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The organic anion balance is critical for metabolic, bioenergetic, and electrochemical processes in plant cells, controlling the quality and quantity of yield and plant stress resistance. Nevertheless, the redistribution and membrane transport of these substances in plant tissues have not been investigated in detail. The mechanism of passive anion efflux from a plant cell through the ion channels has not been established so far. Here, using the patch-clamp technique, we have characterized the ion channel-mediated conductances of ascorbate, malate, gluconate, citrate, fumarate, and pronionate in the root cells of Arabidopsis thaliana, Triticum aestivum, and Helianthus annuus. These conductances showed high permeability to ascorbate, malate, and citrate, as well as low permeability to fumarate, propionate, and gluconate. Anion channel conductances of root cells showed rapid activation kinetics and low potential dependence. They were also inhibited by 9-anthracenecarboxylic acid, suggesting that they belong to the ALMT family of anion channels found only in higher plants. Aequorin chemilu minometry was used to test the effect of organic anions on the Ca2+ signaling in root cells. Among four organic anions tested, only ascorbate induced a significant increase in the cytosolic Ca2+ activity at physiological levels (1 and 10 mM). This effect may underlie the previously unknown functions of exogenous ascorbate related to short- and long-distance signaling in higher plants.

Phytopathogenic micromycetes induce dangerous crop diseases. Traditionally, fungicides have been used to prevent these diseases. In recent years, environmentally friendly non-chemical methods for combating fungal infections have been developed. In particular, ultraviolet (UV) treatment of various wavelengths, intensities and origins has been shown to be effective. In this work, we have analysed the effect of diode low-intensity shortwave UV radiation (UV-C) on the growth and potential viability of the mycelium of Alternaria radicina and A. alternata, the pathogens of important crops. It was shown that irradiation by UV-C diode inhibited growth of Alternaria species at the early stages of development. This effect was high in the first 3 d after UV exposure; however, after 5 d after irradiation, the growth of pathogenic fungi fully restored, suggesting that UV can be only used for partial removal of Alternaria. It was found that A. alternata is less sensitive to diode UV irradiation. The obtained results indicate that the Alternaria micromycetes, particularly, A. alternata, containing high melanin levels, are resistant to low-intensity UV-C diode irradiation.

Phenotyping platforms is hardware and software systems that provide collection and processing of information about plant or other organism phenotypes. In the presented study, an analytical review of the phenotyping platforms of the world’s major manufacturers, their components, characteristics and applications in biology, agriculture and biotechnology was carried out. A comparison of the phenomics attitudes by key parameters was made, trends in the development of their sensory and program-analytical components were revealed. The assessment of the world market of phenotyping systems current state was made. The most important examples of phenomics facilities use for phenotyping in the field and laboratory conditions, fundamental and practical research in various fields of plant biology are given.

Potassium is indispensable for plant growth. Recently, a role of K+ channels has emerged in sensing and transducing stress and nutrient status. Tetraethylammonium (TEA+) is a specific blocker of K+ transport and affects K+ channel gene expression. Two barley varieties with contrasting salinity tolerance, and a chlorophyll b-less mutant, were grown either in the presence of TEA+ alone or combined with NaCl, at two different concentrations of external K+ and Ca2+, and were analyzed nine days after germination. Chlorophyll a transients monitored via JIP-tests were used to evaluate the state of the photosynthetic machinery. In contrast to reported responses to K+ deficiency, TEA+ inhibited shoot growth while inducing root growth and increasing photosynthetic performance. Both TEA+ and NaCl induced the appearance of negative K-bands in OJIP kinetics and an increase in PIABS, indicating a stimulation of photosynthesis by increased sink strength in the context of root to shoot signaling.

In recent years, a new branch of plant physiology, plant phenomics, which focuses on identifying patterns of organization and changes in plant Phenomes, i.e., physical and biochemical characteristics, considered as a set of phenotypes of a plant organism, has emerged. Phenomics is a postgenomic discipline that actively uses the achievements of the genomic era and bioinformatics. It supplements them with standardized and statistically significant factual material on phenotypes with a high degree of detail. The technique of obtaining and analyzing information about phenotypes in phenomics is called phenotyping. High-performance phenotyping, providing digital automated analysis of large data samples, has become widespread. Recent progress in high-performance phenotyping has been associated with the development of image registration systems in various spectral regions, approaches to cultivating plant objects under standardized conditions, sensory technologies, robotics, and methods for data processing and analysis, such as computer vision and machine learning (artificial neural network). Phenomics technologies have a high information content analysis, surpassing human capabilities, performing measurements in the hyperspectral range using X-ray tomography and ultra-precise “thermal” images, and have a number of other low-invasive and precision approaches. Arrays of data obtained using phenomics technologies are recorded and processed automatically and are free from the problems of subjective assessment and inadequate statistical processing. It is assumed that phenotyping will allow for the creation of digital models of the vital activity processes and the “formation” of plant productivity at the organism level in connection with the dynamics of transcriptomes, proteomes, and metabolomes. Phenomics helps researchers transform a large amount of information received from modern sensors into new knowledge using computer data processing and modeling, reducing the distance from basic science to the practical application of results in crop production and breeding. Phenotyping is actively developing both in laboratory and in greenhouse conditions as well as on open agricultural sites, forests, and in real natural phytocenoses. The review analyzes the current state of plant phenomics with a focus on technical aspects, in particular, the design of hardware-software phenotyping complexes, i.e., phenomics platforms, as well as the use of neural networks in phenotyping of plant organisms.

Ascorbic acid (AA) is one of the most important antioxidants and redox-active substances of plants found in the apoplast. In the form of ascorbate anion, it can be exported to the cell wall through anion channels, where it is further oxidized to dehydroascorbic acid (DHA) and in this form is translocated back into the cytoplasm through as yet unidentified transporters. In the apoplast AA is exposed to both enzymatic and non-enzymatic oxidation, participating in the antioxidant and pro-oxidant processes: detoxification of O3, generation of hydroxyl radicals, reduction of phenoxyl radicals, etc. The intermediate product of the AA oxidation in apoplast, monodehydroascorbic acid (MDHA), serves as an extracellular electron acceptor for a number of redox chains localized at the plasma membrane (PM). All three forms of ascorbate, AA, DHA and MDHA, can potentially be involved in the cell signalling. Released from the cell through anionic channels during PM depolarization, AA can stimulate the production of hydroxyl radicals that activate Ca2+-permeable channels, and thus trigger Ca2+-signalling. Due to the ability to generate hydroxyl radicals and reduce phenoxyl radicals, AA affects cell wall extensibility and extension growth. By reducing Fe3+ to a freely soluble transport form of Fe2+, AA is involved in iron uptake by root cells. Apoplastic AA also plays an important role in a number of other processes and is potentially a unique signal-regulatory molecule functioning in the plant cell walls.

Late stage CH functionalization is a powerful tool for modification of natural compounds. Herein we report that the rhodium-catalyzed reaction of brassinosteroids with aryloxysulfonamides proceeds regio- and stereoselectively at C15 position. The derivative obtained from 24-epibrassinolide was easily transformed to the conjugate with a BODIPY dye bearing unaffected functional groups of the native brassinosteroid.

Aluminum (Al) toxicity is the major limiting factor for plant production and crop yield on acid soils. Previous works revealed that ZFP36 played a key role in plant adaptation to water stress and rice blast. Here, we report another function of ZFP36, which is associated with detoxification of Al toxicity through abscisic acid (ABA)- dependent mechanism, in rice. Al exposure significantly increased ZFP36 expression level in an ABA-dependent manner. Knocking down ZFP36 triggered Al sensitivity, while overexpressing ZFP36 resulted in an increased Al tolerance. Chromatin immunoprecipitation-sequencing (ChIP-seq), yeast one-hybrid assay, and dual luciferase test revealed that a tonoplast-localized Al transporter, OsALS1, and antioxidant defense enzymes were targeted by ZFP36. Moreover, by combining yeast two-hybrid assay, bimolecular fluorescence complement (BiFC), GST pull-down and in vitro phosphorylation assay, we showed that ZFP36 could interact with and be phosphorylated by the pyruvate dehydrogenase kinase OsPDK1. A strong association was found between the expression, protein content and kinase activity of OsPDK1 with the responses to ABA and Al. Using transgenic plants, we have showed that OsPDK1 could inhibit OsALS1 expression via regulating ZFP36. Overall, our data demonstrates a new ZFP36-controlled mechanism of Al defense in rice plants, which is mediated by OsPDK1.

Soil salinization is a major threat to global food security and the biodiversity of natural ecosystems. To adapt to salt stress, plants rely on ROS-mediated signalling networks that operate upstream of a broad array of physiological and genetic processes. A key player in ROS signalling is NADPH oxidase, a plasma-membrane-bound enzyme encoded by RBOH genes. In this study, we have conducted a comprehensive bioinformatic analysis of over 50 halophytic and glycophytic species to link the difference in the kinetics of ROS signalling between contrasting species with the abundance and/or structure of NADPH oxidases. The RBOH proteins were predicted in all the tested plant lineages except some algae species from the Rhodophyta, Chlorophyta and Streptophyta. Within the glycophytic group, the number of RBOH copies correlated negatively with salinity stress tolerance, suggesting that a reduction in the number of RBOH isoforms may be potentially related to the evolution of plant salinity tolerance. While halophytes did not develop unique protein families during evolution, they evolved additional phosphorylation target sites at the N-termini of NADPH oxidases, potentially modulating enzyme activity and allowing more control over their function, resulting in more efficient ROS signalling and adaptation to saline conditions.

Boron (B) deficiency and aluminum (Al) toxicity are two major constraints on plants grown in acidic soils. B supply mitigates Al toxicity; however, the underlying mechanisms of this process remain elusive. In this work, Pisum sativum plants were used to address this issue. In the absence of pH buffers, B supply had a better mitigation effect on Al-induced root inhibition at pH 4.0 than pH 4.8. However, in MES buffered solution, mitigating effects of B on Al-induced root inhibition were more pronounced at pH 4.8, indicating a strong pH dependency of this process. Quantification of pH-dependent accumulation of Al in various root zones, modification of root pH by an exogenous addition of rapid alkalization factor (RALF), and measuring changes in the rhizosphere pH by fluorescent dyes have revealed operation of two concurrent mechanisms to explain alleviation of the inhibition of root elongation induced by Al toxicity by boron: (1) via enhancing rhizosphere pH under strong acidic stress (pH4.0), and (2) via stabilizing of cell wall by cross-linking with RGII at relatively higher pH (4.8). These findings provide scientific basis and support for the application of B fertilizers in the regions with inherited soil acidity.

One of the early reactions of plants to high levels of NaCl is the generation of reactive oxygen species (ROS), which is critical for stress signaling, Na+ extrusion, oxidative damage and programmed cell death (PCD) under salt stress. Using moss Physcomitrella patens protonema as a model system, we have tested whether ROS generation and DNA degradation can be induced by salinity in Bryophyta. We have shown with dihydroethidium in vivo test that toxic NaCl concentrations significantly stimulate ROS production. The pharmacological analysis carried out using superoxide dismutase, catalase, dimethyl sulfoxide, thiourea and spermine outlines the superoxide and hydroxyl radicals as major NaCl-induced species. Using Comet assay, we have found that NaCl affects the DNA stability in ROS-dependent manner, increasing both single and double strand DNA breaks (SSBs and DSBs, respectively). Surprisingly, DSBs were induced by NaCl to a lesser extent than SSBs. This is indicative of an unexpected and sophisticated action of NaCl on moss genome including DSBs through PCD and SSBs caused by hydroxyl radical-induced DNA oxidation or transposon rearrangement. We also report a relatively high sensitivity of Physcomitrella patens protonema to NaCl compared to later developmental stages of this moss, suggesting that dividing cells are more sensitive to NaCl than other tissues.

Human beings and plants experience a variety of stress conditions and adapt themselves through novel molecular crosstalk in their cellular constituents. Nitric oxide (NO), haemoglobin and melatonin interact with each other not only in blood stream of human beings, but also in the cells and metabolically active conducting strands of plants. Specialised sites of biosynthesis and differential intracellular spatial distribution of these molecules have been clearly demonstrated by the authors in plant systems. This has led to an understanding of the role of these molecules under salt stress conditions experienced by plants: NO is a modulator of enzyme activity through S-nitrosylation and tyrosine nitration, haemoglobin (phytoglobin) is an NO scavenger, and melatonin is a reactive oxygen species (ROS) scavenger involved in key crosstalk in both plants and humans facing stress. Our recent work on heme oxygenase (HO) activity modulation by stress in plants, and its interaction with NO, further demonstrates common features of molecular crosstalk in protecting plants and human beings from stress.

Phosphatidic acids (PAs) are a key intermediate in phospholipid biosynthesis, and a central element in numerous signalling pathways. Functions of PAs are related to their fundamental role in molecular interactions within cell membranes modifying membrane bending, budding, fission and fusion. Here we tested the hypothesis that PAs are capable of direct transport of ions across bio-membranes. We have demonstrated that PAs added to the maize plasma membrane vesicles induced ionophore-like transmembrane transport of Ca2+, H+ and Mg2+. PA-induced Ca2+ fluxes increased with an increasing PAs acyl chain unsaturation. For all the PAs analysed, the effect on Ca2+ permeability increased with increasing pH (pH 8.0pH 7.2pH 6.0). The PA-induced Ca2+, Mg2+ and H+ permeability was also more pronounced in the endomembrane vesicles as compared with the plasma membrane vesicles. Addition of PA to protoplasts from Arabidopsis thaliana (L.) Heynh. roots constitutively expressing aequorin triggered elevation of the cytosolic Ca2+ activity, indicating that the observed PA-dependent Ca2+ transport occurs in intact plants.

Ion channels activated by reactive oxygen species (ROS) have been found in the plasma membrane of charophyte Nitella flixilis, dicotyledon Arabidopsis thaliana, Pyrus pyrifolia and Pisum sativum, and the monocotyledon Lilium longiflorum. Their activities have been reported in charophyte giant internodes, root trichoblasts and atrichoblasts, pollen tubes, and guard cells. Hydrogen peroxide and hydroxyl radicals are major activating species for these channels. Plant ROS-activated ion channels include inwardly-rectifying, outwardly-rectifying, and voltage-independent groups. The inwardly-rectifying ROS-activated ion channels mediate Ca2+-influx for growth and development in roots and pollen tubes. The outwardly-rectifying group facilitates K⁺ efflux for the regulation of osmotic pressure in guard cells, induction of programmed cell death, and autophagy in roots. The voltage-independent group mediates both Ca2+ influx and K⁺ efflux. Most studies suggest that ROS-activated channels are non-selective cation channels. Single-channel studies revealed activation of 14.5-pS Ca2+ influx and 16-pS K⁺ efflux unitary conductances in response to ROS. The molecular nature of ROS-activated Ca2+ influx channels remains poorly understood, although annexins and cyclic nucleotide-gated channels have been proposed for this role. The ROS-activated K⁺ channels have recently been identified as products of Stellar K⁺ Outward Rectifier (SKOR) and Guard cell Outwardly Rectifying K⁺ channel (GORK) genes.

Silver nanoparticles (Ag NPs) are the world's most important nanomaterial and nanotoxicant. The aim of this study was to determine the early stages of interactions between Ag NPs and plant cells, and to investigate their physiological roles. We have shown that the addition of Ag NPs to cultivation medium, at levels above 300 mg L(-1) , inhibited Arabidopsis thaliana root elongation and leaf expansion. This also resulted in decreased photosynthetic efficiency and the extreme accumulation of Ag in tissues. Acute application of Ag NPs induced a transient elevation of [Ca(2+) ]cyt and the accumulation of reactive oxygen species (ROS; partially generated by NADPH oxidase). Whole-cell patch-clamp measurements on root cell protoplasts demonstrated that Ag NPs slightly inhibited plasma membrane K(+) efflux and Ca(2+) influx currents, or caused membrane breakdown; however, in excised outside-out patches, Ag NPs activated Gd(3+) -sensitive Ca(2+) influx channels with unitary conductance of approximately 56 pS. Bulk particles did not modify the plasma membrane currents. Tests with electron paramagnetic resonance spectroscopy showed that Ag NPs were not able to catalyse hydroxyl radical generation, but that they directly oxidized the major plant antioxidant, l-ascorbic acid. Overall, the data presented shed light on mechanisms of the impact of nanosilver on plant cells, and show that these include the induction of classical stress signalling reactions (mediated by [Ca(2+) ]cyt and ROS) and a specific effect on the plasma membrane conductance and the reduced ascorbate.

Brassinosteroids (BRs) are an important class of plant hormones with a multitude of functions. They have been intensively investigated for their biosynthesis, distribution and physiological functions. The aim of this study was to examine possible effects of BRs on the plant plasma membrane cation conductances and Ca 2+ signalling. The wheat root protoplasts (tested by patch-clamping) and excised arabidopsis roots (analysed by Ca 2+ -aequorin chemiluminometry), were used. In the whole-cell plasma membrane patches, 24-epibrassinolide, 28-homobrassionolide or 24-epicastasterone (1 μM) were applied exogenously. 24-Epicastasterone increased the activity of the K + efflux conductance in 50% of tested protoplasts while 24-epibrassonolide and 28-homobrassionolide did not modify the plasma membrane currents. Addition of 24-epicastasterone at the cytosolic side (to the pipette solution) resulted in dramatic stimulation of a time-dependent K + efflux current (in 30% of protoplasts) and an activation of Ca 2+ influx currents (in 30% of protoplasts). Gadolinium ions, which are blockers of cation channels, inhibited the 24-epicastasterone-induced cation channel activities. In Arabidopsis thaliana plants constitutively expressing aequorin, exogenous 24-epibrassonolide, 28-homobrassionolide and 24-epicastasterone induced a transient elevation of the cytosolic free Ca 2+ , which was inhibited by Gd 3+ and mediated by Ca 2+ influx from the bathing solution. In Ca 2+ -aequorin tests, 10 μM of exogenous BRs was the minimal concentration at which statistically significant changes of the cytosolic Ca 2+ were observed. In conclusion, the obtained results suggest that the plasma membrane of root cells contains the brassinosteroid-activated cation-permeable channels, which can probably be involved in rapid regulation of the K + homeostasis and Ca 2+ signalling.

Oxidative stress is a complex chemical and physiological phenomenon that accompanies virtually all biotic and abiotic stresses in higher plants and develops as a result of overproduction and accumulation of reactive oxygen species (ROS). This review revises primary mechanisms underlying plant oxidative stress at the cellular level. Recent data have clarified the ‘origins’ of oxidative stress in plants, and show that apart from classical chloroplast, mitochondrial and peroxisome sources, ROS are synthesized by NADPH oxidases and peroxidases. ROS damage all major plant cell bio-polymers, resulting in their dysfunction. They activate plasma membrane Ca2+-permeable and K+-permeable cation channels as well as annexins, catalyzing Ca2+ signaling events, K+ leakage and triggering programed cell death. Downstream ROS-Ca2+-regulated signaling cascades probably include regulatory systems with one (ion channels and transcription factors), two (Ca2+-activated NADPH oxidases and calmodulin) or multiple components (Ca2+-dependent protein kinases and mitogen-activated protein kinases). Intracellular and extracellular antioxidants form sophisticated networks, protecting against oxidation and ‘shaping’ stress signaling. Research into plant oxidative stress has shown great potential for developing stress-tolerant crops. This can be achieved through the use of directed evolution techniques to prevent protein oxidation, bioengineering of antioxidant activities as well as modification of ROS sensing mechanisms.

Potassium is the most abundant macronutrient, which is involved in a multitude of physiological processes. Potassium uptake in roots is crucial for plants; however, K(+) efflux can also occur and has important functions. Potassium efflux from roots is mainly induced by stresses, such as pathogens, salinity, freezing, oxidants and heavy metals. Reactive oxygen species (ROS) and exogenous purines also cause this reaction. The depolarisation and activation of cation channels are required for K(+) efflux from plant roots. Potassium channels and nonselective cation channels (NSCCs) are involved in this process. Some of them are 'constitutive', while the others require a chemical agent for activation. In Arabidopsis, there are 77 genes that can potentially encode K(+)-permeable channels. Potassium-selective channel genes include 9 Shaker and 6 Tandem-Pore K(+) channels. Genes of NSCCs are more abundant and present by 20 cyclic nucleotide gated channels, 20 ionotropic glutamate receptors, 1 two-pore channel, 10 mechanosensitive-like channels, 2 mechanosensitive 'Mid1-Complementing Activity' channels, 1 mechanosensitive Piezo channel, and 8 annexins. Two Shakers (SKOR and GORK) and several NSCCs are expressed in root cell plasma membranes. SKOR mediates K(+) efflux from xylem parenchyma cells to xylem vessels while GORK is expressed in the epidermis and functions in K(+) release. Both these channels are activated by ROS. The GORK channel activity is stimulated by hydroxyl radicals that are generated in a Ca(2+)-dependent manner in stress conditions, such as salinity or pathogen attack, resulting in dramatic K(+) efflux from root cells. Potassium loss simulates cytosolic proteases and endonucleases, leading to programmed cell death. Other physiological functions of K(+) efflux channels include repolarisation of the plasma membrane during action potentials and the 'hypothetical' function of a metabolic switch, which provides inhibition of energy-consuming biosyntheses and releasing energy for defence and reparation needs.

Glycation can be defined as an array of non-enzymatic post-translational modifications of proteins formed by their interaction with reducing carbohydrates and carbonyl products of their degradation. Initial steps of this process rely on reducing sugars and result in the formation of early glycation products-Amadori and Heyns compounds via Schiff base intermediates, whereas their oxidative degradation or reactions of proteins with α-dicarbonyl compounds yield a heterogeneous group of advanced glycation end products (AGEs). These compounds accompany thermal processing of protein-containing foods and are known to impact on ageing, pathogenesis of diabetes mellitus and Alzheimer's disease in mammals. Surprisingly, despite high tissue carbohydrate contents, glycation of plant proteins was addressed only recently and its physiological role in plants is still not understood. Therefore, here we summarize and critically discuss the first steps done in the field of plant protein glycation during the last decade. We consider the main features of plant glycated proteome and discuss them in the context of characteristic metabolic background. Further, we address the possible role of protein glycation in plants and consider its probable contribution to protein degradation, methylglyoxal and sugar signalling, as well as interplay with antioxidant defense. ispartof: International Journal Of Molecular Sciences vol:20 issue:9 ispartof: location:Switzerland status: Published online

Electrolyte leakage accompanies plant response to stresses, such as salinity, pathogen attack, drought, heavy metals, hyperthermia, and hypothermia; however, the mechanism and physiological role of this phenomenon have only recently been clarified. Accumulating evidence shows that electrolyte leakage is mainly related to K(+) efflux from plant cells, which is mediated by plasma membrane cation conductances. Recent studies have demonstrated that these conductances include components with different kinetics of activation and cation selectivity. Most probably they are encoded by GORK, SKOR, and annexin genes. Hypothetically, cyclic nucleotide-gated channels and ionotropic glutamate receptors can also be involved. The stress-induced electrolyte leakage is usually accompanied by accumulation of reactive oxygen species (ROS) and often results in programmed cell death (PCD). Recent data strongly suggest that these reactions are linked to each other. ROS have been shown to activate GORK, SKOR, and annexins. ROS-activated K(+) efflux through GORK channels results in dramatic K(+) loss from plant cells, which stimulates proteases and endonucleases, and promotes PCD. This mechanism is likely to trigger plant PCD under severe stress. However, in moderate stress conditions, K(+) efflux could play an essential role as a 'metabolic switch' in anabolic reactions, stimulating catabolic processes and saving 'metabolic' energy for adaptation and repair needs.

Elevation in the cytosolic free calcium is crucial for plant growth, development and adaptation. Calcium influx into plant cells is mediated by Ca2+ depolarisation-activated, hyperpolarisation-activated and voltage-independent Ca2+-permeable channels (DACCs, HACCs and VICCs respectively). These channels are encoded by the following gene families: (1) cyclic nucleotide-gated channels (CNGCs), (2) ionotropic glutamate receptors (GLRs), (3) annexins, (4) 'mechanosensitive channels of small (MscS) conductance'-like channels (MSLs), (5) 'mid1-complementing activity' channels (MCAs), Piezo channels, and hyperosmolality-induced [Ca2+]cyt. channel 1 (OSCA1). Also, a 'tandem-pore channel1' (TPC1) catalyses Ca2+ efflux from the vacuole in response to the plasma membrane-mediated Ca2+ elevation. Recent experimental data demonstrated that Arabidopsis thaliana (L.) Heynh. CNGCs 2, 5-10, 14, 16 and 18, GLRs 1.2, 3.3, 3.4, 3.6 and 3.7, TPC1, ANNEXIN1, MSL9 and MSL10,MCA1 and MCA2, OSCA1, and some their homologues counterparts in other species, are responsible for Ca2+ currents and/or cytosolic Ca2+ elevation. Extrusion of Ca2+ from the cytosol is mediated by Ca2+-ATPases and Ca2+/H+ exchangers which were recently examined at the level of high resolution crystal structure. Calcium-activated NADPH oxidases and reactive oxygen species (ROS)-activated Ca2+ conductances form a self-amplifying 'ROS-Ca2+hub', enhancing and transducing Ca2+ and redox signals. The ROS-Ca2+ hub contributes to physiological reactions controlled by ROS and Ca2+, demonstrating synergism and unity of Ca2+ and ROS signalling mechanisms.

Environmental stresses such as salinity, drought, oxidants, heavy metals, hypoxia, extreme temperatures and others can induce autophagy and necrosis-type programmed cell death (PCD) in plant roots. These reactions are accompanied by the generation of reactive oxygen species (ROS) and ion disequilibrium, which is induced by electrolyte/K+ leakage through ROS-activated ion channels, such as the outwardly-rectifying K+ channel GORK and non-selective cation channels. Here, we discuss mechanisms of the stress-induced ion disequilibrium and relate it with ROS generation and onset of morphological, biochemical and genetic symptoms of autophagy and PCD in roots. Based on our own data and that in the literature, we propose a hypothesis on the induction of autophagy and PCD in roots by loss of cytosolic K+. To support this, we present data showing that in conditions of salt stress-induced autophagy, gork1–1 plants lacking root K+ efflux channel have fewer autophagosomes compared with the wild type. Overall, literature analyses and presented data strongly suggest that stress-induced root autophagy and PCD are controlled by the level of cytosolic potassium and ROS.

Extracellular purine nucleotides are implicated in the control of plant development and stress responses. While extracellular ATP is known to activate transcriptional pathways via plasma membrane (PM) NADPH oxidase and calcium channel activation, very little is known about signal transduction by extracellular ADP. Here, extracellular ADP was found to activate net Ca2+ influx in roots of Arabidopsis (Arabidopsis thaliana) and transiently elevate cytosolic free Ca2+ in root epidermal protoplasts. An inward Ca2+-permeable conductance in root epidermal PM was activated within 1 s of ADP application and repeated application evoked a smaller current. Such response speed and densitization are consistent with operation of equivalents to animal ionotropic purine receptors, although to date no equivalent genes for such receptors have been identified in higher plants. In contrast to ATP, extracellular ADP did not evoke accumulation of intracellular reactive oxygen species. While high concentrations of ATP caused net Ca2+ efflux from roots, equivalent concentrations of ADP caused net influx. Overall the results point to a discrete ADP signaling pathway, reliant on receptor-like activity at the PM.

Phosphatidic acids (PAs) are a key intermediate in phospholipid biosynthesis, and a central element in numerous signalling pathways. Functions of PAs are related to their fundamental role in molecular interactions within cell membranes modifying membrane bending, budding, fission and fusion. Here we tested the hypothesis that PAs are capable of direct transport of ions across bio-membranes. We have demonstrated that PAs added to the maize plasma membrane vesicles induced ionophore-like transmembrane transport of Ca2+, H+ and Mg2+. PA-induced Ca2+ fluxes increased with an increasing PAs acyl chain unsaturation. For all the PAs analysed, the effect on Ca2+ permeability increased with increasing pH (pH 8.0>pH 7.2>pH 6.0). The PA-induced Ca2+, Mg2+ and H+ permeability was also more pronounced in the endomembrane vesicles as compared with the plasma membrane vesicles. Addition of PA to protoplasts from Arabidopsis thaliana (L.) Heynh. roots constitutively expressing aequorin triggered elevation of the cytosolic Ca2+ activity, indicating that the observed PA-dependent Ca2+ transport occurs in intact plants.

Functions of exogenous L-ascorbic acid in plant roots are poorly understood. Recent study by Makavitskaya et al. (doi.org/10.1093/jxb/ery056) has demonstrated that exogenous ascorbate can be released from roots in response to salt stress, and can trigger elevation in the cytosolic free Ca(2+). Here, we report that exogenous ascorbate significantly modifies root elongation in Arabidopsis thaliana. Using a medium exchange technique, we have shown that 10–100 µM ascorbate induces small but significant increase in root elongation while higher levels cause its dramatic decrease. Root border cells of Pisum sativum have been losing viability twice faster in the presence of ascorbate that under control conditions, as tested by the confocal microscopy and a combined staining with propidium iodide and fluorescein diacetate.

FLOWER FLAVONOID TRANSPORTER (FFT) encodes a multidrug and toxin efflux family transporter in Arabidopsis thaliana. FFT (AtDTX35) is highly transcribed in floral tissues, the transcript being localized to epidermal guard cells, including those of the anthers, stigma, siliques and nectaries. Mutant analysis demonstrates that the absence of FFT transcript affects flavonoid levels in the plant and that the altered flavonoid metabolism has wide-ranging consequences. Root growth, seed development and germination, and pollen development, release and viability are all affected. Spectrometry of mutant versus wild-type flowers shows altered levels of a glycosylated flavonol whereas anthocyanin seems unlikely to be the substrate as previously speculated. Thus, as well as adding FFT to the incompletely described flavonoid transport network, it is found that correct reproductive development in Arabidopsis is perturbed when this particular transporter is missing.

Extracellular ATP regulates higher plant growth and adaptation. The signalling events may be unique to higher plants, as they lack animal purinoceptor homologues. Although it is known that plant cytosolic free Ca2+ can be elevated by extracellular ATP, the mechanism is unknown. Here, we have studied roots of Arabidopsis thaliana to determine the events that lead to the transcriptional stress response evoked by extracellular ATP. Root cell protoplasts were used to demonstrate that signalling to elevate cytosolic free Ca2+ is determined by ATP perception at the plasma membrane, and not at the cell wall. Imaging revealed that extracellular ATP causes the production of reactive oxygen species in intact roots, with the plasma membrane NADPH oxidase AtRBOHC being the major contributor. This resulted in the stimulation of plasma membrane Ca2+-permeable channels (determined using patch-clamp electrophysiology), which contribute to the elevation of cytosolic free Ca2+. Disruption of this pathway in the AtrbohC mutant impaired the extracellular ATP-induced increase in reactive oxygen species (ROS), the activation of Ca2+ channels, and the transcription of the MAP kinase3 gene that is known to be involved in stress responses. This study shows that higher plants, although bereft of purinoceptor homologues, could have evolved a distinct mechanism to transduce the ATP signal at the plasma membrane.

Regulation of reactive oxygen species and cytosolic free calcium ([Ca2+]cyt) is central to plant function. Annexins are small proteins capable of Ca2+-dependent membrane binding or membrane insertion. They possess structural motifs that could support both peroxidase activity and calcium transport. Here, a Zea mays annexin preparation caused increases in [Ca2+]cyt when added to protoplasts of Arabidopsis thaliana roots expressing aequorin. The pharmacological profile was consistent with annexin activation (at the extracellular plasma membrane face) of Arabidopsis Ca2+-permeable nonselective cation channels. Secreted annexins could therefore modulate Ca2+ influx. As maize annexins occur in the cytosol and plasma membrane, they were incorporated at the intracellular face of lipid bilayers designed to mimic the plasma membrane. Here, they generated an instantaneously activating Ca2+-permeable conductance at mildly acidic pH that was sensitive to verapamil and Gd3+ and had a Ca2+-to-K+ permeability ratio of 0.36. These results suggest that cytosolic annexins create a Ca2+ influx pathway directly, particularly during stress responses involving acidosis. A maize annexin preparation also demonstrated in vitro peroxidase activity that appeared independent of heme association. In conclusion, this study has demonstrated that plant annexins create Ca2+-permeable transport pathways, regulate [Ca2+]cyt, and may function as peroxidases in vitro.

English Article NEW PHYTOL 325ED; Calcium (Ca2+)-permeable plasma membrane ion channels are critical to root hair elongation and signalling. Arabidopsis thaliana root hair plasma membrane contains a hyperpolarization-activated Ca2+ channel (HACC) conductance. Here, the co-residence of HACC with a depolarization-activated Ca2+ channel (DACC) conductance has been investigated. Whole-cell patch-clamping of apical plasma membrane has been used to study Ca2+ conductances and reveal the negative slope conductance typical of DACCs. Specific voltage protocols, Ba(2+)-permeation and inhibition by the cation channel blocker Gd3+ have been used to identify the DACC conductance. The Gd3+ sensitive DACC conductance was identified in only a minority of cells. DACC activity was quickly masked by the development of the HACC conductance. However, in the period between the disappearance of the negative slope conductance and the predominance of HACC, DACC activity could still be detected. A DACC conductance coexists with HACC in the root hair apical plasma membrane and could provide Ca2+ influx over a wide voltage range, consistent with a role in signalling.

Hydrogen peroxide is an important regulatory agent in plants. This study demonstrates that exogenous H2O2 application to Arabidopsis thaliana root epidermis results in dose-dependent transient increases in net Ca2+ influx. The magnitude and duration of the transients were greater in the elongation zone than in the mature epidermis. In both regions, treatment with the cation channel blocker Gd3+ prevented H2O2-induced net Ca2+ influx, consistent with application of exogenous H2O2 resulting in the activation of plasma membrane Gd3+-sensitive Ca2+-influx pathways. Application of 10 mm H2O2 to the external plasma membrane face of elongation zone epidermal protoplasts resulted in the appearance of a hyperpolarization-activated Ca2+-permeable conductance. This conductance differed from that previously characterized as being responsive to extracellular hydroxyl radicals. In contrast, in mature epidermal protoplasts a plasma membrane hyperpolarization-activated Ca2+-permeable channel was activated only when H2O2 was present at the intracellular membrane face. Channel open probability increased with intracellular [H2O2] and at hyperpolarized voltages. Unitary conductance decreased thus: Ba2+ > Ca2+ (14.5 pS) > Mg2+ > Zn2+ (20 mM external cation, 1 mM H2O2). Lanthanides and Zn2+ (but not TEA+) suppressed the open probability without affecting current amplitude. The results suggest spatial heterogeneity and differential sensitivity of Ca2+ channel activation by reactive oxygen species in the root that could underpin signalling.

Three BS-BODIPY (brassinosteroids-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) conjugates were synthesized and their fluorescent and immunological properties were investigated. Two of the conjugates, having present all the functional groups characteristic of BS, were shown to be potentially useful as biological probes to study involvement of BS into physiological processes in living cells.

Calcium can ameliorate Na+ toxicity in plants by decreasing Na+ influx through nonselective cation channels. Here, we show that elevated external [Ca2+] also inhibits Na+-induced K+ efflux through outwardly directed, K+-permeable channels. Noninvasive ion flux measuring and patch-clamp techniques were used to characterize K+ fluxes from Arabidopsis (Arabidopsis thaliana) root mature epidermis and leaf mesophyll under various Ca2+ to Na+ ratios. NaCl-induced K+ efflux was not related to the osmotic component of the salt stress, was inhibited by the K+ channel blocker TEA+, was not mediated by inwardly directed K+ channels (tested in the akt1 mutant), and resulted in a significant decrease in cytosolic K+ content. NaCl-induced K+ efflux was partially inhibited by 1 mm Ca2+ and fully prevented by 10 mm Ca2+. This ameliorative effect was at least partially attributed to a less dramatic NaCl-induced membrane depolarization under high Ca2+ conditions. Patch-clamp experiments (whole-cell mode) have demonstrated that two populations of Ca2+-sensitive K+ efflux channels exist in protoplasts isolated from the mature epidermis of Arabidopsis root and leaf mesophyll cells. The instantaneously activating K+ efflux channels showed weak voltage dependence and insensitivity to external and internal Na+. Another population of K+ efflux channels was slowly activating, steeply rectifying, and highly sensitive to Na+. K+ efflux channels in roots and leaves showed different Ca2+ and Na+ sensitivities, suggesting that these organs may employ different strategies to withstand salinity. Our results suggest an additional mechanism of Ca2+ action on salt toxicity in plants: the amelioration of K+ loss from the cell by regulating (both directly and indirectly) K+ efflux channels.

Free oxygen radicals are an irrefutable component of life, underlying important biochemical and physiological phenomena in animals. Here it is shown that free oxygen radicals activate plasma membrane Ca(2+)- and K(+)-permeable conductances in Arabidopsis root cell protoplasts, mediating Ca(2+) influx and K(+) efflux, respectively. Free oxygen radicals generate increases in cytosolic Ca(2+) mediated by a novel population of nonselective cation channels that differ in selectivity and pharmacology from those involved in toxic Na(+) influx. Analysis of the free oxygen radical-activated K(+) conductance showed its similarity to the Arabidopsis root K(+) outward rectifier. Significantly larger channel activation was found in cells responsible for perceiving environmental signals and undergoing elongation. Quenching root free oxygen radicals inhibited root elongation, confirming the role of radical-activated Ca(2+) influx in cell growth. Net free oxygen radical-stimulated Ca(2+) influx and K(+) efflux were observed in root cells of monocots, dicots, C3 and C4 plants, suggesting conserved mechanisms and functions. In conclusion, two functions for free oxygen radical cation channel activation are proposed: initialization/amplification of stress signals and control of cell elongation in root growth.

Elevation in the cytosolic free calcium is crucial for plant growth, development and adaptation. Calcium influx into plant cells is mediated by Ca2+ depolarisation-activated, hyperpolarisation-activated and voltage-independent Ca2+-permeable channels (DACCs, HACCs and VICCs respectively). These channels are encoded by the following gene families: (1) cyclic nucleotide-gated channels (CNGCs), (2) ionotropic glutamate receptors (GLRs), (3) annexins, (4) ‘mechanosensitive channels of small (MscS) conductance’-like channels (MSLs), (5) ‘mid1-complementing activity’ channels (MCAs), Piezo channels, and hyperosmolality-induced [Ca2+]cyt. channel 1 (OSCA1). Also, a ‘tandem-pore channel1’ (TPC1) catalyses Ca2+ efflux from the vacuole in response to the plasma membrane-mediated Ca2+ elevation. Recent experimental data demonstrated that Arabidopsis thaliana (L.) Heynh. CNGCs 2, 5–10, 14, 16 and 18, GLRs 1.2, 3.3, 3.4, 3.6 and 3.7, TPC1, ANNEXIN1, MSL9 and MSL10,MCA1 and MCA2, OSCA1, and some their homologues counterparts in other species, are responsible for Ca2+ currents and/or cytosolic Ca2+ elevation. Extrusion of Ca2+ from the cytosol is mediated by Ca2+-ATPases and Ca2+/H+ exchangers which were recently examined at the level of high resolution crystal structure. Calcium-activated NADPH oxidases and reactive oxygen species (ROS)-activated Ca2+ conductances form a self-amplifying ‘ROS-Ca2+hub’, enhancing and transducing Ca2+ and redox signals. The ROS-Ca2+ hub contributes to physiological reactions controlled by ROS and Ca2+, demonstrating synergism and unity of Ca2+ and ROS signalling mechanisms.

Plant signalling is a set of phenomena that serves the transduction of external and internal signals into physiological responses such as modification of enzyme activity, cytoskeleton structure or gene expression. It operates at the level of cell compartments, whole cells, tissues, organs or even plant communities. To achieve this, plants have evolved a network of signalling proteins including plasma membrane receptors and ion transporters, cascades of kinases and other enzymes as well as several second messengers such as cytosolic calcium (Ca2+), reactive oxygen/nitrogen species (ROS/RNS), cyclic nucleotides (cAMP and cGMP) and others. Overall, these systems recognise and decode environmental signals and co-ordinate ontogeny programs. This paper summarises recent progress in the field of plant signalling, which was a major theme of the 4th International Symposium on Plant Signalling and Behaviour, 2016, in Saint Petersburg, Russia. Several novel hypotheses and concepts were proposed during this meeting. First, the concept of ROS-Ca2+ hubs has found further evidence and acceptance. This concept is based on reciprocal activation of NADPH oxidases by cytosolic Ca2+ on the one hand, and Ca2+-permeable channels that are activated by NADPH-produced ROS. ROS-Ca2+ hubs enhance the intensity and duration of originally weak Ca2+ and ROS signals. Hubs are directly involved in ROS- and Ca2+-mediated physiological reactions, such as stress response, growth, programmed cell death, autophagy and long-distance signalling. Second, recent findings have widened the list of cyclic nucleotide-regulated processes and strengthened the biochemical basis of cyclic nucleotide biochemistry by exploring cyclase activities of new receptors such as the Phytosulfokine Receptor 1, the pathogen peptide 1 receptor (atPepR1), the brassinosteroid BRI1 receptor and the cell wall-associated kinase like 10. cGMP and cAMP signalling has demonstrated strong link to Ca2+ signalling, via cyclic nucleotide-gated Ca2+-permeable ion channels (CNGCs), and to ROS and RNS via their nitrosylated forms. Third, a novel role for cytosolic K+ as a regulator of plant autophagy and programmed cell death has emerged. The cell death-associated proteases and endonucleases were demonstrated to be activated by a decrease of cytosolic K+ via ROS-induced stimulation of the plasma membrane K+ efflux channel GORK. Importantly, the origin of ROS for induction of autophagy and cell death varies in different tissues and comprises several pools, including NADPH oxidases, peroxidases, photosynthetic and respiratory electron-transporting chains and peroxisomal enzymes. The peroxisome pool is the ‘latest’ addition to established cellular ROS-producing machineries and is dependent on the state and abundance of catalase in this compartment. Finally, new aspects of phytohormone signalling, such as regulation of root hydraulic pressure by abscisic acid and rate of mitosis by cytokinins, as well as localising cytokinin receptors in endoplasmic reticulum, are reported. Other observations suggest that melatonin is a hormone-like substance in plants, because it targets Ca2+, ROS and RNS.

Calcium is a critical structural and regulatory nutrient in plants. However, mechanisms of its uptake by root cells are poorly understood. We have found that Ca2+ influx in Arabidopsis root epidermal protoplasts is mediated by voltage-independent rapidly activating Ca2+-permeable non-selective cation channels (NSCCs). NSCCs showed the following permeability (P) sequence: PCa (1.00) = PBa (0.93) > PZn (0.51), PCa/PNa = 0.19, PCa/PK = 0.14. They were inhibited by quinine, Gd3+, La3+ and the His modifier diethylpyrocarbonate, but not by the Ca2+ or K+ channel antagonists, verapamil and tetraethylammonium (TEA+). Single channel conductance measured in 20 mm external Ca2+ was 5.9 pS. Calcium-permeable NSCCs co-existed with hyperpolarisation-activated Ca2+ channels (HACCs), which activated 40-60 min after forming the whole-cell configuration. HACCs activated at voltages

In plant cells, Ca(2+) is required for both structural and biophysical roles. In addition, changes in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) orchestrate responses to developmental and environmental signals. In many instances, [Ca(2+)](cyt) is increased by Ca(2+) influx across the plasma membrane through ion channels. Although the electrophysiological and biochemical characteristics of Ca(2+)-permeable channels in the plasma membrane of plant cells are well known, genes encoding putative Ca(2+)-permeable channels have only recently been identified. By comparing the tissue expression patterns and electrophysiology of Ca(2+)-permeable channels in the plasma membrane of root cells with those of genes encoding candidate plasma membrane Ca(2+) channels, the genetic counterparts of specific Ca(2+)-permeable channels can be deduced. Sequence homologies and the physiology of transgenic antisense plants suggest that the Arabidopsis AtTPC1 gene encodes a depolarisation-activated Ca(2+) channel. Members of the annexin gene family are likely to encode hyperpolarisation-activated Ca(2+) channels, based on their corresponding occurrence in secretory or elongating root cells, their inhibition by La(3+) and nifedipine, and their increased activity as [Ca(2+)](cyt) is raised. Based on their electrophysiology and tissue expression patterns, AtSKOR encodes a depolarisation-activated outward-rectifying (Ca(2+)-permeable) K(+) channel (KORC) in stelar cells and AtGORK is likely to encode a KORC in the plasma membrane of other Arabidopsis root cells. Two candidate gene families, of cyclic-nucleotide gated channels (CNGC) and ionotropic glutamate receptor (GLR) homologues, are proposed as the genetic correlates of voltage-independent cation (VIC) channels.

The aim of the present work was to characterize Na(+) currents through nonselective cation channels (NSCCs) in protoplasts derived from root cells of Arabidopsis. The procedure of the protoplast isolation was modified to increase the stability of Arabidopsis root protoplasts in low external Ca(2+) by digesting tissue in elevated Ca(2+). Experiments in whole-cell and outside-out modes were carried out. We found that Na(+) currents in Arabidopsis root protoplasts were mediated by cation channels that were insensitive to externally applied tetraethylammonium(+) and verapamil, had no time-dependent activation (permanently opened or completely activated within 1-2 ms), were voltage independent, and were weakly selective for monovalent cations. The selectivity sequence was as follows: K(+) (1.49) > NH(4)(+) (1.24) > Rb(+) (1.15) approximately equal to Cs(+) (1.10) approximately equal to Na(+) (1.00) > Li(+) (0.73) > tetraethylammonium(+) (0.47). Arabidopsis root NSCCs were blocked by H(+) (pK approximately equal to 6.0), Ca(2+) (K(1/2) approximately equal to 0.1 mM), Ba(2+), Zn(2+), La(3+), Gd(3+), quinine, and the His modifier diethylpyrocarbonate. They were insensitive to most organic blockers (nifedipine, verapamil, flufenamate, and amiloride) and to the SH-group modifier p-chloromercuriphenyl sulfonic acid. Voltage-insensitive, Ca(2+)-sensitive single channels were also resolved. Properties of Arabidopsis root NSCCs are discussed and compared with characteristics of similar conductances studied previously in plants and animals. It is suggested that NSCCs present a distinct group of plant ion channels, mediating toxic Na(+) influx to the cell and probably having other important roles in physiological processes of plants.

Effects of Cu2+ on a non-specific conductance and H+-ATPase activity in the plasma membrane of the freshwater alga Nitella flexilis L. Agardh was studied using a conventional microelectrode voltage-clamp technique. We show that a Cu2+-induced increase in the non-specific conductance is related to the formation of pores in the plasma membrane. Pore formation is the result of unidentified chemical reactions, since the Q10 for the rate of increase of conductance over time was about 3. Various oxidants and antioxidants (10 mmol/l H2O2, 10 mmol/l ascorbate, 100 microg/ml superoxide dismutase, and 100 microg/ml catalase) did not alter Cu2+-induced changes in the plasma membrane conductance, suggesting that the effect of Cu2+ was unrelated to peroxidation of plasma-membrane lipids. In contrast, organic and inorganic Ca2+-channel antagonists (nifedipine, Zn2+, Cd2+, Fe2+, Ni2+) inhibited the Cu2+-induced non-specific conductance increase. This suggests that changes in Ca2+ influx underlie this effect of Cu2+. Decreasing the pH or the ionic strength of external solutions also inhibited the Cu2+-induced plasma-membrane conductance increase. Copper was also found to inhibit plasma-membrane H+-ATPase activity with half-maximal inhibition occurring at about 5-20 micromol/l and full inhibition at about 100-300 micromol/l. The Hill coefficient of Cu2+ inhibition of the H+-ATPase was close to two.

The conventional microelectrode technique was applied to study changes in conductance and activation characteristics of potassium and chloride channels in the plasmalemma of characean alga Nitella flexilis(L.) Agardz. during long-term heat treatment. Measurements were conducted at 18–20°C after preliminary exposure of cells to 33°C for 1–25 days. The conductance of outward- and inward-rectifying potassium channels, as well as the currents of excitable chloride channels, decreased after 2–3 days of heat treatment. By the 15th–17th days, the conductance of potassium channels was reduced by a factor of 3–5, whereas the peak values of the chloride current, associated with the action potential, was reduced by a factor of 8–10. These heat-induced changes were long lasting: the restoration of the initial parameters of transport systems after transferring cells to chilling or room temperature occurred within several days. Moreover, the recovery at chilling temperatures (8–10°C) proceeded nearly two times longer than at room temperature. Prolonged hyperthermia accelerated activation and deactivation of outward-rectifying potassium channels and caused the shift of their activation curve towards positive potentials by 35–40 mV. Analysis of current–voltage relations showed that the inward current in inward- and outward-rectifying potassium channels was reduced to a greater extent than the outward current. At the same time, both inward and outward currents of chloride channels were reduced to an equal extent. It is assumed that the changes observed are involved in thermal adaptation and account for the decrease in the intracellular concentrations of potassium and other cations and anions, which represents a nonspecific response of plant cells to stress.

Calcium is an important macronutrient, which is required for structural and regulatory needs. Elevation of the cytosolic free Ca2+ is widely accepted as a major signalling mechanism in plants, which encodes the information about a multitude of exogenous and endogenous stimuli. Ca2+ uptake by roots from the soil is a passive process, which is catalysed by specialised plasma membrane proteinaceous pores that are called cation channels. These channels demonstrate different biophysical and physiological characteristics and may belong to a number of gene families. This chapter summarises data on physiological techniques and basic concepts for investigation of Ca2+-permeable cation channels in plant root cells. The main focus of the chapter is on patch-clamp technique, ion-selective vibration microelectrodes and Ca2+-aequorin luminometry. In the past two decades, a combination of these techniques allowed to investigate Ca2+-permeable channels in higher plants and establish their crucial roles in plant cell physiology.

Extracellular purine nucleotides appear capable of regulating plant development, defence and stress responses by acting in part as agonists of plasma membrane calcium channels. Factors stimulating ATP release include wounding, osmotic stress and elicitors. Here we show that exogenous abscisic acid and L-glutamate can also cause ATP accumulation around Arabidopsis thaliana roots. Release of ADP from root epidermis would trigger ionotropic receptor-like activity in the plasma membrane, resulting in transient elevation of cytosolic free calcium. Root epidermal protoplasts (expressing aequorin as a cytosolic free calcium reporter) can support an extracellular ADP-induced cytosolic calcium elevation in the presence of an extracellular reductant. This confirms that ADP could elicit calcium-based responses distinct to those of ATP, which have been shown previously to involve production of extracellular reactive oxygen species.

Reactive oxygen species (ROS) are central to plant stress response, signalling, development and a multitude of other processes. In this study, the plasma-membrane hydroxyl radical (HR)-activated K(+) channel responsible for K(+) efflux from root cells during stress accompanied by ROS generation is characterised. The channel showed 16-pS unitary conductance and was sensitive to Ca(2+), tetraethylammonium, Ba(2+), Cs(+) and free-radical scavengers. The channel was not found in the gork1-1 mutant, which lacks a major plasma-membrane outwardly rectifying K(+) channel. In intact Arabidopsis roots, both HRs and stress induced a dramatic K(+) efflux that was much smaller in gork1-1 plants. Tests with electron paramagnetic resonance spectroscopy showed that NaCl can stimulate HR generation in roots and this might lead to K(+)-channel activation. In animals, activation of K(+)-efflux channels by HRs can trigger programmed cell death (PCD). PCD symptoms in Arabidopsis roots developed much more slowly in gork1-1 and wild-type plants treated with K(+)-channel blockers or HR scavengers. Therefore, similar to animal counterparts, plant HR-activated K(+) channels are also involved in PCD. Overall, this study provides new insight into the regulation of plant cation transport by ROS and demonstrates possible physiological properties of plant HR-activated K(+) channels.