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Salinity is a major abiotic stress affecting agricultural production. To understand the genetic behavior of salinity tolerance traits, a half-diallel cross was made among six barley cultivars (Hordeum vulgare L.), with contrasting levels of known tolerance, to study the combining ability of salinity tolerance on the basis of K+ loss from plant roots under saline conditions. The glasshouse pot experiments showed that the six parents were significantly different in salinity tolerance and those tolerances were highly correlated with the K+ flux measurements. The combining ability analysis showed that the variances of both general combining ability (GCA) and specific combining ability (SCA) were highly significant. Two tolerant cultivars, CM72 and Numar, showed significantly higher GCA for salinity tolerance (less K+ loss under salinity stress). Cultivars with medium GCA were YU6472 and Yan90260. Salinity tolerance was mainly controlled by additive effects with the tolerance allele showing partial dominance. High positive SCA was also found between two tolerant cultivars and between tolerant and medium-tolerant cultivars, indicating possible different tolerant genes or some minor genes in these cultivars. The combination of these genes from different sources of tolerant cultivars should produce cultivars with even greater tolerance. © Crop Science Society of America. All rights reserved.

Plants may suffer from ammonium (NH4+) toxicity when NH4+ is the sole nitrogen source. Nitrate (NO3-) is known to alleviate NH4+ toxicity, but the mechanisms are unknown. This study has evaluated possible mechanisms of NO3- alleviation of NH4+ toxicity in canola (Brassica napus L.). Dynamics of net fluxes of NH4+, H+, K+ and Ca2+ were assessed, using a non-invasive microelectrode (MIFE) technique, in plants having different NO3- supplies, after single or several subsequent increases in external NH4Cl concentration. After an increase in external NH4Cl without NO3-, NH4+ net fluxes demonstrated three distinct stages: release (tau1), return to uptake (tau2), and a decrease in uptake rate (tau3). The presence of NO3- in the bathing medium prevented the tau1 release and also resulted in slower activation of the tau3 stage. Net fluxes of Ca2+ were in the opposite direction to NH4+ net fluxes, regardless of NO3- supply. In contrast, H+ and K+ net fluxes and change in external pH were not correlated with NH4+ net fluxes. It is concluded that (i) NO3- primarily affects the NH4+ low-affinity influx system; and (ii) NH4+ transport is inversely linked to Ca2+ net flux.

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.

Hypoxia-induced changes in net H +, K + and O 2 fluxes across the plasma membrane (PM) of epidermal root cells were measured using the non-invasive microelectrode ion flux measurement (MIFE) system in elongation, meristem and mature root zones of two barley (Hordeum vulgare L.) varieties contrasting in their waterlogging (WL) tolerance. The ultimate goal of this study was to shed light on the mechanisms underlying effects of WL on plant nutrient acquisition and mechanisms of WL tolerance in barley. Our measurements revealed that functionally different barley root zones have rather different O 2 requirements, with the highest O 2 influx being in the elongation zone of the root at about 1 mm from the tip. Oxygen deprivation has qualitatively different effects on the activity of PM ion transporters in mature and elongation zones. In the mature zone, hypoxic treatment caused a very sharp decline in K + uptake in the WL sensitive variety Naso Nijo, but did not reduce K + influx in the WL tolerant TX9425 variety. In the elongation zone, onset of hypoxia enhanced K + uptake from roots of both cultivars. Pharmacological experiments suggested that hypoxia-induced K + flux responses are likely to be mediated by both K + -inward- (KIR) and non-selective cation channels (NSCC) in the elongation zone, while in the mature zone K + -outward- (KOR) channels are the key contributors. Overall, our results suggest that oxygen deprivation has an immediate and substantial effect on root ion flux patterns, and that this effect is different in WL-sensitive and WL-tolerant cultivars. To what extent this difference in ion flux response to hypoxia is a factor conferring WL tolerance in barley remains to be answered in future studies. © 2006 Blackwell Publishing Ltd.

Although oscillations in membrane-transport activity are ubiquitous in plants, the ionic mechanisms of ultradian oscillations in plant cells remain largely unknown, despite much phenomenological data. The physiological role of such oscillations is also the subject of much speculation. Over the last decade, much experimental evidence showing oscillations in net ion fluxes across the plasma membrane of plant cells has been accumulated using the non-invasive MIFE technique. In this study, a recently proposed feedback-controlled oscillatory model was used. The model adequately describes the observed ion flux oscillations within the minute range of periods and predicts: (i) strong dependence of the period of oscillations on the rate constants for the H 1 pump; (ii) a substantial phase shift between oscillations in net H 1 and K 1 fluxes; (iii) cessation of oscillations when H 1 pump activity is suppressed; (iv) the existence of some ‘window’ of external temperatures and ionic concentrations, where nondamped oscillations are observed: outside this range, even small changes in external parameters lead to progressive damping and aperiodic behaviour; (v) frequency encoding of environmental information by oscillatory patterns; and (vi) strong dependence of oscillatory characteristics on cell size. All these predictions were successfully confirmed by direct experimental observations, when net ion fluxes were measured from root and leaf tissues of various plant species, or from single cells. Because oscillatory behaviour is inherent in feedback control systems having phase shifts, it is argued from this model that suitable conditions will allow oscillations in any cell or tissue. The possible physiological role of such oscillations is discussed in the context of plant adaptive responses to salinity, temperature, osmotic, hypoxia, and pH stresses.

Development of salt-tolerant genotypes is central both to remediation of salinity-affected land and to meet increasing global food demand, which has been driving expansion of cropping into marginal areas. The bottleneck of any breeding programme is the lack of a reliable screening technique. This study tested the hypothesis that the ability of plants to retain K+ under saline conditions is central to their salt tolerance. Using seven barley cultivars contrasting in salt tolerance (CM72, Numar, ZUG293, ZUG95, Franklin, Gairdner, ZUG403), a comprehensive study was undertaken of whole-plant (growth rate, biomass, net CO2 assimilation, chlorophyll fluorescence, root and leaf elemental and water content) and cellular (net fluxes of H+, K+, Na+ and Ca2+) responses to various concentrations of NaCl (20–320 m m). Na+ selective microelectrodes were found to be unsuitable for screening purposes because of non-ideal selectivity of the commercially available Na+ LIX. At the same time, our results show very strong negative correlation between the magnitude of K+ efflux from the root and salt tolerance of a particular cultivar. K+ efflux from the mature root zone of intact 3-day-old seedlings following 40 min pretreatment with 80 m m NaCl was found to be a reliable screening indicator for salinity tolerance in barley. As a faster and more cost-effective alternative to microelectrode measurements, a procedure was developed enabling rapid screening of large numbers of seedlings, based on amount of K+ leaked from plant roots after exposure to NaCl.

Potassium transport was investigated in the root elongation zone of Arabidopsis seedlings during the first minutes of Al3+ exposure, using the non-invasive MIFE microelectrode technique. To prevent pH changes during Al3+ application, and to separate aluminium from acidic stress, plants were pre-treated with 5 mM homoPIPES before addition of AlCl3 (pH 4.2). The 30-min treatment with 50 or 500 μM AlCl3 led to a significant increase in K+ efflux in solutions containing 100 μM CaCl2. This efflux was suppressed by high concentrations of Ca2+ (10 mM) in the bathing solution. Our results suggest that elevated external Ca2+ activities can sustain K+ influx in the root elongation zone during Al3+ exposure either by maintaining [Ca2+]cyt or by affecting Al3+ uptake across the plasma membrane.

Both parts of the actin-myosin complex involved in cytoplasmic streaming could be regulated by mineral ions. The main goal of this study was to find a relationship between cyclosis and ion transport across the cell wall and plasma membrane. The transport of K(+) and Ca(2+) along pH bands in Chara branchlet internodal cells was characterized by using the MIFE system for non-invasive microelectrode measurement of ion fluxes. Branchlets formed acidic and alkaline bands with the pH ranging from 5 to 8. Different pH patterns were observed for different sides of the branchlets. Sides with cyclosis streaming acropetally generally showed greater variation in the profiles of pH and H(+) fluxes. Although a high correlation was not found between pH bands and Ca(2+) or K(+) fluxes, there was a positive correlation between Ca(2+) and K(+) fluxes themselves for both sides of the branchlets. Application of cytochalasin D, an inhibitor of cyclosis, had no immediate effect on pH and ion fluxes, however, the time of cyclosis cessation corresponded with a dramatic change in Ca(2+) and K(+) fluxes; pH profiles and H(+) fluxes were affected within 2 h. The evidence suggests that, in Chara branchlets, pH band formation and Gd(3+)-insensitive Ca(2+) transport systems are linked to the cyclosis machinery: (i) the pH band amplitude for the acropetally streaming side was larger than that for the basipetally streaming side; (ii) cessation of cytoplasmic streaming after cytochalasin D application resulted in changed pH banding profiles and H(+), Ca(2+) and K(+) fluxes; and (iii) the application of GdCl(3) or incubation in GdCl(3) solutions did not lead to the cessation of cytoplasmic streaming, although external Ca(2+) fluxes changed.

Changes induced by blue light (BL) in ion fluxes and pH in etiolated seedlings of dicotyledons (Cucumis sativus L., Phaseolus lunatus L., Pisum sativum L.) were studied using a non-invasive ion-selective microelectrode technique (MIFE). BL induced significant Ca2+ uptake within the first 5 min after onset in upper parts of the whole cucumber seedling. To locate the sites of the BL perception, studies on excised segments were performed. The data obtained demonstrated that the observed Ca2+ uptake is associated with the very top of the hypocotyl hook of cucumber and butter bean, and with the whole epicotyl of pea, although the response was stronger for the upper parts of the epicotyl. Addition of Gd3+ and La3+ into the bathing solution completely prevented Ca2+ influx, implicating the involvement of Ca2+ channels in the regulation of Ca2+ transport during BL exposure. BL-induced changes in H+ and Ca2+ fluxes were not likely to be linked to the one mechanism, as they were separated spatially and pharmacologically.

Knowledge of the preferred source of N for Eucalyptus nitens will lead to improved fertiliser management practices in plantations. Ion selective microelectrodes were used non-invasively to measure simultaneously net fluxes of NH4+, NO3− and H+ along the tap root of solution-cultured E. nitens. Measurements were conducted in solutions containing 100 μm NH4NO3. The pattern of fluxes was such that there was a large influx of NH4+, a smaller influx of NO3− and large H+ efflux. The ratio of these fluxes was constant, according to the ratio 3:1:−6 (NH4+:NO3−:H+). Within the region 20–60 mm from the root apex of E. nitens seedlings there was spatial and temporal variation in fluxes but flux patterns remained constant. Root hair density did not affect fluxes nor did proximity to lateral roots. Variation was less than that found in previous studies of localised root fluxes using similar high-resolution measurement techniques. It was concluded that small-scale spatial variation in fluxes may have confounded previous studies. There were associations between fluxes of all three ions, the strongest associations being between NH4+ and H+, and NH4+ and NO3−. Overall, these results are consistent with NH4+ being the preferred source N for E. nitens.

and productivity by imposing osmotic stress on plants. To cope with the problem, plant cells must readjust their Transient kinetics of net H+ ,K + ,C a2+, and Cl− fluxes osmotic potential to prevent water losses. That can be were measured non-invasively, using an ion-selective achieved by either uptake of inorganic ions from the microelectrode technique, for bean (Vicia faba L.) leaf external solution, or by de novo synthesis of compatible mesophyll in response to 150 mM mannitol treatment. solutes (amino acids, sugars, polyoles, quaternary amines) In a parallel set of experiments, changes in the plasma acting as osmolytes ( Wyn Jones and Pritchard, 1989; membrane potential and the total proline content in Delauney and Verma, 1993; Bohnert et al., 1995; Bohnert leaves were monitored. Regardless of the ionic com- and Shen, 1999; Serrano et al., 1999). These two processes position of the bath solution, hyperosmotic stress differ significantly in their time scales. Immediate changes caused a significant increase in the K+ and Cl− uptake in ion fluxes are believed to provide quick (within a few into mesophyll cells. At the same time, no significant minutes) osmotic adjustment while a fine ‘tuning’ by proline changes were observed for at least 16 h after means of biochemical synthesis of compatible solutes has the onset of stress. Experiments with inhibitors sug- a scale of hours and days ( Wyn Jones and Pritchard, gested that potassium inward rectifier (KIR) channels, 1989; Lew, 1996). exhibiting mechanosensitive properties and acting as Recent progress in molecular genetics has made bioprimary receptors of osmotic stress, are likely to be chemical mechanisms of osmolyte biosynthesis the priminvolved. Due to the coupling by membrane potential, ary target for genetic engineering of salt and drought changes in K+ and Cl− transport may modify activity tolerance in crops (see Yeo, 1998, for a review). However, of the plasma membrane H+-pump. Such coupling may the progress is disappointingly slow, and an increase in also be responsible for the mannitol-induced oscilla- the osmotic tolerance in a field situation is only marginal tions (period of about 4 min) in net ion fluxes observed (Bohnert and Shen, 1999). It appears that membrane in 90% of plants. Calculations show that influx of K+ transport processes play a more crucial role in plant and Cl− observed in response to hyperosmotic treat- osmoregulation than was believed so far. ment may provide an adequate osmotic adjustment in There is much evidence supporting this statement. bean mesophyll, which suggests that the activity of Cerda et al. concluded that accumulation of inorganic the plasma membrane transporters for these ions ions was sufficient for osmotic adjustment in salt-stressed should be targeted to improve osmotolerance, at least maize cultivars, and that no single organic solute appeared in this crop. to be important in this process (Cerda et al., 1995). Similar conclusions were made by Huang and Redmann

Although salinity is one of the major problems limiting agricultural production around the world, the underlying mechanisms of high NaCl perception and tolerance are still poorly understood. The effects of different bathing solutions and fusicoccin (FC), a known activator of plasma membrane ATPase, on plasma membrane potential (Em) and net fluxes of Na+, K+and H+were studied in wheat suspension cells (Triticum aestivum) in response to different NaCl treatments. Emof cells in Murashige and Skoog (MS) medium was less negative than in cells exposed to a medium containing 10 mM KCl + 0.1 m M CaCl2(KSM) and to a basic salt medium (BSM), containing 1 m M KCl and 0.1 m M CaCl2. Multiphasic Na+accumulation in cells was observed, peaking at 13 min after addition of 120 m M NaCl to MS medium. This time scale was in good agreement with net Na+flux changes measured non-invasively by moving ion-selective microelectrodes (the MIFE system). When 120 m M NaCl was added to all media studied, a quick rise of Na+influx was reversed within the first 20 min. In both 120 and 20 m M NaCl treatments in MS medium, net Na+efflux was observed, indicating that active Na+transporters function in the plant cell response to salt stress. Lower external K+concentrations (KSM and BSM) and FC pre-treatment caused shifts in Na+fluxes towards net influx at 120 m M NaCl stress.

Net fluxes of H a and Ca 2a were measured in the mesophyll tissue of broad bean (Vicia faba L.) leaves and in protoplasts derived from these cells. NaCl at 90 mM enhanced H a extrusion in both protoplasts and tissue, but in diAerent ways. Proton extrusion was inhibited by vanadate, suggesting the involvement of the plasma membrane H a ATPase in cell responses to salinity. There was virtually no eAect of NaCl on the net Ca 2a flux in protoplasts, while in the tissue a large transient Ca 2a eCux followed the salt treatment. Salt-induced Ca 2a eCux was essentially independent of external Ca2a concentrations in the range 0. 1t o 10 mM. Also, Ca2a flux responses were ‘saturated’ above 50 mM NaCl. It is suggested that almost all the measured Ca2a flux originates from Naa/Ca2a and Ha/Ca2a ion exchange in the cell wall. This conclusion was supported by the results of modelling cation exchange in the cell wall. # 2000 Annals of Botany Company

The effects of auxin on net ion fluxes near parenchyma of oat coleoptiles were studied using the non-invasive MIFE system to measure specific ion fluxes using ion selective microelectrodes. Application of 10 μm1-naphthaleneacetic acid (NAA) for 3 h caused doubling of coleoptile segment growth, without changing the pH of the unbuffered bathing solution from pH 5.4 during that time. Short term experiments revealed that auxin led to an immediate three-fold increase of chloride influx to 1200 nmol m−2s−1, maintained for at least 1 h. In the first minutes after auxin application, proton fluxes were small (-25 nmol m−2s−1, an efflux) and tended to decrease, whereas potassium and calcium fluxes changed little, fluctuating from -100 to 0 nmol m−2s−1and from -15 to 0 nmol m−2s−1, respectively. It is suggested that one target of auxin action in plant cells is the plasma membrane chloride transport system mediating increased chloride uptake.

H + flux kinetics were measured in solution around the roots of chilling-tolerant pea (Pisum sativum) and bean (Vicia faba), chilling-sensitive cucumber (Cucumis sativus) and pumpkin (Cucurbita pepo), and intermediate corn (Zea mays) species using a microelectrode technique to measure net flux. As a root warmed to room temperature after 90 min at 4 °C, at which temperature the H + flux was near zero, the flux rose (influx) and then fell. These changes occurred at two apparent critical temperatures, which were higher for the more chilling-sensitive species. The first, lower, apparent critical temperature may represent the start of passive inward H + transport. The higher critical temperature may represent the start of active H + extrusion. From these apparent critical temperatures we have calculated the real critical temperature and the time delay of the chilling signal transduction process. Passive and active H + transporters appear to have the same real critical temperature of chilling sensitivity, about 9 °C, but have, respectively, 4 min and 11 min time delays. Measurement of these apparent critical temperatures may provide quick and reliable screening for chilling sensitivity in plant breeding programmes. Future ion flux studies may show the cellular location of chilling stress perception and the signal transduction pathways.

International audience; Non-invasive microelectrode ion flux measuring (the MIFE system) allows concurrent quantification of net fluxes of several ions with high spatial (several ?m) and temporal (ca 5 s) resolution. Over the last 10 years, the MIFE system has been widely used to study various aspects of salt stress signaling and adaptation in plants. This chapter summarizes some major findings in the area such as using MIFE for deciphering the specific and non-specific components of salinity stress, resolving the role of the plasma membrane H(+)-pump in salinity responses, proving K(+) homeostasis as a key feature of salinity tolerance, and discovering the mechanisms behind the ameliorative effects of Ca(2+) and other mitigating factors (such as polyamines or compatible solutes). The full protocols for microelectrode fabrication, calibration, and use are then given, and two basic routines for measuring net K(+) and Na(+) fluxes from salinity stressed roots are described in the context of plant screening for salt stress tolerance.

Growth and early gravitropic responses of corn roots in solution have been studied using time-lapse photography. Aeration was required for both root growth and gravitropism. The optimum pH for gravitropism was in the range 5 to 6. The bending response seemed to be greater for roots in non-buffered solution than in buffered solution. Fastest growth and maximum curvature occurred with about 0.2 mol m−3 Ca2+. Under some conditions, the gravitropic response started with apparently negligible time delay after the start of the gravitropic stimulus. This may denote graviperception in or near the elongation zone itself. This mechanism for early but relatively weak gravitropism may help to explain a variety of gravitropic responses such as the ‘early wrong way’ curvature, and the behaviour of roots whose columella cells lack amyloplasts. More rapid bending appears to start at about 20 min, which is consistent with observations on roots in humid air and with the accepted statolith model of perception in the root cap.

An account is given of an investigation of net ionic currents and specific ion fluxes occuring during the initiation of organogenesis in detached leaves of Graptopetalum paraguayense E. Walther, in which a dramatic change in growth polarity is cytomorphologically evident 3–5 d after leaf detachment from the plant. Using the vibrating probe, it was possible to identify a peak of ionic current which is focused over the area of the leaf base where organogenesis is initiated. This net current is largest during the initial 12h after leaf detachment. With ion-selective microelectrodes capable of measuring H+, K+ and Ca2+ ion fluxes simultaneously in the same region of the leaf base, H+ and K+ fluxes remain relatively steady during the initial 24 h after detachment, while a large lanthanum-sensitive Ca2+ influx decreases by 50% from 2 to 12h. By 24h, Ca2+ transport is dominated by an efflux. We present evidence from a quantitative comparison of the ion current data collected using these two techniques, that Ca2+, H+ and K+ transport accounts for the major electrogenic ion fluxes during 2 and 12 but not 24 h after leaf detachment. The possibility is addressed that these ion currents, which precede organogenesis, and in particular the predominant Ca2+ flux, play a role in the establishment of growth polarity in higher plant tissues.

Plant salinity tolerance is a polygenic trait with contributions from genetic, developmental, and physiological interactions, in addition to interactions between the plant and its environment. In this study, we show that in salt-tolerant genotypes of barley (Hordeum vulgare), multiple mechanisms are well combined to withstand saline conditions. These mechanisms include: (1) better control of membrane voltage so retaining a more negative membrane potential; (2) intrinsically higher H+ pump activity; (3) better ability of root cells to pump Na+ from the cytosol to the external medium; and (4) higher sensitivity to supplemental Ca2+. At the same time, no significant difference was found between contrasting cultivars in their unidirectional 22Na+ influx or in the density and voltage dependence of depolarization-activated outward-rectifying K+ channels. Overall, our results are consistent with the idea of the cytosolic K+-to-Na+ ratio being a key determinant of plant salinity tolerance, and suggest multiple pathways of controlling that important feature in salt-tolerant plants.

A large-scale glasshouse trial, including nearly 70 barley cultivars (5300 plants in total), was conducted over 2 consecutive years to investigate plant physiological responses to salinity. In a parallel set of experiments, plant salt tolerance was assessed by non-invasive microelectrode measurements of net K+ flux from roots of 3-day-old seedlings of each cultivar after 1 h treatment in 80 mm NaCl as described in our previous publication (Chen et al. 2005). K+ flux from the root in response to NaCl treatment was highly (P < 0.001) inversely correlated with relative grain yield, shoot biomass, plant height, net CO2 assimilation, survival rate and thousand-seed weight measured in glasshouse experiments after 4–5 months of salinity treatment. No significant correlation with relative germination rate or tillering was found. In general, 62 out of 69 cultivars followed an inverse relationship between K+ efflux and salt tolerance. In a few cultivars, however, high salt tolerance (measured as grain yield at harvest) was observed for plants showing only modest ability to retain K+ in the root cells. Tissue elemental analysis showed that these plants had a much better ability to prevent Na+ accumulation in plant leaves and, thus, to maintain a higher K+/Na+ ratio. Taken together, our results show that a plant’s ability to maintain high K+/Na+ ratio (either retention of K+ or preventing Na+ from accumulating in leaves) is a key feature for salt tolerance in barley.

Ion flux kinetics associated with blue light (BL) treatment of two wild types (WTs) and the phot1 , phot2 and phot1/phot2 mutants of Arabidopsis were studied by using the MIFE noninvasive ion-selective microelectrode technique. BL induced significant changes in activity of H + and Ca 2+ transporters within the first 10 min of BL onset, peaking between 3 and 5 min. In all WT plants and in phot2 mutants, BL induced immediate Ca 2+ influx. In phot1 and phot1/phot2 mutants, net Ca 2+ flux remained steady. It is suggested that PHOT1 regulates Ca 2+ uptake into the cytoplasm from the apoplast. Changes in ion fluxes were measured from cotyledons of intact seedlings and from the cut top of the hypocotyl of decapitated seedlings. Thus the photoreceptors mediating BL-induced Ca 2+ and H + fluxes are present in the rest of the decapitated seedling and probably in the cotyledons as well. The H + and Ca 2+ flux responses to BL appear not to be linked because, ( i ) when changes were observed for both ions, Ca 2+ flux changed almost immediately, whereas H + flux lagged by about 1.5 min; ( ii ) in the Wassilewskija ecotype, changes in H + fluxes were small. Finally, wave-like changes in Ca 2+ and H + concentrations were observed along the cotyledon–hook axis regardless of its orientation to the light.

The transport of mineral ions into and out of tissues and cells is central to the life of plants. Ion transport and the plasma membrane transporters themselves have been studied using a variety of techniques. In the last 15 years, measurement of specific ion fluxes has contributed to the characterization of transport systems. Progress in molecular genetics is allowing gene identification and controlled expression of transporter molecules. However the molecular expression of transporter gene products must be characterized at the functional level. The ion-selective microelectrode technique to measure specific ion fluxes non-invasively is ideally suited to this purpose. This technique, its theory, its links with others and its application and prospects in plant science, are discussed. Ions studied include hydrogen, potassium, sodium, ammonium, calcium, chloride and nitrate. Applications discussed include: solute ion uptake by roots; gravitropism and other processes in the root cap, meristematic and elongation zones; Nod factor effect on root hairs; osmotic and salt stresses; oscillations; the effects of light and temperature. Studies have included intact roots, leaf mesophyll and other tissues, protoplasts and bacterial biofilms. A multi-ion capability of the technique will greatly assist functional genomics, particularly when coupled with imaging techniques, patch clamping and the use of suitable mutants.

Recent evidence suggests that in plants, as in vertebrates, natriuretic peptides (NPs) regulate homeostasis. In this study noninvasive ion-selective vibrating microelectrodes were used to measure net fluxes of K+, Na+, and H+ in Zea mays root conductive tissue. Immunoreactant plant natriuretic peptides (irPNP) cause immediate net H+ influx and delayed net K+ and Na+ uptake. Delayed net K+ influx was also observed in response to 8-Br-cGMP, however, not accompanied by significant changes in net H+ fluxes. Furthermore, 8-Br-cGMP does not stimulate the plasma membrane H+-ATPase implying that cGMP directly affects cation channels. The data are consistent with NP and cGMP-dependent stimulation of nonselective cation channels with P(K) > P(Na) and point to a complex role for NPs in plant homeostasis.

Ion-selective microelectrodes were used non-invasively to measure the concentration dependence of NH4+ and NO3– fluxes around the roots of intact solution-cultured Eucalyptus nitens (Deane & Maiden) Maiden. In addition, NH4+ and H+ fluxes were measured simultaneously at a range of NH4+ concentrations, and NO3– and H+ fluxes were measured simultaneously at a range of NO3– concentrations. Nitrogen concentrations ranged from 10–250 μM, i.e. in the range corresponding to the high affinity transport system (HATS). Both NH4+ and NO3– fluxes exhibited saturating Michaelis–Menten-style kinetics. The Km was 16 μM for NH4+ and 18 μM for NO3–. Values of Vmax were 53 nmol m–2 s–1 for NH4+ and 37 nmol m–2 s–1 for NO3–. Proton fluxes were highly correlated with NH4+ and NO3– fluxes, but the relationships were different. Proton efflux increased with increasing NH4+ concentration and mirrored the changing NH4+ fluxes. The ratio between NH4+ and H+ fluxes was 1 : –1.6. Proton influx was evident with initial exposure to NO3–, with the flux stoichiometry for NO3– : H+ being 1 : 1.4. Subsequent increases in NO3– concentration caused a gradual increase in H+ efflux such that the flux stoichiometry for NO3– : H+ became 1 : –0.8. The presence of 100 μM NH4+ greatly reduced NO3– fluxes and caused a large and constant H+ efflux. These results are evidence that E. nitens has a preference for NH4+ as a source of N, and that the fluxes of NH4+ and NO3– are quantitatively linked to H+ flux.

Mechanisms underlying changed K + uptake by plantsat low pH need to be deciphered. One possibility is that K+ acquisition is under the strict control of plasmamembrane potential (E m ), which,in turn, is affected by external pH. To test this hypothesis, we used themicroelectrode ion flux measurement (MIFE ) technique tostudy net K + and H +fluxes near Arabidopsis root hairs at different externalpH, KCl concentrations and clamped Em . Lowering the solution pH led to strong H+ influx, K + efflux andsignificant E m depolarisation.Addition of K + to the bathing media causedsignificant net K + uptake when external pH wasover the range 5.5–6.0. At external pH below 5.0, however, correlationbetween K + availability and net K+ uptake was negative. To explain this apparentparadox, measurements of net K + and H+ fluxes from the root hair surface were performedconcurrently with E m clamped at different values above and below the restingpotential (approx. –180 mV). Our data revealed a strong dependence ofnet K + flux on the clamping voltage. Clamping atvalues more negative than the resting potential caused a significant increasein K + uptake into the root hair; clamping at lessnegative values (–20 and 0 mV) caused significant net K+ efflux from the cell. Qualitatively similarresults were observed for net H + flux. Ourobservations indicate direct control of K + flux bychanging E m , and suggest thatE m depolarisation could be themain reason for the observed K + efflux at low pH.

K+, Ca2+ and H+ flux profiles were studied around the surface of germinating wheat (Triticum aestivum L. cv. Machete) seeds using a non-invasive microelectrode technique. The seed surface was scanned every 500 µm along the longitudinal axis, and net ion fluxes were measured 2 min and 1, 4 and 24 h after seed rehydration com-menced. Functionally different seed zones exhibited different ion exchange patterns, which also changed differently with time. One h after seed rehydration, the peak of H+ extrusion was observed in the embryo-scutellum region. By 24 h this peak had shifted towards the region of radicle emergence, and its magnitude had increased 5-fold. By con-trast, H+ activity in the endosperm region decreased noticeably during this period. At the same time, Ca2+ flux shifted from initial net influx to efflux in the embryo and scutellum zones. Most striking were K+ flux profiles, with large K+ efflux observed at each end and almost zero flux near the geometrical center of the seed. Significant K+ extru-sion from the embryo end was observed for as long as 24 h after seed rehydration and was not related to the loss of membrane integrity. Seed treatment with metabolic inhibitors (vanadate and tetraethylammonium) caused signifi-cant changes in both H+ and K+ fluxes suggesting that at least part of the measured fluxes originated from activities of plasma membrane transporters. We discuss possible mechanisms responsible for the observed regularities and question whether the seed coat in wheat caryopses is as ion-impermeable as previously believed.

Verapamil application in ion transport studies on plant cells is widespread; however, the mechanism of its action is still poorly understood. Net flux of Ca 2+ , K + , Na + , H + and Cl – were measured in solution around oat seedlings using the non-invasive ion-selective microelectrode MIFE technique. The verapamil effect on intact plant tissues was a distinct immediate influx of monovalent cations, H + , K + or Na + . Pre-treatment with tetraethyl-ammonium, Cs + , Ba 2+ or Ca 2+ did not affect K + flux changes, whereas DMSO, Cd 2+ and vanadate increased verapamil-induced K + influx. Verapamil-induced K + flux increased with increased external KCl concentration or pH. Verapamil concentration up to 1 mM failed to shift Ca 2+ flux to efflux. Pre-treatment with adrenaline and dopamine agonists and antagonists led to changes in verapamil-induced ion flux, especially for Ca 2+ . We suggest that a transporter that allows entry of K + and Na + was the main system providing monovalent ion influx after verapamil application.

Changes in the bioelectric activity of maize leaves caused by a single light pulse (6 s; 70 mol m-2 s-1) were used to compare the effects of NaCl treatment (20–200 mM) on plant growth, Na+ accumulation in leaves, chlorophyll fluorescence and pigment composition. Bioelectric responses seemed to be the most sensitive indicator of NaCl effects. Even the weakest salt treatment (20 mM) caused a statistically significant decrease (about 40%) in the amplitude of the bioelectric response. The higher the NaCl concentration, the smaller was the amplitude. Over the full concentration range, the characteristic time of response increased from about 30 to 60 sec, indicating that the rate of bioelectric changes was slowed by increasing salinity. Other reliable characteristics were found to be the fluorescence yield and quenching coefficients. The Fv/Fm ratio was not significantly affected by NaCl treatment. Changes in growth rate, biomass or pigment composition were either insensitive, or showed a plateau over a wide range of NaCl concentrations, and were inappropriate for screening. A possible link between bioelectric and fluorescence characteristics is discussed. We conclude that leaf bioelectric activity can be used together with, or instead of, chlorophyll fluorescence measurements, to screen genotypes for salt tolerance.

Chloride fluxes for oat coleoptile parenchyma were measured using the MIFE system for non-invasive microelectrode ion flux measurement. The effects on chloride fluxes of membrane potential, external chloride concentration, pH, cation composition of the bathing solution, and the anion channel blocker anthracene-9-carboxylic acid were studied. From the experimental data we propose that the step-like appearance of the chloride uptake curve at concentrations higher than 1 mM is due to combined activity of influx and efflux systems. According to this hypothesis the uptake of chloride ions is made by a pH sensitive active transport system with Vmax = 4000 nmol m-2 s-1, Km = 7 mM [Cl-]. Chloride ions are released from the cell through channels with Gaussian type kinetics, the characteristics of which are dependent upon the external bathing solution: Vmax = –2300 nmol m-2 s-1 at 25 mM [Cl-] for CaCl2, and Vmax = –2000 nmol m-2 s-1 at 15 mM [Cl-] for KCl; with both curves having the same width of 17 mM [Cl-]. Key words: Cl- membrane transport, flux kinetics, Avena sativa L.

Although some theoretical predictions have been made, no experimental evidence of chaotic behaviour in plant physiological responses has been reported. Here we present observations of period- doubling and tripling in higher plants. For leaf bioelectric and temperature responses of maize, tomato, and burweed plants to rhythmical light, two different routes to chaos were found experimentally. One was via successive period-doubling and the other via the formation of intermittently chaotic oscillations from a subharmonic synchronisation. Because these effects appeared in intact plants, under conditions close to those found in nature, they may have wide significance, including for plant phylogenesis.

Proton fluxes were measured non-invasively on patch-clamped protoplasts isolated from wheat roots using an external H + electrode to measure the electrochemical gradient in the external solution. Under voltage clamp in the whole-cell configuration, the H + fluxes across the plasma membrane could be measured as a function of voltage and time and correlated with the simultaneous measurements of membrane current. Protoplasts could exist in three states based on the current–voltage (I–V) curves and the flux–V curves. In the pump-state where the membrane voltage (Vm) was more negative than the electrochemical equilibrium potential for potassium (E K ), a net efflux of H + occurred that was voltage-dependent such that the efflux increased as Vm was clamped more positive. In the K-state, where Vm was close to E K , similar flux–V curves were observed. In the depolarised state where Vm was greater than E K the proton flux was characterised by a net influx of H + (H + -influx state) that reversed direction at more positive values of Vm. The inhibitory effect of DCCD and stimulatory effect of fusicoccin were used to correlate current and H + flux through the H + -ATPase for which there was reasonably good agreement within the limits of the flux measurements. Some protoplasts were kept in the whole-cell configuration for up to 3 h revealing slow sustained oscillations (period about 40 min) in H + flux that were in phase with oscillations in free-running Vm. These oscillations were also observed under voltage clamp, with membrane current in phase with H + flux, but which became damped out after a few cycles. The oscillations encompassed the pump-state, K + -state and H + -influx-state. The H +- flux–V curves and I–V curves were used to model the electrical characteristics of the plasma membrane with H + -ATPase, inward and outward K + rectifiers, a linear conductance, and a passive H + influx possibly through gated proton channels.