion uptake regulator Fur. The second type of Ni²⁺ transporter is represented by HoxN of Ralstonia eutropha, the prototype of a novel family of transition metal permeases. Members of this family have been identified in gram-negative and gram-positive bacteria and recently also in a fission yeast. They transport Ni²⁺ with very high affinity, but differ with regard to specificity. Site-directed mutagenesis experiments have identified residues that are essential for transport. Besides these uptake systems, different types of metal export systems, which prevent microorganisms from the toxic effects of Ni²⁺ at elevated intracellular concentrations, have also been described. Received: 14 July / Accepted: 8 October 1999