Mechanism for Cd2+ inhibition of (K++ Mg2+)ATPase activity and K+ (86Rb+) uptake join roots of sugar beet (Beta vulgaris)

Steady state kinetics were used to examine the influence of Cd²⁺ both on K⁺ stimulation of a membrane-bound ATPase from sugar beet roots (Beta vulgaris L. cv. Monohill) and on K⁺(⁸⁶Rb⁺) uptake in intact or excised beet roots. The in vitro effect of Cd²⁺ was studied both on a 12000–25000 g root fraction of the (Na⁺+K⁺+Mg²⁺)ATPase and on the ATPase when further purified by an aqueous polymer two-phase system. The observed data can be summarized as follows: 1) Cd²⁺ at high concentrations (>100 μM) inhibits the MgATPase activity in a competitive way, probably by forming a complex with ATP. 2) Cd²⁺ at concentrations <100 μM inhibits the specific K⁺ activation at both high and low affinity sites for K⁺. The inhibition pattern appears to be the same in the two ATPase preparations of different purity. In the presence of the substrate MgATP, and at K⁺ <5 mM, the inhibition by Cd²⁺ with respect to K⁺ is uncompetitive. In the presence of MgATP and K⁺ >10 μM, the inhibition by Cd²⁺ is competitive. 3) At the low concentrations of K⁺, Cd²⁺ also inhibits the 2,4-dinitrophenol(DNP)-sensitive (metabolic) K⁺(⁸⁶Rb⁺) uptake uncompetitively both in excised roots and in roots of intact plants. 4) The DNP-insensitive (non metabolic) K⁺(⁸⁶Rb⁺) uptake is little influenced by Cd²⁺. As Cd²⁺ inhibits the metabolic uptake of K⁺(⁸⁶Rb⁺) and the K⁺ activation of the ATPase in the same way at low concentrations of K⁺, the same binding site is probably involved. Therefore, under field conditions, when the concentration of K⁺ is low, the presence of Cd²⁺ could be disadvantageous.     

Effects of cadmium and lead on the accumulation of Ca2+ and K+ and on the influx and translocation of K+ in wheat of low and high K+ status

The effects of cadmium and lead on the internal concentrations of Ca²⁺ and K⁺, as well as on the uptake and translocation of K(⁸⁶Rb⁺) were studied in winter wheat (Triticum aestivum L. a. MV-8) grown hydroponically at 2 levels of K⁺ (100 uM and 10 mM). Cd²⁺ and Pb²⁺ were applied in the nutrient solution in the range of 0.3 to 1000 u.M. Growth was more severely inhibited by Cd²⁺ and in the high-K⁺ plants as compared to Pb^z+ and low-K⁺ plants. Ions of both heavy metals accumulated in the roots and shoots, but the K⁺ status influenced their levels. Ca²⁺ accumulation was increased by low concentrations of Cd²⁺ mainly in low-K⁺ shoots, whereas it was less influenced by Pb²⁺. The distribution of Cd²⁺ and Ca²⁺ in the plant and in the growth media indicated high selectivity for Cd²⁺ in the root uptake, while Ca²⁺ was preferred in the radial and/or xylem transport. Cd²⁺ strongly inhibited net K⁺ accumulation in high-K⁺ plants but caused stimulation at low K⁺ supply. In contrast, the metabolis-dependent influx of K⁺(⁸⁶Rb⁺) was inhibited in low-K⁺ plants, while the passive influx in high-K⁺ plants was stimulated. Translocation of K⁺ from the roots to the shoots was inhibited by Cd²⁺ but less influenced in Pb²⁺-treated plants. It is concluded that the effects of heavy metals depend upon the K⁺-status of the plants.     

Changes in Rb+ and Ca2+ influx in winter wheat roots before, during and after exposure to low temperature and short days

Influx of Rb⁺(⁸⁶Rb⁺) and Ca²⁺ (⁴⁵Ca²⁺) in roots of intact winter wheat (Triticum aestivum L. cv. Weibulls Starke II) was determined at intervals before, during and after exposure to cold acclimation conditions (2°C and 8 h light period). The plants were grown in nutrient medium of two ionic strengths. During the initial two weeks of growth at 16°C and 16 h light period, Rb⁺ influx into roots decreased with increasing age, probably as a consequence of a decreasing proportion of metabolically active roots. The presence of 10^?4M 2,4-dinitrophenol (DNP) reduced Rb⁺ influx to a low and constant level, indicating that metabolic influx was the dominant process. In contrast, Ca²⁺ influx in plants grown in full strength nutrient solution was higher in the presence than in the absence of DNP. This effect may have been due to an active extrusion mechanism mediating re-export of absorbed Ca²⁺(⁴⁵Ca²⁺) during the uptake experiment. With the metabolic uncoupler inhibiting such extrusion the Ca²⁺(⁴⁵Ca²⁺) influx mesured would increase. During cold treatment, Rb⁺ influx remained at a low level, and was further decreased when DNP was present in the uptake solution. This effect may have been due to inhibition of residual active influx of Rb⁺ at 2°C by the uncoupler and/or to a decrease in membrane permeability. In contrast to Rb⁺, Ca²⁺ influx increased during cold treatment, which could again be explained as inhibition of re-export. The presence of DNP reduced Ca²⁺ influx at 2°C, indicating decreased membrane permeability by DNP at low temperature. After transfer of plants from cold acclimation conditions to 16°C, Rb⁺ and Ca²⁺ influx increased in plants grown at both ionic strengths. Influx levels were independent of the length of the cold acclimation period (1, 6 and 8 weeks), but the patterns were different for the two ions. After each of the cold acclimation periods, Rb⁺ influx increased during the first week and decreased or remained at the same level during the second week, while Ca²⁺ influx always decreased during the second week of post-cold treatment.     

Control of K+ (Rb+) influx and transport with changed internal K+ concentration and age in two varieties of barley

The influx of Rb⁺ into the roots of two barley varieties (Hordeum vulgare L. cv. Salve and cv. Ingrid) from a K⁺-free ⁸⁶Rb-labelled nutrient solution with 2.0 mM Rb⁺, was checked at intervals from day 6 to day 18. The control plants were continuously grown in complete nutrient solution containing 5.0 mM K⁺, while two other groups of plants were grown in K⁺-free nutrient solution starting on day 6 and between day 6 and day 9, respectively. The pattern of Rb⁺ influx was similar for both varieties, although their efficiencies in absorbing Rb⁺ were different. The relationship between Rb⁺ influx and K⁺ concentration of the root could be interpreted in terms of negative feedback through allosteric control of uptake across the plasmalemma of the root cells. Hill plots were bimodal, but in the opposite direction. The Hill coefficients, reflecting the minimum number of interacting allosteric binding sites for K⁺ (Rb⁺), were low (≤–3.0). It is discussed whether the threshold value, that is the breaking point in the Hill plot, is indicative of a changed efficiency of transporting units for K⁺ (Rb⁺) transport to the xylem. Moreover, feedback regulation might be involved in transport of K⁺ between root and shoot. The variation in K⁺ concentrations in the roots and shoots of control plants were cyclic but in phase opposition despite an exponential growth. The average K⁺ concentration varied only slightly with age.     

High salinity tolerance in the stl2 mutation of Ceratopteris richardii is associated with enhanced K+ influx and loss

The roles of K⁺ uptake and loss in the salinity response of the wild type and the salt-tolerant mutant stl2 of Ceratopteris richardii were studied by measuring Rb⁺ influx and loss and the effects of Na⁺, Mg²⁺, Ca²⁺ and K⁺-transport inhibitors. In addition, electrophysiological responses were measured for both K⁺ and Rb⁺ and for the effects of Na⁺ and NH₄⁺ on subsequent K⁺-induced depolarizations. stl2 had a 26–40% higher uptake rate for Rb⁺ than the wild type at 0.5–10 mol m^?3 RbCl. Similarly, membrane depolarizations induced by both RbCl and KCl were consistently greater in stl2. In the presence of 0–180 mol m^?3 NaCl, stl2 maintained a consistently greater Rb⁺ influx than the wild type. stl2 retained a greater capacity for subsequent KCl-induced depolarization following exposure to NaCl. Five mol m^?3 Mg²⁺ decreased Rb⁺ uptake in stl2; however, additional Mg²⁺ up to 40 mol m^?3 did not affect Rb⁺ uptake further. Ca²⁺ supplementation resulted in a very minor decrease of Rb⁺ uptake that was similar in the two genotypes. Tetraethylammonium chloride and CsCl gave similar inhibition of Rb⁺ uptake in both genotypes, but NH₄Cl gave substantially greater inhibition in the wild type than in stl2. NH₄Cl resulted in a greater membrane depolarization in the wild type and the capacity for subsequent depolarization by KCl was markedly reduced. stl2 exhibited a higher Independent loss of Rb⁺ than the wild type, but, in the absence of external K⁺, loss of Rb⁺ was equivalent in the two genotypes. Since constitutive K⁺ contents are nearly identical, we conclude that high K⁺ influx and loss exact a metabolic cost that is reflected in the inhibition of gametophytic growth. Growth inhibition can be alleviated by reduced supplemental K⁺ or by treatments that slightly reduce K⁺ influx, such as moderate concentrations of Na⁺ or Mg²⁺. We propose that high throughput of K⁺ allows maintenance of cytosolic K⁺ under salt stress and that a high uptake rate for K⁺ results in a reduced capacity for the entrance and accumulation of alternative cations such as Na⁺ in the cytosol.     

Influence of low temperature on regulation of Rb+ and Ca2+ influx in roots of winter wheat

Influx of Rb⁺(⁸⁶Rb⁺) and Ca²⁺(⁴⁵Ca²⁺) was determined in roots of winter wheat (Triticum aestivum L. cv. Weibulls Starke II) after 14 days at 16°C/16 h light, after 1 and 8 weeks of cold acclimation (2°C/8 h light) and at intervals after deacclimation (16°C/16 h light) for up to 14 days. The plants were cultivated at 3 ionic strengths: 100, 10 and 1% of a full strength nutrient solution, containing 3.0 mM K⁺ and 1.0 mM Ca²⁺. K⁺ concentrations in roots and shoots increased during cold treatment, while Ca²⁺ in the roots decreased. In the shoots Ca²⁺ concentrations remained the same. Influx of Rb⁺ as a function of average K⁺ concentration in the roots of 14-day-old, non-cold-treated plants was high at a certain K⁺ level in the root and decreased at higher root K⁺ levels (negative feedback). The pattern for Ca²⁺ influx versus average concentration of Ca²⁺ in the root was the reverse. Independent of duration of treatment (1–8 weeks), cold acclimation partly changed the regulation of Rb⁺ influx, so that it became less dependent upon negative feedback and more dependent on the ionic strength of the cultivation solution. After exposure to 2°C, Ca²⁺ influx increased at high Ca²⁺ concentrations in the root as compared with influx in roots of 14-day-old non-cold-treated plants. Under deacclimation, Ca²⁺ influx gradually decreased again, and reached the level observed before cold treatment within 7–14 days at 16°C; the number of days depending on the exposure time at 2°C. It is suggested that Rb⁺(K⁺) influx became adjusted to low temperature and that abscisic acid (ABA) may be involved in this mechanism. It is also suggested that extrusion of Ca²⁺ was impaired and/or Ca²⁺ channels were activated at 2°C in roots of plants grown in the full-strength solution and that extrusion was gradually restored and/or Ca²⁺ channels were closed under deacclimation conditions.     

Interactions between K+ and NH4+: effects on ion uptake by rice roots

Interactive effects of K⁺ and N (principally NH₄⁺) on plant growth and ion uptake were investigated using hydroponically grown rice (Oryza sativa L. cv. M202) seedlings by varying the availability of NH₄⁺ or NO₃^? and K⁺ during an 18d growth period, a 3d pretreatment period and during flux measurements. Plants grew best in media containing 100 mmol m^?3 NH₄⁺ and 200mmolm^?3 K⁺ (N100/K200), followed by N2/K200 < N100/K2 < N2/K2. ⁸⁶Rb⁺(K⁺) fluxes were increased by exposure to N during the 18 d growth period and the 3 d of pretreatment, but decreased by the presence of NH₄⁺ during flux measurements. This inhibition was a function of prior N/K provision and the [NH₄⁺]₀ present during flux determinations. NH₄⁺ was least inhibitory to 86Rb⁺(K⁺) influx in high-N/low-K plants. Pretreatments with K⁺ failed to stimulate NH₄⁺ uptake, and the presence of K⁺ in the uptake solutions reduced NH₄⁺ fluxes only in high-N/low-K plants.     

Compartmental analysis of efflux of K+(86Rb+) and Rb+(86Rb+) from roots of intact plants of barley (Hordeum vulgare) of high and low K+ status, and after excision of the shoot

The classic compartment analysis of ion efflux from roots is often applied with the assumption that there is a system of 3 compartments in series. However, complex ion transport across the root tissues, as well as influences from the shoot, may complicate the picture. The present experiments were performed to study the immediate effects that excision of the shoot before the experiment exerts on the efflux of Rb⁺(⁸⁶Rb⁺) and of K⁺(⁸⁶Rb⁺) from 9-day-old roots of plants of barley (Hordeum vulgare L. cv. Salve). The efflux from high K⁺ and low K⁺ roots of intact and detopped plants were compared. After excision of the shoot of high K⁺ plants, a marked increase in efflux was observed after 2.5 h with a maximum at about 7 h. The increase in efflux was seen as a peak in plots of efflux versus time. Excision of the shoot from low K⁺ roots did not give rise to a consistent increase in efflux. Regular K⁺ ion efflux curves were observed from roots of intact plants of high or low K⁺ status. Furthermore, after a pulse treatment of 9-day-old roots of intact plants of high or low K⁺ status with a solution containing Rb⁺(⁸⁶Rb⁺), the Rb⁺(⁸⁶Rb⁺) transport to the shoots was not reduced during the following 3 h in unlabelled solution. It is suggested that both the peak appearing in the efflux plots and the maintained tracer transport to the shoots after transfer of the roots to an unlabelled solution indicate the existence of a K⁺/Rb⁺ transport system in the symplasm of the roots that has only a slow exchange with the bulk cytoplasm and vacuoles.     

K+(Rb+) and Ca2+ fluxes in young winter wheat plants exposed to sub-zero temperatures

Ice crystal formation temperature was determined in the region of the crown in one group of 7-day-old intact unhardened high-salt plants of winter wheat (Triticum aestivum L. cv. Weibulls Starke II) with TA (Thermal Analysis) and DTA (Differential Thermal Analysis) methods. After exposure of another group of plants, grown for the first 7 days in the same way as the first group, to various sub-zero temperatures (-1 to 5°C), influx in roots of Rb⁺(⁸⁶Rb⁺) and Ca²⁺(⁴⁵Ca²⁺) and contents of K⁺ and Ca²⁺ were determined at intervals during 7 days of recovery. Ice crystal formation in the crown tissue was probably extracellular and took place at about -4°C. There was a large loss of K⁺ from the roots after treatment at sub-zero temperatures. This loss increased as the temperature of the sub-zero treatment decreased. During recovery, roots of plants exposed to -1, -2 and -3°C gradually reabsorbed K⁺. Reabsorption of K⁺ in roots of plants exposed to -4°C was greatly impaired. Rb⁺ influx decreased and Ca²⁺ influx increased after sub-zero temperature treatments of the plants. Active Rb⁺ influx mechanisms and active extrusion of Ca²⁺ were impaired or irreversibly damaged by the exposure. While Rb⁺ influx mechanisms were apparently repaired during recovery in plants exposed to temperatures down to -3°C, Ca²⁺ extrusion mechanisms were not. The temperature for ice crystal formation in the region of the crown tissue coincides with the temperature at which the plants lost the ability to reabsorb K⁺ and to repair Rb⁺ influx mechanisms during the recovery period. Plants were lethally damaged at temperatures below ?4°C.     

Differential effects of Na+, Mg2+, K+, Ca2+ and osmotic stress on the wild type and the NaCl-tolerant mutants stl1 and stl2 of Ceratopteris richardii

In order to identify physiological components that contribute to salinity tolerance, we compared the effects of Na⁺, Mg²⁺ and K⁺ salts (NaCl, Na₂SO₄, MgCl₂, MgSO₄, KCl and K₂SO₄), Ca₂₊ (CaSO₄), mannitol and melibiose on the wild type and the single-gene NaCl-tolerant mutants stl1 and stl2 of Ceratopteris richardii. Compared with gametophytic growth of the wild type, stl2 showed a low level of tolerance that was restricted to Na⁺ salts and osmotic stress. stl2 exhibited high tolerance to both Na⁺ and Mg²⁺ salts, as well as to osmotic stress. In response to short-term exposure (3 d) to NaCl, accumulation of K⁺ and Na⁺ was similar in the wild type and stl1. In contrast, stl2 accumulated higher levels of K⁺ and lower levels of Na⁺. Ca²⁺ supplementation (1.0 mol m^?3) ameliorated growth inhibition by Na⁺ and Mg²⁺ stress in wild type and stll, but not in stl2. In addition, under Na⁺ stress (175 mol m^?3) wild-type, stll and stl2 gametopbytes maintained higher tissue levels of K⁺ and lower levels of Na⁺ when supplemented with Ca²⁺ (1.0 mol m^?3). stl2 gametophytes were extremely sensitive to K⁺ supplementation. Growth of stl2 was greater than or equal to that of the wild type at trace concentrations of K⁺ but decreased substantially with increasing K⁺ concentration. Supplementation with K⁺ from 0 to 1.85 mol m^?3 alleviated some of the inhibition by 75 mol m^?3 NaCl in the wild type and in stl1. In stl2, growth at 75 mol m^?3 NaCl was similar at 0 and 1.85 mol m^?3 K⁺ supplementation. Although K⁺ supplementation above 1.85 mol m^?3 did not alleviate inhibition of growth by Na⁺ in any genotype, stl2 maintained greater relative tolerance to NaCl at all K⁺ concentrations tested.     

Effects of Monovalent and Divalent Cations on Ca2+ Fluxes Across Chromaffin Secretory Membrane Vesicles

Abstract: Bovine chromaffin secretory vesicle ghosts loaded with Na⁺ were found to take up Ca²⁺ when incubated in K⁺ media or in sucrose media containing micromolar concentrations of free Ca²⁺. Li⁺- or choline⁺loaded ghosts did not take up Ca²⁺. The Ca²⁺ accumulated by Na⁺-loaded ghosts could be released by the Ca²⁺ ionophore A23187, but not by EGTA. Ca²⁺ uptake was inhibited by external Sr²⁺, Na ⁺, Li ⁺, or choline ⁺. All the ⁴⁵Ca²⁺ accumulated by Na⁺-dependent Ca²⁺ uptake could be released by external Na ⁺, indicating that both Ca²⁺ influx and efflux occur in a Na⁺-dependent manner. Na ⁺ -dependent Ca²⁺ uptake and release were only slightly inhibited by Mg²⁺. In the presence of the Na⁺ ionophore Monensin the Ca²⁺ uptake by Na ⁺-loaded ghosts was reduced. Ca²⁺ sequestered by the Na⁺-dependent mechanism could also be released by external Ca²⁺ or Sr²⁺ but not by Mg²⁺, indicating the presence of a Ca²⁺/Ca²⁺ exchange activity in secretory membrane vesicles. This Ca²⁺/Ca²⁺ exchange system is inhibited by Mg²⁺, but not by Sr²⁺. The Na ⁺ -dependent Ca²⁺ uptake system in the presence of Mg²⁺ is a saturable process with an apparent K_m of 0.28 μM and a V_max= 14.5 nmol min^?1 mg protein^?1. Ruthenium red inhibited neither the Na⁺/Ca²⁺ nor the Ca²⁺/Ca²⁺ exchange, even at high concentrations.     

Effects of interrupted K+ supply on growth and uptake of K+, Ca2+, Mg2+ and Na+ in spring wheat

Effects of interrupted K⁺ supply on different parameters of growth and mineral cation nutrition were evaluated for spring wheat (Triticum aestivum L. cv. Svenno). K⁺ (2.0 mM) was supplied to the plants during different periods in an otherwise complete nutrient solution. Shoot growth was reduced before root growth after interruption in K⁺ supply. Root structure was greatly affected by the length of the period in K⁺ -free nutrient solution. Root length was minimal, and root branching was maximal within a narrow range of K⁺ status of the roots. This range corresponded to cultivation for the last 1 to 3 days, of 11 in total, in K⁺ -free nutrient solution, or to continuous cultivation in solution containing 0.5 to 2 mM K⁺. In comparison, both higher and lower internal/external K⁺ concentrations had inhibitory effects on root branching. However, the differing root morphology probably had no significant influence on the magnitude of Ca²⁺, Mg²⁺ and Na⁺ uptake. Uptake of Ca²⁺ and especially Mg²⁺ significantly increased after K⁺ interruption, while Na⁺ uptake was constant in the roots and slowly increased in the shoots. The two divalent cations could replace K⁺ in the cells and maintain electroneutrality down to a certain minimal range of K⁺ concentrations. This range was significantly higher in the shoot [110 to 140 μmol (g fresh weight)^?1] than in the root [20 to 30 μmol (g fresh weight)^?1]. It is suggested that the critical K⁺ values are a measure of the minimal amount of K⁺ that must be present for physiological activity in the cells. At the critical levels, K⁺ (⁸⁶Rb) influx and Ca²⁺ and Mg²⁺ concentrations were maximal. Below the critical K⁺ values, growth was reduced, and Ca²⁺ and Mg²⁺ could no longer substitute for K⁺ for electrostatic balance. In a short-term experiment, the ability of Ca²⁺ to compete with K⁺ in maintaining electroneutrality in the cells was studied in wheat seedlings with different K⁺ status. The results indicate that K⁺, which was taken up actively and fastest at the external K⁺ concentration used (2.0 mM), partly determines the size of Ca²⁺ influx.     

Involvement of Ca2+-calmodulin in Cd2+ toxicity during the early phases of radish (Raphanus sativus L.) seed germination

The toxicity of Cd²⁺in vivo during the early phases of radish (Raphanus sativus L.) seed germination and the in vitro Cd²⁺ effect on radish calmodulin (CaM) were studied. Cd²⁺ was taken up in the embryo axes of radish seeds; the increase in fresh weight of embryo axes after 24 h of incubation was inhibited significantly in the presence of 10 mmol m^?3 Cd²⁺ in the external medium, when the Cd²⁺ content in the embryo axes was c. 1.1 μmol g^?1 FW. The reabsorption of K⁺, which characterizes germination, was inhibited by Cd²⁺, suggesting that Cd²⁺ affected metabolic reactivation. The slight effect of Cd²⁺ on the transmembrane electric potential of the cortical cells of the embryo axes excluded a generalized toxicity of Cd²⁺ at the plasma membrane level. After 24 h of incubation, Cd²⁺ induced no increase in total acid-soluble thiols and Cd²⁺-binding peptides able to reduce Cd²⁺ toxicity. Ca²⁺ added to the incubation medium partially reversed the Cd²⁺-induced inhibition of the increase in fresh weight of embryo axes and concomitantly reduced Cd²⁺ uptake. Equilibrium dialysis experiments indicated that Cd²⁺ bound to CaM and competed with Ca²⁺ in this binding. Cd²⁺ inhibited the activation of Ca²⁺-CaM-dependent calf-brain phosphodiesterase, inhibiting the Ca²⁺-CaM active complex. Cd²⁺ reduced the binding of CaM to the Ca²⁺-CaM binding enzymes present in the soluble fraction of the embryo axes of radish seeds. The possibility that Cd²⁺ toxicity in radish seed germination is mediated by the action of Cd²⁺ on Ca²⁺-CaM is discussed in relation to the in vivo and in vitro effects of Cd²⁺.     

Aluminium interactions with K+(86Rb+) and 45Ca2+ fluxes in three cultivars of sugar beet (Beta vulgaris)

Three cultivars of sugar beet (Beta vulgaris L.), which are sensitive to aluminium (Al) in the order Primahill > Monohill > Regina, were grown in water culture for 2 weeks. Nutrients were supplied at 15% increase of amounts daily, corresponding to the nutrient demand for maximal growth. The 2.4-dinitrophenol (DNP)-sensitive (metabolic) and DNP-insensitive (non-metabolic) uptake of aluminium, phosphate. ⁴⁵Ca²⁺ and K⁺(⁸⁶Rb⁺) in roots were measured as well as transport to shoots of intact plants. All 3 cultivars absorbed more aluminium if DNP was present during the aluminium treatment than in its absence. It is suggested that sugar beets are able to extrude aluminium activity or that they possess an active mechanism to keep Al outside the cell. The presence of Al in the medium during the 1-h experiment affected the metabolic and non-metabolic fluxes of ⁴⁵Ca²⁺ and K⁺(⁸⁶Rb⁺) in different ways. In the presence of DNP, the influx of both ⁴⁵Ca²⁺ and K⁺(⁸⁶Rb⁺) and the efflux of ⁴⁵Ca²⁺ were inhibited by Al in a competitive way. At inhibition of ⁴⁵Ca²⁺ influx, 2 Al ions are probably bound per Ca²⁺ uptake site in cv. Regina (Al-tolerant), but in cvs Primahill and Monohill only one Al ion is bound (more Al sensitive). Aluminium competitively inhibited the active efflux of ⁴⁵Ca²⁺ (absence of DNP) in almost the same way in the 3 cultivars. In contrast, aluminium stimulated the influx of K⁺(⁸⁶Rb⁺) in cvs Primahill, Monohill and Regina in the absence of DNP. Thus, the Al effects on active and passive K⁺(⁸⁶Rb⁺) influx are different. The total influx of K⁺(⁸⁶Rb⁺) increased in the presence of Al and might be connected to an active exclusion of Al. Regina is the least Al-sensitive cultivar, probably because Al interferes less with the Ca²⁺ fluxes and because this cultivar actively excludes phosphate in the presence of Al. Thus Al-phosphate precipitation within the plant could be avoided.     

Effects of Cations on (Ca2++ Mg2+)-Activated ATPase from Rat Brain

Abstract: With a partially purified, membrane-bound (Ca + Mg)-activated ATPase preparation from rat brain, the K_0.5 for activation by Ca²⁺ was 0.8 p μm in the presence of 3 mm -ATP, 6 mm -MgCl₂, 100 m_M-KCI, and a calcium EGTA buffer system. Optimal ATPase activity under these circumstances was with 6-100 μm -Ca²⁺, but marked inhibition occurred at higher concentrations. Free Mg²⁺ increased ATPase activity, with an estimated K_0.5, in the presence of 100 μm -CaCl₂, of 2.5 mm ; raising the MgCl₂ concentration diminished the inhibition due to millimolar concentrations of CaCl₂, but antagonized activation by submicromolar concentrations of Ca²⁺. Dimethylsulfoxide (10%, v/v) had no effect on the K_0.5 for activation by Ca²⁺, but decreased activation by free Mg²⁺ and increased the inhibition by millimolar CaCl₂. The monovalent cations K⁺, Na⁺, and TI⁺ stimulated ATPase activity; for K⁺ the K_0.5 was 8 mm , which was increased to 15 mm in the presence of dimethylsulfoxide. KCI did not affect the apparent affinity for Ca²⁺ as either activator or inhibitor. The preparation can be phosphorylated at 0°C by [γ-³²P]-ATP; on subsequent addition of a large excess of unlabeled ATP the calcium dependent level of phosphorylation declined, with a first-order rate constant of 0.12 s^?1. Adding 10 mm -KCI with the unlabeled ATP increased the rate constant to 0.20 s^?1, whereas adding 10 mm -NaCl did not affect it measurably. On the other hand, adding dimethyl-sulfoxide slowed the rate of loss, the constant decreasing to 0.06 s^?1. Orthovanadate was a potent inhibitor of this enzyme, and inhibition with 1 μm -vanadate was increased by both KCI and dimethylsulfoxide. Properties of the enzyme are thus reminiscent of the plasma membrane (Na + K)-ATPase and the sarcoplasmic reticulum (Ca + Mg)-ATPase, most notably in the K⁺ stimulation of both dephosphorylation and inhibition by vanadate.     

Distribution of NO3− reduction between roots and shoots of peachtree seedlings as affected by NO3− uptake rate

The distribution of NO₃^? reduction between roots and shoots was studied in hydro-ponically-grown peach-tree seedlings (Prunus persica L.) during recovery from N starvation. Uptake, translocation and reduction of NO₃^?, together with transport through xylem and phloem of the newly reduced N were estimated, using ¹⁵N labellings, in intact plants supplied for 90 h with 0.5 mM NH₄⁺ and 0.5, 1.5 or 10 mM NO₃^?. Xylem transport of NO₃^? was further investigated by xylem sap analysis in a similar experiment. The roots were the main site of NO₃^? reduction at all 3 levels of NO₃^? nutrition. However, the contribution of the shoots to the whole plant NO₃^? reduction increased with increasing external NO₃^? availability. This contribution was estimated to be 20, 23 and 42% of the total assimilation at 0.5, 1.5 and 10 mM NO₃^?, respectively. Both ¹⁵N results and xylem sap analysis confirmed that this trend was due to an enhancement of NO₃^? translocation from roots to shoots. It is proposed that the lack of NO₃^? export to the shoots at low NO₃^? uptake rate resulted from a competition between NO₃^? reduction in the root epidermis/cortex and NO₃^? diffusion to the stele. On the other hand, net xylem transport of newly reduced N was very efficient since ca 70% of the amino acids synthesized in the roots were translocated to the shoots, regardless of the level of NO₃^? nutrition. This net xylem transport by far exceeded the net downward phloem transport of the reduced N assimilated in shoots. As a consequence, the reduced N resulting from NO₃^? assimilation, principally occurring in the roots, was mainly incorporated in the shoots.     

Effects of ionic strength and relative humidity on the efflux of K+ (86Rb) and Ca2+ (45Ca) from roots of intact seedlings of cucumber, oat and wheat

Spring wheat (Triticum aestivum L. cv. Svenno), oat (Avena sativa L. cv. Brighton) and glasshouse cucumber (Cucumis sativus L. cv. Bestseller F1) were cultured for a week after germination on complete nutrient solutions of three different dilutions (1, 25 and 50% of the full strength medium). K⁺(⁸⁶Rb) and ⁴⁵Ca were present during the whole culture period. Relative humidity (RH) was 50% except during the last day, when half the material was transferred to 90% RH. Efflux of labelled ions was then followed during eight hours on unlabelled solutions of the same composition as before, and at both 50% and 90% RH in the atmosphere. – Uptake of K⁺(⁸⁶Rb) during growth tended to be saturated in the 25% medium. Contrariwise, the level of Ca²⁺ in the roots increased continuously with strength of the medium. At low concentrations cucumber roots were higher in Ca²⁺ than roots of oat or wheat, whereas all three species showed similar levels of Ca²⁺ in 50% medium. – At the lowest ionic strength, smooth efflux curves were obtained that could be resolved according to the three-compartment theory. At higher ionic strength, irregularities were observed, and more for Ca²⁺ than for K⁺; but for practical purposes compartment analysis with the same time constants could be applied as for the lowest concentration. – Discrimination between K⁺ and Rb⁺ differed between the roots, but not much between the shoots of different species. The roots of oat and wheat took up Rb⁺ preferentially over K⁺ in the 25% and 50% media; whereas K⁺ was preferred over Rb⁺ or little discrimination made in 1% medium and for cucumber. The shoots generally showed less discrimination than the roots. The main variability in discrimination between K⁺ and Rb⁺ thus appears to be localized in the tonoplasts of the roots cells. – Low RH around the shoots increased efflux of K⁺(⁸⁶Rb) from the cytoplasm and vacuoles of the root cells as compared to the efflux at high RH. DNP (2,4-dinitrophenol) in the medium had the same effect as high RH around the shoots. The signal system that must exist between shoots and roots is discussed as a response to “drought” conditions. In relation to investigations of others, it is assumed that the effect of DNP may indicate that part of the chain between roots and shoots consists of metabolically influenced sites, whose output is influenced by the rate of water transport.     

The use of reconstitution and inhibitor/ion interaction assays to distinguish between Ca2+/H+and Cd2+/H+ antiporter activities of oat and tobacco tonoplast vesicles

Tonoplast, ion antiport activities are critical to ion homeostasis and sequestration in plants. The biochemical properties of these activities, and the enzymes that catalyse them, are little characterized. Here we applied biochemical approaches to study some characteristics and to distinguish between Ca²⁺/H⁺ and Cd²⁺/H⁺ antiporter activities of tonoplast vesicles from non‐transformed, wild‐type plants. Solubilization and reconstitution of oat‐seedling (Avena sativa L.) root tonoplast vesicles resulted in about a 6‐fold loss of protein, about a 6‐fold enhancement of Cd²⁺/H⁺ antiport specific activity (at 10 µM Cd²⁺), and almost complete loss of Ca²⁺/H⁺ antiport activity. Similar results were found for vesicles from mature tobacco (Nicotiana tabacum) roots. Cd²⁺ concentration‐dependent proton efflux was similar and linear with both oat vesicles and proteoliposomes. In contrast, Ca²⁺ concentration‐dependent proton efflux of oat vesicles was easily observed while that with proteoliposomes was minimal and non‐linear. Cd²⁺ pre‐treatment of oat vesicles reduced verapamil inhibition of Cd²⁺/H⁺ activity and verapamil binding to vesicles, while Ca²⁺ pre‐treatment was much less protective of Ca^2+/H⁺ activity and verapamil binding. Results show the usefulness of reconstitution, and also inhibitor/ion interaction assays for distinguishing between transporter activities in vitro, but they do not resolve the question of whether there are separate enzymes for Cd²⁺/H⁺ and Ca²⁺/H⁺. Our observation that solubilization and reconstitution have similar effects on both Cd²⁺/H⁺ and Ca²⁺/H⁺ activities of root tonoplast vesicles from immature oat and mature tobacco roots suggests that the transporters involved are similar in young and mature roots, and in roots of different species.     

The effect of divalent cations on Na+ tolerance in Charophytes. I: Char a buckellii

Abstract The comparative Na⁺ tolerance of Chora buckellii cultured in freshwater (FW) or artificial Waldsea water (AWW, which contains about 110 mol m^?3 each Na ⁺, Mg²⁺, Cl^? and SO^2-₄ was tested with respect to the external Na⁺ to Ca²⁺ ratio (Na: Ca). Fifty per cent of FW cells subjected to 70 mol m^?3 NaCl, which raised Na:Ca from 10: 1 to 700: 1 and the external osmotic pressure from 0.024 to 0.402 MPa, died within 6 d. Death was associated with the loss of Na/K selectivity, H⁺ -pump activity and turgor. Restoration of Na:Ca to 10:1 in high Na⁺ medium with CaCl₂ ensured 100% survival and maintained H⁺-pump activity and Na/K selectivity of FW cells. Turgor was regulated within 3 d with net uptake of Na ⁺, K⁺ and Cl^? in the vacuolc. Mg²⁺ was not as effective as Ca²⁺ in enhancing survival or maintaining H⁺ -pump activity and Na/K selectivity of FW cells in the presence of elevated Na⁺. However, turgor was regulated within 3 d by accumulation of Cl^? and an unknown cation in the vacuole. All AWW cells subjected to an increase of 70 mol m ^?3 NaCl, which raised Na: Ca from 16:1 to 25: 1 and the external osmotic pressure from 0.915 to 1.22 MPa, survived and maintained H ⁺ -pump activity. Turgor was regulated within 6d by accumulating Na ⁺, K⁺ and Cl^? in the vacuole. All AWW cells subjected to 70molm^?3 NaCl in a medium in which Na:Ca was equal to 700:1 survived and maintained H ⁺ -pump activity, but showed loss of Na/K selectivity. Turgor was regulated with an unknown osmoticum(a) within 6 d.     

Growth and Major Nutrient Concentrations in Brassica campestris Supplied with Different NH4^＋/NO3 Ratios

In the present study, we investigated whether growth and main nutrient ion concentrations of cabbage （Brassica campestris L.） could be increased when plants were subjected to different NH4^＋/NO3- ratios. Cabbage seedlings were grown in a greenhouse in nutrient solutions with five NH4^＋/NO3- ratios （1：0; 0.75：0.25; 0.5：0.5; 0.25：0.75; and 0：1）. The results showed that cabbage growth was reduced by 87% when the proportion of NH4^＋-N in the nutrient solution was more than 75% compared with a ratio NH4^＋/NO3- of 0.5：0.5 35 d after transplanting, suggesting a possible toxicity due to the accumulation of a large amount of free ammonia in the leaves. When the NH4＋/NO3- ratio was 0.5：0.5, fresh seedling weight, root length, and H2PO4- （P）, K^＋, Ca^2＋, and Mg^2＋ concentrations were all higher than those in plants grown under other NH4^＋/NO3- ratios. The nitrate concentration in the leaves was the lowest in plants grown at 0.5： 0.5 NH4^＋/NO3-. The present results indicate that an appropriate NH4^＋/NO3- ratio improves the absorption of other nutrients and maintains a suitable proportion of N assimilation and storage that should benefit plant growth and the quality of cabbage as a vegetable.