Ion-selective Electrode Technique Research Articles

Cold tolerance of Drosophila species is tightly linked to the epithelial K+ transport capacity of the Malpighian tubules and rectal pads.

Insect chill tolerance is strongly associated with the ability to maintain ion and water homeostasis during cold exposure. Maintenance of K+ balance is particularly important due to its role in setting the cell membrane potential that is involved in many aspects of cellular function and viability. In most insects, K+ balance is maintained through secretion at the Malpighian tubules, which balances reabsorption from the hindgut and passive leak arising from the gut lumen. Here, we used the scanning ion-selective electrode technique (SIET) at benign (23°C) and low (6°C) temperatures to examine K+ flux across the Malpighian tubules and the rectal pads in the hindgut in five Drosophila species that differ in cold tolerance. We found that chill-tolerant species were better at maintaining K+ secretion and suppressing reabsorption during cold exposure. In contrast, chill-susceptible species exhibited large reductions in secretion with no change, or a paradoxical increase, in K+ reabsorption. Using an assay to measure paracellular leak, we found that chill-susceptible species experience a large increase in leak during cold exposure, which could explain the apparent increase in K+ reabsorption found in these species. Our data therefore strongly support the hypothesis that cold-tolerant Drosophila species are better at maintaining K+ homeostasis through an increased ability to maintain K+ secretion rates and through reduced movement of K+ towards the hemolymph. These adaptations are manifested both at the Malpighian tubule and at the rectal pads in the hindgut, and ensure that cold-tolerant species experience less perturbation of K+ homeostasis during cold stress.

Characteristics of cadmium uptake and membrane transport in roots of intact wheat (Triticum aestivum L.) seedlings

Wheat is one of several cereals that is capable of accumulating higher amounts of Cd in plant tissues. It is important to understand the Cd2+ transport processes in roots that result in excess Cd accumulation. Traditional destructive technologies have limited capabilities in analyzing root samples due to methodological limitations, and sometimes may result in false conclusions. The mechanisms of Cd2+ uptake into the roots of wheat seedlings (Triticum aestivum L.) were investigated by assessing the impact of various inhibitors and channel blockers on Cd accumulation as well as the real-time net Cd2+ flux at roots with the non-destructive scanning ion-selective electrode technique. The P-type ATPase inhibitor Na3VO4 (500 μM) had little effect on Cd uptake (p < 0.05) and the kinetics of transport in the root of wheat, suggesting that Cd2+ uptake into wheat root cells is not directly dependent on H+ gradients. While, the uncoupler 2,4-dinitrophenol significantly limited Cd2+ uptake (p < 0.05) and transport kinetics in the root of wheat, suggesting the existence of metabolic mediation in the Cd2+ uptake process by wheat. The Cd content at the whole-plant level in wheat was significantly (p < 0.05) decreased upon pretreatment with the Ca2+ channel blockers La3+ or Gd3+ and Verapamil, but not in case of pretreatment with the K+ channel blocker tetraethylammonium (TEA). In addition, the inhibitors of the Ca2+ channel, as well as high concentrations of Ca2+, reduced the real-time net Cd2+ fluxes at the root surface in SIET experiments. These results indicate that Cd2+ moves across the plasma lemma of the wheat root via Ca2+ channels. In addition, our results suggested a role for protein synthesis in mediating Cd2+ uptake and transport by wheat.

Characteristics of NH4+ and NO3- fluxes in tea (Camellia sinensis) roots measured by scanning ion-selective electrode technique.

As a vital beverage crop, tea has been extensively planted in tropical and subtropical regions. Nitrogen (N) levels and forms are closely related to tea quality. Based on different N levels and forms, we studied changes in NO3− and NH4+ fluxes in tea roots utilizing scanning ion-selective electrode technique. Our results showed that under both single and mixed N forms, influx rates of NO3− were much lower than those of NH4+, suggesting a preference for NH4+ in tea. With the increase in N concentration, the influx rate of NO3− increased more than that of NH4+. The NH4+ influx rates in a solution without NO3− were much higher than those in a solution with NO3−, while the NO3− influx rates in a solution without NH4+ were much lower than those in a solution with NH4+. We concluded that (1) tea roots showed a preference for NH4+, (2) presence of NO3− had a negative effect on NH4+ influx, and (3) NH4+ had a positive effect on NO3− influx. Our findings not only may help advance hydroponic tea experiments but also may be used to develop efficient fertilization protocols for soil-grown tea in the future.

Determining the fluxes of ions (Pb2+, Cu2+ and Cd2+) at the root surface of wetland plants using the scanning ion-selective electrode technique

Background and aims Measuring specific ion fluxes from different regions of the root under practical physiological conditions is crucial for understanding metal uptake mechanisms by plants.

Pathways of root uptake and membrane transport of Cd2+ in the zinc/cadmium hyperaccumulating plant Sedum plumbizincicola.

Uptake and membrane transport of cadmium (Cd) in roots of the hyperaccumulator Sedum plumbizincicola X.H. Guo et S.B. Zhou ex L.H. Wu was characterized by assessing the impact of various inhibitors and ion channel blockers on Cd accumulation as well as the real-time net Cd2+ flux at the roots with application of the scanning ion-selective electrode technique. The uncouplers 2,4-dinitrophenol and P-type adenosine triphosphatase inhibitor Na3 VO4 significantly limited Cd2+ uptake and transport kinetics in the root of S. plumbizincicola. These findings indicate that Cd is actively taken up into the roots. The Cd content in plant was significantly decreased with pretreatments of the Ca2+ channel blocker La3+ or Gd3+ and the K+ channel blocker tetraethylammonium, as well as in the presence of higher concentration of Ca2+ and K+ . These findings indicated that uptake of Cd2+ into the root of S. plumbizincicola proceeds through ion channels that are permeable to both Ca2+ and K+ as confirmed by the direct evidence of real-time net Cd2+ fluxes at the root surface in the treatments with ion channel inhibitors, as well as in the presence of elevated concentrations of Ca2+ and K+ . In addition, the results suggested a role for phytochelatin and protein synthesis in mediating Cd2+ uptake by S. plumbizincicola. These findings increase the understanding of Cd2+ uptake and membrane transport pathways in roots of the Zn/Cd hyperaccumulator S. plumbizincicola. Environ Toxicol Chem 2017;36:1038-1046. © 2016 SETAC.

Salt secretion is linked to acid-base regulation of ionocytes in seawater-acclimated medaka: new insights into the salt-secreting mechanism.

Ionocytes in the skin and gills of seawater (SW) teleosts are responsible for both salt and acid secretion. However, the mechanism through which ionocytes secrete acid is still unclear. Here, we hypothesized that apical Na+/H+ exchangers (NHE2/3), carbonic anhydrase (CA2-like), and basolateral HCO3−/Cl− exchanger (AE1) are involved in acid secretion. In addition, the hypothesized involvement of basolateral AE1 suggested that acid secretion may be linked to Cl− secretion by ionocytes. The scanning ion-selective electrode technique (SIET) was used to measure H+ and Cl− secretion by ionocytes in the skin of medaka larvae acclimated to SW. Treatment with inhibitors of NHE, CA, and AE suppressed both H+ and Cl− secretion by ionocytes. Short-term exposure to hypercapnic SW stimulated both H+ and Cl− secretion. mRNA of CA2-like and AE1 were localized to ionocytes in the skin. Branchial mRNA levels of NKCC1a, CA2-like, and AE1a increased together with the salinity to which fish were acclimated. In addition, both AE1a and AE1b mRNA increased in fish acclimated to acidified (pH 7) SW; NKCC1a mRNA increased in fish acclimated to pH 9 SW. This study reveals the mechanism of H+ secretion by ionocytes, and refines our understanding of the well-established mechanism of Cl− secretion by ionocytes of SW fish.

Localized electrochemical characterization of organic coatings: A brief review

Abstract Local electrochemical techniques such as scanning electrochemical microscopy, scanning vibrating electrode technique, local electrochemical impedance spectroscopy, scanning Kelvin probe technique, and scanning ion-selective electrode technique have gained significant attention in organic coating research. These techniques have enhanced our understanding of the fundamental processes of corrosion at defects and underneath coatings. Each of these techniques employ unique measurement strategy to provide important local information about coatings, their protective properties, defects, and failure mechanisms. In this brief review, the basic principles of these techniques and the nature of information that has been extracted from these techniques to study organic coatings are discussed.

Efficient transmembrane anion transport mediated by a bis(imidazolyl)-functionalized bis(choloyl) conjugate

A bis(imidazolyl)-functionalized bis(choloyl) conjugate was synthesized and assessed for its transmembrane anionophoric activity by means of chloride ion selective electrode technique and pyranine assays. The results indicate that under the assay conditions, this conjugate was capable of mediating the symport of proton and anions, presumably via a channel mechanism. In addition, this compound was found to exhibit much higher anionophoric activity than the analogue without imidazolyl groups, revealing the significant role of the imidazolyl groups in the anion transport process.

Study of the formation of a protective layer in a defect from lithium-leaching organic coatings

Abstract Lithium salts were investigated as leachable corrosion inhibitor and potential replacement for hexavalent chromium in organic coatings. Coatings loaded with lithium carbonate or lithium oxalate demonstrated active corrosion inhibition by the formation of a protective layer in a damaged area. The present paper provides more insight into the formation and composition of the protective layer in a damaged area generated from the lithium salt loaded coatings when exposed to neutral salt spray testing conditions (ASTM B-117). Lithium-ion leaching from the coating matrix was demonstrated with atomic absorption spectroscopy and the pH conditions in the damaged area were determined with a scanning ion-selective electrode technique. Additionally, the formation of the protective layer was studied with microscopic and surface analytical techniques. Scanning electron micrographs and Auger electron spectroscopy depth profiles revealed the process of coverage and growth of the protective layer in the damaged area. Furthermore, X-ray photoelectron spectroscopy analysis indicated that the protective layer likely consists of a hydrated oxide in the form of a (pseudo) boehmite with lithium distributed in its matrix.

Boron Toxicity Causes Multiple Effects on Malus domestica Pollen Tube Growth.

Boron is an important micronutrient for plants. However, boron is also toxic to cells at high concentrations, although the mechanism of this toxicity is not known. This study aimed to evaluate the effect of boron toxicity on Malus domestica pollen tube growth and its possible regulatory pathway. Our results showed that a high concentration of boron inhibited pollen germination and tube growth and led to the morphological abnormality of pollen tubes. Fluorescent labeling coupled with a scanning ion-selective electrode technique detected that boron toxicity could decrease [Ca2+]c and induce the disappearance of the [Ca2+]c gradient, which are critical for pollen tube polar growth. Actin filaments were therefore altered by boron toxicity. Immuno-localization and fluorescence labeling, together with fourier-transform infrared analysis, suggested that boron toxicity influenced the accumulation and distribution of callose, de-esterified pectins, esterified pectins, and arabinogalactan proteins in pollen tubes. All of the above results provide new insights into the regulatory role of boron in pollen tube development. In summary, boron likely plays a structural and regulatory role in relation to [Ca2+]c, actin cytoskeleton and cell wall components and thus regulates Malus domestica pollen germination and tube polar growth.

Addition of Phenylboronic Acid to Malus domestica Pollen Tubes Alters Calcium Dynamics, Disrupts Actin Filaments and Affects Cell Wall Architecture.

A key role of boron in plants is to cross-link the cell wall pectic polysaccharide rhamnogalacturonan-II (RG-II) through borate diester linkages. Phenylboronic acid (PBA) can form the same reversible ester bonds but cannot cross-link two molecules, so can be used as an antagonist to study the function of boron. This study aimed to evaluate the effect of PBA on apple (Malus domestica) pollen tube growth and the underlying regulatory mechanism. We observed that PBA caused an inhibition of pollen germination, tube growth and led to pollen tube morphological abnormalities. Fluorescent labeling, coupled with a scanning ion-selective electrode technique, revealed that PBA induced an increase in extracellular Ca2+ influx, thereby elevating the cytosolic Ca2+ concentration [Ca2+]c and disrupting the [Ca2+]c gradient, which is critical for pollen tube growth. Moreover the organization of actin filaments was severely perturbed by the PBA treatment. Immunolocalization studies and fluorescent labeling, together with Fourier-transform infrared analysis (FTIR) suggested that PBA caused an increase in the abundance of callose, de-esterified pectins and arabinogalactan proteins (AGPs) at the tip. However, it had no effect on the deposition of the wall polymers cellulose. These effects are similar to those of boron deficiency in roots and other organs, indicating that PBA can induce boron deficiency symptoms. The results provide new insights into the roles of boron in pollen tube development, which likely include regulating [Ca2+]c and the formation of the actin cytoskeleton, in addition to the synthesis and assembly of cell wall components.

Segment-specific Ca2+ transport by isolated Malpighian tubules of Drosophila melanogaster: A comparison of larval and adult stages

Haemolymph calcium homeostasis in insects is achieved through the regulation of calcium excretion by Malpighian tubules in two ways: (1) sequestration of calcium within biomineralized granules and (2) secretion of calcium in soluble form within the primary urine. Using the scanning ion-selective electrode technique (SIET), basolateral Ca2+ transport was measured at the distal, transitional, main and proximal tubular segments of anterior tubules isolated from both 3rd instar larvae and adults of the fruit fly Drosophila melanogaster. Basolateral Ca2+ transport exceeded transepithelial secretion by 800-fold and 11-fold in anterior tubules of larvae and adults, respectively. The magnitude of Ca2+ fluxes across the distal tubule of larvae and adults were larger than fluxes across the downstream segments by 10 and 40 times, respectively, indicating a dominant role for the distal segment in whole animal Ca2+ regulation. Basolateral Ca2+ transport across distal tubules of Drosophila varied throughout the life cycle; Ca2+ was released by distal tubules of larvae, taken up by distal tubules of young adults and was released once again by tubules of adults ⩾168h post-eclosion. In adults and larvae, SIET measurements revealed sites of both Ca2+ uptake and Ca2+ release across the basolateral surface of the distal segment of the same tubule, indicating that Ca2+ transport is bidirectional. Ca2+ uptake across the distal segment of tubules of young adults and Ca2+ release across the distal segment of tubules of older adults was also suggestive of reversible Ca2+ storage. Our results suggest that the distal tubules of D. melanogaster are dynamic calcium stores which allow efficient haemolymph calcium regulation through active Ca2+ sequestration during periods of high dietary calcium intake and passive Ca2+ release during periods of calcium deficiency.

An animal homolog of plant Mep/Amt transporters promotes ammonia excretion by the anal papillae of the disease vector mosquito Aedes aegypti.

The transcripts of three putative ammonia (NH3/NH4 (+)) transporters, Rhesus-like glycoproteins AeRh50-1, AeRh50-2 and Amt/Mep-like AeAmt1 were detected in the anal papillae of larval Aedes aegypti Quantitative PCR studies revealed 12-fold higher transcript levels of AeAmt1 in anal papillae relative to AeRh50-1, and levels of AeRh50-2 were even lower. Immunoblotting revealed AeAmt1 in anal papillae as a pre-protein with putative monomeric and trimeric forms. AeAmt1 was immunolocalized to the basal side of the anal papillae epithelium where it co-localized with Na(+)/K(+)-ATPase. Ammonium concentration gradients were measured adjacent to anal papillae using the scanning ion-selective electrode technique (SIET) and used to calculate ammonia efflux by the anal papillae. dsRNA-mediated reductions in AeAmt1 decreased ammonia efflux at larval anal papillae and significantly increased ammonia levels in hemolymph, indicating a principal role for AeAmt1 in ammonia excretion. Pharmacological characterization of ammonia transport mechanisms in the anal papillae suggests that, in addition to AeAmt1, the ionomotive pumps V-type H(+)-ATPase and Na(+)/K(+)-ATPase as well as NHE3 are involved in ammonia excretion at the anal papillae.

Application of scanning electrode techniques for the evaluation of iron–zinc corrosion in nearly neutral chloride solutions

A model cut edge consisting of a zinc anode and a split iron cathode was used for studying corrosion mechanisms and assessing the suitability of scanning electrochemical techniques for corrosion studies at the microscale. Scanning electrochemical microscope (SECM), localized electrochemical impedance spectroscopy (LEIS) and scanning vibrating electrode technique coupled with the scanning ion-selective electrode technique (SVET/SIET) were applied. The corrosion products precipitated on the cathode once the critical pH and solubility limits were reached, which resulted from the diffusion of hydroxyl and zinc ions across the cathode. There was no evidence of self-healing by the corrosion products.

Effect of H3BO3 on corrosion in 0.01 M NaCl solution of the interface between low alloy steel A508 and alloy 52 M

The corrosion of the low alloy steel A508-alloy 52M interface region in buffered and non-buffered chloride containing solutions was studied by using the scanning vibrating electrode technique (SVET), scanning ion-selective electrode technique (SIET), and time of flight secondary ion mass spectroscopy (ToF-SIMS). The typical pit in low alloy steel A508 formed in NaCl solution was stopped immediately when H3BO3 was added to the NaCl solution, and the pit became cathodic. With increasing immersion time, H3BO3 entered into the pit, and the corrosion of A508 became general corrosion.

Localized corrosion of the Mg alloys with inhibitor-containing coatings: SVET and SIET studies

The way of self-healing coating formation at the surface of magnesium alloys on the base of plasma electrolytic oxidation method (PEO) has been suggested. Scanning Vibrating Electrode Technique (SVET) and Scanning Ion-Selective Electrode Technique (SIET) were used for determining the kinetics and mechanism of the self-healing process. The treatment of the PEO-layer by the solution containing 8-hydroxyquinoline enables one to decrease the current density of the composite coating in 30 times (3.2μAcm−2) in the corrosion-active environment in comparison with the base PEO-coating (100μAcm−2) and avert the intensive destruction of the material.

The rectal complex and Malpighian tubules of the cabbage looper (Trichoplusia ni): regional variations in Na+ and K+ transport and cation reabsorption by secondary cells.

In larvae of most Lepidoptera the distal ends of the Malpighian tubules are closely applied to the rectal epithelia and are ensheathed within the perinephric membrane, thus forming the rectal complex. The cryptonephric Malpighian tubules within the rectal complex are bathed in fluid within a functional compartment, the perinephric space, which is separate from the haemolymph. In this study, the scanning ion-selective electrode technique (SIET) was used to measure transport of Na(+) and K(+) across the rectal complex and across multiple regions of the Malpighian tubules of larvae of the cabbage looper Trichoplusia ni. Measurements were made in an intact preparation in which connections of the tubules upstream to the rectal complex and downstream to the urinary bladder and gut remained intact. SIET measurements revealed reabsorption of Na(+) and K(+) across the intact rectal complex and into the bath (haemolymph), with K(+) fluxes approximately twice as large as those of Na(+). Analyses of fluxes in larvae with empty guts, found in recently moulted larvae, versus those with full guts highlighted differences in the rates of K(+) or Na(+) transport within tubule regions that appeared morphologically homogeneous, such as the rectal lead. The distal rectal lead of larvae with empty guts reabsorbed K(+), whereas the same region secreted K(+) in tubules of larvae with full guts. SIET measurements of the ileac plexus also indicated a novel role for secondary (type II) cells in cation reabsorption. Secondary cells reabsorb K(+), whereas the adjacent principal (type I) cells secrete K(+). Na(+) is reabsorbed by both principal and secondary cells, but the rate of reabsorption by the secondary cells is approximately twice the rate in the adjacent principal cells.

Inhibitor-Containing Composite Coatings on Mg Alloys: Corrosion Mechanism and Self-Healing Protection

The way of self-healing coating formation at the surface of magnesium alloys by means of plasma electrolytic oxidation method (PEO) with subsequent filling of the obtained layer with inhibitor has been suggested. The electrochemical properties of such coatings have been described in details. The obtained experimental results indicate that the protective properties of the samples with inhibitor-containing coating were increased (IC = 8.6×10–8 A/cm2) in comparison with the samples without coating (5.3×10–5 A/cm2) and the base coating obtained by plasma electrolytic oxidation method (PEO) (3.4×10–7 A/cm2). The local scanning electrochemical methods of surface investigation, notably Scanning Vibrating Electrode Technique (SVET) and Scanning Ion-Selective Electrode Technique (SIET) were used for determining the kinetics and mechanism of the self-healing process. The treatment by the solution containing 8-hydroxyquinoline, which inhibits the corrosion process, enables one to increase the protective properties of the composite coating in 30 times in the corrosion-active environment in comparison with the base PEO-coating and avert the intensive destruction of the material.

Oxalate secretion from the root apex of Sedum alfredii contributes to hyperaccumulation of Cd

Organic acids play an important role in metal detoxification in plant, but accumulation of heavy metals with the help of organic acids secretion in hyperaccumulators has not been well documented. The aim of this study was to investigate the contribution of oxalate secretion to cadmium (Cd) hyperaccumulation in S. alfredii. Hydroponic experiments were conducted to investigate the characteristics of Cd-induced secretion of oxalate in terms of pattern, location and oxalate-induced Cd translocation pathway, using ion channel inhibitors, scanning ion-selective electrode technique (SIET) and fluorescence imaging. Hyperaccumulating ecotype (HE) had nearly 2-fold higher oxalate secretion than non-hyperaccumulating ecotype (NHE). Phenylglyoxal effectively blocked Cd-induced oxalate secretion and decreased Cd concentration in HE while exogenous oxalate supply promoted Cd accumulation efficiently. SIET analysis indicated that Cd2+ influx into roots of HE mainly occurred at zone 0–10 mm from root apex where oxalate secretion was localized. Cd was distributed preferentially to the root stele of the HE but not the NHE, and was significantly increased with the secreted oxalates in HE. Cd hyperaccumulation by HE S. alfredii is partially associated with oxalate secretion from the root apex, which mediates the loading of Cd from the xylem parenchyma cells.

Aquaporin 1 Is Involved in Acid Secretion by Ionocytes of Zebrafish Embryos through Facilitating CO2 Transport

Mammalian aquaporin 1 (AQP1) is well known to function as a membrane channel for H2O and CO2 transport. Zebrafish AQP1a.1 (the homologue of mammalian AQP1) was recently identified in ionocytes of embryos; however its role in ionocytes is still unclear. In this study, we hypothesized that zebrafish AQP1a.1 is involved in the acid secretion by ionocytes through facilitating H2O and CO2 diffusion. A real-time PCR showed that mRNA levels of AQP1a.1 in embryos were induced by exposure to 1% CO2 hypercapnia for 3 days. In situ hybridization and immunohistochemistry showed that the AQP1a.1 transcript was highly expressed by acid-secreting ionocytes, i.e., H+-ATPase-rich (HR) cells. A scanning ion-selective electrode technique (SIET) was applied to analyze CO2-induced H+ secretion by individual ionocytes in embryos. H+ secretion by HR cells remarkably increased after a transient loading of CO2 (1% for 10 min). AQP1a.1 knockdown with morpholino oligonucleotides decreased the H+ secretion of HR cells by about half and limited the CO2 stimulated increase. In addition, exposure to an AQP inhibitor (PCMB) for 10 min also suppressed CO2-induced H+ secretion. Results from this study support our hypothesis and provide in vivo evidence of the physiological role of AQP1 in CO2 transport.