Cation-stimulated H+ efflux by intact roots of barley

Abstract. Rates of proton extrusion and potassium (⁸⁶Rb) influx by intact roots of barley ( Hordeum vulgare cvs . Fergus, Conquest and Betzes) plants were simultaneously measured in short-term (15min) experiments. The nature and extent of apparent coupling between these ion fluxes was explored by manipulating conditions of temperature, pH and cation composition and concentration during flux determinations. In addition, the influence of salt status upon these fluxes was examined. At low K⁺ concentrations (0.01 to 1 mol m⁻³), H⁺ efflux and K⁺ influx were strongly correlated in both low- and high-K⁺ roots, although K⁺: H⁺ exchange stoichiometries were almost consistently greater than 2:1. At higher concentrations (1 to 5 mol m⁻³), H⁺ efflux was either reduced or remained unchanged while K⁺ influxes increased. In the presence of Na₂SO₄, rates of H⁺ extrusion demonstrated similar cation dependence, although below 10 mol m⁻³ Na₂SO₄, H⁺ fluxes were generally 50% lower than in equivalent concentrations of K₂SO₄. These observations are considered in the context of current hypotheses regarding the mechanisms of k⁺/H⁺ exchange.     

Screening plants for salt tolerance by measuring K+ flux: a case study for barley

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.     

Photosystem I acceptor side limitation is a prerequisite for the reversible decrease in the maximum extent of P700 oxidation after short-term chilling in the light in four plant species with different chilling sensitivities

Changes in the extent of P700 oxidation (P700⁺) were investigated after chilling of barley, rice, pumpkin, and cucumber leaf segments at 4°C for 1 h under light with various photon flux densities. At 50 µmol photons m⁻² s⁻¹, the decrease in P700⁺ was observed only in cucumber, but at 150 µmol photons m⁻² s⁻¹, it was found in all plants except barley, revealing their expected chilling sensitivities. However, the decrease in P700⁺ by this short-term chilling was reversible in the presence of 3-(3',4'-dichlorophenyl)-1,1-dimethylurea or methyl viologen, and it did not show any causal relationship with the decrease in the electron transfer rate nor with the down-regulation of photosystem II through the accumulation of zeaxanthin and the development of non-photochemical quenching. These results led to the suggestion that photosystem I (PSI) acceptor side limitation is a prerequisite for the decrease of P700⁺. Furthermore, PSI acceptor side limitation could be mainly due to limitation of electron-sink pathways such as CO₂ assimilation and ascorbate–glutathione cycle, because treatment with glycolaldehyde which inhibits the former pathway, and with KCN which inhibits both pathways, decreased P700⁺ by 20–30% in barley leaves after chilling in the light.     

Effect of ultraviolet radiation on accumulation and leakage of 86Rb+ in guard cells of Vicia faba

The effects of UV-C (254 nm), UV-A (365 nm) and broad-band UV (280–380 nm) on guard cells of Vicia faba L. cv. Long Pod were investigated in the presence of white light (450 μmol m⁻² s⁻¹). UV-C (7 μmol m⁻² s⁻¹) was found to cause leakage of ⁸⁶Rb⁺ from guard cells, while UV-A (0.3 μmol m⁻² s⁻¹) stimulated increased uptake in these cells. A relatively small stimulatory effect was observed by broad-band UV (3 μmol m⁻² s⁻¹) during the first 30 min of irradiation with an apparent equilibration of influx and efflux thereafter. Leakage of ⁸⁶Rb⁺ from guard cells continued despite the removal of UV-C and an increase in the amount of white light from 450 to 1500 μmol m⁻² s⁻¹, suggesting that membranes were irreversibly damaged. Irradiation of guard cells with UV-C for 30, 45 and 90 min indicated that these cells began to be affected already by 30 min UV-C irradiation.     

Energy balance and temperature relations of Azorella compacta, a high-elevation cushion plant of the central Andes

The environmental relationships and ecophysiology of Azorella compacta, a giant cushion plant, were investigated in Parque Nacional Lauca, Chile (18°10'–18°25' S and 69°16' W, 4400 m asl). The diurnal temperature range can reach 42 °C on some days of the year. The surface temperature of A. compacta was 13 °C below that of the air temperature of −7 °C at dawn, but from midmorning to late afternoon, the plant surface temperature remained within a few degrees of the air temperature. Soil surface temperatures did not differ between north- and south-facing slopes, but a model showed an increase in radiation reception by north-facing slopes throughout most of the year. Gas exchange measurements of A. compacta measured at the onset of the wet season ranged from −0.6662 to 11.4 μmol·m⁻²·s⁻¹, and maximum stomatal conductance (Gs) was 410 mmol·m⁻²·s⁻¹. The estimated light compensation point was 89 μmol·m⁻²·s⁻¹ and estimated light saturation occurred at about 1280 μmol·m⁻²·s⁻¹. Diurnal water potential measurements for A. compacta ranged from −1.67 to −2.65 MPa. This is one of the first ecophysiological studies of a tropical alpine cushion plant.     

Effect of desiccation on potassium and anion currents from young root hairs: Implication on tip growth

Little is known about the early response of roots to desiccation. Young growing root hairs of Arabidopsis thaliana , Vigna unguiculata and Phaseolus vulgaris were used to study the early response of roots to desiccation since they behave like sensors that are able to perceive environmental signals. In control conditions, root hairs were polarized around −120 mV and displayed inward rectifying K⁺ currents. When submitted to short-term desiccation, root hairs stopped their tip growth and their membrane became depolarized. Under these conditions, the K⁺ influx carried by the inward rectifying K⁺ channels was not maintained and instead slow deactivating anion channels were recorded. The inhibition of K⁺ influx and the large anion efflux due to the activation of slow anion currents could participate in the inhibition of tip growth.     

Promoting effect of fusicoccin on Na+ efflux in barley roots: evidence for a Na+−H+ antiport

Abstract. The effect of fusicoccin (FC) on the K⁺stimulated Na⁺ efflux in root cells of Na⁺ loaded barley roots was studied. FC (0.02 mM) stimulated Na⁺ efflux in the presence of K⁺ and its effect was synergistic with that of K⁺, in a similar way as its effect on proton extrusion. Decreasing the pH of the elution medium promoted Na⁺ efflux and partially replaced the effect of FC. As FC is known to increase the electrochemical proton gradient at the plasmalemma level, these results are consistent with the hypothesis that Na⁺ is extruded in exchange for H⁺. A further support to this view came from the finding that Na⁺ efflux was also promoted by a lipophilic cation, tributylbenzylammonium (TBBA ⁺), which stimulates H ⁺ extrusion and is generally accepted not to enter the cells by means of the same carrier as K ⁺.     

The effect of xanthoxylin (2-oxy, 4-6-dimethoxyacetophenone) on potassium-dependent acid: extrusion in wheat and maize root segments

Abstract. Xanthoxylin is a cytotoxic and fungicidal compound with the characteristics of a typical phytoalexin. At a concentration of 0.3 mol m⁻³ it inhibits K⁺-dependent acid extrusion and K⁺ net uptake (or uptake of equivalent alkaline cations such as Rb⁺ and CS⁺) by up to about 80% and hyperpolarizes by about 20% the membrane electrical potential. Its inhibition of the acid extrusion does not depend on altered ion exchange involving the anions in the media, a reduction of the metabolic energy available, or detectable changes in the permeability of the cell membrane to H⁺ ions. The drop in K⁺ net uptake depends on a decrease in the influx of K⁺ into the cell. In functional terms, xanthoxylin is an inhibitor of the K⁺ permeation mechanism and does not appear to interact with the mechanisms creating the electrochemical energy gradient.     

Requirement for far-red light to maintain secondary needle extension growth in northern but not southern populations of Pinus sylvestris (Scots pine)

Extension growth of secondary needles is under photoperiodic control in Pinus sylvestris . To test for the effects of far-red light on maintaining this extension growth, seedlings of six populations originating from latitudes between 57° and 67°N were raised for 11 weeks in continuous incandescent (metal halogen) light at 300 µmol m⁻² s⁻¹ and 20°C and then transferred at the same temperature to a daily regime of 8 h incandescent light (230 µmol m⁻² s⁻¹) followed by a 16 h day extension with cool white fluorescent light (40 µmol m⁻² s⁻¹, R/FR ratio 7.5) or with incandescent lamps (20 µmol m⁻² s⁻¹, R/FR ratio 2.0). For the seedlings from the three populations north of 64°, needle extension growth over 42 days in the FR-poor day extension treatment was lower by up to 40% than in the FR-rich day extension treatment, whereas for the seedlings from the three southern populations the needle extension growth was similar in both day extension treatments. The requirement for FR in day extensions is characteristic of 'light-dominant' photoperiodic control mechanisms. It appears that P. sylvestris changes from dark-dominant night timekeeping to light-dominant day timekeeping with increasing latitude, as with the photoperiodic control of budset in Picea abies .     

Puccinellia tenuiflora maintains a low Na+ level under salinity by limiting unidirectional Na+ influx resulting in a high selectivity for K+ over Na+

Puccinellia tenuiflora is a useful monocotyledonous halophyte that might be used for improving salt tolerance of cereals. This current work has shown that P. tenuiflora has stronger selectivity for K⁺ over Na⁺ allowing it to maintain significantly lower tissue Na⁺ and higher K⁺ concentration than that of wheat under short- or long-term NaCl treatments. To assess the relative contribution of Na⁺ efflux and influx to net Na⁺ accumulation, unidirectional ²²Na⁺ fluxes in roots were carried out. It was firstly found that unidirectional ²²Na⁺ influx into root of P. tenuiflora was significantly lower (by 31–37%) than in wheat under 100 and 150 m m NaCl. P. tenuiflora had lower unidirectional Na⁺ efflux than wheat; the ratio of efflux to influx was similar between the two species. Leaf secretion of P. tenuiflora was also estimated, and found the loss of Na⁺ content from leaves to account for only 0.0006% of the whole plant Na⁺ content over 33 d of NaCl treatments. Therefore, it is proposed that neither unidirectional Na⁺ efflux of roots nor salt secretion by leaves, but restricting unidirectional Na⁺ influx into roots with a strong selectivity for K⁺ over Na⁺ seems likely to contribute to the salt tolerance of P. tenuiflora .     

Inhibition of the glucose and amino-acid carriers of Lemna gibba by pretreatment with HgCl2

The electrical resting potential across the plasmalemma of Lemna gibba L. (G 1) cells is −230 to −250 mV and the diffusion potential in the presence of 1 mol m⁻³ KCN + 1 mol m⁻³ salicylhydroxamic acid is about −100 mV. A concentration of 0.01 mol m⁻³ HgCl₂ depolarises the transmembrane electrical potential in a largely reversible way. When the cells after 16 min of HgCl₂-application are returned to Hg-free solution, the transmembrane electrical potential is only depolarised by 24 × 13 mV (SD, n = 13) compared with the potential prior to HgCl₂ treatment. In contrast, a 16 min pretreatment with HgCl₂ followed by a wash with mercury-free solution reduces the transient depolarisations of transmembrane potential observed after addition of 5 mol m⁻³ D-glncose or 1 mol m⁻³ L-alaoine to about 60% of controls. These transient depolarisations are due to the onset of solute uptake. Accordingly, HgCl₂-pretreatment inhibits uptake of ¹⁴C-3-O-methyl- d -glucose by more than 50% and uptake of ¹⁴C- l -alanine by more than 70%. Washing with 1 mol m⁻³ 1,4-dithiothreitol does not reverse this inhibition. It is, therefore, concluded that Hg²⁺ irreversibly binds to essential SH-groups of the H⁺-hexose and the H⁺-amino-acid cotransport carriers of Lemna gibba and inhibits these carriers without appreciably affecting the electrogenic proton-extrusion pump.     

The pha gene cluster of Rhizobium meliloti involved in pH adaptation and symbiosis encodes a novel type of K+ efflux system

The fix-2 mutant of Rhizobium meliloti affected in the invasion of alfalfa root nodules (Inf⁻/Fix⁻) is K⁺ sensitive and unable to adapt to alkaline pH in the presence of K⁺. Using directed Tn 5 mutagenesis, we delimited a 6 kb genomic region in which mutations resulted in both Inf⁻/Fix⁻ and K⁺-sensitive phenotypes. In this DNA region, seven open reading frames (ORFs) were identified and the corresponding genes were designated phaA , B , C , D , E , F and G . The putative PhaABC proteins exhibit homology to the subunits of a Na⁺/H⁺ antiporter from an alkalophilic Bacillus strain. Moreover, PhaA and PhaD also show similarity to the ND5 and ND4 subunits of the proton-pumping NADH:ubiquinone oxidoreductase respectively. Computer analysis suggests that all seven proteins are highly hydrophobic with several possible transmembrane domains. Some of these domains were confirmed by generating active alkaline phosphatase fusions. Ion transport studies on phaA mutant cells revealed a defect in K⁺ efflux at alkaline pH after the addition of a membrane-permeable amine. These results suggest that the pha genes of R. meliloti encode for a novel type of K⁺ efflux system that is involved in pH adaptation and is required for the adaptation to the altered environment inside the plant.     

Irradiance levels affect growth parameters and carotenoid pigments in kale and spinach grown in a controlled environment

Carotenoids play critical roles in both light harvesting and energy dissipation for the protection of photosynthetic structures. However, limited research is available on the impact of irradiance on the production of secondary plant compounds, such as carotenoid pigments. Kale ( Brassica oleracea L.) and spinach ( Spinacia oleracea L.) are two leafy vegetables high in lutein and β-carotene carotenoids. The objectives of this study were to determine the effects of different irradiance levels on tissue biomass, elemental nutrient concentrations, and lutein β-carotene and chlorophyll (chl) pigment accumulation in the leaves of kale and spinach. 'Winterbor' kale and 'Melody' spinach were grown in nutrient solution culture in growth chambers at average irradiance levels of 125, 200, 335, 460, and 620 μmol m⁻² s⁻¹. Highest tissue lutein β-carotene and chls occurred at 335 μmol m⁻² s⁻¹ for kale, and 200 μmol m⁻² s⁻¹ for spinach. The accumulations of lutein and β-carotene were significantly different among irradiance levels for kale, but were not significantly different for spinach. However, lutein and β-carotene accumulation was significant for spinach when computed on a dry mass basis. Identifying effects of irradiance on carotenoid accumulation in kale and spinach is important information for growers producing these crops for dry capsule supplements and fresh markets.     

Changes in sodium and potassium in Nitellopsis cells treated with transient salt stress

Abstract. Nitellopsis cells grown in fresh water have a relatively low cytoplasmic Na⁺ (11 mol m⁻³) and high cytoplasmic K⁺ (90 mol m⁻³) content. A 30-min treatment with 100 mol m⁻³ external NaCl resulted in a high [Na⁺]_c (90 mol m⁻³) and a low [K⁺]_c (33 mol m⁻³), Subsequent addition of external Ca²⁺ (10 mol m⁻³) prevented Na⁺ influx and then [Na⁺]_c decreased slowly. Changes in [K⁺]_c were opposite to [Na⁺]_c. During the recovery time vacuolar Na⁺ increased, while vacuolar K⁺ decreased. Since all these processes proceeded also under ice-cold conditions, the restoration of original cytoplasmic ion compositions is suggested to be a passive nature. The notion that the passive movement of ions across the tonoplast can act as an effective and economic mechanism of salt tolerance under transient or under mild salt stress conditions is discussed.     

Holocene carbon burial by lakes in SW Greenland

The role of the Arctic in future global change processes is predicted to be important because of the large carbon (C) stocks contained in frozen soils and peatlands. Lakes are an important component of arctic landscapes although their role in storing C is not well prescribed. The area around Kangerlussuaq, SW Greenland (66–68°N, 49–54°W) has extremely high lake density, with ∼20 000 lakes that cover about 14% of the land area. C accumulation rates and standing stock (kg C m⁻²), representing late- to mid-Holocene C burial, were calculated from AMS ¹⁴C-dated sediment cores from 11 lakes. Lake ages range from ∼10 000 cal yr  bp to ∼5400 cal yr  bp , and reflect the withdrawal of the ice sheet from west to east. Total standing stock of C accumulated in the studied lakes for the last ∼8000 years ranged from 28 to 71 kg C m⁻², (mean: ∼42 kg C m⁻²). These standing stock determinations yield organic C accumulation rates of 3.5–11.5 g C m⁻² yr⁻¹ (mean: ∼6 g C m⁻² yr⁻¹) for the last 4500 years. Mean C accumulation rates are not different for the periods 8–4.5 and 4.5–0 ka, despite cooling trends associated with the neoglacial period after 4.5 ka. We used the mean C standing stock to estimate the total C pool in small lakes (<100 ha) of the Kangerlussuaq region to be ∼4.9 × 10¹³ g C. This C stock is about half of that estimated for the soil pool in this region (but in 5% of the land area) and indicates the importance of incorporating lakes into models of regional C balance at high latitudes.     

Effects of amiodarone on K+, internal pH and Ca2+ homeostasis in Saccharomyces cerevisiae

In this study, amiodarone, at very low concentrations, produced a clear efflux of K⁺. Increasing concentrations also produced an influx of protons, resulting in an increase of the external pH and a decrease of the internal pH. The K⁺ efflux resulted in an increased plasma membrane potential difference, responsible for the entrance of Ca²⁺ and H⁺, the efflux of anions and the subsequent changes resulting from the increased cytoplasmic Ca²⁺ concentration, as well as the decreased internal pH. The Δ tok1 and Δ nha1 mutations resulted in a smaller effect of amiodarone, and Δ trk1 and Δ trk2 showed a higher increase of the plasma membrane potential. Higher concentrations of amiodarone also produced full inhibition of respiration, insensitive to uncouplers and a partial inhibition of fermentation. This phenomenon appears to be common to a large series of cationic molecules that can produce the efflux of K⁺, through the reduction of the negative surface charge of the cell membrane, and the concentration of this cation directly available to the monovalent cation carriers, and/or producing a disorganization of the membrane and altering the functioning of the carriers, probably not only in yeast.     

Kinetics of net H+, Ca2+, K+, Na+, NH+4, and Cl- fluxes associated with post-chilling recovery of plasma membrane transporters in Zea mays leaf and root tissues

Although temperature-induced changes in membrane structure and activity seem to be central to chilling stress perception, the specific details of temperature effects on plant nutrient acquisition remain obscure. In this study, we have undertaken a comparative study of low temperature effects on the activity of plasma membrane transporters of different ions in corn ( Zea mays L.) leaf and root tissues by non-invasive measurements of net ion fluxes using ion-selective microelectrode (the MIFE) technique. Kinetics of net H⁺, Ca²⁺, K⁺, Na⁺,     and Cl^– fluxes were measured as plant tissues recovered after short-term (3 h) chilling stress. The major findings can be summarized as follows: (1) The critical temperatures, under which the recovery of the activity of plasma membrane transporters took place, were found to be the same for all ions measured and are likely to be associated with the phase transition of membrane lipids. (2) The most pronounced was the reduction in net uptake of K⁺ and     (3) Chilling treatment caused a significant net influx of Cl^– and Na⁺ in the leaf tissue. (4) For the same species, the critical temperatures for membrane-transport processes in roots were 2–2.5°C lower than in leaves. Possible physiological significance of these findings is discussed.     

Extracellular Calcium Has Distinct Effects on Fast and Slow Components of the Depolarization-Induced Secretory Response from Chromaffin Cells

Abstract: An increase in extracellular Ca²⁺ concentration from 0.25 to 10 m M enhanced secretion of norepinephrine and epinephrine induced by a high extracellular K⁺ concentration (75 m M ). The increment in extracellular Ca²⁺ concentration also increased the observed peak inward Ca²⁺ current in response to long (10-s) depolarizing pulses from a holding potential of −55 mV to +5 mV, from about −26 to −400 pA. However, the total amount of Ca²⁺ influx into the cell only increased when the extracellular Ca²⁺ concentration was raised from 0.25 to 1 m M and then remained constant up to 10 m M extracellular Ca²⁺. ATP is cosecreted with catecholamines following a depolarizing stimulus. Kinetic studies indicated that ATP secretion had two components with time constants, in the presence of 2.5 m M extracellular Ca²⁺, of ∼4 and 41 s, being the fast component of secretion produced by the exocytosis of ∼220 chromaffin granules. The results suggest that, for a given depolarizing stimulus, the size and rate of release for the fast and slow components of secretion are dependent on extracellular Ca²⁺ concentration.     

An electrogenic pump in the xylem parenchyma of barley roots

Analysis of an electrogenic pump in the plasma membrane of xylem-parenchyma protoplasts from barley roots was performed using the patch-clamp technique in the whole-cell configuration. Particularly with regard to understanding xylem loading and unloading, the study of the electrogenic pump from this cell type is important; its functional confirmation was lacking to date. About one-half of the investigated protoplasts displayed current responses with reversal potentials between −80 and −200 mV. The application of fusicoccin, an H⁺-pump stimulator, caused an increase in currents recorded at a membrane potential of 0 mV and a shift of the reversal potential by about −50 mV. Treatment with dicylohexylcarbodiimid, an H⁺-pump inhibitor, resulted in the reduction of the current at 0 mV. The Ca²⁺-pump inhibitor, erythrosin B, showed no effect on current density at 0 mV and on the polarisation of the membrane potential. Enlarging the transmembrane pH gradient by raising the pH of the extracellular solution from 5.8 to 8.8 stimulated the currents. These are strong indications that the electrogenic pump was an H⁺-pump. Neither intracellular pH nor the intracellular Ca²⁺ concentration affected its activity. Simultaneous activity of the electrogenic pump and anion conductances could produce states in which protoplasts exhibited 'intermediate' reversal potentials. It was concluded that the electrogenic pump was not directly involved in the loading of KCl and KNO₃ into the xylem but, in combination with anion channel activities, contributed to the establishment of membrane potentials at which electroneutral salt transport and acid release can proceed.     

Growth, contents of K+ and kinetics of K+(86Rb) uptake in barley cultured at different low supply rates of potassium

Plants of barley ( Hordeum vulgare L. cv. Salve) were grown with 6.5–35% relative increase of K⁺ supply per day (RKR) using a special computer-controlled culture unit. After a few days on the culture solution the plants adapted their relative growth rate (RGR) to the rate of nutrient supply. The roots of the plants remained in a low salt status irrespective of the rate of nutrient supply, whereas the concentration of K⁺ in shoots increased with RKR. Both V_max and K_m for K⁺(⁸⁶Rb) influx increased with RKR. It is concluded that with a continuous and stable K⁺ stress, the K⁺ uptake system is adjusted to provide an effective K⁺ uptake at each given RKR. Allosteric regulation of K⁺ influx does not occur and efflux of K⁺ is very small.