Microelectrode Ion Flux Estimation Research Articles

K+ efflux as an ionic response to Phytophthora capsici in seedlings of Capsicum annuum

Mexico is one of the main producers of green chili in the world, and one of the most important diseases affecting this crop is chili pepper wilt caused by Phytophthora capsici. This oomycete is a plant pathogen that causes extensive damage to solanaceous vegetable crops. An understanding of the biochemical events that occur in the early interaction phase would be essential for describing the initial mechanism of pathogen-host interactions. The ion fluxes that mediate the initial host-pathogen interaction are critical for activating the immune response of plants, so it is important to study the response of plants to their attack at the physiological and ionic levels. Seedlings of Capsicum annuum L. cultivars were inoculated with P. capsici zoospores and the severity of damage and K+ transport using the Microelectrode Ion Flux Estimation technique (MIFE™) were monitored. The susceptibility of C. annuum L. Jalapeño genotype to P. capsici was confirmed and a lower K+ efflux in the sensitive Jalapeño cultivar than in the tolerant cultivar CM33 was demonstrated as an early response to P. capsici zoospores.

AtCIPK16, a CBL-interacting protein kinase gene, confers salinity tolerance in transgenic wheat.

Globally, wheat is the major source of staple food, protein, and basic calories for most of the human population. Strategies must be adopted for sustainable wheat crop production to fill the ever-increasing food demand. Salinity is one of the major abiotic stresses involved in plant growth retardation and grain yield reduction. In plants, calcineurin-B-like proteins form a complicated network with the target kinase CBL-interacting protein kinases (CIPKs) in response to intracellular calcium signaling as a consequence of abiotic stresses. The AtCIPK16 gene has been identified in Arabidopsis thaliana and found to be significantly upregulated under salinity stress. In this study, the AtCIPK16 gene was cloned in two different plant expression vectors, i.e., pTOOL37 having a UBI1 promoter and pMDC32 having a 2XCaMV35S constitutive promoter transformed through the Agrobacterium-mediated transformation protocol, in the local wheat cultivar Faisalabad-2008. Based on their ability to tolerate different levels of salt stress (0, 50, 100, and 200 mM), the transgenic wheat lines OE1, OE2, and OE3 expressing AtCIPK16 under the UBI1 promoter and OE5, OE6, and OE7 expressing the same gene under the 2XCaMV35S promoter performed better at 100 mM of salinity stress as compared with the wild type. The AtCIPK16 overexpressing transgenic wheat lines were further investigated for their K+ retention ability in root tissues by utilizing the microelectrode ion flux estimation technique. It has been demonstrated that after 10min of 100 mM NaCl application, more K+ ions were retained in the AtCIPK16 overexpressing transgenic wheat lines than in the wild type. Moreover, it could be concluded that AtCIPK16 functions as a positive elicitor in sequestering Na+ ions into the cell vacuole and retaining more cellular K+ under salt stress to maintain ionic homeostasis.

The sugar transporter ZmSUGCAR1 of the nitrate transporter 1/peptide transporter family is critical for maize grain filling.

Maternal-to-filial nutrition transfer is central to grain development and yield. nitrate transporter 1/peptide transporter (NRT1-PTR)-type transporters typically transport nitrate, peptides, and ions. Here, we report the identification of a maize (Zea mays) NRT1-PTR-type transporter that transports sucrose and glucose. The activity of this sugar transporter, named Sucrose and Glucose Carrier 1 (SUGCAR1), was systematically verified by tracer-labeled sugar uptake and serial electrophysiological studies including two-electrode voltage-clamp, non-invasive microelectrode ion flux estimation assays in Xenopus laevis oocytes and patch clamping in HEK293T cells. ZmSUGCAR1 is specifically expressed in the basal endosperm transfer layer and loss-of-function mutation of ZmSUGCAR1 caused significantly decreased sucrose and glucose contents and subsequent shrinkage of maize kernels. Notably, the ZmSUGCAR1 orthologs SbSUGCAR1 (from Sorghum bicolor) and TaSUGCAR1 (from Triticum aestivum) displayed similar sugar transport activities in oocytes, supporting the functional conservation of SUGCAR1 in closely related cereal species. Thus, the discovery of ZmSUGCAR1 uncovers a type of sugar transporter essential for grain development and opens potential avenues for genetic improvement of seed-filling and yield in maize and other grain crops.

An Improved Transwell Design for Microelectrode Ion-Flux Measurements.

The microelectrode ion flux estimation (MIFE) is a powerful, non-invasive electrophysiological method for cellular membrane transport studies. Usually, the MIFE measurements are performed in a tissue culture dish or directly with tissues (roots, parts of the plants, and cell tissues). Here, we present a transwell system that allows for MIFE measurements on a cell monolayer. We introduce a measurement window in the transwell insert membrane, which provides direct access for the cells to the media in the upper and lower compartment of the transwell system and allows direct cell-to-cell contact coculture. Three-dimensional multiphoton lithography (MPL) was used to construct a 3D grid structure for cell support in the measurement window. The optimal polymer grid constant was found for implementation in transwell MIFE measurements. We showed that human umbilical vein endothelial cells (HUVECs) efficiently grow and maintain their physiological response on top of the polymer structures.

Triangulation of methods using insect cell lines to investigate insecticidal mode-of-action.

This study investigated three in vitro models to assist in elucidating possible mode-of-action, which could be adopted to evaluate insecticidal activity of complex, unknown, or multi-constituent formulations. We used a combination of absorbance spectrometry, confocal scanning laser microscopy and microelectrode ion flux estimation (MIFE) to provide insight into potential target sites for insecticides. This study used two insect cell lines and evaluated three pyrethroid insecticides. We observed that the two cell lines produced distinctly different responses. Drosophila melanogaster D.mel-S2 cell line was a useful model to monitor ion flux changes, resulting from insecticides with neural toxicity; however, it was less useful to determine some metabolic pathway indicators of toxic stress. Conversely, the Spodoptera frugiperda Sf9 cell line produced acute reactive oxygen species (ROS) in response to insecticide treatments, but was not highly responsive in electrophysiological experiments. We also showed that the natural, multi-constituent botanical extract of pyrethrum elicited different Na+ , Cl- and Ca2+ ion fluxes than its synthetic, single constituent analogues, α-cypermethrin and esfenvalerate. These two methods used in combination with absorbance spectrometry measuring cell growth inhibition plus cell mortality assays shed some light on cytotoxic responses in differing model cell lines. This research highlights the importance of using multiple cell types and interdisciplinary methods to provide a better insight into mode of insecticidal action. This is especially pertinent to novel biopesticide discovery, as the underlying mechanisms for toxicity in initial screening processes are likely to be unknown.

Lipid kinases PIP5K7 and PIP5K9 are required for polyamine-triggered K+ efflux in Arabidopsis roots.

SUMMARYPolyamines, such as putrescine, spermidine and spermine (Spm), are low‐molecular‐weight polycationic molecules present in all living organisms. Despite their implication in plant cellular processes, little is known about their molecular mode of action. Here, we demonstrate that polyamines trigger a rapid increase in the regulatory membrane lipid phosphatidylinositol 4,5‐bisphosphate (PIP2), and that this increase is required for polyamine effects on K+ efflux in Arabidopsis roots. Using in vivo 32Pi‐labelling of Arabidopsis seedlings, low physiological (μm) concentrations of Spm were found to promote a rapid PIP2 increase in roots that was time‐ and dose‐dependent. Confocal imaging of a genetically encoded PIP2 biosensor revealed that this increase was triggered at the plasma membrane. Differential 32Pi‐labelling suggested that the increase in PIP2 was generated through activation of phosphatidylinositol 4‐phosphate 5‐kinase (PIP5K) activity rather than inhibition of a phospholipase C or PIP2 5‐phosphatase activity. Systematic analysis of transfer DNA insertion mutants identified PIP5K7 and PIP5K9 as the main candidates involved in the Spm‐induced PIP2 response. Using non‐invasive microelectrode ion flux estimation, we discovered that the Spm‐triggered K+ efflux response was strongly reduced in pip5k7 pip5k9 seedlings. Together, our results provide biochemical and genetic evidence for a physiological role of PIP2 in polyamine‐mediated signalling controlling K+ flux in plants.

Zinc alleviates cadmium toxicity by modulating photosynthesis, ROS homeostasis, and cation flux kinetics in rice

Understanding of cadmium (Cd) uptake mechanism and development of lower Cd crop genotypes are crucial for combating its phytotoxicity and meeting 70% increase in food demand by 2050. Bio-accumulation of Cd continuously challenges quality of life specifically in regions without adequate environmental planning. Here, we investigated the mechanisms operating in Cd tolerance of two rice genotypes (Heizhan-43 and Yinni-801). Damage to chlorophyll contents and PSII, histochemical staining and quantification of reactive oxygen species (ROS), cell viability and osmolyte accumulation were studied to decipher the interactions between Cd and zinc (Zn) by applying two Cd and two Zn levels (alone as well as combined). Cd2+ and Ca2+ fluxes were also measured by employing sole Cd100 (100 μmol L−1) and Zn50 (50 μmol L−1), and their combination with microelectrode ion flux estimation (MIFE) technique. Cd toxicity substantially reduced chlorophyll contents and maximal photochemical efficiency (Fv/Fm) compared to control plants. Zn supplementation reverted the Cd-induced toxicity by augmenting osmoprotectants and interfering with ROS homeostasis under combined treatments, particularly in Yinni-801 genotype. Fluorescence microscopy indicated a unique pattern of live and dead root cells, depicting more damage with Cd10, Cd15 and Cd15+Zn50. Our results confer that Cd2+ impairs the uptake of Ca2+ whereas, Zn not only competes with Cd2+ but also Ca2+, thereby modifying ion homeostasis in rice plants. This study suggests that exogenous application of Zn is beneficial for rice plants in ameliorating Cd toxicity in a genotype and dose dependent manner by minimizing ROS generation and suppressing collective oxidative damage. The observations confer that Yinni-801 performed better than Heizhan-43 genotype mainly under combined Zn treatments with low-Cd, presenting Zn fortification as a solution to increase rice production.

Root vacuolar Na+ sequestration but not exclusion from uptake correlates with barley salt tolerance.

Soil salinity is a major constraint for the global agricultural production. For many decades, Na+ exclusion from uptake has been the key trait targeted in breeding programs; yet, no major breakthrough in creating salt-tolerant germplasm was achieved. In this work, we have combined the microelectrode ion flux estimation (MIFE) technique for non-invasive ion flux measurements with confocal fluorescence dye imaging technique to screen 45 accessions of barley to reveal the relative contribution of Na+ exclusion from the cytosol to the apoplast and its vacuolar sequestration in the root apex, for the overall salinity stress tolerance. We show that Na+ /H+ antiporter-mediated Na+ extrusion from the root plays a minor role in the overall salt tolerance in barley. At the same time, a strong and positive correlation was found between root vacuolar Na+ sequestration ability and the overall salt tolerance. The inability of salt-sensitive genotypes to sequester Na+ in root vacuoles was in contrast to significantly higher expression levels of both HvNHX1 tonoplast Na+ /H+ antiporters and HvVP1 H+ -pumps compared with tolerant genotypes. These data are interpreted as a failure of sensitive varieties to prevent Na+ back-leak into the cytosol and existence of a futile Na+ cycle at the tonoplast. Taken together, our results demonstrated that root vacuolar Na+ sequestration but not exclusion from uptake played the main role in barley salinity tolerance, and suggested that the focus of the breeding programs should be shifted from targeting genes mediating Na+ exclusion from uptake by roots to more efficient root vacuolar Na+ sequestration.

Developing a high-throughput phenotyping method for oxidative stress tolerance in barley roots

BackgroundMore than 20% of the world’s agricultural land is affected by salinity, resulting in multibillion-dollar penalties and jeopardising food security. While the recent progress in molecular technologies has significantly advanced plant breeding for salinity stress tolerance, accurate plant phenotyping remains a bottleneck of many breeding programs. We have recently shown the existence of a strong causal link between salinity and oxidative stress tolerance in cereals (wheat and barley). Using the microelectrode ion flux estimation (MIFE) method, we have also found a major QTL conferring ROS control of ion flux in roots that coincided with the major QTL for the overall salinity stress tolerance. These findings open new (previously unexplored) prospects of improving salinity tolerance by pyramiding this trait alongside with other (traditional) mechanisms.ResultsIn this work, two high-throughput phenotyping methods—viability assay and root growth assay—were tested and assessed as a viable alternative to the (technically complicated) MIFE method using barley as a check species. In viability staining experiments, a dose-dependent H2O2-triggered loss of root cell viability was observed, with salt sensitive varieties showing significantly more damage to root cells. In the root growth assays, relative root length (RRL) was measured in plants grown in paper rolls under different H2O2 concentrations. The biggest difference in RRL between contrasting varieties was observed for 1 mM H2O2 treatment. Under these conditions, a significant negative correlation in the reduction in RRL and the overall salinity tolerance is reported.ConclusionsThese findings offer plant breeders a convenient high throughput method to screen germplasm for oxidative stress tolerance, targeting root-based genes regulating ion homeostasis and thus conferring salinity stress tolerance in barley (and potentially other species).

Fish gill damage by harmful microalgae newly explored by microelectrode ion flux estimation techniques

Harmful algal blooms (HAB) are responsible for massive mortalities of wild and aquacultured fish due to noticeable gill damage, but the precise fish-killing mechanisms remain poorly understood. A non-invasive microelectrode ion flux estimation (MIFE) technique was successfully applied to assess changes in membrane-transport processes in a model fish gill cell line exposed to harmful microplankton. Net Ca2+, H+, K+ ion fluxes in the rainbow trout cell line RTgill-W1 were monitored before and after addition of lysed cells of this Paralytic Shellfish Toxins (PST) producer along with purified endocellular dinoflagellate PST. It was demonstrated that PST alone do not play a role in fish gill damage during A. catenella outbreaks as previously thought, but that other ichthyotoxic metabolites from lysed algal cells (i.e. lipid peroxidation products or other unknown metabolites) result in net K+ efflux from fish gill cells and thereby gill cell death.

Genotypic difference in the influence of aluminum and low pH on ion flux, rhizospheric pH and ATPase activity between Tibetan wild and cultivated barley

Low-pH and Al3+ toxicity are the major factors causing the inhibition of root elongation and plant growth in acid soils. However, it is still unclear about the adaptive mechanisms for plant roots to cope with simultaneous low-pH and Al3+ toxicity in the acid soil. In this study, changes in root tip H+, K+, and Ca2+ fluxes and rhizospheric pH of three barley genotypes, including two Tibetan wild barley (XZ16, Al-tolerant; XZ61, Al-sensitive), and Al-tolerant cv Dayton, were investigated using microelectrode ion flux estimation (MIFE) technology. The results showed that Al-induced ion flux changes were mainly occurred at the elongation zone. Under low-pH treatment, at 24 h, the rhizosphere pH of low-pH tolerant XZ16 and Dayton were 0.7 and 0.3 units higher than low-pH sensitive XZ61, respectively. The H+ influx of tolerant XZ16 and Dayton was higher than that of sensitive XZ61 during the whole process of both low-pH and Al treatments. Furthermore, the Al stress significantly increased the Ca2+ efflux, inhibited the K+ efflux and ATPase activities, especially in the Al-sensitive XZ61. These results indicated that the higher low-pH tolerance in XZ16 was connected with the higher ability of H+ uptake and rhizospheric alkalization. The different mechanisms between low-pH and Al tolerance were probably tightly modulated by Al-induced increase of Ca2+ efflux and inhibition of ATPase activities.

Piriformospora indica improves salinity stress tolerance in Zea mays L. plants by regulating Na+ and K+ loading in root and allocating K+ in shoot

Piriformospora indica is known as a fungus that can easily colonize a wide range of plants and enhance host’s growth and tolerance to abiotic stresses, including salinity. The mechanistic basis behind this phenomenon remains poorly understood. This work was aimed to fill in this gap and reveal mechanisms enhancing salinity tolerance in maize roots colonised by P. indica. A range of agronomic and physiological characteristics were compared between inoculated and non-inoculated maize plants under 0/100/200 mM NaCl conditions. The impact of P. indica inoculation or root’s cytosolic K+ retention ability were also assessed using micro-electrode ion flux estimation technique. The results showed that inoculated plants had higher biomass, higher stomatal conductance, lower K+ efflux from roots and higher potassium content in shoots than non-inoculated plants under salt stress. Collectively, the results indicated that the beneficial effects of inoculation on plant performance under saline conditions were mainly attributed to the improved stomata operation associated with higher rate of K delivery into the shoots.

Boron Alleviates Aluminum Toxicity by Promoting Root Alkalization in Transition Zone via Polar Auxin Transport.

Boron (B) alleviates aluminum (Al) toxicity in higher plants; however, the underlying mechanisms behind this phenomenon remain unknown. Here, we used bromocresol green pH indicator, noninvasive microtest, and microelectrode ion flux estimation techniques to demonstrate that B promotes root surface pH gradients in pea (Pisum sativum) roots, leading to alkalization in the root transition zone and acidification in the elongation zone, while Al inhibits these pH gradients. B significantly decreased Al accumulation in the transition zone (∼1.0-2.5 mm from the apex) of lateral roots, thereby alleviating Al-induced inhibition of root elongation. Net indole acetic acid (IAA) efflux detected by an IAA-sensitive platinum microelectrode showed that polar auxin transport, which peaked in the root transition zone, was inhibited by Al toxicity, while it was partially recovered by B. Electrophysiological experiments using the Arabidopsis (Arabidopsis thaliana) auxin transporter mutants (auxin resistant1-7; pin-formed2 [pin2]) and the specific polar auxin transporter inhibitor1-naphthylphthalamic acid showed that PIN2-based polar auxin transport is involved in root surface alkalization in the transition zone. Our results suggest that B promotes polar auxin transport driven by the auxin efflux transporter PIN2 and leads to the downstream regulation of the plasma membrane-H+-ATPase, resulting in elevated root surface pH, which is essential to decrease Al accumulation in this Al-targeted apical root zone. These findings provide a mechanistic explanation for the role of exogenous B in alleviation of Al accumulation and toxicity in plants.

Evaluation of salt tolerance and contributing ionic mechanism in nine Hami melon landraces in Xinjiang, China

Melon (Cucumis melo L.) is an important horticultural crop in the world. The objective of this work was to evaluate salinity stress tolerance and understand the ionic mechanism conferring this trait in nine Hami melon landracesfrom Xinjiang, China. Plants were grown in a potting mix (peat: vermiculite: perlite = 1:1:1, v/v) under glasshouse conditions and irrigated with Hoagland’s nutrient solution containing either 0 (control) or 100 mM NaCl. Plant dry weight, single fruit weight, Na+ and K+ content of all leaves, stem and roots of the whole plant were measured at day 70 after commencing the treatment. Also, Na+ and K+ content of the second fully expanded leaf from the top were measured at day 20, 40 and 60 after stress onset. Net fluxes of K+ and Na+ were measured from plant roots in laboratory experiments using a noninvasive MIFE (microelectrode ion flux estimation) technique, and expression levels of the leaf tonoplast Na+/H+ antiporters (NHX) were investigated. Salt injury index and leaf chlorophyll content were also measured. The results showed that, according to the relative (NaCl/Control) plant dry weight and a single fruit weight, the salt tolerance of the nine landraces can be classified into three types: ‘Huangdanzi’, ‘Zajiaojiashigua’, ‘Kalakesai’ and ‘Paotaihong’ are salt tolerant; ‘Laotiepi’, ‘Kaeryunxi’ and ‘Sairekekekouqi’ are intermediate in salt tolerance; and ‘Akekekouqi’ and ‘Paodangua’ are salt sensitive. This conclusion was also supported by the results of measuring of the salt injury index and leaf chlorophyll content. The salt tolerance of landraces was positively correlated with the relative K+ content of all leaves, but correlated negatively with the relative Na+ content of the second fully expanded leaf, and relative K+ content of the root. Compared with salt sensitive landraces, salt tolerant genotypes had significantly lower relative Na+ content of the second fully expanded leaf, lower relative root K+ content (except Paotaihong), and higher relative K+ content of all leaves. They also displayed lower root net K+ efflux and higher Na+ efflux (except Zajiaojiashigua) capacity. The intermediate landrace ‘Laotiepi’ had very high Na+ content and high level of NHX2/3/6 expression in leaves but still possessed high chlorophyll content, suggesting that this landrace had an efficient vacuolar Na+ sequestration mechanism in the leaf mesophyll. Taken together, our results showed that Hami melon landraces in Xinjiang showing significant difference in salt tolerance, and that K+ retention in the shoot and Na+ exclusion from young leaves are the main mechanisms conferring salinity stress tolerance of Hami melon landraces in Xinjiang.

Application of non-invasive microelectrode ion flux estimation technique in crop stress physiology.

Non-invasive microsensing technique has been widely used in evaluating the adaptive responses of plant cells and tissues to abiotic stresses. One of the representative techniques is the microelectrode ion flux estimation (MIFE), which allows concurrent quantification of net fluxes with high spatial and temporal resolution. More importantly, this technique permits simultaneous recording of ion concentration and mobility with less intervention to the in situ physiological status. With the availability of such advanced technique, the last three decades have seen a significant progress towards the role of ion signaling in a variety of abiotic stresses including salinity, extreme temperature, osmotic stress, hypoxia, and drought. In this review, we gave a brief introduction of the MIFE working principles and focused on its applications in detecting ion responses to various abiotic stresses.

Development and Characterization of Needle‐type Ion‐selective Microsensors for in situ Determination of Foliar Uptake of Zn2+ in Citrus Plants

AbstractFor over a decade, the incidence of Huanglongbing (HLB) has grown at an alarming rate, affecting citrus crops worldwide. Current methods of nutrient therapy have little to no effect in alleviating symptoms of HLB, and scarce research has been put forth towards non‐destructive tools for monitoring zinc transport in citrus plants. Here, we have developed and characterized a solid contact micro‐ion‐selective electrode (SC‐μ‐ISE) for the determination of zinc transport in sour orange seedlings using a non‐invasive microelectrode ion flux estimation (MIFE) technique. The SC‐μ‐ISE displayed a 26.05±0.13 mV decade−1 Nernstian response and a LOD of (3.96±2.09)×10−7 M. Results showed a significant Zn2+ uptake in the leaves and roots of sour orange seedlings when bulk concentrations were higher than 5.99 mM. Above this concentration, a linear relationship between flux and bulk Zn2+ concentration was observed. This relationship suggests passive diffusion may be a key mechanism for Zn transport into plants. Overall, this study is the first to use a Zn2+ SC‐μ‐ISE for the determination of ion transport processes in plants. This novel tool can be used to further knowledge the effect of nutrient therapy and disease progression on HLB infected citrus plants.

The coordinated regulation of Na+ and K+ in Hordeum brevisubulatum responding to time of salt stress

Hordeum brevisubulatum, called as wild barley, is a useful monocotyledonous halophyte for soil improvement in northern China. Although previously studied, its main salt tolerance mechanism remained controversial. The current work showed that shoot Na+ concentration was increased rapidly with stress time and significantly higher than in wheat during 0–168h of 100mM NaCl treatment. Similar results were also found under 25 and 50mM NaCl treatments. Even K+ was increased from 0.01 to 50mM in the cultural solution, no significant effect was found on tissue Na+ concentrations. Interestingly, shoot growth was improved, and stronger root activity was maintained in H. brevisubulatum compared with wheat after 7days treatment of 100mM NaCl. To investigate the long-term stress impact on tissue Na+, 100mM NaCl was prolonged to 60 days. The maximum values of Na+ concentrations were observed at 7th in shoot and 14th day in roots, respectively, and then decreased gradually. Micro-electrode ion flux estimation was used and it was found that increasing Na+ efflux while maintaining K+ influx were the major strategies to reduce the Na+ concentration during long-term salt stress. Moreover, leaf Na+ secretions showed little contribution to the tissue Na+ decrease. Thereby, the physiological mechanism for H. brevisubulatum to survive from long-term salt stress was proposed that rapid Na+ accumulation occurred in the shoot to respond the initial salt shock, then Na+ efflux was triggered and K+ influx was activated to maintain a stable K+/Na+ ratio in tissues.

Mechanisms underlying turgor regulation in the estuarine alga Vaucheria erythrospora (Xanthophyceae) exposed to hyperosmotic shock.

Aquatic organisms are often exposed to dramatic changes in salinity in the environment. Despite decades of research, many questions related to molecular and physiological mechanisms mediating sensing and adaptation to salinity stress remain unanswered. Here, responses of Vaucheria erythrospora, a turgor-regulating xanthophycean alga from an estuarine habitat, have been investigated. The role of ion uptake in turgor regulation was studied using a single cell pressure probe, microelectrode ion flux estimation (MIFE) technique and membrane potential (Em ) measurements. Turgor recovery was inhibited by Gd(3+) , tetraethylammonium chloride (TEA), verapamil and orthovanadate. A NaCl-induced shock rapidly depolarized the plasma membrane while an isotonic sorbitol treatment hyperpolarized it. Turgor recovery was critically dependent on the presence of Na(+) but not K(+) and Cl(-) in the incubation media. Na(+) uptake was strongly decreased by amiloride and changes in net Na(+) and H(+) fluxes were oppositely directed. This suggests active uptake of Na(+) in V. erythrospora mediated by an antiport Na(+) /H(+) system, functioning in the direction opposite to that of the SOS1 exchanger in higher plants. The alga also retains K(+) efficiently when exposed to high NaCl concentrations. Overall, this study provides insights into mechanisms enabling V. erythrospora to regulate turgor via ion movements during hyperosmotic stress.

MIFE Technique-based Screening for Mesophyll K+ Retention for Crop Breeding for Salinity Tolerance

Potassium is known as a rate-limiting factor for crop yield and plays an important role in plants response under abiotic stresses. Recently, cytosolic K+ retention ability in leaf mesophyll has emerged as an important component of plant salt tolerance mechanism (Wu et al., 2013; Wu et al., 2014; Wu et al., 2015). In this protocol, the procedure for screening leaf mesophyll for K+ retention by the MIFE (microelectrode ion flux estimation) technique is described in detail using wheat as an example. By measuring NaCl-induced K+ efflux in leaf mesophyll, a large number of plant accessions can be screened and categorised according to their salinity stress tolerance. The method provides a rapid and reliable tool that targets the activity of specific membrane transporters directly contributing to salinity tolerance trait and, because of this, has a competitive advantage over traditional whole-plant phenotyping. While the focus of this protocol is on wheat, the suggested method may be adopted for screening K+ retention in leaf mesophyll in any other crop species.

Mechanisms of salt tolerance in habanero pepper plants (Capsicum chinense Jacq.): Proline accumulation, ions dynamics and sodium root-shoot partition and compartmentation.

Despite its economic relevance, little is known about salt tolerance mechanisms in pepper plants. To address this question, we compared differences in responses to NaCl in two Capsicum chinense varieties: Rex (tolerant) and Chichen-Itza (sensitive). Under salt stress (150 mM NaCl over 7 days) roots of Rex variety accumulated 50 times more compatible solutes such as proline compared to Chichen-Itza. Mineral analysis indicated that Na+ is restricted to roots by preventing its transport to leaves. Fluorescence analysis suggested an efficient Na+ compartmentalization in vacuole-like structures and in small intracellular compartments in roots of Rex variety. At the same time, Na+ in Chichen-Itza plants was compartmentalized in the apoplast, suggesting substantial Na+ extrusion. Rex variety was found to retain more K+ in its roots under salt stress according to a mineral analysis and microelectrode ion flux estimation (MIFE). Vanadate-sensitive H+ efflux was higher in Chichen-Itza variety plants, suggesting a higher activity of the plasma membrane H+-ATPase, which fuels the extrusion of Na+, and, possibly, also the re-uptake of K+. Our results suggest a combination of stress tolerance mechanisms, in order to alleviate the salt-induced injury. Furthermore, Na+ extrusion to apoplast does not appear to be an efficient strategy for salt tolerance in pepper plants.