Voltage-dependent Ca2+ influx into right-side-out plasma membrane vesicles isolated from wheat roots: characterization of a putative Ca2+ channel.

We report on the identification of a voltage-dependent Ca2+ transport system that mediates Ca2+ influx across the plasma membrane (PM) of wheat (Triticum aestivum) root cells. The experimental approach involved the imposition of transmembrane electrical potentials (via K+ diffusion potentials) in populations of purified, right-side-out PM vesicles isolated from wheat roots. Using 45Ca2+ to quantify Ca2+ influx into the PM vesicles, the voltage-dependent characteristics of Ca2+ transport were found to be similar to those exhibited by L-type voltage-gated Ca2+ channels in animal cells. The putative PM Ca2+ channel opened upon depolarization of the membrane potential, and Ca2+ flux increased to a maximum upon further depolarization and then decreased back to zero upon further successive depolarizations. This channel was found to be selective for Ca2+ over Mg2+, Sr2+, K+, and Na+; was blocked by very low concentrations of La3+; was unaffected by high concentrations of the K+ channel blocker tetraethylammonium; and exhibited Michaelis-Menten-type transport kinetics. Based on these transport properties, we argue that this transport system is a PM Ca2+ channel. We suggest that the use of radiotracer flux analysis of voltage-clamped PM vesicles derived from plant roots is a straightforward approach for the characterization of certain voltage-gated ion channels functioning in cellular membranes of higher plant cells.

Aluminum Effects on Calcium (45Ca2+) Translocation in Aluminum-Tolerant and Aluminum-Sensitive Wheat (Triticum aestivum L.) Cultivars (Differential Responses of the Root Apex versus Mature Root Regions).

The influence of Al exposure on long-distance Ca2+ translocation from specific root zones (root apex or mature root) to the shoot was studied in intact seedlings of winter wheat (Triticum aestivum L.) cultivars (Al-tolerant Atlas 66 and Al-sensitive Scout 66). Seedlings were grown in 100 [mu]M CaCl2 solution (pH 4.5) for 3 d. Subsequently, a divided chamber technique using 45Ca2+-labeled solutions (100 [mu]M CaCl2 with or without 5 or 20 [mu]M AlCl3, pH 4.5) was used to study Ca2+ translocation from either the terminal 5 to 10 mm of the root or a 10-mm region of intact root approximately 50 mm behind the root apex. The Al concentrations used, which were toxic to Scout 66, caused a significant inhibition of Ca2+ translocation from the apical region of Scout 66 roots. The same Al exposures had a much smaller effect on root apical Ca2+ translocation in Atlas 66. When a 10-mm region of the mature root was exposed to 45Ca2+, smaller genotypic differences in the Al effects effects on Ca2+ translocation were observed, because the degree of Al-induced inhibition of Ca2+ translocation was less than that at the root apex. Exposure of the root apex to Al inhibited root elongation by 70 to 99% in Scout 66 but had a lesser effect (less than 40% inhibition) in Atlas 66. When a mature root region was exposed to Al, root elongation was not significantly affected in either cultivar. These results demonstrate that genotypic differences in Al-induced inhibition of Ca2+ translocation and root growth are localized primarily in the root apex. The pattern of Ca2+ translocation within the intact root was mainly basipetal, with most of the absorbed Ca2+ translocated toward the shoot. A small amount of acropetal Ca2+ translocation from the mature root regions to the apex was also observed, which accounted for less than 5% of the total Ca2+ translocation within the entire root. Because Ca2+ translocation toward the root apex is limited, most of the Ca2+ needed for normal cellular function in the apex must be absorbed from the external solution. Thus, continuous Al disruption of Ca2+ absorption into cells of the root apex could alter Ca2+ nutrition and homeostasis in these cells and could play a pivotal role in the mechanisms of Al toxicity in Al-sensitive wheat cultivars.

Aluminum effects on calcium fluxes at the root apex of aluminum-tolerant and aluminum-sensitive wheat cultivars.

The role of Ca(2+) transport in the mechanism of Al toxicity was investigated, using a Ca(2+)-selective microelectrode system to study Al effects on root apical Ca(2+) fluxes in two wheat (Triticum aestivum L.) cultivars: Al-tolerant Atlas 66 and Al-sensitive Scout 66. Intact 3-day-old low-salt-grown (100 micromolar CaCl(2), pH 4.5) wheat seedlings were used, and it was found that both cultivars maintained similar rates of net Ca(2+) uptake in the absence of Al. Addition of Al concentrations that were toxic to Scout (5-20 micromolar AlCl(3)) immediately and dramatically inhibited Ca(2+) uptake in Scout, whereas Ca(2+) transport in Atlas was relatively unaffected. The Al-induced inhibition of Ca(2+) uptake in Scout 66 was rapidly reversed following removal of Al from the solution bathing the roots. Similar studies with morphologically intact root cell wall preparations indicated that the Al effects did not involve Al-Ca interactions in the cell wall. These results suggest that Al inhibits Ca(2+) influx across the root plasmalemma, possibly via blockage of calcium channels. The differential effect of Al on Ca(2+) transport in Al-sensitive Scout and Al-tolerant Atlas suggests that Al blockage of Ca(2+) channels could play a role in the cellular mechanism of Al toxicity in higher plants.

Aluminum effects on the kinetics of calcium uptake into cells of the wheat root apex : Quantification of calcium fluxes using a calcium-selective vibrating microelectrode.

The effects of aluminum on the concentration-dependent kinetics of Ca(2+) uptake were studied in two winter wheat (Triticum aestivum L.) cultivars, Al-tolerant Atlas 66 and Al-sensitive Scout 66. Seedlings were grown in 100 µM CaCl2 solution (pH 4.5) for 3 d. Subsequently, net Ca(2+) fluxes in intact roots were measured using a highly sensitive technique, employing a vibrating Ca(2+)-selective microelectrode. The kinetics of Ca(2+) uptake into cells of the root apex, for external Ca(2+) concentrations from 20 to 300 µM, were found to be quite similar for both cultivars in the absence of external Al; Ca(2+) transport could be described by Michaelis-Menten kinetics. When roots were exposed to solutions containing levels of Al that were toxic to Al-sensitive Scout 66 but not to Atlas 66 (5 to 20 µM total Al), a strong correlation was observed between Al toxicity and Al-induced inhibition of Ca(2+) absorption by root apices. For Scout 66, exposure to Al immediately and dramatically inhibited Ca(2+) uptake over the entire Ca(2+) concentration range used for these experiments. Kinetic analyses of the Al-Ca interactions in Scout 66 roots were consistent with competitive inhibition of Ca(2+) uptake by Al. For example, exposure of Scout 66 roots to increasing Al levels (from 0 to 10 µM) caused the K m for Ca(2+) uptake to increase with each rise in Al concentration, from approx. 100 µM in the absence of Al to approx. 300 µM in the presence of 10 µM Al, while having no effect on the V max. The same Al exposures had little effect on the kinetics of Ca(2+) uptake into roots of Atlas 66. The results of this study indicate that Al disruption of Ca(2+) transport at the root apex may play an important role in the mechanisms of Al toxicity in Al-sensitive wheat cultivars, and that differential Al tolerance may be associated with the ability of Ca(2+)-transport systems in cells of the root apex to resist disruption by potentially toxic levels of Al in the soil solution.

Plant contents of magnesium, calcium and potassium in relation to ruminant nutrition.

Grass tetany and wheat pasture poisoning are metabolic diseases of mature, lactating beef cattle. In some other countries, similar problems occur with dairy cattle. In grass tetany, the animals generally are grazing cool-season forages in which Mg concentration or bioavailability of plant Mg is low. Levels of Mg in the blood serum also generally are low. Grass tetany can occur in beef cattle fed hay made from grass, small grains or alfalfa Medicago sativa L.). In wheat pasture poisoning where animals are grazing small grains forages, blood levels of Ca often are low, and blood Mg also may be low. Both grass tetany and wheat pasture poisoning occur when plants are growing rapidly in the spring, at the time of heavy lactation demand by ruminants for Mg and Ca. When the temperature increases and plants start to grow rapidly in the spring, concentrations of K, N, organic acids, and the ratio of K/(Ca + Mg) all increase, and the percent dry matter decreases. Much research remains to be done to understand all the soil, plant, and animal aspects of grass tetany and wheat pasture poisoning.

Relation of soil and plant magnesium to nutrition of animals and man.

The importance of Mg for the production and quality of crops grown on soils, as well as the health and productivity of animals and humans that consume these crops, is the focus of this paper. The Mg requirements and resulting concentrations of most crops are sufficiently high that primary Mg deficiency in animals consuming these crops is rare, providing the crops receive adequate Mg from soil solution for normal growth. Hypomagnesemic grass tetany or conditioned Mg deficiency occurs in mature ruminant lactating females grazing cool season forages. Grass tetany occurrence is seasonal, and is often related to heavy lactation demand for Mg. Another factor is reduced bioavailability of Mg to the ruminant associated with high concentrations of crude protein and K, as well as associated changes in plant constituents such as lowered soluble carbohydrate content and increased levels of higher fatty acids and organic acids. The high water content in the immature grass tissue being grazed may also result in less efficient Mg absorption by animals. Grass tetany studies at Watkinsville, Ga., revealed that high N fertilization of tall fescue pastures from poultry manure, or inorganic fertilizer increased incidence of hypomagnesemic grass tetany, and hypomagnesemia. Regression analyses indicate highly significant correlations between Mg levels in blood serum and forage Mg, K and crude protein. The results between years and experimental pastures clearly suggested that individual years and experimental pasture relationships were different. The following was concluded with regard to the relationship of soil and plant Mg to human nutrition: first, surveys of agricultural raw products indicate that genetic factors (species and cultivars) appear to have more effect on plant Mg composition than do soil and environmental factors. Second, patterns of Mg intake by humans in the USA suggest that about 50% of their intake is from foods of plant origin. About half of this intake is from vegetables, where increases in Mg concentration may be achieved by changing cultural practices. While direct supplementation with Mg appears more efficacious in prevention of Mg deficiency in humans, research with susceptible ruminants indicates that complete protection can only be assured by adequate daily intake of bioavailable Mg in their consumed food. Similar situations would be expected to prevail in humans.

Wheat pasture poisoning. I. An evaluation of cereal pastures as related to tetany in beef cows.

Forage samples were obtained from three wheat and rye mixed pastures (replications) from December 1979, to April 1980 (140 d), while they were being grazed by mature cows at the Southwestern Livestock and Forage Research Station, El Reno, Oklahoma. These samples were taken three times during December, twice monthly in January and February and weekly in March and April. Samples were analyzed for dry matter, ash, N, K, Ca, Mg, P, ash alkalinity, aconitic, malic and citric acids, total lipids, NO3-N, Na and total nonstructural carbohydrates (TNC). In vitro digestibility was also determined. The N:TNC ratio, estimated plasma Mg levels and K:(Ca + Mg) ratio (tetany ratio) were calculated. On d 105 (March 19), 16% (five of 32 head) of the cows developed tetany. At tetany, the forage K, protein, digestibility, ash alkalinity, aconitic acid and total lipids increased suddenly and markedly. Forage dry matter, TNC and ash decreased. Forage Ca and Mg were slightly below or equal to the animals' requirements and remained relatively constant during the period of tetany. Forage P increased about 2 wk before tetany and decreased after tetany occurred. The levels of forage NO3-N and Na did not appear to be related to the incidence of tetany. The N:TNC ratios exceeded .4 immediately before tetany and two sampling periods thereafter. The estimated plasma Mg levels were lowest at tetany and shortly thereafter and tetany ratio exceeded 2.2 during most of the study, with peaks of 3.2 in December, at tetany and in early April.

Magnesium absorption from leafy vegetables intrinsically labeled with the stable isotope 26Mg.

Five leafy vegetables were grown in nutrient solutions in which the natural magnesium was replaced by the stable isotope, 26Mg. They were fed to rats in a test meal together with the extrinsic tracer 28Mg: a) to determine to what extent the instrinsic tracer (26Mg) was exchangeable with extrinsic 28Mg during the digestion and absorption processes, and b) measure the relative Mg availability from the different vegetables. The two tracers, 26Mg and 28Mg, were close to 100% exchangeable, as judged by the ratio of 26Mg/28Mg in the livers. Mean relative Mg absorption from the various vegetables ranged from 108 to 118% of the Mg absorbed from a standard test meal containing MgSO4. There were no statistically significant differences between the rates of Mg absorption from the five vegetables although two of the vegetables tested contained oxalate. The usefulness of stable 26Mg as a tracer in Mg bioavailability tests is discussed.