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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.